Detection of Hepatitis C Virus Antibody in the Absence of Viral RNA in Patients with Autoimmune Hepatitis Shuhei Nishiguchi, MD, PhD; Tetsuo Kuroki, MD, PhD; Tadashi Ueda, MD; Katsuhiko Fukuda, MD; Tadashi Takeda, MD, PhD; Shinya Nakajima, MD, PhD; Susumu Shiomi, MD, PhD; Kenzo Kobayashi, MD, PhD; Shuzo Otani, MD, PhD; Nakanobu Hayashi, MD, PhD; and Toshio Shikata, MD, PhD

• Objective: To determine whether laboratory findings showing antibodies to hepatitis C virus (HCV) in patients with autoimmune hepatitis represent falsepositive results and to identify possible explanations for true-positive results in these patients. • Design: Cross-sectional. • Setting: University-based hospital. • Patients: Fifty-two patients with non-A, non-B chronic hepatitis as a control group and 26 patients with classic chronic active autoimmune hepatitis. • Measurements: Comparison of the results of five kinds of assays of HCV antibodies and HCV RNA. • Main Results: Of 52 patients with non-A, non-B chronic hepatitis, HCV antibodies (anti-HCV) were detected in 42 patients (81%; 95% CI, 67% to 90%) by a first-generation enzyme-linked immunosorbent assay (ELISA-I), in 39 patients (75%) by Sp42 ELISA, in 37 patients (71%) by RIA-I, in 49 patients (94%) by ELISAII, and in 48 patients (92%) by RIBA-II. We found HCV RNA in 47 patients (90%; CI, 79% to 97%). Of the 26 patients with autoimmune hepatitis, anti-HCV were detected in 23 patients (88%; CI, 70% to 98%) by ELISA-I, in 12 (46%) by both RIA-I and Sp42 ELISA, in 20 (77%) by ELISA-II, and in 9 (35%) by RIBA-II. However, HCV RNA was found in only five of these patients (19%; CI, 7% to 39%). None of our patients, including controls, had antibodies to superoxide dismutase. Of the 21 patients who had autoimmune hepatitis that was completely responsive to steroid therapy, 18 had anti-HCV by ELISA-I, but 13 of these patients had negative results by RIBA-II, and only two patients had HCV RNA. Of the five patients who did not respond to steroid treatment, all had anti-HCV by ELISA-I, four had negative results by RIBA-II, and three had HCV RNA. • Conclusions: Testing for HCV antibodies in patients with autoimmune hepatitis frequently elicits positive results when the ELISA-I or ELISA-II tests are used. Most of these appear to represent false-positive results because HCV RNA is usually absent from the serum. Such false positivity may result from previous infection with HCV or from cross-reaction of an epitope of HCV. Other patients with apparent autoimmune hepatitis who fail to respond to corticosteroid therapy may actually have chronic hepatitis C (or other non-A, non-B hepatitis) infection.

Annals of Internal Medicine. 1992;116:21-25. From Osaka City University Medical School, Osaka; and Ninon University School of Medicine, Tokyo, Japan. For current author addresses, see end of text.

