In this prospective, observational study of 3620 reproductiveage women, we found that BV diagnosed on the basis of Gram stain analysis of vaginal fluid was associated with a 1.4–2-fold increased risk for acquisition of gonococcal, chlamydial, or trichomonal genital infection. Our data are consistent with those from other studies of high-risk women.
Numerous longitudinal studies have found that BV or absence of vaginal Lactobacillu
s spp. are associated with incident STI regardless of whether vaginal microbiota were measured by Gram stain or culture [6
]. In a longitudinal study of 657 female sex workers in Kenya, Martin et al [11
] reported that intermediate and BV states as assessed by Gram stain were associated with incidence of trichomoniasis (AHR, 1.8 [95% CI, 1.3–2.4]), and absence of vaginal lactobacilli on culture was associated with the acquisition of gonococcal infection (AHR, 1.7 [95% CI, 1.3–2.4]) and HIV infection (AHR, 2.0 [95% CI, 1.2–3.5]). BV-associated microorganisms have also been found to be associated with a 2-fold increased risk for both incident HSV-2 infection (hazard ratio, 2.1 [95% CI, 1.0–4.5]) [6
] and development of pelvic inflammatory disease (rate ratio, 2.03 [95% CI, 1.16–3.53]) [32
]. Schwebke et al [10
] demonstrated in a randomized trial of 107 women that treatment and prophylaxis of asymptomatic women who had atypical Gram stain smears resulted in a significantly decreased incidence of chlamydial infection compared with that among a group of control women who were under observation. Wiesenfeld et al [5
] also found in a cross-sectional study of women who reported recent exposure to a male partner with urethritis that BV was a strong predictor of gonococcal infection (odds ratio, 4.1 [95% CI, 1.7–9.7]) and chlamydial infections (odds ratio, 3.4 [95% CI, 1.5–7.8]).
In contrast to these studies, a large prospective study, the GYN Infections Follow-Through (GIFT) Study, found the association between BV and incident gonococcal and chlamydial infection was not statistically significant (adjusted relative risk, 1.52 [95% CI, 0.74–3.13]) [23
]. However, the point estimate (1.52) is similar to those noted in the present study for gonococcal and chlamydial infection. There may have been increased power to detect significant associations with sexually transmitted disease outcomes in the LSVF, as the GIFT study observed 46 cases of incident gonococcal and chlamydial infection and the LSVF modeled the analysis on 176 cases of gonococcal infection and 678 cases of chlamydial infection.
The hazard ratios for incident trichomonal infection also demonstrated a dose-response trend for increasing risk with higher Nugent scores. A study of 7918 pregnant women reported by Hillier et al [33
] found trichomonal infection was more strongly associated with intermediate Gram stain smears in a cross-sectional analysis. The data suggest the epidemiology of intermediate status may be unique. We hypothesize that T. vaginalis
may influence its environment. In vitro data suggest that T. vaginalis
grows better in an elevated pH [34
], and BV provides this high-pH environment. Both the intermediate state and the BV state are associated with decreased abundance of protective lactobacilli [15
]. Recent data from Ravel et al [15
] also suggest that intermediate Nugent scores may represent a bacterial community of unique taxa as compared to higher Nugent scores.
