Journal of Gastroenterology and Hepatology (1992) 7, 393-395
LIVER A N D BILIARY
Prevalence of hepatitis C virus antibodies in chronic liver disease and hepatocellular carcinoma patients in India R A J A G O P A L R A M E S H , A N U P A M A M U N S H I A N D S U B R A T K. P A N D A
Department of Pathology, All India Institute of Medical Sciences, New Delhi, India Abstract The prevalence of antibodies to hepatitis C virus (HCV) was investigated in 129 patients with chronic liver disease (85 with chronic active hepatitis and 44 with cirrhosis) and 53 patients with hepatocellular carcinoma. The commercially available second generation anti-HCV enzyme immunoassay kit was used. Antibodies to hepatitis C virus were detected in 16.2% of the patients with chronic liver disease and in 15.1% with hepatocellular carcinoma. Of the HCV positive patients in all groups 51.7% were positive for hepatitis B virus (HBV) markers indicating present or past infection. Prevalence of HBV markers in all the three groups (CAH, cirrhosis and HCC) was higher as compared with anti-HCV prevalence. These results suggest that HCV infection may not be a major cause of chronic liver disease and hepatocellular carcinoma in India and indicate the presence of other aetiological agents. Key words: chronic liver disease, hepatitis B virus, hepatitis C virus, anti-hepatitis C virus, hepatocellular carcinoma.
INTRODUCTION Hepatocellular carcinoma (HCC) is a common neoplasm world-wide.' Epidemiological and molecular hybridization studies demonstrate a causal relationship between hepatitis B virus (HBV) and hepatocellular In countries such as India with an intermediate HBV carrier rate (2-4O/0),~ the incidence of hepatocellular carcinoma (HCC) has been more commonly related to other factors, especially cirrhosis and aflatoxin level in the food Recently, Gilliam et a1.8 and Okuda' have suggested that non-A, non-B hepatitis (NANBH) viruses may play a role in the development of HCC in patients with cirrhosis. The genome of transfusion associated NANBH virus designated as hepatitis C virus (HCV) has been cloned" and has emerged as the major cause of post-transfusion associated chronic hepatitis. "J' Studies from many parts of the world demonstrate the presence of anti-HCV antibodies in the serum of both HBsAg positive and negative chronic hepatitis (50% and 90%) and HCC patients (50 and 70% respectively) and suggests the involvement of HCV in liver carcin~genesis.'~-'~ Thus it is important to know the prevalence of HCV in chronic liver disease and HCC for any geographical region.
METHODS Serum samples were obtained from 53 patients with hepatocellular carcinoma (HCC) and 129 patients with
chronic liver disease (CLD) (85 with chronic active hepatitis [CAH] and 44 with cirrhosis) who attended All India Institute of Medical Sciences (AIIMS), Govind Balabh Pant Hospital (G.B.Pant) New Delhi and Madras General Hospital, Madras between 1988 and 1990. T h e sera were tested for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. All the cases of hepatocellular carcinoma diagnosed clinically as well as by ultrasound were confirmed by histopathological examination of the needle biopsies. These patients comprised 45 males and 8 females whose mean age was 44.7 years (range 10-70 years). T h e 129 cases of chronic liver disease were diagnosed clinically and the diagnosis was confirmed by laboratory investigation followed by histopathological examination of the needle biopsies. T h e mean age in this group was 42.3 years (range 15-60 years) with a sex distribution of 98 males and 31 females. None of the cases included in this study had a history of alcoholism, drug intake or evidence of other known cause of liver disease. At least 5 m L of blood was collected from each of these patients aseptically, serum was separated and stored at - 70°C until further use. All the serum samples were tested for HBV infection (present or past infection) by testing for HBsAg, anti-HBsAg and anti-HBc by using commercial micro ELISA test kits (Hepanostika, Organon Teknika, Netherlands) according to the manufacturer's guidelines. Detection of HBV-DNA in the serum was carried out by dot blot hybridization according to the method described by Scotto et al. l 7 Current infection with HBV was assessed by serological tests for HBsAg, IgM, anti-HBc and/or HBV-DNA. T h e past infection for HBV
Correspondence: Dr S. K. Panda, Department of Pathology, All India Institute of Medical Sciences, New Delhi 110 029, India, Accepted for publication 13 February 1992.
