Hepatitis C virus infection in Greece and its role in chronic liver

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3): $72-$77. © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00. HEPAT 01302. Hepatitis C virus infection in Greece and ...


Journal of Hepatology, 1993; 17 (Suppl. 3): $72-$77 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00

HEPAT 01302

Hepatitis C virus infection in Greece and its role in chronic liver disease and hepatocellular carcinoma S.J. Hadziyannisa, G. Giannoulis a, E. Hadziyannis a, E. K a k l a m a n i c, A. Alexopoulou a, S. Dourakis b and D. Trichopoulos c *Academic Department of Medicine and bNational Center for Communicable Liver Diseases, Hippokration General Hospital and CDepartment of Hygiene and Epidemiology, Athens Medical School, Athetts, Greece

The aim of this study was to evaluate the prevalence of hepatitis C virus infection (HCV) in Greece, to estimate its frequency in chrooic liver disease and to explore the role of HCV infection in the aetiology of hepatoceilular carcinoma. A series of 1034 patients with chronic liver disease of various aetiologies and 299 patients with hepatocellular carcinoma allocated to two case-control studies was tested for anti-HCV. Twelve recent reports on HCV infection in Greece were reviewed and analyzed. The results of the present study indicate the existence of a large pool of HCV infection in Greece and an impressive spread of the virus in high-risk groups. Chronic HCV infection was found to account for 83.6% of patients with chronic non-A, non-B hepatitis parenteraily transmitted, 56.5% of cases of sporadic communityacquired disease and for almost i/4 of all patients with chronic liver disease. The relative risks for development of hepatocellular carcinoma of patients with chronic HCV infection was 6.3 in the first and 13.7 in the second casecontrol study, increasing to 20.0 and 18.7, respectively, when hepatitis B surface antigen (HBsAg) was positive. Serum HBV-DNA was positive and/or anti-HBc IgM levels were high in 12 of 15 (80%) patients with hepatocellular carcinoma positive only for HBsAg, and in 7 of 15 (47%) positive both for HBsAg and antibodies to HCV. The present data support the view that hepatitis B and C virus have an interacting role in the origin of hepatocellular carcinoma.

Key words." Hepatitis C virus (HCV); Epidemiology of HCV; Hepatocellular carcinoma

Chronic liver disease (CLD) and hepatocellular carcinoma (HCC) have been found to be associated with chronic hepatitis B virus (HBV) infection in more than 50% of the cases in Greece (1,2). Hepatitis delta virus (HDV) infection has also been found to play an important role and antibodies to HDV (anti-HD) have been detected in 9-35% of patients with viral B CLD (3) and in 10-19.5% of patients with HCC (3,4). Data on hepatitis C virus (HCV) infection in CLD in Greece are still limited (5), but from a recent report an important role for HCV infection in the origin of HCC (6) has emerged as both an independent and interactive factor with HBV. In this article data will be presented on: (a) the prevalence of HCV infection in Greece, (b) the frequency of markers of HCV infection in CLD and (c) the role of HCV infection in the aetioiogy of HCC.

Materials and Methods

Data analyzed in this article come from: (a) the National Center for Communicable Liver Diseases of Greece, functioning in the frame of the Academic Department of Medicine at the Hippokration General Hospital of Athens and (b) recent Greek reports published in local and international medical journals. The patients studied in the National Reference Center for Communicable Liver Diseases were divided in two groups: (a) With established CLD of various aetioiogies followed as in- or out-patients during 1989-1991. This group consisted of 1034 patients and served to evaluate the prevalence of anti-HCV in the various aetiologies of CLD in Greece. (b) With HCC allocated to two independent case-control

Correspondence to: Prof. Stephanos J. Hadziyannis, Academic Department of Medicine, Hippokration General Hospital, II4 Vas. Sophias Ave., Athens 115 27, Greece.

