There are health risks associated with wastewater and fecal sludge management and use, but little is known about the magnitude, particularly in rapidly growing urban settings of low- and middle-income countries. We assessed the point-prevalence and risk factors of intestinal parasite infections in people with different exposures to wastewater and fecal sludge in Kampala, Uganda.A cross-sectional survey was carried out in September and October 2013, enrolling 915 adults from five distinct population groups: workers maintaining wastewater facilities; workers managing fecal sludge; urban farmers; slum dwellers at risk of flooding; and slum dwellers without risk of flooding. Stool samples were subjected to the Kato-Katz method and a formalin-ether concentration technique for the diagnosis of helminth and intestinal protozoa infections. A questionnaire was administered to determine self-reported signs and symptoms, and risk factors for intestinal parasite infections. Univariate and multivariate analyses, adjusted for sex, age, education, socioeconomic status, water, sanitation, and hygiene behaviors, were conducted to estimate the risk of infection with intestinal parasites and self-reported health outcomes, stratified by population group.Trichuris trichiura, Schistosoma mansoni, Ascaris lumbricoides, and Entamoeba histolytica/E. dispar was 15% and above. For all investigated parasites, we found significantly higher odds of infection among urban farmers compared to the other groups (adjusted odds ratios ranging between 1.6 and 12.9). In general, female participants had significantly lower odds of infection with soil-transmitted helminths and S. mansoni compared to males. Higher educational attainment was negatively associated with the risk of intestinal protozoa infections, while socioeconomic status did not emerge as a significant risk factor for any tested health outcome.The highest point-prevalence of intestinal parasite infections was found in urban farmers (75.9%), whereas lowest point-prevalence was found in workers managing fecal sludge (35.8%). Hookworm was the predominant helminth species (27.8%). In urban farmers, the prevalence of S. mansoni, and intestinal protozoa. Hence, our findings call for public health protection measures for urban farmers and marginalized communities, going hand-in-hand with integrated sanitation safety planning at city level.Urban farmers are particularly vulnerable to infections with soil-transmitted helminths,  Schistosoma mansoni and soil-transmitted helminths in urban farmers and slum dwellers after adjusting for age, sex, and educational attainment. Our data suggest that urban farmers are especially vulnerable to infections with intestinal parasites, which may play an important role in the transmission through contamination of their living and working environments. In view of our results, the control of schistosomiasis and soil-transmitted helminthiasis should be accelerated in Kampala.Urban wastewater and fecal sludge use is of growing importance all over the world. However, unsafe management and inappropriate use might exacerbate the transmission of infectious diseases, including those caused by intestinal protozoa  and parasitic worms . People living and working in densely populated and rapidly transforming cities in Africa and Asia are especially vulnerable. We conducted a cross-sectional survey and assessed people’s risk of intestinal parasitic infections due to exposure to wastewater and fecal sludge management and use in Kampala, the capital of Uganda. We collected data on the prevalence, intensity, and risk factors of infections with parasitic worms and intestinal protozoa among slum dwellers, urban farmers, and workers maintaining the sanitation system. We found high infection prevalence of  Schistosoma mansoni was found with specific farming activities in a medium-sized town in Côte d’Ivoire [Africa and Asia are urbanizing faster than any other region of the world and an increase of 16% of the urban population is predicted for 2050 . In the d’Ivoire . Additiod’Ivoire ,15.For prevention and control of these infectious diseases, the provision of basic sanitation infrastructure, coupled with education and promotion in hygiene practices, and targeted drug administration proofed effective ,16,17. TIn Kampala, the capital of Uganda, more than 90% of the 1.8 million inhabitants rely on onsite sanitation facilities, such as pit latrines and septic tanks. A small portion of wastewater is conveyed to treatment plants, while most of the generated wastewater and fecal sludge is discharged, without treatment, in open storm water channels ,22. AlonSchistosoma spp. are of particular concern, as Kampala is endemic for both soil-transmitted helminthiasis and schistosomiasis [S. mansoni and hookworm prevalence of 89% and 43%, respectively [Giardia intestinalis and Entamoeba histolytica/E. dispar was 12% and 10%, respectively [Infections with soil-transmitted helminths and somiasis ,28. Receectively . The preectively . At the ectively ,32. Hencectively ,33. Popuectively .We report findings from a cross-sectional parasitologic survey in selected population groups  exposed to wastewater and fecal sludge management and use activities in the Nakivubo area in Kampala. We aimed to determine the prevalence rates of intestinal parasitic infections and to assess the associations of disease risks with socioeconomic, environmental, and lifestyle factors in the different population groups. Our investigation is part of a larger study, comprising of an environmental assessment, a quantitative microbial risk assessment to determine the health risks related to microbial contamination, and the development and validation of a sanitation safety planning manual ,21,26.ISRCTN13601686).This manuscript has been developed according to the consolidated standards of reporting trials (CONSORT). The study protocol was approved by the institutional research commission of the Swiss Tropical and Public Health Institute  and the Uganda National Council for Science and Technology . Ethical approval was obtained from the ethics committee of the cantons of Basel-Stadt and Basel-Landschaft  and the Higher Degrees Research and Ethics Committee of Makerere University School of Public Health . This study is registered with the clinical trial registry ISRCTN .2), where approximately 600 farmers pursue urban farming for their livelihood. Informal slum communities are at highest risk of flooding events, as they live along both sides of the wetland . The water from the Nakivubo wetland is ultimately discharged into the Inner Murchison Bay at the shores of Lake Victoria, some 4 km ahead of where the water is pumped and treated to supply Kampala city with drinking water , which receives most of Kampala’s wastewater. The area is located at an altitude of 1,140 m above the mean sea level at latitude 0° 18’ 48.1” N and longitude 32° 36’ 43.86” E. Domestic and industrial wastewater is derived from the central division, while the treated effluent of the Bugolobi Sewage Treatment Works (BSTW) is collected in the Nakivubo channel, a 12.3 km long open storm water channel. This channel enters into the Nakivubo wetland (5.3 kmng water . The stuWe focused on adults (aged ≥18 years) living and working in the Nakivubo area. According to the level of exposure to wastewater, the study participants were stratified into five groups, as follows:comexposed”, slum dwellers at risk of flooding living along the Nakivubo wetland at altitudes ranging between 1,140 m and 1,160 m above mean sea level (AMSL). The communities are characterized by poor housing and unimproved sanitation and unsafe water supply;“comcomparison”, slum dwellers living in similar communities as comexposed without risk of flooding (comparison group) living at least 2 km away from the Nakivubo wetland at altitudes between 1,160 and 1,201 m AMSL;“farmer”, urban farmers using informally and indirectly wastewater to grow sugar cane, yams, and maize within the Nakivubo wetland and living in the same communities as comexposed;“workerww”, workers employed by NWSC who maintain the drainage channels and operate the BSTW; and“workerfs”, workers organised under the pit emptier association managing fecal sludge .“comcomparison” = 350, “comexposed” = 250, “farmer” = 275, “workerww” = 50, and “workerfs” = 100). We aimed at a power of 95%, to ensure that a reduction in effective exposure variance by 35% following confounder adjustment would still leave 80% power. Our assumptions were that the prevalence rate of intestinal parasitic infections is at least 20% in “comcomparison” and the difference in odds ratio (OR) to “farmer”, “workerww”, and “workerfs” is at least 2.5. We also assumed that the final sample size would be reduced by 15% due to non-response and missing data.Our intended sample size was 1,000 participants .We used a questionnaire to determine exposure pathways to wastewater and fecal sludge, potential confounding factors , risk variables  and self-reported health outcomes. Study participants were asked about signs and symptoms experienced over the past 2 weeks before the interview took place, using a pre-tested questionnaire . DiarrheAscaris lumbricoides, hookworm, Trichuris trichiura, S. mansoni, and other helminths) and intestinal protozoa .Participants were invited to provide a fresh morning stool that was subject to the Kato-Katz technique   and a fo2 test. Univariate logistic regression was applied to investigate the potential association between dependent  any intestinal parasite, (ii) intestinal helminth, (iii) soil-transmitted helminth, (iv) intestinal protozoa, (v) A. lumbricoides, (vi) hookworm, (vii) T. trichiura, (viii) S. mansoni, (ix) 14-day diarrhoea, (x) skin problems, and (xi) eye problems) and 49 independent variables . People’s socioeconomic status was determined using principal component analysis and participants were grouped into three categories, as indicated in Helminth- and intestinal protozoa-specific proportions between the five exposure groups were compared with Pearson’s χOdds ratios are reported to compare risks, while differences and associations are considered as statistically significant if p-values are below 0.05, and indicating a trend if p-values are between 0.05 and 0.1. Statistical analyses were done using STATA version 12.0 . Maps, including geographic coordinates of the interviews, were established in ArcMap version 10 . Kato-Katz thick smear and FECT readings were double-entered and validated.comexposed”, 331 “comcomparison”, 245 “farmer”, 43, “workerww”, and 67 “workerfs”.Among the 1,156 people invited, 1,076 fulfilled inclusion criteria, had written informed consent, and completed the questionnaire interview . Stool scomexposed”, “comcomparison”, “farmer”, and “workerww”, respectively, whereas no woman was in the “workerfs” group. Socioeconomic status was highest in “workerfs” and “workerww” with 83% and 74%, respectively, classified as less poor. The lowest socioeconomic status was observed in “comcomparison”, “comexposed”, and “farmer” with 30%, 38%, and 45% classified as most poor, respectively.farmer”, and “comexposed” . 