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Background HIV drug resistance (HIV-DR) is rising in sub-Saharan Africa in both ART-naive and ART-experienced patients. Objectives To estimate the level of acquired DR (ADR) and pre-treatment DR (PDR) across selected urban and rural sites in Southern Africa, in Mozambique. Methods We conducted two cross-sectional surveys among adult HIV patients (October 2017–18) assessing ADR and PDR. In the (ADR) survey, those on NNRTI-based first-line ART for ≥6 months were recruited (three sites). In the PDR survey, those ART-naive or experienced with ≥3 months of treatment interruption prior were enrolled (eight sites). Results Among 1113 ADR survey participants 83% were receiving tenofovir (TDF)/lamivudine (3TC)/efavirenz (EFV). The median time on ART was 4.5 years (Maputo) and 3.2 years (Tete), 8.3% (95% CI 6.2%-10.6%, Maputo) and 15.5% (Tete) had a VL ≥ 1000 copies/mL, among whom 66% and 76.4% had NNRTI+NRTI resistance, and 52.8% and 66.7% had 3TC+TDF-DR. Among those on TDF regimens, 31.1% (Maputo) and 42.2% (Tete) were still TDF susceptible, whereas 24.4% and 11.5% had TDF+zidovudine (ZDV)-DR. Among those on ZDV regimens, 25% and 54.5% had TDF+ZDV-DR. The PDR survey included 735 participants: NNRTI-PDR was 16.8% (12.0–22.6) (Maputo) and 31.2% (26.2–36.6) (Tete), with a higher proportion (≥50%) among those previously on ART affected by PDR. Conclusions In Mozambique, viral failure was driven by NNRTI and NRTI resistance, with NRTI DR affecting backbone options. NNRTI-PDR levels surpassed the WHO 10% ‘alert’ threshold. Replacing NNRTI first-line drugs is urgent, as is frequent viral load monitoring and resistance surveillance. Changing NRTI backbones when switching to second-line regimens may need reconsideration.

High level of HIV drug resistance informs dolutegravir roll-out and optimized NRTI backbone strategy in Mozambique