Relationship of Specific Vaginal Bacteria and Bacterial Vaginosis Treatment Failure in Women Who Have Sex with Women

Context Bacterial vaginosis, a condition that is particularly common among women who have sex with women, is an overgrowth of anaerobic bacteria and depletion of normally occurring lactobacilli. Bacterial vaginosis persists or recurs in 11% to 29% of women at 1 month after treatment. Contribution This study identified that the presence of specific vaginal bacteria at baseline (Clostridia BVAB1, BVAB2, or BVAB3; Peptoniphilus lacrimalis; or Megasphaera phylotype 2) and lower adherence to treatment were the only factors associated with persistence of bacterial vaginosis 1 month after treatment among women who have sex with women. Caution The results might not apply to women who have sex only with men. The Editors Bacterial vaginosis is characterized by depletion of hydrogen peroxideproducing lactobacilli that characterize normal vaginal flora and profound overgrowth of anaerobic bacteria (1). Bacterial vaginosis is the most prevalent vaginal infection in reproductive-age women, affecting 8% to 29%, and is the most common cause of vaginal symptoms prompting medical care (2). Of 3739 women enrolled during 2001 to 2004 in a nationally representative sample of the U.S. civilian noninstitutionalized population, almost 1 in 3 (29.2% [95% CI, 27.2% to 31.3%]) had bacterial vaginosis, as determined by Gram stain of vaginal fluid (3, 4). Bacterial vaginosis has been consistently associated with adverse outcomes related to the upper genital tract and with increased risk for HIV acquisition (57). Treatment of bacterial vaginosis targets the abundance of anaerobes that define the condition. With oral metronidazole used for 7 days or vaginal metronidazole for 5 days, symptoms improve in 83% to 87% of women by 2 to 3 weeks (8, 9). The improvement rate is similar for women who use vaginal clindamycin regimensboth antibiotics are recommended (10)and the restoration rate of vaginal lactobacilli at 30 days is similar (11, 12). Although short-term treatment response is acceptable, bacterial vaginosis persists or recurs in 11% to 29% of women at 1 month (8, 13, 14) and long-term recurrence rates exceed 70% (1517). Few studies have reported factors associated with bacterial vaginosis recurrence after successful treatment, such as black race, older age, higher Nugent score at enrollment (17), history of bacterial vaginosis, regular sex partner during study, female sex partner, and hormonal contraception (15). Even fewer studies have assessed risks associated with bacterial vaginosis persistence at 1 month after treatment. Available data suggest that condom use in the period immediately after treatment might support maintenance of normal vaginal flora and thus increase cure rates (16, 18). For unknown reasons, women who have sex with women have a high prevalence of bacterial vaginosis (25% to 52%) (3, 19). We hypothesized that this population might provide a unique opportunity to study the response to treatment of bacterial vaginosis because the potentially confounding exposure of unprotected vaginal intercourse was not likely to be common. In a cohort study of vaginal flora in this population, we assessed the incidence of bacterial vaginosis persisting at 1 month after treatment with vaginal metronidazole. In addition to measuring the contribution of recognized risk factors for bacterial vaginosis, including race, sexual behaviors, and douching, we assessed the contribution of specific species of bacterial vaginosisassociated bacteria (BVAB) present at initiation of bacterial vaginosis treatment. These bacteria include fastidious anaerobes detected by species-specific polymerase chain reaction (PCR) assays, some of which have not yet been cultivated and include 3 recently identified bacteria in the Clostridiales order (BVAB1, BVAB2, and BVAB3) that are highly specific (>97%) for bacterial vaginosis (20). Methods Participants and Clinical Definitions The study population comprised women age 16 to 30 years who reported sex with at least 1 woman in the previous year and who responded to recruitment through advertisements, media, and community referral between October 2004 and December 2006. Participants completed an extensive computer-assisted self-interview on demographic characteristics and medical, reproductive, and sexual history and underwent a standardized examination, including collection of vaginal fluid for Gram stain, saline microscopy, pH measurement, potassium hydroxide evaluation, and culture of Trichomonas vaginalis. We asked all participants to return for 4 quarterly visits or at any time if genital symptoms developed. To obtain specimens for bacterium-specific PCR assays, we brushed a polyurethane foam swab (Catch-All, Epicentre Biotechnologies, Madison, Wisconsin) against the lateral vaginal wall, resheathed it, and froze it immediately in a 80 C freezer until DNA extraction. We diagnosed bacterial vaginosis if 3 of 4 clinical (Amsel) criteria (vaginal pH >4.5, clue cells on saline microscopy >20% of epithelial cells, amine odor on addition of potassium hydroxide, or homogeneous vaginal discharge) were present (21) and Gram stain of vaginal fluid confirmed abnormal flora (Nugent score >3) (4). We treated women who had bacterial vaginosis with vaginal metronidazole gel (37.5 mg nightly for 5 days) and asked them to return in 1 month for test of cure, when we repeated examination and collected vaginal fluid for Gram stain in all women. Initially, we collected vaginal fluid samples for bacteria-specific PCR assays at test-of-cure visits for all women whose vaginal pH was greater than 4.0 and thus had suspected bacterial vaginosis or trichomoniasis. After approximately one half of the study participants were enrolled, we collected these samples routinely in all participants at the test-of-cure visit. For the analysis, we used the first visit at which a woman was found to have bacterial vaginosis (whether at the initial enrollment visit, at a later quarterly routine visit, or at a visit self-initiated for vaginal symptoms). We used test-of-cure visits after a woman's first bacterial vaginosispositive visit that were completed before 31 March 2007 to examine incidence of persistent bacterial vaginosis and abnormal vaginal flora. We defined persistent bacterial vaginosis by Amsel criteria. We confirmed persistent bacterial vaginosis by Nugent score of vaginal fluid greater than 6 at the 1-month follow-up visit and confirmed abnormal vaginal flora by Nugent score greater than 3. We obtained written informed consent from all participants. Conduct of the study adhered to standard guidelines for research involving human participants and was approved by the University of Washington and Fred Hutchinson Cancer Center Human Subjects Review Committees. Microbiological Analysis For DNA extraction, we placed vaginal swabs for bacterial PCR assay in 15-mL conical vials with 2 mL of saline and vortex-mixed them for 1 minute to dislodge cells. We centrifuged the solution at 14000 rpm for 10 minutes and resuspended the pellet in 100 L of supernatant. We extracted DNA from the pellet by using the Ultra Clean Soil DNA Kit (MoBio, Carlsbad, California) according to the manufacturer's instructions. We eluted DNA from silica columns in a 150-L volume buffer. We performed sham digests by using a swab without human contact with each round of DNA extraction (every 10 to 25 samples) to control for possible contamination from kit reagents or collection swabs. We developed bacterium-specific PCR assays to detect species-specific regions of the 16S rRNA gene. We aligned 16S rDNA sequences from vaginal bacteria detected by broad-range 16S rDNA PCR assays (22). We designed primers to target highly variable regions of the bacterial 16S rRNA gene that seem to be unique for each species. We developed PCR assays for 17 bacterial species that were commonly detected in vaginal samples (23). Each 50-L PCR reaction contained 1PCR Buffer II, 2 mmol of magnesium chloride, 0.8 mmol of deoxyribonucleotide triphosphate mix, and 1 U of AmpliTaq Gold DNA Polymerase (all from Applied Biosystems, Foster City, California), as well as 0.2 mol each of forward and reverse primer and 1 L of template DNA. The PCR conditions included a premelt at 95C for 10 minutes, then 40 to 45 cycles of 95C for 30 seconds (melt), 53C to 62C for 30 seconds (annealing), and 72C for 30 seconds (extension), followed by a final extension at 72C for 7 minutes. We visualized PCR products after electrophoresis in 2% agarose gels and stained them with ethidium bromide. We optimized bacterial PCR assays so that each could detect as few as 100 molecules of cloned 16S rDNA per reaction, although most assays could detect 1 to 10 molecules and thus had even lower detection thresholds. We sequenced every PCR assay with a visible band of the expected size on gel electrophoresis (BigDye, version 3; Applied Biosystems) to confirm that the PCR product had at least 99% similarity with the expected bacterial target, thereby assuring bacterial specificity. We considered PCR reactions to be negative if they did not have visible bands on gel electrophoresis or confirmed sequence homology. We ran no-template PCR controls (consisting of master mix, primers, and water) and sham digest controls (template consisting of water subjected to DNA extraction) with each PCR assay to monitor for contamination. We subjected each participant's extracted DNA to a human -globin PCR assay to assure that amplifiable DNA was successfully extracted from the sample and to monitor for PCR inhibitors (24). The -globin PCR protocol used is the same as that listed for bacterial PCR, with the exception that the following primers were used: GH2O-5-GAAGAGCCAAGGACAGGTAC-3 and PCO4-5-CAACTTCATCCACGTTCACC-3. In addition, we performed an internal amplification control quantitative PCR assay by using DNA from each vaginal sample to detect more subtle PCR inhibitors by monitoring the amplification of an exogenously added template (jellyfish aequorin