serves a 750-bed university hospital, for measurement of Tokyo, Japan), the Abbott enzyme immunoassay (EIA; EIA

Transcription

1 JOURNAL OF CLNCAL MCROBOLOGY, Nov. 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 31, No. 11 Analysis of Discordant Test Results among Five Second-Generation Assays for Anti-Hepatitis C Virus Antibodies Also Tested by Polymerase Chain Reaction-RNA Assay and Other Laboratory and Clinical Tests for Hepatitis. NAKAGR,1 K. CHHARA,l* K. OHMOTO,2 M. HROKAWA,3 AND N. MATSUDA' Department of Clinical Pathology, 1 Department of Gastroenterology,2 and Department of Pathology,3 Kawasaki Medical School, Okayama, Japan Received 6 April 1993/Returned for modification 27 May 1993/Accepted 29 July 1993 The diagnostic performances of five commercially available second-generation assays for anti-hepatitis C virus antibody, two enzyme-linked immunosorbent assays, one enzyme immunoassay, and two particle agglutination assays (passive hemagglutination assay and particle agglutination assay), were evaluated. Among 104 samples from healthy subjects and 300 consecutive samples from patient ordered for routine determinations of anti-hepatitis C virus antibody in serum, assay results showed variable degrees of discordance for 17 samples (4.2%). These 17 samples were further tested by an immunoblot assay, the polymerase chain reaction-rna assay, and the hemagglutination inhibition assay. Four of the 17 samples were regarded as true positive, since all supplementary assays and clinical data indicated active hepatitis C virus infection. Another five samples were considered false positive because no confirmatory evidence was obtained from the laboratory analysis or clinical data. The remaining eight samples were negative for hepatitis C virus RNA, but the results of the other supplementary tests were indeterminate. Some of these samples with indeterminate results may have been from patients with subclinical cases of disease who spontaneously recovered from hepatitis with persistent anti-hepatitis C virus antibody in their sera. Shortly after the nucleotide sequence of the hepatitis C virus (HCV) genome was identified by Choo et al. (1) in 1989, the first enzyme-linked immunosorbent assay (ELSA) system for the detection of anti-hcv antibody (anti-hcv- Ab) was developed by Kuo et al. (5) by using the recombinant HCV C100-3 antigen. Anti-HCV-Ab was detected in a majority of patients with non-a, non-b hepatitis. The assay contributed greatly not only to clarification of the behavior of the elusive HCV but also to the identification of people who are possibly at risk of transmitting or acquiring HCV infection through blood products. The specificity and sensitivity of the assay, however, are not sufficient, and many false-positive results have been reported, especially in patients with autoimmune hepatitis (2, 4, 7). Furthermore, detection of seroconversion in patients with acute HCV infection is often delayed until 3 months to 1 year after the exposure (8). To overcome these problems, new assay systems for anti-hcv-ab (second-generation assays) have recently been developed and have become available for routine use. The assays use various mixtures of recombinant HCV antigens and can detect a broader range of anti-hcv-abs. n the study described here, the diagnostic performances of five commercially available second-generation assays for anti-hcv-ab were evaluated and compared with the results of the polymerase chain reaction (PCR) for serum HCV and with those of other supplemental assays and clinical evidence of hepatitis. * Corresponding author MATERALS AND METHODS Materials. For the analysis we used 300 consecutive serum samples routinely submitted to our clinical laboratory, which serves a 750-bed university hospital, for measurement of anti-hcv-ab. Many of the serum samples were from the departments of gastroenterology, urology, and surgery. Samples were also taken from 104 healthy adults (30 males, 74 females; age range, 22 to 40 years) with normal alanine aminotransferase and aspartate aminotransferase levels and with no previous history of hepatobiliary disorders or blood transfusion. All of the samples were stored at -80 C until the time of the assays. Reagents and assay methods. All samples were tested for anti-hcv-ab by the following five assays: the mucheck ELSA (nternational Reagents Corp., Kobe, Japan), the Ortho ELSA (ELSA ; Ortho Diagnostic Systems nc., Tokyo, Japan), the Abbott enzyme immunoassay (EA; EA ; Abbott Diagnostics Division), a passive hemagglutination assay (PHA; Dainabot Corp. Ltd., Tokyo, Japan), and a particle agglutination assay (PA; Ortho Diagnostic Systems nc.). A map of the amino acid sequences of the recombinant HCV antigens used in each assay system is shown in Fig. 1. ELSA and EA. n both the mucheck and ELSA ELSAs, microplate wells coated with the HCV antigens were used. n the mucheck ELSA, 20,ul of a serum sample was added to 200,u of dilution buffer in the wells. After incubation for 1 h at 37 C, the wells were thoroughly washed to remove the unbound antibodies and serum, and then 100 p,l of a peroxidase-conjugated antibody solution specific for human immunoglobulin G (gg) was added to each well. After another incubation for 30 min at 37 C, the unbound

2 VOL. 31, 1993 DSCORDANCE AMONG FVE ANT-HCV ANTBODY ASSAYS ' - C El/S E2 / NS1 NS2 NS3 NS NS5 K3' 301 laa Ortho ELSA C c cloo c33c Ortho PA Abbott EA Dainabot PHA mucheck c PHCV c c33c BCD phcv cloo C-11 C ? ? FG. 1. Epitope maps of the recombinant antigens used in the five anti-hcv-ab assays. The distribution of the epitopes corresponding to the recombinant antigens is shown for the five anti-hcv-ab assay systems. The abbreviations and the amino acid sequence numbers were adopted from those shown by Houghton et al. (3a); C, core region; El, envelope; NS, nonstructural region. fraction of the conjugate was washed away and 100,ul of a solution of hydrogen peroxide and o-phenylenediamine was added to each well. n the presence of a bound conjugate, the o-phenylenediamine was oxidized to give a colored end product. The enzyme-substrate reaction was terminated by the addition of 100,u of 2 N sulfuric acid. The A492 of the final solution was measured. A sample was considered to be positive when the absorbance exceeded the cutoff value determined from the following formula: cutoff value = NCx (0.2)- PCx, where NCx and PCx represent the mean absorbance of three negative controls and that of three positive controls, respectively. n the ELSA, 20,ul of the serum sample was added to 200,ul of dilution buffer in the wells. After incubation for 1 h at 37 C, the wells were thoroughly washed to remove the unbound fraction, and then 200 p1 of a peroxidase-conjugated anti-human gg solution was added to each well. After another incubation for 1 h at 37 C, the microplate was washed and 200 pl of a solution of hydrogen peroxide and o-phenylenediamine was added to each well. The enzymesubstrate reaction was terminated after 30 min by the addition of 50,ul of 4 N sulfuric acid. The A492 of the sample was measured. The cutoff value was set by the following formula: cutoff value = NCx 0.600, where NCx represents the mean of three negative control readings. The constant was set by the manufacturer to keep the frequency of false-positive results at an optimal level. n the EA, 10 pu1 of the serum sample was diluted 41-fold in a polyethylene test tube. Two hundred microliters of the diluted serum was transferred to a special Abbott plate well. Then, a polystyrene bead coated with HCV antigens was added and the mixture was incubated for 1 h at 40 C. After washing the bead, a solution of peroxidase-conjugated antibody directed against human gg was added, and the mixture was incubated for an additional 30 min at 40 C. The unbound conjugate was then removed and a solution containing o-phenylenediamine and hydrogen peroxide was added. The reaction was terminated by adding 1 ml of 1 N sulfuric acid. The color intensity was measured as described above for the other two methods. Samples were considered positive when their absorbances exceeded the cutoff values determined from the following formula: cutoff value = NCx (0.25)PCx, where NCx and PCx represent the mean absorbances of three negative controls and three positive controls, respectively. PHA and PA assays. The PHA and PA assays were performed in microplates with V-shaped and U-shaped wells, respectively. A fuzzy layer of agglutinated particles is formed at the bottom of the wells when anti-hcv-ab is present in serum, whereas a tight button pattern appears in the absence of the antibody. For the PHA assay, 25 pul of serum was diluted 32-fold in a well by using a micropipetter. Twenty-five microliters of the diluted serum was then transferred to another well, and 25 pul of erythrocytes coated with the HCV antigens was added by using a microdropper. n the PA assay, 25 p,l of serum was diluted 8- or 16-fold. Twenty-five microliters of serum diluted to 1:16 was transferred to a well and was mixed with 25 p,l of gelatin particles coated with the HCV antigens (sensitized gelatin particles). As a control for each sample, 25 p,l of serum diluted to 1:8 was reacted with unsensitized gelatin particles specially coated with superoxide dismutase, a protein used in the production of the recombinant HCV antigens and, therefore, a possible target of a nonspecific reaction in the assay. After the plate was shaken, it was kept at room temperature for 2 h, and then the agglutination pattern was judged by inspection. All the reagents were used within 3 days after preparation

