Comparison of Performance Characteristics of Three Real-Time Reverse Transcription-PCR Test Systems for Detection and Quantification of Hepatitis C Virus

Abstract

We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log 10 IU/ml with a linear dynamic range of 1.0 to 7.0 log 10 IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log 10 IU/ml with a linear dynamic range of 2.0 to 7.0 log 10 IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy Hepatitis C virus (HCV) is still a major health care problem worldwide, including the United States. It is estimated that more than 170 million people worldwide are infected with HCV. HCV is one of the leading indications for liver transplantation in the United States (11). Prospective studies have shown that 60 to 85% of HCV-infected individuals develop chronic disease. Current recommendations for treatment are combination therapy with pegylated interferon and ribavirin (18). Treatment guidelines and monitoring the response to therapy rely heavily on viral load testing and genotype information. Genotyping should be performed before starting treatment to determine duration of treatment and dosage of ribavarin and also to provide prognostic information. The recommended durations of combination therapy are 24 weeks for genotypes 2 and 3 and 48 weeks for genotypes 1, 4, and 6 (18). Responses to treatment vary greatly, from approximately 40% for those infected with HCV genotypes 1 and 4 to about 80% for those infected with HCV genotypes 2 and 3.Quantitative testing should be performed by methods with a wide enough dynamic range for accurate assessment of both pretreatment viral loads as well as an early virologic response, which is defined as a fall in the HCV RNA levels by at least 2 log 10 units or to an undetectable level at week 12 of treatment. Currently, the best indicator of effective treatment is a sustained virologic response (SVR), defined by the absence of detectable HCV RNA in the serum when determined 6 months after the end of treatment. A sensitive method with a low limit of detection of 50 IU/ml or less should be used to assess SVR (18).A variety of assays are commercially available to detect and quantify HCV RNA. They are based on three specific methodologies: PCR, transcription-mediated amplification (TMA) and the branched-DNA technique (4,5,15,20). The most commonly used PCR-based assay for HCV quantification is the COBAS Amplicor HCV Monitor v2.0, with a lower limit of quantification of 600 IU/ml, which is inadequate to define an end-of-treatment response or ...

Performance of the Abbott RealTime and Roche Cobas TaqMan Hepatitis C Virus (HCV) Assays for Quantification of HCV Genotypes

“…However, for subtypes 1a and 2b, the mean difference in VL was not significantly different from non-1a or non-2b specimens, respectively, indicating that the underlying reason for the large discrepancy in VL results for these specimens was not related to subtype. The observed precision, linearity, and sensitivity of the ART assay reported here are similar to results from other laboratories and testing environments (2,5,15,(17)(18)(19)22). In addition, the negative bias of results from the CAP-CTM assay compared to those from the ART assay that we described is consistent with most (14-16), although not all (2, 17), published studies.…”

Evaluation of the Performance Characteristics of an In-House One Step TaqMan Real Time-Polymerase Chain Reaction Assay for Detection and Quantification of Hepatitis C Virus

“…The other study on an in-house real-time reverse transcriptase polymerase chain reaction reported 50 IU/mL as the lower limit of detection (18). We did not examine linearity with reference material with lower and higher concentrations than those used in this study, thus for accurate quantification of HCV RNA in clinical samples with baseline viral load higher than the upper limit of the assay, the samples should be diluted and retested (12,17). The efficiency of the assay was acceptable, due to the slope of the log 10 -linear phase (20).…”

“…In the last decade, several commercial or in-house TaqMan real time systems have been introduced with acceptable performance characteristics (8,(10)(11)(12)(13)(14)(15)(16)(17)(18). Due to increasing sensitivity of quantitative assays, they are recommended for detection and quantification of HCV RNA in seropositive patients (7).…”

An Overview of the Laboratory Assay Systems and Reactives Used in the Diagnosis of Hepatitis C Virus (HCV) Infections

“…20 The Abbott RealTime™ HCV Test provides a lower limit of detection of 12 IU/mL, a specificity of more than 99.5 % and a linear amplification range from 12 to 10,000,000 IU/mL independent of the HCV genotype. 27,39 VERSANT kPCR Molecular System Siemens Healthcare Diagnostics is also avialable as a real time PCR system for quantification of HCV RNA. Rotor Gene Q real time PCR device and Qiagen HCV RNA kits (Qiagen GmbH, Germany) are used for quantification of HCV RNA by real time PCR method with specificity of 99.0 %, a lower limit of detection 34 IU/ml and capable to detect up to 10, 000,000 IU/mL.…”