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To assess the incidence and spectrum of AIDS-defining opportunistic illnesses in the highly active antiretroviral therapy (cART) era.A prospective cohort study of 8070 participants in the HIV Outpatient Study at 12 U.S. HIV clinics.We calculated incidence rates per 1000 person-years of observation for the first opportunistic infection, first opportunistic malignancy, and first occurrence of each individual opportunistic illness during 1994–2007. Using stratified Poisson regression models, and adjusting for sex, race, and HIV risk category, we modeled annual percentage changes in opportunistic illness incidence rates by calendar period.Eight thousand and seventy patients (baseline median age 38 years; median CD4 cell count 298 cells/μl) experienced 2027 incident opportunistic illnesses during a median of 2.9 years of observation. During 1994–1997, 1998–2002, and 2003–2007, respectively, rates of opportunistic infections (per 1000 person-years) were 89.0, 25.2 and 13.3 and rates of opportunistic malignancies were 23.4, 5.8 and 3.0 (P for trend <0.001 for both). Opportunistic illness rate decreases were similar for the subset of patients receiving cART. During 2003–2007, there were no significant changes in annual rates of opportunistic infections or opportunistic malignancies; the leading opportunistic illnesses (rate per 1000 person-years) were esophageal candidiasis (5.2), Pneumocystis pneumonia (3.9), cervical cancer (3.5), Mycobacterium avium complex infection (2.5), and cytomegalovirus disease (1.8); 36% of opportunistic illness events occurred at CD4 cell counts at least 200 cells/μl.Opportunistic illness rates declined precipitously after introduction of cART and stabilized at low levels during 2003–2007. In this contemporary cART era, a third of opportunistic illnesses were diagnosed at CD4 cell counts at least 200 cells/μl.