Scand J Gastroenterol 1992;27:337-342.
REVIEW
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Hepatitis C: Virology, Epidemiology, Clinical Course, and Treatment
In 1989 a clone derived from hepatitis C virus (HCV), the major agent causing blood-transmitted forms of non-A, nonB hepatitis (NANBH), was identified (1). Long before that, the existence of NANBH had been recognized-in Sweden, for instance, since the early 1950s (2). Two major sources of parenterally transmitted NANBH cases are transfusions of blood or blood products and intravenous drug abuse. Other transmission routes exist besides overt inoculation, and these are important, since approximately 50% of acute NANBH cases in the USA have no obvious route of transmission, so-called sporadic cases. Most of these, in western Europe and the USA, are caused by HCV (3). HCV virology The identification of a cDNA clone, 5-1-1, which encoded at least one epitope that specifically identified antibodies in serum from patients with blood-transmitted NANBH, was reported in 1989 by scientists at the Chiron Corporation in California (1). By using a hybridization technique, a larger overlapping clone, c100-3, producing a non-structural protein consisting of 363 amino acids, was constructed (1). This clone was derived from a virus, which was named hepatitis C virus (HCV) (1,4,5). A first-generation antibody test for diagnosing HCV (anti-HCV) was developed by using recombinant c100-3, derived from the NS3/NS4 region of the genome, as antigen. By using this antibody test, it was recognized that HCV was the major agent causing both parenteral and sporadic forms of NANBH (6). HCV has now been shown to be a small, enveloped RNA virus with similarities to the flavi/pesti viruses (5). The genome codes for approximately 3000 amino acids in one single openreading frame. At the 5' end of the genome there is a highly conserved structural region encoding for the nucleocapsid protein, which is followed by five less conserved non-structural regions, NS1-NSS, towards the 3' end (Fig. 1). Between the structural and non-structural regions there is a hypervariable region encoding for the envelope ( E l , E2/NS1) (4,5,7). Recombinant proteins and synthetic peptides derived from both the structural region and NS3/NS4 (c22, c33, ~ 1 0 0 have ) now been included in second-generation tests for HCV (8,9). At least two different HCV genotype groups exist, originally isolated in Japan and the USA, respectively, which is of importance when choosing primers for diagnosing HCV RNA in serum and liver tissue by the polymerase chain reaction (PCR) method (4,5,10,11).
Clinical course of hepatitis NANB/C Most patients with acute hepatitis NANB/C infection are asymptomatic, and only some 1@-15% will become icteric; nevertheless, 50% will progress to chronic liver disease with fluctuating transaminase levels over decades (12-14). Histologically, some 20% seem to develop chronic active hepatitis with signs of cirrhosis within 5 years of follow-up (15). Generally, progression of the chronic disease seems to be slow and most often asymptomatic initially, with symptomatic cirrhosis developing late, after one or more decades (16, 17). A more rapid disease progression has been seen in patients with hypogammaglobulinemia, hemophilia, human immunodeficiency virus (HIV), and immunosuppression associated with organ transplants (18-21). Some patients without underlying immunosuppressive disease will, however, develop signs of cirrhosis already within a few years (22). Recently, data have accumulated suggesting that hepatocellular carcinoma (HCC) may be a late consequence of a chronic HCV infection. Thus a high prevalence of antiHCV has been found among patients with HCC (23,24). Furthermore, in a retrospective study Kiyosawa et al. (17) reported that 21 Japanese patients who had acute transfusion-associated NANBH with chronic evolution later, often after decades, developed HCC. The mean interval between the transfusion(s) and symptomatic chronic hepatitis, symptomatic cirrhosis, and HCC in their study was 10, 21, and 29 years, respectively. Interestingly, antibodies to HCV in these Japanese patients were documented 2-14 years before the diagnosis of HCC (17). HCV infection thus causes a slowly evolving disease with major importance for morbidity and mortality of chronic liver disease. Possibly, yet another infection may contribute to the development of HCC, as is the case with chronic hepatitis B virus infection. Epidemiology and diagnostic methods Transmission routes for HCV infections. The major transmission routes are inoculations and blood transfusions (25); furthermore, vertical transmission has also been documented (26,27). The efficacy of heterosexual transmission seems to be rather low as compared with that of hepatitis B virus (2830). The transmission of HCV by needle sticks was recently shown to be rather inefficient among hospital personnel in Japan, where 4% of 110 recipients of anti-HCV-positive needle sticks developed hepatitis (31). The transmission
338
Reuiew C33
c22
j . J .
1 5‘
C
5-1-1 ~100-3
El
E2INS-1 NS2
NS-3
NS-4
NS-5
3.-end
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Fig. 1. The gene map of hepatitis C virus. The localization of the antigens used in diagnostic tests for hepatitis C virus are indicated with arrows. C = core region; E = envelope; NS = non-structural region. route for the large number of sporadic cases, however, is at present unknown. By PCR the presence of HCV RNA in saliva has been documented (32,33), and the possible transmission of HCV by a human bite has been reported in one case (34). Whether saliva is important for the transmission of HCV in sporadic cases remains to be proven. Finally, since HCV shows homology with flaviviruses, one can speculate whether insect vectors can transmit HCV between humans. This, however, seems to be a remote possibility, since the number of viral particles in blood is low
(35). Seroprevalence of anti-HCV in selected patient categories. By using a first-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) test utilizing the recombinant antigen c100-3, the anti-HCV prevalence in different selected populations has been studied. Generally speaking, very high prevalence figures have been found among patients with chronic posttransfusion NANBH (70-100%) (6,25,36,37), intravenous drug addicts (50-90%) (25,37-40), and hemophiliacs (6443%) (25,29,37,41). Among patients with acute resolving posttransfusion NANBH variable prevalence figures depending on the follow-up time have been recorded (15-76%) (6,42). High prevalence figures have also been found in patients with sporadic NANBPI without obvious transmission routes (5472%) (3,37,39). Among homosexual males rather low prevalence figures have been recorded (8-18%) (25,40,41, 43). Seroprevalence of anti-HCV in blood donors, The seroprevalence of anti-HCV c100-3 among blood donors has been found to differ with geographic location and character of the investigated blood donor population. Thus, higher prevalence figures have been found in Asia (Taiwan and Japan) and Southern Europe (Italy, Spain) (0.9-2%) than in Scandinavia (Norway, Denmark, Sweden) (0.2-0.5%) (44-50). Prevalence figures somewhere between these figures have been reported from central Europe (France and Germany) (51,52). Paid donors have higher prevalence figures than voluntary donors (53,54). Seroconversion for anti-HCV in acute hepatitis NANRIC. The incidence of seroconversion for anti-HCV c100-3 in prospectively followed patients with postttransfusion and community-acquired NANBH has varied geographically, being higher in Italy, Spain, and the USA (90%) than in Scandinavia (50%) (42,47,55, 56). This could be due to the selection of patients or, possibly, due to different HCV
genomic variants. These differences tend to diminish when second-generation tests including other antigens from both the structural and non-structural regions of the HCV genome are included in the anti-HCV tests (9). In an early study seroconversion for anti-HCV c100-3 appeared rather late (mean, 15 weeks after onset of hepatitis) and for some patients not until after 1 year after onset of hepatitis (36). In an Italian study with prospectively followed patients with posttransfusion NANBH seroconversion occurred with 1, 3, 6, and 12 months in 29%, 61%, 79%, and 90%, respectively (42). The specificity and sensitivity of the anti-HCV c100-3 test. On the basis of results of three studies o n blood donations only one of five blood donors (17-26%) reactive for antiHCV c100-3 will transfer HCV infection to their blood recipients (57-59). An exception, however, is a Spanish study in which 88% of the anti-I-ICV (C100-3)-positive donors were implicated in transmission of HCV to their donors (47). It is obvious, however, that many blood donors who are reactive for anti-HCV c100-3 are non-infectious, possibly owing to a false-positive reactivity or a long past infection and acquired immunity. The sensitivity of the anti-HCV c100-3 test could also be improved, since almost half of the transfusion-associated hepatitis C cases would not have been prevented by testing the blood donors in the aforementioned Spanish study (47). A false-positive anti-HCV c100-3 reaction may also occur in patients with hypergammaglobulinemia and autoimmune type-I chronic active hepatitis (CAII) (60-62), primary biliary cirrhosis (62-64), rheumatoid arthritis (65), malaria (66), paraproteinemia (67) and could also be due to cross-reactive antibodies to superoxide disrnutase, a component of the assay (68). Prolonged storage, repeated freezing and thawing, and heat inactivation of sera have also been associated with a false-positive reactivity (62, 69-71). It is therefore obvious that the anti-HCV c100-3 test is not ideal (Table I); it does not differentiate an infectious from a non-infectious patient or an ongoing infection from a remote and resolved infection, and it does not discover all patients with hepatitis C infection (that is, it has a low sensitivity). Furthermore, it is hampered by a long time lag before seroconversion occurs and by the possibility of a false-positive reaction. To circumvent these drawbacks with the anti-HCV c1003 test, different ‘confirmatory’ tests have been used like the Abbott neutralization test and the Chiron/Ortho KIBA-1 test using the 5-1-1 and the c100-3 antigens attached to a
Review
