HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma.
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.
Malmsten
Anders
A
Department of Genetics and Pathology, Uppsala University, and Cavidi Tech AB, Uppsala Science Park, SE-751 83 Uppsala, Sweden. anders@cavidi.se
Shao
Xing-Wu
XW
Aperia
Kajsa
K
Corrigan
Gary E
GE
Sandström
Eric
E
Källander
Clas F R
CF
Leitner
Thomas
T
Gronowitz
J Simon
JS
D000818:Animals; D019380:Anti-HIV Agents / Q000493:pharmacokinetics; D002415:Cats; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D015658:HIV Infections / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D016089:Immunodeficiency Virus, Feline / Q000502:physiology; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D019562:Viral Load; D014771:Virion / Q000378:metabolism
D019380:Anti-HIV Agents; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D004228:Dithionitrobenzoic Acid; D054303:HIV Reverse Transcriptase