2 2. Methods 2.1. Sample Collection and Characterization. This was a secondary analysis of samples collected as part of a prospective cohort study of nonpregnant HIV-1-infected women in Providence, Rhode Island, evaluating the eﬀect of antiretroviral therapy (ART) on HIV-1 RNA shedding in the female genital tract [16]. In the parent study, women were seen at baseline, 2 weeks, one month, and every 6 months for 36 months. All patients gave informed consent to participate in the study; the study protocol was approved by the Institutional Review Board of The Miriam Hospital, Providence, RI. At each visit clinical data and exam ﬁndings were recorded, and blood and cervicovaginal lavage (CVL) samples were collected. Women were asked not to douche, have sex, or use any intravaginal product for the 48 hours prior to the visit. The CVL was collected by infusing 10 cc of normal saline into the vagina with a syringe and then aspirating back the ﬂuid. For this subanalysis samples were randomly selected to represent approximately equal numbers of samples from women with and without BV, and from women who were on antiretroviral therapy and those who were not. Not all selected samples were available for analysis, and so the ﬁnal numbers in each category are not exactly equal (e.g., 34 samples from subjects with BV and 30 samples from subjects without BV). Some women contributed samples from more than one visit, but each sample was analyzed as an individual measurement, and GEE equations were used in the statistical analysis to account for this. Bacterial vaginosis was diagnosed using Amsel’s clinical criteria [17]: clinicians assessed vaginal discharge, pH, and presence of clue cells and ﬁshy odor with addition of KOH and patients with at least 3 of 4 criteria were considered to have BV. Trichomonas and Candida vaginitis were also diagnosed clinically using wet mount. Previous studies in this population had shown that the prevalence of syphilis, gonorrhea, and chlamydia were quite low; so testing was only done annually [18]. HIV-1 RNA was quantiﬁed using the nucleic acid sequence-based assay (Nuclisens, bioMerieux, Durham, NC), with a sensitivity of 400 copies/mL. Cells in the vaginal ﬂuid were classiﬁed by an experienced Medical Technologist as white blood cells, red blood cells, or nucleated cells (primarily vaginal epithelial cells). Manual cell counts were performed on a hemocytometer, counting cells in ﬁve 1 mm squares and averaging the results. 2.2. Sample Processing and Testing. The CVL sample was thawed and then centrifuged for 10 minutes at 14000 ×g, and the supernatant removed. The remaining pellet underwent DNA extraction with the MoBio UltraClean Soil DNA Isolation Kit (MoBio,Calsbad, CA) following the manufac- turer’s instructions. A clean swab was taken through the DNA extraction process as a negative extraction control. All extracted DNA samples and the extraction control were tested in a quantitative PCR using primers targeting the human 18S rRNA gene to validate that successful DNA extraction occurred. An internal ampliﬁcation control PCR using exogenous DNA from a jellyﬁsh gene was used to Infectious Diseases in Obstetrics and Gynecology test for presence of PCR inhibitors [19]. The presence of inhibition is deﬁned as an internal ampliﬁcation control qPCR threshold cycle value that was 2 cycles higher than that of the no-template control. Patient samples were then subjected to eight separate taxon-directed 16S rRNA gene quantitative PCR assays for the detection and quantiﬁcation of individual bacteria: Lac- tobacillus genus, Lactobacillus crispatus, Gardnerella vaginalis, Leptotrichia/Sneathia, Megasphaera, and Bacterial Vaginosis- Associated Bacterium (BVAB) 1, BVAB2, and BVAB3. One assay detects two bacterial species (Leptotrichia and Sneathia) which are closely related. The BVAB are related to bac- teria in the Clostridiales order and have been found to be highly speciﬁc for BV [7]. Each assay has previously been validated and proven to be sensitive (to a level of 1–10 DNA copies/reaction, or 150–1500 copies/mL) and speciﬁc (does not detect other bacteria at a concentration of 106 copies/rxn) [20]. The assays use a TaqMan format and were run on an ABI 7500 Thermocycler (Applied Biosystems, Foster City, CA). Plasmids containing bacterial 16S rRNA genes were used to generate standard curves for quantiﬁ- cation. The standards were generated by cloning bacterial 16S rRNA genes into E. coli and then purifying plasmids. The plasmids were quantiﬁed using a ﬂuorimeter and the Quant-iT Pico Green assay kit (Invitrogen, Carlsbad, CA) to determine the number rRNA gene copies per microliter. 2.3. Statistical Analysis. All analyses were carried out using Stata v9.2 (StataCorp, College Station, Texas). Demographic variables were compared between groups using the Mann- Whitney U-test for continuous variables and Independent samples t -test for categorical variables. Log-transformed concentrations of bacteria were compared between women with and without BV using a t-test. The relationships between presence and absence of bacteria and detection of HIV and sensitivity and speciﬁcity for diagnosis of BV were modeled using logistic regression and generalized estimating equations to account for repeat measures. Linear regression was used to model the relationship between bacterial species and continuous variables such as quantity of nucleated cells. For all regression analyses, the method of generalized estimating equations (GEEs) with robust standard errors was used to account for residual correlation due to the fact that some observations were repeated measures on the same women over time. Our power calculation was based on the 70 samples initially selected, and we estimated an 80% power to detect a twofold increase in the rate of HIV-1 shedding in women with detectable BV-associated bacteria. 3. Results Sixty-four CVL samples from 51 women were analyzed. One woman contributed three samples, and eleven women contributed two samples. When comparing only the ﬁrst sample from each subject, 24 women with BV and 27 women without BV were similar in terms of age, race, time since HIV-1 diagnosis, antiretroviral treatment status, CD4 count, and plasma viral load (Table 1). Initially 65 samples were processed, but one sample did not contain adequate material