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Vaginal microbes, inflammation, and HIV risk in African women - Comment
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"Concentrations of key vaginal bacteria were strongly associated with women's risk of acquiring HIV. High concentrations of some bacteria were substantially more predictive of HIV risk compared with a microscopic diagnosis of bacterial vaginosis. Key bacteria could increase HIV susceptibility through multiple potential pathways, including inflammation, production of HIV inducing factors, and disruption of physical and chemical barriers to infection. Defining vaginal bacterial taxa associated with HIV risk could point to mechanisms influencing HIV susceptibility and provide important targets for future prevention research."
Lancet Infect Dis January 25, 2018 - Jo-Ann S Passmore, Heather B Jaspan
Institute of Infectious Diseases and Molecular Medicine, University of Cape Town Medical School, Cape Town 7925, South Africa
(JSP, HBJ); NRF-DST CAPRISA Centre of Excellence, Cape Town, South Africa (JSP); National Health Laboratory Service, Cape
Town, South Africa (JSP); Seattle Children's Research Institute, Seattle, WA, USA (HBJ); and Department of Global Health, University of Washington, Seattle, WA, USA (HBJ)
Women from sub-Saharan Africa have a disproportionately higher risk of becoming infected with HIV than their male counterparts. Having bacterial vaginosis, a heterogeneous vaginal microbial dysbiosis, increases a woman's risk of acquiring HIV infection1 and the risk of transmitting the virus to their partners2 or their children during childbirth.3 Although bacterial vaginosis is highly prevalent and recurrent in reproductive-aged women worldwide, the composition of organisms that constitute this condition might differ regionally and ethnically.4, 5 Therefore, it is important to understand whether particular vaginal commensals or categories of dysbiosis contribute to HIV acquisition and transmission risks.
In The Lancet Infectious Diseases, Scott McClelland and colleagues6 eloquently show that presence of specific bacterial vaginosis-associated taxa predicted the risk of acquiring HIV infection in five cohorts of women, representing three distinct risk groups recruited from six sub-Saharan African countries. Using both 16S rRNA gene sequencing to explore the relative abundance of vaginal microbes associated with HIV infection, and subsequent real-time PCR to evaluate absolute concentrations of the 20 most influential bacterial species, they showed that Parvimonas species type 1 and Gemella asaccharolytica were the strongest predictors of HIV infection. Healthy lactobacillus quantities were, however, not protective in the primary analysis. Given that parvimonas and gemella are common constituents of bacterial vaginosis and their presence has previously been associated with genital inflammation in heterosexual South African women,5, 7 this study by McClelland and colleagues suggests that both the presence but also the absolute quantity of these particular organisms might be important drivers of inflammation and HIV risk in sub-Saharan African women.
Genital inflammation increases HIV infection risks either by attracting HIV target cells into the vaginal mucosa or by damaging the mucosal barrier in ways that facilitate HIV penetration.8 Therefore, the genital inflammation that is commonly associated with bacterial vaginosis might contribute to increasing HIV acquisition risks. Many bacterial vaginosis-associated organisms, besides parvimonas and gemella, have been associated with genital inflammation.5 Additionally, the presence of some of these organisms in the female genital tract has been further associated with impaired wound healing and decreasing the integrity of the mucosal barrier,9 potentially through the production of sialidases that degrade protective cervical mucous that would normally trap HIV particles.8
By including a wide representation of high-risk women from several countries in eastern and southern Africa—including female sex workers, pregnant and post-partum women, and women in serodiscordant relationships—McClelland and colleagues6 generalise their findings that certain bacterial vaginosis-associated bacterial genera are major drivers of HIV risk throughout this region. It is nevertheless apparent that certain species might be more influential in some groups of women than in others. For example, although high concentrations of Mycoplasma hominis in vaginal swabs from female sex workers had almost a seven-times increased risk of HIV infection, it was not associated with such an effect in the other groups of women. Because pregnant women have less diverse vaginal biomes,10 it was unsurprising that McClelland and colleagues found that only Gemella species were associated with increased HIV acquisition risk during pregnancy. It remains to be determined whether bacterial vaginosis-associated species such as Parvimonas and Gemella are also important contributors to women-to-men or mother-to-child HIV transmission, or both.
