Oligonucleotide Ligation Assay Detects HIV Drug Resistance Associated With Virologic Failure Among Antiretroviral-Naive Adults in Kenya

Abstract

Background Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at time of ART initiation. Methods Pre-ART plasmas from ART-naive Kenyans initiating an NNRTI-based fixed-dose combination ART in a randomized adherence trial conducted in 2006 were retrospectively analyzed by OLA for mutations conferring resistance to NNRTI (K103N, Y181C, and G190A) and lamivudine (M184V). Post-ART plasmas were analyzed for virologic failure (≥1,000 copies/mL) at 6 month intervals over 18-month follow-up. Pre-ART plasmas of those with virologic failure were evaluated for drug resistance by consensus and 454-pyrosequencing. Results Among 386 participants, TDR was detected by OLA in 3.89% [95% Confidence Interval (CI), 2.19-6.33], and was associated with a 10-fold higher rate of virologic failure [Hazard Ratio (HR), 10.39; 95% CI, 3.23-32.41; p<0.001) compared to those without TDR. OLA detected 24 TDR mutations (K103N, n=13; Y181C, n=5; G190A, n=3; M184V, n=3) in 15 subjects (NNRTI, n=15; 3TC, n=3). Among 51 participants who developed virologic failure, consensus sequencing did not detect additional TDR mutations conferring high-level resistance, and pyrosequencing only detected additional mutations at frequencies <2%. Mutant frequencies <2% at ART initiation were significantly less likely to be found at the time of virologic failure compared to frequencies ≥2% (22% vs. 63%; p<0.001). Conclusions Detection of TDR by a point mutation assay may prevent use of sub-optimal ART.

HIV-1 drug resistance and resistance testing

“…It was then re-enforced in subsequent studies in which initial therapy was selected without the availability of genotypic resistance testing but for which the presence of resistance mutations prior to initiation of therapy was detected retrospectively through the analysis of cryopreserved blood samples (Borroto-Esoda et al, 2007; Chaix et al, 2007; Chung et al, 2014; Hamers et al, 2012; Kuritzkes et al, 2008; Lee et al, 2014; Little et al, 2002; Wittkop et al, 2011). …”

Increasing HIV-1 pretreatment drug resistance among antiretroviral-naïve adults initiating treatment between 2006 and 2014 in Nairobi, Kenya

“…Sociodemographic information, medical records, and blood samples were analyzed, including baseline pre-treatment plasma (2006) and peripheral blood mononuclear cells ( PBMC ) (2014). Following quantification of amplifiable copies of HIV cDNA and DNA by realtime PCR [4], ≥100 HIV templates from each participant underwent nested PCR of HIV pol to generate amplicons for OLA. OLA examined point mutations at K103N, Y181C, G190A, and M184V across all specimens [5].…”

“…OLA examined point mutations at K103N, Y181C, G190A, and M184V across all specimens [5]. K65R was evaluated by OLA in 2014 and pyrosequencing was performed on enrollment plasma from those with subsequent virologic treatment failure in 2006 [4]. The proportion of mutant in each subject’s HIV population was quantified by comparing optical densities to standards containing 0%, 2%, 5%, 10%, 25%, 50%, 75% or 100% mutant, with PDR defined by ≥2% mutant [4].…”

“…In 2006, 24 PDR mutations were detected in 15 participants at baseline (Table 1) [4]. Fifteen of the participants had mutations only to NNRTI (K103N, Y181C, G190A), and three had mutations to NNRTI plus lamivudine (M184V).…”

“…The study only surveyed drug resistant mutations at codons K103N, Y181C, G190A, M184V, and K65R, which has the potential to underestimate, not overestimate the prevalence of PDR. Testing PDR in PBMC by OLA and defining resistance at ≥2% detects mutant frequencies below those discernable by consensus sequencing [11]; however, low frequency mutations are associated with greater virologic failure and therefore remain clinically relevant [4]. Despite recruitment at one clinic, several sociodemographic differences existed between the cohorts and likely reflected Kenya’s economic development.…”