65%, 49%, and 56% of the participants from “comexposed”, “farmer”, and “comcomparison” had a toilet at home. Overall, 29% of all participants reported having taken a deworming drug within the past 6 months with the highest proportions reported by “workerfs” (61%).Parameters for exposure to wastewater, access to drinking water, sanitation, and hygienic behaviors are summarized in farmer”, “workerww”, and “workerfs”. While most of the workers are officially contracted (97%), the opposite is seen among “farmer”, as most lack an official employment status (95%). 81%, 63%, and 49% of “workerww”, “workerfs”, and “farmer” wear boots, respectively. Only 4% of “farmer” use gloves, whilst over 80% of the workers use them.farmer”, “comexposed”, “comcomparison”, “workerww”, and “workerfs”, respectively. One quarter (25%) of all participants was found infected with at least two species of intestinal parasites. The highest prevalence of soil-transmitted helminth infection was found in “farmer” . S. mansoni was detected in all exposure groups with prevalences of 5% and above; while the highest prevalence was found in “farmer” (23%). Nine participants were infected with Hymenolepis nana (six cases occurred in “farmer”), two with Taenia spp., and one with Strongyloides spp. Overall, 11 participants were found with heavy S. mansoni infection (≥400 eggs per gram of stool). Forty percent of all participants were infected with intestinal protozoa; the highest prevalence rates occurred in “farmer” and “comexposed” . We found a prevalence of E. histolytica/E. dispar of 15%, 12%, and 7% in “farmer”, “workerww”, and “workerfs”, respectively. Nine people had an infection with G. intestinalis, five of them in “comcomparison”.The prevalence and intensity of parasitic infections, stratified by exposure group, are summarized in Figs comcomparison”, 25% in “farmer”, and 33% in “workerfs”. General skin problems were reported by between 28% (“comexposed” and “comcomparison”) and 39% (workerfs). More specific skin irritation was reported by 19%, 16%, 13%, and 12% in “workerfs”, “workerww”, “comexposed”, and “farmer”, whereas a considerable lower rate of 4% was found in “comcomparison”. Eye problems were most frequently reported by “farmer” (37%), followed by “workerww” and “workerfs” (33% each).Self-reported signs and symptoms are summarized in farmer” had a higher odds of all measured helminth and intestinal protozoa infections, compared to the other groups (adjusted OR between 1.6 and 12.9). Workers  had lower adjusted odds compared to “comcomparison” for intestinal parasite infections, except for hookworm, where “workerfs” had increased risk . However, for 14-day diarrhea prevalence, skin and eye problems, workers had similar or higher risks. For soil-transmitted helminth “comexposed” showed lower infection risks compared to “comcomparison” , while the risk of intestinal protozoa infection was elevated . Compared to “comcomparison”, “comexposed” were at higher risk of S. mansoni infection , but at lower risk of hookworm infection . Moreover, 14-day prevalence rates of diarrhea and skin symptoms according to self-reports were higher among “workerfs” and “farmer” compared to the other groups.Figs S. mansoni infections. Relying on pit latrines or having no toilet facility was associated with significantly increased risk of “any parasitic infection”, soil-transmitted helminth, and T. trichiura infections. Moreover, hand washing after defecation and work was negatively associated with T. trichiura infections . Higher level of education was negatively associated with intestinal protozoa infections . On the other hand, 14-day prevalence rates of diarrhea and skin symptoms according to self-reports were higher among participants with higher socioeconomic status.S. mansoni were the predominant infections; 28% and 23%, respectively. We found significantly higher odds of infection, regardless of the parasite species for urban farmers compared to other population groups (adjusted OR between 1.6 and 12.9).We report data on prevalence and risk factors for intestinal parasitic infections in exposed adult population groups along the major wastewater and fecal sludge use system in Kampala, Uganda. Urban farmers had the highest prevalence rate and higher ORs compared to the other exposure groups for the majority of measured health outcomes. Indeed, urban farmers had a point-prevalence of intestinal parasites of 76%. Hookworm and S. mansoni prevalence in urban farmers are in line with previous reports from other studies in Africa and elsewhere in the tropics [A. lumbricoides (0.2 eggs/l) and the absence of T. trichiura eggs do not correlate with the respective prevalence in the exposure groups [S. mansoni in slum dwellers. However, prevalence obtained in farmers match the model-based predictions for soil-transmitted helminths and S. mansoni and are even exceeding predicted values up to a factor five for T. trichiura and A. lumbricoides [E. histolytica/E. dispar and G. intestinalis among all study participants was considerably lower than in a study of rural communities along Lake Victoria[The high hookworm and  tropics ,13,15,41 tropics ,20. The e groups . Compariricoides ,28. Henc Victoria. Multiva Victoria, our resOur study has five main limitations. First, due to its cross-sectional design, this study only reflects one point in time, i.e., the rainy season, and thus, we may underestimate seasonal patterns of intestinal parasite infections and other diseases that may give rise to diarrhea such as seasonal outbreaks of cholera and typhoid –44. SecoS. mansoni prevalence rates in excess of 20% in urban farmers call for preventive chemotherapy, at least in this population group. Third, we observed differences between self-reported signs and symptoms and actual prevalence of intestinal parasitic infections measured in the different exposure groups, and hence other factors  might have considerable implications in these exposure groups.Despite these limitations, our findings raise a number of important issues. First, urban farmers, living within marginalized slum communities, appear to be most exposed and vulnerable for intestinal parasites and might contribute to their transmission in urban environments. Second, we did not find any significant positive association between current deworming practices and intestinal parasitic infection. However, our findings with hookworm and S. mansoni, most likely through contamination of their living and working environment. We recommend longitudinal monitoring of parasitic infections and diarrhea alongside with targeted interventions in exposed population groups. Altogether, this calls for increased public health protection measures for urban farmers and marginalized communities and integrated sanitation safety planning at city level.Taken together, our results show that urban farmers are especially vulnerable and may play an important role in the transmission of soil-transmitted helminths and S1 Checklist(DOCX)Click here for additional data file.S2 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ13CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following 13C stable isotope analysis. Termite carcass samples  were significantly different from termite gut filtrate samples (−27.53 ± 0.5‰), but not the wood diet (−26.0 ± 0.5‰)  = 6, P < 0.0052). δ13CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ13CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function.Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite  An addiprotists . Togetheprotists .de novo  is attributed to proctodeal trophallaxis and subsequent digestion of microbial fractions  because these cannot be generated by the host de novo . Proctodde novo . Brieflyde novo . Inoculade novo . Similarractions . It stilReticulitermes flavipes, using naturally occurring variations in the 13C/12C ratios of EAAs from bacteria, fungi, and plants. The premise of this approach is two-fold. First, because insects are incapable of de novo EAA biosynthesis and rely solely on dietary sources, the 13C-signature (determined via isotope ratio mass spectrometry) of an EAA in an insect consumer (δ13CConsumer EAA) is expected to approximate that of the diet (δ13CDietary EAA), with little change in the ratio of 13C/12C stable isotopes in the carbon skeleton of that particular EAA  is estimated to within 1‰ of the dietary δ13CEAA. Any significant deviation from the expected Δδ13C of 1 suggests the possibility of alternate or additional sources of EAAs  greater than 1 between the δ13CEAA of a consumer and its diet, suggests the possibility of an additional/alternate source of EAAs for the consumer. The second premise enables the identification of the possible contributing source, within a predictive model framework, based on the unique δ13CEAA signatures of plants, bacteria, and fungi. It is important to stress that, the 13C-offset determination serves only to determine the use of non-dietary EAAs if any, and not to identify biosynthetic origin of such non-dietary EAAs.Thus, according to the first premise, a determined discrimination/offset factor  and were maintained in a laboratory colony in a plastic container fitted with a lid and provisioned with wood (a mixture of wood mulch and pine (Pinus spp.)), that was moistened periodically with distilled water. These colonies were maintained in the laboratory of Dr. Susan Jones. The termite colony originated from a single inbred colony that had been established during May 2010 by pairing a single male and female de-alate, and was maintained at room temperature (∼22 °C ± 2 °C) in the dark, under ambient laboratory conditions.n = 5, each made up of 10 pooled workers) were obtained. The entire alimentary system was removed from each worker and placed in a 1x phosphate buffered saline solution (PBS). The remaining termite carcass was place in a 1.5 ml Eppendorf tube . Hence, each termite sample was subdivided into the termite carcass (n = 5) and its gut fraction (n = 5). Pooled termite guts were homogenized in PBS and filtered through a 0.45 µm membrane filter  to eliminate insect debris. Gut filtrates were stored at −80 °C for 48 h prior to lyophilization. Wood samples (n = 4) from the termite colony were also ground into a coarse powder in a coffee mill and frozen at −80 °C for 48 h before lyophilization. Termite and wood samples then were sent to the Stable