Clinical evaluation of the COBAS Ampliprep™/COBAS TaqMan™ for HCV RNA quantitation in comparison with the branched-DNA assay

Laboratory of Microbiology, Molinette Hospital, University of Turin, Turin, Italy.
Journal of Medical Virology (Impact Factor: 2.35). 02/2008; 80(2):254-60. DOI: 10.1002/jmv.21073
Source: PubMed


Diagnosis and monitoring of HCV infection relies on sensitive and accurate HCV RNA detection and quantitation. The performance of the COBAS AmpliPrep/COBAS TaqMan 48 (CAP/CTM) (Roche, Branchburg, NJ), a fully automated, real-time PCR HCV RNA quantitative test was assessed and compared with the branched-DNA (bDNA) assay. Clinical evaluation on 576 specimens obtained from patients with chronic hepatitis C showed a good correlation (r = 0.893) between the two test, but the CAP/CTM scored higher HCV RNA titers than the bDNA across all viral genotypes. The mean bDNA versus CAP/CTM log10 IU/ml differences were -0.49, -0.4, -0.54, -0.26 for genotype 1a, 1b, 2a/2c, 3a, and 4, respectively. These differences reached statistical significance for genotypes 1b, 2a/c, and 3a. The ability of the CAP/CTM to monitor patients undergoing antiviral therapy and correctly identify the weeks 4 and 12 rapid and early virological responses was confirmed. The broader dynamic range of the CAP/CTM compared with the bDNA allowed for a better definition of viral kinetics. In conclusion, the CAP/CTM appears as a reliable and user-friendly assay to monitor HCV viremia during treatment of patients with chronic hepatitis. Its high sensitivity and wide dynamic range may help a better definition of viral load changes during antiviral therapy.

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Available from: Francesco Cerutti, Nov 27, 2014
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    • "Serum samples were stored at −80°C until they were analyzed. HCV RNA levels were measured using a quantitative real-time PCR-based method (COBAS AmpliPrep/ COBAS TaqMan HCV Test) [29,30]. The reduction in HCV RNA 4 and 12 weeks after initiation of therapy was calculated. "
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    ABSTRACT: Background The importance of the reduction in hepatitis C virus (HCV) RNA levels 4 and 12 weeks after starting peginterferon (PEG-IFN) and ribavirin combination therapy has been reported to predict a sustained virologic response (SVR) in patients infected with HCV genotype 1. We conducted a multicenter study to validate this importance along with baseline predictive factors in this patient subpopulation. Methods A total of 516 patients with HCV genotype 1 and pretreatment HCV RNA levels ≥5.0 log10 IU/mL who completed response-guided therapy according to the AASLD guidelines were enrolled. The reduction in serum HCV RNA levels 4 and 12 weeks after starting therapy was measured using real-time PCR, and its value in predicting the likelihood of SVR was evaluated. Results The area under the receiver operating characteristics (ROC) curve was 0.852 for 4-week reduction and 0.826 for 12-week reduction of HCV RNA levels, respectively. When the cut-off is fixed at a 2.8-log10 reduction at 4 weeks and a 4.9-log10 reduction at 12 weeks on the basis of ROC analysis, the sensitivity and specificity for SVR were 80.9% and 77.9% at 4 weeks and were 89.0% and 67.2% at 12 weeks, respectively. These variables were independent factors associated with SVR in multivariate analysis. Among 99 patients who showed a delayed virologic response and completed 72-week extended regimen, the area under ROC curve was low: 0.516 for 4-week reduction and 0.482 for 12-week reduction of HCV RNA levels, respectively. Conclusions The reduction in HCV RNA levels 4 and 12 weeks after starting combination therapy is a strong independent predictor for SVR overall. These variables were not useful for predicting SVR in patients who showed a slow virologic response and experienced 72-week extended regimen.
    BMC Infectious Diseases 11/2012; 12(1):324. DOI:10.1186/1471-2334-12-324 · 2.61 Impact Factor
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    • "The sensitivity of the system is 1.5 × 10 1 IU/mL. This assay has been reported as a sensitive, precise, and robust assay with a broad dynamic range that is suitable for HCV RNA quantification in patients infected with HCV genotypes 1–6 (Bossler et al., 2011; Pittuluga et al., 2008; Sarrazin et al., 2008). In our study, HCV RNA concentrations were reasonably stable when specimens were exposed to up to 10 FT cycles in both serum and plasma samples. "
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    ABSTRACT: To assess the stability of various sample types and storage conditions for quantitative detectability of hepatitis C virus (HCV) RNA viral loads, we studied serum and EDTA/citrate plasma samples obtained from 10 patients known to be positive for HCV RNA. Samples were subjected to the following conditions: 1) 10 freeze-thaw (FT) cycles, and 2) storage at room temperature for 24, 48, and 72 h. Detection of HCV RNA was performed by COBAS AmpliPrep/COBAS TaqMan HCV. The following conclusions were reached: 1) no significantly different viral loads were observed in different blood compartments; 2) no significantly different viral loads were observed after 24, 48, and 72 h at room temperature; 3) no significantly different viral loads were observed after 10 FT cycles in serum and plasma samples; and 4) HCV RNA is quite stable in serum and plasma (EDTA/citrate) samples.
    Diagnostic microbiology and infectious disease 10/2012; 75(1). DOI:10.1016/j.diagmicrobio.2012.09.017 · 2.46 Impact Factor
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    ABSTRACT: Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties. This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.
    PLoS Medicine 03/2009; 6(2):e31. DOI:10.1371/journal.pmed.1000031 · 14.43 Impact Factor
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