The clinical utility of HCV RNA quantification is well established (7
). Indeed, viral load monitoring before, during, and after antiviral therapy is crucial for the management of hepatitis C. Among other aspects, HCV RNA assays ideally should be sensitive, offer precise and reproducible quantification results, and be reliable across all the different HCV genotypes.
The lack of standardization among HCV quantification assays has been overcome, in part, by the development of an IU standard (22
). However, the results differ significantly between assays, despite the standardization to IU (5
), and it remains unknown which assay calibration best matches the standard. In addition, relative quantification results and LODs for different genotypes may vary among assays, since standardization to IU and the calibration of assay sensitivity are based on genotype 1a (22
In this study, we evaluated and compared the performance characteristics of two quantitative real-time reverse transcription-PCR-based assays (RealTime HCV and CAP/CTM) and one signal amplification-based assay (the bDNA assay).
The quantification of undiluted clinical specimens by RealTime HCV in comparison to that by the bDNA assay displayed a high correlation and good agreement among all genotypes tested. Mean differences were below ±0.3 log10 IU/ml.
Comparison of RealTime HCV and CAP/CTM showed a good correlation only for samples harboring genotypes 2, 3, and 5. The mean difference between RealTime HCV and CAP/CTM for specimens harboring genotype 1 was 0.72 log10 IU/ml; i.e., the quantification results obtained by the CAP/CTM assay were found to be higher than those obtained by RealTime HCV. In contrast, the quantification results for samples harboring genotype 4 obtained by the CAP/CTM assay were lower than those obtained by RealTime HCV (−1.27 log10 IU/ml).
Comparison of CAP/CTM and the bDNA assay again showed a high concordance for samples harboring genotypes 2, 3, and 5, whereas a discrepancy for samples harboring genotypes 1 and 4 similar to that found for RealTime HCV and CAP/CTM was observed (higher levels of quantification for genotype 1 and lower levels of quantification for genotype 4 by CAP/CTM; 0.74 and −1.08 log10 IU/ml, respectively).
Taken together, our results demonstrated an overall good correlation of the results of all assays. However, when the HCV RNA levels in samples harboring genotype 1 were measured by CAP/CTM, they were always greater than the corresponding levels obtained by both RealTime HCV and the bDNA assay. In addition, a lower level of quantification by CAP/CTM was observed for samples harboring genotype 4. Despite the small number of samples available, these results are in line with those of a previous comparative study of Cobas TaqMan assays, the Cobas Amplicor Monitor assay, and the bDNA assay (24
). More recently, Chevaliez et al. have described the overestimation of HCV RNA levels by CAP/CTM (7
). However, in that study the global overquantification of HCV RNA of approximately 0.6 log10
IU/ml was claimed for all genotypes. In the present study, as well as in other previous studies, major differences in HCV RNA quantification between CAP/CTM, the bDNA assay, and RealTime HCV were restricted only to HCV genotype 1 and/or 4 (5
To date, the underquantification obtained for samples harboring genotype 4 has not been fully understood (7
). The mismatch of primers and/or the TaqMan probe and the target viral sequence is unlikely to be the only reason, since the recent introduction of a revised version of the manual High Pure system for specimen extraction, which is used together with the CTM amplification and detection system (version 2), has overcome the prior underestimation of the amounts of genotypes 2 through 5 without changing the set of primers and/or probes used. In fact, only changes in the ethanol concentration of the wash buffer used for sample preparation and the temperature of the reverse transcription step were introduced (8
). However, it has been suggested that suboptimal binding of oligonucleotides due to the secondary structure of the internal ribosome entry site and/or genotype-specific polymorphisms within the highly conserved 5′ nontranslated region may be responsible for underestimation of the quantity of genotype 4 by CAP/CTM (24
The clinical impact of genotype 4 underestimation by CAP/CTM may be the greatest in Egypt and the Middle East, where this genotype is prevalent. However, this technical issue may have minor implications (i.e., the use of different cutoff values for low versus high viral loads at the baseline) for patient management if viral load testing before, during, and after antiviral therapy is always performed by the same assay and in the same laboratory.