R. Ramesh et al.
394 was assessed by the presence of antibodies to the surface antigen (anti-HBs) and/or to the core antigen (IgG, anti-HBc). Anti-HCV was tested by using the second generation ABBOTT-HCV enzyme immunoassay kit (ABBOTT Co., Chicago, IL, USA). This indirect assay uses recombinant HCV antigens (covers the structural and non-structural region) coated on polystyrene beads. Anti-HCV in the serum was detected with an antibodyenzyme conjugate comprised of goat monoclonal antibody against human IgG conjugated with horseradish peroxidase (HRPO). T h e end product was read by a Philips spectrophotometer (PUS800 UV/Visible) at 492 nm. T h e results were interpreted according to the manufacturer's guidelines. Each of the test samples was tested in duplicate and the test was carried out according to the manufacturer's guidelines.
seen in 9/54 (16.6%) of chronic liver disease and 4/35 (11.4%) of HCC (Table 2). There was no statistical (Chi-squared test) difference in HCV positivity between HBV infected or non-infected individuals. Of the patients positive for antibodies to HCV 51.7% were also positive for current HBV infection. On the other hand only 18.8% of current HBV infection cases had antibody against HCV (including all the three groups; Table 2). Among hepatocellular carcinoma patients, three had markers for past HBV infection, characterized by the presence of antiHBsAg and/or IgG anti-HBc. One of them was positive for anti-HCV (Table 2). There were no markers for either HBV or HCV in 34.8% of chronic liver disease patients and 58.4% of hepatocellular carcinoma patients.
DISCUSSION
RESULTS The prevalence of HBsAg/or HBV-DNA and antibodies to HCV in patients with chronic liver disease and hepatocellular carcinoma is shown in Table 1. There is a significant difference (P< 0.001) in current HBV infection marker prevalence between cirrhosis and hepatocellular carcinoma (Table 1). The prevalence of antibodies to HCV was in 16.2% (21/129) of chronic liver disease patients (chronic hepatitis and cirrhosis put together) and in 15.1% (8/53) of hepatocellular carcinoma (Table 1). The status of HBV infection along with positivity for antibodies to HCV is shown in Table 2. Anti-HCV was positive in 12/75 (16%) chronic liver disease patients and 4/18 (22.2%) of HCC patients positive for present or past HBV infection. The positivity for anti-HCV alone was Table 1 Relationship between current HBV infection versus anti-HCV in patients with chronic liver disease (CLD) and hepatocellular carcinoma (HCC) No. samples HBsAg/HBV-DNA Anti-HCV positivity/total (YO) tested positive (%)* CAH Cirr HCC
85 44 53
30 (44.7) 27 (61.4) 15 (28.0)
13 (15.3) 8 (18.0) 8 (15.1)
* Indicates current HBV infection.
