Background: The role of minor drug-resistant variants in anti-retroviral treatment failure is uncertain. To address this question, methods must be developed and validated for their ability to identify minor drug-resistant variants. Therefore, we evaluated the ability of several different methods to detect and quantify a non-nucleoside RT inhibitor (NNRTI)-resistant variant of HIV-1.

Methods: Infectious plasmid clones of HIV-1_LAI, either wild-type or encoding the NNRTI resistance mutation K103N (AAA to AAC), were used to generate a panel of virus mixtures in seronegative human plasma with the % mutant (103N) being 0, 0.01, 0.1, 0.4, 1, 2, 5, 10, 25, 50, or 100%. The mixtures were blinded and distributed to 13 labs that responded to a solicitation of interest. Methods used to test the panel were: 1) allele-specific real-time RT PCR (ASRT, 3 labs); 2) a Ty1/HIV-1RT yeast hybrid assay (Ty1, 1 lab); 3) RT-PCR with allele-specific hybridization (LiPA assay [Bayer Diagnostics], 1 lab); 4) oligonucleotide ligase-based assays (OLA, 2 labs); 5) a limiting dilution RT PCR assay (LD PCR, 1 lab); 6) RT PCR with pyrosequencing (Pyroseq, 1 lab); and 7) RT PCR with direct sequencing of bulk PCR product (Applied Biosystems ViroSeq v2.0, 3 labs; Visible Genetics HIV-1 Truegene, 1 lab).

Results: ASRT assays developed by 3 different laboratories quantified 103N down to a frequency of 0.1, 0.4, or 25%, respectively. The Ty1 system quantified 103N to 0.4%, with direct confirmation of the mutant allele by DNA sequencing of individual yeast clones. LD PCR and Pyroseq assays detected 103N at 2% frequency but were not quantitative below 10%. The OLA and RT PCR/population sequencing assays quantified 103N to 5%–10%. The LiPA detected 103N at 2% frequency but was not quantitative.

Conclusions: Blind testing of a panel of plasmid-derived virus mixtures showed that 2 of 3 ASRT assays and the Ty1 assay could detect and quantify the 103N variant at frequencies < 1%. A potential advantage of Ty1 assay over ASRT is that the mutant allele is cloned and its presence can be confirmed by DNA sequencing. The other methods evaluated had detection and quantification limits ranging from 2%–10% mutant. These findings suggest that several technologies are available to address the role of minor drug-resistant variant in antiretroviral treatment failure.