Since the bDNA assay has a reported lower LOD of 615 IU/ml, it was not included in our sensitivity experiments. In the present study, RealTime HCV had a sensitivity of 16.8 IU/ml for the second international WHO standard HCV RNA (code 96/798) that was comparable to that of the CAP/CTM assay (10.3 IU/ml) and that correlated with previous results (17
The sensitivity of RealTime HCV was even lower for clinical specimens harboring genotypes 1 to 6 (4.7 to 9.0 IU/ml). Interestingly, CAP/CTM had a limit of detection between 3.4 and 14.1 IU/ml for samples harboring genotypes 1, 3, 5, and 6, whereas samples harboring genotype 2 and 4 yielded higher probit values of 44.4 and 40.5 IU/ml, respectively. However, the results for the lower LODs of clinical specimens may vary between different samples of different HCV subtypes and origins. In a recent publication by Sizmann et al., equal lower LODs between 6.5 and 15.8 IU/ml for HCV genotypes 1 to 6 were reported (29
). In that study, however, no comparison with other real-time PCR-based assays was performed, and the results may vary depending on the method used for assessment of the HCV RNA concentration in the original undiluted sample.
Generally, patients who may have tested HCV RNA negative during or after antiviral therapy by older assays with lower LODs of ≥50 IU/ml may test HCV RNA positive by highly sensitive HCV RNA assays. Highly sensitive HCV RNA assays are now used in clinical practice to define a virologic nonresponse to antiviral therapy and to predict relapses after antiviral therapy (10
). In addition, the large dynamic range of the real-time PCR-based assays allows precise HCV RNA quantification without predilution, which was frequently required with previous standard PCR-based assays.
As discussed above, previous studies have shown differences of 0.5 to 0.7 log10
IU/ml for clinical specimens harboring genotype 1 between different assays (7
). Although the bDNA assay, RealTime HCV, and CAP/CTM are calibrated to the WHO standard, it remains unclear which of the assays corresponds best to the WHO standard on direct comparison. In order to investigate whether one of the assays over- or underestimates the true HCV RNA concentration, we performed for the first time a direct comparison of the two real-time PCR-based assays with the WHO standard.
The standard, produced by the National Institute for Biological Standards and Control (South Mimms, United Kingdom), is available only at an assigned unitage of 50,000 IU/ml. Two concentrations (25,000 and 1,500 IU/ml) were chosen and tested in multiple aliquots in a single run each.
The present analysis revealed a consistently lower level of quantification of the WHO standard by RealTime HCV (−0.2 to −0.3 log10
IU/ml) and a consistently higher level of quantification of the WHO standard by CAP/CTM (+0.2 to +0.3 log10
IU/ml). Thus, the total difference adds up to approximately 0.5 log10
IU/ml, and this corresponds to the findings of previous clinical studies that evaluated CAP/CTM versus the bDNA assay and RealTime HCV (7
In conclusion, the real-time PCR-based HCV RNA assays showed comparable, linear HCV RNA quantification abilities and a comparable sensitive detection of all HCV genotypes, with the exception of genotypes 1 and 4. The previously reported differences in the absolute quantification of samples harboring HCV genotype 1, which showed higher quantification results by CAP/CTM and lower quantification results by the bDNA assay and/or RealTime HCV, resulting in a total difference of approximately 0.5 to 0.7 log10
IU/ml, were confirmed (7
). Comparative analysis with the current WHO standard suggests that the differences may be explained by the different calibration methods used and/or the different WHO standards used (RealTime HCV is standardized against the second international WHO standard HCV RNA [code 96/798], whereas CAP/CTM is standardized against the first international WHO standard HCV RNA [code 96/790]).