Alteration of the gut fecal microbiome in children living with HIV on antiretroviral therapy in Yaounde, Cameroon

Abstract

Multiple factors, such as immune disruption, prophylactic co-trimoxazole, and antiretroviral therapy, may influence the structure and function of the gut microbiome of children infected with HIV from birth. In order to understand whether HIV infection altered gut microbiome and to relate changes in microbiome structure and function to immune status, virological response and pediatric ART regimens, we characterized the gut microbiome of 87 HIV-infected and 82 non-exposed HIV-negative children from Yaounde, a cosmopolitan city in Cameroon. We found that children living with HIV had significantly lower alpha diversity in their gut microbiome and altered beta diversity that may not be related to CD4+ T cell count or viral load. There was an increased level of Akkermansia and Faecalibacterium genera and decreased level of Escherichia and other Gamma proteobacteria in children infected with HIV, among other differences. We noted an effect of ethnicity/geography on observed gut microbiome composition and that children on ritonavir-boosted protease inhibitor (PI/r)-based ART had gut microbiome composition that diverged more from HIV-negative controls compared to those on non-nucleoside reverse-transcriptase inhibitors-based ART. Further studies investigating the role of this altered gut microbiome in increased disease susceptibility are warranted for individuals who acquired HIV via mother-to-child transmission.

Figure 1

Cohort demographics. (A) Gives the breakdown by HIV status and ART regimen. (B–E) The distributions of participant age, duration of ART treatment, CD4 concentrations, and HIV titers. Vertical red lines depict clinical thresholds of severe immunodeficiency (> 200 cells/ mm³) and viremic control (> 1000 genome copies/mL). Limit of detection for HIV genomes is 150 genomes per mL.

Figure 2

Taxonomic and compositional description of microbiomes in Cameroonian children. In (A), the taxonomic composition at the class and family levels is given for prevalent lineages (ASVs present in at least 20% of samples). Bacterial families with a median relative abundance less than 1% were removed for ease of visualization. (B) The PCoA decomposition of unweighted and weighted UniFrac distances for ethnic groups with greater than 5 samples. When controlling for HIV infection status, ADONIS analysis showed differential clustering by ethnicity (p < 0.05).

Figure 3

Changes in alpha and beta diversity by HIV infection status. In (A), we show that HIV infection and treatment were associated with decreases in community diversity using the Inverse Simpson, Shannon, and phylogenetic diversity (Faith’s PD) measures. (B) Plots the PCoA for the first two principal coordinates of weighted and unweighted UniFrac distances. ADONIS testing revealed differences in clustering by HIV status for both weighted and unweighted measures (p < 1e−5). Differences in bacterial composition between HIV-infected and HIV non-infected are captured in the first two principal coordinates as shown in (C).

Figure 4

Differential abundance of ASVs and microbiome function by HIV status. Differentially represented ASVs in the HIV infected and HIV non infected cohorts were identified via paired, Wilcoxon tests. Mean relative abundances were calculated for these ASVs for the HIV-negative and positive cohorts. In (A), the log-ratio of those mean abundances are plotted on the x axis, with ASVs more abundant in the HIV infected cohort having a ratio greater than 0. ASVs are grouped at the class level, and the family-level taxonomic assignment is given on the y axis. ASVs that were predicted to produce butyrate are shown as large circles, while small circles donate ASVs that are not capable of butyrate production. ASVs mapped to A. muciniphila and F. prausnitzii are colored red and blue, respectively. ASVs with genus-level taxonomic assignments were filtered against the “List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism” and were assigned a binary value for predicted butyrate production and obligate anaerobic growth. Relative abundances for ASVs predicted to have these traits are shown in (B). There were differing relative abundances of butyrate producers and obligate anaerobes by HIV status (p < 0.001). (C) Depicts the log-linear relationships between the relative abundance of these traits and the corresponding alpha diversity (p < 0.001).

Figure 5

ART regimen-specific effects on diversity and composition. (A) Shows that PI treatment is associated with reduced bacterial evenness and richness when compared to HIV non-infected individuals and those on NNRTI regimens. ADONIS testing of weighted and unweighted UniFrac distances indicated differential clustering when comparing NNRTI to PI-based regimens (p < 0.05). Differences in ordination of the UniFrac distances by ART regimen are detailed in (C). (D) Depicts the relationships between UniFrac ordinations and alpha diversity.

Figure 6

Differential abundance of ASVs and microbiome function by ART regimen. Differentially represented ASVs in the HIV-positive NNRTI and PI/r cohorts were identified via paired, Wilcoxon tests. Mean relative abundances were calculated for these ASVs for the NNRTI and PI/r cohorts. In (A), the log-ratio of those mean abundances are plotted on the x axis, with ASVs less abundant in the PI/r cohort having a ratio less than 0. ASVs are grouped at the family level, and the genus-level taxonomic assignment is given on the y axis. ASVs with genus-level taxonomic assignments were filtered against the “List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism” and were assigned a binary value for predicted butyrate production and obligate anaerobic growth. Relative abundances for ASVs predicted to have these traits are shown in (B). There was a trending difference relative abundances of butyrate producers and obligate anaerobes by HIV status (p = 0.06). (C) Depicts the log-linear relationships between the relative abundance of these traits and the corresponding alpha diversity (p < 5e−8).