339
Table I. Drawbacks with the first-generation anti-HCV ~ 1 0 0 - enzyme-linked 3 immunosorbent assay test ~~
~
Does not differentiate an ongoing infection from a resolved infection Long ‘window-phase’ before seroconversion Does not identify all patients with HCV (that is, low sensitivity) Does not differentiate an infectious patient from a non-infectious patient False-positive reactions ____~
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Table 11. Advantages of the second-generation anti-hepatitis C virus (HCV) tests over the first-generation test Reduces the ‘window-phase’ to seroconversion Increases the sensitivity in diagnosing HCV infection Offers a better means to differentiate infectious from non-infectious blood donors Diminishes the number of false-positive reactions
nitrocellulose strip. The RIBA-1 test was found to be more specific but somewhat less sensitive in predicting infectivity among blood donors (72), and the neutralization test was better at identifying false-positive reactions due to hypergammaglobulinemia (73). The second-generation anti-HCV tests. By including antigens, synthetic or recombinant, derived from other regions of the HCV genome, improvements have been achieved with the second-generation anti-HCV tests (Table 11). Thus antigens derived from the structural region (core), c22, and from the NS3 region, c33, have been added to the original antigens 5-1-1 and c100-3 derived from the NS3/NS4 region (Fig. 1) (9,74). The seroconversion rate in patients with posttransfusion NANBH improved from 54% with the first-generation test to 82% with a second-generation anti-HCV ELISA test, and the time lag to seroconversion decreased from a mean of 6.1 weeks after onset of hepatitis with the first-generation test to a mean of 2.3 weeks with the second-generation test (75). The additional antigens utilized in the second-generation ELISA have also been included in a second-generation recombinant immunoblot assay (RIBA), whereby the sensitivity in diagnosing chronic hepatitis C has substantially increased (76,77). The specificity has also substantially increased with the second-generation tests. Thus all 4-RIBA- and anti-HCV c100-3-positive blood donors in a Dutch study were implicated in the transmission of posttransfusion NANBH, whereas none of the anti-HCV c100-3-positive but 4-RIBAnegative donors seemed to transmit HNANB (8). The second-generation tests thus reduce the time lag for seroconversion during acute HCV infection, seem to offer a better possibility to differentiate an infectious blood donor from a non-infectious one, and, furthermore, diminish the number of false-negative reactions in patients with HCV infection. Whether peptides derived from other regions of the HCV genome, such as the envelope and NS5 regions, can be utilized for diagnosing HCV infections or to differentiate
patients who will recover from those who will develop chronic disease after an acute HCV infection is currently under investigation (9). Detection of HCV RNA in serum. So far the diagnostic approach to HCV infections has largely been based on the detection of antibodies. Assays that can directly identify the virus itself in serum and in the liver would, however, much improve the understanding of HCV infections and would theoretically offer a means to differentiate infectious viremic patients from non-infectious patients. HCV antigens will be difficult to detect in serum since their concentration generally is extremely low. However, by using the PCR, HCV-RNA sequences can be detected in serum. Owing to the variability of parts of the genome, it is of vital importance to choose primers for the PCR from a highly conserved region of the HCV genome (78). The usefulness of PCR has recently been proved (Table 111). Thus, HCV RNA was detected in serum 1-2 weeks after transfusion(s) before the clinical onset of posttransfusion HCV infection and long before serum antibodies appeared (35). In addition, HCV RNA seemed to be only transiently present in serum from patients with acute resolving hepatitis but to persist, during prolonged time periods, in most patients with chronic evolution (35, 58,79, 80). Furthermore, HCV RNA in serum detected by PCR is a better predictor of infectivity than anti-HCV (11). PCR may also be useful for diagnosing vertical transmission of HCV from chronically infected mothers to their offspring (26). Finally, testing for HCV RNA offers a possibility to monitor antiviral treatment for chronic HCV infection and seems to differentiate a successful treatment from a nonsuccessful one, since HCV RNA will disappear permanently from serum after a successful course but will reappear if relapse occurs (80). Therapy of chronic hepatits NANBIC The goals of antiviral therapy for chronic HCV infection includes a reduction and/or elimination of the hepatic inflammation and the ensuing fibrosis/cirrhosis, a reduction and/or elimination of the contagiousness, and the prevention
340
Review
Table 111. The clinical usefulness of the polymerase chain reaction for the detection of hepatitis C virus (HCV) RNA
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
To differentiate an acute resolving hepatitis C from one with chronic evolution To shorten the ‘window-phase’ when diagnosing acute HCV infection To better detect infectivity of anti-HCV-positive patients To diagnose vertical transmission of HCV To monitor response to antiviral therapy for chronic HCV infection
of a later development of HCC. So far, treatment response has mostly been evaluated by monitoring the normalization rate of alanine aminotransferase (ALAT) levels in serum during and after treatment and by evaluating the histologic findings in liver biopsy specimens taken before and after treatment. Recently, however, PCR was used to monitor HCV RNA levels in serum during and after treatment (80-82). Results with interferon. In 1986 it was first reported that alpha interferon seemed to be effective for the treatment of chronic NANBH (83). Since then several randomized controlled clinical trials have proved the efficacy of alphainterferon treatment, at a dose of 3 MU thrice weekly for &9 months, for chronic HCV infection (84-87). In a recent meta-analysis of randomized controlled clinical trials on interfernn treatment the rate of s-ALAT normalization in trcated patients was approximately 5070 (88). Responders to interfcron will also improve histologically (85,89). After the end of treatment 50% of responding patients will relapse, leaving only one of four patients with a sustained response remaining also after cessation of treatment (88). A dose of 3 MU has been shown to be superior to 1 MU (84, 86, 90). Augmenting the dose further to 6 MU in ‘nonresponders’ to lowcr doses, however, did not seem to induce a biochemical response (91). A predictor of interferon response was either less hepatic inflammation or absence of cirrhosis histologically in some studies (84, 89, 92, 93), whereas no such predictors were found in other studies. The presence or not of anti-HCV c100-3 antibodies did not seem to affect the outcome of treatment (87,94). The importance of neutralizinginterferon antibodies as the cause for treatment failures in chronic HCV infections has been discussed but has not been shown as indisputable as with treatment failures in chronic hepatitis B (95). Nearly all treated patients will have an initial flu-like syndrome during the first few injections, which thereafter subsides. Other side effects are bone marrow suppression, hair loss, myalgias, and fatigue, causing dose reduction in somc 15% o f the patients and withdrawal in some 5% (85-87). Lymphoblastoid interferon also appears to be effective (96), whereas gamma interferon seems to be ineffective (97). Extended interferon treatment periods to improve the rate of sustained response is currently under investigation in clinical trials. Ofher treatments. Corticosteroids and acyclovir seem to
be ineffective (98-100). Oral ribavirin has shown promising results in a pilot study, in which 10 patients with chronic hepatitis C were treated for 12 weeks with ribavirin in a daily dose of 1000-1200mg (101). After the end of treatment, however, the ALAT levels increased to pretreatment levels (101). Recently, a case report was published indicating temporary response to inosine pranobex (102). In the future further compounds and combinations of different drugs are likely to be evaluated for treatment of chronic HCV infection. OLAWEILAND ROBERT SCHVARCZ
Dept. of Infectious Diseases Karolinska Institute Roslagstull Hospital S-114 89 Stockholm Sweden REFERENCES
1. Choo Q, Kuo G, Weiner A , Overby L, Bradley D, Houghton M. Isolation of a cDNA clone derived from a blood-home nonA, non-B viral hepatitis genome. Science 1989;244:35942. 2. Weiland 0, Berg J, Bjorvatn B, Flehmig B, Lundbergh P. Acute viral hepatitis A , B and non-A, non-B i n Stockholm in the 1950s and in the 1970s: acomparison. Infection 1981;Y:26874. 3. Alter M, Hadler S, Judson F, ct al. Risk factors for acute iio~iA , non-B hepatitis in the United States and association with hepatitis C virus infection. JAMA 199O;264:223 1-5. 4. Choo Q-L. Richman K, Han J H , et al. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci 1991;88:2451-5. 5. Houghton M, Weincr A , Han J , Kuo G, Choo QL. Molecular biology of the hepatitis C virusis: implications for diagnosis, dcvclopment and control of viral disease. Hepatology 1991;14:381-8. 6 . Kuo G, Choo Q, Alter H, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-4. 7. Rradley D. Hepatitis non-A, non-B viruscs become identified as hepatitis C and E viruses. Prog Med Virol 1990;37:101-35. 8. Van Der Poel C, Cuypers H, Reesink H, ct al. Confirmation of hepatitis C virus infection by new lour-antigen recombinant imrnunoblot assay. Lancct 1991;337:3 17-9. 9. Mattsson L, Gutierrez R, Dawson G, Lesniewski R, Mushahwar I, Weiland 0. Antibodies to recombinant and synthetic peptides dcrived from the hepatitis C virus genome in long-term-studied patients with posttransfusion hepatitis C . Scand J Gastroenterol 1991;26:1257-62. 10. Kato N, Ohkoshi S , Shimotoho K. Japanese isolates of the non-A, non-B hepatitis viral genome show sequence variations from the original isolate in the USA. Proc Jpn Acad 1989;658:219-23, 1 1 . Simmonds P, Zhang L, Watson, et al. Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users. Lancet 1990;336: 1469-72.
