ABSTRACT: BACKGROUND:Initiation of antiretroviral therapy (ART) following diagnosis of HIV infection at birth is an emerging area of paediatric HIV care. We present outcomes of HIV-infected infants identified at birth at Rahima Moosa Mother and Child Hospital in Johannesburg, South Africa. METHODS:From September, 2013 (era 1), only high-risk HIV-exposed infants were offered diagnostic HIV PCR tests at birth. From June, 2014 (era 2), all HIV-exposed infants were offered laboratory-based diagnostic PCR tests. From October, 2014 (era 3), point of care (POC) diagnostic PCR tests were also done if staff availability allowed. We describe time to ART initiation, mortality, retention in care, and viral suppression among the HIV-infected infants identified across these eras. FINDINGS:We tested 5449 HIV-exposed infants who were born between Sept 1, 2013, and June 30, 2016. 88 neonates with confirmed HIV infection were identified and included in the study, of which 86 (98%) started ART. Median age at ART initiation decreased from 9 days (IQR 6-25) in eras 1 and 2 to 2 days (1-8) in era 3. In era 3, more neonates who were co-tested with POC testing started ART within 48 h of birth (29 [83%] of 35; median 1 day [IQR 1-2]) than infants who were not co-tested (one [4%] of 29; median 6 days [5-10]). The probability of mortality by 12 months across the eras was 14% (95% CI 8-24) and did not differ by era. Of the 72 infants who survived and initiated ART at the site, 56 (78%) were retained at 12 months. Of the 56 infants retained in care, 40 (71%) had a viral load less than 400 copies per mL at 12 months, with no differences between eras (p=0·23). INTERPRETATION:HIV-infected infants can be identified at birth and ART can be initiated within hours to days. Although most infants in our cohort started ART, mortality remained unacceptably high with suboptimal retention and viral suppression. Reducing mortality and improving retention and viral suppression remain urgent priorities. FUNDING:Eunice Kennedy Shriver National Institute of Child Health and Human Development/National Institute of Allergy and Infectious Disease, National Institutes of Health, USAID/PEPfAR, and the South African National HIV Programme.