Information on HCV infection is available from many parts of the world since the identification of its genome and availability of a specific antibody test.'*-*' The present study reports the prevalence of HCV antibody in chronic liver disease and hepatocellular carcinoma in India. T h e observations indicate a low prevalence of HCV antibody (15-18%) among chronic liver disease and HCC patients as compared with other studies from Japan, Italy and Spain where the frequency of anti-HCV is more than 80% in chronic liver disease and HCC patient^.'^"^ When HBV-infected individuals were excluded from the group of chronic liver diseases and hepatocellular carcinoma it was found that only 13/89 (14.6%) of the cases had antibody to HCV. However while interpreting the results of the anti-HCV test, the low sensitivity of the test has to be kept in mind. The findings suggest that HBV may be a more important cause of chronic liver diseases and hepatocellular carcinoma in India. This is similar to what has been observed in South Africa.I6 In an earlier study hepatocellular carcinoma was demonstrated in cases with past HBV infection although its exact significance is not known.21 In the present study three cases of hepatocellular carcinoma with past HBV infection were detected and one of them was positive for anti-HCV. T h e higher prevalence of HBV in chronic liver disease in comparison with hepatocellular carcinoma may be related to early development of cirrhosis leading to death where adequate time for the development of hepatocellular carcinoma is not available. Absence of HBV or HCV markers in nearly half (41.7%) of the patients suggests the possible role of
Table 2 HBV markers status in relation to anti-HCV positivity in different categories of chronic liver disease Type of HBV markers present
Active or recent HBV infection (HBsAg/HBV-DNA+) Past HBV infection No HBV infection Total
CAH (n = 85) Cirrhosis (n = 44) HCC (n = 53) anti-HCV + (%) anti-HCV - (%) anti-HCV + (%) anti-HCV - (%) anti-HCV + (%) anti-HCV - (%) 6 (7.0)
32 (37.6)
6 (13.6)
21 (47.0)
3 (5.6)
12(22.6)
0 (0)
6 (7.0) 34 (40.0)
0 (0)
7 (8.2)
2 (4.5)
4 (9.0) 11 (25.0)
l(1.8) 4 (7.5)
2 (3.7) 31 (58.4)
13 (15.3)
72 (84.7)
8 (18.2)
36 (81.8)
8 (15.1)
45 (84.9)
Anti-HCV in chronic liver disease aetiological agents, such as yet unidentified virus(es) or chemical carcinogens including aflatoxins.
395 9. OKUDA K. Primary liver cancer. Dig. Dis. Sci. 1986; 31: 133-46s. 10. CHOOQ. L., KUOG, WEINERA. J. er al. Isolation of a cDNA
clone derived from a blood borne non A, non B viral hepatitis genome. Science 1989; 244: 359-61. ACKNOWLEDGEMENTS R. H., SHIHJ. W. et al. Detection of 11. ALTERH. J., PURCELL antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non A, non B The authors acknowledge that this study has been con. hepatitis. N. Engl. J. Med. 1989; 321: 1494-500. ducted with financial assistance from the Indian Council 12. Kuo G., CHOOQ. L., ALTER H. J. et al. An assay for of Medical Research (New Delhi) and the Overseas circulating antibodies to major etiologic virus of human non Development Administration (UK). A, non B hepatitis. Science 1989; 244: 362-4. 13. KIOSAWA K., SODEYAMA T., TANAKA er al. Interrelationship of blood transfusion, non A, non B hepatitis and hepatocellular carcinoma analysis by detection of antibody to hepatitis C REFERENCES virus. Hepatology 1990; 12: 671-5. M., Kuo G., CHOOQ. L. er al. Prevalence of 14. COLOMBO antibodies to hepatitis C virus in Italian patients with 1. PERKIN D. M., STJENSWARD J. & MUIRC. S. Estimates of the hepatocellular carcinoma. Lancet 1989; ii: 1006-8. world wide frequency of twelve major cancers. Bull. WHO 15. BRUIXJ., BARRERA J. M., CALVET X. er al. Prevalence of 1984; 62: 163-82. antibodies to hepatitis C virus in Spanish patients with 2. SMUZNESS W. Hepatocellular carcinoma and hepatitis B hepatocellular carcinoma. Lancet 1989; ii:1004-6. virus: evidence for a causal association. Prog. Med. Virol. 