Meta-taxonomic analysis of prokaryotic and eukaryotic gut flora in stool samples from visceral leishmaniasis cases and endemic controls in Bihar State India

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon’s, Faith’s and Pielou’s indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally.

India. The primary aim was an observational study to determine the composition of gut prokaryotic and eukaryotic microflora in this region of India endemic for VL, with a small case-control comparison to determine whether this microflora differs between VL cases and non-VL endemic controls. We compare microscopic determination of helminth infections with 18S rRNA data, and report on secondary aims of determining the potential influence of prokaryotic or eukaryotic taxa known to contain pathogenic species on gut microbial diversity. The table below provides the line numbers in the manuscript that address items on the STROBE checklist [1]. Note: Due to the nature of our study, many of the criteria are not relevant to our type of study.

Results
Participants 13* (a) Report numbers of individuals at each stage of study-eg numbers potentially eligible, examined for eligibility, confirmed eligible, included in the study, completing follow-up, and analysed. Single time point of collection of samples; sample size stayed the same as recruitment size.
(b) Give reasons for non-participation at each stage There we no non-participants.
(c) Consider use of a flow diagram Descriptive data 14* (a) Give characteristics of study participants (eg demographic, clinical, social) and information on exposures and potential confounders. Provided in the methods section.
(b) Indicate number of participants with missing data for each variable of interest. See lines 306-309 for numbers of individuals not take forward in part of the analysis due to low sequence reads.
Outcome data 15* Report numbers in each exposure category, or summary measures of exposure. Results provide details of results of sequence analysis for different groups, and numbers contributing as indicated under point 14 above.
Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their precision (eg, 95% confidence interval). Make clear which confounders were adjusted for and why they were included. Not applicable to our kind of data analysis.
(b) Report category boundaries when continuous variables were categorized. Not applicable to our kind of analysis.
(c) If relevant, consider translating estimates of relative risk into absolute risk for a meaningful time period. Not relevant to our study.
Other analyses 17 Report other analyses done-eg analyses of subgroups and interactions, and sensitivity analyses This is not relevant to our study analysis. We provided detailed description of the analysis of sequence data for microbes in the gut of cases and controls.

Discussion (lines 497-643)
Key results 18 Summarise key results with reference to study objectives. We described the microbiomes of individuals living in this region of India. We provided information on interesting differences in gut microbes for VL cases compared to endemic controls.
Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias. This was a scoping study that demonstrates the value of the technology employed, and indicates where further larger scale studies would be required.
Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, multiplicity of analyses, results from similar studies, and other relevant evidence. We have been cautious in the interpretation of our data.
Generalisability 21 Discuss the generalisability (external validity) of the study results. We have discussed the applicability of our methods to the study of NTDs generally.

Other information
Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the present article is based. We have provided the information on funding.
*Give information separately for cases and controls.