Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics

Abstract

Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2. 0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.

FIG. 1

Linear range for the COBAS AMPLICOR (A) and AMPLICOR HCV MONITOR (B) v2.0 tests. Twenty to 48 replicate tests were performed for each input viral RNA concentration. For expected RNA concentrations below 1,600 IU/mL, some of the replicates gave negative results and were excluded from the data analysis. At each expected RNA concentration, the mean of the log₁₀ measured RNA concentrations (circles) and the standard deviations (error bars) were determined. The closed circles, representing measured RNA concentrations between 500 and 500,000 IU/ml, show the results for concentrations used for linear regression analysis. The open circles show results for concentrations that were not included in the regression analysis. The regression analysis was performed using the averaged result for each expected RNA concentration.

FIG. 2

Comparison of COBAS AMPLICOR HCV MONITOR test, v2.0, and AMPLICOR HCV MONITOR test (microwell plate [mwp]), v2.0, values for HCV RNA concentration in 51 clinical specimens of genotypes 1a (□), 1b (▵), 2 (◊), 3 (×), 4 (○), 5a (∗), and 6a (+). For each specimen, the difference between the values reported by the COBAS AMPLICOR format and the AMPLICOR HCV (MWP) format is plotted against the average of the two values.

FIG. 3

Precision profile of COBAS AMPLICOR and AMPLICOR HCV MONITOR v2.0 tests. For the COBAS AMPLICOR format, each of the six sets of values plotted represents results from 40 replicates for each of the six samples tested. For the AMPLICOR format, each of the 16 sets of values plotted represents results from 20 replicates for each of the 16 samples tested. The lower portion of the graph shows the CV (■), and the upper portion of the graph shows the mean (○), the minimum and maximum values obtained (×), and the 95% confidence interval (mean ± 2 standard deviations [−]) for a single measurement.