Editorial. Hepatitis C: From Laboratory to Bedside

Transcription

1 Editorial Hepatitis C: From Laboratory to Bedside It has been 15 years since Feinstone and coworkers' at the National Institutes of Health described a type of hepatitis occurring in recipients of blood transfusions that was clearly not type A or type B; until recently, that condition had been referred to as "non-a, non-b" hepatitis.'^ During the past 18 months, the agent responsible for this type of hepatitis, now referred to as "hepatitis C" virus (HCV), has been identified and characterized." A diagnostic antibody test has been developed to detect hepatitis C.'' Previously, the diagnosis of non-a, non-b hepatitis necessitated exclusion of other causes of liver injury and substantiation of exposure to possibly contaminated blood products such as transfused blood or shared needles in intravenous drug users. In addition, sporadic cases of non-a, non-b hepatitis were recognized when exposure to an obvious source of hepatitis was absent.'"' Within the past year, two large clinical trials have demonstrated the efficacy of recombinant interferon-α in the treatment of chronic hepatitis C.^' With the advent of routine diagnostic testing for hepatitis C and the availability of an efficacious therapeutic modality, clinicians will now need to understand the interpretation and limitations of this diagnostic test as well as the indications for and benefits of therapy. Earlier work involving inoculation of sera in chimpanzees demonstrated that posttransfusion non-a, non-b hepatitis was also caused by a Address reprint requests to Dr. Paul Martin, Division of Gastroenterology and Hepatology, Jefferson Medical College, Room 901, 1025 Walnut Street, Philadelphia, PA transmittable agent that seemed to be a small enveloped virus.* Prospective studies in transfusion recipients drew attention to the propensity to chronicity and the insidious progression of non-a, non-b hepatitis to cirrhosis.'^ Despite the recognition of the infectious nature of non- A, non-b hepatitis and the description of the clinical syndrome, isolation of the causal agent proved difficult. Many investigators hoped to identify viral antigens and antibodies with techniques found useful in the discovery of the hepatitis A and Β viruses. Although several investigators reported diagnostic tests, none of these withstood vigorous scrutiny. Scientists at the Chiron Corporation, working in collaboration with Bradley from the Centers for Disease Control in Atlanta, adopted a different approach." They reasoned that failure to identify the viral antigen was related to its low concentration in infectious serum. In a series of elegant experiments in which the techniques of molecular biology were used, they were finally able to identify the agent (HCV) responsible for most, if not all, non-a, non-b hepatitis. With use of chimpanzee serum known to be highly infectious in the experimental transmission of non-a, non-b hepatitis, they first concentrated whatever quantities of virus were present by ultracentrifugation. Because they did not know whether the virus was an RNA or a DNA virus, they extracted all the nucleic acid present. Using reverse transcriptase with random primers, they then constructed a cloned DNA library complementary (cdna) to all the nucleic acids present in the concentrated pellet. The cdna was then expressed in a bacteriophage vector to allow expression of peptides produced by the DNA. A serum specimen from a patient with a firm clinical diagnosis of chronic non-a, non-b hepatitis was used as a possible source of antibody to the virus. After the screening of approximately Mayo Clin Proc 6,5: ,

2 Mayo Clin Proc, October 1990, Vol 65 EDITORIAL million clones expressed in the bacteriophage vector, clone was found to react with the patient's serum. This 155-base pair clone was then extracted from its vector and used to hybridize to and identify a larger complementary clone in the original cdna library. This latter clone, clone 81, did not hybridize to DNA from either chimpanzee or human liver, an indication that it was not derived from the host genome, but did react with total nucleic acids extracted from the original highly infectious chimpanzee sera. Abolishment of this reaction by ribonuclease but not dideoxynuclease implied that the viral genome being detected by the clone was RNA and not DNA. These investigations also suggested that this RNA virus was single-stranded because it was bound by only one of the two strands in the clone 81 cdna. Furthermore, on the basis of these hybridization studies, the size of the RNA virus was estimated to be in the range of 5,000 to 10,000 nucleotides. Because previous studies had indicated that this virus was sensitive to organic solvents, a lipid envelope was thought to be present.