Pollinators are a major part of global biodiversity and they provide ecosystem services important for the production of many crops. Their abundance and diversity have declined steadily in recent years. Loss of foraging resources through degradation and fragmentation of natural habitats has been a major factor. Enhancing floral resources in the environment can mitigate this decline. Cowpea nectar has been reported to make the crop attractive to pollinators. We evaluated twenty-four cowpea varieties for pollinator abundance and diversity using pan traps, sticky traps, and direct visual counts. Sticky traps captured the highest number of pollinators and pan traps the least. The highest number of pollinators was recorded on Penny Rile cowpea and the lowest on Iron and Clay which had no flowers. Whippoorwill had the most flowers and ranked third in number of pollinators. Our findings indicate that cowpeas can be used to improve pollinator efficiency. Intercropping pollinator-dependent crops with cowpea varieties such as Penny Rile, Dixielee, and Whippoorwill will not only provide resources for the pollinators but can also be effective in increasing pollinator number and activity to increase crop yields.r2 = 0.3; p = 0.009). Cowpea can increase resources for pollinators and could be used to improve pollinator abundance and diversity in different farming systems.Pollinators are on the decline and loss of flower resources play a major role. This raises concerns regarding production of insect-pollinated crops and therefore food security. There is urgency to mitigate the decline through creation of farming systems that encourage flower-rich habitats. Cowpea is a crop that produces pollen and nectar attractive to pollinators. Twenty-four cowpea varieties were planted, and the number of pollinators were counted using three sampling methods: pan traps, sticky traps, and direct visual counts. Five pollinator types , 11 and 16 pollinator families were recorded from direct visual counts, pan and sticky traps, respectively. Pollinator distribution varied significantly among varieties and sampling methods, with highest number on Penny Rile (546.0 ± 38.6) and lowest (214.8 ± 29.2) in Iron and Clay. Sticky traps accounted for 45%, direct visual counts (31%), and pan traps (23%) of pollinators. Pollinators captured by pan traps were more diverse than the other methods. The relationship between number of pollinators and number of flowers was significant ( Pollinators are a key component of global biodiversity, providing vital ecosystem services to crops and wild plants. There is evidence that pollination of crops benefit food production through yield quantity and/or quality ,2,3,4. GVigna unguiculata (L.) Walp., have been reported to be attractive to pollinators [As a result of these findings, the United States Department of Agriculture  has promlinators . Cowpea,linators ,21 and, linators ,23. It ilinators ,25 becaulinators . An incrlinators ,28,29,30linators , amarantlinators , maize wlinators , and caslinators ,35. ThesCowpea floral resources, especially nectar, are attractive to pollinators . InformaIn 2017, a field experiment was carried out at the North Carolina Agricultural and Technical State University (NCA & TSU) Research Farm in Greensboro, North Carolina, USA . The treatments used for this study included twenty-four cowpea varieties . These cowpeas are among the most popular varieties in many southern states. Seeds were obtained from commercial seed houses. The experiment was set up as a randomized complete block design (RCBD) with four replications and each experimental unit consisted of two 5 m row of each cowpea variety. The seeds were planted manually at 0.01–0.015 m below the soil surface, 0.4 m apart in row and 1 m between rows from 22 to 25 May 2017. General agronomic practices such as weed control (weed eater) and irrigation (drip irrigation system) were carried out as needed. No insecticide and fertilizer were applied.At the beginning of flowering, plants were monitored daily. For each cowpea variety, data was recorded on the number of flowers and the number of days to first flower. The number of days to when the first pod was observed was also recorded for each variety.Three sampling methods  were used to sample pollinators. Cowpeas flowering started six weeks after planting. Sampling took place on 3, 10 and 14 July 2017 for sticky traps, pan traps, and direct visual counts, respectively, and continued weekly for five weeks.A total of six weeks after planting, five random flowering plants for each variety were selected and the number of flowers on each plant counted and recorded. A total of twenty-five fresh pods at the R6 stage (50% of pods with fully developed seeds) from each cowpea variety were harvested and weight recorded.Pollinators were sampled using three colored  pan traps. Sampling began on 10 July 2017 and was repeated weekly for five weeks. Traps consisted of 16 oz. squat polypropylene deli bowls  painted with UV-bright fluorescent blue (blue trap) or yellow paint (yellow trap) and unpainted 12 oz. white styrofoam bowls (white trap) . Trap setup was made by gluing individual unpainted 16 oz. polypropylene deli bowls onto a 36” plant prop and three of these placed 0.2 m apart between the 2 rows of each treatment during the entire sampling period. Each colored bowl  was then placed inside one of the unpainted bowls on the prop and filled with approximately 250 mL of soapy water solution . The traps were placed such that they were at the same level as the crop canopy. Traps were set out weekly from 13 July to 10 August between 8:00–10:00 a.m. and removed after 24 h. Each colored pan trap was drained, and contents placed in vials containing 70% ethanol and taken to the laboratory where they were stored in a refrigerator for later identification with a microscope  to the family level. Traps were collected in the order they were placed to ensure that all traps were available to insects for roughly the same duration.2)  adhered to a metal plant stake and placed at the center of each 5 m row. This was aimed at capturing smaller insects that may have been missed with visual and pan trap sampling. They also provided an alternative sampling method for comparison. We adjusted trap height to keep them just below canopy height. Traps were removed after 24 h and replaced weekly from 3 July to 9 August. All samples were transferred into labelled Ziploc® bags and taken to the laboratory where they were identified to the family level using a microscope .Sticky trap sampling began on 3 July 2017 and was carried out by placing one two-sided yellow sticky trap . For each sampling method, cowpea variety, weekly totals were calculated for pollinator types/families and used to determine abundance per treatment. The data were first tested for the assumptions of ANOVA. Where, the assumptions of ANOVA were violated, the data were further transformed and tested again for the assumptions of ANOVA. Since the transformed data improved the data in terms of meeting the assumption of normal distribution, the transformed data were then analyzed using a mixed model with sampling week, sampling method , treatment (cowpea variety), and the interactions between sampling method and treatment as fixed effects and sampling week as a random effect. If the interaction between sampling week, sampling method and treatment was not significant, the data were pooled  and analyzed by sampling week, sampling method and treatments using ANOVA. On the other hand, if interaction between sampling week, sampling method, and treatment was significant, the data was analyzed separately for each sampling week for the trap type and treatment using ANOVA. Means were separated using Tukey–Kramer Honestly Significant Difference at pi ln(pi)The diversity of pollinators reported from pan traps, sticky traps, and through direct visual counts of different cowpea varieties was assessed using the Shannon–Weaver Diversity Index (H′) . The Indwhere H’ = Shannon–Weaver diversity indexpi = the proportion of individuals found in species ipi estimated as: pi = ni/Nwhere ni = number of individuals in species iN = total number of individuals in the communityThe Shannon Equitability (Evenness) Index (E) normalizes the Shannon Diversity Index (H’) to a value between 0 and 1. It provides an idea about the evenness of the distribution of groups of organisms in a community. An index value of 1 means that all groups have the same frequency.The Evenness Index (E), is expressed as: E = H′/log(k)where E = Evenness,H′ = Shannon–Weaver Diversity Indexk = number of species/groups in the communityFor each sampling method , the total number of pollinators in each family or pollinator types captured on each variety for the entire sampling period was used to calculate the Diversity and Evenness indices.F21, 66 = 2.83, p = 0.0007) among the varieties. The first flower was observed on Early Scarlet 42.0 ± 1.2 days after planting. Purple Hull Big Boy and CBE5 were the last to flower at 55.5 ± 2.1 and 55.5 ± 3.7 days after planting, respectively. There were significant differences  among the varieties in the number of flowers recorded. No flower was recorded on Tohono O’Odham and Iron and Clay varieties. The lowest number of flowers were recorded on CBE5 (18.6 ± 1.5) and Purple Hull Big Boy (19.2 ± 1.3). Whippoorwill (56.4 ± 4.2) and Early Scarlet (55.5 ± 3.9) had the highest number of flowers. It was observed that most of the varieties had purple flowers with about a third producing white flowers .The number of days to flower, number of flowers, days to first podding, and color of flowers for each variety are presented in  flowers . Early SThe mixed model analyses revealed significant effects of week, sampling methods and treatment effect on pollinators captured . Based op = 0.02) among the 24 cowpea varieties with high numbers 10.0 ± 4.1, 8.5 ± 3.3 and 6.5 ± 4.6 recorded on Penny Rile, Whippoorwill Steel Black and Dixielee, respectively. Even though Halictidae was the most abundant pollinator family in the yellow pan trap on each of the 24 cowpea varieties, this number was significantly different (p = 0.02) among the cowpea varieties with more recorded on Penny Rile (17.5 ± 49) and Black Crowder (17.3 ± 3.2). In addition, there was a significant difference (p = 0.03) in the number of Crabronidae among the varieties with the highest capture recorded on Penny Rile (7.3 ± 2.1) and Dixielee (5.5 ± 1.8). For all the insect families captured in the blue colored pan trap, there was no significant difference in the number of pollinators recorded among the 24 cowpea varieties.Eleven pollinator families were identified from the three colored pan traps deployed on the twenty-four cowpea varieties. These were Andrenidae, Apidae, Crabronidae, Formicidae, Halictidae, Hesperiidae, Pompilidae, Sphecidae, Syrphidae, Tachinidae, and Vespidae. Of these families, Apidae, Crabronidae, Halictidae, and Tachinidae, were the four most abundant families . The othF23, 72 = 1.52, p = 0.09) among the varieties nonetheless, Whippoorwill Steel Black recorded the highest number (82.5 ± 15.6) and Running Conch the lowest (29.0 ± 5.4). Among the traps, blue pan trap recorded the lowest number of pollinators on each of the 24 cowpeas  in the number of pollinators among the varieties. The numbers ranged from 5.3 ± 1.7 on Tohono O’Odham to 21.3 ± 7.0 on Black Crowder  among the 24 cowpea varieties. However, Black Crowder recorded the highest number (35.5 ± 7.5) and Tohono O’Odham the least (9.3 ± 1.3)  and ranged from 50.8 ± 10.1 on Running Conch to 134.5 ± 29.3 on Whippoorwill Steel Black  c. Combinel Black d.F4, 15 = 21.8, p < 0.0001) among the sampling weeks, with a steady increase from the first sampling week (146.0 ± 26.2) to the last (635.3 ± 75.2) . However, during the second through the fifth week, white pan traps captured significantly (p < 0.01) more pollinators compared to the other traps (Combined pollinator catches for each sampling week indicated a significant difference ( ± 75.2) a. In theer traps a. Differer traps a. Howeveer traps a.F2, 9 = 99.1, p < 0.0001), among the three colored pan traps with white pan trap capturing the highest (1330.5 ± 83.6) number of pollinators and the least (295.3 ± 12.3) in blue colored pan trap  of pollinators were recorded among the 24 cowpea varieties, the highest number recorded on Vietnamese Black (50.0 ± 14.1) and Zipper Cream (49.8 ± 6.9)  from what was recorded on the other varieties  among the cowpeas at this time  among the cowpea varieties. The final sampling week has the most diverse pollinator families recorded   among the varieties (Pollinators captured on sticky traps on each of the 24 cowpea varieties for each sampling week is presented in 8 ± 6.9) a. This s8 ± 6.9) a. In thearieties b. By weehis time c. Similahis time d that in4 total) e. The hiarieties e.F4, 15 = 11.0, p = 0.0002) among the five sampling weeks with the lowest (423.5 ± 67.8) in week four followed by (522.5 ± 55.7) in week five, then (849.3 ± 127.645.0) in three and (945.0 ± 72.6) in week one and the highest (1392.0 ± 194.1) in week two. In addition, five pollinator families  were prominent and captured every week.Overall, the total number of pollinators captured from all 24 cowpea varieties each week was significantly different  among the 24 cowpea varieties  among the five weekly samples.The weekly visual direct counts of the distribution and diversity of pollinators on the 24 cowpea varieties presented in F23, 72 = 15.42, p < 0.0001) in the number of pollinators among the cowpea varieties  among the different sampling methods used. The highest number of pollinators was recorded in sticky traps (4132.3 ± 386.2), and the lowest number was captured in pan traps (2119.0 ± 104.7). Visual observation recorded (2853.8 ± 145.2) pollinators  in the number of pollinators among the 24 cowpea varieties. The highest number of pollinators was recorded on Penny Rile (546.0 ± 38.6), and the lowest number was recorded on Iron and Clay (214.8 ± 29.2)  .A total of 16,529 insects representing 16 families were collected from sticky traps on all the 24 cowpea varieties over the entire sampling period revealed. The number of Tachinidae recorded on each cowpea variety averaged about 589 insects compared to an average range of 0.1 to 38 insects per variety for the remaining families. Therefore, Tachinidae was not included in the Diversity Index calculations. A total of 2389 insects representing 15 families was used. From visual observations, 11,415 insects, representing five pollinator types, were recorded. Of the 11 insect families identified from all pan traps, the number of Halictidae recorded on each cowpea variety averaged about 183 insects compared to an average range of 2–59 insects per cowpea variety for the other families. The family Halictidae was, therefore, not included in the Diversity Index calculations; a total of 4078 insects representing 10 families was used. The Diversity index (H’) and Evenness Indices (E) of insects captured in pan traps, sticky traps or counted visually on each of the cowpea varieties are shown on F1, 20 = 8.13, p = 0.01), with a correlation (r) of 0.54. The results of the regression indicated that approximately 30% of the variation in the number of pollinators is explained by the number of flowers. The relationship between yield (as pod weight) and the number of pollinators (r = 0.06), non-significant  relationship between yield and the number of pollinators.A linear regression was calculated to show the relationship between the number of flowers and the number of pollinators captured in traps a. The nulinators b is weakWe have demonstrated in this study, through the use of three sampling methods including pan traps, sticky traps and direct visual counts, that some cowpea varieties could be used to increase and improve the abundance and diversity of pollinators. Each method provided a unique picture of pollinator diversity and abundance among the cowpeas. The most prominent pollinator in pan traps was Halictidae, while Tachinidae was the most abundant in sticky cards, and honey bees the most recorded from direct visual counts. The reason for this may likely be because honey bees are easily identifiable and large enough to be counted. Nonetheless, these findings confirm previous studies that reported that honey bees were the most abundant pollinators on cowpeas and other crops from direct visual counts ,42,43,44Among the three colored traps used in our study, there were differences in pollinator abundance and diversity among cowpea varieties. White pan traps caught the highest number of pollinators on each of the 24 cowpea varieties b and amoPollination of flowering plants represents a critical ecosystem service. Plant–pollinator interactions are usually mutual, with pollinators foraging for floral resources such as nectar and pollen that are used as food and in the process transfer pollen from anthers to stigmas thereby facilitating plant reproduction. About 75% of crops rely partially or completely on insects for pollination, accounting for ~$600 billion worth of annual global food production . The decAlthough many insect species are known to provide pollination services, honey bees and other bees are often assumed to be the main providers of these services in agricultural ecosystems . For exaVarroa destructor and a number of other pests and diseases [Prescottia densiflora, a flowering plant from the orchid family [The global reliance on honey bees as the single most effective pollinator species is a risky strategy, especially given the current global trends showing a decline in honey bees resulting from poor nutrition, ectoparasitic mite diseases . Howeverdiseases , pollinadiseases . Halictidiseases , and in d family . Some std family ,61 and od family . Meanwhid family . In suchFlower color is one of the most important visual traits exploited by pollinating insects . The disOther workers have suggested that flower number (resource concentration) may also be critical in attraction ,71,72,73Pan traps and sticky traps remain effective sampling tools for estimating the diversity of insects such as pollinators. However, direct visual counts are a quicker sampling method when targeting specific insect groups that are easily assessed. A combination of these sampling methods may provide detailed information on insect diversity in the community and thus particularly useful in pollination studies. Cowpea is a crop that has traditionally been grown as an intercrop, mainly as a source of protein. In some cases, where cowpea has been used as an intercrop, it serves to manage insect pests and diseases as well as improve soil fertility and control weeds, culminating in increased crop yield . Its use


School health has been considered as a high priority intervention in developing countries but it has not been prioritized in Nepal. The objectives of the study are to find out the prevalence of morbidity and nutritional status in school children.A descriptive cross-sectional study was conducted in a private school located at Panauti Municipality of Kavrepalanchowk district for one week of November 2019. A convenient sampling technique was used. From a selected school, a total number of 356 students studying from Grade I to X were included in the study using. Confidence Interval at 95% was calculated along with frequency and proportion for binary data using Statistical Package for Social Science (SPSS) software.Among 356 students, the most common three morbidities were dental caries 43 (18.1%), tonsillitis 26 (6.2 %), and headache 18 (7.1 %). Based on weight for age, 43 (23.9%) boys and 22 (12.5%) girls were underweight and 12 (6.7%) boys and 4 (2.7%) girls were overweight and 6 (2%) were obese. Thus school health programs should give more emphasis on oral health, nutrition, personal hygiene, etc.The health and nutritional status of school children in this study were found to be satisfactory compared to other studies. The present study emphasized oral health. The school health program is important in the school for the prevention of diseases like a parasitic infestation, improving personal hygiene, and nutritional status. Nepal's poverty was 21% in 2018.2 Poor people are more likely to be affected by different forms of malnutrition. Malnutrition increases health care costs, reduces productivity and slows economic growth, which can perpetuate a cycle of poverty and ill health.3World Health Organization's Global School Health Initiative, seeks to mobilize and strengthen health promotion and education activities at the local, national, regional and global levels. The Initiative is designed to improve the health of students, school personnel, families and other members of the community through schools.The objectives of the study are to find out the prevalence of morbidity and nutritional status in school children.A descriptive cross-sectional study was conducted in a school of Panauti Municipality, Kavrepalanchowk district for 10th to 15th November 2019. Ethical approval was taken from the Institutional Review Committee of Kathmandu Medical College. The purpose of the study was explained to the school Principal and permission was taken from the Principal and parents of students to conduct the study. Students studying from grade I to X were taken into the study. Those who were absent or were not willing to participate were excluded from the study.The structured questionnaire was developed. The questionnaire consisted of demographic information, physical and clinical examination. The instruments used were a weighing machine and measuring tape. Each child's weight was measured in kilograms. The scale was calibrated against known weight regularly. Zero error was checked for and removed every day if present. Students wore only light clothes while measuring weight in the classroom. The weights were recorded to the nearest 100g. Height in centimeters was marked on the wall in school with the help of a measuring tape. The students were asked to remove their shoes and stand with heels together and head positioned in such a way that the line of vision was perpendicular to the body. A scale was brought down to the topmost point on the head of students standing against the wall where the calibration was done. The height was measured to the nearest 0.5cm. Health Examination of all students underwent a thorough physical and systemic examination including medical history. The Convenient sampling technique was used. Selection bias and measurement bias has been addressed as far as possible.Collected data were entered into Microsoft Excel. Data analysis was done using SPSS version 20. Frequency and percentage were calculated. BMI for age was computed using Group BMI Calculator, Metric, v1.0-CDC.Out of 356 students, the morbidity was present in 101 (28.37%) school children, commonest was Dental caries 42 (11.08 %), and followed by Tonsillitis 26 (7.3%). Headache and Eye problems were found to be 18 (5.06%) and 17 (4.78%) school children respectively. The morbidities were found higher in boys compared to girls except for ear problems .There were 180 (50.6%) boys and 176 (49.4%) girls included in the study. The mean age of students was 11.38±2.83 years. The age of the students ranged from 6 to 17 years . The majThe mean and standard deviation of height, weight, and Body Mass Index (BMI) were 1.40m±0.16m, 33.82 kg±12.01kg and 16.53 kg/m2 ±2.98 kg/m2 respectively. The proportion of school children with normal BMI was 269 (76%), out of which 124 (68.9%) were boys and 145 (82.4%) were girls. Sixteen (4.5%) of the students were overweight, 12 (6.7%) were boys. Under the obese category, there were 6 (1.7%) students in which 5 (2.8%) were boys .4 The study conducted by Gokhale et al. found that the most common morbidities were dental caries (61.1%) followed by ear wax (38.1%), URTI (19%) and Ear discharge (13.5%).5 The study conducted by Mayavati et al. observed dental caries (65.1%), upper respiratory tract infections (38.2%), ear wax (29.9%) and myopia (10.0%) in urban school children.6 A study conducted in Madhya Pradesh, India by Shinde reported that 31.83% were having morbidity associated with dental carries followed by anemia (15.69%), vitamin A deficiency (6.25%), worm infestation (4.94%), scabies (4.06%), URI (3.77%), defective vision (0.58%), conjunctivitis (0.44%) and congenital malformation (0.15%).7 A study conducted by Bhandari et al. in Kavrepalanchowk district reported pediculosis 42 (26.2 %), dental caries 29 (18.1%), and waxy ear 27 (17.1 %) were three major morbidity in school children.8 Syed et al. conducted the study in Hyderabad, India found most common morbidities were Dental caries (56.24%), anemia (33.8%), worm infestation (48.25%) and Refractive error (11.22%).9 Kalyani et al. found most common morbidities were anemia (40%), Dental caries (61.6%), and pediculosis (40%). URT infection (32.8%) Refractive error (4%).10The present study was carried out to find out the morbidity patterns of the school children. School health survey gives a good chance to screen a large number of school children with minimum resources. In present study dental caries was found in 11.08%, followed by Tonsillitis 7.3%, Headache 5.06% and Eye problem 4.78%. The survey conducted by Shrestha et al. in Pokhara, found the prevalence of dental caries (41.5%), worm infestation (33.7%) and pediculosis (32.6%) in both sexes in six governmental primary school.11 Kausar et al,12 Shakya et al.13These results were higher than that of the present study findings where dental caries was 11.08%, followed by Tonsillitis 7.3%, Headache 5.06% and Eye problem 4.78%. These differences may be due to the fact that the present study conducted in only a private school and improving health status as change with time period. The present study results were consistent with a study conducted by Pandey et al,14 In Nepal, more than 80% of the population lives in villages. Nepal Demographic and Health Survey, 2016 reported that 27% of children under 5 years were underweight, and 5% were severely underweight. The proportion of children who were underweight was greater in rural areas (31%) than urban areas (23%). Children of mothers with no education were more than twice as likely to be underweight as children whose mothers were educated up to SLC level. Underweight was inversely related to wealth quintile; 33% of children in the lowest wealth quintile were underweight, as compared with 12% of children in the highest quintile. 