Performance characteristics of the QUANTIPLEX HIV-1 RNA 3.0 assay for detection and quantitation of human immunodeficiency virus type 1 RNA in plasma

Abstract

The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log(10) estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log(10) in most comparisons. Interlaboratory differences across runs were </=0.10 log(10) at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log(10) HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had >/=50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml; P = 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.

FIG. 1

Box and whiskers plots of log₁₀ estimated HIV-1 RNA concentration at nominal HIV-1 RNA concentrations up to 100 HIV-1 RNA copies/ml after recalculation of censored observations by using the standard curve for the bDNA 3.0 assay. Boxes extend from the 25th percentile (lower edge) to the 75th percentile (upper edge) HIV-1 RNA levels. The horizontal lines in the middle of the boxes indicate median HIV-1 RNA values. Whiskers are the vertical lines that end in brackets above and below the boxes. The horizontal lines beyond the brackets represent outliers (more than 1.5 times the interquartile range below the 25th percentile or above the 75th percentile). The brackets represent the largest and smallest observed values that are not outliers.

FIG. 2

Median difference between log₁₀ estimated HIV-1 RNA concentrations and log₁₀ nominal HIV-1 concentrations from the bDNA 3.0 assay plotted against log₁₀ nominal HIV-1 RNA concentrations. Censored observations were set at the censoring point of the assay (50 or 500,000 HIV-1 RNA copies/ml).

FIG. 3

Intra-assay standard deviation of log₁₀ estimated HIV-1 RNA concentrations versus log₁₀ nominal HIV-1 RNA concentrations.

FIG. 4

Regressions of log₁₀ estimated HIV-1 RNA concentrations on log₁₀ nominal HIV-1 RNA concentrations for the bDNA 3.0 assay, using nominal concentrations of 100 to 500 HIV-1 RNA copies/ml.

FIG. 5

Regressions of log₁₀ estimated HIV-1 RNA concentrations on log₁₀ nominal HIV-1 RNA concentrations for the US-RT-PCR assay, using nominal concentrations of 100 to 500 HIV-1 RNA copies/ml.

FIG. 6

Regressions of log₁₀ estimated HIV-1 RNA concentrations on log₁₀ nominal HIV-1 RNA concentrations for data combined across runs within each laboratory.