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To study temporal changes in HIV incidence, HIV transmission routes, and both injecting and sexual risk behaviour in the open Amsterdam Cohort Study (ACS) among drug users. Initiated in 1985, the ACS enables us to study changes in trends since HAART became widespread in 1996.Person-time techniques were used to study the trend in HIV incidence among HIV-negative drug users. HIV transmission routes were determined using detailed standardised questionnaires. Trends in injecting and sexual risk behaviours were evaluated with a logistic regression model adjusted for correlations between visits of the same individual.The 1315 HIV-negative individuals, of whom 93 seroconverted for HIV, yielded 6970 HIV-negative person-years of follow-up. The HIV incidence was seven per 100 person-years in 1986 and varied between 0 and 0.5 per 100 person-years after 1999. The odds ratio was 15.6 (95% confidence interval, 2.6–94.6) for HIV transmission through unprotected heterosexual contact versus injecting after 1996 compared with the period before. Reports of both injecting and borrowing needles significantly declined over the period 1985–2004. Reports of sexual risk behaviour and sexually transmitted infections at follow-up visits decreased before 1996, but not after 1996.The HIV incidence among drug users in the ACS has declined since 1985. Accompanied by a reduction in injecting drug use and needle sharing, this decline occurred despite continued sexual risk behaviour. At present, new HIV seroconversions are related mainly to unprotected heterosexual contacts. Therefore, HIV prevention programmes for drug users should pay specific attention to the importance of safe sex practices.