C677T polymorphism, circulating levels of homocysteine (Hcy), and the severity of coronary lesion in patients with acute coronary syndrome (ACS) remains unknown.The association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms were determined via amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). Gensini scores were used to evaluate the severity of coronary lesions.Consecutive ACS patients were included. MTHFR C677T polymorphism variant: CC , CT , and TT  groups. No significant differences were detected with respect to baseline characteristics. Patients in TT group had significantly higher Hcy, and significantly lower folic acid (FA) levels as compared with those in the other 2 groups (P < .05 for both). More importantly, patients with TT had more severe coronary lesions as compared with those from the other 2 groups, as evidenced by higher Gensini scores (P < .05 for both); however, no significant differences were observed with respect to the numbers of affected coronary arteries, or the number, length, and diameter of stents implanted in each group . On multivariate logistic regression analysis, presence of a T allele in MTHFR C677T was found to be independently associated with higher circulating Hcy , and higher Gensini scores .Three hundred ten ACS patients were included, and grouped according to the MTHFR C677T TT polymorphism was associated with higher Hcy levels and more severe coronary lesions in patients with ACS.MTHFR  The overall survival of patients with ACS has improved with the use of antiplatelet drugs and statins, and more importantly, with timely revascularization via percutaneous coronary intervention (PCI) and coronary artery bypass graft. However, primary prevention of ACS deserves more efforts as it is a fundamental strategy to lower the morbidity burden attributable to CAD. Therefore, early identification of patients at risk of development of severe CAD is important in clinical practice.Despite significant advances in the diagnosis and treatment for atherosclerosis, coronary artery disease (CAD) continues to be one of the leading causes of morbidity and mortality worldwide. Moreover, epidemiological studies have proved that hyperhomocysteinemia (HHcy) is an important risk factor for stroke. A recent randomized controlled trial in China demonstrated that additional administration of folic acid, an intervention for lowering circulating Hcy level, to hypertensive patients on enalapril significantly reduced the risk of first stroke in patients. Although pilot studies have suggested that HHcy may be a risk factor for CAD, the association between homocysteine and the severity of coronary lesions, particularly in patients with ACS, has rarely been reported.Evidence from experimental studies suggests that homocysteine (Hcy) may be involved in the pathogenesis of atherosclerosis.C677T in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the catabolism of Hcy, were shown to be associated with increased circulating Hcy, since the catabolic activity of the enzyme was reduced to 65% and 30% in CT and TT carriers as compared with that in wild type CC. A meta-analysis of case-control studies found that MTHFR C677T polymorphism was associated with risk of MI in young and middle-aged populations, particularly among Caucasians. However, the potential influence of MTHFR C677T polymorphism on the association between Hcy level and the severity of coronary lesions in patients with ACS has not been determined. Hence, we performed a pilot study to evaluate the potential association between MTHFR C677T polymorphism and the severity of coronary lesions in ACS patients. In addition, the potential role of circulating Hcy, folic acid (FA), and their associations with the anatomic and clinical severity of ACS were also analyzed.Accumulating evidence also suggests various gene polymorphisms may also influence the association between Hcy levels and CAD risk. Variants of 22.1The study protocol was approved by the Ethical Committee at the First Affiliated Hospital of Bengbu Medical College . The study included consecutive adult patients with ACS who underwent coronary angiography (CAG) percutaneous coronary intervention (PCI) at the Cardiovascular Department, the First Affiliated Hospital of Bengbu Medical College  between December 1, 2015 and April 30, 2016. Written consent was obtained from all patients prior to their enrollment. The diagnosis of ACS was in accordance to the current guidelines. All patients were of Han ethnic origin. Patients with hepatic or renal failure, severe hematopoietic diseases, severe infection, tumor or other malignant diseases, as well as diseases of endocrine system that may affect the serum Hcy, were excluded from the study. To minimize the potential influence of medications on circulating Hcy, patients receiving folic acid (FA), vitamin B6 or vitamin B12 were also excluded.2.2C677T genotyping, and for measurement of circulating Hcy and FA levels. Hypertension was diagnosed based on the recommendation of the Chinese Guideline for the Diagnosis and Management of Hypertension (2010), as well as the World Health Organization and International Society of Hypertension (WHO/ISH) Guidelines for the Management of Hypertension, with a systolic blood pressure (BP) ≥140 mmHg and (or) a diastolic BP ≥90 mmHg. All patients were under intensive monitoring and care during their hospitalization.All patients underwent comprehensive assessments including the demographic information, medical history, and physical examination before the CAG. Medications for secondary prevention of CAD were prescribed as soon as the diagnosis of ACS was confirmed; these included aspirin, clopidogrel or ticagrelor, and statins. Other medications, including nitrates, angiotensin converting enzyme inhibitors or angiotensin II receptor blockers, and β blockers were prescribed and adjusted according to the heart rate and blood pressure of the patients. Fasting whole blood samples were collected on the second day of admission for routine blood biochemical tests, MTHFR 2.3Plasma biochemical parameters, including fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), serum creatinine (SCr), serum uric acid (SUA), and C reactive protein (CRP) levels were measured using an Olympus AU5800 autoanalyzer  at the Department of Clinical Laboratory at our institution.2.4Circulating levels of Hcy and FA were determined via high performance liquid chromatography (HPLC) and electrochemiluminescence methods at the Cardiopulmonary Laboratory at our institution.2.5C677T polymorphisms were then determined via amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) at the Cardiopulmonary Laboratory at our institution. Patients were grouped according to the MTHFR C677T polymorphisms into CC, CT, and TT groups.Deoxyribonucleic acid (DNA) was extracted from whole blood samples using a DNA extraction kit, according to the manufacturer's instructions . MTHFR 2.6C677T polymorphisms tests. Results of CAG were evaluated and judged according to the criteria of ACC/AHA Guidelines for PCI (2001). For patients with severe coronary stenosis , a drug-eluting stent (DES) was placed. Success of the PCI process was defined by a residual stenosis <20% and thrombolysis in myocardial infarction (TIMI) 3 flow. The severity of the coronary lesions was assessed using Gensini scores. By 2 experienced cardiovascular specialists independently and the average Gensini score was used for the subsequent analyses.Independent cardiovascular specialists who performed the CAG and PCI procedure were blinded to the results of the MTHFR 2.7P value <.05 was considered to be statistically significant.Continuous variables are presented as mean ± standard deviation (SD), while categorical variables are presented as frequencies and percentiles. If normally distributed, the analysis of variance (ANOVA) was applied for comparison of continuous variables among multiple groups, and Dunnett test was used to assess differences between 2 groups. However, for continuous variables associated with a non-normal distribution, Kruskal–WallisH test was used. Between-group differences with respect to categorical variables were assessed with chi-squared test. Multivariate logistic regression analysis was performed to evaluate the association between the clinical characteristics and presence of a T allele in MTHFR C677T. All statistical analyses were performed using SPSS 17.0 software . A 2-tailed 33.1C677T (25.2%), 137 patients were classified as CT (44.2%), and 95 patients were classified as TT (30.6%). The allele frequencies for the C and T alleles were 47.3% and 52.7%, respectively. Baseline characteristics of the included patients according to the distributions of MTHFR C677T gene polymorphism are shown in Table P > .05 for all).Overall, our study included 310 patients with ACS (179 men and 131 women) (mean age: 62.5 ± 10.8 years). Based on the results of genotyping, 78 patients were classified as CC for MTHFR 3.2C677T polymorphism had different baseline levels of circulating Hcy (P < .001 on ANOVA) and FA (P = .004 on ANOVA). Specifically, patients in the TT group showed significantly higher circulating Hcy levels (23.25 ± 13.58 μmol/L) as compared with those in patients in the CC (14.52 ± 4.86 μmol/L) and CT (14.53 ± 4.55 μmol/L) groups  were significantly higher than those in the CC (30.60 ± 23.38) and CT (35.18 ± 25.90) groups .PCI perioperative characteristics of ACS patients with different MTHFR 3.5C677T genotypes and other related risk factors according the subtype of ACS are shown in Table C677T genotypes were observed between patients with UA and those with AMI, circulating Hcy levels as well as the levels of FBG, TG, and CRP were significantly higher in patients with AMI . These results suggest that patients with AMI may have more remarkably increased Hcy, and more severe metabolic imbalance, and enhanced inflammatory response.Distribution of MTHFR 3.6P = .024), and higher Gensini scores .We performed multivariate logistic analysis to explore the potential association between patient characteristics and the presence of a T allele in MTHFR C677T Table . The res4C677T polymorphism were associated with significantly higher circulating Hcy and significantly lower FA levels as compared with those with CC or CT variants. In addition, patients with TT variants had significantly higher Gensini scores as compared with that in patients with CC or CT, which indicates that the TT variants of the MTHFR C677T polymorphism was associated with more severe coronary lesions, although the baseline characteristics and other perioperative characteristics were comparable between the groups. Moreover, presence of a T allele in MTHFR C677T was independently associated with higher circulating Hcy and higher Gensini scores. Collectively, these results suggest that the TT variants of the MTHFR C677T polymorphism may be an important marker of CAD owing to its’ association with increased circulating Hcy and more severe coronary lesions.In a cohort of ACS patients who had undergone CAG, we found that patients with TT variants of the MTHFR C677T polymorphism was associated with more severe coronary lesions in patients with ACS. These results are consistent with those of some earlier studies. In a previous case-control study, a T genotype of MTHFR C677T gene polymorphism was associated with higher incidence rate of vulnerable plaque and higher Hcy level, which were both considered as risk factors for more severe coronary artery disease. In another case-control study , the frequency of T genotype of MTHFR C677T gene polymorphism among patients with CAD was higher than that in controls. More importantly, the T genotype was more prevalent in ACS patients as compared with that in patients with stable CHD. Also, higher Hcy was noticed in CHD than controls. Our study further extended these results by using the overall severity of the coronary lesions as evaluated by Gensini score of CAG results. Overall, these results indicate that the T genotype of MTHFR C677T gene may be a risk factor for the prevalence of more severe coronary lesions, which