Immune Response and Microbiota Profiles during Coinfection with Plasmodium vivax and Soil-Transmitted Helminths

Abstract

The role of the gut microbiota during coinfection with soil-transmitted helminths (STH) and Plasmodium spp. is poorly understood. We examined peripheral blood and fecal samples from 130 individuals who were either infected with Plasmodium vivax only, coinfected with P. vivax and STH, infected with STH alone, or not infected with either P. vivax or STH. In addition to a complete blood count (CBC) with differential, transcriptional profiling of peripheral blood samples was performed by transcriptome sequencing (RNA-Seq), fecal microbial communities were determined by 16S rRNA gene sequencing, and circulating cytokine levels were measured by bead-based immunoassays. Differences in blood cell counts, including an increased percentage of neutrophils, associated with a transcriptional signature of neutrophil activation, were driven primarily by P. vivax infection. P. vivax infection was also associated with increased levels of interleukin 6 (IL-6), IL-8, and IL-10; these cytokine levels were not affected by STH coinfection. Surprisingly, P. vivax infection was more strongly associated with differences in the microbiota than STH infection. Children infected with only P. vivax exhibited elevated Bacteroides and reduced Prevotella and Clostridiaceae levels, but these differences were not observed in individuals coinfected with STH. We also observed that P. vivax parasitemia was higher in the STH-infected population. When we used machine learning to identify the most important predictors of the P. vivax parasite burden (among P. vivax-infected individuals), bacterial taxa were the strongest predictors of parasitemia. In contrast, circulating transforming growth factor β (TGF-β) was the strongest predictor of the Trichuris trichiura egg burden. This study provides unexpected evidence that the gut microbiota may have a stronger link with P. vivax than with STH infection.IMPORTANCEPlasmodium (malaria) and helminth parasite coinfections are frequent, and both infections can be affected by the host gut microbiota. However, the relationship between coinfection and the gut microbiota is unclear. By performing comprehensive analyses on blood/stool samples from 130 individuals in Colombia, we found that the gut microbiota may have a stronger relationship with the number of P. vivax (malaria) parasites than with the number of helminth parasites infecting a host. Microbiota analysis identified more predictors of the P. vivax parasite burden, whereas analysis of blood samples identified predictors of the helminth parasite burden. These results were unexpected, because we expected each parasite to be associated with greater differences in its biological niche (blood for P. vivax and the intestine for helminths). Instead, we find that bacterial taxa were the strongest predictors of P. vivax parasitemia levels, while circulating TGF-β levels were the strongest predictor of helminth parasite burdens.

Keywords: Colombia; Plasmodium vivax; STH; Trichuris trichiura; malaria; microbiota; soil-transmitted helminths.

FIG 1

P. vivax parasitemia is higher in STH-coinfected individuals. Box plots show the P. vivax parasitemia levels (expressed as the number of parasites per microliter) of children with both P. vivax and STH infections (red circles) and children with P. vivax infections only (orange circles). P. vivax parasitemia is significantly higher in individuals who are coinfected with STH (P, 0.04 by the Wilcoxon signed-rank test). These box plots (and all other box plots shown) represent the median, interquartile range (box), and 95% confidence interval (whiskers).

FIG 2

CBC w/diff analysis can distinguish individuals with P. vivax infection but not those with STH infection. (A) Principal-component analysis based on results from CBC w/diff analysis. Each point represents the result for one child and is color-coded by infection status, as throughout the figures. Data are shown in red for children coinfected with P. vivax and STH, in orange for children infected with P. vivax only, in dark blue for children with STH alone, and in light blue for children who were not infected with either parasite. Ellipses show the areas covering 90% of the samples from each group. (B) The factors loading for PC1 reflect the amounts of variance shared by these parameters (either negatively [in blue] or positively [in red]) with the PC1 values. MCV, mean corpuscular volume. (C) Box plots of the two variables most negatively associated with PC1 (platelets and hematocrit) as well as the two variables most positively associated with PC1 (the percentages of monocytes and neutrophils) are shown with the same color scheme. Box plots for the remaining 12 clinical variables can be found in Fig. S2.