1 he pathogenesis of autoimmune hepatitis is unknown. Antibodies to hepatitis C virus (HCV) are found in large proportions of several groups of patients with autoimmune hepatitis, suggesting a relation between HCV and autoimmune hepatitis (1, 2), but the nature of this relation is uncertain. Does the finding of antibodies in these patients indicate the presence of HCV infection? Did previous infection with HCV lead to the development of autoimmune hepatitis? MacFarlane and colleagues (3) suggested that the finding of HCV antibodies (antiHCV) in patients with autoimmune hepatitis is a falsepositive result. They found that 20 of 31 (65%) of their patients with autoimmune hepatitis had anti-HCV when tested with a CI00-3 enzyme-linked immunosorbent assay (ELISA) kit based on recognition of the CI00-3 region of the virus. Such antibodies were undetected in only 1 of 22 patients in remission, and correlation existed between the titer of anti-HCV and the gammaglobulin level. To determine whether viremia accompanies the finding of anti-HCV, the presence of HCV RNA in the serum must be ascertained with the polymerase chain reaction (4). A positive result from this test is convincing evidence of HCV infection. Tests designed to detect a particular antibody can yield inaccurate results in infected patients in whom the antibody is not produced and in patients with early-stage disease. For example, HCV RNA can be detected in patients during the early phase of transfusion-related hepatitis, when test results for anti-HCV are negative (5). In patients with HCV infection who respond to interferon treatment, antibodies can be detected after HCV RNA becomes undetectable by the polymerase chain reaction. We report the finding of HCV infection in patients with autoimmune hepatitis. In most of these patients, however, only HCV antibodies were detected, without the presence of HCV RNA. These findings are consistent with those of MacFarlane and coworkers and explain the spontaneous resolution of infection seen in these patients. Ikeda and associates (6) have suggested that these false-positive results are associated with the presence of antibodies to superoxide dismutase in the blood of patients with autoimmune hepatitis. The most widely used assay for HCV antibodies, ELISA-I, incorporates this enzyme, which can therefore give falsepositive results if it, rather than anti-HCV, is detected. We obtained serum samples from patients with autoimmune hepatitis and tested each sample using four types of assay kit. One kit (Sp42 ELISA) does not use superoxide dismutase as a fusion protein; therefore, antibodies to this enzyme cannot be detected using this kit. ©1992 American College of Physicians

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Table 1. Clinical and Laboratory Characteristics of Patients with Autoimmune Hepatitis and Controls with Non-A, Non-B Chronic Hepatitis* Variable

Autoimmune Non-A. Non-B Hepatitis Chronic (n = 26) Hepatitis (n = 52)

Mean age, v Sex Women Men Serum ALT, fikat/L Serum gammaglobulin concentration, f>/L

P Value

52 ± 11

48 ± 13

0.13+

23 3 5.5 ± 4.5

16 36 3.4 ± 2.5

< 0.01$

35 ± 1 1

19 ± 3

< 0.01

RIA-I HCV antibodies were also measured with a C100-3 radioimmunoassay (R1A) (Ortho RIA-1, Ortho and Ohtsuka Assay, Tokushima. Japan) that also used the C100-3 antigen and superoxide dismutase as a single fusion protein. The Ortho RIA-I was a modification of the method of Kuo and colleagues (11).

0.08+

* ALT = alanine aminotransferase: data are given as mean ± SD. + By Wilcoxon rank-sum test. ? By Fisher exact test.

This assay d e t e c t s the p r e s e n c e of antibodies to Sp42, a s e q u e n c e of 42 a m i n o acids in H C V (7). T h e c a u s e s of a u t o i m m u n e hepatitis are not k n o w n , and the search for an etiologic agent c o n t i n u e s . S o m e r e p o r t s suggest that viral infections, such as measles (8) or hepatitis A virus (9), may c a u s e a u t o i m m u n e hepatitis. Examination of the w a y s in which anti-HCV is detected in patients with a u t o i m m u n e hepatitis may determine w h e t h e r a relation exists b e t w e e n H C V and this disease. Methods Patient Sample Study patients had autoimmune hepatitis, were hospitalized in our hepatic care unit, and fulfilled the following criteria: The diagnosis of classic autoimmune chronic active hepatitis was histologically confirmed after liver biopsy. The serum gammaglobulin concentration was at least 20 g/L. and antinuclear antibodies and smooth-muscle antibodies were present. Results of tests for hepatitis B surface antigen (HBsAg) were negative. We selected twenty-six patients for study: 23 women and three men, ranging in age from 27 to 69 years (mean. 52 years: SD, 11 years). Of these 26 patients, 13 had lupus erythematosus cells. Five of these patients had had blood transfusions. We studied a control group of fifty-two patients (16 women. 36 men: range. 22 to 75 years of age: mean ± SD. 48 ± 13 years) with clinical and pathologic findings compatible with non-A, non-B chronic active hepatitis who did not have the autoantibodies named above. We selected every eighth patient chronologically from all patients with non-A. non-B chronic active hepatitis (about 400 patients) who had liver biopsy between 1981 and 1991. Eighteen of these control patients had received blood transfusions. All patients with autoimmune hepatitis or non-A. non-B chronic hepatitis denied intravenous drug use. Table 1 shows patient profiles for both groups. A larger proportion of women existed among the patients with autoimmune hepatitis (P < 0.01). and this group had higher serum gammaglobulin levels (P < 0.01) than did controls. Serum samples obtained from patients with autoimmune hepatitis during the active period of liver damage but before the administration of steroids (and. in some cases, other immunosuppressant drugs) were stored frozen before being tested for HCV antibodies and HCV RNA. Serum samples were obtained from control patients before treatment for HCV. H C V Antibody Testing ELISA-I HCV antibodies were measured with a commercially available kit (ELISA-I, Ortho Diagnostics KK. Tokyo. Japan) ac22

