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The changing relative prevalence of hepatitis C virus genotypes: Evidence in hemodialyzed patients and kidney recipients
GASTROENTEROLOGY1995;108:581-583
RAPID COMMUNICATIONS The Changing Relative Prevalence of Hepatitis C Virus Genotypes: Evidence in Hemodialyzed Patients and Kidney Recipients STANISLAS POL,*'* VALI~RIE THIERS, § JEAN-BAPTISTE NOUSBAUM, II CHRISTOPHE LEGENDRE, ¶ PIERRE BERTHELOT,* HENRI KREIS ¶ and CHRISTIAN BRECHOT *'§'11 *Unit6 d'H6patologie et ~Servicede TransplantationR6nale, INSERM*Unit6 99 et llUnit6370, HSpital Necker, LaboratoireHybridotest, and §lnstitut Pasteur, Paris, France
Bacl~round/Aims: Hepatitis C virus (HCV) infection by the genotype l b is significantly associated with a lower rate of response to interferon alfa and with severe liver disease (cirrhosis and hepatocellular carcinoma). This may reflect different intrinsic properties of this genotype l b and/or chronological differences in the epidemiology of HOV genotypes. To address the issue of variations in genotypes prevalence, we studied in the present report the HCV genotypes of 60 hemodialyzed and kidney recipients according to the date of hemodialysis. Methods: Anti-HCV antibodies were tested by a second-generation assay (enzyme-linked immunosorbent assay 2 and recombinant immunoblot assay 2). HCV RNA was detected by reverse-transcription polymerase chain reaction. Genotyping was performed by hybridization of type-specific probes to the amplified product from the 5' untranslated region. Results:Genotype l b accounted for more than two thirds of HCV infection in patients who underwent dialysis before 1977 but less than one third in those hemodialyzed after 1985. In contrast, other genotypes (3a, 4a, 5a) appeared in the 1980s. Conclusions: These data, obtained in an homogenous group of patients, show a changing pattern of HCV genotype prevalence over time and should be considered when discussing the potential clinical implications of HCV genetic variability.
" epatitis C virus (HCV) shows an important genetic .variability, and at least 12 genotypes and subtypes of H C V have now been identified. ~ Several reports have shown that there is a mixing of different H C V types in a given area, although some of them still show preferential geographical distribution. 2 3 In addition, some reports from Japan and Europe have suggested that infections by genotype l b (II) have a lower rate of response to interferon than other genotypes, such as la, 2a, or 3a. 4-5 W e have recently obtained data in France and Italy that support this hypothesis. 6 In addition, we have shown the
H
association of genotype l b (II) infection to severe liver disease (cirrhosis and hepatocellular carcinoma) and provided evidence in liver transplant recipients for a more cytopathic effect ofgenotype lb. 7 A major problem, however, for the interpretation of such data is the possibility of a changing pattern of the relative prevalence of H C V genotypes over time. In this view, we recently obtained, in the general population of subjects with H C V infection, data consistent with a decline over time of the genotype l b relative prevalence. 6 Therefore, the aim of the present study was to analyze the prevalence of H C V genotype according to the time of contamination in an homogenous population. W e thus investigated a group of hemodialyzed patients and kidney transplant recipients because anti-HCV antibodies are highly prevalent (around 25%) in these settings and associated with a high rate of active infection 8 and because the time of contamination can be roughly evaluated.
Materials
and Methods
Patients Sixty anti-HCV-positive French kidney allograft recipients and hemodialyzed patients (39 men and 21 women) were selected on the basis of a positive second-generation HCV serological assay and the detection of HCV RNA by reversetranscription polymerase chain reaction. According to the date of hemodialysis, patients were divided in five groups: patients who underwent hemodialysis before 1974, between 1974 and 1977, between 1978 and 1981, between 1982 and 1985, and after 1983. HCV infection in hemodialyzed patients is associated with the duration of the dialysis period and the number of blood transfusions. 9 Because most acute HCV infections are asymptomatic and because HCV chronic hepatitis rarely leads to increased levels of aminotransferase activities in hemodia-
© 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
582
POL ET AL.