is probably related to a higher circulating Hcy value in these patients.Results of our study indicate that the TT variant of the MTHFR  These results were further confirmed in subsequent case-control studies which suggest a possible independent association between Hcy and severity of CAD.,21 A recently published study from China that included 292 CAD patients of different categories, and 100 non-CAD controls concluded that HHcy is independently associated with the severity of CHD, and significantly correlated with low folic acid levels in CAD patients. The above findings suggest a possible association between T genotype of MTHFR C677T gene, higher circulating Hcy, lower FA levels, and more severe coronary lesions. These results warrant interventions aimed at lowering of Hcy levels in high-risk patients, particularly in those with T genotype of MTHFR C677T gene. Although previous randomized controlled trials (RCTs) of FA supplementation aimed at lowering Hcy levels did not show significant improvement in cardiovascular outcomes,,23 these studies were limited to low-risk patients; moreover, studies that have evaluated the genotype of MTHFR C677T gene have been rare. In fact, higher Hcy levels in patients with high-risk CAD have been shown to be associated with poor outcomes. In a cohort of 587 patients with CAG-confirmed CAD (318 underwent CABG and 120 received PCI), higher baseline Hcy was shown to be a strong predictor of mortality over a follow-up duration of 4.6 years. Therefore, we hypothesize that interventions aimed at lowering Hcy levels in high-risk patients (such as ACS patients after PCI with T genotype of MTHFR C677T gene) may improve cardiovascular outcomes. Further studies are needed to evaluate the above hypothesis.An earlier small-scale study of 53 patients with CAD showed that higher circulating Hcy was related to the severity of coronary disease, as evaluated by atheroma burden index. Subsequent studies found Hcy may induce apoptosis of endothelial progenitor cells via enhancement of endoplasmic reticulum stress-mediated activation of caspase-3. Moreover, HHcy was also shown to be related to impaired endothelial function as evidenced by reduced levels of nitric oxide,,28 which is a key factor involved in the pathogenesis and progression of atherosclerotic vascular disease. Besides, Hcy may also deteriorate the status of CAD via its interaction with cardiomyocytes. In a rat model of myocardial ischemia, Hcy was shown to reduce homing of cardiac stem cells, and subsequently restrain myocardial repair mediated by these cells. Moreover, HHcy was shown to lead to dysfunction and apoptosis of cardiomyocytes, an effect that was mediated via other signaling pathways, such as activation p38 MAPK.,31 Therefore, current evidence suggests that HHcy may aggravate CAD via its interaction with both the endothelial cells and cardiomyocytes.Although the potential mechanisms underlying the association between higher circulating Hcy and more severe coronary lesions are not fully understood, results from experimental studies may provide some clues. Early in vitro studies suggest that Hcy may cause endothelial insult via enhanced inflammatory and oxidative stress related response.C677T polymorphism and the severity of the coronary lesions. Therefore, we were not able to conclude that the TT variant of the MTHFR C677T polymorphism is an independent determinant of the severity of coronary lesions in patients with ACS. Further studies with adequate sample size are needed to determine the influence of conventional risk factors for CAD on the observed association of TT variants with severity of CAD. Further, whether the association between the TT variant and severity of coronary lesion persists after adjusting for circulating Hcy levels also needs to be determined. Secondly, similar to other observational studies, our study design does not allow us to draw causal inferences from the observed association between MTHFR C677T polymorphism, higher circulating Hcy, and more severe coronary lesions. Moreover, we focused our study on the outcome of severity of coronary artery disease. Whether MTHFR C677T polymorphism is associated with other clinical outcomes, such as the risk for the development CAD, and prognosis of CAD patients also deserves evaluation. Finally, although our study confirmed that patients with higher circulating Hcy are associated with severer coronary lesions, the efficacy of Hcy-lowering therapy for the prevention and treatment of CAD remains to be evaluated. Current evidence from meta-analyses of RCTs does not support an effective role of Hcy-lowering therapy for reducing the risk of cardiovascular events.,23 However, whether Hcy-lowering interventions may improve the cardiovascular prognosis in high-risk population, such as those with ACS after PCI, should be further investigated.Some limitations of our study need to be considered while interpreting the results. Firstly, since the sample size of the included patients was relatively small, we did not perform a multivariate analysis of the association between the distribution of TT variants of the MTHFR C677T TT polymorphism was associated with higher Hcy levels and more severe coronary lesions in patients with ACS. Whether Hcy-lowering therapies could improve clinical outcomes in these patients requires further investigation.In conclusion, MTHFR


Saccade slowing has been proposed as endophenotype marker in Spinocerebellar Ataxia type 2 (SCA2), nevertheless the heritability of this trait has not been properly demonstrated. Thus the present paper was aimed to assess the heritability of different saccadic parameters in SCA2.Forty-eight SCA2 patients, 25 preclinical carriers and 24 non-SCA2 mutation carriers underwent electronystagmographical assessments of saccadic eye movements as well as neurological examination and ataxia scoring. Estimates of heritability based on the intraclass correlation coefficients were calculated for saccade velocity, accuracy and latency as well as for age at disease onset from 36, 17 and 15 sibling pairs of SCA2 patients, preclinical carriers and controls, respectively.Saccade velocity was significantly reduced in SCA2 patients and preclinical carriers, whereas decreased saccade accuracy and increased saccade latency were only observed in the patients cohort. Intraclass correlation coefficient for saccade velocity was highly significant in SCA2 patients, estimating a heritability around 94%, whereas for the age at ataxia onset this estimate was around 68%.Electronystagmographical measure of saccade velocity showed higher familial aggregation between SCA2 patients leading the suitability of this disease feature as endophenotype marker, with potential usefulness for the search of modifier genes and neurobiological underpinnings of the disease and as outcome measure in future neuroprotective clinical trials. Endophenotypes are measurable components of a disease that have simpler associations to genetic underpinnings than the disease syndrome itself . The terEndophenotypes must to fit some criteria that define its validity and usefulness , such asATXN2 gene, with the consequent expression of abnormally large polyglutamine tract in the ataxin-2 protein. SCA2 patients present a progressive cerebellar syndrome, usually accompanied by a combination of other non-cerebellar features such as slow saccades, peripheral neuropathy and others [Among the hereditary ataxias, endophenotype term has been only applied in the Spinocerebellar Ataxia type 2 (SCA2) . This aud others , 9. Seved others , 11. SCAd others , 13, ideElectronystagmographical assessments have led to characterize the saccade slowing as SCA2 endophenotype due to this trait affects almost all patients , 14 and Forty-eight patients and 25 preclinical carriers were admitted to the Centre for Research and Rehabilitation of Hereditary Ataxias in Holguín (CIRAH) for this study. Twenty-four non-SCA2 mutation carriers from Holguin province were admitted as control group. The main demographical, clinical and molecular data of each group is shown in the Table All subjects underwent standardized neurological examination  and the Horizontal saccades were recorded binocularly with a 2-channel electrooculography (EOG) device , using Ag-AgCl electrodes over right and left outer canthus of the eyes. The EOG signal was amplified and bandpass filtered (0.2–70 Hz). The data were sampled at a frequency of 200 Hz with a time base of 1000 ms/division, sensitivity of 200 μV/division and a time constant of 8 s. To minimize head movements of participants during the recordings, a forehead and chin rest were used in all cases.Subjects were asked to fixate the target in the central position and to redirect their gaze to the new location of the target as soon as it appeared in the periphery, and later back in the central position. Ten centrifugal saccades in both horizontal directions were registered at 60° predictable amplitudes of stimulus. EOG signals were calibrated for a horizontal angle of 30°. Each session of EOG lasted around only 6 min. Saccade latency, accuracy and maximal saccade velocity were analyzed.The heritability estimates were obtained by analysis of variance components of the saccade parameters and age at onset among sibships and the error variance within the sibships, allowing for the computation of the sibling intraclass correlation (ICC) . ICC valHeritability analyses in SCA2 patients were conducted in 21 sibling groups, 17 of them had two members, three had three members and only one had five members, which yielded a total of 36 sibling pairs. Among preclinical carriers, a total of 17 sibling pairs were obtained from 11 sibling groups, eight of them with two members and three with three members. Finally, 15 sibling pairs of non-SCA2 mutation carriers were obtained from nine siblings groups with two members and two siblings groups with three members.2) tests. One-way ANOVAs, followed by Fisher-LSD post hoc tests, were carried up to compare the saccade variables between groups. Correlation analyses (followed by Bonferroni correction) were performed by the Pearson correlation test. All analyses were conducted using the STATISTICA software package .Intergroup differences of frequencies for clinical features were computed by Chi-square  and SARA score  were significantly correlated to expanded CAG repeats in the patients’s cohort.The frequency comparisons of main clinical features between the groups are showed in the Fig. Similar to preceding works , 14, 15,r = 0.60; p = 0.0001), age at onset , aging  and SARA score  in SCA2 patients.Correlation analyses only disclosed significant associations of saccade velocities with expanded CAG repeats  to expanded ataxin-2 . Within Therefore, the differences observed in the saccade velocity between SCA2 patients with relatively similar mutation sizes could result from differential effects of modifier genes on the PolyQ toxicity in the EBN cells. Hence, the study of patients who have saccade velocities out of the mean range adjusted on CAG repeats offers important advantages for the identification of these modifying genes and the understanding of its mechanisms of actions.Then, with the higher heritability estimates observed in this paper for saccade slowing, we satisfy all criteria defining endophenotypes, which were shown in Introduction and thus we offer new evidences about the conceptualization of this oculomotor trait as endophenotype marker in SCA2 (Fig. A limitation of this paper was the inclusion of a relative small number of subjects. Nevertheless, even in this small cohort, we obtained significant heritability estimates for saccade slowing, suggesting the robustness of this heritable measure.As conclusion, saccade slowing, as assessed by electronystagmography, showed higher familial aggregation between SCA2 patients, supporting the suitability of this oculomotor trait as disease endophenotype, a sensitive tool for the search of modifier genes and neurobiological underpinnings of the disease, as well as to evaluate the efficacy of future neuroprotective clinical trials.