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Review 12. Dienstag J. Non-A, non-B hepatitis I. Recognition, epidemiology, and clinical features. Gastroenterology 1983;85: 439-62. 13. Berman M, Alter H, Ishak K, Purcell R, Jones E. The chronic sequelae of non-A, non-B hepatitis. Ann Intern Med 1979; 91:1-6. 14. Mattsson L, Aberg B, Weiland 0, Sellman M, Daviltn J. NonA. non-B hepatitis after open-heart surgery in Stockholm: declining incidence after introduction of restrictions for blood donations due to the human immunodeficiency virus Scand J Infect Dis 1988;20:371-6. 15. Mattsson L. Chronic non-A, non-B hepatitis with special reference to the transfusion-associated form. Scand J Infect Dis 1989;21 SUPPI59:1-55. 16. Koretz R, Coleman E , Abbey H, Gitnick G. Non-A, nonB post-transfusion hepatitis: When is a disease a dis-ease? [Abstract]. Hepatology 1989;10:645. 17. Kiyosawa K, Sodeyama T, Tanaka E, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990;12:671-5. 18. Bjorkander J , Cunningham-Rundles C, Lundin P, Olsson R, Soderstrom R, Hanson L. Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinemia or IgG subclass deficiency. Am J Med 1988;84: 107-1 1. 19. Hay C, Preston F, Triger D, Underwood J. Progressive liver disease in haemophilia: an underestimated problem? Lancet 1985;1: 1495-8. 20. Martin P, Di Bisceglie A, Kassianides C, Lisker-Melman M, Hoofnagle J. Rapidly progressive non-A, non-B hepatitis in patients with human immunodeficiency virus infection. Gastroenterology 1989;97:155$61. 21. Dienstag J, Alter H . Non-A, non-B hepatitis: evolving epidemiologic and clinical perspective. Semin Liv Dis 1986;6:67R1.
22. Wejstil R , Hermodsson S, Norkrans G. Long-term follow-up of chronic hepatitis non-A, non-B-with special reference to hepatitis C. Liver 1991;11:143-8. 23. Colombo M, Kuo G , Choo Q, et al. Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 1989;2:1006-8. 24. Bruix J, Barrera J, Calvet X , et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989;2:1004-6. 25. Esteban J, Esteban R, Viladomiu L, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;2:294-7. 26. Thaler M, Park C-K, Landers D, et al. Vertical transmission of hepatitis C virus. Lancet 1991;338:17-8. 27. Wejstil R, Hermodsson S, Iwarson S, Norkrans G. Mother to infant transmission of hepatitis C virus infection. J Med Virol 1990;30:178-80. 28. Cilla G, Ptrez-Trallero E, Iturriza M, Arrizabalaga J, Iribarren J . Possibility of heterosexual transmission of hepatitis C virus. Eur J Clin Microbiol Infect Dis 1991;10:533-4. 29. Schulman S, Grillner L. Antibodies against hepatitis C in a population of Swedish haemophiliacs and heterosexual partners. Scand J Infect Dis 1990;22:393-7. 30. Alter M, Coleman P, Alexander J, et al. Importance of heterosexual activity in the transmission of hepatitis B and nonA, non-B hepatitis. JAMA 1989;262:1201-5. 31. Kiyosawa K, Sodeyama T, Tanaka E, et al. Hepatitis C in hospital employees with needlestick injuries. Ann Intern Med 19Y 1;115:367-Y. 32. Takamatsu K, Koyanagi Y, Okita K, Yamamoto N. Hepatitis C virus RNA in saliva. Lancet 1990;336:1515. 33. Wang J-T, Wang T-H, Lin J-T, Sheu J-C, Lin S-M, Chen D-S. Hepatitis C virus RNA in saliva of patients with posttransfusion hepatitis C infection. Lancet 1991;337:48. 34. Dusheiko G, Smith M, Scheuer P. Hepatitis Cvirus transmitted by human bite. Lancet 1990;336:503-4. 35. Farci P, Alter H, Wong D, et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991:325:98- 104.
341
36. Alter H, Purcell R, Shih J, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321: 1494-1500. 37. Roggendorf M, Deinhardt F, Rasshofer R, et al. Antibodies to hepatitis C virus. Lancet 1989;2:324-5, 38. Sonnerborg A, Abebe A, Strannegird 0 . Hepatitis C virus infection in individuals with or without human immunodeficiency virus type 1 infection. Infection 1990;18:347-51. 39. Weiland 0, Lindh G , Mattsson L, Schvarcz R, von Sydow M. Hepatit C en vanlig orsak till kronisk hepatit i Sverige [in Swedish]. Lakartidningen 1990;34:2567-8. 40. Tor J, Llibre J, Carbonell M, et al. Sexual transmission of hepatitis C virus and its relation with hepatitis B virus and HIV. Br Med J 1990;301:1130-3. 41. Widell A, Hansson B, Berntorp E , et al. Antibody to a hepatitis C virus related protein among patients at high risk for hepatitis B. Scand J Infect Dis 1991;23:19-24. 42. Tremolada F, Casarin C, Tagger A, et al. Antibody to hepatitis A virus in post-transfusion hepatitis. Ann Intern Med 1991; 114:277-81. 43. Kiese M, Lenz K, Guggenmoos-Holzmann I, Stark K, Bienzle U. Epidemiology of hepatitis C in homosexual men. KIin Wochenschr 1990;68: 1082. 44. Lin-Chu M, Tsai S , Watanabe J, Nishioka K. The prevalence of anti-HCV among Chinese voluntary blood-donors in Taiwan. Transfusion 1990;30:47 1-3. 45. Nishimura Y, Yamagushi K, Williams N, et al. Antibodies to hepatitis C virus in Japanese blood donors and in hospital personnel. Transfusion 1990;30:667-8. 46. Sirchia G, Almini D, Bellobuono A, et al. Prevalence of hepatitis C virus antibodies in Italian blood donors. Vox Sang 1990;59:2&9. 47. Esteban J, Gonzalez A, Hernandez J, et al. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990;323:1107-12. 48. Hetland G , Skaug K, Larsen J, Maeland A, Stromme J, Storvold G. Prevalence of anti-HCV in Norwegian blood donors with anti-HBc or increased ALT levels. Transfusion 1990; 30:776-9. 49. Wantzin P, Krogsgaard K, Dickmeiss E. Screening af danske bloddonorer for antistof rettet mod Hepatitis C virus [in Danish]. Ugeskr Laeger 1990;152:284&8. 50. Lindholm A, Hermodsson S, Norkrans G , Iwarson S. Nytt serologiskt test ger mojlighet att diagnosticera hepatit C [in Swedish]. Lakartidningen 1990;87:40-1. 51. Janot C, Courouct A, Maniez M. Antibodies to hepatitis C virus in French blood donors. Lancet 1989;2:796-7. 52. Kiihnl P, Seidl S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors. Lancet 1989;2: 324. 53. Menitove J, Richards W, Destree M. Early US experience with anti-HCV kit in blood donors. Lancet 1990;336:244-5. 54. Stevens C, Taylor P, Pindyck J, et al. Epidemiologyof hepatitis C virus: a preliminary study in volunteer blood donors. JAMA 1990;263:49-53. 55. Mattsson L, Grillner L, von Sydow M, Bergdahl S, Weiland 0. Seroconversion to hepatitis C virus antibodies in patients with acute posttransfusion non-A, non-B hepatitis in Sweden. Infection 1991;19: 30S12. 56. Krogsgaard K, Wantzin P, Mathiesen L, Sonne J, Ring-Larsen H. The Copenhagen Hepatitis Acuta Programme. Early appearance of antibodies to hepatitis C virus in community acquired acute non-A, non-B hepatitis is associated with progression to chronic liver disease. Scand J Infect Dis 1990; 22:399-402. 57. Van der Poel C, Reesink H, Schaasberg W, et al. Infectivity of blood seropositive for hepatitis C virus antibodies. Lancet 1990;335:558-60. 58. Garson J, Tedder R, Briggs M, et al. Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 141%22.
