Determination of the impact of Antiretroviral therapy in the proportion and genetic diversity of diarrheal associated gut microbiota among HIV infected population

Abstract

Background: Human gut microbiota are microorganisms that reside in the gastrointestinal tract of humans. Gut microbiota provide various functions in the gut including protection from invasion of pathogenic microbes, shaping host immunity and substrate metabolism. HIV which targets the CD4+ T cells in the gut associated lymphoid tissue (GALT), causes gut microbiota alteration due to disruption of the gut barrier. This result in microbial translocation and pathogen invasion which lead to decreased diversity and proportion of gut microbial and increase in pathobionts and pathogens. As a result, HIV infected patients suffer from diarrhoea due to compromised gut microbiota. Antiretroviral therapy (ART) suppresses viral replication and lead to an undetectable viral load, thus restoring the immune system. Previous studies show that ART does not entirely restore the depleted gut microbiota, hence this study aims at determining the change in proportion and genetic diversity of diarrheal associated gut microbiota in HIV infected patients. Hypothesis: Antiretroviral therapy causes changes in the proportion and diversity of diarrheal causing gut microbiota Objective: The objective of the study was to determine the impact of ART in the proportion and genetic diversity of gut microbiota among HIV infected patients. Methodology: Paired blood and stool samples were collected from 17 HIV infected and 11 HIV uninfected individuals (controls). Samples were collected during baseline (before ART initiation); and samples were collected after every three months thereafter, for a total of 12 months. Blood samples were used for CD4 and viral load measurements using BD FACSPresto machine (BD Biosciences) and HIV Qualitative PCR, respectively. Stool samples were used for extraction of total nucleic acid (TNA) using a modified Qiagen QIAamp Fast DNA Stool Mini Kit. Purification of TNA was done using Ampure XP bead. DNA library preparation kit (Illumina) was used for DNA library preparation. Illumina miniseq sequencing was used for sequencing and the obtained sequence reads were analysed using Geneious prime software for trimming and filtering low sequence reads and to determine the genetic diversity of diarrheal causing gut microorganisms. DRAGEN metagenomics was used for taxonomical classification to generate the proportion of diarrheal causing gut microorganism. GraphPad prism was used to generate graphs and for statistical analysis to generate the association between CD4+ T Cell/viral load and diarrheal causing gut microbiota. Results: Escherichia coli (82.33%), Bacteroides fragilis(2.46%), Shigella spp.(1.96%), Salmonella enterica (1.07%), Clostridioides difficile(0.99%), Campylobacter jejuni (0.20%) were the most prevalent detected microoganisms from HIV infected individuals at prior ART initiation. Among HIV negative individuals, Escherichia coli (62.52%), Bacteroides fragilis(18.43%), Shigella spp.(0.40%), Salmonella enterica (3.05%), Clostridioides difficile(15.06%), Campylobacter jejuni (0.54%) were found. The proportion of Escherichia coli (90.77%), Clostridioides difficile (2.20%), Shigella spp. (2.73%),and Salmonella enterica (1.28%) had increased with treatment at three months post treatment in HIV infected individuals. At six months post ART, there was an increase in the proportion of Bacteroides fragilis (88.65%) and Vibrio cholera (0.01%) when compared to HIV positive samples at three months post-treatment. Escherichia coli (9.74%), Cryptosporidium parvum (1.74%), and shigella spp. (0.35%) increased with treatment from six months to nine months post-treatment. Escherichia coli (58.58%), Shigella spp. (0.81%) and Clostridioides difficile (24.77%) had increased with ART at twelve months post treatment. The proportion of diarrheal causing gut microbiota were fluctuating throughout the intake of ART at different time points. There was no significant correlation between CD4/viral load and diarrheal causing microorganisms, as a result ART did not have an effect in the proportion of diarrheal causing gut microorganisms. Genetic diversity of diarrheal causing gut microbiota was higher in HIV positive individuals prior ART than in HIV negative individuals except for Salmonella typhi. Among HIV infected individuals, genetic diversity of most diarrheal causing gut microbiota was higher at three months post treatment. The fluctuation of diarrheal causing gut microbiota’s genetic diversity from six months to twelve months post treatment may be due to inconsistent change in number of viral loads and CD4 counts at different time points of ART. However, this was statistically insignificant. As a result, the change in the genetic diversity of diarrheal causing gut microorganisms was not due to ART. Conclusion: ART does not eradicate diarrheal causing gut microbiota. However, ART encourages a low genetic diversity of diarrheal causing gut microbiota.