The importance of McClelland and colleagues' study6 is that it should directly inform future HIV risk-mitigation interventions in Africa. None of the present approaches to treat bacterial vaginosis have long-term efficacy, including the use of lactobacilli-containing probiotics and antibiotics that target anaerobic bacteria such as metronidazole. Whereas most of the probiotics that are used to promote vaginal health do not contain the most common vaginal Lactobacillus species, antibiotics used to treat bacterial vaginosis also kill beneficial anaerobes. Furthermore, although resistance to metronidazole is rare it can occur with gemella and parvimonas.11 Despite the inability to effectively manage bacterial vaginosis, in the context of preventing HIV transmission, it is essential that studies direct us towards novel methods to improve the vaginal health of women.
Evaluation of the association between the concentrations of key vaginal bacteria and the increased risk of HIV acquisition in African women from five cohorts: a nested case-control study
Jan 25 2018 Lancet Inf Dis - R Scott McClelland*, Jairam R Lingappa*, Sujatha Srinivasan, John Kinuthia, Grace C John-Stewart, Walter Jaoko, Barbra A Richardson, Krista Yuhas, Tina L Fiedler, Kishorchandra N Mandaliya, Matthew M Munch, Nelly R Mugo, Craig R Cohen, Jared M Baeten, Connie Celum, Julie Overbaugh, David N Fredricks

Disruptions of vaginal microbiota might increase women's susceptibility to HIV infection. Advances in molecular microbiology have enabled detailed examination of associations between vaginal bacteria and HIV acquisition. Therefore, this study aimed to evaluate the association between the concentrations of specific vaginal bacteria and increased risk of HIV acquisition in African women.
We did a nested case-control study of participants from eastern and southern Africa. Data from five cohorts of African women (female sex workers, pregnant and post-partum women, and women in serodiscordant relationships) were used to form a nested case-control analysis between women who acquired HIV infection versus those who remained seronegative. Deep sequence analysis of broad-range 16S rRNA gene PCR products was applied to a subset of 55 cases and 55 controls. From these data, 20 taxa were selected for bacterium-specific real-time PCR assays, which were examined in the full cohort as a four-category exposure (undetectable, first tertile, second tertile, and third tertile of concentrations).
Conditional logistic regression was used to generate odds ratios (ORs) and 95% CIs. Regression models were stratified by cohort, and adjusted ORs (aORs) were generated from a multivariable model controlling for confounding variables. The Shannon Diversity Index was used to measure bacterial diversity. The primary analyses were the associations between bacterial concentrations and risk of HIV acquisition.
Between November, 2004, and August, 2014, we identified 87 women who acquired HIV infection (cases) and 262 controls who did not acquire HIV infection. Vaginal bacterial community diversity was higher in women who acquired HIV infection (median 1•3, IQR 0•4-2•3) than in seronegative controls (0•7, 0•1-1•5; p=0•03). Seven of the 20 taxa showed significant concentration-dependent associations with increased odds of HIV acquisition: Parvimonas species type 1 (first tertile aOR 1•67, 95% CI 0•61-4•57; second tertile 3•01, 1•13-7•99; third tertile 4•64, 1•73-12•46; p=0•005) and type 2 (first tertile 3•52, 1•63-7•61; second tertile 0•85, 0•36-2•02; third tertile 2•18, 1•01-4•72; p=0•004), Gemella asaccharolytica (first tertile 2•09, 1•01-4•36; second tertile 2•02, 0•98-4•17; third tertile 3•03, 1•46-6•30; p=0•010), Mycoplasma hominis (first tertile 1•46, 0•69-3•11; second tertile 1•40, 0•66-2•98; third tertile 2•76, 1•36-5•63; p=0•048), Leptotrichia/Sneathia (first tertile 2•04, 1•02-4•10; second tertile 1•45, 0•70-3•00; third tertile 2•59, 1•26-5•34; p=0•046), Eggerthella species type 1 (first tertile 1•79, 0•88-3•64; second tertile 2•62, 1•31-5•22; third tertile 1•53, 0•72-3•28; p=0•041), and vaginal Megasphaera species (first tertile 3•15, 1•45-6•81; second tertile 1•43, 0•65-3•14; third tertile 1•32, 0•57-3•05; p=0•038).