10% of children were wasted and 2% severely wasted. 36% of children were stunted, and 12% were severely stunted in Nepal .15 A survey done by Shrestha et al found that the prevalence of wasting, stunting and malnutrition in the school children were 10.3%, 14.9% and 85% respectively with 31.8% of boys and 89.4% of girls students were malnourished.4 Study conducted by Bhandari et al found that 55.3% boys and 35.8% girls fell under 1st-degree malnutrition and 23.07% boys and 46.3% girls fell under 2nd-degree malnutrition, 7.2% girls fell under 3rd-degree malnutrition based on weight for age.8 The study conducted by Gokhale et al. found that the majority of school children (69.8%) were normal and 7.1% were severely underweight.5 The study carried out in Sullia town, South India by Amruth et al revealed the prevalence of underweight and stunting were 26.5% and 19.2% respectively.16 The nutritional status of government school children was comparatively poorer than private school children. Boys had a higher prevalence of malnutrition than girls. These findings were consistent with the present study where 24% of children were underweight and boys were more underweight than girls.In the World, the prevalence of malnutrition in term of Underweight, stunting and wasting are 27%, 31% and 10% respectively.Gokhale et al. conducted the study in Maharashtra, India found 73% children had some morbidity. A study conducted in Madhya Pradesh, India reported morbidities were present in 58.72% of school children. The present study found that morbidities were present in 28.37% school children which were lower as compared to the other studies. This difference probably may be due to the present study conducted in grade I to X of urban area and in a private school where children were from educated and well up families. Overall present health status is also improving compared to past health status.Among different morbidities, dental caries was found more common. The health and nutritional status of school children were found to be satisfactory in this study compared to other studies. The present study emphasized the need for the initiation of oral health programs in schools. The study also reflects the importance of improving personal hygiene, prevention of disease like parasitic infection/infestation and improvement of their nutritional status. Today's children are the future of the country and the development of a country depends upon the development of children thus we should invest in school children.


IntroductionIn this study, we reported a single surgeon experience of total laparoscopic hysterectomy (TLH) in terms of intraoperative and early postoperative outcomes and complications. In addition, we compared our results with published literature for a reevaluation of complications and outcomes.Material and methodsThis present prospective study was conducted on 50 patients who underwent TLH due to benign causes. Patients diagnosed with abnormal uterine bleeding (AUB), uterine fibroids, and post-menopausal bleeding (PMB) were included in this analysis. Patients were discharged after 24 hours of surgery if there were considered fit for discharge. The patients' age, co-morbidities, size of the uterus, additional procedure along with TLH, and postoperative complications were collected and analyzed. The follow-up period was three months, done on the tenth day after surgery, the thirtieth day, and then at three months.ResultsThe mean age of our patients was 46.42±5.01 years. The major indication of hysterectomy was fibroids diagnosed in 27 (54.0%) patients and AUB in 18 (36.0%) patients. Out of 50, 10 (20.0%) patients had a previous cesarean section, and 4 (8.0%) had a bilateral tubal ligation (BTL). Mean surgery duration was 124.26±44.74 minutes. Mean hospital stay was 2.18±0.39 days. Total complications occurred in five (10.0%) patients, ureteric injury in one (2.0%) patient, port-site infections in 2 (4.0%), and vault infections in 2 (4.0%) patients.ConclusionTLH is a safe procedure and can be performed with minimal complications in patients with benign uterine etiology. After a cesarean section, total laparoscopic hysterectomy (TLH) is the second most common procedure in gynecology departments . AbnormaHowever, despite these benefits and acceptable clinical outcomes, TLH has not gained widespread importance. Even in the USA, the number of TLHs procedures has decreased instead of increasing. In the USA, the number of TLHs performed was 15.5% in 2006, and it reduced to 8.6% in 2010 . FurtherWith the passage of time, gynecology consultants have gained more experience, therefore, the operative time and complications of TLH have decreased. In the present study, we reported a single surgeon experience of TLH in terms of intraoperative and early postoperative outcomes and complications. In addition, we compared our results with published literature for the reevaluation of complications and outcomes after TLH.This present prospective study was conducted on 50 patients who underwent TLH due to benign causes. Patients diagnosed with AUB, uterine fibroids, and postmenopausal bleeding (PMB) were included in this analysis while cases with malignancy were excluded. The study duration was from August 2018 to December 2019. Before surgery, all patients were informed about TLH and its potential benefits and written consent was taken for participation in the study.Routine investigations, including complete clinical history, complete blood profile, ultrasonography, and biopsy studies, were done to rule out any malignant pathology.A single consultant performed all laparoscopic procedures. All procedures were done by a consultant gynecologist. The procedure was done after inducing general anesthesia by placing the patient in the lithotomy position, with legs spread apart with small flexion at the hip joint . The following steps were followed during hysterectomy: coagulation of upper pedicles, opening of vesicoperitoneal folds, skeletonization of uterine vessels, coagulation of vessels, colpotomy, followed by specimen retrieval vaginally. For keeping the pneumoperitonimum, vaginal occlusion was done. The vaginal vault was closed by continuous suturing by laparoscopy.After surgery, patients were discharged after 24 hours of surgery if they were considered as fit for discharge. The patients' age, co-morbidities, size of the uterus, additional procedure along with TLH, and postoperative complications were collected and analyzed. The follow-up period was three months, done on the tenth day after surgery, the thirtieth day, and then at three months.For data analysis, we used the Statistical Package for the Social Sciences (SPSS) v23 . The analysis of variance (ANOVA) test was applied to compare mean operative time and hospital stay in patients with isolated TLH and those with concomitant procedures by considering p-value ≤0.05 as significant statistically.The mean age of patients was 46.42±5.01 years. Hepatitis was the most common morbidity presented in nine (18%) patients; hypertension and diabetes mellitus presented in five (10%) and five (10%) patients, respectively. The major indication of hysterectomy was fibroids diagnosed in 27 (54%) patients and AUB in 18 (36%). The full etiologic spectrum is given in Table Total complications occurred in five (10%) patients; there were no major complications and only minor complications were diagnosed. Out of five, a ureteric injury occurred in one 2%) patient, port-site infections were diagnosed in two (4%), and vault infections in two (4%) patients. The mean hospital stay was 2.18±0.39 days (Table % patientThe mean hospital stay was not influenced by the type of surgical procedure Table .Laparoscopic procedures are routine practice in gynecology departments for nearly the last 30 years -9. In soIn the present study, we reported a single surgeon’s experience of intraoperative and postoperative outcomes of TLH in benign diseases. In the present study, the mean age was 46.42±5.01 years. Mereu et al. reported a mean age of 49.6 ± 6.5 years among patients who underwent TLH due to benign diseases . Shim etThe most common indication of TLH in our study was uterine fibroids presented in 54% cases and AUB in 36% cases. Agarwal et al. reported fibroids in 51.2% of patients, AUB in 28%, adenomyosis in 19.2%, and hyperplasia in 1.6% of patients who underwent TLH. While for TAH, they reported fibroids in 48%, AUB in 30.4%, adenomyosis in 20%, and hyperplasia in 5.6% of patients [In our study, the mean operative time was 124.26±44.74 minutes. Studies have reported differences in mean operative time. Kim et al. reported a mean operative’ time of 149.3 ± 59 minutes . Their oRegarding complications, we did not encounter any incidence of conversion to laparotomy and hospital mortality in our case series. There were only three types of complications that we encountered: ureteric injury in 2%, port-site infections in 4%, and vault infections in 4% of patients with an overall complication rate of 10%.A study by Mereu et al. reported bladder injury in 0.3%, and blood transfusion in 0.3% of patients, port-site hernia in 0.3%, wound infections in 1.1%, vaginal cuff dehiscence in 0.3%, and vaginal cuff bleeding in 0.8% after TLH. The overall complication rate was 4.4%. They did not report an incidence of conversion to laparotomy or any hospital mortality .A study by Agarwal et al. reported that years of experience of TLH has a significant influence on the complication rate of TLH. They reported complications of 14% in the first year of TLH, and it reduced to only 4.3% after three years of experience. The rate of conversion to laparotomy in the first year was 9.6%, and it decreased significantly within four years [A study by Twijnstra et al. reported a need for laparotomy in 4.8% of patients, bladder injury in 1.4%, ureteral injury in 0.4%, and bowel injury in 0.5% of patients .The mean hospital stay in our study was 2.18±0.39 days. Our length of stay was shorter when compared to Kim et al. and Wallwiener et al., who reported a hospital stay of 5.5±2.0 days and 4.9±2.8 days, respectively ,14. ThisThe major limitation of this study is its non-randomized nature and small sample size because of the low volume of patient flow for hysterectomies in our hospital. However, we have compared our results with international literature, which has enabled us to verify the outcomes of TLH in our patients.TLH is a safe procedure and can be performed with a minimal number of complications. Initial studies reported a higher complication rate, but the latest studies, including the present one, have found a very low rate of procedural complications. So this procedure can be adopted as the preferred approach in patients planned for hysterectomy due to benign diseases.


