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To evaluate a phenotype assay based on plasma reverse transcriptase (RT) to assess HIV susceptibility to nonnucleoside RT inhibitors (NNRTIs). To compare RT-based phenotype with recombinant virus assay (RVA) phenotype- and genotype-based analysis. To assess group O and HIV-2 susceptibility to NNRTIs in correlation with genotype polymorphisms.RT activity was quantified and its susceptibility to efavirenz, nevirapine, and delavirdine measured as drug concentration resulting in 50% inhibition. RT phenotype was compared with genotype analysis. Eighteen plasma samples from 14 group M- and culture supernatants from 4 group M-, 9 group O-, and 7 HIV-2-infected patients were investigated. RT-based and RVA-based phenotypes were compared for identical plasma from 9 group M-infected patients.RT-based and RVA-based phenotypes were in complete agreement. RT-based phenotype- and genotype-predicted susceptibility were concordant for all but 1 group M samples. One plasma showed susceptibility to 3 NNRTIs by phenotypes, despite the presence of 101E and 106I/V residues. The HIV-2 RTs were totally resistant to the NNRTIs tested. Among HIV-1 group O, 6 were totally resistant to NNRTIs independently of the presence of the 181C mutation and 3 were susceptible to some NNRTIs.Plasma RT-based phenotype could be useful as a simple alternative for monitoring resistance to NNRTIs. This assay is suitable for highly divergent strains. It would be particularly useful for large epidemiologic survey of the natural HIV polymorphism and the potential impact in emergence of drug resistance, particularly to nevirapine, widely used to prevent mother-to-child transmission.