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To investigate the relationship between male involvement in prevention of mother-to-child HIV transmission services and infant HIV acquisition and mortality, a prospective cohort study was undertaken between 1999 and 2005 in Nairobi, Kenya.HIV-infected pregnant women were enrolled and followed with their infants for 1 year with infant HIV DNA testing at birth, 1, 3, 6, 9, and 12 months postpartum. Women were encouraged to invite male partners for prevention counseling and HIV testing.Among 456 female participants, 140 partners (31%) attended the antenatal clinic. Eighty-two (19%) of 441 infants tested were HIV infected by 1 year of age. Adjusting for maternal viral load, vertical transmission risk was lower among women with partner attendance compared with those without [adjusted hazard ratio (aHR) = 0.56, 95% confidence interval (CI): 0.33 to 0.98; P = 0.042] and among women reporting versus not reporting previous partner HIV testing (aHR = 0.52, 95% CI: 0.32 to 0.84; P = 0.008). The combined risk of HIV acquisition or infant mortality was lower with male attendance (aHR = 0.55; 95% CI: 0.35 to 0.88; P = 0.012) and report of prior male HIV testing (aHR = 0.58; 95% CI: 0.34 to 0.88; P = 0.01) when adjusting for maternal viral load and breastfeeding.Including men in antenatal prevention of mother-to-child HIV transmission services with HIV testing may improve infant health outcomes.