FIG 3

Elevation of plasma cytokine levels in some individuals infected with P. vivax. (A) Principal-component analysis of levels of 13 different circulating cytokines measured by bead-based immunoassays. Each point represents the result for one child and is color-coded by infection status, including children coinfected with P. vivax and STH (red), children infected with P. vivax only (orange), children with STH alone (dark blue), and children who were not infected with either parasite (light blue). Ellipses show the areas covering 90% of the samples from each group. (B) The factors positively loading for PC1 reflect the amounts of variance shared by these parameters with the PC1 values. (C) Box plots of the four variables (IL-6, IL-8, IL-10, and IL-4) most positively associated with PC1.

FIG 4

Effects of STH coinfection on the microbiota of P. vivax-infected patients. (A) NMDS plot based on Jaccard distances between stool microbiota samples (based on 16S rRNA gene sequencing), colored according to STH-P. vivax infection status. Some individuals with P. vivax infections only (orange) had a distinct microbiota and clustered separately along the first axis. (B) LDA effect sizes calculated using LEFSe (58) are shown, comparing samples from individuals infected with P. vivax alone with samples from those coinfected with P. vivax and STH. Microbes that were significantly (P < 0.01) elevated in individuals infected with P. vivax only are shown in orange, and microbes that were significantly elevated in individuals coinfected with P. vivax and STH are shown in red. This comparison was made based on the visual differentiation of these groups in panel A and the comparability of these two hospital-based groups. (C) Box plots for the three top microbes enriched in samples from coinfected individuals and the top microbe enriched in samples from P. vivax-infected individuals.

FIG 5

RNA-Seq of the peripheral blood identifies genes upregulated by P. vivax infection but not by STH infection. (A) PCA plot based on the 50% most variable genes with at least 400 reads across all samples. Ellipses show the areas covering 90% of the samples from each group, revealing the almost complete overlap between groups. (B) Differential abundance analysis by DESeq identifies 30 genes that are upregulated in individuals infected with P. vivax only compared to uninfected individuals (with an adjusted P value of <0.05 and a log₂ fold change of ≥1). (C) Biological processes overrepresented in these 30 genes relative to all genes in the Homo sapiens Gene Ontology database (accessed 8 October 2019), using Fisher’s exact test in PANTHER (27). The top 10 specific subclasses (from hierarchically sorted output) are shown, based on the lowest FDR.

FIG 6

Integrative analysis of heterogeneous data sets using random forest models to identify the strongest predictors of coinfection and parasite burden. (A) The random forest model selected eight microbes (brown) in the top 10 predictors of whether a P. vivax-infected child is also infected with STH. Bars represent the mean decrease in Gini when a variable is removed from the model; a larger decrease means that the variable is more different between individuals infected with P. vivax only and those with both P. vivax and STH infections. Bars are color-coded based on the type of variable: microbes measured by 16S rRNA gene sequencing are shown in brown, genes measured by RNA-Seq in red, cytokines in yellow, and demographic variables from a questionnaire in purple. The model included 4,046 variables: 38 microbial genera, 3,907 genes, 85 measurements from blood tests, including CBC w/diff and cytokine levels, and 16 variables from the demographic questionnaire. Of the 10 variables shown, all were higher in the coinfected group, except for NDUFA6 and Bacteroides, which were higher in the P. vivax-only group. However, the strongest predictors of whether an individual is infected with P. vivax or not (see Fig. S8A) are found in the data from clinical bloodwork and cytokine panels. (B) The random forest model selected seven microbes in the top 10 predictors of P. vivax parasitemia. In this continuous-outcome model, the increase in node purity represents the importance of the variable to the model; higher numbers mean that the variable is more important for predicting P. vivax parasitemia. The model included the same variables as those in panel A, except that the group (the response variable in panel A) was removed and P. vivax parasitemia was made into a response rather than a predictor. (C) To examine one of these important variables, we created a scatter plot, which shows that Prevotella is correlated with P. vivax parasitemia (r² = 0.13; P = 0.005 [among those infected with P. vivax]). Results for samples from children infected with P. vivax only are shown in orange, and those from children coinfected with STH are shown in red. (D) The random forest model selected TGF-β as the top predictor of the T. trichiura egg count. Other variables that are predictive of egg burden include several genes (from RNA-Seq results), the child’s height, and one microbe. The model included the same variables as those in panel A, except that the T. trichiura egg count was made into a response rather than a predictor. (E) TGF-β is correlated with the T. trichiura egg count (r² = 0.16; P = 0.002 [among those infected with T. trichiura]). Results for samples from children infected with STH only are shown in blue, and those from children coinfected with P. vivax are shown in red.