Background:  Understanding the emergence and decay of drug-resistant HIV-1 variants is important for designing optimal antiretroviral treatment strategies. Standard genotyping methods do not reliably detect minor variants comprising less than 25% of the virus population and are not quantitative. We have therefore developed a high-throughput real-time RT-PCR assay that quantifies the NNRTI-resistant variants K103N (AAT or AAC alleles) and Y181C (TGT) at frequencies down to less than 0.1%, and applied it to study the appearance and disappearance of these variants before, during, and after NNRTI therapy.

Methods:  Longitudinal plasma samples were obtained from patients before and during NNRTI therapy and after its cessation. HIV-1 RNA was converted to cDNA and the target sequence region was amplified and quantified by real-time PCR. About 10⁷ copies per reaction were used as template for a second round of real-time PCR, using primers that discriminate between the mutant and wildtype alleles. To confirm amplification specificity, PCR products were subjected to thermal denaturation analysis.

Results:  In the first patient studied, the frequency of 103N increased from a pretherapy baseline of 0.03% to more than 90% following initiation and failure of an efavirenz (EFV)-containing regimen. After discontinuing EFV, the 103N variant persisted at a frequency of 7% for at least 2.5 years. In a second patient discontinuing EFV after failure, the 103N variant persisted at 82% for 5 years. In a third patient, the frequency of 103N rose from the baseline value of 0.085% to around 40% during 6-months of therapy with a nevirapine (NVP)-containing regimen. Despite exposure to NVP the frequency of 181C did not increase above the pretherapy level of less than 0.03%. The frequency of 103N showed little change during 2 additional years of treatment with an EFV-containing regimen. Both the AAC and AAT codons for 103N were present at equal frequency during NVP exposure, but the AAT codon declined to 2% during EFV exposure and the AAC codon increased to approximately 40%.

Conclusions:  Using our allele-specific assay, we could quantify the emergence of NNRTI-resistant variants and their persistence for years after discontinuing NNRTI therapy. These findings demonstrate the value of allele-specific RT-PCR for detecting and quantifying drug-resistant variants in patients on or off antiretroviral therapy.

Keywords: resistance; persistence; variants