Isotope Facility (SIF) at UC Davis, Davis, California, USA, for 13C-stable isotope analysis.After 8 weeks of feeding, 50 individual workers were removed from the colony and surface sterilized by rinsing once in 10x Coverage Plus  and twice in sterile distilled water. A total of 5 termite samples  was performed using the TRACE GC Ultra gas chromatograph  coupled to a Delta V Advantage isotope ratio mass spectrometer via the GC Combustion Interface III  using the high polarity VF-23ms capillary column . Combustion and reduction furnace temperatures were 950 °C and 650 °C, respectively. δ13C isotopic abundances are reported as δ13C values relative to the standard Vienna Pee Dee Belemnite (V-PDB) scale. δ13CEAA quantified from termite carcasses, termite gut filtrates, and wood samples, following a non-analyte correction relative to internal amino acid standards, and used in the statistical analyses.Freeze-dried termite and wood samples were acid hydrolyzed and derivatized resulting in the addition of a known carbon residue to the analytes of interest . Non-anat-test) were carried out on δ13CEAA data using JMP 10 . Overall 13C-offset between termite (δ13CTermite) and wood (δ13CWood) samples was determined as Δδ13C = (δ13CTermite–δ13CWood). Individual patterns of 13C-offset across EAAs between termite and wood samples was determined as Δδ13CEAA = (δ13CTermite EAA–δ13CWood EAA).Mixed model analysis and mean separations (Student’s δ13CEAA data from our study and from n = 9), bacteria (n = 11), and plants (n = 12). The predictive model was validated using the reference bacteria, fungi and plant samples as classifiers to ascertain distinctness of each group. Additionally, δ13CEAA data obtained from the fungus Fusarium solani (n = 2), used in a previous study and analyzed at the same facility as these samples were, was used to further validate the separation of the classifiers by the model based on the particular EAAs used in the study.An inter-lab calibration was performed to minimize instrumental error and/or variability between This was followed by a supervised discriminant analysis to determine group membership of termite samples (carcass and gut filtrate) and wood samples to the respective classifier groups . ClassifNo animal rights were violated in the execution of this study and conditions were within the guidelines of the Ohio State University’s Office of Responsible Research Practices.δ13C of all EAAs were quantified, and only those passing quality checking (having distinct and non-overlapping peaks obtained from the GC capillary column), were selected for further analysis. δ13CEAA data were obtained for isoleucine, leucine, valine, phenylalanine, lysine and threonine from all samples . Similarly, Threonine δ13C data was unavailable for four out of five termite gut filtrate samples. Threonine was excluded from all further analyses. Leucine, however, was used in calculating the 13C-offset between termite samples and the wood diet, because there were two termite gut filtrate samples, for which this could be calculated relative to wood; but was omitted from the predictive model analysis due to the missing data points  = 13.7, P < 0.0001, R-square = 0.80), with significant group  = 6.0, P < 0.0052) and amino acid  = 35, P < 0.0) effects, as well as significant group*amino acid interaction  = 5.1, P < 0.0001). Termite gut filtrate (−27.53 ± 0.5‰) (mean ± s.e) was significantly different from the wood diet (−26 ± 0.5‰) and the termite carcass (−27.0 ± 0.4‰). Termitethe diet .13C-offset of the five-measured EAAs in the termite samples relative to the wood diet is presented in 13C-offsets presented in δ13CEAA between termite samples and wood provides an indication of the use of non-dietary EAAs . Briefly, phenylalanine, lysine and valine from the termite samples (carcass and gut filtrate) and wood diet were not significantly different from the each other; even though lysine in the termite samples was 13C-enriched relative to the lysine in the wood-diet, and phenylalanine and valine in termite samples were 13C-depleted, relative to the wood diet  plots, the 95% confidence limits decision regions for each group/classifier are depicted as ellipses around the classifiers and the decision boundaries between the groups/classifiers as lines. After establishing the discrimination model, we then predicted posterior probabilities, i.e., the probability that a particular sample belonged to one or another of the three groups. The greater the distance of a particular consumer from the centroid of a classification group, i.e., potential EAA source, the greater the probability mixing of EAA sources occurred. Given the distinct discrimination scores between the classification groups, we interpreted discriminant scores of termite samples falling outside the 95% confidence limits of the food source, wood (plants) as strong indications of symbiotic EAA provisioning.n = 11), fungi (n = 9) and plants (n = 12) to their respective groups  = 25, P < 0.0001; Wilk’s lambda = 0.04, a test of appropriateness of classifiers in predicting group membership of predictors), using the δ13CEAA values of the EAAs, isoleucine, phenylalanine, valine and lysine (Fusarium solani) samples were correctly classified as fungi. The classification of these fungal samples further validated the appropriateness of the model using the selected EAAs  = 28.9, P < 0.0001; Wilk’s lambda = 0.06, a test of appropriateness of classifiers in predicting group membership of predictors)  . As withn origin , furthern origin . Four ten origin . Classifn origin  and 3 wan origin . Omittinn origin . Based oR. flavipes is varied and members perform several key important functions related to the host’s nutritional ecology. Essential amino acid provisioning by these gut microbes have been proposed, but remain to be empirically determined. In this study, we sought to determine gut microbial EAA provisioning to termite hosts by taking advantage of the natural variations in the δ13CEAA stable isotope signatures between plants, bacteria, and fungi.The membership of the gut microbiome of wood-feeding termites like δ13CEAA would closely resemble dietary δ13CEAA (wood) in the absence of microbial provisioning. While we did not determine a significant difference between termite carcass and dietary δ13CEAA(Δδ13C = 1)  , there wood diet . Most wotermites . Degradatermites . Howevertermites , which ady diets . While t13C data . Thus foet of 1‰ .δ13CEAA data. Increasing the number of EAAs used in the model, would essentially offer greater resolution as well as increased confidence in the subsequent model classifications.An investigation of the biosynthetic origins of EAAs in termite carcass and gut filtrate samples using the predictive model classified a majority of termite samples as bacterial in origin  and 3, a13C-offsets between termite samples and three of the EAAs in the study , and the classification of diet (wood samples) as fungal in the predictive model , increased sample numbers, and optimized procedures/protocols for δ13CEAA data generation via isotope ratio mass spectrometry, are needed, in order for definitive establishment of the role of gut-associated microbes of termites as sources of EAA.Overall, this study demonstrates the applicability of the δ13C stable isotope analysis.In summation, this study provides promising evidence in support of putative gut microbial  EAA provisioning in termites. Though the results presented herein are not exhaustive, they serve as the baseline for further work, investigating microbial EAA provisioning functions in greater detail. Finally, the results presented provide a framework/approach to, investigating gut microbial EAA provisioning in similar, and other insect-microbe symbiotic system using 10.7717/peerj.1218/supp-1Table S1δ13CEAA) used in this study. Means are based on 2 technical replicates for each biological sample and 3 technical replicates for fungi, bacteria, and plant reference samples.Sample source and all mean inter-lab calibrated essential amino acid values .Posterior probabilities of the classifier samples  and experimental group samples used in the predictive model plot in Click here for additional data file.10.7717/peerj.1218/supp-3Table S3Click here for additional data file.


The adverse events (AEs) of oxycodone in cancer-related pain were controversial, so we conducted a meta-analysis to determine it.PubMed, Embase, CBM, CNKI, WanFang database, The Cochrane library, Web of Science, and the reference of included studies were searched to recognize pertinent studies. Relative risk (RR) with 95% confidence intervals (CIs) for all AEs were all extracted. The fixed-effects model was used to calculate pooled RRs and 95% CIs. Power calculation was performed using macro embedded in SAS software after all syntheses were completed.P = 0.72; nausea RR = 0.88, 95% CI: 0.72–1.07, Z = 1.26, P = 0.21; vomiting RR = 0.89, 95% CI: 0.70–1.15, Z = 0.9, P = 0.37; sleepiness RR = 0.86, 95% CI: 0.38–1.36, Z = 0.36, P = 0.72; constipation RR = 0.98, 95% CI: 0.81–1.19, Z = 0.21, P = 0.83; anorexia RR = 0.97, 95% CI = 0.58–1.62, Z = 0.11, P = 0.91; pruritus RR = 0.76, 95% CI: 0.44–1.30, Z = 1.01, P = 0.31; dysuria RR = 0.33, 95% CI: 0.07–1.62, Z = 1.36, P = 0.1)]. The subgroup analysis shown that Ox controlled-release (CR) had less sleepiness compared with MS-contin (Mc) CR . The power analysis suggests that all AEs have low statistical power.We identified 11 eligible trials involving 1211 patients: 604 patients included in oxycodone group and 607 patients involved in control group. Our quantitative analysis included 8 AEs, and the pooled analyses indicated that oxycodone compared with other opioids in cancer-related pain were not significantly decreased RRs of all AEs (dizziness RR = 0.94, 95% CI: 0.69–1.30, Z = 0.35, The present meta-analysis detected that no statistically significant difference were found among oxycodone and other opioids in all AEs, but Ox CR may had less sleepiness compared with Mc CR when subgroup analysis were conducted. The guideline of treatment for cancer patients with pain was issued by the World Health Organization (WHO), which was the principle was the foundation for the treatment of cancer-related pain. However, WHO was not suitable at present. Now new opioids guidelines2 was published with overcoming some limitations, and was accepted by most oncologists. In the past, opioids have been used in cancer patients who experience moderate and severe pain many years. Oxycodone, 1 of opioids, has been used in clinic since 1917, and a series of randomized controlled trials (RCTs) about oxycodone used in advanced cancer patients with pain.10 Although many meta-analyses and systematic review published,15 and most of meta-analyses focused on