GASTROENTEROLOGY Vol. 108, No. 2
Table 1. P r e v a l e n c e o f HCV G e n o t y p e s A c c o r d i n g t o t h e D a t e o f D i a l y s i s in 6 0 H e m o d i a l y z e d or K i d n e y R e c i p i e n t s
Genotype
Before 1 9 7 4 (%)
Between 1 9 7 4 and 1 9 7 7 (%)
Between 1 9 7 8 and 1 9 8 1 (%)
Between 1 9 8 2 and 1 9 8 5 (%)
la lb 2a 3a 4a 5a Total
3 (18.8) 10 (62.6) 3 (18.8) ---16
2 (25.0) 6 (75.0) ----8
3 (27.3) 7 (63.6) ---1 (9.1) 11
-7 (50) -2 (15.4) 2 (15.4) 2 (15.4) 13
After 1 9 8 5 (%) 2 2 5 2
(16.7) (16.7) (41.7) (16.7) -1 (8.3) 12
NOTE. The nomenclature of HCV genotypes is based on the classification from Stuyer et al. ~
lyzed patients, 7 we assume that the date of initiation of dialysis is the best end point to compare patients. Eleven patients were retrospectively tested on frozen serum samples at the time of transplantation; 3 hemodialyzed patients were tested at the time of the first occurrence of anti-HCV seropositivity, and 46 patients were tested at the time of first admission in the liver unit.
Methods All of the sera were anti-HCV-positive (HCV antibody enzyme-linked immunosorbent assay 2 test and recombinant immunoblot assay 2 test; Ortho Diagnostic Systems, Emeryville, CA), and all contained HCV RNA detected by reverse-transcription polymerase chain reaction. A nested polymerase chain reaction assay using universal primers in the 5' untranslated region was performed using sets of biotinylated primers in which sequence degeneracy was included to allow annealing to all HCV genotypes. Polymerase chain reaction was performed in one-tube nested polymerase chain reaction as described previously to minimize carryover. 6 Polymerase chain reaction products were subjected to electrophoresis on a 2% agarose gel, stained with ethidium bromide, and observed under UV light. The samples visible on agarose gel were then genotyped. Genotyping was performed by using an assay based on probes specific for each HCV type in the 5' untranslated region, referred to as the lime probe assay procedure (Innogenetics, Gent, Belgium). One tenth of biotinylated polymerase chain reaction fragments was hybridized to oligonucleotides directed against the variable regions of the 5' untranslated region immobilized as parallel lines on membrane strips. After hybridization, streptavidin labeled with alkaline phosphatase was added, and incubation with nitroblue retrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate chromogen allowed the development of the colorimetric reaction. The reactivity of the amplified fragments with one or more lines on the strip allows the recognition of the genotype of HCV. Classification of HCV into major genotypes was based on the recently proposed nomenclature of HCV genotypes. 1
Results Main results are summarized in Table 1. Genotype l b accounted for more than two thirds (16 of 24) of H C V infection until 1977; in contrast, it was responsible
for half (14 of 24) of H C V infection between 1978 and 1985 and less than one third (5 of 12) of H C V infection in the dialysis and renal transplantation setting after 1985. Whereas genotype l b was persistently present during the period of study, genotypes la and 2a appeared in the 1970s and genotypes 3a, 4a, and 5a were introduced in the 1980s. The prevalence of the l b H C V genotype was decreasing over time; in contrast, the prevalence of the 2a was increasing after 1985 (5 of 12).
Discussion Our results offer strong evidence for a changing epidemiology of H C V genotypes from the 1960s to the 1980s. W e have indeed analyzed an homogenous, wellcharacterized group of patients with a fair evaluation of the time of contamination. Our study does indicate that the relative prevalence of genotype l b significantly decreased over the last decades in hemodialyzed patients and kidney recipients who are mainly contaminated by blood transfusion. It is important to emphasize that this result is consistent with other recent observations in patients with chronic hepatitis associated with posttransfusional contamination; we also observed a marked decrease of the relative prevalence of genotype i b over time, ranging from 80% to < 2 0 % when the blood transfusion was given more than 15 years ago and < 5 years ago, respectively. 6'1° By contrast, there were significant differences neither in patients with sporadic chronic hepatitis nor in intravenous drug users. 1° Thus, the data shown in the present study point to a significant changing prevalence of H C V genotypes in the case of posttransfusional contamination. Furthermore, we have shown the emergence of new genotypes in France over the last decades. Thus, one may hypothesize that the decrease in relative prevalence of genotype l b reflects the introduction of new H C V genotypes. There is indeed strong evidence from France and Italy suggesting that drug addicts are preferentially infected, for unknown reasons, by "non lb" genotypes (in
February 1995
particular, la and 3a). It is therefore tempting to speculate that the introduction in the blood bank setting of these new genotypes might be due to donation of intravenous drug users in the early 1980s. 1°-11 This previous introduction of the lb HCV genotype might have major implications for interpretation of the data concerning association ofgenotype lb to the severity of liver disease; this might indeed be at least in part related to the duration of HCV infection or to a combination of longer duration and intrinsic biological properties. Along the same line, we observed a better response to interferon alfa in patients who were recently infected than in those with an anscient infection. 6 Finally, the high prevalence of infection by the l b HCV genotype and its longer duration could explain the frequent histopathologic deterioration that we observed in renal transplant recipients before 1985. t2 Altogether, our study underlines the need for careful evaluation of the prevalence of HCV genotype at different periods of time when discussing the potential clinical implications of the HCV genetic variability.