ROLE OF HCV IN LIVER DISEASE IN GREECE studies specifically designed to explore the role of HCV infection in the origin of HCC. The first study was a large-scale case-control study of the period 1976-1984 and comprised 181 patients with HCC, 31 with metastatic liver cancer and 432 hospital patients as controls (6). Sera kept at - 2 5 °C were tested for anti-HCV with the first-generation ELISA. Positive specimens were characterised as strongly positive if the optical density (OD) was 2 or more times higher than the cut-off limit and as weakly positive otherwise. The statistical analysis was done by modelling the data through multiple logistic regression, controlling for age, gender and HBsAg status (6). In the second case-control study of the 1988-1990 period, 118 patients with HCC, 379 with hepatitis B surface antigen (HBsAg)-positive CLD uncomplicated by HCC and 238 hospital patients, without liver disease were enrolled. Fresh sera were tested for anti-HCV and the positive results were subjected to supplemental testing. The total number of patients with HCC enrolled in the two independent case-control studies was 299. The patients with C L D and HCC included in groups (a) and (b) coincide partly only during the 1989-1990 period. All patients in the present study had at least one serum sample tested in duplicate for anti-HCV by a first-generation enzyme immunoassay (Ortho HCV Antibody ELISA Test System, Ranitan, N J, U.S.A.), with a cut-offof 0.400 in optical density (OD). A smaller number of cases positive for anti-HCV was subjected to supplemental testing by the recombinant immunoblot assay (RIBA) with 80% concordance. Sera were tested for HBV-DNA by a solution hybridization assay (Genostics Abbott) and the results were expressed in pg/ml. HBV-DNA values above 5 pg/ml were considered as positive (7). Antibodies to hepatitis B core antigen of IgM class (anti-HBc IgM) were quantitated by a microparticle enzyme immunoassay (MEIA) on an IMx analyzer (Abbott) with a cut-off of 1.400 for acute hepatitis B and of 0.300 for chronic hepatitis with replicating HBV (8). In chronic HBV infection with nonreplicating B virus and normal ALT levels anti-HBc IgM values have been found to be always below 0.200. In chronic hepatitis with HBV-induced liver damage anti-HBc IgM values are practically always above 0.500

(8). The reviewed Greek reports represent 9 full papers recently published in Greek medical journals (5,9-16) and 3 abstracts published in the Journal of Hepatology (17-19). Data were analyzed statistically using Student's t-test and the formula for relative risk (RR) with 95% confidence intervals (CI) (2,6,20).

$73 Results

Prevalence of HCV infection in Greece The prevalence of anti-HCV by first-generation ELISAs in Greek volunteer blood donors is shown in Table !. The prevalence was similar to the anti-HCV frequency reported in the majority of European countries. Second-generation ELISAs have increased the rate of true anti-HCV positivity approximately by 30% (Kontopoulou I, Mandalaki T, Politi C, unpublished observation, December, 1991). Data on the anti-HCV frequency among several high-risk groups in Greece are also shown in Table 1. In acute non-A, non-B (NANB) hepatitis, infection with the HCV was found to account for more than twothirds of parenterally transmitted cases and for about one-fourth of sporadic, community-acquired disease (Table 2).

Frequency of HCV markers in CLD Among 1034 patients with established CLD of various aetiologies, the frequency of anti-HCV ranged from a minimum of 3.3% and 4.2% in the groups of miscellaTABLE I Prevalence of anti-HCV in blood donors and in high-risk groups in Greece Population

Number Anti-HCV Positive tested (n) (%) Volunteer blood donors" 32 038 179 0.56 Chronic haemodialysis ( 13-15) 898 209 23.3 Renal transplant recipients (14) 206 21 10.2 Oncology patients (5) 72 5 7.0 Multiply transfused (9,10) 665 257 38.6 Haemophiliacs (18) 210 198 94.3 Intravenous drug abusersb 159 129 81.1 Prostitutes (12) 203 9 4.4 Homosexuals HIV(+)(11,12) 163 15 9.2 Homosexuals HIV(-) (I 1,12) 263 4 1.5 "Kontopoulou I, Mandalaki T, Politi L., unpublished observations, December, 1991. bMalliori K, Hatzakis A, Stefanis K., unpublished observations, January, 1992. TABLE 2 Prevalence of anti-HCV in acute and chronic and NANB hepatitis in G reece Diagnosis

Number Anti-HCV-positive tested n


Acute NANB hepatitis (total)" 161 84 52.2 Parenterally transmitted 91 66 72.5 Sporadic (community-acquired) 70 18 25.7 Chronic NANB hepatitis (total)b 172 127 73.8 Parenterally transmitted I l0 92 83.6 Sporadic (cryptogenic) 62 35 56.5 "Reported by Tassopouloset al. (19). bReported by Hadziyanniset al. (I 7) and findings in the present study.



