Evaluation of the Abbott Investigational Use Only RealTi m e Hepatitis C Virus (HCV) Assay and Comparison to the Roche TaqMan HCV Analyte-Specific Reagent Assay

Abstract

The accurate and sensitive measurement of hepatitis C virus (HCV) RNA is essential for the clinical management and treatment of infected patients and as a research tool for studying the biology of HCV infection. We evaluated the linearity, reproducibility, precision, limit of detection, and concordance of viral genotype quantitation of the Abbott investigational use only RealTime HCV (RealTime) assay using the Abbott m2000 platform and compared the results to those of the Roche TaqMan Analyte-Specific Reagent (TaqMan) and Bayer Versant HCV bDNA 3.0 assay. Comparison of 216 samples analyzed by RealTime and TaqMan assays produced the following Deming regression equation: RealTime ‫؍‬ 0.940 (TaqMan) ؉ 0.175 log 10 HCV RNA IU/ml. The average difference between the assays was 0.143 log 10 RNA IU/ml and was consistent across RealTime's dynamic range of nearly 7 log 10 HCV RNA IU/ml. There was no significant difference between genotypes among these samples. The limit of detection using eight replicates of the World Health Organization HCV standard was determined to be 7.74 HCV RNA IU/ml by probit analysis. Replicate measurements of commercial genotype panels were significantly higher than TaqMan measurements for most samples and showed that the RealTime assay is able to detect all genotypes with no bias. Additionally, we showed that the amplicon generated by the widely used Roche COBAS Amplicor Hepatitis C Virus Test, version 2.0, can act as a template in the RealTime assay, but potential cross-contamination could be mitigated by treatment with uracil-N-glycosylase. In conclusion, the RealTime assay accurately measured HCV viral loads over a broad dynamic range, with no significant genotype bias.Methods for accurate quantitation of serum and plasma hepatitis C virus (HCV) RNA levels have become key tools both for understanding the biology of HCV infection and for the clinical management of patients under treatment. The ability to predict likelihood of response to combination interferon/ ribavirin therapy by assessing rates of HCV viral load decline has provided a more individualized treatment algorithm that can identify nonresponsive patients early in treatment, sparing them significant morbidity and cost. New algorithms that examine the kinetics of HCV viral decline provide an even more refined tool for management and complement the information provided by HCV genotype determination. Finally, HCV viral kinetics data are essential for the understanding of new therapeutics such as the class of protease inhibitors. For all applications, accurate quantitation of all HCV types over a broad dynamic range is critical. The advent of real-time PCR methods provides a powerful tool for a broad dynamic range of quantitation of viruses; however, targets such as HCV require careful assay design to avoid errors due to sequence variations intrinsic to RNA viruses. Abbott Molecular, Inc. (Des Plaines, IL), recently released the m2000 system and tests for human immunodeficiency virus type 1, HCV, and chlamydia/gonorrhea (Chlamydia trac...

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