In Uganda, where subtypes A and D predominate, the cBED assay estimated an incidence rate of 6.1% and 6.0% in Masaka and Kakira districts respectively. However, prospective incidence rates in these same areas the previous year were 1.7% and 1.4% suggesting an overestimation of recent infection and misclassification by the assay. In high HIV prevalence rural South Africa, where subtype C predominates, the cBED assay was shown to more accurately classify patients if locally measured long-term false positive ratio are used but may underestimate incidence when used with FPR from other settings. FPR is a correction method to account for non-recently infected who are misclassified as recently infected. It is based on the assumption that after infection, there is finite time progression to cBED threshold except in non progressors. The fraction of HIV infected individuals who have been infected beyond the cBED threshold is the long-term FPR. A prospective study conducted in Zimbabwe using specimens from pregnant women with known dates of seroconversion, recommended a cut off of 187 days instead of the 155 days recommended by the cBED kit manufacturer. Studies from South Africa, Uganda and Zambia have used the 155 day cut off value. In the current study, there was a modest decrease in the estimate of recent infection, using the 187 day ZVITAMBO study cut off. Thus to distinguish probable RI from LTI on a intracellular colocalized population basis, it would appear that either of the two cut-offs may be used. There were several limitations encountered in this study. The prevalence of drug resistance mutations in the study population was very low, and thus there was no ability to estimate the frequency of transmitted drug resistance. However, the focus on young, largely primigravida pregnant women was expected to identify recent infections. The younger age and significantly higher CD4 cell numbers among those estimated to have recent infection provide some evidence for the veracity of the BED assays. Only a few of the women reported that they or their partners had been exposed to ART. Of the few women who were exposed to ART for pMTCT of HIV, one of them misclassified by the cBED assay as RI, had a drug resistant mutation. We were not able to obtain direct access to partners of these young women, men who were on average 8 years older than their wives or partners who may have been the source of transmitted infection. As estimated by the BED assay and estimated dates of conception, young pregnant women are at very high risk of acquiring infection around conception and early pregnancy. The high percentage of women infected during pregnancy shows the urgent need for more prevention education in young women, their spouses and partners. There was a low sequencing rate for the sample in this study, which may have been due to storage and shipping conditions. The prevalence of PDR in Chitungwiza, four years after the commencement of the national ART program, is still far below the WHO threshold limit of 5%. To preserve the low prevalence of PDR, proper prescribing practices of ARV drugs, adherence to ART education should be maintained. Patients should be monitored to identify those failing therapy and provided alternative therapies, condom promotion and adherence counseling to prevent the spread of drug resistant viruses.