HIV‐1 viral load determination based on reverse transcriptase activity recovered from human plasma

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Abstract
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. J. Med. Virol. 71:347–359, 2003.

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