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 nonnucleoside 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 the 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 (≥1000 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: 2.19 to 6.33) and was associated with a 10-fold higher rate of virologic failure (hazard ratio: 10.39; 95% confidence interval: 3.23 to 32.41; P < 0.001) compared with 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 with frequencies ≥2% (22% vs. 63%; P < 0.001).

Conclusions: Detection of TDR by a point mutation assay may prevent the use of suboptimal ART.

Figure 1

Trial Profile

Figure 2

Kaplan-Meier curve of time to virologic failure between those with presumed transmitted drug resistance (TDR) detected in codons M184V, K103N, Y181C, and/or G190A by oligonucleotide ligation assay (OLA) (dashed line) and those without TDR by OLA (solid line). Note: The number of participants at risk at each time point is listed and the number experiencing virologic failure is in parentheses.

Figure 3

Drug resistance mutations detected by OLA, pyrosequencing and consensus sequencing at baseline, and by OLA and consensus sequencing at virologic failure. Note: Pre-ART specimens were tested by OLA (O), pyrosequencing (P), and consensus sequencing (CS); virologic failure (VF) specimens were tested by OLA and CS only. Numbers indicate the % mutant virus quantified by OLA (bold font, limit of detection = 2%) or pyrosequencing (regular font, limit of detection 0.1%). Dark grey indicates mutation detected by all methods evaluated. Light grey indicates mutation not detected by consensus sequencing, except as indicated for subject #264. Numbers with no shading indicate mutations detected by pyrosequencing only. NT = not tested (due to undetectable HIV viral load). ind = indeterminate result by OLA due to the presence of different mutation. ^ = mutation detected by OLA and consensus sequencing but not by pyrosequencing due to sequence polymorphisms in the region of the pyrosequencing PCR primers.

Figure 4

Kaplan-Meier curve of time to virologic failure in participants grouped by whether or not M184V, K103N, Y181C, and/or G190A was detected in baseline (pre-ART) viral population, and if detected, the proportion of the mutant population: 0% (solid line), >0% to <2% (short dashed line), and ≥2% (long dash line). Note: The number of participants at risk at each time point is listed and the number experiencing virologic failure is in parentheses.