Abstract:
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.
