In July 2022, the World Health Organization (WHO) reported the estimated global human immunodeficiency virus (HIV) prevalence across all ages; there are 38.4 million (M) people living with HIV (PLWH) (1). Of those, 1.7M are children aged 15 and less. In 2021, there was a 32% and 52% global decrease in new infections and HIV-related deaths relative to 2010, respectively. Still, 650,000 PLWH died of HIV-related complications, of these 98,000 were children. This decline in HIV-related deaths over time is largely due to the increased availability and effectiveness of antiretroviral therapy (ART) and continuous efforts towards reaching the 95-95-95 targets of the HIV service cascade. Globally, 75% (28.7M) of PWLH received ART in 2021. In Mexico, the estimated number of PLWH is 227,378. The cumulative number of perinatally-infected children aged 0 to 14 years is 4,229 (2). In 2023, 14 children were perinatally-infected with HIV [CLWH, (2)]. The estimated ART coverage among adults and children is 61% and 49% respectively, 54% are virally-suppressed, and the mother-to child transmission is 0.3%. Although the gap in life expectancy is gradually decreasing, PLWH (adults and children alike) are still at a higher risk of noncommunicable diseases (NCD) compared to the general population (3). This is partly due to persistent immune dysfunction and chronic inflammation which fail to normalize despite effective suppressive ART (4, 5).

In 2006, a seminal paper published in Nature Medicine concluded that microbial translocation, the passage of microbial products and/or bacteria from the gut to the systemic circulation, is a key driver of chronic inflammation and HIV pathogenesis, linking the gut microbiome to HIV disease progression (6, 7) and NCD (5, 8–10). This study (and others) conceptualized the current paradigm that functional (massive depletion of gut T helper 17 cells) and structural (loss of tight junctions) events secondary to HIV infection cause lasting damage to the gut mucosa, providing favorable conditions for alterations in the composition and function of gut microbial communities to occur (5, 11–14). These alterations are commonly referred to as microbial dysbiosis. Collectively, studies in adults PLWH (ALWH) have shown a decrease in bacterial (12) and fungal diversity (15, 16), an expansion of the virome (9, 17), an increase in pathobionts and a decrease in anti-inflammatory commensals (14), compared with seronegative individuals. Microbial dysbiosis is partially restored by ART. Also, studies have shown that ART is linked to a distinct microbial dysbiosis independent from HIV (18–21). Less is known in CLWH, as only a handful of studies have been published (22–24). Contrary to ALWH, whose studies have been published in high income countries (for the most part), studies in CLWH have been conducted in Africa [Cameron (24), Zimbabwe (25), South Africa (26)], India (22), and Italy (27). Collectively, studies suggest similarities between ALWH and CLWH, despite differences in mode of HIV transmission (vertical versus horizontal), absence of some confounding variables (sexual preference) and the presence of confounders specific to CLWH [use of co-trimoxazole, a prophylactic antibiotic recommended by the WHO in countries with high burden of malaria and bacterial infections (28)].

Understanding of HIV-associated gut microbial dysbiosis in CLWH lags behind that of ALWH. Since childhood represents a critical window for the gut microbiota (development and maturation), and the immune system (education), disturbances are likely to have a significant impact on long-term health, quality of life and longevity (29–31). Also, prolonged exposure to HIV, ART, antibiotics (32) and maternal factors (33), may result in a less resilient gut microbiome and a compromised gut microbiome-immune system axis, increasing tolerance to pathobionts. This, in turn, may lead to increased prevalence of cardiometabolic alterations, and other NCD (and their premature clinical presentation compared to the general population). The objective of our study was to characterize the gut microbiota in CLWH in Mexico, and compare it with HIV-unexposed and -uninfected children (HUU). We used dietary questionnaires and healthy eating index (HEI) scores to assess dietary patterns and quality; flow cytometry to quantify CD4+ and CD8+ T cell immune activation, senescence, exhaustion and cell cycling; enzyme-linked immunosorbent assays to quantify markers of inflammation, bacterial and fungal translocation, enterocyte integrity, and adipose tissue inflammation, multiplex-bead assay to quantify soluble markers of immune activation, and profiled the gut microbiota using 16S rRNA sequencing using fecal samples. We also explored relationships between the gut microbiota, antibiotic exposure, dietary habits, soluble and cellular markers of HIV disease progression and host metadata.

