Metagenomics reveals impact of geography and acute diarrheal disease on the Central Indian human gut microbiome

© 2020, © 2020 The Author(s). Published with license by Taylor & Francis Group, LLC. Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrheal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilized shotgun metagenomics to comprehensively characterize the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrheal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism. Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrheal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural, and genetic factors in contributing to microbiome differences between rural and urban populations

Metagenomics Reveals Impact of Geography and Acute Diarrhoeal Disease on the Central Indian Human Gut Microbiome

Background: The Central Indian gut microbiome remains grossly understudied. Herein, we sought to investigate the burden of antimicrobial resistance and diarrhoeal diseases, particularly Clostridioides difficile, in rural-agricultural and urban populations in Central India, where there is widespread unregulated antibiotic use. We utilised shotgun metagenomics to comprehensively characterise the bacterial and viral fractions of the gut microbiome and their encoded functions in 105 participants. Results: We observed distinct rural-urban differences in bacterial and viral populations, with geography exhibiting a greater influence than diarrhoeal status. Clostridioides difficile disease was more commonly observed in urban subjects, and their microbiomes were enriched in metabolic pathways relating to the metabolism of industrial compounds and genes encoding resistance to 3rd generation cephalosporins and carbapenems. By linking phages present in the microbiome to their bacterial hosts through CRISPR spacers, phage variation could be directly related to shifts in bacterial populations, with the auxiliary metabolic potential of rural-associated phages enriched for carbon and amino acid energy metabolism.Conclusions: We report distinct differences in antimicrobial resistance gene profiles, enrichment of metabolic pathways and phage composition between rural and urban populations, as well as a higher burden of Clostridioides difficile disease in the urban population. Our results reveal that geography is the key driver of variation in urban and rural Indian microbiomes, with acute diarrhoeal disease, including C. difficile disease exerting a lesser impact. Future studies will be required to understand the potential role of dietary, cultural and genetic factors in contributing to microbiome differences between rural and urban populations

Ocular morbidity among children (aged 6-18 yr) of the tribal area of Melghat, India: A community-based study

Background & objectives: Most of the ocular morbidities among school children are preventable or treatable. Melghat, a difficult to access, hilly, forest, tribal area with poorly developed infrastructure in the Amravati district of Maharashtra. Scarcity of ophthalmologists and low health-seeking behaviour of tribal people contributes to the high burden of ocular morbidity. Given the lack of published studies on the ocular morbidity among children in Melghat, outreach programmes are essential to diagnose and treat visual impairments promptly. The objective was to determine the prevalence of ocular morbidity among children in the tribal area of Melghat. Methods: A community-based observational study was carried out in the Chikhaldara and Dharni blocks of Melghat. Children from 15 tribal villages were screened for eye disorders by trained paramedics. Most of the children were examined by an ophthalmologist. We used Chi-square test for categorical variables. Results: A total of 4357 children aged between 6 and 18 yr were examined. Of these 2336 (53.6%) were females and 2021 (46.4%) were males. Out of 4357 children, 507 (11.63%) had an ocular morbidity. The prevalence of ocular morbidity and refractive error increased in the age group of 8-10 yr (P<0.05 and <0.001, respectively). Refractive error was the most common ocular morbidity (n=339; 7.8%), followed by vitamin A deficiency (VAD) (n=120; 2.8%). Interpretation & conclusions: The prevalence of refractive error and VAD in this study was significantly higher than the rest of India and the world. For the prevention of childhood blindness, immediate intervention programme, including eye screening by trained paramedics, treatment by an ophthalmologist and prophylaxis, is crucial

The assessment of cytokines in Quantiferon supernatants for the diagnosis of latent TB infection in a tribal population of Melghat, India

Summary: The tuberculin skin test (TST) and interferon-gamma release assays (IGRA), namely, the QuantiFERON-TB Gold test (QFT), remain the standard immunological diagnostic tools for latent tuberculosis (TB) infection (LTBI). However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma (IFN-γ) as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: (i) QFT+/TST+ (n = 26), (ii) QFT+/TST− (n = 12), (iii) QFT−/TST− (n = 35) and (iv) QFT−/TST+ (n = 1) groups. A panel of cytokines (IL-6, IL-10, TNF-α and IL-2R) was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT−/TST− group. Correlation analysis identified a positive correlation between IL-6 and the QFT response (r = 0.6723, P < 0.0001), while a negative correlation was seen between QFT and IL-10 expression (r = −0.3271, P = 0.0044). Similarly, IL-6 was positively correlated with TST levels (r = 0.6631, P < 0.0001), and conversely, a negative correlation was found between TST and IL-10 expression (r = −0.5698, P < 0.0001). The positive and negative predictive values of IL-6 were found to be 92.59 and 93.33%, respectively, and the positive and negative predictive values of IL-10 were 96.55 and 91.18%, respectively. No significant impact of the demographic characteristics on cytokine positivity was observed. Our preliminary results suggest that the evaluation of additional cytokines in QFT cell-free culture supernatants may be valuable for the identification of LTBI. Combining IL-6 and IL-10 with QFT and/or TST could markedly improve the detection accuracy of LTBI. Our observations require investigation in larger well-characterized cohorts along with follow-up studies to further confirm the study outcome. Keywords: Cytokines, LTBI, TST, QF

