Increased Sensitivity of the Roche COBAS AMPLICOR HCV Test, Version 2.0, Using Modified Extraction Techniques

Abstract

Processing modifications were made to the COBAS AMPLICOR HCV version 2.0 assay to enhance sensitivity. Two methods of specimen concentration, centrifugation ("ultraspin") and cationic detergent plus silica membrane ("ultracolumn"), were compared to the standard method. The effect of these changes on assay sensitivity and specificity was examined using commercial hepatitis C virus (HCV) preparations. The limits of detection (LOD, defined as detection of HCV RNA in > 95% of replicates) of genotype 1a were 50, 12, and 6 by standard method, ultraspin and ultracolumn, respectively. For genotype 1b, the LOD was 25 IU/ml, 12 IU/ml, and 3 IU/ml; for 2b, it was 50, 12, and 3; for 3a, it was 25, 12, and 1.5; for 4 it was 18, 4, and 2; for 5a, it was 38, 9, and 2; and for 6a it was 47, 6, and 3. No false positives were detected after ultraspin when controls containing high or low HCV concentrations were alternated with normal human plasma. Plasmas in which HCV RNA was not detected by the standard assay were re-tested with modified methods to assess the effect of altered processing in clinical specimens. Three of 152 specimens with no detectable HCV RNA by the standard method were positive by ultraspin and 2 of 109 were positive by ultracolumn, suggesting that these methods may increase assay sensitivity in clinical specimens. Accurate, sensitive detection of hepatitis C virus (HCV) in serum or plasma is essential for diagnosis of HCV infections and assessing response to anti-HCV therapy. The standard, FDA approved COBAS AMPLICOR HCV version 2.0 assay has a reported LOD of 50 IU/ml for HCV genotype 1b. 1 This assay detects HCV RNA during active HCV infection. 2 It has been used to diagnose viremia 3,4 and to assess treatment response. 5 Therapy for chronic HCV infection has evolved from interferon alfa (IFN-alfa) alone, to second generation combination therapy with IFN-alfa plus ribavirin for either 24 or 48 weeks, to the latest generation of pegylatedinterferon ␣ with ribavirin (pegIFN-alfa/riba) for either 24 or 48 weeks. The lowest rates of response and highest rates of relapse were observed with IFN-␣ monotherapy. 6 Treatment with pegIFN-␣/riba resulted in the highest rates of response and the lowest rates of relapse. 5,7 Variables that potentially affect rates of virologic relapse include treatment regimen, genetic variability of the virus, and HCV RNA detection assay sensitivity. The ability to detect very low levels of virus may prove useful in identifying patients who may relapse at end of treatment. Transcription-mediated amplification (TMA) has been used to assess the presence of residual HCV RNA in plasma from patients treated with pegINF-alfa-2a and INF-alfa-2a. 8 In patients who relapsed after therapy, residual HCV RNA was detected in 7% of plasma samples at end of treatment with pegINF-alfa-2a and 33% of samples following therapy with INF-alfa-2a.The aim of this study was to increase the sensitivity of COBAS AMPLICOR HCV version 2.0 through alteration of extraction methods. Two modifications were used to extra...

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees

“…Peripheral blood mononuclear cells (PBMCs) from naive and HCV‐infected chimpanzees ( n = 15) were used. HCV chimpanzees had been infected experimentally with HCV genotype 1a or 1b and HCV RNA load was determined with Roche Cobas Amplicor (Roche Diagnostics Limited, Rotkreuz, Switzerland). Virus load in serum of the animals varied between 6667 and > 850 000 IU/ml.…”