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Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Published on: September 26, 2011
Quantitative PCR for the measurement of circulating proviral load in HIV-infected individuals
B Conway1, D S Ko, D W Cameron
1Department of Medicine, Ottawa General Hospital, University of Ottawa, University of Ottawa AIDS Research Group, Ottawa, Ont, K1H8L6, Canada.
Background:
PCR has been applied extensively as a tool for the detection of HIV. We have previously developed a semi-quantitative microtiter plate assay for the specific detection of HIV PCR products.
Objective:
To develop and evaluate a novel quantitative assay for the measurement of circulating proviral load in HIV-infected individuals.
Study Design:
We evaluated 70 consecutive, unselected HIV-infected patients, divided into 3 groups, according to their CD4 cell count: greater than 500 cells/microl (10 subjects); 200-500 cells/microl (31 subjects); less than 200 cells/microl (29 subjects). Peripheral blood mononuclear cell lysates were amplified, and a portion of the product was added to streptavidin-coated wells with a biotinylated capture probe and a horseradish peroxidase-linked reporter probe, complementary to separate regions of the amplified product. Following incubation, readings were taken with an automated plate reader. Products were quantitated by interpolation into a standard curve of serial dilutions of an HIV-containing plasmid, included in each assay.
Results:
HIV sequences were detected in all 70 clinical samples. Within each patient category, a wide range of proviral loads were observed. However, proviral load/10(6) CD4 cells was associated with disease progression when the patient groups were considered (up to 9.6% infected cells in subjects with CD4 cell counts below 200/microl).
Conclusions:
This quantitative PCR assay will allow the measurement of proviral load in clinical samples. It may be useful in the managment of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy.
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