Among women without BV who were subsequently followed for an average of one year, we found that detection of several BV-associated bacteria (BVAB) in vaginal fluid at the enrollment (BV-negative) visit predicted subsequent acquisition of BV. Conversely, detection of L. crispatus
at enrollment was associated with reduced risk of subsequent BV acquisition. Women were also more likely to have BV diagnosed when they presented in the two weeks after menses. The significant association between detection of BVAB at enrollment and BV acquisition remained significant after adjusting for timing of examination relative to participants' menstrual cycles and new sex partner with a history of BV. Notably, although over 80% of participants without BV at enrollment had Lactobacillus crispatus
at enrollment as detected by PCR assay, 20% acquired BV in the year of follow-up. The incidence of BV that we observed in our participants—0.23 episodes/woman-year—was only slightly lower than that reported in a larger yearlong prospective study of young heterosexual women.
Despite collection of extensive sexual risk behaviors using CASI during follow-up, only report of a new female sex partner who provided a history of BV was clearly associated with increased risk of BV acquisition in univariate analysis. The dose-response relationship we observed between risk of BV acquisition and increasing reported number of episodes of receptive vulvovaginal sex is of interest (as is the less significant but directionally similar association with receptive oral-anal sex), although we did not observe these associations when these behaviors were analyzed using other measures (for example, report of any behavior vs. not, or time to last behavior). Thus, these findings support further detailed investigation of the potential role of these behaviors in future BV acquisition studies, to confirm the associations we detected. Given the absence of associations between other recent sexual practices and BV acquisition, the significance of association between BV and report of a new female partner with BV history remains unclear. As we have previously reported, an intervention to prevent sexual transmission of vaginal fluid in this cohort – despite succeeding in reducing the risk of sexual practices targeted by the intervention—failed to reduce the risk of BV acquisition.
Our inability to establish a role for sexual transmission of BV in this cohort may relate to several factors, including imprecise assessment of the time of sexual exposure to acquisition of a “precipitant” for BV, the possibility that multiple sexual practices may be involved and are often practiced concurrently, or, as is highly likely, that BV pathogenesis is multifactorial: while unprotected sex is likely contributory, other factors are probably involved.
We have previously reported that among women with BV in this cohort who were treated with vaginal metronidazole, predictors of treatment failure included detection of specific BVAB at baseline, including the Clostridia-like bacteria BVAB1, BVAB2 and BVAB3, Peptoniphilus lacrimalis
phylotype 2, as well as failure to adhere to five days of vaginal metronidazole.
Importantly, report of no specific sexual practices with either male or female partners in the month after treatment predicted either persistent bacterial vaginosis or abnormal microbiota. The latter finding is notable, as others have reported that unprotected vaginal intercourse is associated with recurrent BV. However, as we note below, our ability to detect such associations was limited by the relatively small number of outcomes we observed and the selected population we studied.
Our observation that detection of specific BVAB in vaginal fluid weeks to months prior to a diagnosis of BV suggests that changes in vaginal microbiota may precede the development of BV by a considerable time period. Bacterial vaginosis is a heterogeneous syndrome characterized by diverse microbiota, some of which might be essential “founder” species necessary to establish an environment favorable to progression to BV. For example, a tenacious biofilm on vaginal epithelium may lead to antibiotic failure or create a persistent reservoir of bacteria further increasing women's risk of treatment failure.
Concentrations of bacteria at the earliest stages of biofilm formation may be insufficient to cause BV, yet may provide a critical layer of bacterial cells that facilitate attachment of other members of the bacterial community. These early colonizers may be detected by species-specific PCR assays as described above. In some women assessed frequently (for example, daily), rapid shifts in vaginal microbiota as measured by quantitative PCR assays and by Nugent score are evident.
Studies focused on the kinetics of acquisition of vaginal bacterial communities are likely to be very useful for understanding the pathogenesis of BV.
In our participants, BV was not associated with risk factors that others have identified, including failure to use condoms
and other sexual behaviors with male partners.
This may partly be explained by a low incidence of sexual contact with men among our subjects in the month after treatment. However, given the high prevalence of BV among lesbians, the frequency of concordant BV in female sex partners, and the observation that report of sex with another woman was associated with increased risk of recurrence in one prospective study,
we were surprised not to find a clearer association between BV acquisition and sexual behaviors with female partners in the month after treatment. This could be explained by small numbers of women in some subgroups, or by an overriding role of baseline vaginal microbiota associated with BV promoting persistence in this group —rather than “reinfection” or exposure to another causative factor through sex
The main limitation of our analysis is that PCR assays for individual BVAB in vaginal fluid were obtained only at the enrollment visit; it is likely that more proximate detection of these bacteria in women without BV to the actual BV episode will demonstrate an even higher risk for subsequent BV acquisition. Prospective studies that apply PCR assays to frequent serial measurements—ideally through self-collection of vaginal swabs—would be the ideal approach to clarifying the temporal sequence of events that lead to BV. Nonetheless, several of the BVAB we detected at the enrollment visit were associated with markedly elevated risk of subsequent BV. Second, the interpretation of our multivariate analysis is limited by two factors: the relatively small numbers of women who had some individual BVAB detected by PCR assay, and the high degree of correlation among BVAB in that each species is frequently found as part of a larger community of BVAB. For this reason, we did not analyze individual BVAB in our multivariate analysis. Third, our subjects were selected on the basis of reporting sex with other women. Although 24% also reported sex with men in the three months prior to enrollment, they are unlikely to be representative of exclusively heterosexual women, a group that should be studied in similar fashion and to whom our findings may not necessarily apply. However, the fact that our subjects infrequently reported vaginal intercourse with male partners in the month after treatment might actually be advantageous in helping to define the role of vaginal microbiology in determining whether antecedent vaginal colonization of these bacteria have an independent role in predicting future incidence of BV. Finally, we relied on self-report of sexual behaviors to estimate subsequent BV risk, although we used CASI to minimize under- or over-reporting of these behaviors.
Our findings raise several important areas for future research. First, comparative assessments of risks for acquisition of BV, including molecular characterization of baseline microbiology of BVAB, need to be performed in heterosexual women; these efforts should help to define whether the molecular epidemiology of bacterial vaginosis differs from that of women who have sex with women, and to further help define a potential role of unprotected vaginal intercourse with male partners in BV incidence. Second, our BVAB-specific assays were qualitative. Quantitative polymerase chain reaction assays applied to vaginal fluid samples obtained in our subjects may offer more precise predictive value for incident BV, though they are typically less specific than performing sequence analysis on the amplicons as was performed in this study. Third, more frequent sampling (for example, daily or weekly) for application of bacterium-specific PCR assays in women without BV should help to better define the evolution of vaginal microbiota towards the “tipping point” of actual BV; these measures should be thoughtfully integrated with concomitant collection of behavioral data to further explore potential links with sexual practices. Finally, the establishment of consensus definitions for incidence, persistence and recurrence of BV would assist in progress towards describing the natural history of this condition. Whether risk factors that promote incidence are the same as those that promote persistence or recurrence is not clear.
In summary, our findings suggest that vaginal colonization with key BVABs among women with no BV is an independent risk factor for incident BV. Because BV may confer an increased risk of poor pregnancy outcome and HIV acquisition and predict upper genital tract disease, defining microbiological risk factors for new episodes of BV may offer new approaches to prevention.