Journal of Medical Virology 37:158-160 (1992)

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Prevalence of Antibody to Hepatitis C Virus in HBsAg-Negative Chronic Liver Disease in Hong Kong Using Different Assays J.Y. Lai, John S. Tam, L.Y. Lam, and Nancy W.Y. Leung Departments of Medicine ( J Y L , N W Y L ) and Microbiology ( J S T , L Y L 1, Faculty of Medicine, Chinese UniuersLty of Hong Kong, Prince o f Wales Hospital, Shatin, N T , Hong Kong The prevalence of anti-HCV antibodies in Chinese patients with HBsAg-negative chronic liver diseases was studied retrospectively. Anti-HCV was detected by t w o different ELISAs. In 97 patients with HBsAg-negative chronic liver disease, 26 (27%) were anti-HCV positive. Of 157 control subjects, only 1 (0.6%) was anti-HCV positive (P < 0.001). Anti-HCV was detected in 18 of 27 (67%) patients with post-transfusion non-A, non-B (PTNANB) chronic hepatitis or cirrhosis, 5 of 25 (20%) patients with cryptogenic chronic hepatitis or cirrhosis, 2 of 33 (6%) patients with alcoholic liver disease, 1 of 5 (20%) patients with autoimmune chronic active hepatitis (AICAH), none of 4 patients with primary biliary cirrhosis (PBC), and none of 3 patients with fatty liver. The prevalence in this group of patients was lower when compared t o reports from other countries. The addition of a urea washing step reduced false-positivity i n alcoholic and AICAH groups. The ELISA that employs three recombinant HCV antigens confirmed all positive results by another ELISA with the exception of one weakly positive result in the AICAH group and one in the alcoholic group. One patient in the PTNANB group was detected i n addition by the second generation assay. In conclusion, ELISA with a urea wash proved to be useful in reducing falsepositivity, and the second generation assay proved to be a sensitive and specific test for antiHCV antibody. D 7992 Wiiey-Liss, inc.

KEY WORDS: Anti-HCV, chronic liver disease, Chinese patients

INTRODUCTION A blood-borne non-A, non-B hepatitis agent, designated hepatitis C virus (HCV) was identified by Choo et al. 119891. The detection of viral nucleic acid sequences C-100-3 led to the development of a recombinant-based immunoassay for detection of specific antiHCV antibodies (Anti-HCV) [Kuo e t al., 19891. Based 0 1992 WILEY-LISS, INC.

on this assay, 89% of post-transfusion hepatitis cases are considered to be caused by HCV in Spain [Esteban et al., 19901 and 86.5% in Taiwan [Lee et al., 19911. Several studies indicated that HCV may have a pathogenic role in many cases of chronic liver disease, but unexpectedly high seropositivity rates were reported in patients with alcoholic liver disease and those with autoimmune chronic hepatitis [Esteban et al., 1989; Brillanti et al., 1989; Lenzi et al., 19901. A strong correlation was found between Anti-HCV reactivity and total serum globulin in patients with autoimmune chronic active hepatitis (AICAH) which raised the possibility of false-positive results with the Ortho C-100-3 ELISA [McFarlane et al., 19901. Further false-positive results were reported on patients with rheumatoid arthritis [Theilmann et al., 19901, paraproteinaemia [Boudart et al., 19901 and malaria [Aceti et al., 19901. Suggested modifications included a n additional washing step using 8 mol/L urea in ELISA procedure to eliminate lowavidity cross-reacting antibodies [Gray et al., 19901 or using recombinant immunoblot assay (RIBA) with the 5-1-1 and C-100-3 antigens IEbeling et al., 1990; Skidmore, 19901. A new three-antigen ELISA has been developed (Abbott HCV ELISA, 2nd Generation) in which two additional HCV recombinant antigens were added to the C-100-3 antigen. One antigen is from the non-structural NS3 region (33c) and the other is a structural antigen from the core region. We studied retrospectively the prevalence of anti-HCV in Chinese patients with hepatitis B virus surface antigen (HBsAg)-negative chronic liver disease using the Ortho C-100-3 ELISA (Ortho ELISA) and compared the results with Ortho ELISA plus urea wash and the Abbott HCV ELISA 2nd Generation (Abbott HCV-2).