Impact of socioeconomic status and living condition on latent tuberculosis diagnosis among the tribal population of Melghat: A cohort study

Aims: To study socioeconomic status (SES) and living conditions (LC) as risk factors for latent tuberculosis infection (LTBI) and their impact on QuantiFERON-TB gold (QFT-G) and tuberculin skin test (TST) outcome for determining a better diagnostic test for LTBI in the malnourished tribal population of Melghat. Settings and Design: Six hundred sixty nine participants matching the inclusion criteria were recruited from 10 tribal villages of Melghat region, India. Subjects and Methods: Complete information related to various risk factors and test outcome was obtained on 398 participants, which was analyzed as per predefined conceptual framework. Factors were classified based on their relevance either at individual or household level, and subsequently based on the possibility of intervention. Data were partitioned into concordant and discordant sets depending on test agreement. Results: In concordant set, the two tests revealed that LTBI was significantly associated with smoking (adjusted odds ratio [aOR]: 2.64 [95% confidence interval [CI]: 1.03-6.79]), tobacco usage (aOR: 2.74 [95% CI: 1.50-4.99]), and malnourishment (aOR: 1.97 [95% CI: 1.12-3.48]) after basic adjustment. Inclusion of latent variable SES and LC in the model has mediating effect on the association of above factors with LTBI. Further, the association of SES and LC with LTBI in concordant set was unaltered in presence of other cofactors. From discordant set, results of QFT-G corroborated with that of concordant set. Conclusions: Poor SES and LC can be considered as strong risk factors linked with LTBI as compared to malnourishment, which is often targeted in such communities. Further, our study showed QFT-G test as a reliable tool in screening of LTBI in the tribal population of Melghat, India

The Molecular Epidemiology of <i>Clostridioides difficile</i> Infection in Central India: A Prospective Observational Cohort Study

This prospective observational cohort study aimed to establish and compare baseline rates of Clostridioides difficile infection (CDI) in community and hospitalized patients in Nagpur and rural Melghat Maharashtra, including adults aged ≥18 years with a diagnosis of diarrhoea as defined as 3 or more loose stools in a 24 h period. All diarrhoeal samples were tested for CDI using the C. diff Quik Chek Complete enzyme immunoassay. C. difficile-positive stool samples were characterised by toxigenic culture, antimicrobial susceptibility testing and PCR ribotyping. C. difficile testing was performed on 1683 patients with acute diarrhoea. A total of 54 patients (3.21%; 95% CI: 2.42–4.17) tested positive for both the GDH antigen and free toxin. The risk factors for CDI included the presence of co-morbidities, antibiotic usage, and immunosuppression. The detected PCR ribotypes included 053-16, 017, 313, 001, 107, and 216. Our findings show that toxigenic C. difficile is an important but neglected aetiologic agent of infective diarrhoea in Central India. These results underscore the need to enhance the awareness and testing of patients with diarrhoea in India regarding the presence of toxigenic C. difficile, particularly in high-risk individuals with multiple co-morbidities, immunosuppression, and recent or ongoing antibiotic exposure or hospitalization

Differential Levels of Alpha-2-Macroglobulin, Haptoglobin and Sero-Transferrin as Adjunct Markers for TB Diagnosis and Disease Progression in the Malnourished Tribal Population of Melghat, India

<div><p>Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to <i>Mycobacterium tuberculosis</i> (<i>M</i>.<i>tb</i>) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis.</p></div

Hematological parameters of the population under study.

<p>Hematological parameters of 275 subjects from Melghat are listed in the table. Complete blood count was done in the Pathology department, CIIMS, Nagpur using commercial kits. The normal detection ranges of each parameter are indicated in the brackets. P < 0.05 was considered statistically significant. Values are reported as Mean ± SD. Statistical analysis was done using GraphPad Prism (version 5.03). Statistical variance between groups was calculated using mean values through ANOVA test with Tukey post test.</p><p>* Statistically significant when the significance level is set as P < 0.05 based on the Tukey correction</p><p>Hematological parameters of the population under study.</p

Evaluation of TB biomarkers in original and follow-up samples of the population under study.

<p>Chart shows a comparison between the levels of TB biomarkers (30 kDa antigen, ADA; Adenosine deaminase, Mycobacterial Dormancy Regulon Protein Rv2623, Multiplex antigens; Ag85B; <i>M</i>.<i>tb</i> Ag85B secretory protein, Hsp-16; 16 kDa heat-shock protein, 45kDa antigen, GroES; <i>M</i>.<i>tb</i> 10kDa chaperonin, ESAT-6; 6 kDa early secretory antigenic target and CFP-10; 10 kDa culture filtrate protein, QFT; QunatiFERON-TB Gold test and TST; Tuberculin Skin Test) in original and follow-up cases of Melghat categorized into three groups; Latent to Active TB group (n = 1), Latent to Latent TB Group (n = 5) and Active to Active TB group (n = 3).</p

Representative 1-DE gel images of identified proteins in different categories.

<p>Boxes indicate the differentially expressed protein bands in different study groups. Gel imaging and analysis was done using Image Lab software (version 4.0, BioRad). The protein bands in the test groups were compared against healthy control group.</p