16. KEWM. C., HOUCHTON M., CHOOQ.L. & Kuo G. Hepatitis 1978; 24: 40-69. C virus antibodies in south African blacks with hepatocellu3. SHAFRITZ D. A. & KEWM. C. Identification of integrated lar carcinoma. Lancer 1990; 335: 873-4. hepatitis B virus DNA sequences in hepatocellular carciM., HERYC. er al. Detection of 17. SCOTTOJ., HADCHOUEL noma. Hepatology 1981; 1: 1-8. hepatitis B virus DNA in serum by simple spot hybridisation 4. BEASLEY R. P. Hepatitis B virus as the etiologic agent in technique: Comparison with results for other viral markers. hepatocellular carcinoma. Epidemiologic considerations. Hepatology 1983; 279-84. Hepatology 1982; 2(Suppl): 2 1-6s. H. W., LELIEP. N. et al. Anti 18. VAN DER POELC. L., REESINK 5. NAYAK N. C., PANDA S. K.,ZUCKERMAN A. J. et al. Dynamics hepatitis C antibodies and non A, non B posttransfusion and impact of perinatal transmission of hepatitis B virus in hepatitis in the Netherlands. Lancet 1989; ii:297-8. north India. 3. Med. Virol. 1987; 21: 137-45. P., SEIDLS., STANGEL W., BEYER J. SIBROWSKI W. & 19. KUHNL 6. JOHNSON P.J. & WILLIAMS R. Cirrhosis and the aetiology of FLIKJ. Antibody to hepatitis C virus in German blood hepatocellular carcinoma. 3. Heparol. 1987; 4: 140-7. donors (letter). Lancet 1989; ii: 324-5. S. J., MOZAFFARI P. C., VANSCHALKURYK20. BORTOLOTTI 7. VAN RENSBURG F., TAGGER A., CADROBBI P., CRIVELLARO C., D. J., VANDERWATTJ. J., VINCENT T. J. & PURCHASE I. F. RIBERO M. L. & ALBERT A. Antibodies to hepatitis C virus in Hepatocellular carcinoma and dietary aflatoxin in Mozamchildren with acute or chronic viral hepatitis (letter). Lancer bique and Transkei. Br. J. Cancer 1985; 51: 713-26. 1989; ii:1162. 8. GILLIAMJ., GEISINGER J. R. & RICHTERJ. E. Primary P., WERNERB., LAROUZE B. et al. Antibody to 21. MAUPAS hepatocellular carcinoma after chronic non A, non B post hepatitis B core antigen in patients with primary hepatic carcinoma. Lancet 1975; i:9. transfusion hepatitis. Ann. Intern. Med. 1984; 101: 794-5.
LIVER A N D BILIARY
Prevalence of hepatitis C virus antibodies in chronic liver disease and hepatocellular carcinoma patients in India R A J A G O P A L R A M E S H , A N U P A M A M U N S H I A N D S U B R A T K. P A N D A
Department of Pathology, All India Institute of Medical Sciences, New Delhi, India Abstract The prevalence of antibodies to hepatitis C virus (HCV) was investigated in 129 patients with chronic liver disease (85 with chronic active hepatitis and 44 with cirrhosis) and 53 patients with hepatocellular carcinoma. The commercially available second generation anti-HCV enzyme immunoassay kit was used. Antibodies to hepatitis C virus were detected in 16.2% of the patients with chronic liver disease and in 15.1% with hepatocellular carcinoma. Of the HCV positive patients in all groups 51.7% were positive for hepatitis B virus (HBV) markers indicating present or past infection. Prevalence of HBV markers in all the three groups (CAH, cirrhosis and HCC) was higher as compared with anti-HCV prevalence. These results suggest that HCV infection may not be a major cause of chronic liver disease and hepatocellular carcinoma in India and indicate the presence of other aetiological agents. Key words: chronic liver disease, hepatitis B virus, hepatitis C virus, anti-hepatitis C virus, hepatocellular carcinoma.
INTRODUCTION Hepatocellular carcinoma (HCC) is a common neoplasm world-wide.' Epidemiological and molecular hybridization studies demonstrate a causal relationship between hepatitis B virus (HBV) and hepatocellular In countries such as India with an intermediate HBV carrier rate (2-4O/0),~ the incidence of hepatocellular carcinoma (HCC) has been more commonly related to other factors, especially cirrhosis and aflatoxin level in the food Recently, Gilliam et a1.8 and Okuda' have suggested that non-A, non-B hepatitis (NANBH) viruses may play a role in the development of HCC in patients with cirrhosis. The genome of transfusion associated NANBH virus designated as hepatitis C virus (HCV) has been cloned" and has emerged as the major cause of post-transfusion associated chronic hepatitis. "J' Studies from many parts of the world demonstrate the presence of anti-HCV antibodies in the serum of both HBsAg positive and negative chronic hepatitis (50% and 90%) and HCC patients (50 and 70% respectively) and suggests the involvement of HCV in liver carcin~genesis.'~-'~ Thus it is important to know the prevalence of HCV in chronic liver disease and HCC for any geographical region.