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
342
Review
59. Eheling F, Naukkarinen R, Lcikola J . Post-transfusion hepdtitis C in Finland. Transfusion 1991;1:109-13. 60. McFarlane I, Smith H , Johnson P, Bray G , Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 1990;33s:754-7. 61. Schvarcz R, von Sydow M, Weiland 0 . Autoimmune chronic active hepatitis: changing reactivity for antibodies to hepatitis C virus after immunosuppressive treatment. Scand J Gastroenterol 1990;25:1175-80. 62. Schrumpf E, Elgjo K, Fausa 0, Haukenes G , Kvale D , Rollag H. The signficance of anti-hepatitis C virus antibodies measured in chronic liver disease. Scand J Gastroenterol 1990;25:1169-74. 63. Di Bisceglie A , Alter H, Kuo G, Houghton M, Hoofnagle J. Detection of antibody to hepatitis C virus in patients with various chronic liver diseases [Abstract]. Hepatology 1989; 10:581. 64. Fusconi M, Lenzi M, Ballardini G , et al. Anti-HCV testing in autoimmune hepatitis and primary hiliary cirrhosis. Lancet 1990;336:823. 65. Theilmann L, Blazek M, Goeser T, Gmelin K, Kommerell B, Fiehn W. False-positive anti-HCV tests in rheumatoid arthritis. Lancct 1990;335:1346. 66. Aceti A. Taliani G , De Bac C, Sebastiani A. Anti-HCV false positivity in malaria. Lancet 1990;336:1442-3. 67. Boudart D, Lucas J-C, Muller J-Y, Lc Carrer D, Planchon B, Harousseau J-L. False-positive hepatitis C virus antibody tests in paraproteinaemia. Lancet 1990;336:63. 68. Ikeda Y, 'I'oda G , Hashimoto N, Kurokawa K. Antibody to superoxide dismntase, autoimmune hepatitis, and antibody tests for hepatitis C virus. Lancet 1990;335:1345-6. 69. Ellis L, Brown D, Conradie J , et al. Prevalence of hepatitis C in South Africa: detection of anti-HCV in recent and stored serum. J Med Virol 1990;32:24Y-51. 70. Mortimcr P, Cohen B, Litton P, et al. Hepatitis C virus antibody. Lancet 1989;2:798. 71. Wong D, Diwan A, Rosen L, et al. Non-specificity of antiHCV test for seroepidemiological analysis. Lancet 1990;336: 7Sc-1. 72. Ebeling F, Naukkarincn R, Leikola J. Recombinant immunoblot assay for hepatitis C virus antibody as predictor of infectivity. Lancet 1990;335:982-3. 73. Schvarcz R, von Sydow M, Weiland 0 . Positive reactivity with anti-HCV ELISA in patients with autoimmune hepatitisnot confirmed with a neutralization test. Scand J Infect Dis 1991;23:127-X. 74. Choo Q-L, Weiner A, Overby L, Kuo G , Houghton M, BradIcy D. Hcpatitis C virus: the causative agent of viral non-A, non-B hepatitis. Br Med Bull 1990;46:423-41. 75. Mattsson L, Grillner L, Weiland 0.Seroconversion to hepatitis C virus antibodies in patients with acute posttransfusion nonA, non-B hepatitis in Sweden with a second generation test. Scand J Infect Dis 1Y92. In press. 76. Marcellin PMM-P, Boyer N , Pouteau M, Aumont P, Erlinger S , Benhamou J-P. Second generation (RIBA) tests in diagnosis of chronic hepatitis C. Lancet 1991;337:551-2. 77. Craxi A, Fiorentino G , Di Marco V, et al. Second generation tests in diagnosis of chronic hepatitis C. Lancet 1991;337:1354. 711. Carson J, Ring C, Tuke P, 'l'cdder R. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990;336:8769. 79. Garson J , Tuke P, Makris M, et al. Demonstration of viremia patterns in haemophiliacs treated with hepatitis-c-virus-contaminated factor VIII concentrates. Lancet 1990;336:1022-5. 80. Schlauder G , Leverenz G , Mattsson L, Weiland 0,Mushahwar I. Detection of hepatitis C viral RNA by the polymerase chain reaction in serum of patients with post-transfusion non- A, nonB hepatitis. J Virol Methods. In press. 81. Brillanti S, Garson J, Tuke P, et al. Effect of alfa-interferon therapy on hepatitis C viraemia in community-acquired chronic non-A, non-B hepatitis: A quantitative polymerase chain reaction study. J Med Virol 1991;34:13&41. 82. Chayama K, Saitoh S, Arase Y, et al. Effect of interferon
administration on serum hepatitis C virus RNA in patients with chronic hepatitis C. Hepatology 1991;13:1040-3. 83. Hoofnagle J, Mullen K, Jones D, et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon. A preliminary report. N Engl J Med 1986;315:1575-8. 84. Causse X, Godinot H, Chevalier M, et al. Comparison of 1 or 3 MU of interferon alfd-2b and placebo in patients with chronic non-A, non-B hepatitis. Gastroenterology 1991;101:497-502. 85. Di Bisceglie A, Martin P, Kassianides C, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med 1989; 321:3so6-10. 86. Davis G , Balart L, Schiff E , et al. Treatment of chronic hepatitis C with recombinant interferon alfa. A multicenter randomized, controlled trial. N Engl J Med 1989;321:1501-6. 87. Schvarcz R, Weiland 0, Wejstdl R, Norkrans G , Foberg U, Fryden A. A randomized controlled open study of interferon alpha-2h treatment of chronic non-A. non-B posttransfusion hepatitis: no correlation of outcome to presence of hepatitis C virus antibodies. Scand J Infect Dis 1989;21:617-25. 88. Tine F, Magrin S, Craxi A, Pagliaro L. Interferon for non-A, nun-B chronic hepatitis. A meta-analysis of randomised clinical trials. J Hepatol 1991;13:192-9. 89. Schvarcz R, Glaumann H, Weiland 0 , Norkrans G , Wejsrdl R, FrydCn A. Histological outcome in interferon alpha-2b treated patients with chronic posttransfusion non-A, non-B hepatitis. Liver 1991;11:3&8. 90. Marcellin P, Boyer N. Giostra E, et al. Recombinant human alfa-interferon in patients with chronic non-A, non-B hepatitis: a multicenter randomized controlled trial from France. Hepatology 1991;13~393-7. 91. Schvarcz R , Glaumann H, Weiland 0 , Norkrans G , Wejstil R, Fryden A . Interferon alpha-2b treatment of chronic posttransfusion non-A, non-B/C hepatitis: long-term outcome and effect of increased interferon doses in non-responders. Scand J Infect Dis 1991;23:413-20. 92. Ferenci P, Vogel W, Pristautz H, et al. One-year treatment of chronic non-A. non-B hepatitis with interferon alfa-2b. J Hepatol 1990;ll Suppl:SO-3. 93. Saracco G , Rosina M, Torrani Cerenzia M, et al. A randomized controlled trial of interferon alfa-2b as therapy for chronic nonA, non-B hepatitis. J Hepatol 1990;ll Suppl:4>9. 94. Marcellin P, Giostra E , Boyer N , Loriot M-A, MartinotPeignoux M, Benhamou J-P. Is the response to recornbinant alpha interferon related to the presence of antibodies to hepatitis C virus in patients with chronic non-A, non-B hepatitis? J Hepatol 1990;11:77-9. 95. Brook M, McDonald J, Karayiannis P, et al. Randomised controlled trial of interferon alfa 2A (rbe) (Roferon-A) for the treatment of chronic hepatitis B virus (HBV) infection: factors that influence response. Gut 1989;30:111622. 96. Jacyna M, Brooks M, Loke R, Main J, Murray-Lyon I, Thomas H. Randomized controlled trial of interferon alfa (lymfoblastoid interferon) in chronic non-A, non-B hepatitis. Br Med J 1989;298:8&2. 97. Saez-Royuela F, Porres J, Moreno A, et al. High doscs of recombinant alfa-interferon or gamma-interferon for chronic hepatitis C: a randomized, controlled trial. Hepatology 1991;13:327-3 I . 98. Stokes P, Lopez W, Balart L. Effects of short-term corticostcroid therapy in patients with chronic non-A, non-B hepatitis [Abstract]. Gastroenterology 1987;92:1783. 99. Pappas S, Hoofnagle J, Young N , Straus S , Jones E. Treatment of chronic non-A, nun-B hepatitis with acyclovir: pilot study. J Med Virol 198S;lS: 1-9. 100. Hoofnagle J. Chronic hepatitis: the role of corticosteroids. In: Szmuness W, Alter HJ, Maynard JE, editors Viral hepatitis. 1981 international symposium. Philadelphia: Franklin Press, 1982573-83. 101. Reichard 0, Andersson J , Schvarcz R, Weiland 0. Ribavirin treatment for chronic hepatitis C. Lancct 1991;337:105X-61. 102. Prohaska W, Kleesiek K. Treatment of chronic hepatitis C with inosine pranobex. Lancet 1991;338:390-1.