Algae encompass a wide array of photosynthetic organisms that are ubiquitously distributed in aquatic and terrestrial habitats. Algal species often bloom in aquatic ecosystems, providing a significant autochthonous carbon input to the deeper anoxic layers in stratified water bodies. In addition, various algal species have been touted as promising candidates for anaerobic biogas production from biomass. Surprisingly, in spite of its ecological and economic relevance, the microbial community involved in algal detritus turnover under anaerobic conditions remains largely unexplored.Chlorella vulgaris (Chlorophyta), Chara sp. strain IWP1 (Charophyceae), and kelp Ascophyllum nodosum (phylum Phaeophyceae), using sediments from an anaerobic spring , sludge from a secondary digester in a local wastewater treatment plant , and deeper anoxic layers from a seasonally stratified lake  as inoculum sources. Within all enrichments, the majority of algal biomass was metabolized within 13–16 weeks, and the process was accompanied by an increase in cell numbers and a decrease in community diversity. Community surveys based on the V4 region of the 16S rRNA gene identified different lineages belonging to the phyla Bacteroidetes, Proteobacteria , Spirochaetes, and Firmicutes that were selectively abundant under various substrate and inoculum conditions. Within all kelp enrichments, the microbial communities structures at the conclusion of the experiment were highly similar regardless of the enrichment source, and were dominated by the genus Clostridium, or family Veillonellaceae within the Firmicutes. In all other enrichments the final microbial community was dependent on the inoculum source, rather than the type of algae utilized as substrate. Lineages enriched included the uncultured groups VadinBC27 and WCHB1-69 within the Bacteroidetes, genus Spirochaeta and the uncultured group SHA-4 within Spirochaetes, Ruminococcaceae, Lachnospiraceae, Yongiibacter, Geosporobacter, and Acidaminobacter within the Firmicutes, and genera Kluyvera, Pantoea, Edwardsiella and Aeromonas, and Buttiauxella within the Gamma-Proteobaceteria order Enterobacteriales.Here, we characterized the microbial communities mediating the degradation of Our results represent the first systematic survey of microbial communities mediating turnover of algal biomass under anaerobic conditions, and highlights the diversity of lineages putatively involved in the degradation process. Bacillariophyceae, Chrysophyceae, Eustigmatophyceae), Viridiplanta , in addition to exclusively algal phyla such as the Euglenozoa, Cryptomonads, Haptophyta, and Rhodophyta , whose blooms are often encountered in lakes and other freshwater habitats , which seasonally occur in ponds and lakes and cause a strong and unpleasant musky odor (Phaeophyceae), which is believed to be one of the most productive photosynthetic organisms and tend to attain long lengths at a very fast elongation rate (∼50–60 cm/day)  levels in the ecosystem, often resulting from anthropogenic inputs  , as wellhabitats , Chara bsky odor , as wellsky odor . Fast gr cm/day) .Algae provide a large input of organic carbon into aquatic ecosystems such as coastal kelp forests , meromicChlorella sp. , representing green algae that are known to bloom during summer months (June–September) in multiple freshwater bodies within the US. Strain UTEX 2714 was obtained from the University of Texas at Austin Algal Culture collection, and cultures were maintained on proteose medium plates  at 22 ± 3 °C. Fluorescent light was used (3500 lux) on a 16:8 h light to dark cycle. Cultures were harvested by scraping the growth on the surface of agar plates, and the resulting biomass was used as the carbon source. (2) Chara sp. strain IWP: Mats of Chara (class Charophyceae) were obtained from a local pond  in August 2015, and identified using morphological and microscopic analysis as Chara sp. Chara is known to grow locally in ponds in Oklahoma and peak around June–September . Samples collected were thoroughly washed and soaked in DI water for 2 h, to remove other associated biomass, before they were centrifuged and the resulting biomass was used as the carbon source to represent class Charophyceae. (3) Kelp: Due to the geographical location of the study and brown algae being common occurrences in marine environments, we were not able to obtain a fresh kelp sample to be used as a substrate. Alternatively, Ascophyllum nodosum (phylum Phaeophyceae) representing brown algae was obtained as a whole dried powder from a local provider  and directly used as the carbon source.We evaluated the microbial community mediating algal detritus degradation by setting enrichments with various types of algal biomass as the only source of carbon. Three different algal species were examined: (1) Axenic Chlorella culture, the Chara and the kelp samples were not guaranteed axenic and a minimal input of bacteria with the carbon source in these enrichment bottles could not be ruled out.It is worth noting that, with the exception of the axenic 2) man-made lake in Northeastern OK operated by the Grand River Dam Authority, which keeps continuous records of the lake water geochemistry. During summer months (starting in June), large areas of the lake become seasonally stratified, with deeper layers (June–September) becoming completely anoxic. Within these sites, surface chlorophyl1-a concentration peaks in May–July, followed by algal biomass sinking to deeper anaerobic layer in September (Samples were obtained from Grand Lake O’ the Cherokees (hereafter Grand Lake) to investigate the microbial community involved in algal turnover. The lake is a large . Due to the constant ejection of sulfide laden water from the spring source, the shallow spring is light exposed, yet mostly anoxic . The mic2HPO4 3 g l−1), 150 ml of mineral solution II 2SO4, 6; NaCl, 6; MgSO4.7H2O, 0.6, and CaCl2.2H2O, 0.6), 10 ml Balch vitamins solution , 1 ml of Wolin’s metal solution 2, 0.01; Na2MoO4.2H20, 0.01; boric acid, 0.01; Na2SeO4, 0.005; NiCl2.6H20, 0.003; CoCl2.6H20, 0.1). The media were amended with L-cysteine hydrochloride  as a reductant, and resazurin  as a redox indicator, boiled under a stream of N2 gas, dispensed in the serum bottles, autoclaved, cooled, then transferred to an anaerobic chamber  where 5 g of sediment , 5 g of sludge , or 5 ml of concentrated lake water (the pellet obtained after centrifugation of 1.5 L of Grand Lake water (GL)) were added as the inoculum sources. Algal biomass was added as a substrate (∼0.25 g per bottle). In addition, substrate unamended controls  were included. After enrichment preparation in the anaerobic chamber, the bottles were stoppered, sealed, taken out of the chamber and the headspace in the bottles was changed by repeated flushing with 100% N2. Samples were incubated at room temperature (22 °C) in the dark. Enrichments were periodically sampled  for DNA extraction by thoroughly mixing the serum bottle and anoxically withdrawing 3 ml of the enrichment. At the end of the enrichment process , bottles were sacrificed, centrifuged and 3 ml of the pellet was used for DNA extraction. The rest of the pellet was used for chemical analysis of the remaining algal detritus.Enrichments were prepared in 120 ml serum bottles under anaerobic conditions. Nine different treatments  were set up in triplicates. Serum bottles contained 45 ml of an anoxic solution containing (per liter): 150 ml of minerals solution I  as per the manufacturer’s instruction. DNA from triplicate treatments was pooled prior to amplification and sequencing. DNA from substrate-unamended controls was also extracted (hereafter pre-enrichment sample). The extracted and pooled DNA  was quantified using Qubit fluorometer . The genes for the V4 hypervariable region of 16S rRNA were amplified using the prokaryotic-specific primer pair 515F and 806R (SRP083898.DNA was extracted using the PowerSoiland 806R  to avoidand 806R . Both PChttps://hpcc.okstate.edu/). For quality control purposes and to eliminate poor quality sequences, an average quality score of 25 was chosen as the threshold value below which sequences were considered of poor quality and removed from the dataset. In addition, sequences that contained an ambiguous base (N), sequences having a homopolymer stretch longer than 8 bases, and sequences longer than 293 bp were also removed from the datasets.mothur software  was usedhttps://www.arb-silva.de/). The aligned, filtered, de-noised, and chimera-free sequences were used to generate an uncorrected pair wise distance matrix. Sequences were clustered into operational taxonomic units (OTUs) at 0.03% sequence divergence cutoff using the vsearch clustering method employed through mothur. A shared file was created and was used for subsequent analyses. Most of the above steps were derived from the MiSeq SOP available from the mothur website (http://www.mothur.org/wiki/MiSeq_SOP).High-quality reads were aligned in mothur using the Silva alignment database as a template. Aligned sequences were then filtered to remove columns that corresponded to ‘.’ or ‘-’ in all sequences. Filtered alignments were then subjected to a pre-clustering de-noising step using a pseudo-single linkage algorithm with the goal of removing sequences that are likely due to sequencing errors . PossiblPhyla considered significant to the degradation process were empirically defined as those phyla that constituted 5% or more of the community at any time during enrichment. These include phyla that were abundant prior to enrichment and remained abundant during and after enrichment, phyla that transiently increased in abundance during part of the enrichment but then decreased in abundance by the end of enrichment, and phyla that significantly and progressively increased in abundance with enrichment time. Within these abundant phyla, genera considered significant to the degradation process were also empirically defined as those whose percentage abundance represented 1% or more of the total abundance.Various alpha diversity indices  were performed on individual datasets in mothur. When comparing species richness across datasets , numbers were reported per sample size to normalize for the differences in the number of sequences obtained between datasets. Beta diversity based on community structure was assessed by calculating pairwise Bray–Curtis dissimilarity indices and using the output to construct non-metric multidimensional scaling (NMDS) plots in mothur.To study the significance of the effect of inoculum source versus algae type on community structure, we performed an analysis of variance using the function Adonis in the R statistical package vegan. The effect was visualized using the percentage abundances of significant phyla/classes (≥5% as defined above) in a constrained correspondence analysis (CCA) using the function cca in the R statistical package vegan.