We describe a procedure (ExaVir Load) to carry out human immunodeficiency virus-1 (HIV-1) viral load testing using reverse transcriptase (RT) recovered from HIV-1 virions in plasma. Samples from individuals infected with HIV-1 were treated with a sulphydryl-reactive agent to inactivate endogenous polymerases. Virions were then immobilised on a gel and washed in individual mini columns to remove RT-inhibiting antibodies, antiviral drugs, and other RT inhibitors. Immobilised virions were lysed finally, and the viral RT eluted. The amount of RT recovered was quantified by a sensitive RT activity assay using either colorimetry or fluorimetry to detect DNA produced by RT. The "RT load" values of 390 samples from 302 HIV-1 patients living in Sweden were compared to results obtained with an HIV-1 RNA viral load assay. The correlation between the two tests was r = 0.90, P < 0.0001. Four of 202 samples from healthy blood donors gave low positive values in the RT test. All samples in a panel with 10 HIV-1 subtypes were positive by the RT load. The RT load test provides a technically less demanding and cost-effective alternative to methods based on nucleic acid amplification. Being insensitive to genetic drift occurring in HIV, the assay should be of particular use in resource-limited settings, where different subtypes and recombinant HIV strains occur.
Copyright 2003 Wiley-Liss, Inc.