Background HIV drug resistance (HIVDR) remains a major threat to achieving sustainable viral suppression on antiretroviral therapy (ART). In South Africa, dolutegravir (DTG) is the preferred first-line ART backbone since its rollout in December 2019.

Methods We curated HIVDR genotypic data obtained from the National Health Laboratory Service (NHLS) for ART-experienced patients with virological failure (i.e., consecutive viral loads ≥1,000 copies/mL) receiving HIV-care at public-sector health facilities in KwaZulu-Natal (KZN) province, South Africa. We estimated levels of HIVDR from genotypes processed between January 2018 and June 2022, and assessed temporal trends of HIVDR across 11 districts of KZN, prior to- and following DTG-rollout in South Africa.

Results Of 4,069 genotypes curated, 3,511 (86.3% CI 85.2–87.3) had HIVDR mutations, with most resistance mutations occurring among adult females aged >15 years, p=0.01. Despite an annual decrease in protease inhibitor (PI)-specific mutations (p=0.0001), about one-third of genotypes had ≥3 drug-class resistance mutations, mainly nucleoside, and non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations. Only 50 genotypes had integrase resistance data, from which 9 (18.0%) had intermediate to high-levels of resistance to DTG. Overall, rural districts had fewer HIVDR genotypes (598/4069, 15%) but with higher HIVDR prevalence (88.1% CI 85.3–90.6) compared to densely populated peri-urban and urban districts.

Conclusion Six in every seven genotypes from patients with virologic failure had HIVDR mutations despite DTG-rollout, with persistent NNRTI resistance. Thus, whilst introduction of DTG is expected to alleviate HIVDR burden, a sub-population of people may not fully benefit from DTG-use due to multi-drug resistance, at which point PI-based ART is warranted. Higher proportions of HIVDR in rural districts and among adult women, highlight regions and individuals needing priority HIV care. Overall, these findings urge strengthening of HIV services in public healthcare systems to ensure sustainable DTG-use in first-line and subsequent ART regimens.