® Instrument (Life Technologies). Primer pair EUB-338F/UNI518R (mcrA) gene from the total methanogenic community, and primer pair Dsr2060F/Dsr4R (dsrB) gene from the total sulfate reducing community. The 25-µl PCR reaction mixtures contained 0.3 µM of each forward and reverse primers , 2 µl extracted template DNA, and 12.5 µl SYBR GreenER™ qPCR SuperMix. The reactions were heated at 95 °C for 8.5 min, followed by 40 cycles, with one cycle consisting of 30 s at 95 °C, 45 s at 50 °C  or 55 °C (for sulfate reducers), 30 s at 72 °C, and 15 s at 85 °C for signal reading. To calculate the total number of cells belonging to total bacteria, total archaea, methanogens, and sulfate reducers in the enrichments, a standard curve was generated using DNA from Bacillus subtilis strain 168 (ATCC 23857), Haloferax sulfurifontis strain  M6 (DSM 16227), Methanosarcina hungatei strain JF1 (ATCC 27890), and Desulfovibrio desulfuricans strain G20 (ATCC BAA-1058), respectively. To account for the multiple copies of 16S rRNA genes per cell, the number of copies obtained from the standard curve was divided by an empirical value of 3.5 (average of 1–6 copies of rRNA genes in one cell). However, since the mcrA and dsrB genes are known to be present as single copies in methanogens, and sulfate reducers, respectively, no such adjustment of the total number of cells was required when calculating the total number of cells belonging to methanogens and sulfate reducers.We used qPCR to quantify total Bacteria, total Archaea, as well as methanogens and sulfate-reducing bacteria in the enrichments using a MyiQ thermocycler  and SYBR GreenER™ qPCR SuperMix for iCycler/UNI518R  was used/UNI518R  was used/UNI518R  was used0F/Dsr4R  was usedf; final dry weight) and ground to fine material. The ground material was first used for protein extraction using the method described previously  and family  levels were always above 96 and 98.2% , strongly indicating that the communities have been adequately sampled in all enrichments  were retained for further analysis. The average number of sequences per dataset was 26,946. The calculated Good’s coverage for the majority of samples at putative species (OTUichments .Chlorella and Chara) at the conclusion of the experiment. Final time point analysis demonstrated that the majority of the starting dry weight of Chara , Chlorella , and, to a relatively lower extent, kelp  in ZDT, WWT, and GL enrichments, respectively, was metabolized at the conclusion of the experiment. Analysis of the chemical composition of the remaining algal detritus in comparison to the starting material showed that the carbohydrate, lipid, and protein contents of the algal detritus were consumed to varying extents (0.03 and OTUs0.1 as well as the estimated species richness (using both Chao and ACE estimators) decreased compared to the pre-enrichment sample, hence indicating the selection for few taxa  were used to compare and visualize differences in the microbial community structure between all enrichments at all sampled data points. At first glance, it was apparent that the enriched microbial communities  within each algae type-enrichment source combination  versus inoculum source  on the enriched microbial communities revealed that kelp selects for a distinct and highly similar microbial community, regardless of the inoculum source   . On the munities . This ismunities .p-value for algae type = 0.028, p-value for inoculum source = 0.001). To decipher the relative contributions of algae type versus inoculum source on the microbial community composition at the phylum/class level we employed canonical correspondence analysis (CCA) using the enriched phyla/classes relative abundances. The results  showed that both the algae type and the source of inoculum were significant in shaping the microbial community albeit to varying levels . These phyla were: Firmicutes , Bacteroidetes (in six enrichments), Spirochaetes (in five enrichments), and the Gamma (5 enrichments), Delta (6 enrichments), Alpha, Beta, and Epsilon (one enrichment) Proteobacteria –5. HowevChara microcosms, Bacteroidetes, Firmicutes, and Delta-Proteobacteria were consistently abundant , previously identified as an important in-situ fermenter of organic matter-rich soil , previously enriched from anaerobic freshwater sediment (Bacteroides (WWT and GL microcosms), a well-documented complex carbohydrate degrader in a wide range of environments (Barnesiella (GL microcosms), a known fermentative gut microbe , Spirochaetes only enriched in WWT and ZDT microcosms, and Gamma-Proteobacteria enriched in GL (but not ZDT) enrichments. Similar to Chara enrichments, the taxa VadinBC27,  Mangroviflexus, Paludibacter, Barnesiella, and WCHB1-69 within the Bacteroidetes; Desulfovibrio, Desulfobacter, and Desulfomicrobium within the Delta Proteobacteria; Spirochaeta and unclassified SHA-4 within the Spirochaetes were all abundant community members at the end of enrichment. Within the Firmicutes, the family Lachnospiraceae was abundant in all enrichments, similar to what was observed in Chara microcosms. However, apart from this notable exception, the enriched community of Firmicutes genera/families differed in Chlorella microcosms when compared to Chara enrichments. Within the ZDT microcosms on Chlorella, a wide range of Clostridiales-affiliated genera and families were encountered, with members of the genera Geosporobacter (family Clostridiaceae_1), and Acidaminobacter (family Clostridiaceae_4), Youngiibacter (family Clostridiaceae_1), and members of Clostridiales Family XIII constituting ∼34% of total sequences encountered in ZDT microcosms. Further, In contrast to Chara enrichments where Veillonellaceae was only restricted to GL microcosms, Chlorella enrichments selected for members of this family in ZDT and WWT microcosms.Enrichment patterns in Chlorella enrichments selected for members of the Gamma-Proteobacteria only in GL microcosms where they constituted ∼54% of the total taxa in these enrichments. Buttiauxella and Aeromonas were identified as major taxa in GL Chlorella microcosms, similar to what was observed in Chara enrichments. In addition, members of the genus Edwardsiella (family Enterobacteriaceae) were identified as a Chlorella enrichment-specific taxon  previously shown to contribute to the overall cellulolytic activities in barley straw microcosms (Arcobacter and Sulfurospirilum) and Gamma  Proteobacteria, collectively comprising ∼25% of the total enriched taxa. Members of these genera were previously implicated in anaerobic plant biomass degradation  representing the brown algae (Phaeophyceae). We followed the turnover of these algae in enrichments that were set up using three different sources of inoculum: an anoxic freshwater sulfide- and sulfur-rich spring , a wastewater treatment plant , and a seasonally stratified lake that experience seasonal algal blooms . We identified multiple microbial lineages that were significantly enriched in these treatments. Some of these lineages appear to be substrate-specific , habitat-specific , or treatment-specific .In this study we investigated the microbial community mediating algal detritus turnover under anaerobic conditions. We utilized three representative algal species: Within all nine treatments examined, a high level of diversity was invariably retained at the conclusion of the incubation process. We reason that this is a reflection of the complexity of the substrate utilized. Algal detritus harbors multiple complex macromolecules, e.g., proteins, lipids, nucleic acids, and polysaccharide, that vary considerably in structure and hence require multiple enzymes and pathways for their efficient degradation . Such lClostridium and Anaerosporobacter and family Veillonellaceae, regardless of the inoculum source . While only a handful of environments were examined in this study, the consistent selection for members of a specific lineage regardless of the starting inoculum suggests the ecological significance of this lineage in kelp detritus turnover in anaerobic habitats. The reason for this observed pattern of Clostridiales genera/families selection on kelp could only be speculated upon. A possible contributing factor could be the unique cell wall structure of kelp ; multiple cellulose microfibrils layers embedded in large interfibrillar matrices that are mostly composed of alginates and fucans (Clostridiaceae bacterium and a Porphyromonadaceae bacterium (Dysgonomonas capnocytophagoides)) anaerobically degrading alginate. Based on the study by Our results and subsequent community analysis  indicated fucans . Alginatd fucans  and fucad fucans  degradatd fucans . On the d fucans , there iChara or Chlorella as an algal inoculum, the final microbial community enriched was highly divergent, and the final community structure was mostly dependent on the inoculum sources , rather than the type of algal substrate provided  in Charophyta, or sulfated polysaccharides in Chlorophyceae  is a reflection of the relative ubiquity of microbial lineages capable of the anaerobic degradation of cellulose, hemicellulose, and pectin in the algal cell walls, hence allowing ready access to the intracellular substrates within the algal cells.On the other hand, when using provided , Table 3ophyceae . We reasClostridium, Anaerosporobacter, Lachnospiraceae incertae sedis) as shown before (Enterobacteriaceae (Kluyvera)  in kelp enrichment from ZDT  and detected by qPCR  . Producepirillum . Proteincobacter . Additiocobacter . Previoucobacter , which ccobacter . On the  and CO2 . While oanalysis  and