Molecular studies of the vagina based on the analysis of 16S ribosomal RNA gene sequences have shown that in both women presenting with BV and those without BV, the diversity, composition, and relative abundance of microbial species in the vagina varies dramatically between women [14
] and that a significant proportion of women lack appreciable numbers of Lactobacillus spp. [36
]. Lactobacilli have historically been considered keystone species of vaginal communities in reproductive- age women. Lactobacilli benefit the vagina by producing lactic acid as a fermentation product, and it accumulates in the vagina and is associated with a reduced pH [18
]. Low pH and high lactic acid concentration are hypothesized to inhibit BVassociated bacteria and STIs [37
also produce bacteriocins— low-molecular-weight proteins that can inhibit the growth of a variety of bacteria, thus reinforcing the protective role of lactobacilli [38
]. Vaginal communities that lack Lactobacillus spp. may have high numbers of other lactic acid- producing bacteria such as Atopobium
spp., or Leptotrichia
]. Despite the ability of these bacteria to produce lactic acid, the community resilience and infection susceptibility profiles may not be equivalent to those of lactobacilli, which may account for the different microbial community states that are observed between women [15
]. Adding complexity to the problem is the fact that the functional differences between the many different species (or strains) of lactobacilli, the most dominant of which include Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri
, and Lactobacillus jensenii
, are not known and our study based on Gram stain was not able to decipher them. As part of the Human Microbiome Project (http://nihroadmap.nih.gov/hmp/
], genomic studies of the vagina are expected to describe the structure of the complex microbial communities and how they contribute to disease susceptibility. Finally, BV may predispose to acquisition of STIs upon exposure because the local cytokine production associated with BV may facilitate the acquisition of STIs [42
Strengths of our study include the prospective design, which enabled us to evaluate temporal ordering of Gram stained vaginal smears and subsequent incidence of STIs, collection of detailed data on sexual behaviors, and demonstration of a doseresponse relationship between Nugent score and STI outcome. Unlike the high-risk populations in other studies, this study population was drawn from women who presented for routine health care, which may allow the findings to be more generalizable than those of previous studies. In addition, the prevalence of BV among African American and white women in this study was similar to rates reported from nationally representative surveys in the United States [19
A limitation regarding the geneA limitation regarding the generalizability of the study population, however, is the fact that it was composed of women who predominantly were young, were African American, had lower incomes, and were not high school graduates. A critical limitation of this study was the wide sampling interval of 3 months between study visits. Several longitudinal studies have shown that vaginal microbiota fluctuate rapidly and over the time course of the menstrual cycle [43
]. More frequent sampling is necessary to address this time-sensitive sampling issue; however, the interval-censored sampling presented in this study is suggestive, because women with BV episodes often have recurrent episodes [47
]. In addition, although the characterization of the vaginal microbiota was not immediately proximal to the STI acquisition, a woman who lacks vaginal Lactobacillus
spp. is likely to persist or enter that community state again over the time course of the menstrual cycle [44
]. The hazard ratios obtained in this epidemiological study are indeed only estimates, but they are highly indicative of a strong association between BV microbiota and subsequent STI acquisition. It would also be very challenging to achieve the necessary sample size to observe STI outcomes in a study of daily vaginal microbiota sampling, and this interval-censored cohort provides a reasonable estimate of the risk. The results of this analysis were based on Gram stain and would be appreciably bolstered by additional molecular studies, such as information of the bacteria, fungi, and viruses that compose the vaginal microbiome.
Another limitation of this study is the fact that one of the strongest epidemiological risk factors for BV is sexual activity [48
]; therefore, BV may be a marker for having sex partners who are also harboring STIs. Unmeasured confounding may account for the association between BV and STIs. We adjusted for number of sex partners and use of condoms, but high-risk sexual activity or membership in a core group of STI may account for our findings.Women who reported consistent condom use were also observed to have increased risk for incident STIs, confirming that condom use may be subject to self-reporting bias [49
], error in use [50
], or misuse in high-risk situations or that the condom or its components have an effect on the vaginal microbiome.
In summary, vaginal microbiota consistent with BV by Gram stain analysis is significantly associated with increased risk for STI acquisition. Given the strength of evidence in this large, prospective study combined with the findings from a randomized trial that treatment and prophylaxis of asymptomatic women with atypical Gram stain smears was associated with decreased rates of incident chlamydial genital infection [10
], consideration and future research should focus on whether intervention should be recommended to women who are at high risk for STIs and who present with BV or BV-associated microbiota. There also remains an urgent need to developmore effective interventions for BV because the recurrence following current treatments is disappointingly high [47
]. Future research that utilizes molecular phylogenetic technologies will facilitate our understanding of microbial communities and species that are more protective of the vagina and confer protection from STIs. We envision that better knowledge of vaginal microbial communities will lead to the development of interventions, such as antibiotic regimens and probiotic or prebiotic treatments, to drive the vaginal microbiota toward healthier states that are more resilient to pathogens.