Evaluation of the Abbott investigational use only RealTime hepatitis C virus (HCV) assay and comparison to the Roche TaqMan HCV analyte-specific reagent assay

Abstract

The accurate and sensitive measurement of hepatitis C virus (HCV) RNA is essential for the clinical management and treatment of infected patients and as a research tool for studying the biology of HCV infection. We evaluated the linearity, reproducibility, precision, limit of detection, and concordance of viral genotype quantitation of the Abbott investigational use only RealTime HCV (RealTime) assay using the Abbott m2000 platform and compared the results to those of the Roche TaqMan Analyte-Specific Reagent (TaqMan) and Bayer Versant HCV bDNA 3.0 assay. Comparison of 216 samples analyzed by RealTime and TaqMan assays produced the following Deming regression equation: RealTime = 0.940 (TaqMan) + 0.175 log(10) HCV RNA IU/ml. The average difference between the assays was 0.143 log(10) RNA IU/ml and was consistent across RealTime's dynamic range of nearly 7 log(10) HCV RNA IU/ml. There was no significant difference between genotypes among these samples. The limit of detection using eight replicates of the World Health Organization HCV standard was determined to be 7.74 HCV RNA IU/ml by probit analysis. Replicate measurements of commercial genotype panels were significantly higher than TaqMan measurements for most samples and showed that the RealTime assay is able to detect all genotypes with no bias. Additionally, we showed that the amplicon generated by the widely used Roche COBAS Amplicor Hepatitis C Virus Test, version 2.0, can act as a template in the RealTime assay, but potential cross-contamination could be mitigated by treatment with uracil-N-glycosylase. In conclusion, the RealTime assay accurately measured HCV viral loads over a broad dynamic range, with no significant genotype bias.

FIG. 1.

Bland-Altman analysis. The correlation data were analyzed by the method of Bland and Altman (1) by comparing the average log₁₀ HCV RNA IU/ml for the two methods versus the difference in log₁₀ HCV RNA IU/ml of the two methods. The genotypes are indicated where known. The average difference (0.143 log₁₀ HCV RNA IU/ml), average plus 2 standard deviations ([SD] 1.059 log₁₀ HCV RNA IU/ml), and average minus 2 standard deviations (−0.773 log₁₀ HCV RNA IU/ml) are indicated by the heavy solid lines.

FIG. 2.

Genotype panels. Tenfold dilutions of HemaCare Bioscience's HCV genotype panels HCVGTP-004c and HCVGTP-005a containing a total of 15 samples representing genotypes 1a, 1b, 2, 3a, 4, 5a, 6a, 6b, 7c, 8c, and 9b were tested (n = 3, except for TaqMan 1b, where n = 2). Samples with genotypes of 2, 3a, 6a, and 7c are represented twice in these panels. The mean ± standard deviation is shown. The pairs indicated by asterisks are not significantly different (P > 0.05). The remaining samples showed significantly different values between the methods.

FIG. 3.

Cross-contamination and effect of UNG. PCR product produced by the Amplicor assay was preliminarily quantitated by agarose gel electrophoresis, diluted in Basematrix, and then extracted and amplified in the RealTime assay without UNG or with UNG and incubated at room temperature (RT) or 44°C for 10 min prior to thermocycling. The mean ± standard deviation is shown (n = 3, except for the samples without UNG at an input of 5 log₁₀ HCV RNA IU/ml, where n = 2). The fractions in the chart indicate the number of samples that were detected but not quantitated out of the total number of replicates tested at the given input level.