Prevalence of Antibodies to Hepatitis C Virus among Patients with Cryptogenic Chronic Hepatitis and Cirrhosis LENNOX J. JEFFERS,~ FUAD MARIA DE MEDINA,~ RAJENDER REDDY,~ TALLEYPARKER,l LEONARDO MENDEZ,~ EUGENER. SCHIFF,' MICHAELMA"S,' MICHAELHOUGHTON,3 QUI-LIMCHO03 AND GEORGEKU03
'Division of Hepatology, University of Miami School of Medicine and Veterans' Administration Medical Center, Miami, Florida, 331 01; 2Medizinische Klinic und Poliklinik, Johannes Gutenberg-Universitat Mainz, 0-6500Muinz, Germany; and 3the Chiron Corporation, Emeryuille, California 94608
Many cases of chronic hepatitis and cirrhosis cannot be attributed to a known cause and are collectively referred to as cryptogenic chronic liver disease. We have evaluated the role of the hepatitis C virus in the pathogenesis of this condition in a retrospective serum analysis for antibody to hepatitis C virus in 129 patients with cryptogenic liver disease. Other causes of chronic hepatitis and cirrhosis were ruled out by clinical, serum biochemical and serological techniques. All 129 patients were HBsAg negative, but 28 (22%)had antibody to HBcAg. Sera were tested by radioimmunoassays using recombinant peptides for antibodies to nonstructural ((2100-3 and C33c) and structural regions (C22) of HCV. Among the 129 patients, 61 (47%)had antibody to C100-3, 76 (59%)had antibody to C33c and 74 (57%)had antibody to C22. Seventy-nine(61%)were reactive with at least one and 76 (59%)were reactive with at least two HCV peptides (this is the criterion used for hepatitis C virus antibody reactivity). A proportion of patients with chronic hepatitis and cirrhosis (55 of 91; 60%) similar to that of patients without cirrhosis (21of 38; 55%)had hepatitis C virus antibody. No significant clinical, serum biochemical or histological differences were noted between the group of patients with hepatitis C virus antibody and those without this antibody reactivity. Thus more than half the patients with cryptogenic chronic liver disease had hepatitis C virus antibody, suggesting that chronic HCV infection plays a major role in the origin of cryptogenic chronic hepatitis and cirrhosis. (HEPATOLOGY 1992;15:187-190.)
considerable number of patients remains elusive. Non-A, non-B (NANB) viruses have been implicated as a cause of some cases (11, but because of the lack of specific serological markers, the association has been epidemiological. The identification of a blood-borne NANB hepatitis agent designated hepatitis C virus (HCV) by Choo and coworkers led to the development of a recombinant-based immunoassay for the detection of specific antibodies to HCV (anti-HCV) (2, 3). We carried out a study to determine the prevalence of anti-HCV in sera of patients with chronic hepatitis and cirrhosis of unknown origin (cryptogenic chronic liver disease). SUBJECTS AND METHODS
Subjects. The records of all patients with cryptogenic chronic liver dwease seen at the Center for Liver DiseasesUniversity of Miami Hospital and Clinics from January 1978 to March 1989 were reviewed. The following categories of patients were excluded: (a) HBsAg-positive patients; (b) patients who consumed more than 80 gm alcohol/day; (c) patients with histories of blood transfusion; (d) intravenous drug users; (e) male homosexuals; (f) medical and dental personnel; (g) patients with hemochromatosis, PBC, Wilson's disease or a,-antitrypsin; (h) patients who had antinuclear antibody (ANA) in titers greater than 1:40 or anti-smooth muscle antibody (ASMA) in titers greater than 1:20; and (i) patients whose hepatitis responded to corticosteroid therapy. Diagnoses of chronic hepatitis and cirrhosis were based on these criteria: ALT levels 1.5 times the upper limit of normal or greater for more than 6 mo, with biopsy-proven chronic Identifying the origin of chronic hepatitis and cir- hepatitis or cirrhosis (4).A total of 129 patients fulfilled the rhosis can be difficult. In general, the various causes can criteria for inclusion in the study. None of these patients had be distinguished by their clinical and epidemiologic a history of acute hepatitis or jaundice. Ninety-one patients features, viral and autoimmune serologic markers, had cirrhosis, and 38 had chronic hepatitis without cirrhosis. biochemical tests and histological characteristics. Nev- Fifty-nine patients with chronic hepatitis B and 200 normal donors served as controls for serological testing. ertheless, the causeb) of chronic liver disease in a blood Anti-HCV Serological Tests. Serum samples frozen at -20" C were retrieved from storage, coded and tested for anti-HCV by the method of Kuo et al. (3). In this RIA, a recombinant HCV nonstructural polypeptide referred to as Received March 20, 1991; accepted October 8,3991. C-100-3 was used. Sera were tested also by newly developed Address reprint requests to: Lennox J. Jeffers, MD, Center for Liver Diseases, RIAs for antibodies to two other HCV antigens: the C33c University of Miami School of Medicine, P.O. Box 016960 (R-IO), Miami, FL nonstructural and C22 core-associated antigens. Patients were 33101. considered anti-HCV positive if their sera reacted with at least 31/1/34192
JEFFERS ET AL.
