Table 1.
Basic demographic details of study participants.
Full metadata are provided in S1 Table.
Fig 1.
Results of 16S rRNA analysis of ASVs at genus level in the study sample.
(a) Bar plot ordered by relative abundance for prokaryotic taxa colour coded according to the key to the right of the bar plot. (b) Bar plot for prokaryotic mock control samples. (c) Heat map of relative abundance for the 4 taxa associated with previously defined bacterial enterotypes, ordered by relative abundance of Prevotella.
Table 2.
Genus-level community composition, and family to which they belong, for VL cases, endemic controls, and the total study sample.
Abundance at the study group level was calculated for aggregated read counts across all samples for ASVs classified at genus level (i.e. aggregates all taxa with the same genus assignment). Genera with an average group-level abundance <1% are not shown, except where relevant to the results or discussion. Full details are provided in S2 and S3 Tables.
Fig 2.
Results of 18S rRNA analysis of ASVs at genus level in the study sample.
(a) Bar plot ordered by relative abundance for eukaryotic taxa colour coded according to the key to the right of the bar plot. (b) Shows the bar plot for eukaryotic mock control samples; mock controls 1 and 2 were not spiked with Leishmania or Toxoplasma DNA. (c) Heatmap derived from unsupervised cluster analysis of eukaryotic taxa. Red labelling indicates the cluster of 5 most prevalent eukaryotic protozoan taxa for which samples cluster into three eukaryotic enterotypes according to: (A) Blastocystis Ambiguous taxon with/without Dientamoeba and/or Entamoeba; (B) Pentatrichomonas and/or Blastocystis sp. MJ99-568 in the absence of taxa from cluster A; (C) low abundance of these protozoan taxa. Bold labelling indicates the helminth eukaryotic taxa. Other eukaryotic taxa include fungi and a variety of plant taxa derived from the diet of participants. Of note, only one individual was observed with Leishmania DNA in the faeces (which does not show up in the bar plot of abundance for the top 24 taxa).
Fig 3.
Analysis of gut microbial profiles in relation to VL case versus EC status.
On 16S rRNA-determined prokaryotic and 18S rRNA-determined eukaryotic gut microbial profiles. (a) bar plots for relative abundance of 16S rRNA taxa separated for VL cases and EC groups, each ordered by relative abundance of taxa; for colour coding see key to main Fig 1(a). (b) bar plots for relative abundance of 18S rRNA taxa separated for VL cases and EC groups, each ordered by relative abundance of taxa; for colour coding see key to main Fig 2(a). (c) to (f) comparisons of species richness (number of ASVs) and alpha diversity measures (as labelled) for 16S rRNA data. (g) to (j) comparisons of species richness (number of ASVs) and alpha diversity measures (as labelled) for 18S rRNA data.
Fig 4.
Beta diversity comparisons for VL cases and EC.
(a) and (b) are PCoA plots (left to right: PC1xPC2; PC1xPC3; PC2xPC3) for Bray-Curtis dissimilarity measures for 16S rRNA and 18S rRNA data, respectively. (c) and (d) are PCoA plots (left to right: PC1xPC2; PC1xPC3; PC2xPC3) for weighted UniFrac measures for 16S rRNA and 18S rRNA data, respectively. See keys on plots for colour coding of VL cases and EC. Note: the weighted UniFrac distances are skewed due to 2 samples with high worm burdens.
Fig 5.
Results of ANCOM analysis of between-group differences in microbial composition.
(a) and (b) box plots for prokaryotic taxa that differed between EC and VL cases. W-values were 41 for Gastranaerophilales presented in (a) and 61 for Ruminococcaceae presented in (b), indicating that these taxa were significantly higher in the EC group compared to VL in comparison with 41 or 61 other taxa examined, respectively. (c) and (d) ANCOM box plots for prokaryotic taxa that differed between groups of individuals carrying high (46–89%) versus low (<6%) relative abundance of eukaryotic Blastocystis. W-value = 31 (c) indicating that Bacteroides abundance was significantly lower in the high Blastocystis group in comparison with 31 other bacterial families examined. W-value = 30 (d) indicating that Clostridiales vadinBB60 abundance was significantly higher in the high Blastocystis group in comparison with 30 other bacterial families examined.
Fig 6.
Influence of VL on eukaryotic Pentatrichomonas burden.
(a) heatmap of the top 5 eukaryotic taxa separated by VL case versus EC status, each ordered according to Pentatrichomonas load. (b) and (c) box plots comparing Pentrichomonas and Entamoeba log10 counts between EC and VL cases. (d) and (e) show the same comparisons for percent relative abundance. VL cases carry higher loads of Pentatrichomonas but not of Entamoeba.
Fig 7.
Influence of Blastocystis on beta diversity of bacterial taxa.
(a-c) PCoA plots of Jaccard indices for beta diversity of bacterial microflora colour coded (see scales) according to relative abundances for (a) Blastocystis_MJ99-340, (b) Blastocystis_Ambiguous taxon, and (c) all Blastocystis. Results of the biplot analysis are superimposed on the PCoA plots, with length of arrows indicating the degree of influence of different bacterial taxa (see key to biplot arrows) on Blastocystis abundance. (d) PCoA plot of unweighted UniFrac distances colour coded (see scale) according to Blastocystis load, with biplot vectors superimposed as before. (e) graph showing negative correlation between Blastocystis log10 counts and Escherichia-Shigella log10 counts.
Fig 8.
Influence of Blastocystis on alpha diversity of bacterial taxa.
(a-d) box plots comparing influence of high or low Blastocystis MJ99-340 abundance on alpha diversity measures (as labelled) and species richness (as measured by number of ASVs). (e-h) the same comparisons for groups of individuals with high or low Blastocystis_Ambiguous taxon abundance.
Table 3.
Comparison of helminth identification by microscopy (yes/no for presence/absence) with log10 read counts for parallel identification to the appropriate order using 18S rRNA analysis.
Bold indicates where positive microscopy and 18S rRNA concur; italics indicates where they do not concur.