REVIEW
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Hepatitis C: Virology, Epidemiology, Clinical Course, and Treatment
In 1989 a clone derived from hepatitis C virus (HCV), the major agent causing blood-transmitted forms of non-A, nonB hepatitis (NANBH), was identified (1). Long before that, the existence of NANBH had been recognized-in Sweden, for instance, since the early 1950s (2). Two major sources of parenterally transmitted NANBH cases are transfusions of blood or blood products and intravenous drug abuse. Other transmission routes exist besides overt inoculation, and these are important, since approximately 50% of acute NANBH cases in the USA have no obvious route of transmission, so-called sporadic cases. Most of these, in western Europe and the USA, are caused by HCV (3). HCV virology The identification of a cDNA clone, 5-1-1, which encoded at least one epitope that specifically identified antibodies in serum from patients with blood-transmitted NANBH, was reported in 1989 by scientists at the Chiron Corporation in California (1). By using a hybridization technique, a larger overlapping clone, c100-3, producing a non-structural protein consisting of 363 amino acids, was constructed (1). This clone was derived from a virus, which was named hepatitis C virus (HCV) (1,4,5). A first-generation antibody test for diagnosing HCV (anti-HCV) was developed by using recombinant c100-3, derived from the NS3/NS4 region of the genome, as antigen. By using this antibody test, it was recognized that HCV was the major agent causing both parenteral and sporadic forms of NANBH (6). HCV has now been shown to be a small, enveloped RNA virus with similarities to the flavi/pesti viruses (5). The genome codes for approximately 3000 amino acids in one single openreading frame. At the 5' end of the genome there is a highly conserved structural region encoding for the nucleocapsid protein, which is followed by five less conserved non-structural regions, NS1-NSS, towards the 3' end (Fig. 1). Between the structural and non-structural regions there is a hypervariable region encoding for the envelope ( E l , E2/NS1) (4,5,7). Recombinant proteins and synthetic peptides derived from both the structural region and NS3/NS4 (c22, c33, ~ 1 0 0 have ) now been included in second-generation tests for HCV (8,9). At least two different HCV genotype groups exist, originally isolated in Japan and the USA, respectively, which is of importance when choosing primers for diagnosing HCV RNA in serum and liver tissue by the polymerase chain reaction (PCR) method (4,5,10,11).
Clinical course of hepatitis NANB/C Most patients with acute hepatitis NANB/C infection are asymptomatic, and only some 1@-15% will become icteric; nevertheless, 50% will progress to chronic liver disease with fluctuating transaminase levels over decades (12-14). Histologically, some 20% seem to develop chronic active hepatitis with signs of cirrhosis within 5 years of follow-up (15). Generally, progression of the chronic disease seems to be slow and most often asymptomatic initially, with symptomatic cirrhosis developing late, after one or more decades (16, 17). A more rapid disease progression has been seen in patients with hypogammaglobulinemia, hemophilia, human immunodeficiency virus (HIV), and immunosuppression associated with organ transplants (18-21). Some patients without underlying immunosuppressive disease will, however, develop signs of cirrhosis already within a few years (22). Recently, data have accumulated suggesting that hepatocellular carcinoma (HCC) may be a late consequence of a chronic HCV infection. Thus a high prevalence of antiHCV has been found among patients with HCC (23,24). Furthermore, in a retrospective study Kiyosawa et al. (17) reported that 21 Japanese patients who had acute transfusion-associated NANBH with chronic evolution later, often after decades, developed HCC. The mean interval between the transfusion(s) and symptomatic chronic hepatitis, symptomatic cirrhosis, and HCC in their study was 10, 21, and 29 years, respectively. Interestingly, antibodies to HCV in these Japanese patients were documented 2-14 years before the diagnosis of HCC (17). HCV infection thus causes a slowly evolving disease with major importance for morbidity and mortality of chronic liver disease. Possibly, yet another infection may contribute to the development of HCC, as is the case with chronic hepatitis B virus infection. Epidemiology and diagnostic methods Transmission routes for HCV infections. The major transmission routes are inoculations and blood transfusions (25); furthermore, vertical transmission has also been documented (26,27). The efficacy of heterosexual transmission seems to be rather low as compared with that of hepatitis B virus (2830). The transmission of HCV by needle sticks was recently shown to be rather inefficient among hospital personnel in Japan, where 4% of 110 recipients of anti-HCV-positive needle sticks developed hepatitis (31). The transmission
338
Reuiew C33
c22
j . J .
1 5‘
C
5-1-1 ~100-3
El
E2INS-1 NS2
NS-3
NS-4
NS-5
3.-end
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Fig. 1. The gene map of hepatitis C virus. The localization of the antigens used in diagnostic tests for hepatitis C virus are indicated with arrows. C = core region; E = envelope; NS = non-structural region. route for the large number of sporadic cases, however, is at present unknown. By PCR the presence of HCV RNA in saliva has been documented (32,33), and the possible transmission of HCV by a human bite has been reported in one case (34). Whether saliva is important for the transmission of HCV in sporadic cases remains to be proven. Finally, since HCV shows homology with flaviviruses, one can speculate whether insect vectors can transmit HCV between humans. This, however, seems to be a remote possibility, since the number of viral particles in blood is low
(35). Seroprevalence of anti-HCV in selected patient categories. By using a first-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) test utilizing the recombinant antigen c100-3, the anti-HCV prevalence in different selected populations has been studied. Generally speaking, very high prevalence figures have been found among patients with chronic posttransfusion NANBH (70-100%) (6,25,36,37), intravenous drug addicts (50-90%) (25,37-40), and hemophiliacs (6443%) (25,29,37,41). Among patients with acute resolving posttransfusion NANBH variable prevalence figures depending on the follow-up time have been recorded (15-76%) (6,42). High prevalence figures have also been found in patients with sporadic NANBPI without obvious transmission routes (5472%) (3,37,39). Among homosexual males rather low prevalence figures have been recorded (8-18%) (25,40,41, 43). Seroprevalence of anti-HCV in blood donors, The seroprevalence of anti-HCV c100-3 among blood donors has been found to differ with geographic location and character of the investigated blood donor population. Thus, higher prevalence figures have been found in Asia (Taiwan and Japan) and Southern Europe (Italy, Spain) (0.9-2%) than in Scandinavia (Norway, Denmark, Sweden) (0.2-0.5%) (44-50). Prevalence figures somewhere between these figures have been reported from central Europe (France and Germany) (51,52). Paid donors have higher prevalence figures than voluntary donors (53,54). Seroconversion for anti-HCV in acute hepatitis NANRIC. The incidence of seroconversion for anti-HCV c100-3 in prospectively followed patients with postttransfusion and community-acquired NANBH has varied geographically, being higher in Italy, Spain, and the USA (90%) than in Scandinavia (50%) (42,47,55, 56). This could be due to the selection of patients or, possibly, due to different HCV
genomic variants. These differences tend to diminish when second-generation tests including other antigens from both the structural and non-structural regions of the HCV genome are included in the anti-HCV tests (9). In an early study seroconversion for anti-HCV c100-3 appeared rather late (mean, 15 weeks after onset of hepatitis) and for some patients not until after 1 year after onset of hepatitis (36). In an Italian study with prospectively followed patients with posttransfusion NANBH seroconversion occurred with 1, 3, 6, and 12 months in 29%, 61%, 79%, and 90%, respectively (42). The specificity and sensitivity of the anti-HCV c100-3 test. On the basis of results of three studies o n blood donations only one of five blood donors (17-26%) reactive for antiHCV c100-3 will transfer HCV infection to their blood recipients (57-59). An exception, however, is a Spanish study in which 88% of the anti-I-ICV (C100-3)-positive donors were implicated in transmission of HCV to their donors (47). It is obvious, however, that many blood donors who are reactive for anti-HCV c100-3 are non-infectious, possibly owing to a false-positive reactivity or a long past infection and acquired immunity. The sensitivity of the anti-HCV c100-3 test could also be improved, since almost half of the transfusion-associated hepatitis C cases would not have been prevented by testing the blood donors in the aforementioned Spanish study (47). A false-positive anti-HCV c100-3 reaction may also occur in patients with hypergammaglobulinemia and autoimmune type-I chronic active hepatitis (CAII) (60-62), primary biliary cirrhosis (62-64), rheumatoid arthritis (65), malaria (66), paraproteinemia (67) and could also be due to cross-reactive antibodies to superoxide disrnutase, a component of the assay (68). Prolonged storage, repeated freezing and thawing, and heat inactivation of sera have also been associated with a false-positive reactivity (62, 69-71). It is therefore obvious that the anti-HCV c100-3 test is not ideal (Table I); it does not differentiate an infectious from a non-infectious patient or an ongoing infection from a remote and resolved infection, and it does not discover all patients with hepatitis C infection (that is, it has a low sensitivity). Furthermore, it is hampered by a long time lag before seroconversion occurs and by the possibility of a false-positive reaction. To circumvent these drawbacks with the anti-HCV c1003 test, different ‘confirmatory’ tests have been used like the Abbott neutralization test and the Chiron/Ortho KIBA-1 test using the 5-1-1 and the c100-3 antigens attached to a
Review
339