efficacy of this agent, but few of them confirmed the side effects. Wang et al12 detected that oxycodone significantly decreased the incidence of nausea and constipation compared with other opioids, while recently studies5 found that the adverse events (AEs) were similar among oxycodone and other opioids. Oosten et al14 reviewed the common AEs of opioids for cancer-related pain, but the review did not pooled analysis the side effects, the descriptive analyses could not be observed visually by anyone.Pain is 1 of the most common symptoms in cancer patients. Approximately 60% patients experience pain, one-third of the patients who graded their pain as moderate or severe.Therefore, a meta-analysis and power analysis was carried out to compare the all AEs among oxycodone and other opioids in the management of cancer-related pain based on currently available studies.16 and according to Cochrane Handbook for Systematic Reviews of Interventions. The protocol was registered by Centre for Reviews and Dissemination PROSPERO (available at: http://www.crd.york.ac.uk/prospero/register_new_review.asp?RecordID=13401&UserID=7339) (Registration No. CRD 42014013401).Ethical approval and patient written informed consent are not necessary because of this is not primary research. This study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement ,Eligible trials were identified through electronically searching the databases of PubMed, Web of Science, The Cochrane library, and EMBASE, China National Knowledge Infrastructure (CNKI), WanFan Database, China Biomedical Literature database (CBM), and Chinese Science and Technology Periodical Database (VIP) using the following terms: (“Pain Measurement” OR “Pain”) AND (“Tumors” OR “Cancer” OR “Neoplasms”) AND (“Oxycodone” OR “Oxycone” OR “Dinarkon”) . The search strategy for PubMed and Embase were summarized in Supplemental Data 2. Language or date restrictions were not imposed. We manually checked the bibliographies of previous reviews and included trials to identify other potentially eligible trials.All studies focused on the all AEs among oxycodone and other opioids in cancer-related pain were involved by using following selection criteria—Population: patients were diagnosed as cancer, with no other restrictions; Intervention: oxycodone plus other agents or alone, regardless of any formulation and any route of administration; Comparison: other opioids , regardless of extended-release or other formulation; Outcomes: all of the side effects will be evaluated; Study design: RCTs, no matter parallel-or cross-over group.Two reviewers (HM and LH) independently screened the titles and abstracts to exclude studies that were not reach the inclusion criteria, then the full-text articles were read. Finally, data extraction was conducted using a premade data extraction form based on electronic database to collect information as following: authors, the population studied, publication year, country, the formulation of oxymorphone or control, and the detailed information regarding Patient(P), Intervention(I), Comparison(C) and Outcome(O) study design(s) (PICOs). Extracted data were entered into a database, which created by EpiData version 3.1.17 The authors estimated the following domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. Based on the information extracted from included studies, each domain was assigned as a value of “high risk,” “unclear risk,” or “low risk.” Any disagreement between searchers concerning the eligibility of a trial was resolved by consulting a third reviewer (J-GZ).Two reviewers (HM and YL) independently evaluated the risk of bias using the Cochrane Collaboration tool.http://www.gradeworkinggroup.org/).18Two authors (J-GZ and XT) independently evaluated the quality of evidence for all of AEs following the GRADE methodology for risk of bias, inconsistency, indirectness, imprecision, and publication bias; assigned as very low, low, moderate, or high. The summary table of quality of evidence was made using GRADE Profiler  . The presence of publication bias was evaluated by using Begg and Egger regressions.22 We considered a P value of <0.05 to be statistically significant.23Except for publish bias used STATA version 12.0 , all analysis used RevMan  in this meta-analysis. We estimated the relative risk (RR) with 95% confidence intervals (CIs) for dichotomous outcomes. I24 after all syntheses were performed by SAS version 9.21 . Details on the macro and SAS code used were included in the online supplement.Power calculation was performed using the methodology described by Cafri et al26 with 11 trials and 1211 patients were involved in this meta-analysis, and 604 patients included in oxycodone group and 607 patients involved in control group, respectively, and the sample size ranged from 30 to 248. Moreover, 126 patients with cancer-related pain have appeared dizziness, 269 patients have appeared nausea, 188 patients have occurred vomiting, 60 patients with cancer pain have occurred sleepiness, 42 patients have reported pruritus, 260 patients have reported constipation, 49 patients shown anorexia, and 6 patients have reported dysuria, and all side effects being included in the final analysis. The flow diagram of the literature searched and evaluated was presented in Figure A total of 580 unfiled titles and abstracts were identified through database searching and 5 records identified through references searching. Finally 10 studies4 was an RCT with 3-arm design comparing morphine, MS contin and oxycodone in treatment of cancer pain. Nausea, vomiting, and constipation were available in all of trials, dizziness was appeared in 10 trials, anorexia and pruritus were reported in 4 trials, sleepiness was occurred in 5 trials, dysuria was reported in 3 trials, and insolence was appeared in 2 trials. Riley et al3 reported that opioid adverse reaction scores were scored on an 11-point Numerical Rating Scale, Yu et al5 shown that treatment-emergent adverse events (TEAEs) were reported by either patients or interviewers, Heiskanen and Kalso26 used Modified Specific Drug Effect Questionnaire to assessment AEs, Mucci-LoRusso et al10 used the Specific Drug Effect Questionnaire to evaluation side effects, Gabrail et al.8 AEs were rated by investigators; however, the other studies did not report the method to assessment TEAE. Five studies come from China,25 2 come from United States,10 the others come from United Kingdom,3 Finland,26 and Europe.6 Five trials compared oxycodone (Ox) controlled-release (CR) with MS-contin (Mc) CR,25 3 trials compared Ox CR with morphone (Mo) CR,26 the other trials were Ox prolonged-release (PR) vs oxycodone/naloxone (Oxn) PR,6 Ox CR vs oxymorphone (Omo) CR,8 and Ox CR vs DHC-contin (Dc) CR,9 respectively. The main characteristics of the included studies were recorded in Table All eligible studies were published between 2002 and 2015. In total, 10 studies provided outcomes, the trial finished by Zhang et al3 Two of all the studies were assessed as high risk in allocation concealment, 4 as unclear risk, and others as low risk of bias. Two studies have blinding of participants, personnel, and outcome assessment. Only 1 study possessed of incomplete outcome data, and 8 of all eligible studies reported selective reporting. Nevertheless, there are some criteria of assessments judged as high bias; however, those unlikely to affect the quality assessment. The overall methodological quality was generally good and fair.The detail of the risk-of-bias assessment was summarized in Figure 2 = 56%, P = 0.06), so a random model was used. The rest of AEs calculated results are I2 < 50% and P > 0.10, and did not detect significant heterogeneity, so we choose the fixed model to meta-analysis about them. We found no significant difference in the RRs of overall AEs. The results of subgroup analysis are following.There are 8 AEs including in this systematic review to quantitative analysis, and sleepiness found significant heterogeneity (IP = 0.72), the subgroup analysis shown that no statistical difference between differently control groups   Figure , besidesP = 0.37), and the subgroup analysis did not detect superiority of different agents , while Mo CR or Dc CR compared with Ox CR had no statistical difference, respectively  .The publication bias of our meta-analysis was assessed using funnel Begg and Egger regressions. Insufficient evidence of publication bias was found from the formal statistical tests . While subgroup analysis shows that there were significantly different between Ox CR (n = 12) and Mc CR (n = 25) in sleepiness .This systematic review and meta-analysis involved 10 studies and 11 trials, enrolled a total of 1211 patients. Current literature demonstrated that oxycodone had no significant difference in the RR of all AEs compared with other opioids (dizziness: RR = 0.94, 95% CI: 0.69–1.30, Z = 0.35, 12 and our meta-analysis demonstrated that the all AEs of oxycodone were similar to other opioids. Therefore, oxycodone could be a critical opioids in the management of moderate or severe pain in cancer patients in clinic. However, opioids were the second-line drugs in the management of neuropathic, tricyclic antidepressants were more appropriate compared with opioids.32The efficacy of oxycodone in the treatment of cancer-related pain had been proved to be superior to other opioids,12 To our best knowledge, this meta-analysis was the first article based on the current evidence, which focus on the side effects of oxycodone in patients with cancer pain. Besides risk of bias was assessed based on Cochrane Collaboration tool, and the methodological quality of included studies were good and fair. In addition, power analysis for this meta-analysis was conducted. Finally, no significant publication bias about all AEs was found.Our study has several strengths compared early meta-analysis.There were also some limitations in our meta-analysis. First, the statistical power of the RR of all AEs were low, so the results may not be sufficient convinced. Second, only small number of participates were included to evaluate the RRs of all AEs, and may reduce the power of our analysis; therefore, further studies should involve larger patients. Finally, not all types of cancers were involved, and the AEs of oxycodone may be different among various cancers originally.In conclusion, this meta-analysis suggested that there were no statistically different among oxycodone and other opioids in all AEs, subgroup analysis showed that Ox CR may had less sleepiness compare with Mc CR. However, low power analysis not reaches power of test and insufficient patients were existed in our study; this conclusion should be interpreted cautiously. Therefore, further high-quality RCTs are warranted in this field.