neous causes and of primary biliary cirrhosis, respectively, to a maximum of 73.8% in HBsAg-negative chronic hepatitis and cirrhosis (Table 3). Patients with HBsAg(+) chronic hepatitis/cirrhosis had an overall antiHCV prevalence of 4.8%, ranging in the various histological and serological subgroups from i.6% to 5.8%. These differences were not statistically significant. In HBsAg(+)/anti-HD(+) patients the anti-HCV prevalence was 12.7% (7 of 55). Six of the 7 anti-HCV(+)/anti-HD(+) patients had been infected parenterally (intravenous drug abusers). The prevalence of anti-HCV was significantly higher in HBsAg-negative than -positive H C C (Table 3). The anti-HCV prevalence in chronic N A N B hepatitis was 83.6% in parenterally transmitted and 56.5% in sporadic, community-acquired (cryptogenic) disease (Table 2). On the basis of the anti-HCV prevalence rates in Table 3 and of the relative frequencies of various aetiologies of C L D detected during the period 1985-1990 in the National Center for Communicable Liver Diseases, the overall prevalence of HCV infection in C L D in Greece was calculated to be 23.7% (Table 4), with HCV being the only aetiological factor in 19.7% and an aetiological co-factor in 4% of the cases. TABLE 3 Prevalence of serum anti-HCV m patients with chronic liver disease of various aetiologies in Greece Diagnosis

Tested In)

Anti-HCV(+) n

Chronic hepatitis/cirrhosis HBsAg (+) HBsAg [-I Hepatocellular carcinoma HBsAg (+) HBsAg (-) Alcoholic liver disease Primary biliary cirrhosis Autoimrnune chronic active hepatitis Miscellaneous chronic liver diseases Total

458 172

22 127

4.8 73.8

130 50 40 72 22 90 1034

23 16 3 3 3 3 200

17.7 32.0 7.3 4.2 13.6 3.3 19.3

TABLE 4 Relalive frequencies of various aetiologies of chronic liver disease in Greece (1985-1990) and the role of HCV infection Aetiology

Prevalence of Prevalence of aetiology anti-HCV Viral B and D 54.5 4.8 NANB and cryptogenic 26.7 73.8 Alcoholic 6.4 7.5 Primary biliary 6.1 4.2 Autoimmune 3.4 13.6 Miscellaneous 2.9 3.3 All aetiologies 100.0~ 23.7%~ aWeighted mean prevalence in chronic liver disease of any aetiology.

Role of HCV in hepatocelhdar carcinoma Table 5 shows the estimates of the relative risk (RR) linking strongly positive anti-HCV reactions with HCC, after multivariate analysis (6), in the case-control study of the period 1976-1984. Weakly positive assays for anti-HCV had no aetiological implication. Controlling for HBsAg reduced the RR associated with strongly positive anti-HCV from 8.6 to 6.3, and controlling for anti-HCV reduced the RR associated with HBsAg from 13.7 to 11.4. However, in addition to being a confounding factor, the association between anti-HCV and H C C was modified by the presence of HBsAg. As shown in Table 5, the RR for H C C was significantly higher in subjects positive for both anti-HCV and HBsAg than in subjects positive for anti-HCV but negative for HBsAg (p0.500 (Fig. I), indicative of HBV-induced liver damage. HCV-RNA has been detected by others in the serum and liver of anti-HCV-

positive patients with HCC (22,23,29). It is therefore logical to assume that hepatocellular injury, probably induced by a cytopathic action of HCV, may combine, in some patients infected by both viruses, with liver-cell damage induced by immune mechanisms against the replicating HBV. This can represent a particularly highrisk condition for the development of HCC and could give a pathogenetic interpretation to the high probability of occurrence of HCC in patients infected by both viruses. In summary, the results of the present study have shown a large pool of HCV in Greece, have delineated the important role of chronic HCV infection in the aetiology of chronic liver disease in Greece, have documented the high relative risk for development of HCC of patients with viral C chronic liver disease ranging between 6 and 20, and have suggested that HBV and HCV may play an interactive role in hepatocarcinogenesis which, in some patients, appears to be related to underlying replication of both viruses.


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