Table I. Drawbacks with the first-generation anti-HCV ~ 1 0 0 - enzyme-linked 3 immunosorbent assay test ~~
~
Does not differentiate an ongoing infection from a resolved infection Long ‘window-phase’ before seroconversion Does not identify all patients with HCV (that is, low sensitivity) Does not differentiate an infectious patient from a non-infectious patient False-positive reactions ____~
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Table 11. Advantages of the second-generation anti-hepatitis C virus (HCV) tests over the first-generation test Reduces the ‘window-phase’ to seroconversion Increases the sensitivity in diagnosing HCV infection Offers a better means to differentiate infectious from non-infectious blood donors Diminishes the number of false-positive reactions
nitrocellulose strip. The RIBA-1 test was found to be more specific but somewhat less sensitive in predicting infectivity among blood donors (72), and the neutralization test was better at identifying false-positive reactions due to hypergammaglobulinemia (73). The second-generation anti-HCV tests. By including antigens, synthetic or recombinant, derived from other regions of the HCV genome, improvements have been achieved with the second-generation anti-HCV tests (Table 11). Thus antigens derived from the structural region (core), c22, and from the NS3 region, c33, have been added to the original antigens 5-1-1 and c100-3 derived from the NS3/NS4 region (Fig. 1) (9,74). The seroconversion rate in patients with posttransfusion NANBH improved from 54% with the first-generation test to 82% with a second-generation anti-HCV ELISA test, and the time lag to seroconversion decreased from a mean of 6.1 weeks after onset of hepatitis with the first-generation test to a mean of 2.3 weeks with the second-generation test (75). The additional antigens utilized in the second-generation ELISA have also been included in a second-generation recombinant immunoblot assay (RIBA), whereby the sensitivity in diagnosing chronic hepatitis C has substantially increased (76,77). The specificity has also substantially increased with the second-generation tests. Thus all 4-RIBA- and anti-HCV c100-3-positive blood donors in a Dutch study were implicated in the transmission of posttransfusion NANBH, whereas none of the anti-HCV c100-3-positive but 4-RIBAnegative donors seemed to transmit HNANB (8). The second-generation tests thus reduce the time lag for seroconversion during acute HCV infection, seem to offer a better possibility to differentiate an infectious blood donor from a non-infectious one, and, furthermore, diminish the number of false-negative reactions in patients with HCV infection. Whether peptides derived from other regions of the HCV genome, such as the envelope and NS5 regions, can be utilized for diagnosing HCV infections or to differentiate
patients who will recover from those who will develop chronic disease after an acute HCV infection is currently under investigation (9). Detection of HCV RNA in serum. So far the diagnostic approach to HCV infections has largely been based on the detection of antibodies. Assays that can directly identify the virus itself in serum and in the liver would, however, much improve the understanding of HCV infections and would theoretically offer a means to differentiate infectious viremic patients from non-infectious patients. HCV antigens will be difficult to detect in serum since their concentration generally is extremely low. However, by using the PCR, HCV-RNA sequences can be detected in serum. Owing to the variability of parts of the genome, it is of vital importance to choose primers for the PCR from a highly conserved region of the HCV genome (78). The usefulness of PCR has recently been proved (Table 111). Thus, HCV RNA was detected in serum 1-2 weeks after transfusion(s) before the clinical onset of posttransfusion HCV infection and long before serum antibodies appeared (35). In addition, HCV RNA seemed to be only transiently present in serum from patients with acute resolving hepatitis but to persist, during prolonged time periods, in most patients with chronic evolution (35, 58,79, 80). Furthermore, HCV RNA in serum detected by PCR is a better predictor of infectivity than anti-HCV (11). PCR may also be useful for diagnosing vertical transmission of HCV from chronically infected mothers to their offspring (26). Finally, testing for HCV RNA offers a possibility to monitor antiviral treatment for chronic HCV infection and seems to differentiate a successful treatment from a nonsuccessful one, since HCV RNA will disappear permanently from serum after a successful course but will reappear if relapse occurs (80). Therapy of chronic hepatits NANBIC The goals of antiviral therapy for chronic HCV infection includes a reduction and/or elimination of the hepatic inflammation and the ensuing fibrosis/cirrhosis, a reduction and/or elimination of the contagiousness, and the prevention
340
Review
Table 111. The clinical usefulness of the polymerase chain reaction for the detection of hepatitis C virus (HCV) RNA
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
To differentiate an acute resolving hepatitis C from one with chronic evolution To shorten the ‘window-phase’ when diagnosing acute HCV infection To better detect infectivity of anti-HCV-positive patients To diagnose vertical transmission of HCV To monitor response to antiviral therapy for chronic HCV infection
of a later development of HCC. So far, treatment response has mostly been evaluated by monitoring the normalization rate of alanine aminotransferase (ALAT) levels in serum during and after treatment and by evaluating the histologic findings in liver biopsy specimens taken before and after treatment. Recently, however, PCR was used to monitor HCV RNA levels in serum during and after treatment (80-82). Results with interferon. In 1986 it was first reported that alpha interferon seemed to be effective for the treatment of chronic NANBH (83). Since then several randomized controlled clinical trials have proved the efficacy of alphainterferon treatment, at a dose of 3 MU thrice weekly for &9 months, for chronic HCV infection (84-87). In a recent meta-analysis of randomized controlled clinical trials on interfernn treatment the rate of s-ALAT normalization in trcated patients was approximately 5070 (88). Responders to interfcron will also improve histologically (85,89). After the end of treatment 50% of responding patients will relapse, leaving only one of four patients with a sustained response remaining also after cessation of treatment (88). A dose of 3 MU has been shown to be superior to 1 MU (84, 86, 90). Augmenting the dose further to 6 MU in ‘nonresponders’ to lowcr doses, however, did not seem to induce a biochemical response (91). A predictor of interferon response was either less hepatic inflammation or absence of cirrhosis histologically in some studies (84, 89, 92, 93), whereas no such predictors were found in other studies. The presence or not of anti-HCV c100-3 antibodies did not seem to affect the outcome of treatment (87,94). The importance of neutralizinginterferon antibodies as the cause for treatment failures in chronic HCV infections has been discussed but has not been shown as indisputable as with treatment failures in chronic hepatitis B (95). Nearly all treated patients will have an initial flu-like syndrome during the first few injections, which thereafter subsides. Other side effects are bone marrow suppression, hair loss, myalgias, and fatigue, causing dose reduction in somc 15% o f the patients and withdrawal in some 5% (85-87). Lymphoblastoid interferon also appears to be effective (96), whereas gamma interferon seems to be ineffective (97). Extended interferon treatment periods to improve the rate of sustained response is currently under investigation in clinical trials. Ofher treatments. Corticosteroids and acyclovir seem to
be ineffective (98-100). Oral ribavirin has shown promising results in a pilot study, in which 10 patients with chronic hepatitis C were treated for 12 weeks with ribavirin in a daily dose of 1000-1200mg (101). After the end of treatment, however, the ALAT levels increased to pretreatment levels (101). Recently, a case report was published indicating temporary response to inosine pranobex (102). In the future further compounds and combinations of different drugs are likely to be evaluated for treatment of chronic HCV infection. OLAWEILAND ROBERT SCHVARCZ
Dept. of Infectious Diseases Karolinska Institute Roslagstull Hospital S-114 89 Stockholm Sweden REFERENCES
1. Choo Q, Kuo G, Weiner A , Overby L, Bradley D, Houghton M. Isolation of a cDNA clone derived from a blood-home nonA, non-B viral hepatitis genome. Science 1989;244:35942. 2. Weiland 0, Berg J, Bjorvatn B, Flehmig B, Lundbergh P. Acute viral hepatitis A , B and non-A, non-B i n Stockholm in the 1950s and in the 1970s: acomparison. Infection 1981;Y:26874. 3. Alter M, Hadler S, Judson F, ct al. Risk factors for acute iio~iA , non-B hepatitis in the United States and association with hepatitis C virus infection. JAMA 199O;264:223 1-5. 4. Choo Q-L. Richman K, Han J H , et al. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci 1991;88:2451-5. 5. Houghton M, Weincr A , Han J , Kuo G, Choo QL. Molecular biology of the hepatitis C virusis: implications for diagnosis, dcvclopment and control of viral disease. Hepatology 1991;14:381-8. 6 . Kuo G, Choo Q, Alter H, et al. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-4. 7. Rradley D. Hepatitis non-A, non-B viruscs become identified as hepatitis C and E viruses. Prog Med Virol 1990;37:101-35. 8. Van Der Poel C, Cuypers H, Reesink H, ct al. Confirmation of hepatitis C virus infection by new lour-antigen recombinant imrnunoblot assay. Lancct 1991;337:3 17-9. 9. Mattsson L, Gutierrez R, Dawson G, Lesniewski R, Mushahwar I, Weiland 0. Antibodies to recombinant and synthetic peptides dcrived from the hepatitis C virus genome in long-term-studied patients with posttransfusion hepatitis C . Scand J Gastroenterol 1991;26:1257-62. 10. Kato N, Ohkoshi S , Shimotoho K. Japanese isolates of the non-A, non-B hepatitis viral genome show sequence variations from the original isolate in the USA. Proc Jpn Acad 1989;658:219-23, 1 1 . Simmonds P, Zhang L, Watson, et al. Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users. Lancet 1990;336: 1469-72.
