Background: Understanding the selection and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral therapy.
Objective: To develop a high-throughput, real-time reverse transcriptase (RT) polymerase chain reaction (PCR) assay to quantify non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants K103N (AAT or AAC alleles) at frequencies as low as 0.1%, and to apply this to monitor these variants before, during, and after NNRTI therapy.
Methods: HIV-1 RNA in longitudinal plasma samples obtained from patients starting and stopping NNRTI therapy was converted to cDNA and the target sequence region amplified and quantified by real-time PCR. Approximately 10 copies/reaction provided a template for a second round of PCR using primers that discriminated between the mutant and wild-type alleles. Amplification specificity was confirmed by thermal denaturation analysis.
Results: Frequencies of 103N similar to assay background (0.029%) were observed in longitudinal samples from 9 of 12 treatment-naive patients; three patients had transient increases in 103N frequency to a range of 0.21-0.48%, which was 7-16.5 times assay background. Analysis of longitudinal plasma samples from six NNRTI-experienced patients showed three patterns: persistence of 103N variants after stopping NNRTI therapy, codon switching of 103N between AAC and AAT during NNRTI therapy, and decay of 103N variants to below assay background after cessation of NNRTI therapy.
Conclusions: Allele-specific RT-PCR quantified the emergence and decay of drug-resistant variants in patients over a broad range of frequencies (0.1-100%). The rate of decay of K103N variants after stopping NNRTI therapy was highly variable.