A major risk in scaling-up antiretroviral therapy (ART) is the selection and transmission of HIV drug resistance (HIVDR) variants due to poor monitoring and surveillance systems in place (Damond and Descamps, 1998; Grant et al., 2002). As HIVDR represents a major threat in ensuring treatment success (Roquebert et al., 2009), implementation of HIVDR surveillance and testing in clinical practice should be recommended to support thelong-term effectiveness of ART, especially in sub-Saharan Africa (SSA) where therapeutic options are limited, antiretrovirals with poor genetic to resistance are widely used, and several viral clades have been identified (Roquebert et al., 2009). Unlike in the western world, standard methods (i.e. Sanger-sequencing) for detecting drug resistance mutations (DRMs) are not yet routinely implemented in several resource-limited settings (RLS) like in SSA, due to several pitfalls: sequencing is financially unaffordable, its procedure is cumbersome, it requires high-level staff for processing/interpretation, and the maintenance/procurement systems are not available locally (Larder et al., 1991, Brun-Vézinet et al., 2004, Grant et al., 2003). Thus, implementing simple and affordable approaches for HIVDR testing, such as point mutation assays (PMA), might be convenient for RLS scaling-up ART.

Among PMAs, Allele-Specific Polymerase Chain Reaction (ASPCR) is one of the promising techniques for implementation in RLS. Of note, ASPCR was first reported on HIV clinical isolates for detecting DRMs down to≥ 1% of minority variants (Larder et al., 1991). In addition to the aforementioned pitfalls, standard sequencing methods only detects variants present at levels ≥ 20% of the overall viral population(Brun-Vézinet et al., 2004; Grant et al., 2003; Halvas et al., 2006; Palmer et al., 2005). With the advent of Real-Time PCR, the diagnostic performance of ASPCR has been improved for possible use in clinical practice (Halvas et al., 2006, Metzner et al., 2005, Metzner et al., 2003, Palmer et al., 2005).

In September 2017 a breakthrough pricing agreement was reached to provide generic dolutegravir for HIV treatment in low- and middle-income countries (LMICs). Combined with tenofovir disoproxil fumarate and lamivudine, this single pill, fixed-dose combination will cost approximately USD 75 per person per year and is likely to be cost effective when compared to current non-nucleoside reverse transcriptase inhibitor (NNRTI) first-line regimens (Jienchi et al. Lancet HIV. 2018 Jul). So, in March 2019, the Chadian Ministry of Health approved the induction of dolutegravir (DTG) based regimens as per WHO recommendations in their settings where the first packs of dolutegravir arrived in 2020 (Abderrazzack et al., 2021). But in clinical practice, the impact of minority DRMs (i.e. variants present at levels below 20% of the overall viral population) of human immunodeficiency virus type 1 (HIV-1) could influence the virological response to treatment based on non-nucleoside reverse transcriptase inhibitors (NNRTIs) not rilpivirine in which data on minority rilpivirine resistant variants are scarce.(Raymond S et al., 2018). In this context, ASPCR might be useful for detecting HIVDR to first-line ART in RLS consisting mainly of NNRTI-based regimens (Eshleman et al., 2001, Jackson et al., 2000). However, HIVDR testing using ASPCR has not been substantially ascertained on non-B viral populations, especially within the SSA context (Gutsche et al., 2004, Lecossier et al., 2005).

Following the “proof of principle” of PMA, the footprint in a successful implementation of ASPCR relies on knowledge of emerging DRMs (Grant et al., 2002). In several SSA countries, NRTI and NNRTI are the most commonly used antiretrovirals. Regarding NRTIs, cytidine analogues are found in almost all drug combinations and select for M184V mutation, thus causing high-level resistance to lamivudine (3TC) and emtricitabine (FTC) while decreasing the viral replicative fitness (Wei et al., 2002, Wei et al., 2003). Thymidine analogue mutations (TAMs) are class of variants selected by two NRTIs, zidovudine (AZT) and stavudine (d4T), and subcategorised as either TAMs-1 (M41L, L210W and T215Y) or TAMs-2 (D67N, K70R, T215F and K219Q/E),each having distinct effects on resistance to AZT and D4T (Bennett et al., 2005, Yeni, 2008). Furthermore, TAMs might also cause cross-resistance to other NRTIs (abacavir [ABC], didanosine [ddI], tenofovir [TDF]), especially when selected cumulatively (Calvez et al., 2002, Johnson et al., 2010, Kellam et al., 1992, Adult ACTG Protocol 306 370 Teams, 2004, Larder and Kemp, 1989, Whitcomb et al., 2003). Regarding NNRTIs, commonly selected mutations are K103N and Y181C causing high-level resistance tonevirapine (NVP) and efavirenz (EFV), drugs with low genetic barrier to resistance and present in most first-line ART regimens within SSA countries (Hirsch et al., 2008, Richman et al., 1994, Torti et al., 2001, Yang et al., 2004).

Considering the most used ARV drugs, major DRMs and viral clades found within the Chadian national ART program (Adawaye et al., 2017), we sought to ascertain the performance of ASPCR in detecting HIVDR mutations targeting NNRTIs (K103N, Y181C) and NRTIs (K70R, M184V, T215Y/F) across diverse non-B HIV-1 clades circulating in Chad.