Fortuin, Tumelo L.Nkone, PaballoGlass, Allison J.Viana, RaquelMoeng, KeitumetseLoubser, ShayneTiemessen, Caroline T.Mayaphi, Simnikiwe Horatious2025-01-292025-01-292024-12Fortuin, T.L., Nkone, P., Glass, A. et al. 2024, 'Performance of an in-house multiplex PCR assay for HIV-1 drug resistance testing – a cheaper alternative', Journal of Virological Methods, vol. 330, no. 115034, pp. 1-9. https://DOI.org/10.1016/j.jviromet.2024.115034.0166-0934 (print)1879-0984 (online)10.1016/j.jviromet.2024.115034http://hdl.handle.net/2263/100380DATA AVAILABILITY : All data generated or analysed during this study are included in this manuscript.BACKGROUND : Currently, most HIV drug resistance PCR assays amplify the protease-reverse transcriptase (PR-RT) fragment separately from the integrase (IN) fragment. The aim of this study was to develop a multiplex PCR assay that simultaneously amplifies PR-RT and IN fragments for HIV-1 drug-resistance testing. METHODS : The in-house multiplex PCR assay was evaluated on extracted total nucleic acids obtained from the National Health Laboratory Service (NHLS) and Lancet laboratories. Sanger sequencing was performed on amplicons, and HIV-1 drug-resistance mutations (DRMs) were assessed using HIV Stanford drug resistance database. RESULTS : This study tested 59 patient samples with known HIV-1 viral load and DRM results; 41 from Lancet and 18 from NHLS. In-house multiplex PCR assay detected one or both fragments in most samples but had higher sensitivity for detection of IN fragment (93.2 %) compared to PR-RT fragment (83.1 %). There was 100 % concordance between Lancet assay versus in-house assay sequence data for IN DRMs, but lower concordance with PR-RT (87.0 %). The in-house multiplex PCR assay’s precision and reproducibility analysis showed ≥99.9 % sequence similarity and yielded similar DRM results for both PR-RT and IN fragments. CONCLUSIONS : The in-house multiplex PCR assay demonstrated satisfactory performance and higher sensitivity for IN fragment amplification. This could be a cost-effective method for HIV-1 drug resistance testing as both PR-RT and IN fragments are successfully amplified in one reaction in most samples.en© 2024 The Author(s). This is an open access article under the CC BY-NC-ND license.In-house multiplex PCRHIV-1 drug resistance testingProtease-reverse transcriptase PCR fragmentIntegrase PCR fragmentARV drug resistance mutationsPolymerase chain reaction (PCR)Antiretroviral (ARV)SDG-03: Good health and well-beingArticle