Journal of Medical Virology 37:237-240 (1992)

Improved Serodiagnosis of Chronic Hepatitis C in Japan by a Second-GenerationEnzyme-Linked Immunosorbent Assay Nobukazu Yuki, Norio Hayashi, Hideki Hagiwara, Tetsuo Takehara, Masahide Oshita, Akinori Kasahara, Hideyuki Fusamoto, and Takenobu Kamada First Department of Medicine, Osaka University Medical School, Osaka, Japan To clarify the role of hepatitis C virus (HCV) infection in chronic liver disease, sera from Japanese patients which were negative by the original anti-HCV assay (Ortho) were subjected to a second-generation anti-HCV assay based on a combination of structural (C22) and nonstructural (C200)recombinant HCV proteins. Of 29 patients with chronic non-A, non-B hepatitis, 20 (69%) were anti-HCV-positive by the second-generation enzyme-linked immunosorbent assay (ELISA) and also positive by the reverse transcription-polymerase chain reaction (RT-PCR) which detects the HCV genome. Of 41 chronic hepatitis B virus carriers, 3 (7%) were positive by the second-generation ELISA; 1 of 3 was positive by RT-PCR. The HCV genome was detected in ail cases positive for anti-HCV with high titers. Of 59 healthy subjects negative by the second-generation ELISA, none were positive by RT-PCR. These findings indicate that HCV is a major causative agent of chronic non-A, non-B hepatitis in Japan and that second-generation ELISA is specific and a more sensitive diagnostic assay for chronic hepatitis C. o 1992 Wiley-Liss, Inc.

KEY WORDS: antibodies to hepatitis C virus, hepatitis C virus, hepatitis C virus RNA, non-A, nan-8 hepatitis, polymerase chain reaction

INTRODUCTION The genome of a non-A, non-B hepatitis virus was cloned and termed hepatitis C virus (HCV) [Choo e t al., 19891. Part of the nonstructural region of the genome was expressed in yeast (C100-3 antigen), and a specific assay was established to detect the antibody to this recombinant antigen (anti-C100-3) [Kuo et al., 19891. However, the problem with the assay was its relatively low sensitivity. In some cases, HCV infection can be found in anti-ClOO-3-negative patients by the reverse 0 1992 WILEY-LISS, INC.

transcription-polymerase chain reaction method (RTPCR) which detects the HCV genome [Weiner et al., 19901. The HCV genome is a positive-stranded RNA molecule of approximately 10 kilobases and has a n organization similar to that of the flavivirus family. The 5'terminal sequence is thought to be the structural region coding for core and envelope proteins, and the larger remaining part to be the nonstructural region which is divided into five sections (NSl-NS5)and codes for nonstructural proteins. Recently, a second-generation anti-HCV assay based on a combination of structural (core) and nonstructural (NS3-NS4) recombinant HCV proteins (C22 and C200 antigens, respectively) has been developed, in which the former C100-3 antigen is included as a partial polypeptide of C200 antigen. Using this system and RT-PCR, anti-C100-3-negative sera from Japanese patients with chronic liver disease were tested and the prevalence of HCV infection in this population was evaluated.

MATERIALS AND METHODS Serum Samples Sera were obtained from 29 patients with chronic non-A, non-B liver disease and 41 hepatitis B virus (HBV) carriers who were anti-C100-3-negative by the original anti-HCV assay. The diagnosis of non-A, non-B hepatitis was based on clinical history and laboratory examination. The 29 patients were negative for hepatitis B surface antigen (HBsAg) and showed no evidence of alcoholic, autoimmune, or drug-induced liver disease. Liver histology, available for 18patients, showed the features of chronic persistent hepatitis (CPH) in two cases, chronic active hepatitis (CAH) in ten, and liver cirrhosis in six. The remaining 11patients had liver cirrhosis with hepatocellular carcinoma (HCC), which was demonstrated by ultrasonography, computed tomography, and angiogra-

Accepted for publication January 20,1992. Address reprint requests Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima 1-1-50,Fukushima-ku, Osaka 553, Japan.


Yuki et al.

