DETERMINATION OF HIV-1 SUSCEPTIBILITY TO REVERSE-TRANSCRIPTASE (RT) INHIBITORS BY A QUANTITATIVE CELL-FREE RT ASSAY

Background: Mutations in the human immunodeficiency virus type 1 (HIV- 1) reverse transcriptase (RT) gene confer resistance to antiviral drug s acting on RT. Current methods employed to detect such resistance req uire time-consuming culture techniques during which selective pressure s may affect the outcome of the test. Objectives: We sought to determi ne; whether drug-susceptible and drug-resistant HIV-1 derived from cli nical specimens could be distinguished by the effects of the active fo rm of the drug on the enzyme activity in a quantitative, cell-free RT assay. Study design: Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed vir al extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory conc entration) values were determined by application of the median effect equation. Results: Assays from nine post-nevirapine therapy isolates g ave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sens itive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays. Con clusions: This technique appears to offer a simple, rapid method for d etermining the resistance of HIV-1 isolates to nevirapine, ddI and pos sibly other RT-inhibiting drugs. The method is not useful for identify ing resistance to ZDV. (C) 1996 Elsevier Science B.V.