Since its discovery in 1989, 1 it has been demonstrated that hepatitis C virus (HCV) is a major cause of chronic hepatitis throughout the world. The routine virological diagnosis is based on serological tests detecting antibodies to HCV. However, in the absence of an efficient culture system for HCV, or viral antigen assays, direct detection of HCV is based on nucleic acid amplification and hybridization methods. HCV RNA was principally detected with in-house nested polymerase chain reaction (PCR) methods, but new standardized assays such as Amplicor (Roche Diagnostics Systems, Basel, Switzerland) and nucleic acid sequence-based amplification (NASBA) (Organon Teknika, Boxtel, the Netherlands) have been developed to detect HCV RNA on a routine basis 2,3 or for research purposes. 4,5 Several studies have shown a relation between pretreatment viral load and the response to interferon (IFN) therapy, 6-10 creating a need for standardized HCV-RNA quantification assays. Currently, the most frequently used kit is the branched DNA (bDNA) assay (Quantiplex HCV RNA, Chiron Diagnostics, Emeryville, CA), a well-standardized signalamplification method. Other assays are based on HCV-RNA amplification (Amplicor HCV Monitor, Roche Diagnostics Systems, and NASBA HCV). The aim of this study was to compare the NASBA, bDNA, and Monitor HCV assays in terms of reproducibility, sensitivity, and linearity in various clinical settings and using standardized panels. Two qualitative methods (in-house nested PCR and Amplicor) were used as standards for sensitivity. Specificity was not a focus of this study, because specificity studies were performed by the manufacturers during the test registration procedure, and because we routinely obtain very good specificity in our lab. Moreover, negative samples were included in each series of tests (anti-HCV-and PCR-negative blood donors). MATERIALS AND METHODS Pelicheck HCV-RNA EUROHEP PanelThis panel was used to evaluate the sensitivity and linearity of each assay. It contains two dilution panels of the EUROHEP genotype 1 and 3 plasma standards. The plasma standards have been characterized in two collaborative EUROHEP studies. 11,12 The EUROHEP HCV-RNA genotype 1b and 3a plasma standard dilutions were prepared from a pool of plasma units negative for hepatitis B surface antigen, anti-hepatitis B core antibodies (Ab), anti-HCV Ab, anti-human immunodeficiency virus-1 or -2 Ab, anti-human T-lymphotropic virus type 1 Ab, syphilis, HCV RNA, hepatitis B virus DNA, and human immunodeficiency virus RNA. The undiluted plasma samples are not available. According to the EUROHEP group, the undiluted plasma corresponding to the genotype 1 panel should contain 38 Ϯ 8 ϫ 10 5 genome equivalents per milliliter (eq/mL) in the bDNA 2.0 assay, and 2.8 ϫ 10 5 copies per milliliter (cps/mL) in the HCV RNA Monitor assay. The potency of the
A part of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) amino acid sequence, designated as an interferon (IFN)-sensitive determining region (ISDR), has been shown to be correlated with a response to IFN in Japanese patients. We have shown previously that the presence of NS5A antibodies (Abs) detected by the INNOLIA test (IL-NS5A Ab) is also correlated with a response to IFN. The aim of this study was to investigate, in a wide range of patients, the possible relationship within the NS5A protein between the sequence of ISDR and that used in the INNOLIA test designated as IL3R. Serum samples from 52 patients infected by HCV genotypes 1, 2, and 3 were analyzed before and after treatment. The patients were classified as nonresponders (NRs), responder-relapsers (RRs), or long-term responders (LTRs). We amplified the NS5A region for 42 patients using polymerase chain reaction (PCR), and these amplicons were sequenced directly. The 10 remaining patients were analyzed using PCR with mutation-specific primers. No correlation was found between the IL3R sequence of the HCV strains and the presence of the IL-NS5A Ab for all genotypes. However, for the subtype 1b, only 2 of 11 NR patients tested had an arginin in position 2218 within the ISDR versus 3 of 3 LTR and 10 of 13 RR patients. All patients with R-2218 had IL-NS5A Ab. For the genotype 1a, 2 of 2 LTR and 1 of 3 RR were mutated in position 2216-2218 in comparison to three NR sequences. For the genotype 3, no mutations were found in the region homologous to 1b-ISDR, but 4 of 5 LTR and RR patients had a mutation T-2161 to A or V versus 0 of 3 NR patients. A close correlation was found between arginin in position 2218 in ISDR, the presence of IL-NS5A Ab, and the response to IFN therapy for genotype 1b, but this association did not predict a long-term response. For genotype 3, a potential ISD mutation could be located at the codon 2161. (HEPATOLOGY 1998;28:1674-1679.)Hepatitis C virus (HCV) is the main cause of non-A, non-B hepatitis. 1 The HCV genome is a single strand of positive RNA composed of a region coding for structural proteins (core and glycoproteins E1, E2, and p7) in its first terminal third and region coding for nonstructural proteins (NS2, NS3, NS4A/B, and NS5A/B) in the remaining part. HCV infection can be diagnosed by serological methods and 5Ј-untranslated region polymerase chain reaction (PCR). Third-generation confirmatory assays have been developed to detect antibodies (Abs) against structural and nonstructural antigens as shown by the INNOLIA HCV Ab III test (Innogenetics, Antwerp, Belgium), which detects nonstructural protein 5A Abs (IL-NS5A Ab).Interferon (IFN) has some efficacy on chronic hepatitis C, but relapse is common when treatment is discontinued. 2,3 We have shown previously 4 that sensitivity to IFN is closely associated with the presence of IL-NS5A Ab. In this previous study, 95% of long-term responders (LTRs; 20 of 21) had IL-NS5A Ab versus 13% of nonresponders (NRs; 3 of 23). Concurrent with this work, several studies in Jap...
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