The work presented here characterized the gut microbiota in CLWH, and assessed its relation to markers of HIV disease progression and pathogenesis (and others), dietary patterns and host metadata, comparing it with HUU. We found no clear evidence that HIV impacted the gut microbiota composition, structure or diversity, nor did we find discriminant taxa between CLWH and HUU. Our results are both in agreement and in contrast with previously published studies in CLWH. Kaur and colleagues (22) showed that alpha diversity was decreased in CLWH compared to uninfected children, in both CLWH naïve to ART and CLWH on ART. CLWH naïve to ART had the least alpha diversity. They also reported that CLWH had a distinct pattern of gut microbiota compared to uninfected children, characterized by increased Bacteroidetes, and decreased Firmicutes, and Actinobacteria at phylum level, and increased Prevotella, and decreased Bacteroides, and Ruminococcus at genus level. Abange and colleagues (24) also reported that CLWH had lower alpha diversity and altered beta diversity. They found that CLWH had lower relative fractions of butyrate producers and obligate anaerobes, that Prevotella was decreased, together with gammaproteobacteria (Haemophilus influenzae) and Enterobacteriaceae, while Akkermansia and Faecalibacterium were increased. In contrast, Flygel and colleagues (25) suggested that prolonged ART (> 10 years) might restore microbial richness in CLWH. Flygel and colleagues also reported increased beta-diversity and a rectal microbiota enriched in 3 genera from the phylum Actinobacteria (Corynebacterium, Lawsonella, Collinsella) and 4 genera from the phylum Firmicutes (Finegoldia, Anaerococcus, Erysipelotrichaceae, and Lachnoclostridium). Sessa and colleagues (46) reported higher alpha diversity in CLWH, and two distinct clusters characterized by Akkermansia muciniphila in cluster 1 and Bacteroides, Faecalibacterium (and others) in cluster 2. Goosen et al. (26), reported that alpha diversity did not differ between CLWH and uninfected controls, while beta diversity was significantly higher in CLWH. Finally, Tincati and colleagues (27) found similar bacterial microbiota composition and alpha diversity in fecal samples. They also interrogated the plasma microbiome and found no difference in alpha and beta diversity. However, they did find a predominance of Gammaproteobacteria and Pseudomonas spp. in HIV-exposed infected compared to HIV-exposed uninfected children.

Discrepancies between studies are expected. These could be due to differences in the number of participants included (small versus large cohorts), age of children, inclusion of CLWH naïve to ART, or only CLWH on ART, specific ART regimens and length of ART duration, whether CLWH on ART were virally suppressed or viremic, use of antibiotics, and the choice of HIV-uninfected controls [whether HIV-unexposed and uninfected or HIV-exposed and uninfected (27)]. Furthermore, other variables including geographic location and even ethnic groups within the same country (24) are also likely to impact results, precluding their generalization. Kaur and colleagues included both CLHW naïve to ART and on suppressive ART and compared them to age- and sex-matched uninfected children. In this cohort, children were excluded if antibiotics were used one month prior to sample collection. Abange and colleagues included CLWH on ART, predominantly NNRTI-based regimen, for a median of 5 years and non-exposed HIV-negative children. Some CLWH were viremic (n=13) and most had CD4>500 cells/mm³. Flygel and colleagues investigated the gut microbiota in CLWH on long-term ART (most CLWH had chronic lung disease) and HIV-uninfected children, and used rectal swabs instead of fecal samples. Sessa and colleagues included aviremic CLWH for a median of 9 years (46). Goosen and colleagues (26) included aviremic CLWH aged 8 to 13 years, and excluded children with recent antibiotic use, and those with BMI/A <-3 or >2. Finally, Tincati and colleagues (27) included HIV-exposed infected children all on ART but not all aviremic, and HIV-exposed uninfected children (born from HIV-infected mothers), and excluded children with previous antibiotic exposure (2 weeks), gastrointestinal disorders and co-infections. Compared to other studies, our cohort included a small number of participants, CLWH were all aviremic, on PI-based ART for the most part. Interestingly, Abange and colleagues (24) reported that CLWH on PI-based regimen had lower alpha diversity and altered composition when compared to CLWH on NNRTI, as well as their HIV non-infected counterparts. In particular, they found that Blautia, Prevotella, Oscillospira, and Faecalibacterium were more abundant in NNRTI compared to PI/r, with CLWH on PI/r had a lower abundance of microorganisms predicted to be butyrate producers and obligate anaerobes. Given the predominant use of PI-based regimen in our cohort and the small number of CLWH, subgroup analysis looking at the potential impact of ART on the gut microbiota was not possible. Interestingly, we reported differences in the gut microbiota of Mexican ALWH on PI/r-based and EFV-based regimen (36). Although alpha diversity was not significantly different between ART regimens, we did find higher microbial translocation (assessed by plasma sCD14) and enterocyte damage (assessed by plasma levels of I-FABP) in those ALWH on PI/r-based regimen. Flygel and colleagues found no association between beta diversity and type of ART, nor years on ART (25). Both studies were however in agreement with the fact that duration of ART was not associated with alpha and beta diversity, suggesting that in pediatric HIV and viremia were not predictive of microbiota diversity (12), in contrast with ALWH (47, 48).