** These characteristics are similar to those of togaviruses and flaviviruses. Three overlapping clones isolated by clone were then expressed in bacteria as a fusion polypeptide with human superoxide dismutase, as the latter promotes the efficient expression of foreign proteins in plasmids. These overlapping clones have a common open reading frame that encodes an HCV-related antigen. The fusion polypeptide (C100-3), which contains 363 viral amino acids, was used in a radioimmunoassay to capture circulating anti-hcv antibodies in blood. This radioimmunoassay was then used to validate the specificity of this clone in detecting viral antibodies associated with non-a, non-b hepatitis. Unlike any previously described test used to detect the causative agent for non-a, non-b hepatitis, the anti-hcv radioimmunoassay correctly identified six of seven coded samples of proven infectivity in chimpanzees.'' Normal control samples and sera noninfectious to chimpanzees were negative with this test. This radioimmunoassay was also used to test blood donor and recipient sera in 10 well-characterized cases of chronic posttransfusion non-a, non- B hepatitis. Each of the 10 cases showed seroconversion during the 12 months after transfusion when the specimens were tested retrospectively. Nine of these patients had received at least one donor unit that, on retrospective testing, was positive for anti-hcv. In addition, in this initial study 58% of sporadic cases of non-a, non-b hepatitis from the United States were found to be seropositive. Since these seminal observations, numerous publications have reported results of anti-hcv testing in stored serum banks. In general, at least 80% of patients with a firm clinical diagnosis of posttransfusion non-a, non-b hepatitis are anti-hcv positive." A much lower frequency of seropositivity is noted in acute resolving non-a, non-b hepatitis as low as 15% in the original study."* Various groups at increased risk for hepatitis C, such as intravenous drug abusers, hemodialysis patients, and hemophiliacs, have been shown to have a high frequency of seropositivity.'" A high frequency of anti-hcv has also been reported in patients with hepatocellular carcinoma." These results have led to the introduction into clinical medicine of routine anti-hcv testing with use of an enzyme-linked immunosorbent assay. With the widespread application of anti- HCV testing, certain limitations to this antibody test have emerged. First, even in patients with well-characterized posttransfusion non-a, non-b hepatitis, the antibody may take up to 1 year to develop and remains absent in up to 20% of patients." This result probably reflects the inadequacy of the test rather than the absence of HCV infection. Use of the polymerase chain reaction to amplify minute amounts of virus has indicated that HCV infection can occur without antibody formation." Second, the routinely available anti-hcv test measures an IgG antibody. This antibody is obviously not neutralizing because serum containing anti-hcv is infectious. The antibody may disappear with time in cases of resolving hepatitis and may be present in persons with no biochemical evidence of ongoing hepatic inflam-

3 1374 EDITORIAL Mayo Clin Proc, October 1990, Vol 65 mation. Thus, the test must be interpreted in conjunction with the patient's history, blood test results, or liver biopsy to determine the significance of a positive result. In addition, an immunocompromised state apparently may result in failure to develop or loss of anti-hcv seropositivity,'" inasmuch as patients with both human immunodeficiency virus infection and well-substantiated posttransfusion non-a, non-b hepatitis may be anti-hcv negative. The difficulty in interpreting a positive anti-hcv result is analogous to hepatitis Β virus, in which the presence of an isolated IgG antibody to hepatitis Β core antigen does not enable one to determine whether the patient has active or resolved infection or even a falsely positive result. Third, concern has been raised about the specificity of the anti-hcv test, in addition to its confirmed lack of sensitivity. McFarlane and colleagues"'' reported that 65% of patients with active autoimmune chronic liver disease were seropositive for anti-hcv with the enzyme-linked immunosorbent assay, in comparison with only 5% of patients whose disease was inactive at the time of testing. They related this apparent biologic false-positivity to the hypergammaglobulinemia characteristic of autoimmune chronic active hepatitis. Six patients who had been anti- HCV positive when their autoimmune liver disease was active lost seropositivity once immunosuppressive therapy was begun. Esteban and colleagues'" reported a 44% frequency of anti- HCV seropositivity in Spanish patients with autoimmune chronic active hepatitis. All these patients apparently had active liver disease at the time of testing. In addition to the common type of autoimmune chronic active hepatitis, 78% of patients with so-called type 2 autoimmune hepatitis, characterized by the presence of liver-kidney microsomal type 1 antibody, have been reported to be anti-hcv seropositive."