Differences in the vaginal microbial diversity and concentrations of key bacteria were associated with greater risk of HIV acquisition in women. Defining vaginal bacterial taxa associated with HIV risk could point to mechanisms that influence HIV susceptibility and provide important targets for future prevention research.
National Institute of Child Health and Human Development.

Compared with other parts of the world, where men account for most new infections, 56% of new HIV infections in Africa in 2015 were in women.1 Bacterial vaginosis, a condition characterised by the presence of complex anaerobic vaginal bacterial communities, might contribute to HIV transmission and the disproportionate burden of HIV infection in African women.2 The specific bacteria underlying the association between bacterial vaginosis and HIV infection remain poorly understood.
Advances in molecular microbiology have enhanced the understanding of normal and dysbiotic human microbiota.3 These approaches have facilitated identification of distinct vaginal bacterial community types, ranging from low diversity and lactobacillus-dominated bacterial communities to heterogeneous and highly diverse bacterial vaginosis-associated communities, characterised by an abundance of anaerobic species.3, 4, 5 Bacterial species vary in terms of their associations with bacterial vaginosis,6 particular symptoms,7 and vaginal inflammation.8 A recent study from South Africa showed that young women with high-diversity vaginal bacterial communities had increased numbers of activated genital mucosal CD4-positive T cells and a four-times increased risk of HIV acquisition compared with women with low-diversity Lactobacillus crispatus-dominated communities.9 Increased relative abundance of several bacterial taxa (Prevotella melaninogenica, Veillonella montpellierensis, Mycoplasma, Prevotella bivia, and Sneathia sanguinegens) were also associated with increased risk. One limitation of relative abundance data is that they do not provide absolute concentrations of bacteria, which can vary widely in women with the same relative abundance. Therefore in this study, we test the hypothesis that concentrations of specific vaginal bacteria are associated with increased risk of HIV acquisition in African women.
Using two sophisticated bacterial PCR approaches and data from five cohorts spanning six sub-Saharan African countries, this study was the first to show significant associations between the quantity of specific vaginal bacteria and women's risk of HIV acquisition. Concentrations of Parvimonas species types 1 and 2, G asaccharolytica, M hominis, Leptotrichia/Sneathia, Eggerthella species type 1, and Megasphaera were significantly associated with increased HIV risk. There were strong correlations between concentrations of many of the 20 bacteria evaluated with rtPCR, suggesting they might frequently be found together, establishing high-risk bacterial communities. These findings were consistent across three distinct risk groups: female sex workers, pregnant and post-partum women, and women in serodiscordant relationships.