However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species . Specifically, the kinetic parameters Vmax, Km and intrinsic clearance (CLint) were determined for AFB1 dialdehyde reductase  and AFB1 monoalcohol reductase . In all cases, AFB1 monoalcohol reductase activity saturated at the highest aflatoxin B1 dialdehyde concentration tested (66.4 μM), whereas AFB1 dialdehyde reductase did not. Both activities were highly and significantly correlated and therefore are most likely catalyzed by the same AFAR enzyme. However, it appears that production of the AFB1 monoalcohol is favored over the AFB1 dialcohol. The production of alcohols from aflatoxin dialdehyde showed the highest enzymatic efficiency  in chickens, a species resistant to AFB1; however, it was also high in the turkey, a species with intermediate sensitivity; further, CLint values were lowest in another tolerant species (quail) and in the most sensitive poultry species (the duck). These results suggest that AFAR activity is related to resistance to the acute toxic effects of AFB1 only in chickens and ducks. Genetic selection of ducks for high AFAR activity could be a means to control aflatoxin sensitivity in this poultry species.Aflatoxin B Hepatic biotransformation of AFB1 by cytochrome P450 (CYP) enzymes produces aflatoxin B1-8,9-epoxide (AFBO) with two possible stereoisomers: aflatoxin B1-8,9-exo-epoxide and aflatoxin B1-8,9-endo-epoxide; only aflatoxin B1-8,9-exo-epoxide can react with DNA, producing adducts at position 7 of guanine leading to carcinogenesis /Km + [S], where v is the enzyme reaction velocity, [S] represents substrate concentration, Vmax represents maximal velocity and Km represents the Michaelis-Menten constant. Intrinsic clearance (CLint—mL/mg protein/minute) was calculated as the ratio Vmax/Km. The calculated CLint only applies for the selected enzymatic activity and not for the hepatic clearance, since AFAR enzyme was not purified from liver extracts. In all cases the kinetic parameters are “apparent” because hepatic extracts and not purified enzymes were used. Inter-species differences in enzymatic kinetic parameters were determined by using the Kruskal-Wallis test, while nonparametric multiple comparisons were made by using the Dwass-Steel-Critchlow-Fligner method, with a significance level of 5% (p <0.05). Correlations were estimated by using the Spearman’s rank-order correlation coefficient. All analyses were performed using the Statistical Analysis System software Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This manuscript provides an evidence that differences in the resistance of poultry species to aflatoxin B1 toxicity are associated with differences in AFAR activity.Please consider the following suggestions:Klein et al : 193-201.) show that AFAR acivity increases with age.In this study, chickens were 7 weeks old, turkeys and quails were 8 weeks old, and Peking Duck was 9 weeks old. Please explain whether these age differences affect AFAR activity.Different poultry species may have different feeds.Please explain the potential effects of different feed types, compositions, and feeding patterns on bioenzymatic activity.Ethoxyquin is an ingredient found in commercial feeds. It should be explained whether the diet used in this study contained ethoxyquin.L307-309: The present study... in chickens and ducks....in different poultry species?Reviewer #2: GENERAL COMMENTSThe MS provides a description of a study with potentially very important results. However, before the suggestions of selecting ducks with high AFAR activity, a larger variety of birds may need to be assessed to validate more clearly the population effect across a wider diversity of bird sources.Is there any evidence already that some ducks may be dramatically less susceptible to AF exposure than others?The limitation of the study should be included in the discussion.It is not clear why other routes of detoxification were not discussed and this should be addressed. For example, enzyme activity in other organs may add to that of the activity in the liver (eg GST activity in the kidney).SPECIFIC COMMENTSTITLEChange tolerance to tolerant: “… in poultry species TOLERANT to AFB1 toxic effects”ABSTRACTI suggest that the abstract would invite greater interest from potential readers if it stated more clearly WHAT IS KNOWN and WHAT IS ADDED BY THIS STUDY. It is an important and interesting subject, but one that is familiar to a very limited audience, an audience that could be increased by acknowledging that they are not abreast of the AFB1 literature.As captured later, please consider and respond to the following points:Intrinsic clearance: from a pharmacological perspective, clearance relates to a particular organ. In the biochemical context that is described in the MS it applies only to the selected enzyme. This should be made very clear, as hepatic clearance may be far higher than a single enzyme clearance if more than one enzyme is involved.Highest AF dialdehyde concentration tested (66mcM): how does this concentration relate to the expected exposure concentration in intoxicated birds?The same AFAR enzyme catalyses mono- and di-alcohol AFB1: is this the same enzyme or the same or related or linked enzyme?AFAR activity related to toxic effects in chickens and ducks – not quail and turkey: does not this imply that the relationship is complex and potentially multifactorial in all species?INTRODUCTIONNo comments MATERIALS AND METHODSLines 100-104Details of the breed/source of the turkeys, quail, and Pekin ducks should be provided, especially to allow comparison with any further studies of in vitro or in vivo activity of AFB1.In addition, the composition of the diet of the birds prior to sacrifice should be described, especially the presence of any medications in the diet. For example, it would be common practice to include anticoccidial agents in the diet of chickens and special anticoccidial agents can cause changes in hepatic enzyme activity. Similarly, the main nutritional ingredients in the diet should be identified.Line 128Is there a normal avian body temperature? Measurement of adult/mature bird body temperature has found a temperature of 41 for chickens, 41 for turkeys and 42 for ducks. Your comments on choice of a single temperature for all species and justification for the selection of 39 would be welcome. Younger birds may have a temperature closer to 39.Lines 150-160The statistical significance level should be presented and the actual value in each test presented later in the MSLine 155Intrinsic clearance – the units should be provided. In addition, as each species has a different mass of liver tissue, the total liver clearance for the enzyme of interest is a useful parameter.RESULTSFig 2 and Fig 3 CaptionsWhy is the difference in MW of monoalcohol and dialcohol 2 units and not 1 unit?DISCUSSIONLine 248… its capacity to FORM adduct …Line 264The fact that there is an apparent relationship between AFAR activity and AFB1 susceptibility only in ducks and chickens, and not in quail and turkeys, suggests that toxicity may be multifactorial. Some discussion of this would add increased credibility to the study.Line 273-274Does the correlation described indicate that the same enzyme is involved or suggest that the enzyme activities are related or linked? Why is the only conclusion that they are the same enzyme?Line 276How representative of the concentration present in the liver of intoxicated birds is the highest concentration of AFBI-dialdehyde of 66mcM?Line 298Is the cytotoxic effect of AFB1 exposure in ducks DUE TO or ASSOCIATED WITH the lack of a detoxification pathway? What is the status of GST detoxification pathways in ducks?CONCLUSIONLine 317The mention of individuals with high activity may relate to the SD values in Table 1. It would be useful to provide the individual bird values from which the Table was compiled – in this way the variation by individual birds and the number of birds with significant variation can be seen – this could be included in a supplement.Line 318-Line 319Genetic selection would only be useful if there was a relationship between intoxication and enzyme activity. This would need to be established for the duck and may require challenge of ducks at the extremes (ie high and low) of enzyme activity with AF to see if in fact there is an in vitro in vivo (IVIV) relationship. Furthermore, only a very small sample of ducks from an unknown source has been studied and described herein.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  15 Apr 2020Reviewer #1: This manuscript provides an evidence that differences in the resistance of poultry species to aflatoxin B1 toxicity are associated with differences in AFAR activity.Please consider the following suggestions:Klein et al : 193-201.) show that AFAR activity increases with age.In this study, chickens were 7 weeks old, turkeys and quails were 8 weeks old, and Peking Duck was 9 weeks old. Please explain whether these age differences affect AFAR activity.Reply: We are in complete agreement with the reviewer about the changes on the expression levels of AFAR enzyme associated with the bird’s age, as it was reported by Klein et al. (2002). Unfortunately, the study of Klein et al. (2002) does not present western immunoblot densitometry values to compare between ages; however, their data suggest that AFAR expression reaches a maximum level in turkeys at 41 days of age. Further, other two key enzymes involved in xenobiotic biotransformation (cytochrome P450s and glutathione sulfotransferases) also seem to reach a maximum enzyme activity at 41 days of age in turkeys.Since the turkeys used in our study were 8 weeks old (56 day-old), they were expected to have reached their maximum AFAR activity. To our knowledge there are no data regarding the relationship between age and AFAR activity in the other poultry species studied. A sentence has been inserted indicating the fact that AFAR expression and activity change with age and that consideration must be given to this fact when analyzing the results of the present trial (lines 297 - 300).Different poultry species may have different feeds.Please explain the potential effects of different feed types, compositions, and feeding patterns on bioenzymatic activity.Reply: We agree with the reviewer in that