To explore the associations between different dietary patterns and semen quality in a general Asian male population.Cross-sectional study. Healthy Taiwanese men aged 18 years or older who participated in a standard medical screening program from 2008-2013 run by a private firm were included in this study. Semen parameters including sperm concentration (SC), total sperm motility (TSM), progressive motility (PRM) and normal sperm morphology (NSM) were recorded. A dietary questionnaire was used to categorize the participants into 5 groups: “Healthy diet”, “Western diet”, “High-carbohydrate diet”, “High sweet snacks & sugar-sweetened drinks” and “High-sodium diet”.A total of 7282 men completed the questionnaire regarding dietary pattern, and examination of anthropometric indexes was performed and laboratory data were obtained. A high intake of a “Western diet” resulted in statistically linear declines of SC and NSM (P < 0.001 and P < 0.001). Similarly, a greater intake of “High sweet snacks & sugar-sweetened drinks” was associated with a lower SC (P = 0.001). Increased intake of a “High-carbohydrate diet” was related to higher prevalences of abnormal TSM and PRM (P = 0.012 and P = 0.025). Similarly, a greater intake of a “High-sodium diet” was correlated with an elevated prevalence of abnormal NSM (P = 0.035).This study showed that a greater intake of a “Western diet” is associated with poorer SC and NSM, a “High sweet snacks and sugar-sweetened drinks” intake is correlated with a lower SC, and high-carbohydrate food is related to elevated prevalences of abnormal TSM and PRM. During the past few decades, there has been much discussion about changes in semen quality, with some studies showing its significant decline –3. OtherSeveral studies have indicated not only a decline in sperm concentration in most Western countries, but also poor sperm motility and an abnormal sperm morphology. While genetics, the endocrine system, obesity, age, smoking status and heavy alcohol use are known risk factors for a decreased sperm quality, there is increasing evidence that nutrition could play an important role in influencing semen quality –9. Over In addition to some studies that have demonstrated that a “western” diet, which is high in animal foods, processed foods, fats and sugar, might have a negative effect on male fertility, other recent studies have also indicated that a "prudent" diet, in which more fish/seafood, poultry, walnuts, vegetables and whole grain foods are consumed, may benefit sperm quality –13. HoweIn this cross-sectional cohort study, the cohort initially consisted of 7941 healthy male individuals aged 18 years or older who participated in a standard medical screening program run by a private firm  between January 2008 and May 2013. The firm attracted paying participants from all over Taiwan because of its known quality services, operational efficiency, and key facilities that were easily accessible. Membership of the program was required, with discounts in examination fees offered for people with a large-sized family or related individuals and for regular members who came back for repeat examinations in subsequent years, and these incentives succeeded in attracting and sustaining a large number of customers. Each participant signed a consent form authorizing MJ Health Management Institution to process the data generated from medical screening. Ethical reviews  were processed and approved at MJ Health Management Institution and at Tri-Service General Hospital in Taiwan. Data related to individual identification were removed and remained anonymous during the entire study process.3 and 0.4% (v/v) formaldehyde in distilled water. Sperm motility was classified as PRM (WHO class A+B) and TSM (WHO class A+B+C). Briefly, 10 microliters of well-mixed semen were placed on a clean glass slide that had been kept at 37°C and covered with a 22 × 22 mm coverslip. The slide was placed on the heating stage of a microscope at 37°C and immediately examined at ×400 magnification. Owing to the studied subjects being a general population, the semen parameters of most subjects were within the normal range. In particular, the abnormal NSM rate according to the latest WHO 2010 criteria (< 4%) was too low to enable us to evaluate the trend according to different anthropometric indexes. Therefore, the sperm morphology was assessed using WHO 1999 classification and LIFE data-based study [Semen samples were collected via masturbation following at least 3 days of abstinence using home-collection kits with a sterile plastic container, and the samples were sent to the laboratory for analysis within one hour. Four dependent semen parameters, including sperm concentration (SC), total sperm motility (TSM), progressive motility (PRM) and normal sperm morphology (NSM), were recorded. SC was evaluated using a hemocytometer . Samples were diluted in a solution of 0.6 M NaHCOed study , and theIn addition, all subjects completed a questionnaire related to dietary pattern and had their biochemical data recorded in order to analyze the relationships between different dietary patterns and semen quality in a large cohort study of the general population. Most food frequency questionnaires have been designed following the principles developed by American researchers, which may not be completely applicable in Taiwan. Therefore, in this study, we used a questionnaire based on the characteristics of Taiwanese dietary patterns according to the results of the Nutrition and Health Survey conducted by the government in Taiwan. This was a computerized questionnaire and was designed to display comprehensive food lists for self-selection by the participants. The questionnaire included the frequency and amount of consumption for each food group. There were 19 items in the questionnaire that covered the major Taiwanese dietary patterns. For analysis, the dietary patterns were then assessed and divided into five groups: “Healthy diet”, “Western diet”, “High-carbohydrate diet”, “High sweet snacks & sugar sweetened drinks” and “High-sodium diet”. The “Healthy diet” contained three different foods in total: light-color vegetables, dark-color vegetables and fruits. The definition of a “Western diet” was seven food groups, including milk, cheese, yogurt, meat, seafood, salad and fried food. The “High-carbohydrate diet” included three foods: refined grains, whole grains and root vegetables. “High sweet snacks & sugar sweetened drinks” included three types of food: cake or cookies, additional sugars and sweetened beverages. The “High-sodium diet” involved three food items: processed canned food, instant noodles and condiments. All items on the questionnaire were scored 1, 2, 3, 4 or 5, representing no intake, less than one exchange/week, one to three exchange(s)/week, four to six exchanges/week, one exchange/day and more than two exchanges/day, respectively. Exchanges are foods that are grouped together into categories or lists according to similarities in nutritional values; for example, meats, starches, dairy, vegetables and fruit. Measured portions of foods within the categories may be exchanged or traded when planning meals. Each serving of a food has about the same amount of carbohydrate, protein, fat, and calories as the other foods on the same list. Mean scores were calculated for all items of each dietary pattern, with higher scores indicating a greater intake of an individual dietary pattern. In this study, Cronbach’s alpha for the overall scale was 0.895 and Cronbach’s alphas for the individual domains ranged from 0.703–0.873.Owing to the properties of the questionnaire, we applied the quartile method to assess the relationships between intakes of all these food items and semen quality. Differences in the sperm parameters related to various food intake amounts as assessed by the food frequency questionnaire were compared using analysis of variance (ANOVA). To illustrate the real association between semen quality and obesity, all subjects were categorized by quartile according to each anthropometric index, and the severities and prevalences of abnormal sperm parameters were analyzed while increasing the intake of some dietary patterns. Abnormal sperm parameters were defined as SC < 15 M/ml, TSM < 40%, PRM < 32% and NSM < 30%. The frequencies of abnormal sperm parameters in each quartile were compared by the chi-square test. To categorize each dietary pattern, we assessed their relationships with semen outcomes by first conducting a logistic trend analysis and then estimating the odds ratios (ORs). Moreover, the topic of smoking is popular in relation to public health, and therefore we applied a questionnaire to assess the relationship between smoking and male infertility. According to the questionnaire, smoking duration was assessed as “0”: non-smoker, “1”: smoking < 1 year, “2”: 1 year to < 3 years, “3”: 3 years to < 5 years, “4”: 5 years to < 10 years, “5”: 10 years to < 20 years, “6”: 20 years or more. We then explored the association between smoking and semen quality by the quartile method . All analyses were conducted using SPSS statistical software .After excluding those with incomplete records and subjects with a major systemic disease or a history of reproductive organ disorder, a total of 7282 subjects were enrolled in the study. All subjects were aged between eighteen and seventy-five years and the semen quality, including sperm concentration , total sperm motility , progressive sperm motility  and normal sperm morphology , was examined.2, 81.06 ± 8.52 cm, 96.24 ± 6.44 cm, 0.84 ± 0.14, 0.47 ± 0.05 and 23.47 ± 5.49%, respectively , waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), waist-to-height ratio (WHtR) and body fat percentage, were 23.79 ± 3.30 kg/mectively . We disc< 0.001) with each quartile of the different dietary patterns (< 0.001 and P = 0.002).The results showed that the SC and NSM of each upper quartile for the “Western diet” and “High sweet snacks & sugar sweetened drinks” were lower than those of the lower quartile indexes. The mean SC values of the individuals with a “Western diet” and those with a high intake of “Highly sweet snacks & sugar-sweetened drinks” had significantly negative correlations . Our analyses showed that there was a linear association between a greater intake of a “High-carbohydrate diet” and increased prevalences of abnormal TSM  included in this study may not apply to other populations, particularly if the dietary pattern is dissimilar or foods are produced and processed in different ways. The strengths of this study were its cross-sectional design and the relatively large study sample size of 7282 men. We concluded that a higher “Western diet” intake, including dairy food, meat and saturated trans fatty acid intake, is associated with a poorer SC and NSM. A higher intake of “Highly sweet snacks & sugar-sweetened drinks” is related to a decline in SC. A greater intake of “High-carbohydrate food” is correlated with increased prevalences of abnormal total sperm motility and progressive sperm motility. However, some limitations of our epidemiological study existed, which included the fact that the questionnaire was simply a rough estimation of the connective food groups and was not typical of the food frequency questionnaire (FFQ), and data regarding semen volume and total sperm count were not available due to the protocol of the physical health examination. In addition, no assessments of related endocrine variations were made. Also, the participants had to pay a fee (~US$100) in order to participate in the medical screening program, which, although acceptable for the middle classes of the general population in Taiwan, may exclude poorer populations. In addition, as the participants had only been asked to undertake at least 3 days of abstinence, longer abstinence durations were not recorded in the data obtained from MJ Health Management Institution. Therefore, the effect of abstinence duration could not be examined. Furthermore, as this was a large sample size study, it was difficult to perform on-site collection of semen samples. Therefore, semen samples were collected using home-collection kits. As the semen samples were requested to be sent to the laboratory within one hour, the quality may not be equal to that resulting from on-site collection.Intake of a “Western diet” is linked to a poorer SC and NSM, a “Highly sweet snacks and sugar-sweetened drinks” intake is associated with a lower SC, and high-carbohydrate food is related to elevated prevalences of abnormal TSM and PRM. The observed relationships are biologically plausible; nevertheless, data regarding the relationships of diet in general and groups of food in particular with semen quality or male factors of infertility remain limited. Therefore, additional prospective studies, including studies exploring the biological mechanisms, may be needed to explain the associations between dietary patterns and male fertility.