^ Again, these patients apparently had active liver disease at the time of testing. Ikeda and associates'** suggested that the high rate of anti-hcv seropositivity reported in autoimmune liver diseases is related to reactivity to the superoxide dismutase moiety of the recombinant C100-3 fusion polypeptide used in the enzjrmelinked immunosorbent assay. With use of superoxide dismutase alone as the antigen in an enzyme-linked immunosorbent assay, they demonstrated a reaction in all nine patients with autoimmune chronic active hepatitis with anti- HCV seropositivity. In contrast, none of the 10 patients with anti-hcv seropositivity and posttransfusion hepatitis reacted to superoxide dismutase alone. Recently, investigators have described a more specific test for HCV infection the recombinant immunoblot assay. This test detects antibody directed against three recombinant antigens: the HCV-derived polypeptide in a fusion protein with superoxide dismutase; C100-3, the antigen present in the enzyme-linked immunosorbent assay; and the superoxide dismutase protein itself. Preliminary experience with this test indicates that it is generally positive in patients with an unequivocally positive anti-hcv enzyme-linked immunosorbent assay. Furthermore, the recombinant immunoblot assay seems to be a strong predictor of infectivity, as experienced in recipients of blood products. In one small study, all six blood donor samples testing positive with the recombinant immunoblot assay were implicated in posttransfusion hepatitis." In this issue of the Proceedings (pages 1303 to 1313), Czaja and colleagues report on their experience with anti-hcv testing in patients with "severe cryptogenic chronic active hepatitis" that is, patients with chronic liver disease but no evidence of autoimmunity, viral hepatitis, or other cause of a specific type of liver injury. They tested 17 such patients at the Mayo Clinic, 3 of whom were anti-hcv positive on enzymelinked immunosorbent assay. Two of these patients were also positive on recombinant immunoblot assay; the third patient reacted to only the C100-3 antigen in the recombinant immunoblot assay and was regarded as having an indeterminate result. Interestingly, this third patient had had a remote history of a blood transfusion. The two patients who were positive on both the enzyme-linked immunosorbent assay and the recombinant immunoblot assay remained positive during immunosuppressive

4 Mayo Clin Proc, October 1990, Vol 65 EDITORIAL 1375 therapy. Also noted was that only 5 of 85 patients with autoimmune chronic active hepatitis were seropositive for anti-hcv. Although these patients with autoimmune chronic active hepatitis are not the main focus of this report, the low rate of seropositivity is obviously in distinct contrast to some of the early European reports. The reason for this discrepancy is somewhat unclear; however, in some of the Mayo patients with autoimmune chronic active hepatitis, the serum samples may have been obtained when the patients were already in remission from immunosuppressive therapy, a situation that McFariane and associates''' suggested leads to disappearance of anti-hcv seropositivity in this condition. These considerations about the presence or absence of anti-hcv seropositivity in various types of liver disease are of more than pathophysiologic interest. There is a widespread clinical impression that patients with chronic liver disease due to HCV infection do poorly with corticosteroid therapy."' Conversely, a few reports have suggested that patients with non- HCV-related chronic active hepatitis will deteriorate if treated with recombinant interferon-α, which is of demonstrated efficacy in chronic hepatitis C* Therefore, effective therapy depends on an accurate diagnosis. The indications for therapy in autoimmune chronic active hepatitis and chronic hepatitis C are broadly similar. Therapy is directed at reducing necroinflammatory activity in the liver and halting progression of chronic active hepatitis to cirrhosis. Unfortunately, as previously noted, clear-cut distinctions among various types of chronic hepatitis may not always exist. In particular, in an individual patient with both markers of autoimmune chronic active hepatitis and a positive enzyme-linked immunosorbent assay for anti-hcv, determining appropriate therapy may be difficult. Most clinicians do not have ready access to either recombinant immunoblot assay or polymerase chain reaction testing and must therefore rely on their clinical judgment. The patient described by Czaja and co-workers who was anti-hcv positive by enzyme-linked immunosorbent assay and had had a blood transfusion 18 years before the current examination did poorly with corticosteroid treatment and died of liver failure. It is tempting to speculate that this patient's liver disease was, in fact, due to chronic hepatitis C, which deteriorated as a result of corticosteroid treatment. Unfortunately, the indeterminate result on recombinant immunoblot assay does not settle the issue. On the basis of our current state of knowledge, it seems reasonable to treat patients with chronic hepatitis who have an "autoimmune flavor" with immunosuppressive therapy either prednisone alone or