METHODS Serum samples were obtained from 53 patients with hepatocellular carcinoma (HCC) and 129 patients with
chronic liver disease (CLD) (85 with chronic active hepatitis [CAH] and 44 with cirrhosis) who attended All India Institute of Medical Sciences (AIIMS), Govind Balabh Pant Hospital (G.B.Pant) New Delhi and Madras General Hospital, Madras between 1988 and 1990. T h e sera were tested for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. All the cases of hepatocellular carcinoma diagnosed clinically as well as by ultrasound were confirmed by histopathological examination of the needle biopsies. These patients comprised 45 males and 8 females whose mean age was 44.7 years (range 10-70 years). T h e 129 cases of chronic liver disease were diagnosed clinically and the diagnosis was confirmed by laboratory investigation followed by histopathological examination of the needle biopsies. T h e mean age in this group was 42.3 years (range 15-60 years) with a sex distribution of 98 males and 31 females. None of the cases included in this study had a history of alcoholism, drug intake or evidence of other known cause of liver disease. At least 5 m L of blood was collected from each of these patients aseptically, serum was separated and stored at - 70°C until further use. All the serum samples were tested for HBV infection (present or past infection) by testing for HBsAg, anti-HBsAg and anti-HBc by using commercial micro ELISA test kits (Hepanostika, Organon Teknika, Netherlands) according to the manufacturer's guidelines. Detection of HBV-DNA in the serum was carried out by dot blot hybridization according to the method described by Scotto et al. l 7 Current infection with HBV was assessed by serological tests for HBsAg, IgM, anti-HBc and/or HBV-DNA. T h e past infection for HBV
Correspondence: Dr S. K. Panda, Department of Pathology, All India Institute of Medical Sciences, New Delhi 110 029, India, Accepted for publication 13 February 1992.
R. Ramesh et al.
394 was assessed by the presence of antibodies to the surface antigen (anti-HBs) and/or to the core antigen (IgG, anti-HBc). Anti-HCV was tested by using the second generation ABBOTT-HCV enzyme immunoassay kit (ABBOTT Co., Chicago, IL, USA). This indirect assay uses recombinant HCV antigens (covers the structural and non-structural region) coated on polystyrene beads. Anti-HCV in the serum was detected with an antibodyenzyme conjugate comprised of goat monoclonal antibody against human IgG conjugated with horseradish peroxidase (HRPO). T h e end product was read by a Philips spectrophotometer (PUS800 UV/Visible) at 492 nm. T h e results were interpreted according to the manufacturer's guidelines. Each of the test samples was tested in duplicate and the test was carried out according to the manufacturer's guidelines.
seen in 9/54 (16.6%) of chronic liver disease and 4/35 (11.4%) of HCC (Table 2). There was no statistical (Chi-squared test) difference in HCV positivity between HBV infected or non-infected individuals. Of the patients positive for antibodies to HCV 51.7% were also positive for current HBV infection. On the other hand only 18.8% of current HBV infection cases had antibody against HCV (including all the three groups; Table 2). Among hepatocellular carcinoma patients, three had markers for past HBV infection, characterized by the presence of antiHBsAg and/or IgG anti-HBc. One of them was positive for anti-HCV (Table 2). There were no markers for either HBV or HCV in 34.8% of chronic liver disease patients and 58.4% of hepatocellular carcinoma patients.
