Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 to 5

Abstract

The key parameter for diagnosis and management of hepatitis C virus (HCV) infection is HCV RNA. Standardization of HCV RNA assays to IU is mainly based on genotype 1 panels. Little is known about the variability of commercially available HCV RNA assays for quantification of different genotypes. Two real-time reverse transcription (RT)-PCR assays (COBAS TaqMan HCV Test for use with the High-Pure System [HPS/CTM] and COBAS Ampliprep/COBAS TaqMan HCV Test [CAP/CTM]), one standard RT-PCR assay (COBAS Amplicor HCV Monitor 2.0 [CAM]), and one signal amplification assay (Versant Quantitative 3.0 [branched DNA [bDNA]]) were compared for quantification of genotypes 1 to 5 (n = 108). Using CAM as a reference assay for genotype 1-infected patients, the mean interassay differences compared with CAP/CTM, HPS/CTM, and bDNA were 0.16, -0.13, and -0.48 log(10) IU/ml HCV RNA, respectively. Comparison of CAM with CAP/CTM, HPS/CTM, and bDNA for the remaining genotypes showed the following results, respectively: 2a/c, -0.24, -0.78, and -0.49; 2b, -0.21, -0.18, and -0.64; 3a, 0.13, -1.04, and -0.55; 4, -0.52, -1.51, and -0.05; and 5, -0.28, -1.00, and -0.24 log IU/ml HCV RNA. A correct decision for treatment discontinuation in genotype 1 patients at week 12 was possible only when the same assay was used at baseline and week 12. Comparison of CAM with the CAP/CTM assay showed equal quantifications of genotype 1, 2, 3, and 5 samples, while genotype 4 samples were slightly underestimated. For the HPS/CTM assay, a significant underestimation of the HCV RNA concentrations of genotypes 2a/c, 3, 4, and 5 was observed. For the bDNA assay, a constant lower quantification of genotypes 1 to 3 was detected.

FIG. 1.

Correlation of HCV RNA concentrations of clinical samples between the CAM and the CAP/CTM assays. Results are shown separately for HCV genotype 1a/b, 2a/c, 2b, 3a, 4, and 5 samples. In addition to the single HCV RNA concentrations, the identity line and the regression line (boldface) are shown.

FIG. 2.

Correlation of HCV RNA concentrations of clinical samples between the CAM and the HPS/CTM assays. Results are shown separately for HCV genotype 1a/b, 2a/c, 2b, 3a, 4, and 5 samples. In addition to the single HCV RNA concentrations, the identity line and the regression line (boldface) are shown.

FIG. 3.

Correlation of HCV RNA concentrations of clinical samples between the CAM and the bDNA assays. Results are shown separately for HCV genotype 1a/b, 2a/c, 2b, 3a, 4, and 5 samples. In addition to the single HCV RNA concentrations, the identity line and the regression line (boldface) are shown.