different types of feed may affect the patterns of bioenzymatic activity. However, all diets were formulated with the same macro-ingredients , adjusted for the specific requirements of each poultry species. Therefore, we do not expect an effect of the diet on the results obtained.Ethoxyquin is an ingredient found in commercial feeds. It should be explained whether the diet used in this study contained ethoxyquin.Reply: No ethoxyquin was added to the diets provided to the experimental birds.L307-309: The present study... in chickens and ducks. ...in different poultry species?Reply: The words “in chickens and ducks” have been replaced by “in different poultry species” (lines 335 - 336). Reviewer #2: GENERAL COMMENTSThe MS provides a description of a study with potentially very important results. However, before the suggestions of selecting ducks with high AFAR activity, a larger variety of birds may need to be assessed to validate more clearly the population effect across a wider diversity of bird sources. Is there any evidence already that some ducks may be dramatically less susceptible to AF exposure than others?The limitation of the study should be included in the discussion.Reply: We agree with the reviewer in that the population of ducks used in our study is very limited. However, we chose the white Pekin duck in our study, which it is the type of duck most commonly used in commercial duck meat and egg production. We have added a sentence addressing this issue in the revised manuscript (lines 347 - 353).It is not clear why other routes of detoxification were not discussed and this should be addressed. For example, enzyme activity in other organs may add to that of the activity in the liver (eg GST activity in the kidney).Reply: We completely agree with the reviewer in that there are other routes of detoxication that are also important in AF metabolism. However, due to the fact that aflatoxin exposure is through the gastrointestinal tract and because all of the absorbed compounds from the GIT must pass first through the liver , it is the liver the organ that is most important in AF biotransformation and detoxication. GST detoxication of the electrophilic metabolite of AFB1  is a key detoxication pathway that is also being investigated by our research group. What we want to point out in the present study is that tolerant poultry species like the chicken have a higher AFAR enzyme activity than sensitive poultry species like the duck. We do not intend to ignore the possible role of other metabolic pathways in AFB1 sensitivity. We only want to postulate that AFAR activity is probably contributing to the higher resistance of chickens to the adverse effects of AFB1.SPECIFIC COMMENTSTITLEChange tolerance to tolerant: “… in poultry species TOLERANT to AFB1 toxic effects”Reply: “Tolerance” has been replaced by “tolerant” in the title. ABSTRACTI suggest that the abstract would invite greater interest from potential readers if it stated more clearly WHAT IS KNOWN and WHAT IS ADDED BY THIS STUDY. It is an important and interesting subject, but one that is familiar to a very limited audience, an audience that could be increased by acknowledging that they are not abreast of the AFB1 literature.Reply: This is a very important suggestion and we would like to thank the reviewer for it. In order to create a greater interest in the article the following sentence has been added at the beginning of the abstract: “Aflatoxin B1 aldehyde reductase (AFAR) enzyme activity has been associated to a higher resistance to the aflatoxin B1 (AFB1) toxicity in ethoxyquin-fed rats. However, no studies about AFAR activity and its relationship with tolerance to AFB1 have been conducted in poultry. To determine the role of AFAR in poultry tolerance, the hepatic in vitro enzymatic activity of AFAR was investigated in liver cytosol from four commercial poultry species ”As captured later, please consider and respond to the following points:Intrinsic clearance: from a pharmacological perspective, clearance relates to a particular organ. In the biochemical context that is described in the MS it applies only to the selected enzyme. This should be made very clear, as hepatic clearance may be far higher than a single enzyme clearance if more than one enzyme is involved.Reply: We are in complete agreement with the reviewer’s comments regarding CLint. We have inserted a sentence explaining that the calculated intrinsic clearance only applies for the selected enzymatic activity (not for the liver) and that due to the fact that we did not purify AFAR enzyme from hepatic cytosolic extracts, the calculated CLint values are only “apparent” [Apparent Intrinsic Clearance (CLintapp)] .Highest AF dialdehyde concentration tested (66mcM): how does this concentration relate to the expected exposure concentration in intoxicated birds?Reply: The AFB1 concentration expected to occur in the hepatocyte upon AFB1 exposure is in the nanomolar or even the femtomolar order . In chickens dosed with a single intra-crop bolus of 2 mg of AFB1 per kg of body weight (a very large amount compared to what it is expected from a contaminated diet) AFB1 plasma concentrations have been only 96 nM . Therefore, it is likely that under natural conditions AFB1 dialdehyde concentrations are not going to reach the 66 µM concentration, because the cytosolic AFB1 concentration is expected to be in the nanomolar range. We used high AFB1 dialdehyde concentrations in our in vitro model because of the need to saturate the enzymes; also we needed to be able to detect the analytes . The same AFAR enzyme catalyzes mono- and di-alcohol AFB1: is this the same enzyme or the same or related or linked enzyme?Reply: The search conducted in databases like the NCBI or Kegg pathways indicates that the poultry AKR7A2 enzyme is responsible for the reduction of AFB1 dialdehyde and AFB1 monoalcohol. Because we found a strong relationship between these two enzyme activities (high Spearman’s rank-order correlation coefficient), we postulated that both activities are carried out by the same enzyme.AFAR activity related to toxic effects in chickens and ducks – not quail and turkey: does not this imply that the relationship is complex and potentially multifactorial in all species?Reply: We completely agree with the reviewer. AFAR activity is expected to depend on other reactions like glutathione sulfotransferase (GST) enzyme activity. In separate studies conducted by our research group we have found significant differences in GST activity among the same poultry species studied. Differences in the enzymatic nucleophilic trapping of AFBO will affect the production of AFB1 dihydrodiol and therefore the concentration of AFB1 dialdehyde. The overall metabolism of AFB1 most likely depends on several enzymatic reactions. A sentence has been added to clarify the multifactorial nature of the AFB1 detoxication pathways (lines 282 - 289).INTRODUCTIONNo comments MATERIALS AND METHODSLines 100-104Details of the breed/source of the turkeys, quail, and Pekin ducks should be provided, especially to allow comparison with any further studies of in vitro or in vivo activity of AFB1.Reply: Details of the poultry used in the study have been provide as follows: Nicholas turkeys, Japanese quail (Coturnix coturnix japonica) (there are no Bobwhite quail -Colinus virginianus- in Colombia) and meat-type Pekin ducks (there are both meat-type and egg-type Pekin ducks (lines 102 - 104).In addition, the composition of the diet of the birds prior to sacrifice should be described, especially the presence of any medications in the diet. For example, it would be common practice to include anticoccidial agents in the diet of chickens and special anticoccidial agents can cause changes in hepatic enzyme activity. Similarly, the main nutritional ingredients in the diet should be identified.Reply: No additives or medication were added to the diets provided to the birds. The diets were formulated with the same ingredients  formulated to reach or exceed the nutrient requirements of each poultry species studied. The ingredients and lack of use of non-nutritive additives have been indicated in the revised manuscript (lines 104 - 109).Line 128Is there a normal avian body temperature? Measurement of adult/mature bird body temperature has found a temperature of 41 for chickens, 41 for turkeys and 42 for ducks. Your comments on choice of a single temperature for all species and justification for the selection of 39 would be welcome. Younger birds may have a temperature closer to 39.Reply: We agree with reviewer comment. Because the birds used in this trial were 7–9 weeks old birds, the expected temperature was around 39 degrees Celsius. The revised manuscript has been added with the words “the normal body temperature for the age of the birds used” (line 138 - 139).Lines 150-160The statistical significance level should be presented and the actual value in each test presented later in the MSReply: The following sentence has been added to the “Statistical analysis” section: “with a significance level of 5% (p<0.05).” (line 173). The actual p-value obtained in each test has been presented in the “Results” section where appropriate. Line 155Intrinsic clearance – the units should be provided. In addition, as each species has a different mass of liver tissue, the total liver clearance for the enzyme of interest is a useful parameter.Reply: The CLint units have been indicated: “mL/mg protein/minute” (lines 166-167). We agree with the reviewer comment about the concept of total liver clearance; however, since we did not purify the hepatic AFAR enzyme, we are not able to calculate the total liver clearance. We used the CLint value to express the enzyme efficiency in the same way that it has been reported in previous studies . Further, as it was mentioned previously, the CLint value obtained corresponds to the apparent CLint.RESULTSFig 2 and Fig 3 CaptionsWhy is the difference in MW of monoalcohol and dialcohol 2 units and not 1 unit?Reply: AFB1 monoalcohol has an aldehyde function (R – CHO) that is reduced to alcohol, thus producing AFB1 dialcohol. In this carbonyl function, there is a double bond between carbon and oxygen (R – CH = O). When the carbonyl function is reduced, this C = O double bond becomes R – CH – O. One hydrogen is received