Field evaluation of the gut microbiome composition of pre-school and school-aged children in Tha Song Yang, Thailand, following oral MDA for STH infections

Abstract

Soil-transmitted helminths, such as roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma spp.), are gastrointestinal parasites that occur predominantly in low- to middle-income countries worldwide and disproportionally impact children. Depending on the STH species, health status of the host and infection intensity, direct impacts of these parasites include malnutrition, anaemia, diarrhoea and physical and cognitive stunting. The indirect consequences of these infections are less well understood. Specifically, gastrointestinal infections may exert acute or chronic impacts on the natural gut microfauna, leading to increased risk of post-infectious gastrointestinal disorders, and reduced gut and overall health through immunomodulating mechanisms. To date a small number of preliminary studies have assessed the impact of helminths on the gut microbiome, but these studies are conflicting. Here, we assessed STH burden in 273 pre-school and school-aged children in Tha Song Yang district, Tak province, Thailand receiving annual oral mebendazole treatment. Ascaris lumbricoides (107/273) and Trichuris trichiura (100/273) were the most prevalent species and often occurred as co-infections (66/273). Ancylostoma ceylanicum was detected in a small number of children as well (n = 3). All of these infections were of low intensity (<4,999 or 999 eggs per gram for Ascaris and Trichuris respectively). Using this information, we characterised the baseline gut microbiome profile and investigated acute STH-induced alterations, comparing infected with uninfected children at the time of sampling. We found no difference between these groups in bacterial alpha-diversity, but did observe differences in beta-diversity and specific differentially abundant OTUs, including increased Akkermansia muciniphila and Bacteroides coprophilus, and reduced Bifidobacterium adolescentis, each of which have been previously implicated in STH-associated changes in the gut microfauna.

Fig 1. Bacterial gut community abundance to phyla and genera level, hierarchical clustering, soil-transmitted helminth infection status by microscopy (Kato Katz thick smear) and multiplexed-tandem qPCR (MT-PCR), sex, age and faecal score are depicted in the above figure (A) (adapted from Rosa et al. 2018 [11]).

Infection intensity classification is depicted in MT-PCR gene copy number output and cycle threshold (Ct) values separated after species-specific infections: Ascaris lumbricoides (Alu), Necator americanus (Nam), Ancylostoma ceylanicum (Ace), Ancylostoma duodenale (Adu) and Trichuris trichiura (Ttr) (from left to right). Scale bar represents evolutionary distance. Relative abundance is visualised in percentage of all bacterial phyla (B) and the top 50 bacterial genera (C). Abbreviations within the figure for schools are KK: Krae Khi, MN: Mae Nil, MNK: Mae Nil Khi, HM: Huay Manhok, HMK: Huay Manhok Khi, MSK: Mae Salid Khi, MK: Mae Kho, MSN: Mae Salid Noi, MS: Mae Song, MSL: Mae Salid Luang and TMKT: Thi Mo Ko Tha; and for diagnostics K: Kato Katz thick smear, M: MT-PCR, G: gene copy number and C: Ct value.

Fig 2

Abundance of significantly increased and decreased operational taxonomic units (OTU) as per log fold change influenced by soil-transmitted helminth (STH) infection status estimated by multiplexed-tandem qPCR (MT-PCR) (A) for Ascaris lumbricoides (a) and Trichuris trichiura (aa) and sex (B). Green or orange OTUs are found to be significantly more abundant and purple or blue OTUs significantly less abundant (ns = not significant). For STH infection status (A) OTU 91 Akkermansia muciniphila, OTU 214 Prevotella, OTU 65 Bacteroides coprophilus, OTU 118 Ruminococcaceae, OTU 9 Tenericutes are more and OTU 527 Bifidobacterium adolescentis is less abundant. For sex (B) OTU 65 Bacteroides coprophilus, OTU 9 Mollicutes, OTU 214 Prevotella, OTU 84 Parabacteroides, OTU 89 Bacteroides uniformis, OTU 23 Bacteroides fragilis are more and OTU 200 Ruminococcus, OTU 20 Lactococcus garvieae are less abundant.

Fig 3. Alpha-diversity measures with analysis of variance (ANOVA) statistics of the complete cohort of faecal samples preserved in DESS (n = 273).

Statistical analysis was performed using a p-value cut-off of 0.05 (*0.05 **0.005 ***0.0005) for soil-transmitted helminth (STH) infection status as estimated by multiplexed-tandem qPCR (MT-PCR) (0 uninfected, 1 infected) (A), age (3 to 6 years) (B), school (C) and sex (female, male) (D).

Fig 4

Beta-diversity measure as calculated by principal coordinates analysis (PCoA) with Bray-Curtis distance clustering (A) and non-metric multi-dimensional scaling (NMDS) using weighted UniFrac distance measure (B) of the complete cohort of faecal samples preserved in DESS (n = 273). PCoA with Bray-Curtis distance clustering for sex (C), soil-transmitted helminth (STH) infection status (D), age (E) and school (F) are shown (p-values 0.571; 0.021; 0.778; 0.032).