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Background  We assessed the integration of early infant HIV diagnosis with the expanded programme for immunization in a rural Zambian setting with the aim of determining whether infant and postpartum maternal HIV testing rates would increase without harming immunization uptake.    Methods  In an unblinded, location stratified, cluster randomised controlled trial, 60 facilities in Zambia’s Southern Province were equally allocated to a control group, Simple Intervention group that received a sensitization meeting and the resupply of HIV testing commodities in the event of a stock-out, and a Comprehensive Intervention group that received the Simple Intervention as well as on-site operational support to facilitate the integration of HIV testing services with EPI.    Findings  The average change in number of first dose diphtheria, pertussis, and tetanus vaccine (DPT1) provided per month, per facility was approximately 0.86 doses higher [90% confidence interval (CI) -1.40, 3.12] in Comprehensive Intervention facilities compared to the combined average change in the Simple Intervention and control facilities. The interventions resulted in a 16.6% [90% CI: -7%, 46%, P-value = 0.26] and 10% [90% CI: -10%, 36%, P-value = 0.43] greater change in average monthly infant DBS testing compared to control for the Simple and Comprehensive facilities respectively. We also found 15.76 (90% CI: 7.12, 24.41, P-value < 0.01) and 10.93 (90% CI: 1.52, 20.33, P-value = 0.06) additional total maternal re-tests over baseline for the Simple and Comprehensive Facilities respectively.    Conclusions  This study provides strong evidence to support Zambia’s policy of integration of HIV testing and EPI services. Actions in line with the interventions, including HIV testing material supply reinforcement, can increase HIV testing rates without harming immunization uptake. In response, Zambia’s Ministry of Health issued a memo to remind health facilities to provide HIV testing at under-five clinics and to include under-five HIV testing as part of district performance assessments.    Trial Registration  ClinicalTrials.gov Registration Number:  NCT02479659

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A Cluster Randomised Trial on the Impact of Integrating Early Infant HIV Diagnosis with the Expanded Programme on Immunization on Immunization and HIV Testing Rates in Rural Health Facilities in Southern Zambia