the from ZDT . The lacfrom ZDT  argues a by qPCR  in the p by qPCR , where nChara and Chlorella enrichments, complex carbohydrate  degradation to sugar monomers could be mediated by members of the Bacteroidetes uncultured groups VadinBC27  and WCHB1-69 , as well as the Spirochaetes (Spirochaeta and the uncultured group SHA-4 enriched on Chara and Chlorella from ZDT and WWT sources). These lineages have been consistently enriched in anaerobic sludge digestors , Family Veillonellaceaea (in Chara enrichments from GL), Family Lachnospiraceae (in Chara and Chlorella enrichments from GL), and Family Clostridiaceae genera Yongiibacter, Geosporobacter, Acidaminobacter (in Chloreela enrichments from ZDT)), as well as Enterobacteriales (Genera Kluyvera (Chara enrichments from ZDT), Pantoea (Chara enrichments from GL), Edwardsiella and Aeromonas (Chlorella enrichment from GL), and Buttiauxella (Chara and Chlorella enrichments from GL)) could potentially mediate complex carbohydrate degradation  or incompletely  by sulfate-reducing bacteria , or the incomplete oxidizers ) when sulfate is available. The produced acetate, H2 and CO2 could either be metabolized to methane by aceticlastic or hydrogenotrophic methanogenic lineages observed in the enrichments  enrichments from ZDT), or metabolized by the aceticlastic autotrophic SRBs in the presence of sulfate. The increase in dsr copy numbers in Chara and Chlorella enrichments as measured by qPCR, the utilization of the available substrates in these enrichments , as wel sulfate  evidence sulfate . It is wcrocosms  and coulIn conclusion, our work represents the first systematic survey of microbial communities mediating turnover of algal biomass under anaerobic conditions, and highlights the diversity of lineages putatively involved in the degradation process. The results presented here could certainly open the door for future studies that investigate the interactions between the abundant genera identified as significant for the degradation process, as well as for targeted isolation studies for algal detritus degraders.10.7717/peerj.2803/supp-1Supplemental Information 1Includes two supplementary tables and two supplementary figuresTable S1Table S2Figure S1Figure S2Click here for additional data file.


Eyelid surgery is done to correct trachomatous trichiasis to prevent blindness. However, recurrent trichiasis is frequent. Two procedures are recommended by WHO and are in routine practice: bilamellar tarsal rotation (BLTR) and posterior lamellar tarsal rotation (PLTR). This study was done to identify which procedure gives the better results.A randomised, controlled, single masked clinical trial was done in Ethiopia. Participants had upper lid trachomatous trichiasis with one or more eyelashes touching the eye or evidence of epilation, in association with tarsal conjunctival scarring. Exclusion criteria were age less than 18 years, recurrent trichiasis after previous surgery, hypertension, and pregnancy. Participants were randomly assigned (1:1) to either BLTR or PLTR surgery, stratified by surgeon. The sequences were computer-generated by an independent statistician. Surgery was done in a community setting following WHO guidelines. Participants were examined at 6 months and 12 months by assessors masked to allocation. The primary outcome was the cumulative proportion of individuals who developed recurrent trichiasis by 12 months. Primary analyses were by modified intention to treat. The intervention effect was estimated by logistic regression, controlled for surgeon as a fixed effect in the model. The trial is registered with the Pan African Clinical Trials Registry (number PACTR201401000743135).vs 63/496 [13%]; adjusted odds ratio [OR] 1·96 [95% CI 1·40–2·75]; p=0·0001), with a risk difference of 9·50% (95% CI 4·79–14·16).1000 participants with trichiasis were recruited, randomly assigned, and treated (501 in the BLTR group and 499 in the PLTR group) between Feb 13, 2014, and May 31, 2014. Eight participants were not seen at either 6 month or 12 month follow-up visits and were excluded from the analysis: three from the PLTR group and five from the BLTR group. The follow-up rate at 12 months was 98%. Cumulative recurrent trichiasis by 12 months was more frequent in the BLTR group than in the PLTR group (110/496 [22%] PLTR surgery was superior to BLTR surgery for management of trachomatous trichiasis, and could be the preferred procedure for the programmatic management of trachomatous trichiasis.The Wellcome Trust. Chlamydia trachomatis, is the leading infectious cause of blindness.Trachoma, a neglected tropical disease caused by Unfortunately, trichiasis frequently recurs after surgery. This outcome represents a substantial limitation in preventing sight loss from trachoma. Studies have reported trichiasis recurrence rates between 10% at 3 months and up to 60% at 3 years, with an average of around 20% at 1 year.Evidence before this studyClinicalTrials.gov, and WHO ICTRP. We searched until May 7, 2015, using the search terms “trachoma” and “trichiasis”. See the review's appendix for full search methods for each database. We identified one previous randomised trial , which compared variants of the BLTR and PLTR procedure done by ophthalmologists in a teaching hospital environment in Ethiopia; 153 patients were randomly assigned and followed for 3 months. No evidence of a difference in outcome was found. However, this earlier study was constrained by a small sample size and short duration. The surgery was performed in a teaching hospital setting by ophthalmologists, in contrast to the health centre provision by non-physicians typical of trachoma control programmes, limiting the conclusions that can been drawn.Members of our study group recently published a systematic review of the management of trachomatous trichiasis . When preparing this systematic review, we searched CENTRAL, Ovid MEDLINE, Embase, ISRCTN registry, Added value of this studyOur trial was designed to compare the two most common operations used to treat trachomatous trichiasis to determine which gives the best results in terms of disease recurrence and complications in a programmatic setting. The results show that the PLTR was superior to BLTR because it had a substantially lower trichiasis recurrence rate by 1 year and fewer intraoperative and immediate postoperative complications.Implications of all the available evidenceThis study provides evidence of superiority of PLTR, suggesting that it could be the best procedure for the programmatic management of trachomatous trichiasis. We suggest that new surgical trainees in both established and new programmes be trained in the PLTR procedure. Another trial examining the outcomes of PLTR surgery done by surgeons previously trained in BLTR surgery should be considered.About 20 years ago several procedures were compared with the BLTR operation in randomised controlled trials.There is an unprecedented effort to scale up global trichiasis surgery output and improve outcomes, to clear the huge trichiasis backlog. This effort requires training many trichiasis surgeons on the easiest, safest, and most successful operation with the least recurrence and complications. There is an urgent need to examine rigorously which of these two most frequently performed operations has the best outcomes in a programmatic setting, with an adequate sample size and follow-up period. This question was identified as a research priority several years ago by the WHO Alliance for the Global Elimination of Trachoma by 2020 (GET2020).This was a single-masked, individual-randomised, controlled trial done in Ethiopia. Adults with trachomatous trichiasis were randomly allocated to either BLTR or PLTR surgery, stratified by surgeon, and followed up for 1 year. The study was approved by the Ethiopian National Health Research Ethics Review Committee, the London School of Hygiene & Tropical Medicine Ethics Committee, Emory University Institutional Review Board, and the Ethiopia Food, Medicine and Healthcare Administration and Controls Authority. The trial was done in compliance with the Declaration of Helsinki and International Conference on Harmonisation–Good Clinical Practice. An independent data and safety monitoring committee oversaw the trial.Participants had upper lid trachomatous trichiasis with one or more eyelashes touching the eye or evidence of epilation, in association with tarsal conjunctival scarring. We excluded people with trichiasis due to other causes, recurrent trichiasis after previous surgery, hypertension, pregnancy, and those under 18 years. Patients were recruited mainly through community-based screening in three districts of West Gojam Zone, Amhara Region, Ethiopia. Recruitment and surgery were performed in community level health centres. Written informed consent in Amharic was obtained before enrolment from participants. If a participant was unable to read and write, the information sheet and consent form were read to them and their consent recorded by thumbprint.Participants were randomly assigned (1:1) to either PLTR or BLTR surgery for each surgeon, with random block sizes of 4 or 6. Randomisation was stratified by surgeon because of potential intersurgeon variability. The sequences were computer-generated by an independent statistician. Separate allocation sequences for each surgeon were concealed in sequentially numbered, sealed, opaque envelopes. A person independent of all other aspects of the trial prepared these envelopes.On most recruitment days, two surgeons operated simultaneously. Following baseline examination, participants were allocated to the next available surgeon. A fieldworker was responsible for implementing the intervention assignment in a dedicated area. The fieldworker and surgeon jointly confirmed the allocation and recorded this in the surgical logbook. The different surgical equipment sets for the two procedures were kept separately. The randomisation fieldworker and surgeon jointly collected the appropriate surgical set for the allocated procedure. Surgeons and patients were aware of the allocation. The two examiners  who were responsible for clinical observations at baseline, 6 months, and 12 months were masked to the allocation. They