cording to the manufacturer's instructions. The C100-3 antigen used in this kit is a sequence of 363 amino acids, part of which is in the nonstructural (NS) region 3 and part of which is in the NS4 region: the antigen was identified in the genome of HCV by Choo and colleagues (10) and was expressed in yeast. In ELISA-I. the C100-3 antigen and superoxide dismutase are fused and are used to coat the wells of a microplate.

ELISA (Sp42) A sequence of 51 amino acids in the C100-3 antigen was expressed in Escherichia coli and was named the 5-1-1 antigen. We prepared a new assay that uses as the epitope a chemically synthesized peptide with 42 amino acids (Sp42) from the 5-1-1 antigen of HCV (7). This peptide was not fused with superoxide dismutase, but was used alone to coat the microplate wells, allowing the assay to measure antibodies to Sp42. ELISA-II Recently, a second-generation ELISA for anti-HCV (ELISA-II. Ortho) has become available. The kit combines the expression peptide C22-3 from the core region and the expression peptide C33c from the NS3 region with the C100-3 antigen and detects antibodies for these three antigens (12). These four assays for anti-HCV were used to test each serum sample. RIBA-/I Test results were checked with a second-generation recombinant immunoblot assay (RIBA-II: Chiron Corporation, Emeryville. CA: and Ortho. Tokyo, Japan). The RIBA-II, recommended as a confirmatory test, was used to check the specificity of the results from the other tests. This method uses four peptides with sequences of 5-1-1, C100-3, C33c, and C22-3. coded for by HCV. and fixed to a strip. Antibodies to superoxide dismutase are also detectable with this assay (13) (Table 2). All positive results were checked by retesting on two subsequent occasions. H C V R N A Testing We extracted RNA using the method of Chomczynski and Sacchi (14). and complementary DNA (cDNA) was prepared with reverse transcriptase. Primers and a probe were constructed for the 5'-noncoding region (where the base sequence is highly conserved) based on sequence data for HCV (15). The outer primers used were 5'-ACTCCACCATAGATCAC TCCC-3' (sense) and 5-CCCAACACTACTCGGCTAGCA-3' (antisense). The cDNA was amplified by 30 cycles of the polymerase chain reaction. One portion of the products of this reaction was sampled and used with another pair of primers designed to be inside those named above. Another 30 cycles of the polymerase chain reaction was done. We used a sense primer of 5'-TTAGTATGAGTGTCGTGCAGC-3\ and an antisense primer of 5-GGCGCGCCCAAATCTCCAGG-3'. The product obtained was treated by electrophoresis and then by Southern blotting using a probe. 5'-GCAATTCCGGTGTACT CACC-3'. labeled with 32 P. All polymerase chain reactions were initially done in duplicate. This reaction was done a third time with the same serum sample after one week. Each polymerase chain reaction included three negative controls and three positive controls. Using this method, we were able to check for false-positive results caused by contamination and for false-negative results Taq polymerase activity or similar reactions. Statistical Analysis The Wilcoxon rank-sum test, chi-square test, and the Fisher exact test were used in the analysis of data. A P value less than 0.05 was considered significant. Ninety-five percent CIs were used.

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Results

of 5 patients (100%) by Sp42 ELISA, 5 of 5 patients (100%) by RIA-I, 4 of 5 patients (80%) by ELISA-I, 2 of 5 patients (40%) by ELISA-II, and 2 of 5 patients (40%) by RIBA-II.

