Recent advances in laboratory diagnosis of hepatitis C virus infection

T HE DISCOVERY OF HEPATITIS C VIRUS (1-ICV) (1) HAS LED TO the development of serological tests (2) for the detection of antibody to this newly discovered virus. HCV is the primary etiological agent of the parenterally transmitted non-A, non-B hepatitis (3). In Canada, groups at greater risk of acquiring HCV infection include intravenous drug users, hemodialysis patients, hemophiliacs and blood transfusion recipients (4). The screening of blood donors for anti-HCV has greatly reduced the incidence of post-transfusion hepatitis (5). One of the major problems with HCV infection is the development of chronic hepatitis in 50 to 60% of cases, which could lead to cirrhosis and hepatocellular carcinoma (6). HCV is a single-stranded RNA virus (9400 nucleotides) related to the family Flaviviridae (7). The 5' end codes for core and envelope proteins followed by nonstructural proteins NS2, NS3, NS4 and NS5. There is also a noncoding region at the 5' end (Figure 1). This paper briefly reviews the available serological and molecular diagnostic tests for the detection of 1-1cv antibody and viral RNA. Serological assays for detection of HCV infection: The first-generation enzyme immunoassay (EIA) was developed by Chiron Corporation (California) to detect antibody against HCV in 1989 (2). Later, Abbott Laboratories (Illinois) also developed a first-generation EIA test for the detection of anti-HCV under licence from Chiron

Corporation.Chiron developed a recombinant immunoblot assay (RJBA 1.0) as a supplemental test.The first-generation tests have become obsolete (8) because they lacked sensitivity and specificity, and were soon replaced by second-and third-generation tests.The details of the different EIAs are given in Table 1 and of immunoblot assays in Table 2.
Third-generation assays are more sensitive and specific for the detection of anti-HCV, and they should be the test of choice.In Canada anti-HCV test kits are available mainly from Ortho Diagnostics (New Jersey), Abbott Laboratories and Organon Teknika (The Netherlands).
The diagnosis of HCV infection depends mainly on detecting circulating antibodies to this virus.EIA 2.0 detects anti-HCV in approximately 90% of cases (9).The third-generation EIA 3.0 is more sensitive than EIA 2.0 and the predictive positive values are 0 .52versus 0.23 (10).EIA 3.0 detects antibody earlier in the course of infection (11), five to six weeks after the onset of hepatitis in 80% of patients.Some limitations have been observed with EIA tests in that they could not differentiate among acute, chronic and past infection.In some acute cases there could be a long interval before seroconversion.In low risk groups such as blood donors, even the third-generation EIAs produce false positive results.
Most of the false positive EIA results could be resolved by supplemental testing with immunoblot assays.RJBA 3 .0 is more sensitive and specific than RJBA 2.0, and 60 to 95% of the indeterminates could be resolved by the later test (10 , 12-14).RIBA 3.0 contains two recombinant (c33[NS3], NS5) and two synthetic (c22[core], c 100[NS4]) antigens.The concentration of antigens was optimized to improve sensitivity and specificity.Improvement was observed with RJBA 3.0 and was obtained mostly by the synthetic peptides but was not due to NS5 antigen (10).A comparative study (12)    The diagnosis of chronic or acu te infection is still restricted due to th e lack of a test for the detection of vira l antigen.However.recent developm ents in the polymerase chain reaction (PCR) techniqu e h ave made it possible to detect HCV RNA in serum or p lasma ( 15-l 7).
Primers specific for 5' untranslated region (UTR) are the most sensitive because this region is h ighly conserved (15).HCV RNA could b e detected many weeks before the appearance of anti-HCV (6) a nd in some cases this m ay be the only evidence of HCV infection.Nested PCR is the most sensitive technique for the detection of HCV RNA .In our study ( l 7) 67% of the RIBA r eactive samples were positive for HCV RNA by n ested PCR with primers from the 5' UTR .On the other hand, 100% of RIBA reactive samples from h igh risk groups such as transfused patients, h emodialysis p a tients and intra-260 Anti enic composition R; c 100-3 (NS4), c200 (NS3 + NS4), c22 (core) R; c200 (NS3 + NS4), c22 (core), NS5 R; c 100 (NS4), 33c (NS3) , HC34 (core) R; clOD CNS4), HC43 (core+ NS3), H34 (core), NS5 R; Microparticles; c200 (NS3 + NS4) , HC34 (core) R; Probe; HC31 (NS3 + NS4) , c200 (NS3 + NS4), c22 (core) Core (P) , NS3 (R) , NS4 (P), NS5 (P) Core (P), NS3 (P), NS4 (P), NS5 (P) venous drug users were PCR positive .A commercial PCR kit is available for the fast detection of HCV RNA (Amplicor HCV) from Roche Diagnostic Systems (New J ersey) .Th e Amplicor HCV test uses 5' UTR specific primers, the antisense primer is biotinylated at the 5' end, and a thermostable polymerase from Thermus thermophilus is used for both reverse transcription and PCR steps.The a mplified product is detected by hybridization to a specific n u cleic acid probe (18) .We have tried this kit a nd our prelimary data indicate that it is a useful assay for the detection of HCV RNA.
The oth er assay that is commercially available for the detection of HCV RNA is called 'branched-DNA signal amplification a ssay' (Chiron).In this case, HCV RNA is hybridized directly to synthetic oligonucleotides from the highly conserved 5' UTR and core gene of HCV immobilized on a microwell plate.Synthetic bONA a mplifier molecules and multiple copies of an alkaline-phosphatase linked probe are hybridized to the immobilized