Accepted for publication December 12, 1991. Address reprint requests to Dr. Nancy W.Y. Leung, Department of Medicine, Prince of Wales Hospital, Shatin, NT, Hong Kong.

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TABLE I. Anti-HCV Antibody Results Anti-HCV positive Aetiology PTNANB Cryptogenic Alcoholic AICAH PBC Fatty liver Total Control

Patient Ortho No. ELISA 27 18 (67%) 25 5 (20%) 33 2 (6%) 5 1(20%) 4 0 3 0 97 26 (27%) 157 1(0.6%)

PATIENTS AND METHODS Patients Ninety-seven patients aged 24 to 77 (mean 54), 58 males and 39 females, with HBsAg-negative chronic liver diseases from various causes were studied. This group included 27 patients with post-transfusion non-A, non-B (FTNANB) chronic hepatitis or cirrhosis; 25 patients with cryptogenic chronic hepatitis or cirrhosis; 33 patients with alcoholic liver disease; 5 patients with autoimmune chronic active hepatitis (AICAH); 4 patients with primary biliary cirrhosis (PBC); and 3 patients with fatty liver (diabetes mellitus in one patient and obesity in two patients). Thirty-nine patients had a diagnostic liver biopsy. Diagnosis of cryptogenic chronic hepatitis or cirrhosis was based on no history of alcohol, blood transfusion or drug abuse, negative autoantibodies, and absence of aetiological clue on liver histology. A total of 157 patients, aged 16 to 88 (mean 50), 118 males and 39 females, with respiratory tract infections and without clinical evidence of liver disease were included a s controls. Antibody Testing HBsAg was assayed by reversed passive haemagglutination and monoclonal-based ELISA (Abbott). AntiHCV was detected by the recombinant c-100-3 ELISA (Ortho)on serum stored at -20°C. Specimens were considered positive if the sample OD/cut off ratio were greater than 1. Anti-HCV positive samples were retested with 8 mol/L urea wash during the first ELISA washing procedure to dissociate low-avidity antibodies from the antigen [Gray e t al., 19901. All serum samples of patients were tested further in duplicate with another ELISA (Abbott HCV-2). RESULTS Using Ortho ELISA, 26 of 97 patients with HBsAgnegative chronic liver diseases, had anti-HCV antibodies. By comparison, there was only one positive subject among 157 controls (P< 0.001). Eighteen (67%) of the 27 patients with PTNANB chronic liver disease and 5 (20%) of the 25 patients with cryptogenic chronic liver disease were anti-HCV positive. Among the 33 patients with alcoholic liver disease, 5 patients with AICAH, 4 patients with PBC, and 3 patients with fatty liver, HCV

Ortho ELISA plus urea wash 18 (67%) 4 (16%) 1(3%) 1 (20%) 0 0 24 (25%) 1 (0.6%)

Abbott HCV-2 19 (70%) 5 (20%) 1(3%) 0 0 0

25 (26%) Not tested

antibody was detected in 5, 2, 5, 0, and 0 patients, respectively (Table I). On repeat testing with Ortho ELISA and the addition of a urea washing step, the serology in one cryptogenic cirrhotic and one alcoholic patient became negative. The HCV-2 assay detected one additional patient in the PTNANB group. However, one anti-HCV positive alcoholic patient and the only anti-HCV positive patient in the AICAH group became anti-HCV negative.

