Vol. 30, No. 4

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1992, p. 931-934

0095-1137/92/040931-04$02.00/0 Copyright © 1992, American Society for Microbiology

Detection of Hepatitis C Virus Sequences in Sera with Controversial Serology by Nested Polymerase Chain Reaction YAMINA LAZIZI,1* EMILE ELFASSI,2 AND JACQUES PILLOT"2 Service de Microbiologie et d'Immunologie, H6pital Antoine Beclere, 92141 Clamart, 1 and Unite6 d'Immunologie Microbienne, Institut Pasteur, 75015 Paris,2 France Received 13 September 1991/Accepted 30 December 1991 The specificity of first-generation enzyme-linked immunosorbent assays (ELISAs) for antibody detection in individuals with hepatitis C virus (HCV) infection has been questioned in some pathological situations. We observed a surprisingly high prevalence of anti-HCV antibodies in alcoholic patients, and thus, false-positive reactions in anti-HCV tests were strongly suspected. The introduction of new epitopes, particularly a core protein, C22 (second-generation tests), seems to increase the sensitivity of anti-HCV detection. In order to study the specificity of the second-generation tests, 60 serum samples from alcoholic patients found to be positive by the first-generation anti-HCV ELISA (Ortho) were reexamined by a second-generation anti-HCV enzyme immunoassay (Abbott) and a recombinant immunoblot assay (RIBA II; Chiron). Fifteen serum samples gave contradictory results when they were tested by the two assays. We performed nested polymerase chain reactions (PCRs) to confirm that the discrepancies that we observed could be due to the presence of low levels of anti-HCV antibodies, which were detected by a more sensitive test, or to unspecific positive reactions. Nested PCR revealed the presence of HCV RNA sequences in all anti-HCV-positive sera or sera that were weakly positive by ELISA. Anti-HCV positivity by RIBA II was always correlated with the presence of viral RNA in serum, but HCV RNA was detected in RIBA II-negative sera. These results indicate that the specificity of the second-generation tests is an important improvement but that an HCV infection can still persist without detectable antibodies. PCR remains the reference assay to clear up controversial serology results and to detect HCV infection in patients with no anti-HCV-detectable immune response.

epitope C33-c in the second-generation assay has been considered an important advance in the sensitivity of antiHCV detection (16). If the sensitivities are improved by those second-generation assays, their specificities remain to be studied. Therefore, we selected 60 serum samples from a cohort of alcoholic patients that were weakly positive for anti-C100-3 antibodies by the first-generation anti-HCV ELISA (Ortho Diagnostic Systems) and were found to be negative or weakly positive by another commercial ELISA (EIA; Abbott) using the same recombinant HCV proteins. These sera were retested by the second-generation ELISA, the Abbott HCV EIA and by a recombinant immunoblot assay (RIBA II; Chiron). Discrepancies between the first- and second-generation tests and between both the second-generation tests were found. The polymerase chain reaction (PCR) technique was used as a reference test of the controversial sera to confirm the presence or absence of HCV RNA.

Hepatitis C virus (HCV) is considered to be the major etiological agent of 90 to 95% of posttransfusion non-A, non-B hepatitis cases (17). This virus has homologies with the Flaviridae. The HCV genome is a linear single-stranded RNA molecule (positive sense) of about 10,000 nucleotides (14). Several serological assays based on the nucleotide sequence of the HCV genome have been developed. In a first step, anti-HCV antibodies were detected by an enzymelinked immunosorbent assay (ELISA), based on the HCV C100-3 antigen, in the sera of a large number of patients with chronic liver diseases (10). The HCV recombinant antigen used in this first-generation test was produced in yeasts (C100-3 antigen) and Escherichia coli (5-1-1 antigen) from the nonstructural region NS4. One of the most important findings of this first HCV serology was that a high proportion of anti-HCV-positive cases was related to parenteral transmission of the virus. Nevertheless, screening of blood donors showed the existence of asymptomatic HCV carriers who had no history of transfusion or intravenous drug use (7). Furthermore, antiHCV antibodies were also observed independently of any transfusion history in patients with sporadic hepatitis and in some pathological situations, such as in patients with autoimmune hepatitis (9) and hepatocarcinoma (6) and alcoholic patients (12). This observation suggests that HCV can be transmitted other than by the parenteral route and that HCV could be implicated in different pathologies. However, nonspecific ELISA reactions have been described for anti-HCV antibodies, and doubts have been raised about the validity of this test (11; 18). The introduction of the structural epitope C22-3 and the nonstructural *

MATERIALS AND METHODS Serum samples and serological assays. Serum samples from 1,259 alcoholic patients were stored at -25°C. We first tested for the presence of anti-HCV antibodies using the first-generation anti-HCV ELISA (Ortho). Then, positive samples from alcoholic patients were retested by the firstgeneration HCV EIA (Abbott). According to the optical densities (ODs) of samples in the anti-HCV ELISA (Ortho), three categories of anti-HCV-positive patients were defined: (i) patient serum samples with ODs of less than 1 (cutoff < OD < 1), (ii) patient serum samples with ODs of between 1 and 2 (1 < OD < 2), and (iii) patient serum samples with ODs greater or equal to 2 (OD, >2). For this study 60 serum samples were selected from weakly positive sera (cutoff

Detection of hepatitis C virus sequences in sera with controversial serology by nested polymerase chain reaction.

The specificity of first-generation enzyme-linked immunosorbent assays (ELIAs) for antibody detection in individuals with hepatitis C virus (HCV) infe...
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