DISCUSSION
RESULTS The prevalence of HBsAg/or HBV-DNA and antibodies to HCV in patients with chronic liver disease and hepatocellular carcinoma is shown in Table 1. There is a significant difference (P< 0.001) in current HBV infection marker prevalence between cirrhosis and hepatocellular carcinoma (Table 1). The prevalence of antibodies to HCV was in 16.2% (21/129) of chronic liver disease patients (chronic hepatitis and cirrhosis put together) and in 15.1% (8/53) of hepatocellular carcinoma (Table 1). The status of HBV infection along with positivity for antibodies to HCV is shown in Table 2. Anti-HCV was positive in 12/75 (16%) chronic liver disease patients and 4/18 (22.2%) of HCC patients positive for present or past HBV infection. The positivity for anti-HCV alone was Table 1 Relationship between current HBV infection versus anti-HCV in patients with chronic liver disease (CLD) and hepatocellular carcinoma (HCC) No. samples HBsAg/HBV-DNA Anti-HCV positivity/total (YO) tested positive (%)* CAH Cirr HCC
85 44 53
30 (44.7) 27 (61.4) 15 (28.0)
13 (15.3) 8 (18.0) 8 (15.1)
* Indicates current HBV infection.
Information on HCV infection is available from many parts of the world since the identification of its genome and availability of a specific antibody test.'*-*' The present study reports the prevalence of HCV antibody in chronic liver disease and hepatocellular carcinoma in India. T h e observations indicate a low prevalence of HCV antibody (15-18%) among chronic liver disease and HCC patients as compared with other studies from Japan, Italy and Spain where the frequency of anti-HCV is more than 80% in chronic liver disease and HCC patient^.'^"^ When HBV-infected individuals were excluded from the group of chronic liver diseases and hepatocellular carcinoma it was found that only 13/89 (14.6%) of the cases had antibody to HCV. However while interpreting the results of the anti-HCV test, the low sensitivity of the test has to be kept in mind. The findings suggest that HBV may be a more important cause of chronic liver diseases and hepatocellular carcinoma in India. This is similar to what has been observed in South Africa.I6 In an earlier study hepatocellular carcinoma was demonstrated in cases with past HBV infection although its exact significance is not known.21 In the present study three cases of hepatocellular carcinoma with past HBV infection were detected and one of them was positive for anti-HCV. T h e higher prevalence of HBV in chronic liver disease in comparison with hepatocellular carcinoma may be related to early development of cirrhosis leading to death where adequate time for the development of hepatocellular carcinoma is not available. Absence of HBV or HCV markers in nearly half (41.7%) of the patients suggests the possible role of
Table 2 HBV markers status in relation to anti-HCV positivity in different categories of chronic liver disease Type of HBV markers present
Active or recent HBV infection (HBsAg/HBV-DNA+) Past HBV infection No HBV infection Total
CAH (n = 85) Cirrhosis (n = 44) HCC (n = 53) anti-HCV + (%) anti-HCV - (%) anti-HCV + (%) anti-HCV - (%) anti-HCV + (%) anti-HCV - (%) 6 (7.0)
32 (37.6)
6 (13.6)
21 (47.0)
3 (5.6)
12(22.6)
0 (0)
6 (7.0) 34 (40.0)
0 (0)
7 (8.2)
2 (4.5)
4 (9.0) 11 (25.0)
l(1.8) 4 (7.5)
2 (3.7) 31 (58.4)
13 (15.3)
72 (84.7)
8 (18.2)
36 (81.8)
8 (15.1)
45 (84.9)
Anti-HCV in chronic liver disease aetiological agents, such as yet unidentified virus(es) or chemical carcinogens including aflatoxins.
