Clinical evaluation of an in-house reverse transcription-competitive PCR for quantitation of human immunodeficiency virus type 1 RNA in plasma

Abstract

An in-house reverse transcription (RT)-competitive PCR (RT-cPCR) for the quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma samples was developed and validated. The procedure involves (i) extraction of RNA with spin columns, (ii) ready-to-use bead-mediated RT, (iii) competitive PCR in a microtiter plate, (iv) agarose gel electrophoresis of the reaction products, and (v) densitometric analysis of the digitized image of the gel. Quadruplicate tests and dilution studies showed that the sensitivity and intertest coefficient of variability of the RT-cPCR are comparable to those of the reference AMPLICOR HIV-1 MONITOR test. The results obtained by the two assays with a panel of 45 clinical samples were in good agreement (mean difference, 0.36 +/- 0.25 log units). Analysis of 1,982 clinical samples by the in-house RT-cPCR yielded the typical range of plasma HIV-1 RNA levels with the expected inverse correlation between CD4 counts and HIV-1 RNA titers. In addition, testing of plasma from 36 subjects at weeks 0 and 4 with respect to the time of initiation of protease inhibitor therapy detected a significant decrease in HIV-1 viremia. The mean reduction in the HIV-1 RNA level was 0.914 log unit for those receiving saquinavir (P = 0.0210), 1.584 log units for those receiving indinavir (P = 0.0047), and 1.904 log units for those receiving ritonavir (P < 0.0001). The in-house RT-cPCR assay is simple to develop and perform and allows quantitation of HIV-1 RNA in 100 to 200 samples per operator per week. Since the cost is 1/8 to 1/10 of those of reference commercial assays, this procedure could be conveniently used in medium-scale laboratories.

FIG. 1

Relative location of HIV-1-specific primers on the HIV-1 pol gene (thick lines). Sense and antisense HIV-1 DNA strands are labeled (+) and (−), respectively. Note that since the sequence of the 5′ tail of primer T52 is identical to that of primer LR52, PCR products obtained with primer pair LR52-LR62 and with primer pair T52-LR62 differ in length but are delimited by the same sequences at the 5′ end (LR52) and the 3′ end (LR62). Primers, target DNA, and PCR products are not drawn to scale.

FIG. 2

Quantitation of a wt HIV-1 RNA standard by RT-cPCR. HIV-1 RNA (5,000 nominal copies) was reverse transcribed with primer LR62. The reaction mixture was split into four aliquots and subjected to amplification with primers LR52 and LR62 in the presence of 40, 240, 1,440, and 8,640 RNA equivalents (eq.) of the competitor (comp) T52-LR62 DNA. The wt target copy number was estimated by plotting the log ratio between wt and competitor intensities (intens) (after correction for the difference in base pairs) versus the log number of competitor target copies. The result for this control experiment is 1,375 × 4 = 5,500 copies.

FIG. 3

Representative quantitative analysis of plasma HIV-1 RNA by the in-house RT-cPCR assay. Competitive amplification products obtained from six clinical samples were loaded as subsequent three-sample series on the same gel with a 10-min delay. Each cDNA was amplified in the presence of 40, 240, 1,440, and 8,640 competitor RNA equivalents. wt and comp, the amplification products generated from wt and competitor templates, respectively. Data analysis for the samples indicated the following numbers of HIV-1 RNA copies per milliliter: 100,166 for sample 1, 138,086 for sample 2, 4,300 for sample 3, 546,999 for sample 4, 30,567 for sample 5, and 33,703 for sample 6.

FIG. 4

Scatter plot showing the correlation between HIV-1 RNA levels and CD4 counts in the population whose plasma samples were analyzed by the in-house RT-cPCR assay. Samples with undetectable HIV-1 RNA levels (<400 copies/ml) are shown as containing 200 copies/ml.

FIG. 5

Effect of saquinavir, ritonavir, and indinavir therapy on HIV-1 RNA load in three groups of 12 patients as measured by the in-house RT-cPCR assay. Each line represents a different subject. Weeks refer to the time from the initiation of protease inhibitor therapy. For graphical representation and statistical analysis, samples with <400 HIV-1 RNA copies/ml are considered to contain 200 copies/ml.