were not involved in the allocation process, surgery, postoperative care, or the 10 day follow-up. The intraoperative and 10 day observations were made by separate fieldworkers who could not be masked to the allocation.At the preoperative assessment before randomisation, demographic characteristics were recorded. Presenting logMAR (logarithm of the minimum angle of resolution) visual acuity at 2 m was measured using PeekAcuity software on a smartphone in a dark room.Eyes were examined by a single examiner (EH) using 2·5× binocular loupes and torch, and graded using the Detailed WHO FPC Grading System.Before recruitment, nine experienced trichiasis nurse-surgeons, already trained, certified, and regularly performing PLTR surgery were trained in BLTR surgery. We followed the procedures described in the WHO Trichiasis Surgery for Trachoma manual.The procedures are described in detail in the WHO manual.Participants were examined at 10 days, 6 months, and 12 months after operation. At 10 days, data were collected on patient-reported outcomes . Participants were examined for recurrence, degree of lid eversion, infection, granulomata, and eyelid contour abnormality (ECA) before suture removal.At 6 months and 12 months participants were re-examined following the same procedures as for baseline (SA at 6 months and EH at 12 months). The examiners were standardised and had very strong agreement for the primary outcome in grading validation studies (κ=0·95). Based on severity, trichiasis cases were categorised into minor trichiasis with less than six lashes or evidence of epilation in less than one third of the lid margin, and major trichiasis with six or more lashes or evidence of epilation in one third or more of the lid margin. The degree of entropion correction was graded as follows: (grade 1) extra eversion: main lashes point superiorly, whole lid margin visible, and tarsal plate surface visible; (grade 2) lid margin eversion: lashes point superiorly, whole lid margin visible, and tarsal surface not visible; (grade 3) partial lid margin entropion: some parts of the lashes might point anteriorly and some part of the lid margin not visible; (grade 4) total lid margin entropion: lashes might point inferiorly or towards the globe and lid margin is not visible. We considered grade 1 over-correction, grade 2 normal correction, and grades 3 and 4 under-correction. Granulomata were defined as fleshy tissue growth of at least 2 mm on the tarsal conjunctiva or at the edge of the eyelid.High-resolution digital photographs of upper eyelid, cornea, and tarsal conjunctiva were taken at 6 months and 12 months.The primary outcome was the cumulative proportion of individuals who developed recurrent trichiasis by 12 months. Recurrent trichiasis was defined as one or more lashes touching the eye or clinical evidence of epilation, or a history of repeat trichiasis surgery by 12 months. A-priori defined secondary outcome measures were: recurrent trichiasis at 6 months and 12 months; trichiasis recurrence difference by surgeon; trichiasis recurrence difference by baseline disease severity; number, type, and location of recurrent lashes at 12 months; corneal opacity, vision, and contrast sensitivity changes at 12 months; intraoperative, immediate, and late postoperative surgical complications ; ECA at 12 months; and patient-reported outcomes.vs 10%). Therefore, we aimed to recruit 1000 cases (500 in each group), to allow for about 15% loss to follow-up.In the STAR trial, the 1 year trichiasis recurrence rate using BLTR surgery was about 10% (tetracycline group).Data were double-entered into Access 13 (Microsoft) and transferred to Stata 11 (StataCorp) for analysis. For participants who had bilateral surgery, we randomly designated one eye to be the study eye for the analysis. A modified intention-to-treat analysis was done, with primary outcome data analysed on all participants seen at either the 6 month or 12 month follow-up or both. Those not seen at either of these follow-up visits were excluded from the analysis.The primary outcome and binary secondary outcomes were compared between the two surgical groups with logistic regression analyses to estimate the odds ratio (OR) and 95% CI. All comparisons between the two surgical procedures were controlled for surgeon as a fixed effect in the model to account for the stratified randomisation. The risk difference in the primary outcome (recurrent trichiasis by 12 months) between BLTR and PLTR procedures was estimated. The possibility of effect modification between group and a-priori defined factors such as surgeon, preoperative trichiasis severity, papillary inflammation, age, and sex was investigated by including interaction terms in the model and using a likelihood ratio test to assess statistical significance of the interaction term. Ordered categorical secondary outcomes  were compared between the two surgical interventions using ordinal logistic regression. Categorical secondary outcomes  were analysed using multinomial logistic regression to estimate relative risk ratio (RRR) and 95% CI. Negative binomial regression was used to analyse the difference in the number of recurrent lashes touching the eye between the two intervention groups. The signed-rank test was used to analyse visual acuity and contrast sensitivity changes between baseline and the 12 month follow-up. The risk of trichiasis recurrence difference by surgeon between the two surgical interventions was analysed using logistic regression adjusted for baseline disease severity such as entropion and trichiasis. To investigate the possibility of a learning curve effect during recruitment, the trichiasis recurrence rates for the first 50% of cases to be recruited versus the second 50% of cases recruited for each surgeon were compared using logistic regression adjusted for baseline disease severity such as entropion and trichiasis. The trial is registered at the Pan African Clinical Trials Registry (PACTR201401000743135).The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between Feb 13, 2014, and May 31, 2014, 5168 people were examined for eligibility, of whom 1314 (25%) had trachomatous trichiasis . The remParticipants were reassessed at 10 days (range 7–14) for suture removal, 6 months, and 12 months after enrolment. Almost all (98%) participants were examined at each follow-up. At 10 days, two people had travelled to another region, and had sutures removed in their new locality. Eight (1%) participants were not seen at either 6 month or 12 month follow-up visits and were therefore excluded from the analysis: three from the PLTR group and five from the BLTR group. Hence, primary outcome data were available and analysed for 992 (99%): 496 in each group.vs 16 min 39 s; p<0·0001).Baseline demographic and clinical characteristics were balanced between the trial groups . The majBy 12 months, the primary outcome, cumulative recurrent trichiasis, had developed in 173 (17%) of 992 study eyes. Cumulative recurrence was significantly more frequent in the BLTR group (110/496 [22%]) than in the PLTR group (63/496 [13%]); after adjusting for surgeon, the OR was 1·96 . The risk difference for recurrent trichiasis between BLTR and PLTR procedures was 9·50% (95% CI 4·79–14·16). There was no evidence of effect modification between group and a-priori defined other factors on the primary outcome, including surgeon.vs 19%; OR 1·97 [95% CI 1·47–2·65]; p<0·0001). The risk difference for recurrent trichiasis between BLTR and PLTR procedures was 12·5% (95% CI 7·2–17·8).The primary outcome analysis using the photograph grading results was similar to the field grading. By 12 months, cumulative recurrent trichiasis was recorded for 250 (25%) of 992 study eyes. Recurrence was significantly more frequent in the BLTR group than in the PLTR group . At 12 months, recurrent trichiasis was present in 131 (13%) of 981 study eyes and again remained significantly more frequent in the BLTR group than the PLTR group .At 10 days, recurrent trichiasis was present in three study eyes, one in the PLTR group and two in the BLTR group. At 6 months, recurrent trichiasis was present in 114 (12%) of 983 study eyes, and was significantly more frequent in the BLTR group than the PLTR group (71 [14%] There was no evidence of a difference in the risk of trichiasis recurrence between surgeons by 12 months for either PLTR (p=0·80) or BLTR (p=0·44), or for a learning curve during the course of the trial for either procedure. For PLTR, recurrence risks during the first and second half of recruitment were 32 (13%) of 248 and 31 (12%) of 248, respectively (p=0·68). For BLTR, recurrence risks during the first and second half of recruitment were 55 (22%) of 247 and 55 (22%) of 249, respectively (p=0·93).vs 12 month median logMAR, 0·5 [0·2–0·7]; signed-rank test, p<0·0001) and contrast sensitivity  in the entire combined study sample.The number, type, and location of recurrent lashes were comparable between the two groups , Table 2After adjusting for surgeon, there was evidence of a difference in odds of intraoperative, immediate, or late postoperative complications between the two surgical interventions . There wvs PLTR, 5%; RRR 0·76 [95% CI 0·41–1·44]; p=0·40). There was evidence of more under-correction at 12 months with BLTR surgery than PLTR surgery  ECA at 12 months was lower in the BLTR surgery group than the PLTR surgery group  offer the operational advantage of not needing to be removed, we have previously found in a randomised trial that silk and absorbable sutures have comparable outcomes, and therefore it is unlikely that the outcome of this present study would be modified by their use.Overall, the PLTR procedure was superior to the BLTR in terms of lower trichiasis recurrence and fewer intraoperative and immediate postoperative complications. All other factors being equal, PLTR could be the preferred procedure for the programmatic management of trachomatous trichiasis. We suggest new surgical trainees in both established and new programmes should be trained in the PLTR procedure. Additionally, consideration could be given to further research to investigate whether individuals previously trained to do BLTR surgery need to be re-trained in PLTR surgery.