DISCUSSION Since the isolation of the HCV genome and the development of a specific antibody test, clinical data on the epidemiology of HCV infection have accumulated rapidly. The worldwide prevalence of anti-HCV in volunteer blood donors is 0.3-6% [Sherlock, 19901 and 6090% in transfusion-associated NANB hepatitis [Dienstag, 19901.Intravenous drug abusers and haemophiliacs also had high positive rates. In most reported series, about half of the patients with anti-HCV had no identifiable risk factors. Most of the data had been reported from western countries. A recent report from Taiwan showed anti-HCV prevalence of 0.95% in volunteer blood donors, 28 of 43 (65%)in HBsAg-negative patients with chronic hepatitis, 13 of 30 (43%) in HBsAg-negative cirrhotics, i.e., overall 41 of 73 (56%) in HBsAg-negative chronic liver disease [Chen et al., 19901. In this study the prevalence of anti-HCV was 0.6% among control subjects without evidence of liver disease. Among the patients with HBsAg-negative chronic liver diseases, 26% were anti-HCV positive by Ortho ELISA, with 67% in PTNANB group, 20% in cryptogenic group, 6% in alcoholic group, and none in PBC and fatty liver. Anti-HCV prevalence of 20% in the AICAH group was not confirmed by the second generation assay. The prevalence in this series is generally much lower than reported from other countries which employed the Ortho ELISA or Chiron RIA [Esteban et al., 1989; Sansonna and Dammacco, 1989; Par, 1990; Chen et al., 19911. Repeating anti-HCV tests in the patients by the Ortho ELISA with the addition of a urea wash step only resulted in minor reduction of the prevalence rates. This implies that false-positivity due to low-avidity antibodies is not a n important factor in our

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patients. However, there were only small numbers of patients in the AICAH and PBC groups because these diseases are uncommon in Chinese patients in Hong Kong. The prevalence of anti-HCV in our patients was highest in the PTNANB group (67%).The consistently antiHCV positive patient with alcoholic cirrhosis received a blood transfusion for bleeding duodenal ulcer four years earlier. Our results confirmed t h a t parenteral transmission is the most important route for HCV infection. The anti-HCV positive patients in the cryptogenic group may represent sporadic cases of HCV infection. One patient in the cryptogenic group positive for anti-HCV by Ortho ELISA became negative after the urea wash and was again positive by Abbott HCV-2. Urea washing probably eliminates false positivity and its incorporation in the HCV-2 assay may be useful for avoiding low-avidity antibodies. One patient in the alcoholic group with anti-HCV by Ortho ELISA with a sample ODicut off ratio of 2.2 became negative after the urea wash. This was confirmed a s negative by the HCV-2 assay, demonstrating a n increased specificity of Ortho ELISA after urea wash. The patient in the AICAH group who was anti-HCV positive by Ortho ELISA had a sample OD/cut off ratio of 1.5 before and 1.1after urea wash, implying weak positivity only. The negative result with the Abbott HCV-2 test in this patient strongly suggests false-positivity. Based on the results shown in Table I, the Ortho ELISA plus urea wash had a sensitivity of 92%, specificity of loo%, and percentage agreement of 98%. One patient in the PTNANB group was detected in addition by the Abbott HCV-2 assay. One patient in the alcoholic group with a definite history of blood transfusion was consistently anti-HCV positive by both Ortho ELISA and Abbott HCV-2. The patient in the AICAH group with weakly positive anti-HCV by Ortho ELISA was negative by Abbott HCV-2. These were examples of improved sensitivity and specificity of HCV-2 assay. The HCV-2 assay had a sensitivity of 93%, specificity of 99%, and percentage agreement of 97%. The accuracy in the diagnosis of HCV-related liver disease is obviously a n important first step in the understanding of the clinicopathological features of HCV infection. Further improvement in serological assays in both sensitivity and specificity is essential. Recently a second generation four-antigen recombinant immunoblot assay (4-RIBA) has been developed a s a confirmatory test for HCV infection [Van der Poel et al., 19911. Detection of HCV RNA by the technique of complementary DNAipolymerase chain reaction is another major serological advance in the detection of the replicating hepatitis C virus [Weiner et al., 19901.

ACKNOWLEDGMENTS The authors thank Abbott Diagnostic Division for confirmation of our Abbott HCV ELISA 2nd Generation test results and Miss Phyllis Yiu and Miss Hidy Y.L. Yeung for technical and secretarial assistance.

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Prevalence of antibody to hepatitis C virus in HBsAg-negative chronic liver disease in Hong Kong using different assays.

The prevalence of anti-HCV antibodies in Chinese patients with HBsAg-negative chronic liver diseases was studied retrospectively. Anti-HCV was detecte...
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