by oxygen to produce the alcohol function (R – CH – OH) and the carbon atom receives the other hydrogen to complete its 4 bonds (R – CH2 – OH). That is why the molecular weight difference between the monoalcohol and the dialcohol is 2 units.DISCUSSIONLine 248… its capacity to FORM adduct …Reply: The word “form” has been inserted as it was suggested (line 265)Line 264The fact that there is an apparent relationship between AFAR activity and AFB1 susceptibility only in ducks and chickens, and not in quail and turkeys, suggests that toxicity may be multifactorial. Some discussion of this would add increased credibility to the study.Reply: We completely agree with the reviewer. The following paragraph has been added to the Discussion section: “It is important to highlight that AFAR enzyme activity cannot be considered as the only reaction capable of explaining the differences in sensitivity among different poultry species. For example, glutathione sulfotransferase (GST) enzyme activity is capable of affecting the production of AFB1 dihydrodiol  through AFBO nucleophilic trapping. Therefore, the toxicity of AFB1 should be considered as a multifactorial mechanism in which different metabolic pathways in AFB1 biotransformation are interconnected, including AFAR activity” (lines 282 - 289).Line 273-274Does the correlation described indicate that the same enzyme is involved or suggest that the enzyme activities are related or linked? Why is the only conclusion that they are the same enzyme?Reply: A linear correlation between two enzyme activities has been used in different experimental models to assume that a single enzyme is responsible for both enzyme activities . Because of the high correlation between AFB1 dialdehyde reduction and AFB1 monoaldehyde reduction, we have hypothesized that the AKR7A2 member is likely responsible for both activities. Line 276How representative of the concentration present in the liver of intoxicated birds is the highest concentration of AFBI-dialdehyde of 66mcM?Reply: As it was explained previously, the 66 µM concentration of AFB1 dialdehyde was used due to the conditions required by the experimental model . However, because reduction products are normalized to the amount of cytosolic protein used, the kinetic parameters obtained are expected to reflect what actually happens in the hepatocyte. Line 298Is the cytotoxic effect of AFB1 exposure in ducks DUE TO or ASSOCIATED WITH the lack of a detoxification pathway? What is the status of GST detoxification pathways in ducks?Reply: Currently there is no information on the enzyme kinetic parameters of GST activity against AFB1 or its metabolite AFBO in ducks. Kinetic parameters for this enzyme activity have been determined by our research group and these results are currently under consideration for publication. These unpublished studies have shown that the duck GST activity against AFBO is so low that the cytotoxic effects of AFB1 are most likely due to the lack of an appropriate detoxication pathway.CONCLUSIONLine 317The mention of individuals with high activity may relate to the SD values in Table 1. It would be useful to provide the individual bird values from which the Table was compiled – in this way the variation by individual birds and the number of birds with significant variation can be seen – this could be included in a supplement.Reply: We agree with the reviewer regarding the identification of possible “high AFAR activity” individuals. Individuals with a dhd/dial or dhd/mono ratio value below average minus SD reflect individuals with the highest AFB1 dialdehyde reduction or AFB1 monoalcohol reduction efficiency (inactivation reactions) relative to the AFB1-dhd efficiency (activation reaction). The following chart has been added as supplementary material:Species Sex Individual dhd/dial dhd/mono dial/mono mono/dialross female 1 2.73 2.72 1.00 1.00ross female 2 1.09 0.74 0.67 1.48ross female 3 1.74 1.57 0.90 1.11ross female 4 1.40 0.64 0.46 2.20ross female 5 2.88 1.29 0.45 2.23ross female 6 3.99 2.31 0.58 1.73ross male 1 1.27 0.94 0.74 1.36ross male 2 1.21 1.16 0.96 1.04ross male 3 7.27 2.13 0.29 3.41ross male 4 4.73 2.70 0.57 1.75ross male 5 1.69 1.04 0.62 1.62ross male 6 3.25 2.66 0.82 1.22island female 1 11.14 5.02 0.45 2.22island female 2 11.13 3.49 0.31 3.19island female 3 7.97 3.06 0.38 2.61island female 4 5.74 4.04 0.70 1.42island female 5 6.11 4.53 0.74 1.35island female 6 6.16 3.73 0.61 1.65island male 1 5.25 3.29 0.63 1.60island male 2 3.53 3.84 1.09 0.92island male 3 6.83 4.00 0.59 1.71island male 4 10.08 4.23 0.42 2.38island male 5 7.45 3.15 0.42 2.37island male 6 7.93 5.76 0.73 1.38quail female 1 18.22 18.44 1.01 0.99quail female 2 6.73 6.22 0.92 1.08quail female 3 9.67 9.28 0.96 1.04quail female 4 8.18 6.94 0.85 1.18quail female 5 29.19 17.06 0.58 1.71quail female 6 6.34 3.76 0.59 1.68quail male 1 40.84 29.43 0.72 1.39quail male 2 17.77 12.99 0.73 1.37quail male 3 14.70 7.25 0.49 2.03quail male 4 34.83 10.65 0.31 3.27quail male 5 19.59 9.87 0.50 1.98quail male 6 33.03 24.02 0.73 1.38turkey female 1 3.58 3.92 1.09 0.92turkey female 2 9.48 7.64 0.81 1.24turkey female 3 2.59 3.08 1.19 0.84turkey female 4 3.63 2.85 0.78 1.27turkey female 5 3.15 3.90 1.24 0.81turkey female 6 3.05 3.38 1.11 0.90turkey male 1 3.95 2.33 0.59 1.69turkey male 2 3.63 2.36 0.65 1.54turkey male 3 2.69 1.67 0.62 1.61turkey male 4 2.83 2.31 0.82 1.22turkey male 5 5.32 3.82 0.72 1.39turkey male 6 2.81 2.35 0.84 1.20duck female 1 208.75 81.43 0.39 2.56duck female 2 228.83 59.49 0.26 3.85duck female 3 361.73 84.35 0.23 4.29duck female 4 177.11 85.12 0.48 2.08duck female 5 44.56 23.13 0.52 1.93duck female 6 101.50 82.99 0.82 1.22duck male 1 181.93 182.60 1.00 1.00duck male 2 60.95 87.24 1.43 0.70duck male 3 102.01 139.39 1.37 0.73duck male 4 58.63 38.46 0.66 1.52duck male 5 142.59 81.06 0.57 1.76duck male 6 345.03 245.13 0.71 1.41S1 table. Values of CLint AFB1-dhd enzyme production / CLint AFB1 dialcohol enzyme production , CLint AFB1-dhd enzyme production / CLint AFB1 monoalcohol enzyme production (dhd/mono), CLint AFB1 monoalcohol enzyme production / CLint AFB1 dialcohol enzyme production  and the inverse  ratios. Values in bold represent individuals which ratio value is below SD. Line 318-Line 319Genetic selection would only be useful if there was a relationship between intoxication and enzyme activity. This would need to be established for the duck and may require challenge of ducks at the extremes (ie high and low) of enzyme activity with AF to see if in fact there is an in vitro in vivo (IVIV) relationship. Furthermore, only a very small sample of ducks from an unknown source has been studied and described herein.Reply: We agree with the reviewer comment. We are fully aware of the limitations of the present study. Nonetheless we believe that we can postulate that research about genetic selection could potentially be used as a possible means of enhancing species tolerance. S1 Table gives additional support regarding the possibility of selecting individuals with a particularly high AFAR activity that could be selected for potentially higher tolerance to AFB1. As it has been mentioned in the first comment of reviewer #2, a sentence has been added to clarify the limitations of the present study in regard to the population studied (lines 347 - 353).AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 19 May 2020PONE-D-19-32966R1Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effectsPLOS ONEDear Mr. Murcia,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Please review the referee comments on the term and giving supplemental data on the actual feed ingredients (mixing ratio and ME), feed intake, body weight, origin of rearing and notable clinical symptoms.https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.We would appreciate receiving your revised manuscript by Jul 03 2020 11:59PM. 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Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********3. 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Reviewer #1: Thank you for responding to my comments and taking appropriate opinions.However, in order to objectively understand the views expressed in this paper, there seems to be insufficient information on the experimental conditions as background.To the author's opinion that dietary effects on the results obtained are not expected, it is necessary to be objective by presenting as much evidence as possible of each poultry rearing status in this study.The authors should give supplemental data on the actual feed ingredients (mixing ratio and ME), feed intake, body weight, origin of rearing and notable clinical symptoms.Reviewer #2: Thankyou for your detailed responses to the various questions raised.The only matter you may wish to consider (and it is minor) is the use of DETOXICATION or DETOXIFICATION. The latter term is by far the most common term (a pubmed or a google search has around 10X as many entries for DETOXIFICATION compared with DETOXICATION).