Performance of an in-house multiplex PCR assay for HIV-1 drug resistance testing – A cheaper alternative

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.