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Objectives: Mother-to-child transmission (MTCT) of HIV can be minimized using elimination of MTCT (eMTCT) services. This study aimed to determine the proportion of infants who tested positive for HIV despite receiving eMTCT services in a rural setting in Northern Uganda., Methods: We retrospectively reviewed the early infant diagnosis register for the year January 2019 through June 2021 to collect data on participants enrolled in eMTCT services at Lalogi Health Centre IV in Omoro district, Uganda. Breakthrough HIV infection was defined as a positive HIV RNA on a dried blood sample at 18 months in a patient who received eMTCT services as recommended by national guidelines., Results: A total of 118 infants were enrolled in the study, 64 (54.2%) of whom were female. Most of the participants (n = 111, 94.1%) were on nevirapine prophylaxis for at least 6 weeks, 115 (97.5%) were exclusively breastfed, two (1.7%) were on complementary feeding, and one (0.8%) was not breastfed. Only five (4.2%) infants were lost to follow-up, four (3.4%) had incomplete data, and three (2.5%) had breakthrough HIV infections (positive HIV RNA and HIV antibody tests). All three cases of breakthrough HIV infection (one male and two female infants) were born to mothers who were diagnosed with HIV at delivery and were on nevirapine prophylaxis for less than 6 weeks., Conclusion: Our findings indicate that while eMTCT services were largely successful in minimizing vertical transmission of HIV in the rural setting in Northern Uganda, there were still some cases of breakthrough HIV infection associated with non-adherence to nevirapine prophylaxis and delayed maternal HIV diagnosis. Therefore, adhering to the national guidelines on nevirapine prophylaxis for at least 6 weeks for children born to mothers with HIV is recommended to further reduce the risk of vertical transmission of HIV., Competing Interests: The authors have no competing interests to declare., (© 2023 The Author(s).)