Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds

Abstract

Methods for the absolute quantitation of nucleic acids present in small amounts in biological samples (competitive PCR and competitive reverse transcription PCR) were applied to the direct monitoring of specific anti-human immunodeficiency virus type 1 (HIV-1) therapy. With these techniques, different parameters of HIV-1 activity (including genomic RNA copy numbers in plasma, proviral and late transcript copy numbers in peripheral blood lymphocytes, and mean transcriptional activity per each HIV-1 provirus) were monitored during therapy with azidothymidine or ddI. In most of these treated patients, a direct response to the antiretroviral compounds employed was detected during the first few weeks of treatment, as documented by a fast decrease of all molecular indexes of HIV-1 activity. However, residual viral replication (albeit at minimal levels) was documented during therapy in all subjects monitored in this study. In a minority of the patients under study (3 of 12), the drug-dependent viral inhibition was maintained throughout the observation time (213 to 791 days), but in 9 patients a rebound in viremia level was detected during therapy with competitive reverse transcription PCR. Sequencing analysis of a portion of the HIV-1 gene pol from cell-free virions showed that circulating viral variants bearing at least two mutations compatible with azidothymidine or ddI resistance were detectable in the patients who exhibited a rebound in cell-free HIV-1 genomic RNA copy numbers in plasma but not in one patient who maintained (for 455 days) lowered levels of viral load during ddI treatment.

Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HIV monitor, and QUANTIPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma

“…A relationship between HIV-1 RNA levels in plasma and clinical progression has been found by these assays (12,30,33,35). Moreover, several investigators reported a decrease in plasma RNA levels in patients starting antiretroviral therapies, suggesting that this viral surrogate marker may be useful for predicting the clinical response to therapy, although it is not yet known how the results obtained by these assays correlate with the clinical outcome (3,7,11,13,17,22,24,25,40).…”

Quantitative Molecular Analysis of Virus Expression and Replication

“…A major consequence of this new scenario will be the acute need for reliable parameters to evaluate the efficacy of therapies in real time and to monitor them (in some cases for months or years). Theoretical studies (57,97) and early experimental evidence (4,7,11,32,39,45,59,67,95) have indicated unambiguously that most quantitative molecular methods are able to provide information on changes in systemic viral activity and that they are thus suitable for following up infected patients treated with antivirals. While there is no doubt of the usefulness of these methods in evaluating the efficacy of any antiviral treatment in vivo, several new questions have been raised.…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…No matter what controls are used to generate quantitative data or monitor successful amplification, it is imperative to accurately determine their concentration and to ensure that internal controls are added at suitable levels in order to prevent extreme competition for reagents with the wild-type template (Zimmermann and Manhalter, 1996;Brightwell et al, 1998). A spectrometer alone is inadequate for quantifying a control molecule; however, in combination with experimental and statistical analysis, the reliability of the data is greatly enhanced (Glasel, 1995;Bagnarelli et al, 1995;Wang and Spadoro, 1998;Rodrigo et al, 1997;Taswell, 1981;Sykes et al, 1998). Finally, one must remember that the results of quantitation using a control need to be expressed relative to a suitable biological marker, e.g.…”