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To estimate the effects of reproductive tract infections (RTIs) on HIV acquisition among Zimbabwean and Ugandan women.A multicenter prospective observational cohort study enrolled 4439 HIV-uninfected women aged 18 to 35 attending family planning clinics in Zimbabwe and Uganda. Participants were interviewed, and tested for HIV and RTIs every 3 months for 15 to 24 months. They received HIV risk reduction counseling, male condoms, and treatment for curable RTIs.Despite HIV risk reduction counseling and regular screening and treatment for RTIs, the HIV incidence did not decline during the study. Positive HSV-2 serostatus at baseline (hazard ratio [HR] = 3.69, 95% confidence interval = 2.45–5.55), incident HSV-2 (HR = 5.35, 3.06–9.36), incident Neisseria gonorrhoeae (HR = 5.46, 3.41–8.75), and altered vaginal flora during the study (bacterial vaginosis [BV]: HR = 2.12, 1.50–3.01; and intermediate flora: HR = 2.02, 1.39–2.95) were independently associated with HIV acquisition after controlling for demographic and behavioral covariates and other RTIs (Treponema pallidum, Chlamydia trachomatis, Trichomonas vaginalis, and vaginal yeasts). For N. gonorrhoeae, C. trachomatis, T. vaginalis, and vaginal yeasts, the risk of HIV acquisition increased when the infection was identified at the visit before the HIV-detection visit or with the duration of infection. Population attributable risk percent (PAR%) calculations show that HSV-2 contributes most to acquisition of new HIV infections (50.4% for baseline HSV-2 and 7.9% for incident HSV-2), followed by altered vaginal flora (17.2% for bacterial vaginosis and 11.8% for intermediate flora).A substantial proportion of new HIV infections in Zimbabwean and Ugandan women are attributable to RTIs, particularly HSV-2 and altered vaginal flora.