Performance Characteristics of a Quantitative TaqMan Hepatitis C Virus RNA Analyte-Specific Reagent

Both qualitative and quantitative hepatitis C virus (HCV) RNA tests are used in diagnosis and management of patients with hepatitis C, because no single commercially available test combines high analytical sensitivity with a broad dynamic range. Qualitative nucleic acid amplification tests for detection of HCV RNA in serum are used to confirm the diagnosis of hepatitis C, distinguish active from resolved infection, assess virological response to therapy, and screen blood donors (3, 18, 23). Quantitative tests are used in evaluation of patients being considered for therapy and to assess early response to therapy. A pretreatment viral load of less than 800,000 IU/ml is one of several predictors of a sustained virological response (20, 24). Viral load testing has also been used in early assessment of treatment response. Patients who fail to achieve at least a 2-log10 decline in viral load after 12 weeks of treatment have little chance of a sustained response and can be spared the cost and toxicity of a complete treatment course (9, 17). However, viral load does not predict the progression of hepatitis C and is not associated with the severity of liver disease. A variety of tests for detection and quantitation of HCV RNA based on different nucleic acid amplification technologies are commercially available. The qualitative AMPLICOR HCV and quantitative AMPLICOR HCV MONITOR version 2.0 tests (Roche Diagnostics Corporation, Indianapolis, Ind.) are based on conventional reverse transcription-PCR in a heterogeneous format (17). The VERSANT HCV RNA qualitative and VERSANT HCV RNA 3.0 quantitative assays (Bayer Healthcare, Tarrytown, N.Y.) are based on transcription-mediated amplification and branched DNA signal amplification, respectively (10, 15). These tests also differ in their lower limits of detection and dynamic ranges. The qualitative AMPLICOR and VERSANT tests have lower limits of detection of 50 and 5 IU/ml, respectively. Although the lower limits of detection for the quantitative AMPLICOR and VERSANT tests are both approximately 600 IU/ml, the dynamic ranges differ by approximately 1 log10 and are 3.1 and 4.1 log10, respectively. Because of the differences in sensitivity between the qualitative and quantitative assays, many clinical laboratories use a quantitative test to determine viral load and a sensitive qualitative test for diagnosis and test-of-cure. A single test with sensitivity similar to the qualitative tests that accurately quantitates high viral loads would be beneficial for clinical laboratories. A number of homogeneous TaqMan reverse transcription-PCR assays for detection and quantitation of HCV RNA have been described (12, 13, 19, 21, 29). These tests are very sensitive, have broad dynamic ranges, and provide precise quantitation of viral load. These tests also generate results more rapidly than the earlier heterogeneous tests and are not prone to amplicon carry-over contamination, since the amplification and detection steps are combined in a single closed tube. Roche Diagnostics Corp. recently developed a TaqMan HCV analyte-specific reagent (ASR). It was designed for the newly released COBAS TaqMan analyzer, a real-time PCR instrument developed for the clinical laboratory. An ASR may be sold to clinical laboratories regulated under the Clinical Laboratory Improvement Amendments (CLIA) of 1998 as qualified to do high-complexity testing. The laboratory is responsible for verifying and validating the test, and the reports should be appended with a standard disclaimer stating that the test was developed by and its performance characteristics determined by the laboratory and that the test has not been cleared or approved by the U.S. Food and Drug Administration (FDA). This study is the first to report the dynamic range, sensitivity, reproducibility, accuracy, and genotype bias of the ASR. The performance characteristics of the ASR were also compared with those of the qualitative AMPLICOR and quantitative AMPLICOR MONITOR HCV version 2.0 tests.