Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-free RT assay

Abstract

Background: Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test.

Objectives: We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form 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 viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC₅₀ (50% inhibitory concentration) values were determined by application of the median effect equation.

Results: Assays from nine post-nevirapine therapy isolates gave 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-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.

Conclusions: This technique appears to offer a simple, rapid method for determining the resistance of HIV-1 isolates to nevirapine, ddI and possibly other RT-inhibiting drugs. The method is not useful for identifying resistance to ZDV.