T-LE 1. Prevalence of antibodies to three HCV epitopes among 129 patients with cryptogenic liver disease Pattern (interpretation)
Positive Positive Indeterminate Positive Positive Indeterminate Indeterminate Negative
+ + + +
+ + -
+ = positive test result; -
Cryptogenic chronic hepatitis (n = 38)
Cryptogenic cirrhosis (n = 91)
18 (47%) 2 1 (55%) 21 (5%) 2 1 (55%)
43 (47%) 55 (60%) 53 (58%) 55 (60%)
NS NS NS NS
Anti-C100-3 Anti-C33c Anti-C22 Anti-HCV“
+ + +
56 (43%) 2 (2%) 3 (2%) 0 18 (14%) 0 0 50 (39%) 129 (100%)
negative test result.
TABLE 2. Prevalence of anti-HCV reactivities
NS = not significant. “Reactive on at least two assays
two of the three recombinant antigens. Sera that reacted with one antigen alone were considered indeterminate in anti-HCV reactivity. Sera that did not react with any of the three antigens were considered anti-HCV negative. Other Serological Tests. All sera were tested for HBsAg (Auszyme) and antibody to HBcAg (anti-HBc) (Corzyme; Abbott Laboratories, North Chicago, IL) by enzyme immunoassay, ANA was tested by standard immunofluorescence; antibodies to liver-kidney microsomal antigen (anti-LKM-1) were tested by immunofluorescence and by RIA specifically measuring LKM- 1autoantibodies, which were directed against human cytochrome P-450 dbl(llD6). Sera were also tested for antibodies to soluble liver antigen (anti-SLA) by RIA (5, 6). Histological confirmation was obtained in all patients with chronic hepatitis and in all but one patient with cirrhosis who had clinically apparent ascites and esophageal varices. The x2 test and Student’s t test were used for statistical comparisons.
RESULTS Among the 129 patients with cryptogenicchronic liver disease, 61 (47%) were positive for antibody to C100-3 (anti-C100-3), 76 (59%) were positive for antibody to C33c (anti-C33c) and 74 (57%) were positive for antibody to C-22 (anti-C22). Of the 129 patients, 79 (61%) were reactive with at least one of the three assays. However, using the criterion of reactivity on at least two assays, 76 (59%) patients were considered to have anti-HCV. The remaining three patients had antiC100-3 reactivity only. Results of testing by the eight possible combinations of the test results are shown in Table 1. Anti-HCV reactivity was no more common among patients with cryptogenic cirrhosis (60%)than in patients with chronic hepatitis without cirrhosis (55%).
Thus anti-C100-3, anti-C33c and anti-C22 were detected in 47%, 60% and 58%, respectively, of patients with cirrhosis and in 47%, 55%and 5596, respectively, of patients with chronic hepatitis without cirrhosis (Table 2). All three patients with anti-C100-3 alone (indeterminate pattern) had cirrhosis. No significant differences were noted in the prevalence of anti-HBc among patients who were anti-HCV positive (21%)compared with those who were anti-HCV negative (23%)(Table 3). Conversely, 61 of 101 patients (60%) without anti-HBc were positive for anti-HCV, a prevalence similar to that among patient with anti-HBc (15 of 28; 54%). Only 10 of 59 patients (17%) with chronic hepatitis B-related liver disease tested positive for anti-C100-3, compared with 61 of 129 patients (47%) with equally severe chronic cryptogenic liver disease (p < 0.01). Among the 200 volunteer blood donors, only one (0.5%)was positive for anti-C100-3 (p < 0.01). All sera tested negative for ANA (in titers 2 1:40), anti-LKM1 and anti-SLA. Nine patients had ANA in low titers ( < 1:40). The sera of six of these patients tested positive for antibodies to all three HCV epitopes. Two of the remaining three patients were negative, and one was indeterminate (anti-C100-3 only). Demographical, Biochemical and Histological Features. Comparison of demographic, clinical, serum biochemical and histological features of patients with and without anti-HCV revealed no significant differences between the two groups (Table 3). Patients with anti-HCV had levels of serum aminotransferases, bilirubin, albumin and globulin similar to those of patients without anti-HCV (Table 3). DISCUSSION
In many patients with chronic liver disease, the origin of the liver disorder cannot be established. In an epidemiological study from the United Kingdom, 35%of cirrhotic patients studied between 1959 and 1970 were classified as having cryptogenic liver disease (7). In the United States, it has been estimated that 10%of cases of cirrhosis are of no determinable origin (8). An important agent that may cause many cases of cryptogenic liver disease is community-acquired or sporadic hepatitis C. Several studies have shown that sporadic NANB or type C hepatitis can evolve into
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ANTI-HCV IN CRYF'TOGENIC CHRONIC LIVER DISESASE
