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Comparative Study
. 2011 Sep;49(9):3309-15.
doi: 10.1128/JCM.00602-11. Epub 2011 Jul 13.

Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity

Johannes Vermehren  1 , Giuseppe Colucci, Peter Gohl, Nabila Hamdi, Ahmed Ihab Abdelaziz, Ursula Karey, Diana Thamke, Heike Zitzer, Stefan Zeuzem, Christoph Sarrazin
Affiliations
Comparative Study

Development of a second version of the Cobas AmpliPrep/Cobas TaqMan hepatitis C virus quantitative test with improved genotype inclusivity

Johannes Vermehren et al. J Clin Microbiol. 2011 Sep.

Abstract

Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log(10) HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were -0.05, 0.05, -0.12, -0.10, -0.44, and -0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5' untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.

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Figures

Fig. 1.
Fig. 1.
Bland-Altman analysis of CAP/CTM versus CAM (A), prototype CAP/CTM v2/MMx1 versus CAM (B), and prototype CAP/CTM v2/MMx2 versus CAM (C).
Fig. 1.
Fig. 1.
Bland-Altman analysis of CAP/CTM versus CAM (A), prototype CAP/CTM v2/MMx1 versus CAM (B), and prototype CAP/CTM v2/MMx2 versus CAM (C).
Fig. 2.
Fig. 2.
Regression analysis of CAP/CTM versus CAM (A), prototype CAP/CTM v2/MMx1 versus CAM (B), and prototype CAP/CTM v2/MMx2 versus CAM (C).
Fig. 3.
Fig. 3.
Detection and quantification of one GT1 transcript and three GT4 transcripts by CAP/CTM and the prototype CAP/CTM v2/MMx1 and v2/MMx2 assays. GT4 transcripts 1, 2, and 3 contain the following amino acid substitution(s): transcript 1, A165T; transcript 2, A165T + G145A; and transcript 3, A165T + G145A + A142G.
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