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Early initiation of antiretroviral therapy depends on an early infant diagnosis and is critical to reduce HIV-related infant mortality. We describe the implementation of a routine prevention of mother-to-child transmission program and focus on early infant diagnosis to identify opportunities to improve outcomes.HIV-exposed infants and their mothers were enrolled in a prospective, observational cohort study at a routine, hospital-based prevention of mother-to-child transmission and HIV treatment service in Johannesburg, South Africa. Infant HIV status was determined by testing samples collected between birth and 6 weeks and searching the national laboratory information system for polymerase chain reaction results of defaulting infants who accessed testing elsewhere.Of 838 enrolled infants, HIV status was determined for 606 (72.3%) by testing at the study site, 85 (10.1%) by accessing test results from other facilities, 19 (2.3%) by testing stored samples and remained unknown in 128 (15.3%) infants. In total, 38 perinatally HIV-infected infants were identified. Thirty (79%) HIV-infected infants accessed 6-week testing and initiated antiretroviral therapy at a median age of 16.0 weeks, but only 14 were in care a median of 68 weeks later and 4 had died. Eight (21%) HIV-infected infants, 2 of whom died, escaped identification by routine testing. Their mothers were younger, more likely to be foreign and accessed less optimal antenatal care.Six-week testing delayed antiretroviral therapy initiation beyond the time of early HIV-related infant mortality and missed one-fifth of perinatally HIV-infected infants. Earlier diagnosis and improved retention in care are required to reduce infant mortality and accurately measure elimination of mother-to-child transmission.