ULTRASENSITIVE REVERSE TRANSCRIPTION PCR ASSAY FOR QUANTITATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA IN PLASMA

With the recent introduction of combination therapy, human immunodefic iency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the;AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Syste ms). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Tes t, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. Thi s ''ultrasensitive'' method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml, A precision study yielded av erage within-run and between-run coefficients of variation (CV) of 24. 8 and 9.6%, respectively. A multicenter reproducibility study demonstr ated that the laboratory-to-laboratory reproducibility of this assay i s good, with an average CV of 32%. The linear range of this test is be tween 50 and 50,000 copies/ml of plasma. RNA concentrations measured b y the ultrasensitive and standard HIV-I MONITOR tests exhibited good a greement within the shared linear range of the two methods. The two me asurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being onl y slightly lower (median, 22% lower), Preliminary studies suggest that this assay will prove to be useful for predicting the stability of vi ral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretraviral therapy.