395 9. OKUDA K. Primary liver cancer. Dig. Dis. Sci. 1986; 31: 133-46s. 10. CHOOQ. L., KUOG, WEINERA. J. er al. Isolation of a cDNA
clone derived from a blood borne non A, non B viral hepatitis genome. Science 1989; 244: 359-61. ACKNOWLEDGEMENTS R. H., SHIHJ. W. et al. Detection of 11. ALTERH. J., PURCELL antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non A, non B The authors acknowledge that this study has been con. hepatitis. N. Engl. J. Med. 1989; 321: 1494-500. ducted with financial assistance from the Indian Council 12. Kuo G., CHOOQ. L., ALTER H. J. et al. An assay for of Medical Research (New Delhi) and the Overseas circulating antibodies to major etiologic virus of human non Development Administration (UK). A, non B hepatitis. Science 1989; 244: 362-4. 13. KIOSAWA K., SODEYAMA T., TANAKA er al. Interrelationship of blood transfusion, non A, non B hepatitis and hepatocellular carcinoma analysis by detection of antibody to hepatitis C REFERENCES virus. Hepatology 1990; 12: 671-5. M., Kuo G., CHOOQ. L. er al. Prevalence of 14. COLOMBO antibodies to hepatitis C virus in Italian patients with 1. PERKIN D. M., STJENSWARD J. & MUIRC. S. Estimates of the hepatocellular carcinoma. Lancet 1989; ii: 1006-8. world wide frequency of twelve major cancers. Bull. WHO 15. BRUIXJ., BARRERA J. M., CALVET X. er al. Prevalence of 1984; 62: 163-82. antibodies to hepatitis C virus in Spanish patients with 2. SMUZNESS W. Hepatocellular carcinoma and hepatitis B hepatocellular carcinoma. Lancet 1989; ii:1004-6. virus: evidence for a causal association. Prog. Med. Virol. 16. KEWM. C., HOUCHTON M., CHOOQ.L. & Kuo G. Hepatitis 1978; 24: 40-69. C virus antibodies in south African blacks with hepatocellu3. SHAFRITZ D. A. & KEWM. C. Identification of integrated lar carcinoma. Lancer 1990; 335: 873-4. hepatitis B virus DNA sequences in hepatocellular carciM., HERYC. er al. Detection of 17. SCOTTOJ., HADCHOUEL noma. Hepatology 1981; 1: 1-8. hepatitis B virus DNA in serum by simple spot hybridisation 4. BEASLEY R. P. Hepatitis B virus as the etiologic agent in technique: Comparison with results for other viral markers. hepatocellular carcinoma. Epidemiologic considerations. Hepatology 1983; 279-84. Hepatology 1982; 2(Suppl): 2 1-6s. H. W., LELIEP. N. et al. Anti 18. VAN DER POELC. L., REESINK 5. NAYAK N. C., PANDA S. K.,ZUCKERMAN A. J. et al. Dynamics hepatitis C antibodies and non A, non B posttransfusion and impact of perinatal transmission of hepatitis B virus in hepatitis in the Netherlands. Lancet 1989; ii:297-8. north India. 3. Med. Virol. 1987; 21: 137-45. P., SEIDLS., STANGEL W., BEYER J. SIBROWSKI W. & 19. KUHNL 6. JOHNSON P.J. & WILLIAMS R. Cirrhosis and the aetiology of FLIKJ. Antibody to hepatitis C virus in German blood hepatocellular carcinoma. 3. Heparol. 1987; 4: 140-7. donors (letter). Lancet 1989; ii: 324-5. S. J., MOZAFFARI P. C., VANSCHALKURYK20. BORTOLOTTI 7. VAN RENSBURG F., TAGGER A., CADROBBI P., CRIVELLARO C., D. J., VANDERWATTJ. J., VINCENT T. J. & PURCHASE I. F. RIBERO M. L. & ALBERT A. Antibodies to hepatitis C virus in Hepatocellular carcinoma and dietary aflatoxin in Mozamchildren with acute or chronic viral hepatitis (letter). Lancer bique and Transkei. Br. J. Cancer 1985; 51: 713-26. 1989; ii:1162. 8. GILLIAMJ., GEISINGER J. R. & RICHTERJ. E. Primary P., WERNERB., LAROUZE B. et al. Antibody to 21. MAUPAS hepatocellular carcinoma after chronic non A, non B post hepatitis B core antigen in patients with primary hepatic carcinoma. Lancet 1975; i:9. transfusion hepatitis. Ann. Intern. Med. 1984; 101: 794-5.