**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  2 Jun 2020Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Thank you for responding to my comments and taking appropriate opinions. However, in order to objectively understand the views expressed in this paper, there seems to be insufficient information on the experimental conditions as background. To the author's opinion that dietary effects on the results obtained are not expected, it is necessary to be objective by presenting as much evidence as possible of each poultry rearing status in this study. The authors should give supplemental data on the actual feed ingredients (mixing ratio and ME), feed intake, body weight, origin of rearing and notable clinical symptoms.Reply: We want to thank again reviewer #1 for his constructive comments. In order to clarify rearing origin and poultry clinical signs , the following phrase has been added to manuscript: “Poultry were obtained from local commercial poultry suppliers and at the moment of sacrifice no noticeable clinical signs were observed “ (lines 109 - 111). In regard to the feed ingredient’s composition and calculated analysis (mixing ratio and ME), feed intake and body weight, tables S1 and S2 have been added as supplementary information. Former S1 table has been renumbered as S3 Table. Feed ingredients (%) Chicken diet Turkey diet Duck diet Quail dietCorn 55.0 49.1 51.5 47.5Corn gluten meal --- 4.5 5.0 3.5Wheat bran --- --- 18.0 ---Full-fat soybean (extruded) 11.7 6.0 2.0 6.8Soybean meal (48%) 30.0 34.9 20.5 37.5Vegetable oil 0.1 2.8 --- 1.0Calcium carbonate 0.92 1.00 1.25 1.40Calcium phosphate (20% P) 1.48 1.00 1.30 1.75Iodized salt 0.3 0.2 0.2 0.35Vitamin:mineral premix 1.0 1.0 1.0 1.0Methionine 0.32 0.12 0.05 0.15Lysine 0.10 0.25 0.12 ---Threonine 0.03 --- --- ---Calculated analysis (%)Crude protein 25.1 25.5 20.1 26.1ME  3103 3120 2800 2909Ether extract 5.13 3.95 3.61 4.07Crude fiber 2.77 3.24 4.08 3.33Linoleic acid 1.25 1.49 1.42 1.29 �-Linolenic acid 0.22 0.15 0.08 0.16Calcium 0.95 0.88 0.99 1.24Total phosphorus 0.62 0.54 0.66 0.69Available phosphorus 0.30 0.25 0.33 0.34Digestible lysine 0.74 1.41 0.96 1.50Digestible methionine 1.36 0.48 0.38 0.49Total sulphur amino acids 0.48 0.83 0.72 0.85S1 Table. Feed ingredients and nutritional content of the diets fed to the experimental birds.Species Sex Body weight (g) Feed Intake (kg/bird) Species Sex Body weight (g) Feed Intake (kg/bird)Rhode Island Red chicks Female 750 1,07 Quail Female 89,1 0,84 720 1,19 80,8 0,86 625 0,99 79,2 0,86 590 1,16 80,5 0,81 630 1,18 83,0 0,85 630 1,26 83,6 0,81 Male 865 1,29 Male 92,9 0,81 850 1,41 86,8 0,79 830 1,37 96,1 0,82 750 1,43 78,5 0,78 810 1,27 73,6 0,81 790 1,26 73,4 0,85Ross chicks Female 1600 4,25 Turkey Female 2613 4,15 2850 4,15 2674 4,25 3175 4,23 3167 4,28 2175 4,48 2600 4,26 2450 4,15 2817 4,22 3200 3,81 2627 4,26 Male 3180 4,61 Male 3269 3,37 2900 4,55 2912 3,28 2750 4,78 2892 3,65 2850 4,50 3036 3,56 2550 4,78 2740 3,02 2760 4,53 3197 3,44Duck Female 1850 8,82  2640 8,69  2170 8,89  2300 8,92  2000 8,64  2100 8,70  Male 2700 7,77  2500 7,65  3100 7,79  2800 7,68  2600 7,95  2350 7,71 S2 Table. Final body weight and total feed intake at the time of sacrifice of the experimental birds.Reviewer #2: Thank you for your detailed responses to the various questions raised.The only matter you may wish to consider (and it is minor) is the use of DETOXICATION or DETOXIFICATION. The latter term is by far the most common term (a pubmed or a google search has around 10X as many entries for DETOXIFICATION compared with DETOXICATION).Reply: We want to also thank again reviewer #2 for his suggestion. The word “detoxication” has been replaced by “detoxification” were appropriate.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 9 Jun 2020Dealing with aflatoxin B1 dihydrodiol acute effects: impact of aflatoxin B1aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effectsPONE-D-19-32966R2Dear Dr. Murcia,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Best regards,Reviewers' comments: 11 Jun 2020PONE-D-19-32966R2 1 dihydrodiol acute effects: impact of aflatoxin B1-aldehyde reductase enzyme activity in poultry species tolerant to AFB1 toxic effects Dealing with aflatoxin BDear Dr. Murcia:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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OS for stage IB cervical cancer (n = 1101) was 90.4% (95% CI: 88.3–92.2%). Survival from stage IB was worse for women diagnosed age 20–24 years compared to those diagnosed 25–29 years at diagnosis (p < 0.0001), but no difference was observed by age at first invitation for screening, p = 0.8575. OS for stage II  and stage III+  were poorer. Survival from stage I cervical cancer in young women in England is excellent: mortality in women with stage IA cancer is akin to that of the general population regardless of age at first invitation to screening.Age at which women are first invited to attend cervical screening in England has changed twice: in 2004, women under 25 years were no longer invited; and in 2012, first invitations were sent six months earlier (at age 24.5 years). Concomitantly, a dramatic increase in screen-detected cervical cancer was observed, and their survival had not been documented. Diagnoses of invasive cervical cancer at ages 20–29 years in 2006–2016 in England were followed until the end of 2018 for deaths. We estimated 8-year overall survival (OS) by International Federation of Gynecology and Obstetrics (FIGO) stage and age at first screening invitation. Overall and relative survival for stage IA cervical cancer for women diagnosed aged 20–29 years in England ( The National Health Service (NHS) Cervical Screening Program in England invites women aged 25–49 years for cytology screening every three years and women aged 50 to 64 years every five years. Over the last 15 years, the age at which women get their first invitation to attend cervical screening has changed twice: once in 2004, and then again toward the end of 2012. In 2004, the age of first cervical screening invitation in England was increased from 20 to 25.0 years .p < 0.001) [Inviting women at age 25.0 years was associated with an increase of 43.7 cancers diagnosed at age 25 years per 100,000 women-years : 37.4–49.9, Here, we aim to explore the effect of the changes to the age at first screening invitation on survival from cervical cancer diagnosed in women under the age of 30. Given the changes in age of first invitation for screening, survival was both explored by screening cohort  and by age at diagnosis (20–24.5 (or 25.0) and 25 (or 24.5)–29 years). It is worth noting that the cut-off age for the groups by age at diagnosis will depend on the age at which women were first invited to screening. Finally, we explored the survival from stage IB cervical cancer among those invited at age 24.5 to 29.9 years and grouped them based on whether the cancer was diagnosed as a result of screening or not.n = 734) or a FIGO stage I not otherwise specified (n = 167). Forty-four percent of women were known to have a stage IA (n = 1905), 25% a stage IB (n = 1101), 6.2% a stage II (n = 268), and 3.4% a stage III+ (n = 147) cervical cancer. Overall survival at eight years for women diagnosed age 20–29 years with stage IA cervical cancer was excellent , whereas eight-year survival for those with stage III+ cancer was just 36.6% (95% CI: 28.4–44.7%), as shown in Between 2006 and 2016, there were 4322 eligible cervical cancers diagnosed in women aged 20–29 years. Of these cases, 20.8% either had an unknown or missing FIGO stage  and relative survival  were excellent. In fact, observed survival was ≥99% regardless of age at first invitation or age at diagnosis .p = 0.86). However, survival was significantly worse for women diagnosed under age 24.5 (or 25.0) compared to women diagnosed 25 (or 24.5)–29 years, chi2 (1) = 18.2, p < 0.0001. Worst differences in 8-year survival between women diagnosed age 20–24 years compared to those diagnosed 25–29 years were observed in those first invited for screening from age 20: 75% vs. 90.7%, chi2 (1) = 5.1, p = 0.024 and among those first invited at age 25.0: 65.1% vs. 91.9%, chi2 (1) = 21.6, p < 0.0001  diagnosed with stage IB cervical cancer were known to be screen-detected cancers, 29.5% (n = 325) had no information on screening status, and 34.6% (n = 381) were not screen detected. Here, we restricted the analysis to include only those cases that would have been due a screening test at the time of diagnosis, which we classified in three groups. Hence only 70% (n = 278/395) of women identified as being screen-detected and 31% (n = 120/381) of those not screen-detected were included in this analysis. Eight-year survival was similar among all screen-detected groups. It was 94.6% (95% CI: 88.9–97.4%) in the group of women first invited at ages 24.5–25.0 years and diagnosed age of 24.5–25.9. It was 94.5% (95% CI: 88.1–97.5%) in women first invited at ages 24.5–25.0 years and diagnosed age 27.5–28.9 (around their second invitation). Among those diagnosed age 24.5–29.9 years during the Jade Goody period, survival was 94.1% (95% CI: 78.5–98.5%). The much-publicized diagnosis and death from cervical cancer of celebrity Jade Goody led to a 70% increase in attendance to screening (between September 2008 and June 2009) in England, leading to a substantial increase in the diagnosis of cervical cancer [p = 0.04.A total of 35.9% of all cervical cancers . There was no evidence that survival from stage II or worse cervical cancer differed by age at first invitation, regardless of age at diagnosis, p = 0.7083.Survival among the 268 women with FIGO stage II cervical cancer was similar at 5-years  and eight years post diagnosis , regardless of the age at first invitation for screening. There is no evidence that mortality rates within eight years of stage IA cervical cancer diagnosis was any worse than among all women aged 20–29 years living in England. Although 8-year survival from stage IB cervical cancer in women aged 20 to 29 was 90%, survival was significantly poorer for those diagnosed under age 25 (between 65–75%) compared to those diagnosed at ages 25–29 (around 92%). There was also evidence that survival from IB cervical cancer was better among the screen-detected women. Survival for stage II or worse cervical cancer was poorer. However, since most of these women will be unscreened, one would not expect differences in survival by age of invitation and indeed, there is no difference.Strengths of the study include the use of population-based data from the National Cancer Registration Dataset, which ensures the inclusion of almost all cervical cancers diagnosed in England over the period of study.Although cancer registry data are not linked to screening history information, there were data on whether the cancer was screen-detected or not, which enabled us to carry out a sub analysis. The results presented here suggest better survival from FIGO IB cervical cancer when it is screen-detected. A difference in survival between screen-detected and symptomatic cancers of all stages has been reported previously . SurvivaThe proportion of women whose cancer stage was FIGO I or unknown differed between screening cohorts and could be a source of bias. The proportion of cancers with FIGO I or unknown stage ranged from 33% among women invited from age 20 years to 9% among those invited at age 24.5 years. The lack of staging information is directly linked to the stage at diagnosis: the more advanced cancers are the least likely to be staged . In thisStatistics for England report 5-year net survival from