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Ontlametse T Bareng,1,2 Wonderful T Choga,1,2 Segomotso T Maphorisa,3 Sekgabo Seselamarumo,1 Kaelo K Seatla,1 Patrick T Mokgethi,1,4 Dorcas Maruapula,1,4 Mompati L Mogwele,1 Doreen Ditshwanelo,1,5 Natasha O Moraka,1 Irene Gobe,2 Modisa S Motswaledi,2 Joseph M Makhema,1,6 Rosemary Musonda,1 Roger Shapiro,1,6 Max Essex,1,6 Vlad Novitsky,1 Sikhulile Moyo,1,2,6 Simani Gaseitsiwe1,6 1Botswana Harvard AIDS Institute Partnership, Gaborone, Botswana; 2School of Allied Health Professions, Faculty of Health Sciences, University of Botswana, Gaborone, Botswana; 3Ministry of Health and Wellness, Republic of Botswana, Gaborone, Botswana; 4Department of Biological Sciences, Faculty of Science, University of Botswana, Gaborone, Botswana; 5Department of Biological Science and Biotechnology, Botswana International University of Science and Technology, Palapye, Botswana; 6Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USACorrespondence: Simani Gaseitsiwe, Botswana Harvard AIDS Institute Partnership, Private Bag BO320, Bontleng, Gaborone, Botswana, Tel +267 390 2671, Fax +267 390 1284, Email sgaseitsiwe@bhp.org.bwPurpose: Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401– 999 copies/mL) due to the use of a threshold of VL ≥ 1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV.Methods: We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401– 999 copies/mL, and 46 had ≥ 1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test.Results: The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥ 1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI- and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI- and NRTI-associated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥ 1000 copies/mL.Conclusion: The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.Keywords: in-house genotyping, low-level viremia, samples, HIV-1C drug resistance testing