We evaluated subsequent virologic outcomes in individuals experiencing low-level virem ia (LLV) on dolutegravir (DTG)-based first-line antiretroviral therapy (ART) in Botswana. We used a national dataset from 50,742 adults who initiated on DTG-based first-line ART from June 2016–December 2022. Individuals with at least two viral load (VL) measurements post three months on DTG-based first-line ART were evaluated for first and subsequent episodes of LLV (VL:51–999 copies/mL). LLV was sub-categorized as low-LLV (51–200 copies/mL), medium-LLV (201–400 copies/mL) and high-LLV (401–999 copies/mL). The study outcome was virologic failure (VF) (VL ≥ 1000 copies/mL): virologic non-suppression defined as single-VF and confirmed-VF defined as two-consecutive VF measurements after an initial VL < 1000 copies/mL. Cox regression analysis identified predictive factors of subsequent VF. The prevalence of LLV was only statistically different at timepoints >6–12 (2.8%) and >12–24 (3.9%) (p-value < 0.01). LLV was strongly associated with both virologic non-suppression (adjusted hazards ratio [aHR] = 2.6; 95% CI: 2.2–3.3, p-value ≤ 0.001) and confirmed VF (aHR = 2.5; 95% CI: 2.4–2.7, p-value ≤ 0.001) compared to initially virally suppressed PLWH. High-LLV (HR = 3.3; 95% CI: 2.9–3.6) and persistent-LLV (HR = 6.6; 95% CI: 4.9–8.9) were associated with an increased hazard for virologic non-suppression than low-LLV and a single-LLV episode, respectively. In a national cohort of PLWH on DTG-based first-line ART, LLV > 400 copies/mL and persistent-LLV had a stronger association with VF. Frequent VL testing and adherence support are warranted for individuals with VL > 50 copies/mL.

HIV is known to accumulate escape mutations in the gag gene in response to the immune response from cytotoxic T lymphocytes (CTLs). These mutations can occur within an individual as well as at a population level. The population of Botswana exhibits a high prevalence of HLA*B57 and HLA*B58, which are associated with effective immune control of HIV. In this retrospective cross-sectional investigation, HIV-1 gag gene sequences were analyzed from recently infected participants across two time periods which were 10 years apart: the early time point (ETP) and late time point (LTP). The prevalence of CTL escape mutations was relatively similar between the two time points—ETP (10.6%) and LTP (9.7%). The P17 protein had the most mutations (9.4%) out of the 36 mutations that were identified. Three mutations (A83T, K18R, Y79H) in P17 and T190A in P24 were unique to the ETP sequences at a prevalence of 2.4%, 4.9%, 7.3%, and 5%, respectively. Mutations unique to the LTP sequences were all in the P24 protein, including T190V (3%), E177D (6%), R264K (3%), G248D (1%), and M228L (11%). Mutation K331R was statistically higher in the ETP (10%) compared to the LTP (1%) sequences (p < 0.01), while H219Q was higher in the LTP (21%) compared to the ETP (5%) (p < 0.01). Phylogenetically, the gag sequences clustered dependently on the time points. We observed a slower adaptation of HIV-1C to CTL immune pressure at a population level in Botswana. These insights into the genetic diversity and sequence clustering of HIV-1C can aid in the design of future vaccine strategies.