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Background: Approximately 15-20 million people worldwide are infected with hepatitis delta virus (HDV), which is approximately 5 % of people with chronic hepatitis B virus (HBV). Sub-Saharan Africa has high HDV prevalence, leading to worse clinical outcomes among people who are HIV/HBV/HDV tri-infected. There are limited data on HDV prevalence among people with HIV (PWH) who are HBV-infected and uninfected in Botswana. We, therefore, determined HDV prevalence among PWH in Botswana., Methods: This was a retrospective cross-sectional study utilizing archived plasma samples from PWH with results for HBV markers such as hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), immunoglobulin M antibody to hepatitis B core antigen (IgM anti-HBc) and hepatitis B e antigen (HBeAg). Samples were categorized according to their HBsAg status and screened for anti-HDV antibodies. Total nucleic acid was extracted from samples with a single positive anti-HDV result, and HDV ribonucleic acid (RNA) load was quantified using the Altona Diagnostic RealStar® HDV RT-PCR kit. Statistical analysis was performed using STATA version 14.0 where p-values < 0.05 were considered statistically significant., Results: The study cohort (n = 478) included both HBsAg positive (44 %) and negative (56 %) participants, with a median age of 42 [IQR; 41-43]. Anti-HDV prevalence of (15/211) [7.1 %, 95 % CI: 4.4 - 11.4] was recorded among HBsAg positive participants, all of whom were IgM anti-HBc negative, while 5/6 participants were HBeAg negative. HDV RNA load was detected in 11/12 (92 %) anti-HDV-positive participants. No HDV prevalence was recorded among participants who were HBsAg negative, therefore, the overall HDV prevalence was (15/478) [3.1 %, 95 % CI: 1.9 - 5.1]. HIV viral load suppression was statistically insignificant, irrespective of HDV status., Conclusions: We report high HDV prevalence among HBsAg-positive PWH in Botswana. Most HDV-positive participants had active HDV infection, therefore, we recommend HDV screening in this cohort to guide their clinical care., Competing Interests: Declaration of Competing Interest We have no conflict of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)