Some studies, including ours, suggest a beneficial impact of long-term ART on the gut microbiota. Some genera associated with intestinal health were also found in CLWH [Akkermansia, Faecalibacterium, (24)]. In agreement with other studies (25–27), we found that long-term ART might restore all metrics of alpha diversity. The median time on ART was similar between these studies and ours. We found no differences in alpha and beta diversity, and a high abundance of Faecalibacterium. Akkermansia was not among the top 20 genera in our cohort, but we were able to detect this genus, the mean overall abundance was 0.2% in CLWH and 0.7% in HUU. Faecalibacterium (Firmicutes) and Akkermansia (Verrucomicrobia) are two genera thought to be beneficial, due to their protective role in metabolism, immune system and gut barrier (49). Kaur and colleagues found decreased abundance of Faecalibacterium in CLWH on ART compared to HIV-uninfected controls (22). Interestingly, Sessa and colleagues found that the gut microbial group represented predominantly by Akkermansia (A. muciniphila) was linked to higher markers of inflammation, vascular endothelial activation, microbial translocation, but not T cell activation (46). Their results suggest that overabundance of a beneficial commensal could have negative implications. Indeed, Akkermansia has been linked to deleterious effects (50).

We report that markers linked to HIV disease progression and pathogenesis were similar to those of HUU, which suggests that prolonged ART might normalize their levels. Our results are in agreement with Fitzgerald and colleagues (51) who found that immune activation decreased in pediatric HIV-infected children after ART initiation. Weinberg and colleagues also found that several markers including IFN-, IL-6, TNF-, sCD163, CRP, sCD14 and MCP-1 decreased after virologic control (52). Others found that ALWH on stable ART showed evidence of altered gut permeability and fungal translocation (53) and increased monocyte and T cell activation and higher sCD14 (54). In our study, no differences in inflammation, immune activation, bacterial and fungal translocation, and enterocyte damage were observed between CLWH and HUU. However, our results also differ from Tincati and colleagues (27). They found elevated levels of sCD14, IL-6 in HIV-exposed infected children compared with HIV-exposed uninfected children, however levels of I-FABP were similar. They raise an important point in their discussion regarding the choice of the control uninfected group used to compare with HIV-exposed infected children, as we alluded to earlier. Most studies enroll HIV-unexposed and uninfected children. As shown in ALWH, careful consideration is needed to enroll the most appropriate control group (55). Studies in perinatal HIV should ideally include both HIV-exposed uninfected and HIV-unexposed uninfected children, when possible. We did find increased frequency of exhausted T cell, a marker of HIV disease progression in CLWH. This is in agreement with other studies in CLWH (56, 57). Exhausted T cells are also a marker of disease progression in ALWH (58). In CLWH, PD-1+ T cells are heterogenous population of central and effector memory T cells that have impaired proliferative capacity and secrete proinflammatory cytokines. Interestingly, we also found that Proteobacteria, a phylum known to harbor pathobionts, was associated with the frequency of activated CD4+ T cells and exhausted CD4+ T cells, linking the gut microbiome to persistent immune exhaustion and premature aging. In our cohort, the mean overall abundance of Proteobacteria was 2.40%, when stratifying by HIV, we found an increase in CLWH (2.8%) compared with HUU (2.1%). At genus level, we found an unclassified Alphaproteobacteria, Sutterella (order of Burkholderiales) and Escherichia-Shigella (family Enterobacteriaceae), known to translocate and elicit pro-inflammatory responses (59, 60). Interestingly, Tincati and colleagues (27) found that the plasma microbiome was enriched in Gammaproteobacteria and Pseudomonas spp. in HIV-exposed infected children and Alphaproteobacteria in HIV-exposed uninfected children. They also reported a weak trend towards an inverse correlation between plasma Pseudomonas spp. and CD4+ T cell counts. Collectively, our results suggest that although some markers and gut microbiota metrics might normalize or differences may not be apparent or quantifiable (in particular if studies are underpowered like ours), CLWH are at increased risk of premature aging. Also, the CD4/CD8 ratio did not normalize, which is concordant with the fact that most CLWH in our study started ART later. Our findings could also be attributable, at least in part to the inclusion of CLWH and HUU who had recently taken antibiotics. Interestingly, studies have shown that co-trimoxazole increased alpha diversity (23), decreased inflammation (61) and suppressed the gut-resident viridans group streptococcal species (VGS) associated with intestinal inflammation (62). At the same time, antibiotics are a confounder in gut microbiota studies (63) and have long-term adverse effects of the gut microbiota (32). Most studies report that antibiotic use causes a reduction in microbial diversity and richness, including a decrease in beneficial bacterial taxa, resulting in metabolic alterations, increased susceptibility to colonization by pathogens and an increase in bacterial resistance to antibiotics. Children in this study (CLWH and HUU alike) reported a high number of antibiotics courses, even considering the recall bias that parents might have had at the time of the interview. Antibiotics taken by children in this study were primarily β-lactams, broad-spectrum antibiotics which interrupt bacterial cell-wall formation (64). When assessing the impact of antibiotics on the gut microbiota, we found none. Anecdotally, stool consistency was not indicative of diarrhea associated with antibiotics (type 6-7), most children had normal stool consistency (type 3-4). These results were unexpected as the cumulative effects on the microbiome as a result of repeated administration of multiple courses of antibiotics have been demonstrated^29,30. Consequences have also been reported with children facing the risk of experiencing temporary or chronic alterations in their microbiome, which could predispose them to develop chronic disorders ^27,28. Our findings could be attributed, at least in part, to the fact that amoxicillin-clavulanate was the most frequently prescribed antibiotic, and it has been reported to cause no or only mild to moderate changes in the composition of the intestinal microbiome (65, 66).