buffer (pH 7.5) containing 4 molil guanidinium thiocyanate, 0.5% sodium lauryl sarcosinate, and 1% p-mercaptoethanol. Nucleic acids were extracted with phenolichloroform, and precipitated a t -80°C for 1 h r after addition of 0.1 vol of 3 molil sodium acetate (pH 6.0) and 2.5 vol of absolute ethanol to the aqueous phase. After another centrifugation, the pelleted RNA was washed with 70% ethanol, dried, and dissolved in 7 pl of distilled water. The RNA solution was made up to 20 pl with reaction solution containing 200 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD), 20 pmol of the antisense primer, 1mmolil each dNTP, and 20 U of RNase inhibitor (Promega, Madison, WI). cDNA synthesis was done as instructed by the manufacturer. The cDNA preparations were added to 80 pl of PCR solution containing 2.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT), 20 pmol of sense primer, 1.2 mmolil MgCl,, and 45 mmolil KC1. The amplification mixtures were overlaid with mineral oil and amplified in a DNA thermal cycler (Perkin-Elmer Serological Tests Cetus) for 40 cycles (94°C for 0.5 min; 55°C for 1 min; HBsAg, HBeAg, and anti-HBe were determined us- 72°C for 1rnin), followed by a 10-min final extension a t ing commercially available radioimmunoassays (Dain- 72°C. A 10 ~1 aliquot was fractionated by agarose gel abot Co., Ltd., Tokyo, Japan). Anti-(3100-3 was tested electrophoresis, transferred onto a nylon membrane, by the original Ortho ELISA (Ortho Diagnostic Sys- hybridized to a 32P-labeledHCV cDNA between the two tems Co., Ltd., Tokyo, Japan). Anti-HCV was further primers, and autoradiographed. Because of the extreme examined by second-generation (C200iC22) ELISA (Or- sensitivity of PCR, great care was taken to prevent tho Diagnostic Systems Co., Ltd.). Both assays were false-positive results, and the contamination avoidance carried out according to the manufacturer’s instruc- measures of Kwok and Higuchi [19891 were strictly tions. Microtiter plates, which had been coated with applied throughout this study. recombinant HCV proteids), were incubated with test Statistical Analysis sera a t 37°C for 1hr, followed by incubation with peroxStatistical analysis for group comparisons was done idase-conjugated monoclonal anti-human IgG antibody for another 1 hr. A solution containing o-phenylene by the x2 method and the unpaired Student’s t test. diamine and H,Oz was then added, and the resulting RESULTS optical density a t 492 nm (OD,,,) was measured using a photo-enzyme-linked immunosorbent assay colorimeSera from 70 Japanese patients with chronic liver ter (Titertek Multiscan MCC/340, Flow Laboratories, disease were negative for anti-HCV by the original McLean, VA). The mean value of OD,,, for three nega- (C100-3) ELISA. Table I shows the clinical characteristive controls of the kits plus 0.400 OD units was taken tics and anti-HCV seropositivity by second-generation as the cut-off value. All assays were carried out in du- (C200iC22) ELISA in this population. plicate. To compare ELISA results of different samples, Of the 29 patients with chronic non-A, non-B hepatithe ratio of the mean OD reading of the duplicate re- tis, anti-HCV (C200iC22) was found in 20 (69%) pasults to the cut-off value (cut-off index) was calculated. tients (1/2 of CPH, 8/10 of CAH, 416 of liver cirrhosis, and 7/11 of cirrhosis with HCC), including three of the Detection of HCV RNA Sequences by the PCR five patients who had received blood transfusions previHCV RNA was extracted from serum samples, copied ously. Anti-HCV (C200iC22) was also found in 3 (7%) of the into cDNA by reverse transcription, and amplified by PCR a s described elsewhere [Hagiwara et al., 19921. 41 patients with chronic HBV infection (two liver cirPrimers were derived from the 5’-noncoding region of rhotics and one cirrhotic with HCC) but less frequently the published sequence; antisense primer, 5’CATGGT- than in those with chronic non-A, non-B hepatitis (P < GCACGGTCTACGAG3‘ (nucleotides 308-327); and 0.01). Two of the three were positive for HBeAg, and sense primer, 5’ACTCCACCATAGATCACTCC3’ (nu- one had anti-HBe. Figure 1presents the anti-HCV (C200iC22) titers in cleotides 7-26) [Okamoto et al., 19901. For RNA extraction, 200 pl of serum was mixed with relation to the detection of HCV RNA sequences in sera 100 pl of 21% polyethylene glycol 6000 and 1.5 molil by RT-PCR. Among those with chronic non-A, non-B sodium chloride and left at 4°C for 2 hr. After centrifu- hepatitis, the 20 anti-HCV (C200iC22)-positive cases gation at 18,500g for 20 min at 4”C, the pellet was tested positive with a cut-off index (C.I.) >4; all had resuspended for 15 min in 600 p1 of 0.1 molil Tris-HC1 HCV RNA sequences. As for the three anti-HCV ((22001

phy. Five of the 29 patients had had blood transfusions 7-34 (median 24) years before diagnosis. Sera from 41 patients with chronic HBV infection, who were known to be carriers of HBsAg for more than 12 months were also examined. Twenty-seven patients had hepatitis B e antigen (HBeAg). Antibody to HBeAg (anti-HBe) was detected in 12 of the other 14 patients. Four were asymptomatic HBV carriers with normal liver function tests and 37 were HBV carriers with chronic liver diseases. Liver histology showed the features of CPH in 15 cases, of CAH in 11, and of liver cirrhosis in 6. The remaining five patients had liver cirrhosis with HCC, which was confirmed angiographically. None of these patients had received blood transfusions. All samples were stored at -80°C. The controls consisted of sera from 26 anti-C100-3positive chronic non-A, non-B liver disease patients (six with CPH, seven with CAH, six liver cirrhotics, and seven cirrhotics with HCC) and those from 59 healthy subjects with normal liver function tests.


