11th Conference on Retroviruses and Opportunistic Infections San Francisco, California - February 8 - 11, 2004

Conf Retroviruses Opportunistic Infect. 2004 Feb 8-11;11th:Abstract No. 37

S Palmer*¹, V Boltz¹, F Maldarelli¹, E Halvas², J Mican³, J Mellors², and J Coffin^1
1HIV Drug Resistance Prgm., NCI, NIH, DHHS, Frederick, MD, USA; ²Univ. of Pittsburgh, PA, USA; and ³Lab. of Immunoregulation, NIAID, NIH, DHHS, Bethesda, MD, USA

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.

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