References 1. Stuyver L, Rossau R, Wyseur A, Duhamel M, Vanderborght B, Van Heuverswyn B, Maertens G. Typing of HCV isolates and characterisation of new subtypes using a probe assay. J Gen Virol 1993; 74:1093-1102. 2. Enomoto N, Takada A, Nakao T, Date T. There are two major types of hepatitis C virus in Japan. Biochem Biophys Res Commun 1990; 170:1021-1025. 3. Okamoto H, Kurai K, Okada S-I, Yamamoto K, Itzuka H, Tanaka T, Fukuda S, Tsuda F, Mishiro S. Full-length sequence of hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 1992;188: 331-341.
CHANGING PREVALENCE OF HCV GENOTYPES 583
4. Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M, Higashi Y, Shibata M, Morishima T. Detection of hepatitis C virus by polymerase chain reaction and response to Interferonalpha therapy: relationship to genotypes of hepatitis C virus. Hepatology 1992; 16:293-299. 5. Brouwer J, Nevens F, Kleter G, Eleurant A, Adler M, Brenard R, Chamuleau RAFM, Michielson P, Lerotte J, Hautekeete ML, Arendt, Schalm SW. Which hepatitis C patient will benefic from Interferon? Multivariate analysis of 350 patients treated in a Benelux multicentre study (abstr). J Hepatol 1993;18(Suppl.
l):SlO. 6. Nousbaum J-B, Pol S, Nalpas B, Landeis P, Berthelot P, Br6chot C. Characteristics of hepatitis C virus type II (lb) infection in France and Italy. Ann Intern Med (in press). 7. Feray C, Grigou M, Samuel D, Okamoto H, Reynes M, Mishiro S, Bismuth H, Br6chot C. HCV type II has a more pathogenic course after liver transplantation (abstr). Hepatology 1993; 18:59A. 8. Pol S, Rom6o R, Zins B, Driss F, Lebkiri B, Carnot F, Berthelot P, Br6chot C. Hepatitis C virus (HCV) RNA in anti-HCV positive hemodialyzed patients: significance and therapeutic implications. Kidney Intern 1993;44:1097-1100. 9. Muller GY, Zabaleta ME, Arminio A, Colmenares CJ, Capriles FY, Bianco NE, Machado IV. Risk factors for dialysis-associated hepatitis C in Venezuela. Kidney Intern 1992;42:1055-1058. 10. Pol S, Thiers V, Nousbaum J-B, Legendre C, Berthelot P, Kreis H, Br6chot C. Changing distribution of HCV genotypes in Europe in the last decades (abstr). J Hepatol 1994;21:$13. 11. Silini E, Bono F, Cerino A, Cividini A, Maccabruni A, Babarini G, Mondelli MU. Molecular epidemiology of HCV infection in drug addicts (abstr). J Hepatol 1994;21:$33. 12. Pol S, Debure A, Degott C, Camot F, Legendre C, Br6chot C, Kreis H. Chronic hepatitis in kidney allograft recipients. Lancet 1990; 335:878-880. Received October 11, 1994. Accepted November 16, 1994. Address requests for reprints to: Stanislas Pol, M.D., Ph.D., Service d'H6patologie, H6pital Necker, 149 Rue de S6vres, 75747 Paris Cedex 15, France. Fax: (33) 1-44-4943-30. Supported by a grant from the Commission ~ la Recherche Clinique de I'Assistance Publique. Presented at the 29th annual meeting of the European Association for the Study of the Liver, September 1994, in Athens, Greece.