Chronic Hepatitis C in Japan

TABLE I. Detection of Anti-HCV by Second-Generation ELISA in Patients Negative by First-Generation ELISA Agea Sex Past history of Anti-HCV seropositivity by (M/F) blood transfusion second-generation Ortho ELISA n (years) Patients 20/29 (69%)* 29 56 11* 18/11 5/29 (17%)* Chronic non-A, non-B liver disease 3/41 (7%) 41 42 k 12 32/9 0/41 Chronic HBV carriers



V a l u e s are mean S.D. *P< 0.01 vs. chronic HBV camers.











0 c


2 I



.-c I

c m





0 0




8$ Chronic Non-A, Non-B Liver Disease (n = 29)

Chronic HBV Carriers (n = 41 )

Fig. 1. Titers of second-generation anti-HCV ELISA relative to the detection of HCV RNA sequences. Open circles represent sera negative for HCV RNA sequences, and closed circles represent sera positive for HCV RNA sequences.

C22)-positive HBV carriers, one cirrhotic with HBeAg was positive with a cut-off index >4 and had HCV RNA sequences. The other two were positive for anti-HCV (C2OO/C22)with a cut-off index between 1 and 2. I n these two cases, HCV RNA sequences were not found in the serum by RT-PCR. In all, HCV RNA sequences were detected in 21 (91%) of the 23 patients with anti-HCV (C2OOiC22) and in only one (2%) of the 47 patients negative for it (P < 0.01). All of the 26 anti-C100-3-positive chronic liver disease patients had HCV RNA sequences and were positive for anti-HCV (C200/C22), whereas HCV RNA sequences and anti-HCV (C200/C22)were not detected in the 59 healthy subjects.

DISCUSSION Since the development of a n anti-HCV assay based on C100-3 antigen, HCV has been shown to be the pre-

dominant agent of non-A, non-B hepatitis [Bruix et al., 1989; Colombo et al., 1989; Hopf et al., 1990; Saito et al., 1990; Kiyosawa et al., 1990; Yuki et al., 19921. HCV may also be implicated in chronic liver disease with HBsAg [Fattovich et al., 1989; Bruix et al., 1989; Colombo et al., 1989; Kiyosawa et al., 1990; Kew et al., 19901. However, the problem of sensitivity remains in that anti-C100-3 is directed toward only a small portion of the HCV polyprotein, i.e., toward a 363-amino acid nonstructural polypeptide in a polyprotein of approximately 3,000 amino acids. The present study revealed that a considerable proportion of anti-C 100-3-negative Japanese patients with chronic liver disease is infected with HCV as proved by RT-PCR. HCV RNA was detected in 72% of anti-C1003-negative patients with chronic non-A, non-B hepatitis and in 2.4% (one of 41) HBV carriers. Tests with a second-generation anti-HCV assay based on structural (C22) and nonstructural (C200) HCV proteins revealed a marked correlation between the occurrence of antiHCV (C200iC22) and HCV infection. Approximately 95% of the HCV RNA-positive patients were positive for anti-HCV (C200/C22)whereas 91% of the anti-HCV (C200/C22)-positive patients had detectable levels of HCV RNA in sera. HCV RNA was detected in all cases positive for anti-HCV (C2OOX22) with high titers. These findings suggest that anti-HCV (C200/C22) in chronic liver disease might serve a s a sensitive and specific marker of persistent HCV infection. In conclusion, this study showed that the original anti-HCV (C100-3) assay is not sensitive enough to determine accurately HCV infection in chronic liver disease. HCV was also shown to be a major causative agent of anti-C100-3-negative chronic non-A, non-B hepatitis in Japan. Persistent HCV infection can be confirmed by amplification of HCV RNA sequences with the PCR, but routine techniques are not available. The present study suggests that the second-generation anti-HCV (C2OO/C22) assay with the incorporation of HCV NS3-NS4- and core-associated antigens offers a better way of testing for persistent HCV infection.

ACKNOWLEDGMENTS This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan. REFERENCES Bruix J, Barrera JM, Calvet X, Ercilla G , Costa J, Sanchez-TapiasJM, Ventura M, Val1 M, Bruguera M, Bru C, Castillo R, Rodes J (1989): Prevalence of antibodies t o hepatitis C virus in Spanish patients

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Improved serodiagnosis of chronic hepatitis C in Japan by a second-generation enzyme-linked immunosorbent assay.

To clarify the role of hepatitis C virus (HCV) infection in chronic liver disease, sera from Japanese patients which were negative by the original ant...
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