Rapid screening of phenotypic resistance to nevirapine by direct analysis of HIV type 1 reverse transcriptase activity in plasma

Drug susceptibility testing for the clinical management of human immunodefi ciency virus type 1 (HIV-1)-infected persons is often curtailed because suc h testing is expensive and time consuming. We describe a nonculture-based p henotypic assay for the rapid analysis of HIV-1 resistance to nevirapine, T he assay measures the susceptibility of plasma reverse transcriptase (RT) a ctivity to inhibition by nevirapine by using the PCR-based Amp-RT assay, As say validation was made using two reference wild-type (WT) and six other ne virapine-resistant (>100-fold) HIV-1 isolates. Amp-RT IC50 values were foun d to correlate with those obtained by a conventional replication-based assa y. The results also indicated that 50 mu M nevirapine can be used in a sing le screening test to detect nevirapine resistance. Analysis of virus mixtur es showed a detection threshold of 10% of nevirapine-resistant HIV-1 in a b ackground of WT virus. To evaluate the assay on clinical samples, 30 plasma specimens collected longitudinally from ii patients before and after treat ment with nevirapine were analyzed, and results were compared with codon 18 1 genotypes, Pretreatment samples and those obtained during the first 6 day s of therapy (n = 21) were sensitive to nevirapine, and none had detectable Y181C mutation. Phenotypic resistance was seen in eight samples obtained a fter 1 week of treatment and was correlated with detection of the Y181C mut ation, An increase in the level of phenotypic resistance was seen over time . These data validate this rapid and simple assay for monitoring phenotypic resistance to nevirapine.