Vaginal microbiota could influence women's risk of HIV acquisition at multiple levels.23 First, genital inflammation, mediated by the presence of particular bacterial taxa or communities, is likely to influence HIV susceptibility.9, 24 A recent study identified six vaginal bacterial genera independently associated with proinflammatory cytokines.5 Two of these genera, Sneathia and Gemella, correspond to vaginal bacteria showing concentration-dependent associations with HIV risk in the present analysis. Second, vaginal dysbiosis has been associated with HIV-inducing factors in vaginal fluid.25 Third, many bacteria associated with bacterial vaginosis produce sialidases and mucinases that disrupt the protective cervicovaginal mucus layer.7
In one earlier study, the presence of cultivable Lactobacillus species was associated with a decreased risk of acquiring HIV.26 Additionally, a recent study using molecular characterisation of vaginal microbiota found that women with vaginal bacterial communities deficient in non-iners species of Lactobacillus were at increased risk for HIV infection.9 The present analysis showed an association between lower relative abundance of L iners and HIV acquisition. However, the primary analysis using rtPCR assays in the full dataset showed no significant associations between concentrations of Lactobacillus species (L iners, L crispatus, and L jensenii) and HIV acquisition. Given the negative correlations between concentrations of lactobacilli and bacterial vaginosis-associated species, low relative abundance of Lactobacillus species might simply reflect the presence of high concentrations of bacterial vaginosis-associated bacteria that affect HIV susceptibility. This study used a novel sequential experimental approach that used complementary methods to evaluate the vaginal microbiome. In the first step, relative abundance data were used to show the association between vaginal bacterial diversity and HIV acquisition, and to guide selection of a restricted set of bacteria for further investigation. In the second step, highly sensitive taxon-directed rtPCR assays were used to test the hypothesis that concentrations of 20 key bacteria would be associated with HIV risk. The two steps captured related but distinct exposures. Specifically, relative abundance is not the same as absolute quantity of a bacterial taxon. Additionally, the rtPCR assays are more sensitive but might be less specific for detection of individual bacteria than use of broad range PCR with pyrosequencing.
This study included a validation analysis in a subset of samples that were not included in the pyrosequencing step, facilitating independent testing of hypotheses generated from pyrosequencing data. Two important points should be considered in comparing the primary analysis with the validation analysis. First, because of the smaller sample size in the validation analysis than in the primary analysis, bacterial taxa were modelled in a binary fashion rather than as four quantiles. Second, the validation subset was not a randomly generated group of cases and controls, so distribution across the five cohorts diverged from that of the full dataset. Despite these caveats, the validation confirmed an association between bacterial taxa and HIV acquisition for five of the seven bacteria identified in the primary analysis.
An important strength of this study was the large and geographically diverse sample, with individuals representing three distinct risk groups. A further strength in the study design was the collection of vaginal microbiota samples before HIV acquisition in more than 80% of cases, and shortly after HIV acquisition in the remainder of cases.
Furthermore, the analyses were robust in multiple sensitivity analyses testing assumptions in the experimental approach. This study also had limitations. First, as an observational study, these analyses do not provide definitive evidence that the associations detected are caused by bacteria increasing HIV susceptibility. Second, these analyses did not explore mechanisms through which individual bacterial taxa might increase HIV risk. Such mechanistic data will help to further evaluate the likelihood of a causal link between vaginal bacteria and HIV susceptibility, and will be the focus of future studies. Third, despite adjustment for potential confounding factors, residual confounding is possible because of measurement error or unmeasured confounding factors. Fourth, longer intervals between sample collection and HIV acquisition could attenuate the observed associations,27 although this source of variability was minimised by avoiding sample collection during menses. Fifth, sampling methods and laboratory procedures varied across the five cohorts included in this analysis. Related to this point, laboratory data on sexually transmitted infections and vaginal yeast were not available at all analysis visits, so the primary analysis does not include adjustment for these conditions. Finally, despite inclusion of multiple risk groups in this study, all participants were from eastern and southern Africa. Although this represents the region affected the most by the HIV epidemic, the findings might not be generalisable to all geographical regions.
Higher diversity vaginal bacterial communities not dominated by lactobacilli are more common in African and Hispanic women than in women of Asian or European origin,4, 28 leading to the hypothesis that racial differences in vaginal microbiota might contribute to population-level differences in HIV transmission and prevalence.29 Underlining this point, recent studies suggest that vaginal dysbiosis accounts for 20-30% of the population attributable risk of HIV acquisition in African women.30, 31 Because bacterial vaginosis is an extremely heterogeneous condition,3, 4 defining individual vaginal bacteria that are associated with HIV risk in women could provide additional specific targets and inform future strategies for HIV prevention research.

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