3 2976 NAKAGR ET AL. to ensure their stability. The positive results of the PHA assay were confirmed by an antibody inhibition assay supplied by Dainabot Corp. Ltd. This assay is designed to detect nonspecific reactions by preincubating anti-hcv-ab-positive sera with the recombinant antigens used in the PHA assay. RBA-2. All samples showing positive test results in any one of the five anti-hcv-ab assays were further tested by the second-generation recombinant immunoblot assay (RBA-2; Ortho Co.) to analyze the reactivity of the sample antibodies to four recombinant HCV antigens attached discretely to a nitrocellulose strip. PCR-RNA. The PCR-RNA assay for HCV was performed by using a modification of the procedure described by Garson et al. (3), which took advantage of the highly conserved nucleotide sequences within a 5'-noncoding region of the HCV genome. RNA was extracted from 100,ul of serum by the acid guanidium thiocyanate-phenol-chloroform method and was converted to cdna with reverse transcriptase. The cdna was then amplified by the nested PCR method by using two sets of primers. The sequences of the outer primers were 5'-CTGTCTTCACGCAGAAAGCG-3' (sense; nucleotide positions 41 to 60 in the HCV genome according to the revised numbering system of Okamoto et al. [6]) and 5'-CACTCGCAAGCACCCTATCA-3' (antisense; nucleotide positions 277 to 296). The inner primers were 5'- CGTTAGTATGAGTGTCGTGC-3' (sense; nucleotide positions 72 to 91) and 5'-AGGCATTGAGCGGG1TlATC-3' (antisense; nucleotide positions 184 to 203) (8). Thirty cycles of denaturation for 2 min at 94 C, annealing for 1 min at 60 C, and extension for 2 min at 72 C were carried out. The PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide, and the resultant pattern was photographed. RESULTS Spectrum of anti-hcv-ab specificity in reference to the immunoblot assays. Prior to the present study, we performed the RBA-2 on samples which screened positive for anti- HCV-Ab by the first-generation assay and identified three patterns of combinations of anti-hcv-abs. The first pattern was characterized by the specificity of the anti-hcv-ab almost exclusively to the core antigen C22-3 (Table 1). Results for two typical samples belonging to this pattern are shown in Table 1 along with the corresponding results of the five assays. The two samples were clearly detected by all methods, and there was no major difference in the titer, which was determined by serial dilution from 1:1 to 1:64. The second pattern was a combination of anti-hcv-abs which possess specificity only to the nonstructural (NS4 region) antigen designated C As shown for two typical samples in Table 1, the results for both samples were negative by the mucheck ELSA but were mixed by the EA and PA methods. On the other hand, the ELSA and PHA methods gave clearly positive results even at higher dilutions. Anti-HCV-Abs which belonged to the third pattern showed specificity to all four antigens covered in the RBA-2. All five assays gave positive results for the two typical samples for which the results are shown in Table 1. Summary of the results of the five anti-hcv-ab assays. Comparisons of the results from the five HCV-Ab assays with samples from patients and healthy subjects are shown in Table 2. The test results were negative by all of the assays J. CLN. MCROBOL. for 222 samples. The results were positive by all of the assays for 62 samples. All samples were confirmed to be HCV positive from the positive results of subsequent PCR- RNA assays. For 17 subjects, the results of the five assays showed various degrees of discordance. One of the healthy subjects whose serum was positive by the mucheck ELSA was 1 of the 17 subjects whose sera had various degrees of discordance by the five assays. Analyses of samples with discordant results. All