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
Review 12. Dienstag J. Non-A, non-B hepatitis I. Recognition, epidemiology, and clinical features. Gastroenterology 1983;85: 439-62. 13. Berman M, Alter H, Ishak K, Purcell R, Jones E. The chronic sequelae of non-A, non-B hepatitis. Ann Intern Med 1979; 91:1-6. 14. Mattsson L, Aberg B, Weiland 0, Sellman M, Daviltn J. NonA. non-B hepatitis after open-heart surgery in Stockholm: declining incidence after introduction of restrictions for blood donations due to the human immunodeficiency virus Scand J Infect Dis 1988;20:371-6. 15. Mattsson L. Chronic non-A, non-B hepatitis with special reference to the transfusion-associated form. Scand J Infect Dis 1989;21 SUPPI59:1-55. 16. Koretz R, Coleman E , Abbey H, Gitnick G. Non-A, nonB post-transfusion hepatitis: When is a disease a dis-ease? [Abstract]. Hepatology 1989;10:645. 17. Kiyosawa K, Sodeyama T, Tanaka E, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990;12:671-5. 18. Bjorkander J , Cunningham-Rundles C, Lundin P, Olsson R, Soderstrom R, Hanson L. Intravenous immunoglobulin prophylaxis causing liver damage in 16 of 77 patients with hypogammaglobulinemia or IgG subclass deficiency. Am J Med 1988;84: 107-1 1. 19. Hay C, Preston F, Triger D, Underwood J. Progressive liver disease in haemophilia: an underestimated problem? Lancet 1985;1: 1495-8. 20. Martin P, Di Bisceglie A, Kassianides C, Lisker-Melman M, Hoofnagle J. Rapidly progressive non-A, non-B hepatitis in patients with human immunodeficiency virus infection. Gastroenterology 1989;97:155$61. 21. Dienstag J, Alter H . Non-A, non-B hepatitis: evolving epidemiologic and clinical perspective. Semin Liv Dis 1986;6:67R1.
22. Wejstil R , Hermodsson S, Norkrans G. Long-term follow-up of chronic hepatitis non-A, non-B-with special reference to hepatitis C. Liver 1991;11:143-8. 23. Colombo M, Kuo G , Choo Q, et al. Prevalence of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 1989;2:1006-8. 24. Bruix J, Barrera J, Calvet X , et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989;2:1004-6. 25. Esteban J, Esteban R, Viladomiu L, et al. Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;2:294-7. 26. Thaler M, Park C-K, Landers D, et al. Vertical transmission of hepatitis C virus. Lancet 1991;338:17-8. 27. Wejstil R, Hermodsson S, Iwarson S, Norkrans G. Mother to infant transmission of hepatitis C virus infection. J Med Virol 1990;30:178-80. 28. Cilla G, Ptrez-Trallero E, Iturriza M, Arrizabalaga J, Iribarren J . Possibility of heterosexual transmission of hepatitis C virus. Eur J Clin Microbiol Infect Dis 1991;10:533-4. 29. Schulman S, Grillner L. Antibodies against hepatitis C in a population of Swedish haemophiliacs and heterosexual partners. Scand J Infect Dis 1990;22:393-7. 30. Alter M, Coleman P, Alexander J, et al. Importance of heterosexual activity in the transmission of hepatitis B and nonA, non-B hepatitis. JAMA 1989;262:1201-5. 31. Kiyosawa K, Sodeyama T, Tanaka E, et al. Hepatitis C in hospital employees with needlestick injuries. Ann Intern Med 19Y 1;115:367-Y. 32. Takamatsu K, Koyanagi Y, Okita K, Yamamoto N. Hepatitis C virus RNA in saliva. Lancet 1990;336:1515. 33. Wang J-T, Wang T-H, Lin J-T, Sheu J-C, Lin S-M, Chen D-S. Hepatitis C virus RNA in saliva of patients with posttransfusion hepatitis C infection. Lancet 1991;337:48. 34. Dusheiko G, Smith M, Scheuer P. Hepatitis Cvirus transmitted by human bite. Lancet 1990;336:503-4. 35. Farci P, Alter H, Wong D, et al. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 1991:325:98- 104.
341
36. Alter H, Purcell R, Shih J, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;321: 1494-1500. 37. Roggendorf M, Deinhardt F, Rasshofer R, et al. Antibodies to hepatitis C virus. Lancet 1989;2:324-5, 38. Sonnerborg A, Abebe A, Strannegird 0 . Hepatitis C virus infection in individuals with or without human immunodeficiency virus type 1 infection. Infection 1990;18:347-51. 39. Weiland 0, Lindh G , Mattsson L, Schvarcz R, von Sydow M. Hepatit C en vanlig orsak till kronisk hepatit i Sverige [in Swedish]. Lakartidningen 1990;34:2567-8. 40. Tor J, Llibre J, Carbonell M, et al. Sexual transmission of hepatitis C virus and its relation with hepatitis B virus and HIV. Br Med J 1990;301:1130-3. 41. Widell A, Hansson B, Berntorp E , et al. Antibody to a hepatitis C virus related protein among patients at high risk for hepatitis B. Scand J Infect Dis 1991;23:19-24. 42. Tremolada F, Casarin C, Tagger A, et al. Antibody to hepatitis A virus in post-transfusion hepatitis. Ann Intern Med 1991; 114:277-81. 43. Kiese M, Lenz K, Guggenmoos-Holzmann I, Stark K, Bienzle U. Epidemiology of hepatitis C in homosexual men. KIin Wochenschr 1990;68: 1082. 44. Lin-Chu M, Tsai S , Watanabe J, Nishioka K. The prevalence of anti-HCV among Chinese voluntary blood-donors in Taiwan. Transfusion 1990;30:47 1-3. 45. Nishimura Y, Yamagushi K, Williams N, et al. Antibodies to hepatitis C virus in Japanese blood donors and in hospital personnel. Transfusion 1990;30:667-8. 46. Sirchia G, Almini D, Bellobuono A, et al. Prevalence of hepatitis C virus antibodies in Italian blood donors. Vox Sang 1990;59:2&9. 47. Esteban J, Gonzalez A, Hernandez J, et al. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990;323:1107-12. 48. Hetland G , Skaug K, Larsen J, Maeland A, Stromme J, Storvold G. Prevalence of anti-HCV in Norwegian blood donors with anti-HBc or increased ALT levels. Transfusion 1990; 30:776-9. 49. Wantzin P, Krogsgaard K, Dickmeiss E. Screening af danske bloddonorer for antistof rettet mod Hepatitis C virus [in Danish]. Ugeskr Laeger 1990;152:284&8. 50. Lindholm A, Hermodsson S, Norkrans G , Iwarson S. Nytt serologiskt test ger mojlighet att diagnosticera hepatit C [in Swedish]. Lakartidningen 1990;87:40-1. 51. Janot C, Courouct A, Maniez M. Antibodies to hepatitis C virus in French blood donors. Lancet 1989;2:796-7. 52. Kiihnl P, Seidl S, Stangel W, Beyer J, Sibrowski W, Flik J. Antibody to hepatitis C virus in German blood donors. Lancet 1989;2: 324. 53. Menitove J, Richards W, Destree M. Early US experience with anti-HCV kit in blood donors. Lancet 1990;336:244-5. 54. Stevens C, Taylor P, Pindyck J, et al. Epidemiologyof hepatitis C virus: a preliminary study in volunteer blood donors. JAMA 1990;263:49-53. 55. Mattsson L, Grillner L, von Sydow M, Bergdahl S, Weiland 0. Seroconversion to hepatitis C virus antibodies in patients with acute posttransfusion non-A, non-B hepatitis in Sweden. Infection 1991;19: 30S12. 56. Krogsgaard K, Wantzin P, Mathiesen L, Sonne J, Ring-Larsen H. The Copenhagen Hepatitis Acuta Programme. Early appearance of antibodies to hepatitis C virus in community acquired acute non-A, non-B hepatitis is associated with progression to chronic liver disease. Scand J Infect Dis 1990; 22:399-402. 57. Van der Poel C, Reesink H, Schaasberg W, et al. Infectivity of blood seropositive for hepatitis C virus antibodies. Lancet 1990;335:558-60. 58. Garson J, Tedder R, Briggs M, et al. Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 141%22.
Scand J Gastroenterol Downloaded from informahealthcare.com by University Library at IUPUI on 09/21/12 For personal use only.