chronic liver disease (9-15). Two recent Scandinavian studies of larger groups of patients followed for longer intervals confirmed the development of cirrhosis in more than one fifth of patients (16, 17). Unfortunately, the diagnosis of NANB hepatitis in all these studies was based on the exclusion of other causes of chronic liver disease because serological markers for hepatitis C were not available. Our study showed that anti-HCV is common among patients with cryptogenic chronic liver disease. Similar findings were reported in two European studies (18,19). A third study from Boston has reported somewhat different results (20). The prevalence of anti-HCV among patients with cryptogenic hepatitis was comparable to that observed in our patients (53% and 55%, respectively). However, in the study from Boston, only 8%of patients with cryptogenic cirrhosis were anti-HCV positive, compared with 60% of our patients. The reason for this discrepancy is unclear. The number of patients studied in Boston was small (n = 24), and the criteria for sampling and the epidemiological characteristics of patients were probably quite different between the two studies. Both the Boston study and our Miami-based study demonstrated marked increases in anti-HCV reactivity when tests for anti-C33c and anti-C22 were used instead of just the conventional test for antiC100-3. In the Boston study, anti-HCV reactivity increased by 40%; in this study, the increase was 30% (from 45% to 59%) with the addition of the two new recombinant antigen tests. These results underscore the need to augment current recombinant tests for antiC100-3 with other HCV peptides. Nevertheless, many of our patients tested negative for all three antibodies. Perhaps some of these patients are infected with another NANB (non-U virus that awaits identification. Alternatively, anti-C100-3, anti-C33c and anti-C22 seronegativity testing may still miss some patients who actually have hepatitis C (false-negative assays). In a study by Weiner et al.(21), HCV-RNA was occasionally detected by polymerase chain reaction in the liver and sera of infected patients and in chimpanzees seronegative for anti-C100-3. We have obtained similar data in testing liver tissue or sera of patients from this study who were negative or indeterminate for anti-HCV (Jeffers, L, et al., Unpublished data, 1991). False-positive results with the available ELISA for anti-C100-3 have been reported in sera from patients with chronic liver disease, particularly autoimmine CAH (22). Hy-pergammaglobulinemia or cross-reactivity of the antigenic components used in the assay with antibodies other than anti-HCV may explain false seropositivity. In addition, sera of patients with autoimmune chronic hepatitis may contain antibodies to the superoxide dismutase used in the Ortho Diagnostics ELISA kit (23). In our study, sera from 58 of the 61 patients with anti-C100-3 tested positive for at least one other epitope and therefore were unlikely to be falsely positive (24).Sera from the remaining three patients were considered indeterminate because they were positive for anti-C100-3 only. These results suggest that
TABLE 3. Demographic, serological and histological features of study population Characteristics
Age (yr) Range Mean Male (no.) Race White Hispanic Black Histological diagnosis Chronic hepatitis Cirrhosis ALT (IU/LIb Range Mean f S.D. Bilirubin (mg/dl)" Range Mean f S.D. Albumin (gm/dV Range Mean f S.D. Globulin (gm/dl)* Range Mean S.D. Anti-HBc positive
Anti-HCV positive (n = 76)
Anti-HCV negative/indeterminate (n = 53)
21-75 59 42 (55%)
29-80 62 36 (68%)
42 (55%) 33 (44%) 1(l%)
31 (58%) 19 (36%) 3 (6%)
21 (28%) 55 (72%)"
78-834 117 2 26
90-480 120 t 28
0.2-14 3.0 t 2.6
0.3-26 3.2 t 2.8
1.4-4.8 3.6 2 0.9
1.4-4.7 3.3 k 0.9
1.3-5.4 3.3 k 0.7 16 (21%)
1.4-5.5 2.9 i 0.9 12 (23%)
See text for definition of anti-HCV seropositivity and seronegativity. "Histological diagnosis of cirrhosis was made in 54 patients. The remaining patient had clinical evidence of cirrhosis. bAll comparisons were not statistically significant (p > 0.05).
the rate of false-positive anti-C100-3 reactions was rather low in this population. This may be attributed to the careful exclusion of patients with clinical and serological evidence of autoimmune hepatitis and the use of RIA rather than ELISA for testing. The prevalence of anti-HCV among patients with cryptogenicchronic hepatitis and cirrhosis were similar. Therefore it is likely that patients with chronic hepatitis and cirrhosis are part of a continuum of the same disease process. The differences in histological severity may be primarily a function of time. Finally, the mode of transmission of HCV in patients with cryptogenic liver disease is unclear. Many of the patients in this study were Cuban-American. The practice of administering vitamin and calcium injections was common in Cuba, and the injections were often administered by nonmedical personnel; this represents a potential parenteral mode of transmission of this disease in these patients. Studies of sexual transmission of hepatitis C have conflicting results, indicating that this mode is not very common (25,26). Vertical and perinatal spread of hepatitis C has not been established (27, 28). Nonsexual household contact is also unproved as a mode of spread. Further studies are needed before the mode of transmission of sporadic hepatitis C is definitively characterized.
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