of the 17 samples with discordant results showing positive results by any one of the five assays were further tested for their antibody spectra by the RBA-2 and for the presence of viral RNA in serum by the PCR-RNA assay. Some of them were also tested by the hemagglutination inhibition assay to exclude any nonspecific reaction by the PHA method. The summary of the results for these 17 patients is shown in Table 3. Sera from 4 of the 17 patients were found to be positive for HCV RNA by PCR, and 7 of the 9 serum samples which were tested for anti-hcv-ab by the inhibition assay gave results in agreement with the results of the PHA assay. On the other hand, 5 of the 17 serum samples were nonreactive to all four HCV antigens arranged in the RBA-2. On the basis of the results of the supplemental assays, the validities of the five anti-hcv-ab assay results for those conflicting samples can be interpreted as follows. Since serum positivity by the PCR method rules in active infection and sets a "gold standard," the negative test results for patients 1 through 4 in Table 3 can be regarded as falsenegative ones, whereas the negative results for all three supplemental assays almost certainly rule out actual HCV infection. Hence, the results for patients 13 through 17 in Table 3 indicate false-positive results. On the other hand, we cannot make any definite comments regarding the data for patients 5 through 12 in Table 3 unless we turn to clinical evidence or other laboratory evidence of hepatic infection. By setting a positive PCR-RNA test result as a gold standard for active HCV infection, the sensitivity and specificity of each assay were calculated from the data in Table 2 with the results presented in the boxed area categorized by using the information in Table 3. They were, respectively, 97.0 and 97.9% for the mucheck ELSA method, 97.0 and 98.8% for ELSA, 93.9 and 100% for EA, 98.5 and 98.5% for PHA, and 100 and 99.1% for PA. Clinical and laboratory data for the samples with discordant results. The relevant clinical and laboratory data regarding possible hepatic infection for the 17 patients whose sera showed discordant results by the five assays are shown in Table 4. The first four patients, whose sera gave positive results for viral RNA, were actually diagnosed with acute or chronic hepatitis because of current or previous elevations in the levels of the transaminases. Histological evidence of infection was also obtained for all four of these patients. Among the remaining 13 patients (1 from the healthy group and the others with variable nonliver disorders), 12 patients had no clinical history of hepatitis at the time of the blood test for anti-hcv-ab. Three of the 13 patients had recent histories of blood transfusion, but all donor blood was found to be negative for anti-hcv-ab by a second-generation assay. As a matter of fact, there was no or only a slight elevation in serum transaminase levels in the 13 patients, with the exception of patient 17, who had a well-documented episode of transient hepatic ischemia secondary to circulatory shock. Furthermore, in response to the positive results for anti-hcv-ab, liver biopsies were performed in 3 of the

4 VOL. 31, 1993 DSCORDANCE AMONG FVE ANT-HCV ANTBODY ASSAYS 2977 TABLE 1. Antibody spectra and sensitivities of the five anti-hcv-ab assays compared with the result of an immunoblot assay' Pattern and mucheck ELSA Ortho ELSA Abbott EA Dainabot Ortho RBA-2 sample Dilution PHA PA sample ~~OD CO OD COT OD coi HA P C100-3 C33C C22-3 Core-positive pattern 1 2 NS4-positive pattern 3 4 Both core- and NS4-positive patterns [ 0.938] 2.78U l1.56 j F [ W w W >3.000 > ] Ljj [ _ ] _. - _ W _ >3.000 >10.00 >3.000 >5.00 >2.200 > >3.000 > >3.000 >5.00 >2.200 > >3.000 >10.00 >3.000 >5.00 >2.200 > >3.000 > >3.000 > >3.000 > , w 2 ± a On the basis of reactivity to four distinct epitopes (5-1-1, C100-3, C33C, and C22-3) in the immunoblot assay, anti-hcv-ab-positive sera were grouped into three patterns. The test results of two typical serum samples in the five anti-hcv-ab assays are shown for each pattern: those with almost exclusive specificity to the core antigen C22-3, those with specificity only to the nonstructural (NS4 region) antigen C100-3, and those with specificity to all four antigens distinguishable by RBA-2. Each sample was serially diluted. OD, optical density; CO, cutoff index. Superscript w's indicate a weakly positive reaction. Boxing indicates a positive test result.

