Worldwide Evaluation of DNA Sequencing Approaches for Identification of Drug Resistance Mutations in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Abstract

A panel (ENVA-1) of well-defined blinded samples containing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was analyzed by automated DNA sequencing in 23 laboratories worldwide. Drug resistance mutations at codons 41, 215, and 184 were present in the panel samples at different ratios to the wild type. The presence of mutant genotypes was determined qualitatively and quantitatively. All laboratories reported the presence of sequence heterogeneities at codons 41, 215, and 184 in one or more of the panel samples, though not all reported the correct codon genotypes. Two laboratories reported a mutant genotype in samples containing only the wild type, whereas two and three laboratories failed to detect the mutant genotypes at codons 41 and 215, respectively, in a completely mutant DNA population. Mutations present at relative concentrations of 25% of the total DNA population were successfully identified by 13 of 23, 10 of 23, and 16 of 23 labs for codons 41, 215, and 184Val, respectively. For more than 80% of those laboratories that qualitatively detected the presence of a mutation correctly, the estimated wild type/mutant ratio was less than 25% different from the input ratio in those samples containing 25 to 50% or 75% mutant input. This first multicenter study on the quality of DNA sequencing approaches for identifying HIV-1 drug resistance mutations revealed large interlaboratory differences in the quality of the results. The application of these procedures in their current state would in several cases lead to inaccurate or even incorrect diagnostic results. Therefore, proper quality control and standardization are urgently needed.

Deep sequencing analysis of HIV‐1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE

“…Population-based sequencing (PS) is commonly used in clinical practice to determine the presence of reverse transcriptase (RT) resistance-asso ciated mutations (RAMs). This technology has limitations in its ability to detect variants which represent <25.0% of the viral quasispecies [Schuurman et al, 1999], also referred to as "minority variants." Several deep sequencing (DS) technologies have been developed in recent years claiming to be capable of detecting minority variants that exist at frequencies as low as 0.01-3% of the total viral population [Paredes et al, 2007;Simen et al, 2009].…”

Capacity building and predictors of success for HIV‐1 drug resistance testing in the Asia‐Pacific region and Africa

“…EQA programmes should aim to assess the HIVDR laboratory testing process using clinically relevant sample types. The predominant use of plasma samples in TAQAS enabled assessment of detection of viral mixtures, important in DRM detection [6,8,20,32], and useful in the assessment of inter-laboratory testing variation [33]. The use of a sample type with inherent variability like plasma, in contrast to clones or plasmids, mandated the use of a TG rather than a sequence derived by a reference laboratory [10,25,30].…”

“…Both tools can improve utility of future EQA programmes. The importance of continuous EQA participation to maintain and improve HIVDR genotyping outcome has been validated [8,25,30]. Recent reports provide novel methods to standardize the interpretation of electropherograms for HIVDR testing [38,39].…”

“…Whether using commercial or in-house HIVDR testing, laboratory participation in external quality assessment (EQA) is recommended by expert committees [3Á7]. Available HIVDR EQA programmes include a centralized laboratory certification approach [4], a ''collective'' approach, with no personal communication between evaluator and evaluatees [8,9] and ''within network'' and ''within country'' approaches, where providers liaise with participants to improve test outcome [10Á14].…”

Detection of residual human immunodeficiency virus type 1 reverse transcriptase K103N minority species in plasma RNA and peripheral blood mononuclear cell DNA following discontinuation of non-nucleoside therapy

“…Routine genotypic resistance testing is performed through bulk sequencing of RT-PCR-amplified HIV-1 RNA obtained from plasma. Although effective population sequencing methods have been approved for in vitro diagnostic use, this technology has inherently limited sensitivity, allowing detection of the most prevalent virus species but not of minority species accounting for <25% of the whole population [2].…”