RAPID COMMUNICATIONS The Changing Relative Prevalence of Hepatitis C Virus Genotypes: Evidence in Hemodialyzed Patients and Kidney Recipients STANISLAS POL,*'* VALI~RIE THIERS, § JEAN-BAPTISTE NOUSBAUM, II CHRISTOPHE LEGENDRE, ¶ PIERRE BERTHELOT,* HENRI KREIS ¶ and CHRISTIAN BRECHOT *'§'11 *Unit6 d'H6patologie et ~Servicede TransplantationR6nale, INSERM*Unit6 99 et llUnit6370, HSpital Necker, LaboratoireHybridotest, and §lnstitut Pasteur, Paris, France
Bacl~round/Aims: Hepatitis C virus (HCV) infection by the genotype l b is significantly associated with a lower rate of response to interferon alfa and with severe liver disease (cirrhosis and hepatocellular carcinoma). This may reflect different intrinsic properties of this genotype l b and/or chronological differences in the epidemiology of HOV genotypes. To address the issue of variations in genotypes prevalence, we studied in the present report the HCV genotypes of 60 hemodialyzed and kidney recipients according to the date of hemodialysis. Methods: Anti-HCV antibodies were tested by a second-generation assay (enzyme-linked immunosorbent assay 2 and recombinant immunoblot assay 2). HCV RNA was detected by reverse-transcription polymerase chain reaction. Genotyping was performed by hybridization of type-specific probes to the amplified product from the 5' untranslated region. Results:Genotype l b accounted for more than two thirds of HCV infection in patients who underwent dialysis before 1977 but less than one third in those hemodialyzed after 1985. In contrast, other genotypes (3a, 4a, 5a) appeared in the 1980s. Conclusions: These data, obtained in an homogenous group of patients, show a changing pattern of HCV genotype prevalence over time and should be considered when discussing the potential clinical implications of HCV genetic variability.
" epatitis C virus (HCV) shows an important genetic .variability, and at least 12 genotypes and subtypes of H C V have now been identified. ~ Several reports have shown that there is a mixing of different H C V types in a given area, although some of them still show preferential geographical distribution. 2 3 In addition, some reports from Japan and Europe have suggested that infections by genotype l b (II) have a lower rate of response to interferon than other genotypes, such as la, 2a, or 3a. 4-5 W e have recently obtained data in France and Italy that support this hypothesis. 6 In addition, we have shown the
H
association of genotype l b (II) infection to severe liver disease (cirrhosis and hepatocellular carcinoma) and provided evidence in liver transplant recipients for a more cytopathic effect ofgenotype lb. 7 A major problem, however, for the interpretation of such data is the possibility of a changing pattern of the relative prevalence of H C V genotypes over time. In this view, we recently obtained, in the general population of subjects with H C V infection, data consistent with a decline over time of the genotype l b relative prevalence. 6 Therefore, the aim of the present study was to analyze the prevalence of H C V genotype according to the time of contamination in an homogenous population. W e thus investigated a group of hemodialyzed patients and kidney transplant recipients because anti-HCV antibodies are highly prevalent (around 25%) in these settings and associated with a high rate of active infection 8 and because the time of contamination can be roughly evaluated.
Materials
and Methods
Patients Sixty anti-HCV-positive French kidney allograft recipients and hemodialyzed patients (39 men and 21 women) were selected on the basis of a positive second-generation HCV serological assay and the detection of HCV RNA by reversetranscription polymerase chain reaction. According to the date of hemodialysis, patients were divided in five groups: patients who underwent hemodialysis before 1974, between 1974 and 1977, between 1978 and 1981, between 1982 and 1985, and after 1983. HCV infection in hemodialyzed patients is associated with the duration of the dialysis period and the number of blood transfusions. 9 Because most acute HCV infections are asymptomatic and because HCV chronic hepatitis rarely leads to increased levels of aminotransferase activities in hemodia-
© 1995 by the AmericanGastroenterologicalAssociation 0016-5085/95/$3.00
582
POL ET AL.