5 2978 NAKAGR ET AL. J. CLN. MCROBOL. TABLE 2. Summary of results of the five anti-hcv-ab assays' No. of samples from: mucheck Ortho Abbott Dainabot Ortho Healthy Patient ELSA ELSA EA PHA PA controls samples (n = 104) (n = 300) a Test results that were below the cutoff value are indicated by a minus sign, and those that were above the cutoff value are indicated by a plus sign. The test results for the five assays were classified according to their patterns. The two rightmost columns represent the sums of each pattern for samples from healthy adults and patients. Boxing indicates samples exhibiting various degrees of discordance by the five assays. The sensitivity and specificity of the assay were calculated from the data in this table after classifying the data in the boxed area according to the results in Table 3 by setting a positive PCR-RNA test result as a "gold standard" for HCV infection. 13 patients, but the histological findings were negative for active liver disease for all 3 patients. Accordingly, among the 17 patients whose sera provided discordant results, the first 4 patients can also be regarded as true positive for HCV infection on clinical grounds, while for the remaining patients, the likelihood of active infection seemed to be very low. DSCUSSON Several second-generation assays for anti-hcv-ab detection are now available. Many of them use the principle of TABLE 3. ELSA or EA, but those that use the principle of particle agglutination are also in use. The latter assays use either erythrocytes (PHA) or gelatin particles (PA) as a solid support for the HCV antigens, allowing easy detection of the reaction without special equipment. There have, however, been no reports on comparisons of the ELSA or EA and PHA or PA methods. n the present study, we compared the results of the five anti-hcv-ab assays (two ELSAs, one EA, and two particle agglutination assays [PHA and PA]) that are commercially available in Japan. All of them had sufficient precision; intraassay variations in terms of the coefficient of variation for the two ELSAs and the one EA method were all within 6%. The reproducibilities of the PHA and PA methods were within a difference of plus or minus one well. The performances of the five methods were assessed from the results of RBA-2, the antibody inhibition assay, as well as by the nested PCR-RNA assay. Results of the five assays agreed very well for 387 of 404 samples tested. Among the 17 samples that gave discordant results, 4 were confirmed to be positive for HCV in serum by the PCR-RNA assay, and it was strongly suspected that the patients had active infections on the grounds of clinical information and the positive results by RBA-2. Since the PHA and PA methods consistently gave positive results for the four samples, the chance that these tests would overlook those patients with borderline infections seemed to be low. Samples from another 5 of the 17 patients with discordant results were found to be consistently negative by all the supplemental assays, and the patients had no clinical evidence of hepatitis. On the other hand, interpretation was not easy for the remaining eight patients whose serum samples had discordant results by the five assays. On clinical grounds, seven of them had no apparent history of hepatitis or transfusion of blood products untested for HCV. None underwent any form of interferon therapy, which frequently leads to the disappearance of HCV in serum with or without concomitant lowering of serum anti-hcv-ab levels. Liver tissues were Discordance among the five anti-hcv-ab assays and results of supplemental assaysa Patient mucheck Ortho Abbott Ortho Dainabot Antibody RBA-2 no. ELSA ELSA EA PA PHA inhibition HCV-RNA assay C22-3 C33C C SOD ()b 0.12(-) 0.31(-) W _ () 1.89() 0.62(-) W (-) 0.25(-) 0.33(-) W (-) 4.40() 0.92(-) W 2 _ () 0.01(-) 0.14(-) - - NT (-) 0.18(-) 0.20(-) (-) 0.24(-) 0.22(-) - NT (-) 0.10(-) 0.42(-) (-) 4.42() 0.46(-) 2 (*1) _ (-) 1.36() 0.62(-) - W _ (-) 1.02 () 0.80 (-) W _ NT ± (*2) () 2.04() 0.88(-) W 2 (*3) () 0.07(-) 0.32(-) - - NT () 0.12(-) 0.34(-) - - NT () 0.11(-) 0.13(-) - - NT () 0.14(-) 0.28(-) - - NT () 0.16(-) 0.35(-) - - NT a Test results for the 17 samples which gave discordant results by the five anti-hcv-ab assays. NT, not tested; w, weakly positive; SOD, superoxide dismutase. Some of the results for the core region obtained by RBA-2 were retested later. The indeterminate value () for C22-3 in serum sample 9 was negative 8 months later (*1), and that in serum sample 11 remained at the indeterminate level 6 months later (*2). The positive results for serum sample 12 decreased to the indeterminate level of in 10 months (*3). Repeated PCR-RNA tests carried out simultaneously by RBA-2 were also negative for the three serum b samples. Data in columns 2 through 4 are cutoff indices and their plus/minus judgments.