The vast majority of AIS patients who require surgical intervention are women. Blood loss is a major concern during the operation.The medical records of all female AIS patients who underwent posterior correction and fusion operations using the all-pedicle screw system from January 2012 to January 2014 were reviewed. Patients with irregular menstruation; underwent osteotomy; use coagulants were excluded from the study. The remaining patients were divided into 4 groups according to the operation date in the menstrual cycle . The information of patients from the 4 groups was reviewed. The data was analyzed using analysis of variance, the Student-Newman-Keels test and Kruskal-Wallis Test.A total of 161 patients were included in this study. There were 40 patients included in group A, 38 patients in group B, 41 patients in group C and 42 patients in group D. The 4 groups were matched in age (P = 0.238), body height (P = 0.291), body weight (P = 0.756), Risser sign (P = 0.576), mean curve Cobb angle (P = 0.520), and bending flexibility index (P = 0.547), the number of levels fused (P = 0.397). The activated partial thromboplastin time (P = 0.235) and prothrombin time (P = 0.074) tended to be higher in group A, but the difference was not statistically significant. The fibrinogen level was lower in group B than the other 3 groups (P = 0.039). Blood loss and normalized intraoperative blood loss (NBL) was significantly higher in group A than the other 3 groups (P<0.01).The hemostatic function tended to be lower in the premenstrual phase. The fibrinogen level was lowest in the mid-follicle phase. Female AIS patients tended to endure more intraoperative blood loss when the operation was performed in the premenstrual phase during the menstrual cycle. Adolescent idiopathic scoliosis (AIS) is a structural, lateral, rotated curvature of the spine that arises in otherwise healthy children at or around puberty. AIS may affect 1–3% of the at-risk population when defined as a Cobb angle>10° Intraoperative blood loss (IOBL) is a major concern during the operation and may lead to many complications such as hypotension, anemia, coagulopathy, infection, and the need for transfusion of large volume of blood products with associated risks Sex hormone variations may affect the level of blood coagulation factors, thus, influence the hemostatic function in female adolescents In this retrospective analysis, we investigated the effect of operation time during different phases of menstrual cycle on IOBL in female adolescent idiopathic scoliosis patients who underwent posterior correction and all pedicle screw fixation. Previous studies have shown that increase in blood sex hormone could cause blood coagulation changes. We speculate that natural hormone variation during normal menstrual cycle may affect blood coagulation too. This variation is especially meaningful considering that the majority of AIS patients who need surgical intervention are female and the large IOBL during posterior correction and fusion surgery for AIS patients.Female AIS Patients, who sought medical help and underwent posterior correction and all pedicle screw fixation and fusion surgery in Chinghai hospital between January 2012 and January 2014, were retrospectively reviewed. The diagnosis of AIS followed the description of Weinstein et al Parameters, including: patient age; body weight; body height; the number of levels fused; major curve Cobb angle; major curve bending flexibility index; blood type; albumin (Alb); hemoglobin (Hg); fibrinogen; platelet count (PLT); activated partial thromboplastin time (APTT); prothrombin time (PT); thrombin time (TT); IOBL; operation date and menstrual cycle data were reviewed. Normalized intraoperative blood loss  was calculated using the following equation:The study protocol was approved by the Institutional Review Board of the Second Military Medical University, Shanghai, China. Written informed consent was obtained from every participant. The protocols of this study have been approved by the institutional review board of Changhai Hospital.Data from different groups was analyzed using analysis of variance (ANOVA), the number of levels fused was analyzed by Kruskal-Wallis Test. Differences between the groups were further analyzed using the Student-Newman-Keuls (SNK) test. The data was checked for normality and equal variances. A P value less than 0.05 was considered significant for each individual test in this study. Statistical Package for Social Science software 18.0  was used to perform the statistical analysis. Graphs were drawn using GraphPad Prism 5.0 .A total of 276 female AIS patients underwent posterior correction and spinal fusion were screened. Among them, 115 patients were excluded because of: Ponte Osteotomy during the surgery (38); combined anterior approach (17); diagnosed coagulopathies (9); menstrual cycle problems ; or intraoperative hemostasis (43). The remaining 161 patients were reviewed. There were 40 patients included in group A, 38 patients in group B, 41 patients in group C, 42 patients in group D. ANOVA showed that the 4 groups were matched in age (P = 0.246), body height (P = 0.359), body weight (P = 0.348), Risser sign (P = 0.628), mean curve Cobb angle (P = 0.596), bending Cobb (P = 0.993), bending flexibility index (P = 0.849). Kruskal-Wallis Test showed there was no difference in the number of levels fused (P = 0.497). The APTT (P = 0.168) and PT (P = 0.107) tended to be higher in group A than the other groups; however, the difference was not statistically significant. There were no significant differences in Hg (P = 0.875), PLT (P = 0.517), Alb (P = 0.247), and TT (P = 0.949) between the groups. There were significant differences in fibrinogen (P = 0.039), IOBL (P<0.01), and NBL (P<0.01) between the groups. The details of the 4 groups analyzed by ANOVA are shown in The SNK test  showed that the IOBL and NBL were significantly higher in group A than in other groups; there was no observed difference between groups B, C, and D. Fibrinogen was significantly lower in group B than groups A, C, and D; there was no observed difference between groups A, C, and D. Posterior correction with multilevel spinal fusion (PCSF) has been proven to be the most effective way to treat AIS A significant finding in our study was that patients in group A experienced significantly higher IOBL  than those in group B , group C , and group D . Even in group A, the NBL of patients tended to increase as the operation date approached menstruation. This indicates that patients who undergo surgery before menstruation may experience more blood loss. The differences between the groups may be attributed to the hormone-induced hemostatic function change. Van Roojen et al However, studies on variations in hemostatic factors during the normal menstrual cycle are rare and show contradictory results. Miller et al Another finding of this study was that the fibrinogen level was lowest in the follicle phase , combined anterior and posterior approach, use of antifibrinolytic medication before or during surgery, and use of osteotomy during surgery. Finally, rather than only dividing the patients into follicle and luteal groups as in previous clinical and basic studies related to this field Female AIS patients tended to endure more IOBL when the operation was performed in the premenstrual phase of the menstrual cycle. The hemostatic function tended to be lower in the premenstrual phase; however, statistical significance was not reached. The fibrinogen level was lowest during the mid-follicle phase.


HMIP-2 is a human quantitative trait locus affecting peripheral numbers, size and hemoglobin composition of red blood cells, with a marked effect on the persistence of the fetal form of hemoglobin, HbF, in adults. The locus consists of multiple common variants in an enhancer region for MYB (chr 6q23.3), which encodes the hematopoietic transcription factor cMYB. Studying a European population cohort and four African-descended groups of patients with sickle cell anemia, we found that all share a set of two spatially separate HbF-promoting alleles at HMIP-2, termed “A” and “B.” These typically occurred together (“A–B”) on European chromosomes, but existed on separate homologous chromosomes in Africans. Using haplotype signatures for “A” and “B,” we interrogated public population datasets. Haplotypes carrying only “A” or “B” were typical for populations in Sub-Saharan Africa. The “A–B” combination was frequent in European, Asian, and Amerindian populations. Both alleles were infrequent in tropical regions, possibly undergoing negative selection by geographical factors, as has been reported for malaria with other hematological traits. We propose that the ascertainment of worldwide distribution patterns for common, HbF-promoting alleles can aid their further genetic characterization, including the investigation of gene–environment interaction during human migration and adaptation. HBS1L-MYB intergenic polymorphism (HMIP) on Chromosome 6q23.3, which was first detected in a large Asian Indian family (“Family D”) of Gujarati/North Indian descent , but some individuals, including members of Family D, continue to produce significant amounts of HbF. When such “HPFH” occurs in patients with sickle cell anemia  blocks of common genetic variants associated with the trait , which occupies a 24-kb stretch of DNA that acts as a distal upstream enhancer for MYB  causing HPFH in this family. Subsets of these 12 SNPs have shown association with erythroid traits in every human population studied so far  a cohort of healthy European twins and (2) patients of African descent with SCA. Subsequently, we investigated the prevalence of haplotypes signaling the presence of trait-affecting functional variants in human populations across the world, interrogating data from the 1000 Genomes Project  and of the Tanzanian patient cohort by the Muhimbili University Research and Publications Committee (MU/RP/AEC/VOL XI/33). The African American patient cohort is part of a multicentre study (see below), approved by the Institutional Review Boards at the collaborating institutions. The Nigerian patients are part of an archival cohort that was analyzed anonymously. Study of the Twins UK cohort was approved by the St. Thomas’ Research Ethics Committee (LREC04/015).0 thalassemia hemizygous), from the UK, Nigeria, Tanzania, and the USA (Table S1). For all patients, the HbF levels  were measured by HPLC  from samples obtained during “steady state” outpatient visits, and off hydroxyurea therapy. Common variant genotypes were generated within individual genetic studies taking place at each of the centers involved. A cohort of three hundred African British SCA patients  were from stored material from a previous study of βS-haplotypes involving patients from the Northern  and Southern  parts of the country  are either of Hb SS or Hb S/β0 genotype and have been described previously  were collected from sources including the Cooperative Study of Sickle Cell Disease (CSSCD), Howard University and Children's Hospital Oakland. Of the 254 patients, 111 patients were recorded clinically with HbF < 3.1, whereas 133 patients had HbF > 8.6 and 10 patients with HbF in the intermediate range. We have previously  of healthy British European twins . As HbF levels in non-anemic individuals are below the dynamic range of the HPLC detection system, the trait is represented in the twins by the fraction of red blood cells that carries HbF (“F cells”) enumerated by flow cytometry after anti-HbF staining . For this, PCR reactions were carried out in a volume of 15 μl that contained Ampli Taq Gold , 2.5 mM MgCl2, 0.2mM each dNTP, FAM, and VIC labeled upstream primers and PIG-tailed . Fragment sizing and SNaPshot reaction were both analyzed using GeneMarker software, version 1.95 from SoftGenetics .Genotypes were generated from genomic DNA isolated from peripheral white blood cells. Genotype data from previous studies were included, which had been generated as described  with Q solution (using Qiagen recommended procedures). PCR products were purified using Wizard SV Gel and PCR clean up system and cycle-sequenced with BigDye Terminator v3.1 chemistry (Applied Biosystems). After3130XL electrophoresis, sequencing traces were inspected and scored with Sequencher 4.6 software.To investigate the critical region at the “A/a” sublocus, a 542-bp PCR amplicon  was sequenced in 18 unrelated Europeans  and 15 African British patients with SCA , all selected to be homozygous for HMIP-2 region for this study.Genomic DNA samples from African American patients were genotyped using Illumina HumanOmni1-Quad BeadChip System , which was designed for 76% genomic coverage for people of African ancestry. SNP genotype was called by using Illumina Genome Studio and extracted for the LD between markers was estimated and plotted with Haploview 4.2  and rs9402686 (at the “B/b” sublocus of HMIP-2). Within each clade and each population/patient group, allele frequencies of the remaining genotyped variants were calculated and displayed as “sequence logos” .Phase alignment of variant alleles into haplotypes in the Gujarati family was manually inferred from segregation patterns. In sickle patients, who are unrelated, haplotypes were inferred statistically using Phase 2.1.1  was analyzed by multiple linear regression , with age and sex included as covariates. The unstandardized regression coefficient (“β”) was estimated as a measure of the effect each variant allele has on ln(HbF) levels, independent of sample sizes and allele frequencies, which differ across populations. Meta-analysis of the four groups was conducted, using a fixed-effects (inverse-variance weighted) model and included a test for heterogeneity.In the European twins cohort, regression analysis with ln(%F cells) was carried out using the regress procedure in Stata version 10.1 after imputing missing genotypes with MACH 1.0 (Y Li and G Abecasis). Cotwin clustering was modeled by means of a modified sandwich estimator of the variance.1000 Genomes Project  and http://ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521/phase1_integrated_calls.20101123.ALL.panel (for the Sample-Population Mapping File), and the genomic region to be extracted as between chr6:135,411,228 and 135,465,800 (in hg19 coordinates). This region contains the entire HMIP-2 locus and a 3’ adjacent 30 kb genome segment. Haplotype clades were assembled and displayed as described above for the sickle cell disease patients.The HMIP-2 interval for 53 populations were accessed through the HGDP Selection Browser . Data are from 938 individuals genome-scanned on an Illumina 650K chip platform . Haplotype frequencies (Table S5) were plotted with Inkscape 0.48 into map positions according to sampling location . Thus, in total, two chromosomes were investigated for Denisovans and three, on average for Neanderthals, allowing the detection of major alleles, but making the detection of minor alleles at positions polymorphic in Neanderthals or Denisovans relatively unlikely.Denisova genotypes were accessed through the UCSC genome browser (track: Denisova High Coverage Sequence Reads). These originate from high-coverage genome sequence generated from a single individual , Gorilla , Orangutan , and Baboon  reference sequences were accessed through UCSC genome browser track “Multiz Alignments of 46 Vertebrates.”Chimpanzee  in our cohort of healthy European twins. We detected eight new variants  that were strongly associated with HbF persistence (Table S2). Four markers were not or only weakly associated with the trait and for five markers assays could not be designed or failed. Exploratory sequencing of a selection of twin samples showed that two of the latter  were in close LD with the other associated markers, bringing the total number of strongly trait-associated variants within the HMIP-2 interval to 22 . As expected, all these variants are in close LD , the minor alleles  forming one principal haplotype clade (23% of haplotypes) and the major alleles (associated with low HbF) forming the other principal clade (73%). For the two indels, the shorter alleles are part of the high-HbF clade, while the longer alleles reside within the low-HbF clade . The composition of the (major) low-HbF clade matches the sequence of the homologous chimpanzee positions and also sequence reads available for extinct hominins  at the HMIP-2 in SCA patients and population cohorts of African descent  was detected with 15 of the “European” variants, while seven variants were not associated . SNPs from the two groups contributed separately to the overall association with HbF , suggesting that the genetic architecture of HMIP-2 is similar in the patient cohorts, i.e., in African British patients  and “B/b” (at the 3’ end), each possessing a high-HbF form  or a low-HbF form .The clinical importance of HbF persistence in the β-hemoglobin disorders has led to numerous studies investigating the European-derived association signals at  HbF Fig.. This pars9376090-“C,” which is consistent with the hypothesis that these haplotypes joined the patients’ gene pool through European admixture (11% admixture from a European population carrying this haplotype at 22% would predict a frequency of 2.4%). The majority of HbF-increasing alleles at the 10 critical positions reside in two haplotype clades that contrast with the European-type  situation. One of these clades  contains the high-HbF alleles at three positions within the “A/a” sublocus, but not at the “B/b” sublocus and the other clade  is characterized by a strong consensus for the high-HbF alleles at six positions within the “B/b” sublocus only , rs66650371-“del,” rs9399137-“C” and rs35786788-“A.” Thus the haplotype signature “del-C-A” tags the presence of a functional, HbF-promoting “allele A” at this sublocus in European, North Indian (Gujarati), and diverse African populations. It has previously been suggested that rs66650371-“del” itself might be biologically effective and responsible for HbF association at HMIP-2 , +0.51 for rs9402686-A (tagging “a–B” and “A–B”) and +0.58 for rs9376090-C (tagging “A–B” only). Thus the European clade, containing “A” as well as “B” alleles, appeared to have an effect no larger than the two alleles on their own, though variability across groups was considerable , where the frequency of “A–B” is 23%, and where there are small numbers (∼0.6% each) of chromosomes carrying only either “A” or “B.”The three high-HbF clades  seem to have similar effects on HbF persistence in SCA patients, with similar regression coefficients  for tag SNPs representing the subloci ; access to larger datasets or populations with higher frequencies of “A–b” and “a–B” are required to sufficiently power this test. Homozygotes for “A–B” showed significantly increased trait values  suggests that the various instances of each, “A” and “B” allele, are derived from common ancestors and that they might be a general feature of human populations. To systematically investigate the presence of such alleles in populations across the globe, we looked for the presence of their characteristic haplotype signatures in public data from the 1000 Genomes Project  from the project's public online data repository. This included data on 21 of the 22 variants associated with HbF variants in Europeans. When grouping the haplotypes into clades based on tag SNPs rs9399137 (representing “A/a”) and rs9402686 (representing “B/b”), the remaining seven HbF-associated variants displayed a high-consensus pattern within these clades in all populations, analogous to what had been observed in the sickle patients. That is, in each of the populations sampled, haplotypes carrying the low-HbF variant for both tag SNPs display a high consensus for having low-HbF alleles at the remaining seven positions that define both subloci . Of all groups studied in Phase 1 of the 1000 Genomes project, the Japanese population had the highest frequency (38%) of HbF-increasing haplotypes, contributed from the Eurasian and “a–B”-type clades.The three Latin American populations sampled in the 1000 Genomes project  exhibit a high frequency of rs9399137, tagging “A/a,” and rs4895441, tagging “B/b” , those leading to higher HbF containing either “A” or “B’ (clades “A–b” and “a–B”), and those containing both high-HbF alleles (“A–B”).MYB enhancer , which is missing from the 1000 Genomes dataset. Beyond “A” and “B”, common alleles with comparable impact on erythroid traits are unlikely to exist at this locus, since association studies with haematological traits have consistently identified variants that belong to the “A–b,” “a–B,” or “A–B” clades, including African  high-HbF haplotype, which is common to most populations outside of Africa, is likely to have been prevalent in the founder population that started populating the rest of the world during the last interglacial period >125,000 years ago  of the 1000 Genomes project harbors small amounts of European-like haplotypes: a single “A–B” haplotype and two instances of an “A–b” haplotype with European-like features (rs9376090-C), which could either point towards the possible source of Eurasian haplotypes from within East Africa or, alternatively, be due to back-migration from Eurasia  has no such European-like features. High-HbF haplotypes belonging to “A–b” and “a–B” clades would have existed in Sub-Saharan Africa long before the expansion out of Africa, given their wide, albeit low frequency, distribution on this continent. Presently, it cannot be excluded that these polymorphisms might have predated anatomically modern humans. While the presence of a single read containing an allele belonging to “B” in the Neanderthal sequence pool (Table S3) could be an artifact, it is the prevalence gradient of “a–B” haplotypes (red in Fig.The functionally significant MYB enhancer polymorphisms at Plasmodium vivax, which could have been present in Africa since the divergence of human and chimpanzee lines (Carter & Mendis, HMIP-2-controlled traits, such as red cell number, size, and hemoglobin content, in addition to F cell increase and HbF persistence, are thought to arise through the intervention of cMYB in the kinetics of erythropoiesis (Thein et al., Powerful selection pressure exerted by malaria has shaped the distribution of erythroid trait variants across the world, with the largest impact in tropical regions, which have consistently supported malaria parasites and their insect vectors throughout primate evolution (Carter & Mendis, HMIP-2 variants between populations will be a useful tool in tracing human migration and assimilation through much of our evolutionary history. During these events, environmental challenges might have led to different demands on the generation of red blood cells. Exploring these processes and their genetic consequences will contribute to our understanding of human erythroid biology.We have presented evidence that two functionally similar but evolutionarily distinct enhancer polymorphisms affecting erythroid traits are present in most human populations. Their wide distribution will aid further mapping efforts to identify their biologically functional constituents. We propose that the comparison of haplotypes harboring critical