3 . 0 .
Matrix l , LiaTek III) showed a poor correlation among th e three tests.Molecular assays fo r the detection of HCV infectio n: CAN j INFECT DIS VOL 5 N o 6 NOVEMBER/DECEMBER 1994 of immunoblot assays (RJBA The entire genome is about 10.000 bases.coding for about 3000 amino acids.Only the translated portions of the genome are shown.Not shown is a 5' conserved noncoding region of approximately 300 bases.which is located to the left of core (c) region.and a 3' noncoding reg ion of about 50 bases .which is at the e nd of NS5 region.gp Glycoprotein: PCT Polymerase carboxy terminus: PNT Polymerase amino terminus.Modified from reference 8

TABLE 1 Antigenic
composition of second-and third -generation enzyme immunoassay tests

TABLE 2
10tigenic composition of second -and third -generation immunoblot assays Canadian isolates (25) has shown that 60 .5% are type I. 10.9% type II, 6.7% type III.5.9% type N,10.9% not typeable and 5% type I + II.A commercial test for HCV subtyping has been developed by Innogen etics NV Belgium (26).In this assay, oligonucleotides derived from the 5' UTR act as specific probes for each genotype.The specific probes are immobilized as parallel lines on nitrocellulose strips and then hybridized with the amplified PCR products (26).The subtyping of Canadian HCV isolates by th is line probe assay showed a good correlation wilh other methods we h ave used for genotyping (27).The Laboratory for Viral Hepatitis in th e National Laboratory for Special Pathogens, Bureau of Microbiology , Laboratory Centre for Disease Control provides reference service for HCV testing.This includes co nfirm a tion of EtA-positive samples for anti-HCV by RJBA 3.0.testing for HCV RNA by PCR and genotyping.In addition, we provide a proficiency panel for HCV testing to al l provincial public h ealth laboratories and large h ospital centres.16.Lin H -11. Kao J-H.Leu J-11.et a l.Compa h son of three different immuno assays and PCR for lhe detection of hepatitis C vi rus infection in pregn ant women in Taiwan.Chan S. McOmish F. Holmes E. et a l.Ana lysis of n ew h epati tis C virus type a nd its phy logenetic relation sh ip to exis ting vatiants.J Gen V irol 1992 :73: 11 3 1-4 1. 22. Chyma 1<.Tsubota A. Arase Y. et al.Genotyping subtyping of hepa titis C virus.Gastroenterology I 993:8: 150-6.23.Okamoto H. Sugiyama Y. Ok ada S. et al.Typing h epa titis C virus by polymerase c h ai n rection wiU1 type specific primers: ap plication to clinical surveys and tracing i nfectious sources.J Gen V irol 1992:73 :673-89.24 .T subota A. Ch ayama K. A.rase Y. et al.Factors useful in pred ic ting th e response to interferon therapy in chronic h epati ti s C. J Gastroenterol H epa tol I 993:8:535-9.25.Andonov A. Chaud hary RK Genotyping of Canad ian isolates by PCR.J Cli n Microbial 1994:32:2031 -4. 26.Stuyvcr L. Rossan R. Wyseur A. et al.Typing of h epa titis C virus isolates and ch a.racte tization of n ew subtypes u sing a line probe assay.J Gen Virol 1993:74:1093-102.27.Andonov A. Chaudhary RK Subtyping of h ep atitis C virus isol ates with a line probe assay by hybridiza tion.