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Increased risk of herpes simplex virus 2 (HSV-2) has been proposed as a possible indirect pathway through which hormonal contraceptives (specifically depot medroxyprogesterone acetate (DMPA)) may increase the risk of HIV acquisition among women. We investigated the effects of DMPA on HSV-2 acquisition among female sex workers.Longitudinal data were drawn from a prospective cohort of sex workers in Vancouver, Canada. The primary outcome was HSV-2 seroconversion. Extended Cox regression analyses were used to model the independent effect of DMPA use on HSV-2 acquisition.Between January 2010 and February 2014, 149 HSV-2 seronegative women were enrolled, contributing to 228 person-years (py) of follow-up. Of these, 19 (13.3%) reported DMPA use. There were 39 HSV-2 seroconversions (12 among DMPA users and 27 among non-users) over the study period (median follow-up of 18.6 months (IQR 8.4–29.9)), resulting in an overall incidence rate of 17.1 cases per 100 py (95% CI 12.4 to 23.6). Incidence rates were higher among DMPA users (57.4 cases per 100 py, 95% CI 31.4 to 105.0) compared with non-users (13.1 cases per 100 py, 95% CI 8.9 to 19.1). After adjusting for key confounders, use of DMPA remained an independent predictor of HSV-2 acquisition (adjusted HR 4.43, 95% CI 1.90 to 10.35).The high observed incidence rates of HSV-2, together with a strong association between DMPA exposure and HSV-2 acquisition, raise serious concerns about the provision of optimal reproductive and sexual healthcare to sex workers in this setting. Given the known links between HSV-2 and HIV, our findings underscore the need for further research to better understand the potential association between DMPA and increased risk of HSV-2 and other STIs to help inform the development of safer reproductive choices for women worldwide.