Emergence and Long-Term persistence of Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Variants In Patients

学友会セミナー

2006年開催　学友会セミナー

開催日時：	平成１８年７月２４日（月）　１７：３０～１８：３０
開催場所：	病院棟8階会議室
講　　師：	Dr. Sarah E. Palmer, Ph.D.
所　　属：	HIV Drug Resistance Program, National Cancer Institute, Frederick Maryland
演　　題：	Emergence and Long-Term persistence of Non-Nucleoside Reverse Transcriptase Inhibitor-Resistant Variants In Patients
概　　要：	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 <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/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 and Conclusions: Serial samples from treatment-experienced patients and from mothers who had received single-dose nevirapine revealed three patterns of NNRTI-resistance: persistence of 103N variants after stopping NNRTI treatment; 103N codon switching (between AAC and AAT), and the apparent loss of the 103N mutation. 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.
世 話 人：	○岩本　愛吉、河岡　義裕