Alterations of the upper respiratory microbiome among children living with HIV infection in Botswana

Background Children living with HIV (CLWH) are at high risk of colonization and infection by bacterial respiratory pathogens. Microbes in the upper respiratory microbiome can prevent colonization by these pathogens. The impact of HIV infection on development of the upper respiratory microbiome during childhood is poorly understood. Methods We enrolled healthy CLWH (<5 years) and age- and sex-matched HIV-exposed, uninfected (HEU) and HIV-unexposed, uninfected (HUU) children in a cross-sectional study conducted in Botswana. We used shotgun metagenomic sequencing to compare the nasopharyngeal microbiomes of children by HIV status. Findings Of 143 children, 44 were CLWH, 49 were HEU, and 50 were HUU. Nasopharyngeal microbiome composition differed by HIV status (p=0·043, R2=0·019). The relative abundance of Corynebacterium pseudodiphtheriticum was lower in CLWH compared to HEU and HUU children (p=0·01). Among CLWH, a low (<25%) CD4+ cell percentage was associated with microbiome composition (p=0·009, R2=0·042) and lower relative abundances of Corynebacterium propinquum (p=0·003), C. pseudodiphtheriticum (p=0·007), and Dolosigranulum pigrum (p=0·004). The relative abundances of C. propinquum, C. pseudodiphtheriticum and D. pigrum in the nasopharyngeal microbiome were negatively correlated with the abundances of Streptococcus pneumoniae and Staphylococcus aureus . Interpretation CLWH with HIV-associated immunosuppression have altered nasopharyngeal microbiome composition and lower abundances of bacterial species associated with respiratory health during childhood. These findings suggest that the upper respiratory microbiome may contribute to the high risk of bacterial respiratory infections among CLWH. Funding National Institutes of Health, Duke Center for AIDS Research, Penn Center for AIDS Research Evidence before this study We searched PubMed for research articles published from database inception through November 20, 2022, using the terms (“nasopharyngeal” OR “nasal” OR “upper respiratory”) AND (“HIV” OR “human immunodeficiency virus”) AND (“microbiome” OR “microbiota”) AND (“pediatric” OR “child” OR “children” OR “infants”). This search returned five articles, three of which collected nasopharyngeal specimens from children living with HIV (CLWH). The objective of two of these articles was pathogen identification using culture- and polymerase chain reaction-based methods. The remaining study characterized the nasopharyngeal microbiomes of children with pneumonia, children with upper respiratory infections, and healthy children in Botswana using 16S rRNA sequencing. Genera associated with respiratory health were less abundant in CLWH with pneumonia, but no data was available for healthy CLWH. Thus, it remained unknown if the microbiome alterations observed in CLWH were associated with HIV or with pneumonia. Added value of this study To our knowledge, this is the first study to investigate the nasopharyngeal microbiome in healthy CLWH using metagenomic sequencing. To account for shifts in the microbiome that occur with age, we enrolled age- and sex-matched HIV-exposed, uninfected and HIV-unexposed children for each CLWH. The use of shotgun metagenomic sequencing allowed us to investigate differences in the microbiome at the species level. We found that HIV infection and HIV-associated immunosuppression were associated with an altered nasopharyngeal microbiome and a lower abundance of species associated with respiratory health and resistance to colonization by common bacterial respiratory pathogens. Implications of all the available evidence These findings suggest that HIV-associated alterations in the nasopharyngeal microbiome may predispose CLWH to colonization by bacterial respiratory pathogens responsible for invasive infection and death. Strategies to reduce pathogen colonization through modification of the microbiome hold promise for reducing infectious morbidity and mortality in CLWH. ### Competing Interest Statement KAF is an employee of Merck Research Laboratories, Merck & Co., Inc. No other authors have any conflicts of interest to report. ### Funding Statement SMP was supported by an Institutional Training Grant in Pulmonary and Critical Care Medicine (T32 HL 007538) and a VECD Global Health Fellowship, funded by the NIH Office of AIDS Research and Fogarty International Center (D43 TW009337). SMP, MSK, and CKC received financial support from the Duke University Center for AIDS Research (CFAR), an NIH funded program (5P30-AI064518). MSK was supported by a NIH Career Development Award (K23-AI135090). APS and TAM received financial support from the NIH through the Penn Center for AIDS Research (P30-AI045008). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Health Research and Development Committee (Ministry of Health, Botswana) and institutional review boards at the University of Botswana, the Botswana-Baylor Children's Clinical Centre of Excellence, the University of Pennsylvania, and the Duke University Health System gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All microbial genetic sequences generated as part of this study will be uploaded to the National Center for Biotechnology Information. The R code used for analysis is available for review at . Deidentified participant data can be made available via a shared GitHub repository after investigator approval of a proposal.