We used samples collected as part of a prospective cohort study of HIV-1 seropositive women conducted from 2002–2007 in Seattle, WA and Rochester, NY. The institutional review boards at the University of Washington and University of Rochester approved the study and participants provided written informed consent. At enrollment, eligible women were 18–50 years old, HIV-1 seropositive and not pregnant. Women were not eligible to enroll if they had active substance abuse that would preclude their ability to participate in the study or if they had a hysterectomy. Participants had 4 study visits in the first year and 3 visits per year in subsequent years. Each study visit included a face-to-face interview to ascertain information on demographics, sexual behavior, medication use, and reproductive and medical history. Plasma was obtained for HIV-1 RNA quantification. A pelvic examination was performed with collection of vaginal swabs for diagnosis of vaginal infections and Lactobacillus culture. Vaginal fluid specimens were not collected if the participant was menstruating. Bacterial vaginosis was diagnosed from vaginal Gram stain using Nugent’s criteria . Trichomonas vaginalis was detected by culture using the InPouch system (Biomed Diagnostics, White City, Oregon). The presence and quantity of Lactobacillus was assessed by vaginal culture on Columbia 5% sheep blood and Rogosa agar. Isolates from blood agar were grown in 5-10% CO2. Isolates from Rogosa agar were incubated anaerobically. All colonies with morphology suggestive of Lactobacillus on blood agar as well as any colonies growing on Rogosa were isolated and identified on the basis of colony morphology and Gram stain [8, 9]. These isolates were subcultured on tetramethylbenzidine (TMB) agar containing horseradish peroxidase in order to assess hydrogen peroxide (H2O2) production [10, 11].
Cervicovaginal lavage (CVL) was collected by washing the ectocervix and vaginal walls with 7 mL of 10 mM lithium chloride solution, which was then collected from the vaginal pool, spun at 800 g for 5 minutes to separate the epithelial cells and stored at −80°C. Plasma and CVL HIV-1 RNA were quantified by an independently validated real-time PCR assay described previously , with a lower limit of detection of 30 copies/mL.
The frozen cell pellet from CVL was thawed and underwent DNA extraction with the MO BIO Bacteremia Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). Extracted DNA was tested by quantitative PCR using primers targeting the human 18S rRNA gene to validate that successful DNA extraction occurred and an internal amplification control PCR using exogenous DNA from a jellyfish gene was used to test for presence of PCR inhibitors . Samples were then subjected to taxon-directed 16S rRNA gene qPCR assays for the detection and quantification of L. crispatus and L. jensenii. Each assay had been validated previously and proven to be sensitive (to a level of 1–10 DNA copies/reaction) and specific (does not detect other bacteria at a concentration of 106 copies/reaction) [14, 15]. The assays used a TaqMan format and were run on an ABI 7500 Thermocycler (Applied Biosystems, Foster City, CA) or Eppendorf Mastercycler ep Realplex thermal cycler (Eppendorf, Westbury, NY). Negative assays were assigned a value at the lower limit of detection for that assay.
Based on the findings from our analysis of concordance, we conducted an exploratory analysis using broad range 16S rRNA gene PCR on a subset of culture isolates derived from samples with high quantities of H2O2-producing Lactobacillus by culture (≥ 106 colony forming units [CFUs]), but with negative results for both L. crispatus and L. jensenii by qPCR in CVL. Lactobacillus isolates were retrieved from −80°C storage and streaked to Rogosa agar and Columbia agar with 5% sheep blood. A repeat streak to fresh agar was performed in order to obtain new isolates for broad range PCR testing. Genomic DNA was extracted from single bacterial colonies, single colonies converted to a lawn of bacteria (patches), or streaks of colonies on plates using the BiOstic Bacteremia DNA Isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA). PCR was performed to amplify a portion of the bacterial 16S rRNA gene using combinations of the following broad range bacterial primers: 27 F bact (5'-AGAGTTTGATCMTGGCTCAG-3'), 338 F (5'-ACTCCTRCGGGAGGCAGCAG-3'), 806R (5'-GGACTACCAGGGTATCTAAT-3') and 1407R (5'-GACGGGCGGTGWGTRCA-3'). A range of 10ul neat DNA to 1ul 1:10 diluted DNA was added to 50ul reaction. Thermal cycling consisted of pre-melting at 95°C for 2 minutes, followed by 25 cycles of melting at 95°C for 30 seconds, annealing at 55°C or 60°C for 30 seconds and extension at 72°C for 2 minutes, with a final extension at 72°C for 10 minutes. PCR products were cleaned using the 0.5-mL 100 K Amicon Ultra Centrifugal Filters (Millipore, Billerica, MA). The same PCR primers were used in the sequencing reactions using ABI Prism 3730xI DNA Analyzer and BigDye chemistry. Sequencher software (GeneCodes, Ann Arbor, MI) was used to assemble sequence data. Sequences of the isolates (~800 base pairs in length) were compared to known bacterial sequences in Genbank by BLAST algorithm. Identities of the isolates to known species was defined by having >98% sequence similarity.
We used descriptive statistics for planned and exploratory analyses and calculated a kappa statistic to assess concordance between culture and qPCR using Stata version 11.0 (StataCorp, College Station, TX).