The prognostic significance of 1p19q loss in astrocytic gliomas has been inconclusive.IDH 1/2 mutations, MGMT promoter methylation, and expression of p53 and ATRX were assessed. Survival outcome was compared between the two histological groups.We collected 57 gliomas with total 1p19q loss from among 218 cases of WHO grade-II/III gliomas operated at Keio University Hospital between 1990 and 2010. These tumors were classified as oligodendroglial or “astrocytic” by a WHO-criteria-based institutional diagnosis. Chromosomal copy number aberrations (CNAs), Of the 57 codeleted gliomas, 37, 16, and four were classified as oligodendroglial, “astrocytic”, and unclassified, respectively. Comparative genomic hybridization revealed that although chromosome 7q/7 gain was more frequent in “astrocytic” gliomas, other CNAs occurred at a similar frequency in both groups. None of the “astrocytic” gliomas showed p53 accumulation, and ATRX loss was found in three of the 15 “astrocytic” gliomas. The estimated overall survival (OS) curves in the patients with codeleted oligodendroglial and “astrocytic” gliomas overlapped, and the median OS was 187 and 184 months, respectively. Histopathological re-assessment by a single pathologist showed consistent results.Gliomas with total 1p19q loss with “astrocytic” features have molecular and biological characteristics comparable to those of oligodendroglial tumors. Since the initial discovery of the predictive and prognostic significance of chromosome 1p loss -9. AlthoIn this study, we investigated a consecutive series of diffuse gliomas with an “astrocytic” appearance and that exhibited 1p19q loss, in order to assess their molecular–genetic and clinical–biological characteristics. The deletion status of 1p and 19q was analyzed by comparative genomic hybridization (CGH), which (unlike fluorescence in situ hybridization (FISH)) can accurately distinguish between complete and partial chromosome loss , 20. TheIn total, 218 supratentorial, diffuse gliomas of WHO grade II or III from adult patients without prior chemotherapy or radiotherapy were found in the hospital pathology records. The status of 1p and 19q was examined for clinical purpose in 152 of 218 gliomas. Of these, 53 were oligodendroglial , 93 were astrocytic (astrocytoma or anaplastic astrocytoma), and 6 were unclassified because of small sample size, by the original institutional diagnosis. Fifty-eight of 152 gliomas (38.2%) showed 1p19q loss. Of these, 38 were oligodendroglial , 16 were astrocytic , and 4 were unclassified. Written informed consent for translational research  was obtained for 57 of the 58 codeleted gliomas, and subsequent analysis was performed on these 57 tumors. The 57 patients comprised 35 males and 22 females, with ages ranging from 22 to 63 years. Demographic factors such as age and sex were comparable between the oligodendroglial and astrocytic groups .The 37 codeleted oligodendroglial gliomas comprised 15 oligodendrogliomas, 9 oligoastrocytomas, 9 anaplastic oligodendogliomas, and 4 anaplastic oligoastrocytomas . Nine ofThe 16 codeleted “astrocytomas” comprised 7 diffuse astrocytomas and 9 anaplastic astrocytomas. Five of the 16 cases were diagnosed by biopsy .Histopathological features of the 57 codeleted gliomas were re-assessed by a single pathologist according to current criteria . FollowiIDH mutation status was obtained for 53, and MGMT promoter methylation status was obtained for 51. Evaluation of the status of ATRX and p53 were possible in 15 of the 16 codeleted “astrocytic” gliomas. Mutational screening results along with original institutional diagnoses for each case are found in Of the 57 cases, CGH profiles were obtained for all, IDH1 or IDH2 mutations were detected in all 34 cases (100%) for which both genes were successfully examined. All changes at IDH1 codon 132 comprised a CGT to CAT (R132H) mutation, and all IDH2 codon 172 mutations were AGG to ATG (R172M). Twenty-one of 32 analyzed cases (65.6%) displayed MGMT promoter methylation , followed by loss on 14q , loss of 18q , gain on 11q/11 , loss on 13q , gain on 17q , and gain on 19p   for which both genes were successfully examined. MGMT promoter methylation was found in all 15 cases for which MSP was successfully performed. Expression of p53 was negative in all of the 15 successfully evaluated cases, whereas loss of ATRX expression was detected in three of the 15 , loss on 4q , gain on 11q/11, loss on 18q , gain of 19p , loss on 9p , gain on 9q , loss of 10p , loss on 14q , loss of 15q , and gain on 17q   and loss of 19p (25%).p = 0.0015), and loss of 10p was numerically more frequent in “astrocytic” gliomas (p = 0.0871).Gain on 7q/7 was significantly more frequent in “astrocytic” gliomas (p = 0.828). Eleven patients with “astrocytic” gliomas by resection showed comparable survival times . Moreover, histological grouping by a single pathologist using current criteria demonstrated a consistent result, again showing no significant OS difference between the two glioma categories . Furthermore, those results were confirmed by a comparison between oligodendroglial (33) and “astrocytic” (10) gliomas by consensus of institutional and single-pathologist diagnoses (p = 0.67).To determine whether any distinct clinical or biological characteristics occurred in 1p19q codeleted gliomas with astrocytic features, the survival outcome was compared between the two groups. Although initial treatments varied , treatment intensities during the overall clinical courses were similar between the two histological groups, i.e., 22 of 37 patients with oligodendroglial gliomas and 12 of 16 patients with “astrocytic” gliomas underwent both chemotherapy and radiotherapy in their clinical courses. Kaplan–Meier survival curves overlapped between the groups, and with a median follow-up of 87 months, the median OS in the oligodendroglial and “astrocytic” glioma groups was 187 and 184 months, respectively  exhibiting total 1p19q loss (71.7%) was similar to that observed in previous studies  and evenIDH mutations and MGMT promoter methylation was similar in both the “astrocytic” and oligodendroglioma groups. Because these genetic/epigenetic alterations are strong prognostic factors in diffuse gliomas, their similar frequency supports the hypothesis that both tumor types are likely to exhibit similar biological characteristics.Our study demonstrated that the survival curves of 1p19q codeleted “astrocytic” gliomas and oligodendrogliomas overlapped and that there was no statistical difference in the median survival time between the two groups. The observed median OS of the “astrocytic” gliomas with 1p19q loss (15.5 years) is much longer than that reported for low-grade astrocytomas (5–7 years) , 31. TheA previous study suggested the prognostic relevance of classic oligodendroglial morphology, regardless of the 1p19q status . In contAlong with 1p19q codeletion, loss of chromosome 4q, loss on 14q, and loss on 18q are commonly observed chromosomal alterations in oligodendroglial tumors , 33. In On the other hand, the finding that gain on chromosome 7q/7 was associated with an “astrocytic” phenotype even within codeleted gliomas was intriguing. Gain on chromosome 7q/7 is the most frequent chromosomal alteration in astrocytic tumors, as determined by CGH , 34, 35.TP53 and ATRX genes represent genetic hallmarks of astrocytomas [TP53 and ATRX mutations compared with those of astrocytomas in general [Mutations in the ocytomas , 38. Los general , 38 suggThe histopathological assessments conducted in this study were not performed by a panel of neuropathologists. Moreover, five of the 16 “astrocytic” gliomas were diagnosed by biopsy specimens. Therefore, it is possible that the “astrocytic” gliomas analyzed here could contain a population of “non-classic oligodendrogliomas” with some astrocytic features .The present study was retrospective and treatments were not uniform. Nonetheless, the cohort analyzed here was collected from consecutive cases treated at a single institution, and the survival analyses were based on long-term follow-ups. Moreover, the comparable survival outcomes between the two groups were confirmed in “astrocytic” gliomas by resection, and by comparison of the two groups with consensus diagnoses.In conclusion, gliomas with total 1p19q loss with some or more “astrocytic” features are likely to have comparable biological and prognostic characteristics to those with oligodendroglial features. Although some of the codeleted “astrocytic” gliomas showed molecular markers associated with astrocytic differentiation, such as 7q/7 gain and ATRX loss, the molecular and genetic characteristics of those gliomas were, overall, very similar to codeleted oligodendrogliomas. Thus, total loss of 1p and 19q is a robust molecular and prognostic marker for gliomas, regardless of their histological features. These findings further substantiate the importance of molecular classification of gliomas.Pathology records of brain tumors treated at the Department of Neurosurgery, Keio University Hospital between 1990 and 2010 were reviewed, and patients who fulfilled the following criteria were included: a) age > 18 years; b) supra-tentorial tumor location; c) institutional histopathological diagnosis of diffuse glioma of WHO classification grade II or III; d) no prior chemotherapy or radiotherapy treatment. The institutional histopathological diagnosis was conducted according to WHO criteria , 39-41.Written informed consent was obtained from all 57 patients finally included in the study for translational research approved by the Institutional Review Board at Keio University .Clinical data were obtained from the patients' records and included age at diagnosis, sex, extent of surgery, composition of initial postoperative therapy, time to progression, treatment at recurrence, and date of death or last contact. The survival outcome of patients lost during follow-up was updated for the present study.The histological criteria for defining oligodendroglial tumors have changed over time, partly because of the relevance of the diagnosis to therapeutic decision-making and the estimation of prognosis , 21. AltTumor DNA was extracted from microdissected pieces of formalin-fixed paraffin-embedded (FFPE) tissue . For tisChromosomal number aberrations (CNAs) were assessed by metaphase CGH as described previously , 44. In MGMT promoter methylation was assessed by methylation-specific PCR (MSP) using the EZ DNA Methylation-DirectTM Kit  as described previously [eviously .IDH1/2 genes was assessed in three steps. First, formalin-fixed paraffin (FFP) sections were examined for IDH1R132H by immunohistochemistry with an anti-mutant IDH1 antibody  [IDH1 gene was amplified with previously described primers [IDH1 exon 4 mutation, exon 4 of IDH2 was similarly amplified and sequenced [Mutation of the Germany) . For neg primers . After pequenced .Accumulation of p53 and loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression were assessed by immunohistochemistry in “astrocytic” gliomas by original institutional diagnosis as molecular surrogates of astrocytic nature. FFP sections were examined for ATRX immunohistochemistry with anti-human ATRX  and for p53 immunohistochemistry with anti-human p53 . For the evaluation of p53, FFP sections from anaplastic astrocytoma resected in 1994 were used as positive control; staining of >5% nuclei for p53 was judged as positive. For the evaluation of ATRX, FFP sections from anaplastic oligoastrocytoma resected in 2012 were used as positive control, and staining of endothelial cells was considered as an internal positive control. Staining of >10% nuclei was judged as positive for ATRX .Patients were classified into two histopathological groups . Demographic factors and the treatment and distribution of categorical factors such as CNAs were compared between groups by Fisher's exact test. The overall survival (OS) was calculated from the beginning of treatment, i.e., from the date of initial surgery for resection cases and from the first day of adjuvant therapy for biopsy cases. Survival curves were estimated and compared between groups using the Kaplan–Meier method and the log rank test. Significance levels for all tests were 2-sided and 0.05. All data were analyzed with IBM SPSS statistics 22.