Pelvic Examination

This cross-sectional study enrolled 242 women from the Public Health, Seattle and King County Sexually Transmitted Diseases Clinic (STD clinic) between September 2006 and June 2010. The study was approved by the Institutional Review Board at the Fred Hutchinson Cancer Research Center. All study participants provided written informed consent. Participants were interviewed regarding medical history and sexual behaviors, and underwent a standard physical examination including a pelvic examination with speculum for collection of samples. Swabs of vaginal fluid were collected for Gram staining, pH, saline microscopy and potassium hydroxide preparation. Samples for DNA extraction and PCR were obtained using polyurethane foam swabs (Epicentre Biotechnologies, Madison, WI) that were brushed against the lateral vaginal wall, re-sheathed and frozen immediately at −20°C and subsequently held at −80°C. BV was diagnosed using Amsel’s criteria [8] (link) and confirmed by Gram stains [9] (link). Subjects with BV were treated with 5 g 0.75% intravaginal metronidazole gel used each night for 5 days. Testing for sexually transmitted infections and other vaginal infections was performed as previously described [19] (link).

Between September 2009 and July 2010, 135 nonpregnant women of reproductive age were enrolled in a longitudinal study at the University of Alabama at Birmingham. The clinical study protocol was approved by the Institutional Review Board of the University of Alabama at Birmingham and the University of Maryland School of Medicine. Written informed consent was appropriately obtained from all participants.
At study entry, participants were asked to answer a lengthy questionnaire on demographics as well as medical, dental, obstetric, hygiene, sexual and behavioral histories. They were also asked to self-collect three midvaginal swabs daily for ten weeks. The first Copan ESwab (Copan Diagnostics, Murrieta, CA, USA) was placed in RNAlater (Ambion, Austin, TX, USA) for use in future metatranscriptomics analyses. A second Copan ESwab was placed in Amies liquid transport medium for use later in extracting genomic DNA. Third, a Starplex double-headed Dacron swab (Starplex Scientific, Cleveland, TN, USA) was stored dry in a tube for later use in metabolomic and metaproteomic analyses. One of the Dacron swabs was also used to prepare a smear that was later Gram-stained for Nugent scoring. In addition, the participants measured their vaginal pH using the CarePlan VpH test glove (Inverness Medical Innovations, Waltham, MA, USA). Finally, participants completed a diary each day to record hygienic practices and sexual activities using a standardized form on which all responses were precoded. Pelvic examinations were performed at the time of enrollment and at weeks 5 and 10 or at interim times if the participant reported vaginal symptoms. A diagnosis of SBV was made when three of four Amsel criteria were recorded by the clinician and the participants reported symptoms spontaneously or upon direct questioning. ABV was defined as a finding of three of four Amsel criteria, but without self-report of any vaginal symptoms. Women diagnosed with SBV were treated for BV using standard of care practices [22 (link)]. Women with ABV were not treated. Clinical and selected daily behavioral data are described in Additional file 1.