GASTROENTEROLOGY Vol. 108, No. 2
Table 1. P r e v a l e n c e o f HCV G e n o t y p e s A c c o r d i n g t o t h e D a t e o f D i a l y s i s in 6 0 H e m o d i a l y z e d or K i d n e y R e c i p i e n t s
Genotype
Before 1 9 7 4 (%)
Between 1 9 7 4 and 1 9 7 7 (%)
Between 1 9 7 8 and 1 9 8 1 (%)
Between 1 9 8 2 and 1 9 8 5 (%)
la lb 2a 3a 4a 5a Total
3 (18.8) 10 (62.6) 3 (18.8) ---16
2 (25.0) 6 (75.0) ----8
3 (27.3) 7 (63.6) ---1 (9.1) 11
-7 (50) -2 (15.4) 2 (15.4) 2 (15.4) 13
After 1 9 8 5 (%) 2 2 5 2
(16.7) (16.7) (41.7) (16.7) -1 (8.3) 12
NOTE. The nomenclature of HCV genotypes is based on the classification from Stuyer et al. ~
lyzed patients, 7 we assume that the date of initiation of dialysis is the best end point to compare patients. Eleven patients were retrospectively tested on frozen serum samples at the time of transplantation; 3 hemodialyzed patients were tested at the time of the first occurrence of anti-HCV seropositivity, and 46 patients were tested at the time of first admission in the liver unit.
Methods All of the sera were anti-HCV-positive (HCV antibody enzyme-linked immunosorbent assay 2 test and recombinant immunoblot assay 2 test; Ortho Diagnostic Systems, Emeryville, CA), and all contained HCV RNA detected by reverse-transcription polymerase chain reaction. A nested polymerase chain reaction assay using universal primers in the 5' untranslated region was performed using sets of biotinylated primers in which sequence degeneracy was included to allow annealing to all HCV genotypes. Polymerase chain reaction was performed in one-tube nested polymerase chain reaction as described previously to minimize carryover. 6 Polymerase chain reaction products were subjected to electrophoresis on a 2% agarose gel, stained with ethidium bromide, and observed under UV light. The samples visible on agarose gel were then genotyped. Genotyping was performed by using an assay based on probes specific for each HCV type in the 5' untranslated region, referred to as the lime probe assay procedure (Innogenetics, Gent, Belgium). One tenth of biotinylated polymerase chain reaction fragments was hybridized to oligonucleotides directed against the variable regions of the 5' untranslated region immobilized as parallel lines on membrane strips. After hybridization, streptavidin labeled with alkaline phosphatase was added, and incubation with nitroblue retrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate chromogen allowed the development of the colorimetric reaction. The reactivity of the amplified fragments with one or more lines on the strip allows the recognition of the genotype of HCV. Classification of HCV into major genotypes was based on the recently proposed nomenclature of HCV genotypes. 1
Results Main results are summarized in Table 1. Genotype l b accounted for more than two thirds (16 of 24) of H C V infection until 1977; in contrast, it was responsible
for half (14 of 24) of H C V infection between 1978 and 1985 and less than one third (5 of 12) of H C V infection in the dialysis and renal transplantation setting after 1985. Whereas genotype l b was persistently present during the period of study, genotypes la and 2a appeared in the 1970s and genotypes 3a, 4a, and 5a were introduced in the 1980s. The prevalence of the l b H C V genotype was decreasing over time; in contrast, the prevalence of the 2a was increasing after 1985 (5 of 12).
Discussion Our results offer strong evidence for a changing epidemiology of H C V genotypes from the 1960s to the 1980s. W e have indeed analyzed an homogenous, wellcharacterized group of patients with a fair evaluation of the time of contamination. Our study does indicate that the relative prevalence of genotype l b significantly decreased over the last decades in hemodialyzed patients and kidney recipients who are mainly contaminated by blood transfusion. It is important to emphasize that this result is consistent with other recent observations in patients with chronic hepatitis associated with posttransfusional contamination; we also observed a marked decrease of the relative prevalence of genotype i b over time, ranging from 80% to < 2 0 % when the blood transfusion was given more than 15 years ago and < 5 years ago, respectively. 6'1° By contrast, there were significant differences neither in patients with sporadic chronic hepatitis nor in intravenous drug users. 1° Thus, the data shown in the present study point to a significant changing prevalence of H C V genotypes in the case of posttransfusional contamination. Furthermore, we have shown the emergence of new genotypes in France over the last decades. Thus, one may hypothesize that the decrease in relative prevalence of genotype l b reflects the introduction of new H C V genotypes. There is indeed strong evidence from France and Italy suggesting that drug addicts are preferentially infected, for unknown reasons, by "non lb" genotypes (in
February 1995
particular, la and 3a). It is therefore tempting to speculate that the introduction in the blood bank setting of these new genotypes might be due to donation of intravenous drug users in the early 1980s. 1°-11 This previous introduction of the lb HCV genotype might have major implications for interpretation of the data concerning association ofgenotype lb to the severity of liver disease; this might indeed be at least in part related to the duration of HCV infection or to a combination of longer duration and intrinsic biological properties. Along the same line, we observed a better response to interferon alfa in patients who were recently infected than in those with an anscient infection. 6 Finally, the high prevalence of infection by the l b HCV genotype and its longer duration could explain the frequent histopathologic deterioration that we observed in renal transplant recipients before 1985. t2 Altogether, our study underlines the need for careful evaluation of the prevalence of HCV genotype at different periods of time when discussing the potential clinical implications of the HCV genetic variability.
