Incidence & Predictors of Treatment Failure for Bacterial Vaginosis JM Marrazzo, KK Thomas, K Ringwood, T Fiedler, DN Fredricks University of Washington & Fred Hutchinson Cancer Research Center, Seattle WA

Background l Most common  Cause of vaginal symptoms prompting medical evaluation for vaginitis  Cause of vaginitis  >10% of women experience BV  May cause 11% of preterm deliveries in U.S. l Etiology not understood, but associated with douching, new male partner, unprotected sex in heterosexual women, sex between women l Frequently persists after treatment (15% - 20%) l Very frequently recurs after successful treatment (30% - 60%)  History of BV, regular partner throughout follow-up, and female partner implicated Sobel 2006; Bradshaw 2006

Background l Traditionally defined as overgrowth of commensal anaerobic flora (classically G. vaginalis, Prevotella, Mobiluncus, M. hominis) relative to H 2 O 2 + lactobacilli l Molecular (cultivation-independent) approaches have recently expanded spectrum of BV microbiology l Some bacteria highly specific for BV in recent studies l Some with known high-level resistance to metronidazole  Atopobium vaginae l “New” bacteria not yet incorporated into prospective analyses for BV persistence or recurrence Verstraelen 2004, Ferris 2004, Fredricks 2005

BV-associated bacteria (BVAB 1, 2, 3) and their relationship to bacteria in the Clostridium phylum Clostridium Cluster XIVa BVAB1 BVAB2 BVAB3

Methods l Subjects recruited to research clinic  years  Sex with woman (prior year)  Recruited through ads (self-referred) and partners l Underwent computer-assisted self-interview (CASI) at enrollment l BV diagnosed by Amsel criteria and treated with vaginal metronidazole  BV confirmed by Nugent criteria (later) l Vaginal fluid collected with polyurethane foam swab and saline lavage (0.5 cc)

Methods l All women with BV asked to return at 1 month for repeat procedures (CASI, vaginal fluid collection)  BV persistence = Nugent >6  Abnormal vaginal flora = Nugent >3 l All women asked to return at 3 months  BV recurrence defined by Nugent score >6 among women whose baseline BV was cured at 1 month

Methods Vaginal Fluid Collected at All Visits: l Traditional culture  Used to define H2O2 production by lactobacilli  Typical panel of anaerobic flora l Bacterium-specific PCR assays based on cloned 16s rDNA sequences derived from earlier analysis of clones by RFLP, sequencing; all positive results sequence-confirmed BVAB1Leptotrichia sppMobiluncus curtisii BVAB2Peptoniphilus sppMobiluncus mulieris BVAB3P. lacrimalisPrevotella G1 Megasphaera phylotype 1 (elsdensii-like) Megasphaera phylotype 2 (micronucliformis-like) Lactobacillus crispatus Lactobacillus iners Eggerthella-like uncultured bacterium Gardnerella vaginalisAtopobium spp

Results Baseline and Persistent BV 239 women enrolled BV N = 12 / 72 (17%) No BV N = 60 / 72 (83%) Characteristics: Median age 25 y Nonwhite: 25% Sex with male last 3 mos: 28% Douche last 3 mos: 6.4% At 1 month baseline N = 66 (28%) No BV N = 173 (72%) New BV during study N = % had BV at baseline Overall f/u at 1 month = 90% 83% responded to vaginal MTZ Persistent BV incidence: 17% 72 women with BV

Baseline Vaginal Microbiology (PCRs) Demographics OR (95% CI) Age y3.9 (0.9, 16.4) Black race7.0 (3.9, 12.4) P=0.15 P=0.67 P=0.009 P=0.06 OR (95% CI) BVAB1 2.3 (0.8, 6.5)) BVAB22.2 (0.3, 15.5) BVAB34.4 (1.5, 13.4) Results Factors Associated with BV Persistence Incidence of BV Recurrence (%) P=0.03

OR (95% CI) BVAB12.6 (1.3, 5.2) Adherence0.36 (0.2, 0.7) Any sex4.1 (0.6, 28) Vaginal sex2.1 (1.1, 4.2) P=0.05 *Intercourse with male partner Results Additional Factors Associated with Persistence of Abnormal Flora (N = 22) Incidence of BV Recurrence (%) P=0.02 P=0.05 P=0.09

Results Baseline and Persistent BV 239 women enrolled BV N = 12 / 72 (17%) No BV N = 60 / 72 (83%) At 1 month baseline N = 66 (28%) No BV N = 173 (72%) New BV during study N = % who initially responded to MTZ had BV recurrence 72 women with BV Recurrent BV N = 8 / 53 (15%)

Conclusions Distinct risks predict BV persistence –Demographics Black women consistently at high risk for BV –Reasons unclear; not related to douching in our study Older age –Vaginal microbiology BVAB3, possibly BVAB1 –Sex Vaginal fluid exchange, intercourse with men [Sanchez 2004, Bradshaw 2006] Further study needed –Additional accrual of subjects to substantiate preliminary trends and assess risks for BV recurrence –Cultivation of BVAB to assess pathogenicity and antibiotic susceptibilities

Limitations Small number of subjects who were self- referred or referred by partners –Limits on reproducibility, generalizability –However, excellent retention and diverse sexual practices Development of bacterium-specific PCRs directed by initial clone analysis –May not include all relevant species

Acknowledgements Study personnel Nancy Dorn Dana Varon Lauren Asaba Susan Heideke Corey Fish Kathy Agnew Becca Hutcheson Support Dave Eschenbach Sharon Hillier King Holmes Larry Corey

Associations Between Bacteria Detected and BV Bacterium-specific PCR assays for vaginal fluid samples from 40 subjects with BV and 65 without BV. ORs adjusted for age, site of enrollment, vaginal symptoms, report of sex with men