prednisone plus azathioprine. Thus, patients with pronounced hypergammaglobulinemia as well as the presence of antinuclear antibody and anti-smooth muscle antibodies should be treated with corticosteroids even if they are anti-hcv positive unless, perhaps, they have a clear-cut history of a possible exposure to hepatitis C, such as a blood transfusion or intravenous drug usage antedating the recognition of their liver disease. McFariane and associates''' suggested that these patients will lose anti-hcv seropositivity once their liver disease responds to immunosuppression. In contrast, patients with features more typical of chronic hepatitis C and no markers of autoimmunity should be treated initially with interferon-α. With either group of patients, a poor response to or deterioration during treatment should prompt reconsideration of the original diagnosis and perhaps a switch to the alternative therapy. The optimal dose and duration of interferon-α therapy for chronic hepatitis C remain to be established. This algorithm can be considered only tentative, primarily because absence of anti-hcv antibody currently does not exclude HCV infection" and absence of autoimmune markers does not preclude corticosteroidresponsive severe chronic active hepatitis. With increasingly sophisticated diagnostic tests for HCV antigens and more sensitive and specific antibody tests, it should be easier to make the distinction between true- and falsepositive anti-hcv enzyme-linked immunosorbent assays within the next few years. In the interim, physicians caring for patients with anti-hcv-positive chronic hepatitis will have

5 1376 EDITORIAL Mayo Clin Proc, October 1990, Vol 65 to consider all the relevant clinical information before deciding on a therapeutic regimen. Acknowledgment. I thank Kerry Porter for expert secretarial assistance. Paul Martin, M.D. Division of Gastroenterology and Hepatology Jefferson Medical College Philadelphia, Pennsylvania REFERENCES 1. Feinstone SM, Kapikian AZ, Purcell RH, Alter HJ, Holland PV: Transfusion-associated hepatitis not due to viral hepatitis type A or B. Ν Engl J Med 292: , Dienstag JL: Hepatitis non-a, non-b: C at last (editorial). Gastroenterology 99: , Choo Q-L, 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 244: , Kuo G, Choo Q-L, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee W-S, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M: An assay for circulating antibodies to a major etiologic virus of human non-a, non-b hepatitis. Science 244: , Dienstag JL: Non-A, non-b hepatitis. I. Recognition, epidemiology, and clinical features. Gastroenterology 85: , Di Bisceglie AM, Martin P, Kassianides C, Lisker- Melman M, Murray L, Waggoner J, Goodman Z, Banks SM, Hoofnagle JH: Recombinant interferon alfa therapy for chronic hepatitis C: a randomized, double-blind, placebo-controlled trial. Ν Engl J Med 321: , Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC Jr, Perrillo RP, Carey W, Jacobson IM, Payne J, Dienstag JL, VanThiel DH, Tamburro C, Lefkowitch J, Albrecht J, Meschievitz C, Ortego TJ, Gibas A, Hepatitis Interventional Therapy Group: Treatment of chronic hepatitis C with recombinant interferon alfa: a multicenter randomized, controlled trial. NEnglJMed 321: , Bradley DW, McCaustland KA, Cook EH, Schable CA, Ebert JW, Maynard JE: Posttransfusion non-a, non-b hepatitis in chimpanzees: physicochemical evidence that the tubule-forming agent is a small, enveloped virus. Gastroenterology 88: , Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo Q-L, Kuo G: Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-a, non-b hepatitis. NEnglJMed 321: , Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, Roget M, Vargas V, Genesca J, Buti M, Guardia J: Hepatitis C virus antibodies among risk groups in Spain. Lancet 2: , Colombo M, Kuo G, Choo Q-L, Donate MF, Del Ninno E, Tommasini MA, Dioguardi N, Houghton M: Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 2: , Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo Q-L, Houghton M: Detection of hepatitis C viral sequences in non-a, non-b hepatitis. Lancet 335:1-3, Martin P, Di Bisceglie AM, Kassianides C, Lisker- Melman M, Hoofnagle JH: Rapidly progressive non- A, non-b hepatitis in patients with human immunodeficiency virus infection. Gastroenterology 97: , McFariane 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 335: , Lenzi M, Ballardini G, Fusconi M, Cassani F, Selleri L, Volta U, Zauli D, Bianchi FB: Type 2 autoimmune hepatitis and hepatitis C virus infection. Lancet 335: , Ikeda Y, Toda G, Hashimoto N, Kuro Kawa K: Antibody to superoxide dismutase, autoimmune hepatitis, and antibody tests for hepatitis C virus (letter to the editor). Lancet 335: , Ebeling F, Naukkarinen R, Leikola J: Recombinant immunoblot assay for hepatitis C virus antibody as predictor of infectivity (letter to the editor). Lancet 335: , Vento S, Di Perri G, Garofano T, Cosco L, Concia E, Ferraro T, Bassetti D: Hazards of interferon therapy for HBV-seronegative chronic hepatitis (letter to the editor). Lancet 2:926, 1989