Biological variation and quality control of plasma human immunodeficiency virus type 1 RNA quantitation by reverse transcriptase polymerase chain reaction

Abstract

Quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in the plasma of seropositive individuals was performed by using an external control assay with techniques to standardize and control each measurement. Rigorous study of the variability of the assay showed that the median intraassay reproducibility was log1o 0.15 RNA copies per ml of plasma, while the median interassay reproducibility on replicate plasma samples was log1o 0.25 copies per ml. Specimen stability studies showed reproducible recovery of RNA from plasma stored at-70°C for up to 12 months. In clinically stable patients who were either untreated or taking zidovudine, the average week-to-week variation in plasma RNA levels, measured in real time, was log1o 0.30 RNA copies per ml. In contrast, patients either initiating or changing antiretroviral therapy showed a fall of loglo 0.8 to log1o 2.0 copies per ml in plasma RNA levels. Overall, 105 of 110 (96%) HIV-1-seropositive individuals with CD4 counts of 36 to 868 cells per mm3 had quantifiable HIV-1 RNA over a range of log1o 2.70 to loglo 6.23 RNA copies per ml, including 81% (13 of 16) of the individuals with greater than 500 CD4 cells per mm3. Accurate and reproducible quantitation of plasma viremia in real time by reverse transcriptase polymerase chain reaction, particularly in asymptomatic HIV-1-infected individuals with high CD4 counts, provides a basis for the use of this virologic measure to monitor the short-and long-term effects of early intervention therapeutic strategies on viral burden.

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).…”

Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals: direct assessment of resistance to reverse transcriptase inhibitors in vivo

“…The viral DNA extraction procedures used in these studies have been previously described (23,29). Viral RNA extraction was conducted, as previously described, with minor modifications (2,4,11,25). Briefly, half of an aliquot of the pelleted bead-virion complex was mixed with 400 l of solution D (6.3 M guanidine thiocyanate, 42 mM sodium citrate [pH 4.0], 0.83% N-lauroyl sarcosine, 0.2 mM ␤-mercaptoethanol).…”

Optimization of Specimen-Handling Procedures for Accurate Quantitation of Levels of Human Immunodeficiency Virus RNA in Plasma by Reverse Transcriptase PCR

“…PCR has limitations as a quantitative assay, however, including decreased efficiency at higher initial copy numbers (5) and inhibition by heparin present in blood samples (1). The accuracy of quantitative HIV-1 RNA measurements in plasma specimens can be affected by both the choice of anticoagulant and specimen-handling procedures (13,19,21). In order to determine the effects of specimen collection and handling procedures on quantitation of plasma HIV-1 RNA, we compared anticoagulants and sample processing times with quantitative RT-PCR.…”