Patients with Non-A, Non-B Chronic Hepatitis Antibodies to HCV were found in 42 of 52 patients (81%; 95% CI, 67% to 90%) by ELISA-I; 38 of whom had a high titer (cutoff index of 6.0 or more; data not shown). Of the 52 control patients, anti-HCV was detected in 39 patients (75%) by Sp42 ELISA, in 37 patients (71%) by RIA-I, in 49 patients (94%) by ELISAII, and in 48 patients (92%) by RIBA-II. Forty-seven patients (90%; CI, 70% to 97%) had HCV RNA. Therefore, in 94% to 100% of the control patients (depending on the test), each type of assay showed that the patient had " r e a r anti-HCV coexistant with HCV RNA (Figure 1, top). The sensitivity of each type of assay for anti-HCV was calculated by dividing the number of patients with anti-HCV detected with each assay kit by the number of patients who were found to have HCV RNA. The calculated sensitivities for control patients were 39 of 47 patients (83%) by Sp42 ELISA, 37 of 47 patients (79%) by RIA-I, 42 of 47 patients (89%) by ELISA-I, 47 of 47 patients (100%) by ELISA-II, and 46 of 47 patients (98%; CI, 89% to 100%) by RIBA-II. The specificity of each type of assay was calculated by dividing the number of patients who were anti-HCV negative by the number of patients without HCV RNA. The calculated specificities for control patients were 5

Patients with Autoimmune Hepatitis Of the 26 patients with autoimmune hepatitis, 23 (88%; CI, 70% to 98%) were anti-HCV-positive by ELISA-I; of these, 10 patients had a high titer (cutoff index of 6.0 or more). Twelve patients (46%) had antiHCV detected by both RIA-I and Sp42 ELISA; and 20 patients (77%), by ELISA-II. Of these 26 patients, 9 (35%; CI, 17% to 56%) had HCV antibodies confirmed by RIBA-II, but only 5 (19%; CI, 6% to 40%) had HCV RNA. Therefore, "real" HCV antibodies co-existed with HCV RNA in 20% to 44% of such patients, depending on the test (Figure 1, bottom). None of our patients, including the controls, had antibodies to superoxide dismutase (see Table 2). The sensitivities of the assay kits for the patients with autoimmune hepatitis were 3 of 5 (60%) by Sp42 ELISA, 3 of 5 (60%) by RIA-I, 5 of 5 (100%) by C100 ELISA-I, 4 of 5 (80%) by ELISA-II, and 3 of 5 (60%) by RIBA-II. The sensitivities of the assays in patients with autoimmune hepatitis were usually lower than those in patients with non-A, non-B chronic hepatitis, but the differences were not significant. The specificities of the assay kits in patients with au-

Table 2. Antibodies to Hepatitis C Virus (HCV) and HCV RNA in Patients with Autoimmune Chronic Active Hepatitis* Patient

~\ 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

ELISA-I (C100-3) 6.73 6.73 6.73 6.73 6.61 6.73 6.49 5.55 5.28 5.01 2.72 1.82 1.68 1.50 1.31 1.28 1.10 1.04 0.35 0.33 0.27 6.73 6.73 6.56 6.73 4.50

+ + + + + + + + + + + + + + + + + + + + + + +

RIA-I (C100-3)

ELISA (Sp42)

