for any RTI diagnosed at enrolment, risk reduction counselling, family planning and condoms. All women, including those ineligible to enrol, were offered free HIV voluntary counselling and testing, with onward referral to the nearest centre providing HIV care, including anti-retroviral treatment where appropriate. Laboratory methods Screening sera were tested for HSV-2 using a type-specific IgG ELISA (Kalon Biologicals, Guildford, Surrey, UK). Screening and enrolment sera were tested for HIV by dual ELISA (Murex HIV Ag/Ab Combination ELISA, Murex Biotech, Dartford, UK; Uni- FormII Ag/Ab micro ELISA system, bioM´erieux, Basingstoke, UK). Repeatedly discordant samples were tested by HIV-1 p24 Ag EIA (Biorad Genetic Systems, California, USA) and line immunoassay (INNO-LIA HIV I/II, Innogenetics, Gent, Belgium), as described previously.20 Enrolment samples were tested for syphilis by the rapid plasma reagin (RPR) test (Omega Diagnostics, Alva, Scotland, UK) and Treponema pallidum particle agglutination (TPPA) assay (Fujirebio Inc, Tokyo, Japan), and by fluorescent treponemal antibody assay (Trepo- Spot IF, bioM´erieux, Maray-l’Etoile, France) if RPR positive and TPPA negative. For BV, vaginal smears were heat-fixed and Gram-stained then examined by light microscopy by a single reader and scored using the Nugent criteria.21 External quality control of a random sample of 10% of slides was performed by an external reader (PH). Trichomonas vaginalis was diagnosed by wet preparation and culture (InPouch TV, BioMed Diagnostics, San Jose, California, USA). Wet preparations were examined by light microscopy for Candida albicans spores and hyphae. Chlamydia trachomatis and Neisseria gonorrhoeae were diagnosed by the AmplicorCT/NG PCR assay (Roche Diagnostics Systems, Branchburg, New Jersey, USA). N gonorrhoeae PCR-positive samples were retested in duplicate and classified using a ‘‘two- out-of-three’’ strategy, and an optical density >2 was used as the cut-off for positive rather than the manufacturer’s recommended value of >0.2. Cervico-vaginal lavage (CVL) from all HIV positive women were tested for HIV-1 RNA and HSV DNA by real-time PCR using the ABI-7300 system (Applied Biosystems, Courtabeouf, France) and manual nucleic acid extraction (Nuclisens miniMAG bioM´erieux, Marcy l’Etoile, France). In addition, CVL from a random sample of 449 HIV negative women were tested for HSV using the same method. Statistical methods Risk factors for BV were analysed using odds ratios (OR) and 95% confidence intervals (CI) obtained by logistic regression. Normal and intermediate flora categories (Nugent score 0–6) were combined to form the category ‘‘absence of BV’’. Unclassifiable slides containing blood, mucus and/or pus were excluded. Potential determinants of BV infection were considered using a conceptual framework22 with four groups: socio-economic factors, sexual behavioural factors, IVP and menstruation, and biological factors. Age, considered an a priori confounder, was included in all multivariate models. An initial model included socio-economic factors adjusted for age. Factors whose associa- tion reached statistical significance at p,0.1 were included in a multivariate model and those that remained independently associated with BV (p,0.1) were retained. The association between each determinant in the sexual behaviour group was Sex Transm Infect 2009;85:370–375. doi:10.1136/sti.2008.035543 Epidemiology assessed by adding the single determinant into the multivariate socio-economic model. Sexual behavioural factors whose association reached significance at p,0.1 were included in a multivariate model; those which remained significantly asso- ciated with BV (p,0.1) were retained. Associations with factors in the IVP group and in the biological group were assessed in the same way. The final model was reached by excluding factors one at a time until all remaining factors were significant at the p,0.05 level. RESULTS Study population Results at screening have been described previously.20 In total, 1305 HSV-2 seropositive women were enrolled. Excluding 22 women with unclassifiable vaginal smears, 807 (62.9%) women had BV, 287 (22.4%) had intermediate changes on Nugent score and 189 (14.7%) had normal flora. Among women with BV, 207 (25.7%) had a Nugent score of 9–10. Overall, 37.1% of women were HIV seropositive, 13.8% had evidence of active syphilis, 5.3% had gonorrhoea, 7.0% had C trachomatis and 30.3% had trichomoniasis. HSV genital shedding was detected in 34 (7.6%) HIV negative and 70 (14.5%) HIV positive women. Among HIV seropositive women, 52.6% had detectable HIV shedding. At least one symptom of a RTI was reported by 40.3% of women. There was no association between any reported clinical symptom and BV prevalence, and 60.7% of women with BV claimed to be asymptomatic. On examination, women with BV were more likely to have a diagnosis of vaginal discharge syndrome (OR 1.24; 95% CI 0.99 to 1.57). Socio-economic factors and association with BV The mean age of participants was 27.6 years (SD 4.9); 54% were divorced, widowed or separated, and 86% had at least one child. About half (51%) were local food handlers and 16% were bar workers. Over half (53%) reported drinking alcohol each week. BV was most prevalent in younger women, with 71.8% of those aged 16–19 years having BV compared with 60.9% of those aged over 25 years. Prevalence was highest among bar workers and guesthouse workers (71.0% and 73.2%, respectively, compared with 59.3% of local food handlers; table 1). A multivariate model of socio-economic factors adjusted for age showed that type of facility and decreasing number of dependents were independently associated with BV. Association of BV with sexual and other behavioural factors The majority of women (71%) reported fewer than 10 lifetime partners and 54% reported only 1 partner in the past 3 months. Most participants (64%) reported never or rarely using condoms and 38% reported having had sex in exchange for money or gifts in the past 3 months. At enrolment, 30% were using hormonal contraception; among those, 62% used injectable depot- medroxyprogesterone acetate (DMPA). On univariate analysis, BV was significantly associated with a number of sexual risk factors, including younger sexual debut, increasing partners in the past 3 months, increasing condom use, paid sex in the past 3 months and sex in the past week (table 1). After adjusting for age and socio-economic factors, only younger age at first sex, increasing alcohol consumption and recent sex remained independently associated with BV. There was some evidence of an inverse association between BV and hormonal contraception (adjusted OR 0.80; 95% CI 0.62 to 1.04). 371