Poor vitamin D status has been associated with tuberculosis (TB); whether poor status is cause or consequence of disease is uncertain. We conducted a case-control study and two nested case-control studies to determine whether vitamin D levels were associated with active TB, tuberculin skin test (TST) conversion, and risk of progression to the active TB in prisoners in Brazil. In multivariable conditional logistic regression, subnormal vitamin D levels  were more likely in prisoners with active TB. In contrast, vitamin D was not found to be a risk factor for either TST conversion  or progression to active disease . Black race , less than 4 years of schooling , cigarette smoking  were identified as risk factors for TST conversion. Risk of progression to active TB was found to be associated with cigarette smoking . Our findings in the prison population show that poor vitamin D status is more common in individuals with active TB, but is not a risk factor for acquisition of latent TB or progression to active TB. The World Health Organization estimates that one-third of the world’s population has latent TB1, of which approximately 10% will develop active disease2. Understanding which individuals will progress to TB and which will maintain lifelong immune control or achieve clearance, remains an important scientific question with substantial public health implications. This high-risk group also provides the opportunity to examine host factors that currently lack complete understanding in the disease pathway for active TB.Tuberculosis (TB) is a major global public health problem, causing approximately 1.4 million deaths annually3. Nutritional deficiency, low exposure to sunlight due to spending only short periods of time outdoors, and seasonal changes resulting in reduced ultraviolet radiation type B (UVB) may be associated with subnormal vitamin D levels4. Vitamin D levels of <30 ng/ml, which are characterized clinically as either insufficient (29–20 ng/ml) or deficient (<20 ng/ml). Vitamin D is composed of a group of secosteroid molecules that are predominantly synthesized in the skin following exposure to solar radiation, resulting in vitamin D3 , or obtained via dietary intake, resulting in vitamin D2  or D35. Active vitamin D requires two hydroxylation reactions. The primary circulating form of vitamin D is 25(OH)D3 after vitamin D3 is hydroxylated in the liver. 25(OH)D3 is the precursor for the biologically active form of vitamin D, 1,25(OH)2D3, formed largely in the kidney. It is now known that immune cells (innate and adaptive), e.g., macrophages express enzymes to locally activate 25(OH)D3 to supra-physiological concentrations of 1,25(OH)2D3 for diverse functions. For example, it has been demonstrated that activated vitamin D is required for interferon-γ mediated antimicrobial activity of macrophages against Mycobacterium tuberculosis6.One of the prominent host factors that has been implicated in TB risk is vitamin D levels7. However, isolating the role of vitamin D from other seasonal confounders has been difficult. The majority of studies investigating the relationship between vitamin D levels and TB infection or disease risk have been cross-sectional in nature; as TB may cause micronutrient deficiencies, determining whether there is a causal relationship between vitamin D levels and TB risk remains challenging. One prospective study among household contacts in Spain found that vitamin D deficiency was associated with risk of TST conversion and incident disease among household contacts of TB cases8. Further evidence is needed to understand whether vitamin D levels are predictive of risk of incident TB infection and subsequent risk of disease.Many studies have reported seasonality of TB disease incidence, leading to speculation that seasonal differences in vitamin D levels may play a role in TB infection or disease progression9. In this population, the incidence of active TB is at least 30 times higher than that observed in the general population10. A number of structural and host determinants have been implicated in the high rates of TB infection and disease observed in prisons11. These include factors associated with increased transmissibility of the disease, such as overcrowding and ventilation11, as well host factors associated with both TB risk and incarceration, such as nutritional deficiency12, HIV infection, smoking14 and drug use16. Previous studies have identified low vitamin D levels among prisoners in correctional facilities, likely due to prolonged indoor incarceration with insufficient sunlight exposure or dietary sources of vitamin D7. Whether differences in vitamin D levels among prisoners are an important determinant of elevated TB risk is not understood.Brazil is a medium TB-burden country, which has over 70,000 new cases TB diagnosed annually. Of the 526,000 new cases of TB reported between 2009 and 2014 in Brazil, 38,000 (7.3%) were diagnosed among prisonersM. tuberculosis infection (tuberculin skin test (TST) conversion), and progression to active disease in this high-risk population.The purpose of this study was to investigate the role of subnormal vitamin D in incident TB infection and disease risk among incarcerated individuals. We performed one case-control study and two, nested case-control studies among an observational cohort of Brazilian prisoners. We aimed to determine whether vitamin D levels were associated with three distinct features of TB disease: active TB, 3 level was significantly lower among the cases (27.7 ± 7.85 ng/mL) than controls . In multivariable analysis, only subnormal serum 25(OH)D3 level  was determined to be significantly associated with active TB after adjustment for drug use , previous incarceration  and black race .Bivariable conditional logistic regression analyses were performed to assess the associations between active TB and thirteen potential predictor variables. These variables included drug use over the last year , previous incarceration , black race , and serum 25(OH)DIn the first nested case-control study, we selected individuals who screened negative for TB at baseline by symptoms and sputum culture, and compared those who converted their TST from negative to positive when retested at one year (converters) with those who remained negative upon repeat testing (non-converters). The mean age of the converters was 32 years (±6 years) and 31 years (±7 years) among the non-converters  and serum albumin level  were associated with risk of TST conversion. The multivariable analysis showed black race , less than 4 years of schooling , cigarette smoking  and serum albumin level  were associated with an increased risk of TST conversion , less than 4 years of schooling , cigarette smoking , serum 25(OH)D3 levels was identified between progressors (37.0 ± 14.83 ng/mL) and non-progressors  or did not (“non-progressors”). Thirty-six percent of those who progressed and 50% of those who did not progress had positive TSTs at baseline. No significant difference in baseline serum 25(OH)D3 level <30 ng/mL , cigarette smoking  and black race  were associated with risk of TB progression. However, in the multivariable analysis, only current smoker  was associated with the development of active TB after adjustment for black race , drug use during the last year , and serum 25(OH)D3 level at enrollment was observed among prisoners who progressed to active TB in the first 6 months compared to prisoners who had progressed after 6 months of follow-up .The average time to active TB diagnosis among progressors was 6.9 months. No statistical difference in serum 25(OH)DDespite the low power, this is the largest study ever published that evaluate the association of vitamin D levels with progression to TST conversion and progression of active disease in the prison population. We found that prisoners with active TB had significantly lower vitamin D levels at the time of diagnosis compared with prisons without active TB. However, baseline vitamin D levels were not predictive of TST conversion or progression to active disease. Notably, while 75% of prisoners with active TB had subnormal vitamin D levels at TB diagnosis, less than 33% of all other groups of participants (cases and controls) presented subnormal levels. These findings provide support for a pattern of disrupted vitamin D metabolism that results from the presence of active TB disease, and do not provide evidence for subnormal vitamin increasing susceptibility to infection or disease.26. Furthermore, previous cohort studies conducted among household contacts of TB cases have suggested that low levels of vitamin D may be associated with risk of subsequent M. tuberculosis infection27 and development of active TB29; we did not find these associations in this study when vitamin D levels were examined months prior to diagnosis and found to be largely normal. The number of subjects experiencing the outcome of interest in previous studies was limited, however, ranging from 3 to 18 over follow-up periods 2 to 4 years. These studies have been found to vary with study design, geographical location8, as well as ethnicities30 and gender24 of subjects. Thus, studies with larger sample sizes in different populations are needed to help clarify whether the association between vitamin D and susceptibility to TB infection or disease is causal in nature.Our primary findings of poor vitamin D status associated with active TB among prisoners were consistent with the results of case-control studies conducted in the general population10. These rates provided more robust sample sizes of 30 and 24 cases, respectively, for the nested case-control studies to examine the impact of vitamin D status at baseline. Surprisingly, the majority of healthy prisoners show normal vitamin D. In the individuals without active disease at enrollment, both diet and sun exposures in this incarceration setting appear to be adequate to sustain precursor vitamin D levels in healthy prisoners. Vitamin D status under these conditions appeared not to be associated with either the acquisition of latent infection, or the development of active disease in this prison population. It is possible that there was insufficient heterogeneity in vitamin D in this population and setting to detect such effects. Here, factors other than vitamin D levels (e.g. smoking status) appear to contribute risk along the TB disease pathway.Notably, we previously identified high rates of conversion (28%) and incident active disease (1.39%) over one year in this cohort study3 levels as the precursor supply for activated vitamin D32. The normal immune response against M. tuberculosis produces inflammatory cytokines that activate CYP27B1 (1α-hydroxylase), converting available 25(OH)D3 to its activated form 1,25(OH)2D333 and contributing to the measurement of low 25(OH)D3 levels34. Thus, the low levels of 25(OH)D3 (precursor) identified among prisoners with active TB in our case-control and among in several previous studies may have been associated with high conversion rates to 1,25(OH)2D3 caused by persistent disease. Notably, elevated plasma 1,25(OH)2D3 levels have been found in TB patients compared to healthy individuals36, suggesting hyperresponsive metabolism to form activated vitamin D may be associated with the disease in