cervical cancer diagnosed between 2013 and 2017 at ages 15–44 of 97.2% (95% CI: 96.6–97.8%) for stage I cervical cancer and of 72.0% (95% CI: 67.2–76.8%) for stage II and of 89.1% (95% CI: 88.2–90.2%) overall  using SEThe published literature supports the view that younger age  and early stage  at diagnosis are prognostic factors for improved survival from cervical cancer and that improved survival over time can be attributed to the effect of screening on both prognostic factors ,12,13. Hn = 113) of women eligible for analysis were also eligible for human papilloma virus vaccination, so vaccination cannot have had a substantial impact on incidence in this cohort. Localized cervical cancer is treated using fertility preserving methods such as cone biopsy or LLETZ  [It appears that the change in policy on the age at which women are first invited to screening has increased diagnoses of cervical cancers by 45% [rmation) . LLETZ iData on all cervical cancer tumors (ICD10 code C53) diagnosed between January 2006 and December 2016 in women aged 20 to 29 years and resident in England were extracted from the cancer registration dataset produced by the National Cancer Registration and Analysis Service (NCRAS) . Date ofTwenty-six women had two cervical cancer tumors diagnosed during the study period. Only the first diagnosis was included, and when both tumors were diagnosed on the same day, the tumor with non-missing or more advanced stage was retained. One death certificate only (DCO) and seven women with data quality issues were excluded from the analysis.Women born on or before 25 August 1984 (Invited from age 20)Women born 26 August 1984 to 3 November 1985 (Mix of age at first invitation)Women born 4 November 1985 to 12 June 1988 (Invited at age 25.0)Born on or after 12 June 1988 (Invited at age 24.5)Data were available on whether a cervical cancer was screen detected or not, but we did not have access to full screening history data, and hence did not know the exact dates from which women would have been invited from age 25. To explore the effect of age at first invitation to screening on survival women diagnosed with cervical cancer were grouped in one of the following four screening cohort groups:For women invited at age 24.5 years: 20.0–24.49 years and 24.5–29.9 years.For all other women: 20.0–24.9 years and 25.0–29.9 years.To explore survival by age at diagnosis, we grouped stage IA cervical cancers in four age groups  so that we could compare survival to the general population using lifetables. For stage IB or worse cervical cancer, age at diagnosis was grouped as follows:This is reported in the results as 20–24.5 (or 25) and 25 (or 24.5)–29.Women with age at diagnosis 24.5–25.9 years, which were recorded in the cancer registry as being screen-detected or born on or after 4 November 1985 . Women diagnosed between 1 September 2008 to 30 June 2009 were excluded . Women diagnosed during the Jade Goody period were excluded.Women with age at diagnosis of 24.5–29.99 and diagnosed during the Jade Goody period: 1 September 2008 to 30 June 2009.Not screen-detected included women in the three categories above who were known not to have been screen-detected.To explore survival among those diagnosed with stage IB cervical cancer in more detail, we focused on women invited at age 24.5 to 29.9 years and grouped them based on whether the cancer was diagnosed as a result of screening or not. We used the information in the National Cancer Registration Dataset on whether or not a cancer was screen-detected. We divided these women with screen-detected cervical cancer into three groups as follows:The main outcome was 8-year overall survival among women diagnosed with stage IA or stage IB cervical cancer by age at first invitation for screening. Secondary analyses explored 8-year survival among those diagnosed with stage II or worse cervical cancer and whether being screen-detected influenced survival from stage IB cervical cancer.All-cause mortality was used to calculate survival at 1, 5, and 8 years by FIGO stage, age at diagnosis, and age of first invitation to screening. The censoring date for deaths was the 31 December 2018 or date of death, if earlier. Survival, 95% confidence intervals, women-years at risk, and number of deaths were calculated using Kaplan–Meier survival estimate . When there were no deaths, we used a one-sided 97.5% confidence interval based on a poison observation of 0 deaths in int (PY/K) people, where PY is the number of person-years of follow-up up to K years. Log-rank test was used to assess differences in survival.National life tables for England based on data for the years 2015–2017  were useCervical cancer screening services across the globe have been severely disrupted during the COVID-19 pandemic. The resulting delays to routine screening are likely to result in an increased number of diagnoses of screen-detected (and maybe even symptomatic) cancer. Given the excellent eight-year survival in women diagnosed with stage IA cancer under the age of 30 (regardless of the age of first invitation to screening), that the majority of stage IA cervical cancer is asymptomatic, and that treatment can preserve fertility, diagnoses of stage IA cervical cancer in women under age 30 should be considered a success of the screening program.


Exposure to particulate matter (PM) emitted from biomass burning is an increasing concern, particularly in Southeast Asia. It is not yet clear how the source of PM influences the risk of an adverse health outcome. The objective of this study was to quantify and compare health risks of PM from biomass burning and non-biomass burning sources in northern Thailand.10], PM2.5, Carbon Monoxide [CO], Ozone [O3], and Nitrogen Dioxide [NO2]) from ground-based monitors and daily outpatient hospital visits in Thailand during 2014–2017. Outpatient data included chronic lower respiratory disease (CLRD), ischaemic heart disease (IHD), and cerebrovascular disease (CBVD). We performed an ecological time series analysis to evaluate the association between daily air pollutants and outpatient visits. We used the 90th and 95th percentiles of PM10 concentrations to determine days of exposure to PM predominantly from biomass burning.We collected ambient air pollutant data  between daily PM10 and outpatient visits were elevated most on the same day as exposure for CLRD = 1.020 (95% CI: 1.012 to 1.028) and CBVD = 1.020 (95% CI: 1.004 to 1.035), with no association with IHD = 0.994 (95% CI: 0.974 to 1.014). Adjusting for CO tended to increase effect estimates. We did not find evidence of an exposure response relationship with levels of PM10 on days of biomass burning.There was significant intra annual variation in PM10 to be associated with certain respiratory and cardiovascular outpatient visits. We advise implementing measures to reduce population exposures to PM wherever possible, and to improve understanding of health effects associated with burning specific types of biomass in areas where such large-scale activities occur.We found same-day exposures of PM Ambient air pollution, and most notably particulate matter (PM), causes significant harm on a global scale, including over 4 million attributable deaths  and 5 mi3 rise in ambient PM2.5 ) with a consistent upward trend ; however, on days with higher biomass burning-related PM, there was no clear indication of a concentration response association (1.002 [95% CI: 0.993 to 1.012]). Including CO in the model enlarged the IRR for PM10 (1.026 [95% CI: 1.017 to 1.035])  Fig. . When st10 and IHD visits on non-burning (0.994 [95% CI: 0.974 to 1.014]) or burning days (0.991 [95% CI: 0.964 to 1.019]). By contrast, IRRs for CBVD visits were significantly elevated with PM on non-burning (1.020 [95% CI: 1.004 to 1.035]),but not burning days (0.997 [95% CI: 0.976 to 1.019]); as with the trend for CLRD visits, there was a consistent upward curve with more uncertainty at higher concentrations . When adjusting for the presence of other pollutants, similar trends were observed as those for CLRD visits: IRRs were slightly increased with CO. When stratified by age and sex, there were no clear increased IHD risks with PM10 for both single and multi-pollutant models; coefficients were highest for those aged ≥65 years. Risks with CBVD were elevated for males and for those < 65 years of age, but only when adjusting for CO (Table There was no apparent association between same-day PM10 risk estimate with respiratory visits on non-burning days (1.017 [1.007 to 1.026]) and had little effect on that for burning days (1.002 [0.994 to 1.009]). For the cardiovascular outcomes, the lower burning threshold made little difference to the non-burning day IRR for PM10 and IHD visits (0.996 [95% CI: 0.973 to 1.020]), but increased the IRR on burning days to 1.015 (95%CI: 0.994 to 1.035). For CBVD visits, the effect of PM10 on non-burning days was attenuated to borderline significance (1.017 [95% CI: 0.999 to 1.035]), but there was little difference to the relationship on burning days (0.998 [95% CI: 0.981 to 1.014]).In the sensitivity analysis at lag0, using the 90th percentile as the threshold for burning slightly reduced the PM10 exposures in the upper north of Thailand. These associations were maintained after adjusting for ambient CO concentrations. Evidence of a greater risk with PM10 was apparent for different sexes and health outcomes: females (CLRD) and males (CBVD). Although we identified overall risks of PM10 with CLRD and CBVD visits when exposure was predominantly from other  sources, we did not identify an exposure-response association for these outcomes on days of higher concentrations with a greater proportion of PM from biomass burning exposure.Our study findings indicate increased outpatient hospital visits for CLRD and CBVD, but not IHD, on the same day as PM10 and CLRD visits, which were elevated with both PM10 thresholds used to identify biomass burning days, compared to CBVD visits. This result is in agreement with findings from a recent global systematic review on air pollution and cardiorespiratory diseases ).Additional file 2: Figure S2. The average predicted number of a) Chronic Lower Respiratory Disease (CLRD), b) Ischaemic Heart Disease (IHD), and c) Cerebrovascular Disease (CVD) visits relating to daily mean PM10 exposure.