Diet is one of the key modulators of the gut microbiota (63). In this study, children had a diet that is representative of the Mexican urban population (67). Both diet and its effect on the gut microbiota have clearly been linked to obesity, metabolic syndrome, and type 2 diabetes (68, 69). We found no associations between dietary intake amounts or diet quality (HEI score) and gut microbiota metrics in the context of HIV infection. We also found that markers of adipose tissue inflammation were comparable between CLWH and HUU. Generoso and colleagues also found that sCD163 normalized in Kenyan CLWH on ART (70). According to the Encuestas Nacionales de Salud y Nutrición (Ensanut, National Health and Nutrition Surveys), Instituto Nacional de Salud Pública (INSP, National Institute of Health) in 2018-2019, the prevalence of overweight and obesity in children aged 5 to 11 years was 18.1 and 17.5% (respectively) and in adolescents aged 12 to 19 years was 23.8 and 14.6%, respectively (71). In our study, no CLWH had obesity, compared to 2 HUU who had obesity (no significant differences were observed between groups). Childhood obesity is a growing health problem worldwide including Mexico (72). Childhood obesity in the context HIV infection is further alarming given the increased prevalence of cardiometabolic diseases and other NCD. Improving the health of CLWH (and HUU) is very important. One possible avenue is dietary interventions, which can be very successful short-term, but are not largely sustainable over time (73, 74). Food and nutrition literacy should only be explored, as empowering children with knowledge and skills related to nutrition and dietary habits, are likely to promote healthier eating behaviors from an early age (75). Interventions targeting the gut microbiota using different approaches have had relatively little success so far (13, 76). A recent study reported an attenuation of bacterial dysbiosis in vertically HIV-infected children (n=24), randomized to receive a daily nutritional supplementation or placebo for 4 weeks (77). Authors postulated that the gut microbiota is less resilient in children and better amiable to change and therefore would respond better to interventions. Interventions to promote a healthier gut microbiome, together with healthier lifestyle to increase life expectancy, quality in CLWH is necessary as a pediatric HIV cure is still lacking (78). CLWH will face many challenges as they transition to adulthood, and efforts should be made to minimize NCD and other comorbidities, which will negatively impact their quality of life. Interestingly, no correlations were found between bacterial genera, alpha diversity and dietary variables. We did find that alpha diversity metrics were positively correlated with UCG-002, a genus of the Oscillospiraceae family. UCG-002 has been negatively associated with obesity and insulin resistance, suggesting that UCG-002 may be involved in mechanisms that improve insulin sensitivity (79, 80). Another study reported an enrichment of UCG-002 in pubertal participants, as well as an increased abundance of Bacteroides and Prevotella in individuals at different stages of puberty (81). The authors suggest that these bacterial genera may represent a more mature stage of the gut microbiota. It is therefore plausible, though highly speculative, that the positive association between UCG-002 and alpha diversity may indicate, at least in part, the start of the transitional change from childhood to adulthood, impacting the gut microbiota. The mean of our cohort was 9.53 years.