6 VOL. 31, 1993 DSCORDANCE AMONG FVE ANT-HCV ANTBODY ASSAYS 2979 TABLE 4. Clinical and laboratory data for serum samples from 17 patients with discordant results by the five anti-hcv-ab assaysa Patient ALT AST gm Past History of Crnc Liver no. Age (yr) Sex (U/iter) HA-Ab HBs-Ag history of blood Clinical diagnosis hepatitis biopsy hepatitis transfusion result 1 44 F 1, Acute hepatitis Hepatitis 2 11 F Chronic hepatitis Hepatitis 3 53 M Chronic hepatitis Hepatitis 4 70 F Chronic hepatitis Hepatitis 5 28 F None, found by chance M Head trauma - Normal 7 88 M Unknown fever 8 70 F Unknown fever - Normal 9 42 M Male infertility F Epilepsy F None, found by chance M Angina pectoris F Cholangiocarcinoma F Multiple cerebral infarction M Pulmonary tuberculosis F Multiple cerebral infarction M Lung cancer - Normal a F, female; M, male; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HA-Ab, anti-hepatitis A virus antibody; HBs-Ag, hepatitis B virus surface antigen. obtained from three of the patients, but none was found to have histological evidence of hepatitis. Furthermore, their levels of transaminases were normal or only slightly elevated, and the levels of anti-hcv-ab in serum, in general, were only slightly above the cutoff values. On the basis of this evidence, active HCV infection among the patients seems very unlikely. The results of the supplemental assays, however, were mixed because serum from three of the eight patients showed indeterminate results for a core region antibody by RBA-2. Furthermore, the inhibition assay confirmed the validity of the PHA assay for four of the five PHA-positive samples tested. Consequently, there was still a possibility of actual HCV infection which could not be detected by the primers used for the PCR assay or which took a subclinical course and ameliorated spontaneously but with the persistence of anti-hcv-ab in serum. Although no appreciable differences were observed in the overall diagnostic sensitivity or specificity among the five methods, the numbers of false-positive or false-negative results were slightly lower in the particle agglutination assays. These may be attributable to the fact that the antigen-attached particles can react to both gm and gg and no distinction is possible between them during the detection step. n contrast, since the ELSA and the EA methods use enzyme-labeled anti-human antibodies which are directed against gg, they cannot detect the gm-type anti-hcv-abs. Regarding the supplemental tests for anti-hcv-ab-positive sera, the RBA-2 has been the most widely used (9, 10). There are, however, few reports on its performance for borderline cases such as those described here. Our results suggest that there are still some patients whose infections cannot be clearly classified by RBA-2, despite improvement in its performance from its first-generation counterpart. From a practical point of view, selection of one of the five assays depends on a laboratory's situation and size. n an emergency laboratory or a doctor's office, the PHA or PA assay is the method of choice, because they have sufficient sensitivity and rapidity and are convenient. For quantitative measurement of the antibody titer, however, these methods require serial dilution, and thus, the costs of the reagents cannot be ignored. On the other hand, the ELSAs or EA, which can be performed with fully automated equipment, are suitable for large laboratories. The level of anti-hcv-ab can be obtained as a continuous value within the dynamic ranges of the assays. ACKNOWLEDGMENTS We are grateful to nternational Reagents Corp., Dainabot Corp., and Ortho Diagnostic Systems nc. for generously supplying reagents and technical support. We also express our gratitude to S. Yamamoto and A. Harano, Kawasaki Medical School, for helpful comments on our work. REFERENCES 1. Choo, Q.-L., G. Kuo, A. J. 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7 2980 NAKAGR ET AL. 7. Onji, M., T. Kikuchi, K. Michitaka,. Saito, T. Miyamura, and Y. Ohta Detection of hepatitis C virus antibody in patients with autoimmune hepatitis and other chronic liver diseases. Gastroenterol. Jpn. 26: Saito, M., A. Hasegawa, T. Kashiwakuma, M. Kohara, M. Sugi, K. Miki, T. Yamamoto, H. Mori, Y. Ohta, E. Tanaka, K. Kiyosawa, S. Furuta, M. Wakashima, S. Tanaka, and N. Hattori Performance of an enzyme-linked immunosorbent assay system for antibodies to hepatitis C virus with two new antigens J. CLN. MCROBOL. (cll/c7). Clin. Chem. 38: Yuki, N., N. Hayashi, H. Hagiwara, T. Takehara, M. Oshita, A. Kasahara, H. Fusamoto, and T. Kamada Serodiagnosis of chronic hepatitis C in Japan by second-generation recombinant immunoblot assay. J. Hepatol. 17: Yun, Z. B., G. Lindh, 0. Weiland, B. Johansson, and A. Sonnerborg Detection of hepatitis C virus (HCV) RNA by PCR related to HCV antibodies in serum and liver histology in Swedish blood donors. J. Med. Virol. 39:57-61.