Differences between two real-time PCR-based hepatitis C virus (HCV) assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and one signal amplification assay (Versant HCV RNA 3.0) for RNA detection and quantification

Abstract

Hepatitis C virus (HCV) RNA detection and quantification are the key diagnostic tools for the management of hepatitis C. Commercially available HCV RNA assays are calibrated to the HCV genotype 1 (gt1)-based WHO standard. Significant differences between assays have been reported. However, it is unknown which assay matches the WHO standard best, and little is known about the sensitivity and linear quantification of the assays for non-gt1 specimens. Two real-time reverse transcriptase PCR-based assays (RealTime HCV and Cobas Ampliprep/Cobas TaqMan HCV [CAP/CTM]) and one signal amplification-based assay (the Versant HCV RNA, version 3.0, branched DNA [bDNA] assay) were compared for their abilities to quantify HCV RNA in clinical specimens (n = 65) harboring HCV isolates of gt1 to g5. The mean differences in the amounts detected by RealTime HCV in comparison to those detected by the bDNA assay and CAP/CTM were -0.02 and 0.72 log(10) IU/ml HCV RNA, respectively, for gt1; -0.22 and 0.03 log(10) IU/ml HCV RNA, respectively, for gt2; -0.27 and -0.22 log(10) IU/ml HCV RNA, respectively, for gt3; -0.19 and -1.27 log(10) IU/ml HCV RNA, respectively, for gt4; and -0.03 and 0.09 log(10) IU/ml HCV RNA, respectively, for gt5. The lower limits of detection for RealTime HCV and CAP/CTM were 16.8 and 10.3 IU/ml, respectively, for the WHO standard and in the range of 4.7 to 9.0 and 3.4 to 44.4 IU/ml, respectively, for clinical specimens harboring gt1 to gt6. Direct comparison of the two assays with samples of the WHO standard (code 96/798) with high titers yielded slightly smaller amounts by RealTime HCV (-0.2 log(10) at 1,500 IU/ml and -0.3 log(10) at 25,000 IU/ml) and larger amounts by CAP/CTM (0.3 log(10) at 1,500 IU/ml and 0.2 log(10) at 25,000 IU/ml). Finally, all three tests were linear between 4.0 x 10(3) and 1.0 x 10(6) IU/ml (correlation coefficient, >or=0.99). In conclusion, the real-time PCR based assays sensitively detected all genotypes and showed comparable linearities for the quantification of HCV RNA, with the exception of gt1 and gt4. The previously reported differences in the absolute quantification of samples harboring gt1 were confirmed and may be explained by different calibrations to the WHO standard.

FIG. 1.

Correlation of the results for clinical specimens (n = 65) from patients with chronic HCV infection, as assessed by direct comparison of the results of RealTime HCV versus those of the bDNA assay (A), the results of RealTime HCV versus those of CAP/CTM (B), and the results of CAP/CTM versus those of the bDNA assay (C). The different genotypes (GTs) are represented by different symbols.

FIG. 2.

Bland-Altman analysis of genotype-specific mean differences in HCV RNA quantification by RealTime HCV versus that by the bDNA assay. Due to the relatively low number of samples harboring genotypes 4 and 5, Bland-Altman analysis was performed only for genotypes 1 (A), 2 (B), and 3 (C). The bold lines represent the mean differences for the samples, the thin lines represent the 95% limits of agreement, and the dashed lines are the reference lines.

FIG. 3.

Bland-Altman analysis of genotype-specific mean differences in HCV RNA quantification by RealTime HCV versus that by CAP/CTM. Due to the relatively low number of samples harboring genotypes 4 and 5, Bland-Altman analysis was performed only for genotypes 1 (A), 2 (B), and 3 (C). The bold lines represent the mean differences for the samples, the thin lines represent the 95% limits of agreement, and the dashed lines are the reference lines.

FIG. 4.

Bland-Altman analysis of genotype-specific mean differences in HCV RNA quantification by CAP/CTM versus that the bDNA assay. Due to the relatively low number of samples harboring genotypes 4 and 5, Bland-Altman analysis was performed only for genotypes 1 (A), 2 (B), and 3 (C). The bold lines represent the mean differences for the samples, the thin lines represent the 95% limits of agreement, and the dashed lines are the reference lines.

FIG. 5.

Linearities of RealTime HCV (A), the bDNA assay (B), and CAP/CTM (C), as assessed with five threefold serial dilutions of clinical specimens representing genotypes (GTs) 1 to 5. The expected and the observed HCV RNA concentrations are shown on the basis of known HCV RNA concentrations, as assessed by RealTime HCV.