We found that sex impacted the gut microbiota with female children being Bacteroides predominant and having lesser microbial richness compared to their male counterparts, although after adjusting for HIV status, age, BMI/A and recent antibiotic use, these associations were lost. Recent studies have shown that sex (and sex hormones) contribute to differences in the gut microbiome (82), as early as birth and throughout life. Interestingly, differences in inflammation profiles by sex have also been reported (52, 83). Because our primary objective was to understand the impact of HIV infection, associations between markers of HIV disease progression and sex or age were not explored. Differential effects of age have also been reported (84). We acknowledge that this is a limitation. In HIV infection, Bacteroides is a common enterotype, together with Prevotella, this latter is linked to sexual preference in ALWH (55). Bacteroides abundance has also been linked to consumption of protein- and animal-fat rich diets (85) and an increased Prevotella/Bacteroides has been linked to obesity (86). Interestingly, a study in Mexican children also reported the Bacteroides was highly abundant in children with type 1 diabetes (87). Additionally, we found a negative correlation between Prevotella and Bacteroides, which has been previously reported (86, 88). Our understanding of which variables are responsible for these taxonomic shifts remains incomplete. Indeed, Sessa and colleagues (46) failed to identify clinical variables that would explain the presence of two bacterial clusters in CLWH (A. muciniphila, less diverse, and Bacteroides and other genera, more diverse). Factors that impact the gut microbiota are likely to have synergistic and cumulative effects, making teasing out the impact of each factor difficult. Disentangling these effects are very important in the immediate future to improve our understanding of HIV-associated microbial dysbiosis.

This study has several limitations. First, our cohort included a small number of participants, and is underpowered to detect differences. Also, our cohort was not matched on age. The inability to balance groups on confounders might have introduced bias in our analyses. Second, CWH and HUU were recently exposed to antibiotics, which might have masked differences in bacterial community structure. Also, although our results suggest microbial signatures linked to CD4+ T cell activation and exhaustion (Proteobacteria) and sex (Bacteroides), these should be interpreted carefully given the high degree of interindividual variation in gut microbiota, which are not accurately represented when visualizing data (mean relative abundance) using heatmaps or barplots. Also, we were limited to reporting taxonomic classification to genus level, a limitation inherent to the16S targeted sequencing. Subgroup analyses to explore the impact of different variables (ART, diet) were not possible due to low number of participants in each subgroup. Finally, our study is cross-sectional and does not allow to establish causality. At the same time, our study also has strengths, as we included a comprehensive use of biologics: dietary patterns and quality; several markers of inflammation, immune activation (soluble and cellular), microbial and fungal translocation, enterocyte integrity, and adipose tissue inflammation, host metadata and explored relationships with the gut microbiota.

Given the prolonged exposure to HIV and ART, and prophylactic exposure to antimicrobials, the gut microbiota of CLWH may be less resilient, increasing tolerance to pathobionts. This, in turn, may lead to increased prevalence of cardiometabolic alterations, and NCD, or their premature symptomatic presentation compared to the general population. Here we report that the gut microbiota of CLWH appears similar to that of HUU, and that most markers of HIV disease progression normalize on ART. Our findings suggest that long-term ART administration (8 years) in CLWH, might have a beneficial impact on the gut microbial ecology and immune activation/inflammation. Sex is also likely to impact the gut microbiota. CLWH do exhibit signs of premature aging (frequency of exhausted CD4+ T cells), which puts them at risk for increased comorbidities in adulthood. Interventions to reverse HIV-associated premature aging, in synergy with interventions targeting overweight and obesity are likely to positively impact the quality of life and longevity of CLWH.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PRJNA868762 (SRA).

The studies involving humans were approved by Ethics and Scientific committees of INER and IMSS. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from the recruitment of participants to a research protocol. Written informed consent and age-appropriate assent were obtained from all participants and/or parents before study enrollment.

VB-M, SP-C, CR-P: Designed the study. CR-P, NAMJ, HP-L, JV-R: Recruited participants. CR-P: performed interviews and questionnaires. CR-P, IO-P: Performed dietary assessments. CA-A: Performed laboratory determination of lymphocyte populations. MC-T, NM, SP-C: Performed laboratory experiments. SP-C, CR-P: Analyzed the data. VB-M, GR-T, SA-R: Provided critical intellectual contributions. GRT, VB-M, SP-C: Funding acquisition. CR-P, SP-C: Wrote the manuscript with contributions from all co-authors. All authors approved the final version of this manuscript.