342
Review
59. Eheling F, Naukkarinen R, Lcikola J . Post-transfusion hepdtitis C in Finland. Transfusion 1991;1:109-13. 60. McFarlane I, Smith H , Johnson P, Bray G , Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 1990;33s:754-7. 61. Schvarcz R, von Sydow M, Weiland 0 . Autoimmune chronic active hepatitis: changing reactivity for antibodies to hepatitis C virus after immunosuppressive treatment. Scand J Gastroenterol 1990;25:1175-80. 62. Schrumpf E, Elgjo K, Fausa 0, Haukenes G , Kvale D , Rollag H. The signficance of anti-hepatitis C virus antibodies measured in chronic liver disease. Scand J Gastroenterol 1990;25:1169-74. 63. Di Bisceglie A , Alter H, Kuo G, Houghton M, Hoofnagle J. Detection of antibody to hepatitis C virus in patients with various chronic liver diseases [Abstract]. Hepatology 1989; 10:581. 64. Fusconi M, Lenzi M, Ballardini G , et al. Anti-HCV testing in autoimmune hepatitis and primary hiliary cirrhosis. Lancet 1990;336:823. 65. Theilmann L, Blazek M, Goeser T, Gmelin K, Kommerell B, Fiehn W. False-positive anti-HCV tests in rheumatoid arthritis. Lancct 1990;335:1346. 66. Aceti A. Taliani G , De Bac C, Sebastiani A. Anti-HCV false positivity in malaria. Lancet 1990;336:1442-3. 67. Boudart D, Lucas J-C, Muller J-Y, Lc Carrer D, Planchon B, Harousseau J-L. False-positive hepatitis C virus antibody tests in paraproteinaemia. Lancet 1990;336:63. 68. Ikeda Y, 'I'oda G , Hashimoto N, Kurokawa K. Antibody to superoxide dismntase, autoimmune hepatitis, and antibody tests for hepatitis C virus. Lancet 1990;335:1345-6. 69. Ellis L, Brown D, Conradie J , et al. Prevalence of hepatitis C in South Africa: detection of anti-HCV in recent and stored serum. J Med Virol 1990;32:24Y-51. 70. Mortimcr P, Cohen B, Litton P, et al. Hepatitis C virus antibody. Lancet 1989;2:798. 71. Wong D, Diwan A, Rosen L, et al. Non-specificity of antiHCV test for seroepidemiological analysis. Lancet 1990;336: 7Sc-1. 72. Ebeling F, Naukkarincn R, Leikola J. Recombinant immunoblot assay for hepatitis C virus antibody as predictor of infectivity. Lancet 1990;335:982-3. 73. Schvarcz R, von Sydow M, Weiland 0 . Positive reactivity with anti-HCV ELISA in patients with autoimmune hepatitisnot confirmed with a neutralization test. Scand J Infect Dis 1991;23:127-X. 74. Choo Q-L, Weiner A, Overby L, Kuo G , Houghton M, BradIcy D. Hcpatitis C virus: the causative agent of viral non-A, non-B hepatitis. Br Med Bull 1990;46:423-41. 75. Mattsson L, Grillner L, Weiland 0.Seroconversion to hepatitis C virus antibodies in patients with acute posttransfusion nonA, non-B hepatitis in Sweden with a second generation test. Scand J Infect Dis 1Y92. In press. 76. Marcellin PMM-P, Boyer N , Pouteau M, Aumont P, Erlinger S , Benhamou J-P. Second generation (RIBA) tests in diagnosis of chronic hepatitis C. Lancet 1991;337:551-2. 77. Craxi A, Fiorentino G , Di Marco V, et al. Second generation tests in diagnosis of chronic hepatitis C. Lancet 1991;337:1354. 711. Carson J, Ring C, Tuke P, 'l'cdder R. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990;336:8769. 79. Garson J , Tuke P, Makris M, et al. Demonstration of viremia patterns in haemophiliacs treated with hepatitis-c-virus-contaminated factor VIII concentrates. Lancet 1990;336:1022-5. 80. Schlauder G , Leverenz G , Mattsson L, Weiland 0,Mushahwar I. Detection of hepatitis C viral RNA by the polymerase chain reaction in serum of patients with post-transfusion non- A, nonB hepatitis. J Virol Methods. In press. 81. Brillanti S, Garson J, Tuke P, et al. Effect of alfa-interferon therapy on hepatitis C viraemia in community-acquired chronic non-A, non-B hepatitis: A quantitative polymerase chain reaction study. J Med Virol 1991;34:13&41. 82. Chayama K, Saitoh S, Arase Y, et al. Effect of interferon
administration on serum hepatitis C virus RNA in patients with chronic hepatitis C. Hepatology 1991;13:1040-3. 83. Hoofnagle J, Mullen K, Jones D, et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon. A preliminary report. N Engl J Med 1986;315:1575-8. 84. Causse X, Godinot H, Chevalier M, et al. Comparison of 1 or 3 MU of interferon alfd-2b and placebo in patients with chronic non-A, non-B hepatitis. Gastroenterology 1991;101:497-502. 85. Di Bisceglie A, Martin P, Kassianides C, et al. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med 1989; 321:3so6-10. 86. Davis G , Balart L, Schiff E , et al. Treatment of chronic hepatitis C with recombinant interferon alfa. A multicenter randomized, controlled trial. N Engl J Med 1989;321:1501-6. 87. Schvarcz R, Weiland 0, Wejstdl R, Norkrans G , Foberg U, Fryden A. A randomized controlled open study of interferon alpha-2h treatment of chronic non-A. non-B posttransfusion hepatitis: no correlation of outcome to presence of hepatitis C virus antibodies. Scand J Infect Dis 1989;21:617-25. 88. Tine F, Magrin S, Craxi A, Pagliaro L. Interferon for non-A, nun-B chronic hepatitis. A meta-analysis of randomised clinical trials. J Hepatol 1991;13:192-9. 89. Schvarcz R, Glaumann H, Weiland 0 , Norkrans G , Wejsrdl R, FrydCn A. Histological outcome in interferon alpha-2b treated patients with chronic posttransfusion non-A, non-B hepatitis. Liver 1991;11:3&8. 90. Marcellin P, Boyer N. Giostra E, et al. Recombinant human alfa-interferon in patients with chronic non-A, non-B hepatitis: a multicenter randomized controlled trial from France. Hepatology 1991;13~393-7. 91. Schvarcz R , Glaumann H, Weiland 0 , Norkrans G , Wejstil R, Fryden A . Interferon alpha-2b treatment of chronic posttransfusion non-A, non-B/C hepatitis: long-term outcome and effect of increased interferon doses in non-responders. Scand J Infect Dis 1991;23:413-20. 92. Ferenci P, Vogel W, Pristautz H, et al. One-year treatment of chronic non-A. non-B hepatitis with interferon alfa-2b. J Hepatol 1990;ll Suppl:SO-3. 93. Saracco G , Rosina M, Torrani Cerenzia M, et al. A randomized controlled trial of interferon alfa-2b as therapy for chronic nonA, non-B hepatitis. J Hepatol 1990;ll Suppl:4>9. 94. Marcellin P, Giostra E , Boyer N , Loriot M-A, MartinotPeignoux M, Benhamou J-P. Is the response to recornbinant alpha interferon related to the presence of antibodies to hepatitis C virus in patients with chronic non-A, non-B hepatitis? J Hepatol 1990;11:77-9. 95. Brook M, McDonald J, Karayiannis P, et al. Randomised controlled trial of interferon alfa 2A (rbe) (Roferon-A) for the treatment of chronic hepatitis B virus (HBV) infection: factors that influence response. Gut 1989;30:111622. 96. Jacyna M, Brooks M, Loke R, Main J, Murray-Lyon I, Thomas H. Randomized controlled trial of interferon alfa (lymfoblastoid interferon) in chronic non-A, non-B hepatitis. Br Med J 1989;298:8&2. 97. Saez-Royuela F, Porres J, Moreno A, et al. High doscs of recombinant alfa-interferon or gamma-interferon for chronic hepatitis C: a randomized, controlled trial. Hepatology 1991;13:327-3 I . 98. Stokes P, Lopez W, Balart L. Effects of short-term corticostcroid therapy in patients with chronic non-A, non-B hepatitis [Abstract]. Gastroenterology 1987;92:1783. 99. Pappas S, Hoofnagle J, Young N , Straus S , Jones E. Treatment of chronic non-A, nun-B hepatitis with acyclovir: pilot study. J Med Virol 198S;lS: 1-9. 100. Hoofnagle J. Chronic hepatitis: the role of corticosteroids. In: Szmuness W, Alter HJ, Maynard JE, editors Viral hepatitis. 1981 international symposium. Philadelphia: Franklin Press, 1982573-83. 101. Reichard 0, Andersson J , Schvarcz R, Weiland 0. Ribavirin treatment for chronic hepatitis C. Lancct 1991;337:105X-61. 102. Prohaska W, Kleesiek K. Treatment of chronic hepatitis C with inosine pranobex. Lancet 1991;338:390-1.