ELISAII

5-1-1

C100-3

+

+ + + + +

+ + +

4+ 4+ 4+

4+ 2+ 2+ 2+

+

+

+ +

-

+

-

+

-

+ + + + +

+ + +

+ + + + +

-

-

+ +

+

+ + +

+ -

+

-. +

-

+

+ + + + + +

-

+

-

RIBA-II C22-3 C33c

-

2+

-

4+ 4+ 4+ 4+ 2+

-

4+ 4+ 4+ 4+

-

4+ 2+ 4+ 4+

4+ + 4+ 4+

4+ 4+ 4+ 4+

4+ 4+ 4+ 4+ 4+

-

-

-

-

HCV RNA

SOD

Decision

_ -

+ + + + + I

_ -

-

-

I + + +

+ -

+

+

-

+

+

+

-

* Patients 1 through 21 had a complete response to steroid treatment. Patients 3, 6, 8, 10, and 24 had a history of blood transfusion. Titers of ELISA-I are expressed as the cutoff index, calculated as the ratio of the optical density (OD) of the sample divided by the mean OD of the three negative manufacturer's controls with 0.400 OD units added to the mean. In RIBA-II, four HCV antigens (5-1-1, C100-3, C33c, and C22-3) and superoxide dismutase (SOD) are coated in bands on a nitrocellulose strip. The antigen band intensities were compared with weakly positive (1+) and moderately positive (3+) control bands, and were graded from negative ( - ) to 4+ for each antigen. A response of 1+ or greater to any one HCV antigen was considered an indeterminate result (I), and a response to two or more HCV antigens was considered a positive result (+). The result for HCV RNA was considered positive if staining was detected by Southern blotting. Minus ( - ) = negative result.

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decreased to normal. Three of these five patients, however, remained anti-HCV positive. We detected HCV RNA before and during steroid treatment in two patients who responded completely to therapy; one had indeterminate results by RIBA-II, and the other had negative results by this assay. One patient had a history of blood transfusion. Their serologic profiles were typical of autoimmune hepatitis and their clinical courses did not differ from those of the 19 patients who did not have HCV RNA. Of the five patients who did not respond completely to therapy (levels o^ ALT or gammaglobulin or both decreased but did not reach or maintain normal values), all had anti-HCV detected by RIA-I, ELISA-I, and ELISAII; four patients had anti-HCV detected by Sp42 ELISA and RIBA-II; and three patients had HCV RNA (Table 2). These patients had severe gammaglobulinemia and a high titer of autoimmune antibodies; two patients had lupus erythematosus cells. One patient had a history of blood transfusion. During steroid treatment, the titer of antiHCV decreased but remained positive, and HCV RNA was detected. Based solely on pretreatment clinical data, these patients could not have been distinguished from those who had a complete response to steroid therapy.

Discussion

Figure 1. Percentages of true- and false-positive findings of hepatitis C virus antibodies by Sp42 ELISA, RIA-I, ELISA-I, ELISA-II, and RIBA-II in (top) non-A, non-B chronic hepatitis and (bottom) classic autoimmune chronic active hepatitis. The finding of HCV RNA is the criterion for HCV infection. The black columns show the percentage of patients with true-positive results of anti-HCV: and the white columns, the falsepositive results. The fractions shown in the columns indicate the number of patients with the anti-HCV assayed as the denominator and either the number of patients with HCV RNA assayed (black column) or the number without HCV RNA (white column) as the numerator. In RIBA-II. the overall results (labeled "Decision") are considered to be positive if two or more of the four HCV antibodies are detected. HCV = hepatitis C virus.

toimmune hepatitis were 12 of 21 (579£) by Sp42 ELISA, 12 of 21 (57%) by RIA-I, 3 of 21 (14%; CI, 2% to 379f) by ELISA-I, 5 of 21 (24r/r) by ELISA-II, and 15 of 21 (71%; CI, 489f to 89%) by RIBA-IL The specificities of the ELISA-1 and -II assays were lower than those of the other assays in patients with autoimmune hepatitis. All patients with autoimmune hepatitis received corticosteroid therapy after serum samples were obtained. Twenty-one (81%) had a complete response as shown by normalization of alanine aminotransferase (ALT) and gammaglobulin levels. The ALT levels of the remaining five patients decreased after therapy but did not attain normal values. Of the 21 patients who responded completely to therapy, 18 had anti-HCV (86%; CI, 63% to 96%) by ELISA-L Of these 18 patients, only 5 had anti-HCV confirmed by RIBA-II. These five patients, however, did not have HCV RNA. The titer of antiHCV detected by ELISA-II decreased in these patients during steroid therapy when the gammaglobulin level 24