humans. However, vitamin D supplementation as part of randomized clinical trials during TB treatment has not been shown to reduce time to sputum culture conversion after 8 weeks44.Previous studies have recognized that inflammatory process may lower 25(OH)DM. tuberculosis is associated with Toll-like receptor (TLR)45. Mycobacterial antigens, especially lipoproteins, are recognized by TLR2 or associated with TLR1 or TLR6 which when activated participate in the expression of vitamin D receptor (VDR) and activation of 25(OH)D, assisting in the release of cathelicidin, a peptide capable of inhibiting the intracellular growth of M. tuberculosis, in vivo46 and in vitro45.One of the major defense mechanisms involving vitamin D and 47, and if vitamin D measurement is performed after the initiation of treatment, these levels may be considerably lower among patients with TB51. One strength of our study was that all serum samples used to measure vitamin D levels were collected before treatment was initiated removing this potential factor impacting vitamin D.Vitamin D levels may be influenced by anti-TB drugs, especially rifampicin and isoniazid53. Evidence suggests that smoking decreases TST reactivity55 but increases the risk of progression to active TB56 by directly impairing host immunity, especially the responses of T-cells and macrophages to M. tuberculosis58. An impaired immune response makes it more difficult to eliminate M. Tuberculosis and easier to develop the active disease57. While the variable cigarette smoking was found here to exert a protective effect on risk of TST conversion to latent infection, it dramatically increased risk of progression to active TB. TST conversion was also associated with black race, which increased the odds of infection by M. tuberculosis.Risk of TST conversion was not influenced by baseline vitamin D status in the prisoners, but other risk factors showed associations consistent with the previous reports. For example, previous studies reported an association between being a current smoker and TBM. tuberculosis infection greater than other racial backgrounds59. Although the mechanism underlying this association has not been fully elucidated, it may be attributed to environmental factors, unfavorable socioeconomic conditions61, and genetic factors63. Black skin can impair vitamin D synthesis from UVB exposure, but notably, vitamin D status was found to be an independent risk factor for active TB when controlling for race in the case-control study. Although it is not yet clear why race is a risk factor for TST conversion, we believe that in addition to the polymorphisms vitamin D receptors, other genetic factors may be associated. In our study, in the final model, the level of Vitamin D was adjusted by race and even then, the serum level of vitamin D was not associated with TST conversion. Other polymorphisms associated with black race should be investigated.Evidence suggests that black race may increase risk of 64. The prisoners are combined in cell blocks of mixed socioeconomic conditions, thus increasing the probability of becoming infected with M. tuberculosis66. However, the fact that all prisoners were exposed to the same conditions during incarceration, reinforces the need for further studies to be conducted to explain the reasons behind the racial and education inequalities among prisoners with active TB.Low levels of schooling also showed a tendency toward increased risk and may be directly related with incarceration mixing patterns of high risk individuals during incarcerationWe examined risk of incident TB among all prisoners rather than those who were TST positive at baseline. The majority of TB disease in this high transmission setting occurs due to recent infection rather that late reactivation. Indeed, the majority of incident TB cases over one year in this cohort occurred among individuals who were TST negative at baseline.68. In this study the average age was 32.1  in TST conversion and 30.8  in progression to active TB, it seems unlikely that BCG vaccination interfered with the TST results. As well as, exposure to non-tuberculosis mycobacteria (NTM) may interferes with TST results only in populations with a high prevalence of NTM sensitization and low TB infection, that does not happen in our study population, where the incidence of active TB is at least 30 times higher than that observed in the general population10. Interferon-ɤ release assays (IGRA) to determinate TST conversion may be more useful in populations where the BCG vaccine has not been administered or if the test was performed within 10 years of vaccination, thus, in our study, the TST and IGRAs likely would generate similar findings67.While studies remain challenged by power, this work represents the largest study ever conducted to evaluate TST conversion and progression of active disease. We used the TST conversion to assess TB infections and progression to active TB, however, some studies indicate that TST can cause false-positive results by BCG vaccination and exposure to non-tuberculosis mycobacteria (NTM). In Brazil, BCG vaccination usually performed in the first days of life that does not interfere with the TST result if carried out 10 years or more after vaccination69 and the incarceration time70 may favor a decrease in vitamin D levels. A strength of our design however, was that all prisoners were screened in the same months, and cases and controls were paired according to the prison cell block and incarceration time, having the same time of potential sun exposure and similar diet; these features help minimize confounding in our analysis, however is very difficult to perfectly match due to the prolonged and variable infectious period, variable social mixing, and frequent movement of inmates between cells. Overmatching among cases and controls, may have decreased the variability in vitamin D levels limiting the ability to identify important associations over the spectrum of vitamin D with the disease pathway. As a caveat, as many participants have sufficient vitamin D this may play a minor role in relation to other risk factors.Data suggest that seasonalityWe identified that prisoners with active TB had poor vitamin D status; however, vitamin D levels were not identified to be a risk factor for TST conversion or the development of active TB over a follow-up period of one year in the evaluated prison population. Other risk factors such as black race and low education were found to be risk factors for TST conversion, while smoking was the only variable associated with disease progression. Our findings suggest active TB may alter vitamin D metabolism, leading to compromised precursor supply, but that vitamin D status is not necessarily a risk factor for susceptibility to infection or disease.71. A one-year prospective cohort study was conducted in 2013 in 12 prisons  to identify the incidence of latent and active TB16. The case-study and two nested case-studies here are part of this larger cohort study.In 2013, the state of Mato Grosso do Sul (MS), located in the west-central Brazil, had the country’s seventh largest prison population, including 14,904 prisoners, and the higher rate of national incarceration M. tuberculosis were considered TB cases. A venous blood sample was collected, and plasma was frozen for assessment of vitamin D levels.Prisoners who were over 18 years old and consented to participate in the study were recruited from a randomly stratified sample at the 12 prisons. A structured questionnaire was administered and a (TST)  was performed. Two sputum samples were obtained from all participants reporting cough, and smear microscopy and culture, using the Ogawa-Kudoh technique, was performed. Although X-ray screening shows good sensitivity, and is an important method for screening suspected cases of TB, the specificity of X-ray screening is low. Additionally, chest radiography is not available in the study prisons. For these reasons, we used sputum culture to define TB cases. Individuals with at least one smear or culture result positive for 10.TST positivity was defined as an induration of ≥10 mm measured by a trained TST reader, 48 hours after injected. After one year, in 2014, a second TST was performed, and two sputum samples were collected for smear microscopy and culture from all participants reporting cough at the second time point. TST conversion was defined as TST ≥10 mm and an induration increase of at least 6 mm in an individual with a baseline TST <10 mmIncident TB cases were defined as those occurring after the first screening time point, including those diagnosed between the two screening dates (day 0 and day 365) and those diagnosed on the second screening date. Those diagnosed between the screening dates were diagnosed through routine medical services provided by the prison, in which symptomatic individuals are clinically evaluated and sputum is sent for smear and/or culture as determined the prison clinician. These TB cases occurring between the screening dates were identified through prison medical records and the National Notifiable Disease database .10.A plasma sample for vitamin D levels was collected at the time of enrollment, which was prior to TB diagnosis, and therefore, prior to initiation of treatment. Due to the low rates of TB in the female prisoners, the vitamin D evaluation was conducted in only male prisonersAll eligible participants provided written informed consent prior to study participation. All methods described in this study were approved by Research Ethics Committee of the Federal University of Grande Dourados  and were performed in accordance with the relevant guidelines and regulations. The study was authorized by the State Agency of Administration of the Penitentiary System (AGEPEN) who gave full support during the study period.M. tuberculosis and, therefore, had active TB16  over the course of 12 months were compared with sixty prisoners who had negative TST tests at baseline and repeat testing at 12 months (non-converters)  and deficiency (<20 ng/ml). Serum albumin levels were measured in samples obtained from prisoners included the nested case-control studies using the Cobas Integra® 400 plus analyzer . All procedures were performed in the Laboratory of the University Hospital of the Federal University of Grande Dourados.Vitamin D levels were measured in serum samples, which were stored away from light and at −20 °C until the time of analysis. Levels of 25(OH)D (ng/ml) were identified using the automated electrochemiluminescence immunoassay method and Cobas® e411 analyzer . Serum 25(OH)D levels were considered normal when values were ≥30 ng/mLAll questionnaires were double entered into the Research Electronic Data Capture database (REDCap). SAS version 9.2  was used to perform the analyses. We used the t-test for continuous variables, Chi-square test for categorical variables and bivariable conditional logistic regression analyses to examine the associations between dependent and independent variables. Vitamin D insufficient and all variables with p ≤ 0.10 in the bivariable analyses were included in multivariable conditional logistic regression analysis. P values ≤0.05 were considered statistically significant.The data will not be shared in order to protect the participants’ anonymity.