References 1. Stuyver L, Rossau R, Wyseur A, Duhamel M, Vanderborght B, Van Heuverswyn B, Maertens G. Typing of HCV isolates and characterisation of new subtypes using a probe assay. J Gen Virol 1993; 74:1093-1102. 2. Enomoto N, Takada A, Nakao T, Date T. There are two major types of hepatitis C virus in Japan. Biochem Biophys Res Commun 1990; 170:1021-1025. 3. Okamoto H, Kurai K, Okada S-I, Yamamoto K, Itzuka H, Tanaka T, Fukuda S, Tsuda F, Mishiro S. Full-length sequence of hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 1992;188: 331-341.
CHANGING PREVALENCE OF HCV GENOTYPES 583
4. Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M, Higashi Y, Shibata M, Morishima T. Detection of hepatitis C virus by polymerase chain reaction and response to Interferonalpha therapy: relationship to genotypes of hepatitis C virus. Hepatology 1992; 16:293-299. 5. Brouwer J, Nevens F, Kleter G, Eleurant A, Adler M, Brenard R, Chamuleau RAFM, Michielson P, Lerotte J, Hautekeete ML, Arendt, Schalm SW. Which hepatitis C patient will benefic from Interferon? Multivariate analysis of 350 patients treated in a Benelux multicentre study (abstr). J Hepatol 1993;18(Suppl.
l):SlO. 6. Nousbaum J-B, Pol S, Nalpas B, Landeis P, Berthelot P, Br6chot C. Characteristics of hepatitis C virus type II (lb) infection in France and Italy. Ann Intern Med (in press). 7. Feray C, Grigou M, Samuel D, Okamoto H, Reynes M, Mishiro S, Bismuth H, Br6chot C. HCV type II has a more pathogenic course after liver transplantation (abstr). Hepatology 1993; 18:59A. 8. Pol S, Rom6o R, Zins B, Driss F, Lebkiri B, Carnot F, Berthelot P, Br6chot C. Hepatitis C virus (HCV) RNA in anti-HCV positive hemodialyzed patients: significance and therapeutic implications. Kidney Intern 1993;44:1097-1100. 9. Muller GY, Zabaleta ME, Arminio A, Colmenares CJ, Capriles FY, Bianco NE, Machado IV. Risk factors for dialysis-associated hepatitis C in Venezuela. Kidney Intern 1992;42:1055-1058. 10. Pol S, Thiers V, Nousbaum J-B, Legendre C, Berthelot P, Kreis H, Br6chot C. Changing distribution of HCV genotypes in Europe in the last decades (abstr). J Hepatol 1994;21:$13. 11. Silini E, Bono F, Cerino A, Cividini A, Maccabruni A, Babarini G, Mondelli MU. Molecular epidemiology of HCV infection in drug addicts (abstr). J Hepatol 1994;21:$33. 12. Pol S, Debure A, Degott C, Camot F, Legendre C, Br6chot C, Kreis H. Chronic hepatitis in kidney allograft recipients. Lancet 1990; 335:878-880. Received October 11, 1994. Accepted November 16, 1994. Address requests for reprints to: Stanislas Pol, M.D., Ph.D., Service d'H6patologie, H6pital Necker, 149 Rue de S6vres, 75747 Paris Cedex 15, France. Fax: (33) 1-44-4943-30. Supported by a grant from the Commission ~ la Recherche Clinique de I'Assistance Publique. Presented at the 29th annual meeting of the European Association for the Study of the Liver, September 1994, in Athens, Greece.