Almost all patients with chronic non-A, non-B hepatitis had HCV RNA. Antibodies to HCV were detected at similar rates by the four assays, and almost all these findings were confirmed by RIBA-IL These were therefore probably true-positive results. Results in the patients with autoimmune hepatitis differed widely depending upon the assay kit used. We found HCV RNA in only five of these patients. We cannot exclude the possibility that the polymerase chain reaction failed to detect small amounts of HCV. However, the method we used has a lower limit of detectability ranging from 1 to 10 CID/mL (C1D = chick infective dose), whereas HCV was usually found to be present at levels of I03 to 107 CID/mL in serum samples from 10 patients with chronic hepatitis C (data not shown). The sensitivity o\^ the polymerase chain reaction therefore seemed sufficient to detect viremia in patients with autoimmune hepatitis. Another, perhaps more likely, explanation is that ELISA-I and -II may give more false-positive results than the other kits because of the severe gammaglobulinemia or because of the detection of other antibodies, such as those to anti-superoxide dismutase. Ikeda and associates (6) reported that false-positive results can result from the presence of antibodies to superoxide dismutase, but such antibodies have not been detected in studies done with RIBA (16). All our 26 patients lacked such antibodies. Detection of antibodies by Sp42 ELISA does not involve superoxide dismutase; thus, what seem to be false-positive results in 9 of 12 patients with positive results by Sp42 ELISA (the 9 without HCV RNA) could not have been caused by reactions with superoxide dismutase. Complete response to steroid treatment is sometimes used as a defining characteristic of autoimmune hepatitis. We are unsure whether the inflammation seen in patients with HCV who had incomplete responses to

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steroid treatment was caused by viral activity or by autoimmune response, and perhaps such patients should be excluded when evaluating patients with autoimmune hepatitis. Of the 21 patients who showed complete responses to steroid treatment, only 2 (10%) had HCV RNA. The detection of anti-HCV in patients with autoimmune hepatitis does not prove that the virus is present. If we define a false-positive result as the finding of anti-HCV in the absence of HCV RNA, then most of our patients with autoimmune hepatitis had false-positive results by ELISA-I. Were the results in the five patients who had HCV antibodies as confirmed by RIBA-II nonspecific and therefore false positive? These patients had anti-HCV that were detected by all assay kits involving four antigens in HCV. A possible explanation for these results is that although these patients did not have HCV RNA, the anti-HCV found in these patients reacted with HCV or cross-reacted with autoantibodies with an epitope resembling part of HCV. Mishiro and coworkers (17) reported that an epitope, GOR, was encoded by a host sequence in a chimpanzee infected experimentally with human non-A, non-B hepatitis, and that an antibody to GOR (anti-GOR) was detected in 59 (81%) of 73 patients with this disease. The anti-GOR had affinity to CP10, one of the core peptides of HCV (15, 18). Thus, part of HCV may resemble a peptide produced by the host, and antibodies against this host component, which may appear in autoimmune disease, could cross-react in an assay of anti-HCV. Such a cross-reaction could result in a false-positive result. Previous HCV infection could trigger the production of autoantibodies such as anti-GOR and thus cause autoimmune hepatitis in persons predisposed to this disease. Vento and colleagues (9) recently reported that acute hepatitis A triggered classical chronic active autoimmune hepatitis in 2 of the 58 relatives of 13 patients with autoimmune hepatitis. Immune reactions against viral antigens expressed on cell surfaces occur in patients with hepatitis A, hepatitis B, and non-A, non-B (usually type C) hepatitis. It is therefore possible that helper T cells, reactive to HCV antigens that are exposed on the surface of infected liver cells, assist autoreactive B cells that are specific for an antigen (the asialoglycoprotein receptor) expressed on the surface of the same cell. Querry and Keene (19) proposed a model for one possible etiology of autoimmunity. Antibodies to plOgag, a peptide induced by a retrovirus, crossreact with host snRNP-associated 70K protein. Antibodies to this epitope of the 70K protein bind and allow presentation of immune complexes, causing the pathologic effects associated with autoimmunity. Therefore, in most of our patients with autoimmune hepatitis, the detection of anti-HCV by ELISA-I may be a false-positive result that is nonspecific for HCV. In some patients, however, previous HCV infection seems to have resulted in the presence of anti-HCV, despite the cessation of viremia. In these patients, anti-HCV may cross-react with a host protein such as GOR, leading to pathologic effects, such as those seen when retrovirus antibodies trigger autoimmune disease.

Acknowledgments: The authors thank Ms. H. Fujimoto and Ms. E. Maekawa for technical assistance; Ms. C. Latta for reading the manuscript; and Mr. K. Nukui of Sumitomo Pharmaceutical Company for statistical analysis. Grant Support: In part by a grant from the Ministry of Welfare, Japan. Requests for Reprints: Shuhei Nishiguchi, MD, Third Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahimachi, Abeno-ku, Osaka 545, Japan. Current Author Addresses: Drs. Nishiguchi, Kuroki, Ueda, Fukuda, Takeda, Nakajima, Shiomi, and Kobayashi: Third Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Dr. Otani: Department of Biochemistry, Osaka City University Medical School. 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Drs. Hayashi and Shikata: Department of Pathology, Nihon University, School of Medicine, 30-1, Otaniguchikamimachi, Itabashi-ku, Tokyo 173, Japan. References 1. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet. 1989;2:294-7. 2. Lenzi M, Ballardini G, Fusconi M, Cassani F, Selleri L, Volta U, et al. Type 2 autoimmune hepatitis and hepatitis C virus infection. Lancet. 1990;335:258-9. 3. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet. 1990;335:754-7. 4. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990:335:1-3. 5. Garson J A, Tuke PW, Makris M, Briggs M, Machin SJ, Preston FE, et al. Demonstration of viraemia patterns in haemophiliacs treated with hcpatitis-C-virus-contaminated factor VIII concentrates. Lancet. 1990;336:1022-5. 6. Ikeda Y, Toda G, Hashimoto N, Kurokawa K. Antibody to superoxide dismutase, autoimmune hepatitis, and antibody tests for hepatitis C virus. Lancet. 1990;335:1345-6. 7. Takahashi T. Enzyme-linked immunosorbent assay (ELISA) for hepatitis C using synthetic peptide (in Japanese). Acta Hepatol Jpn. 1990;31:1380-4. 8. Robertson DA, Zhang SL, Guy EC, Wright R. Persistent measles virus genome in autoimmune chronic active hepatitis. Lancet. 1987; 2:9-11. 9. Vento S, Garofano T, Di Perri G, Dolci L, Concia E, Bassetti D. Identification of hepatitis A virus as a trigger for autoimmune chronic hepatitis type 1 in susceptible individuals. Lancet. 1991 ;337: 1183-7. 10. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989;244:359-62. 11. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, et al. An assay for circulating antibodies to a major etiologic virus of human non-A. non-B hepatitis. Science. 1989;244:362-4. 12. Bassetti D, Cutrupi V, Dallago B, Alfonsi P. Second-generation RIBA to confirm diagnosis of HCV infection [LetterJ. Lancet. 1991; 337:912-3. 13. Van der Poel CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, Di Nello R, et al. Confirmation of hepatitis C infection by new four-antigen recombinant immunoblot assay. Lancet. 1991 ;337: 317-9. 14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-9. 15. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, et al. The 5'-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med. 1990;60:167-77. 16. Fusconi M, Lenzi M, Ballardini G, Miniero R, Cassani F, Zauli D, et al. Anti-HCV testing in autoimmune hepatitis and primary biliary cirrhosis [Letter!. Lancet. 1990:336:823. 17. Mishiro S, Hoshi Y, Takeda K, Yoshikawa A, Gotanda T, Takahashi K, et al. Non-A, non-B hepatitis specific antibodies directed at host-derived epitope: implication for an autoimmune process. Lancet. 1990;336:1400-3. 18. Okamoto H, Munekata E, Tsuda F, Takahashi K, Yotsumoto S, Tanaka T, et al. Enzyme-linked immunosorbent assay for antibodies against the capsid protein of hepatitis C virus with a synthetic oligopeptide. Jpn J Exp Med. 1990:60:223-33. 19. Query CC, Keene JD. A human autoimmune protein associated with Ul RNA contains a region of homology that is crossreactive with retroviral p 3 0 j w antigen. Cell. 1987;51:211-20.

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Detection of hepatitis C virus antibody in the absence of viral RNA in patients with autoimmune hepatitis.

To determine whether laboratory findings showing antibodies to hepatitis C virus (HCV) in patients with autoimmune hepatitis represent false-positive ...
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