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Low prevalence of precore mutations in hepatitis B virus DNA in fulminant hepatitis type B in France
Journal of Hepatology, 1993; 18:119-122 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00
1 19
HEPAT 01466
Rapid Publication
Low prevalence of precore mutations in hepatitis B virus DNA in fulminant hepatitis type B in France Cyrille F 6 r a y a'b, Michelle G i g o u a'b, D i d i e r S a m u e l b, J a c q u e s B e r n u a u c, H e n r i B i s m u t h b a n d Christian Br6chot a aHybridotest, Institut Pasteur, Paris, bService de Chirurgie H~pato-biliaire et Transplantation H~patique, H6pital Paul Brousse, Villejuif, cUnit~ d'Hdpatologie, H6pital Beaujon, Clichy and dlNSERM U-370, CHU Necker and Unit~ d'H#patologie, H6pital La#nnec, Paris, France
The presence of mutants of the precore region of the hepatitis B virus genome was investigated in French patients with fulminant hepatitis. Only one of the 10 subjects had a detectable mutation by direct sequencing. On the other hand, 2/10 and 3/10 had evidence of coinfection by hepatitis D virus and hepatitis C virus. These results indicate that the precore stop mutation at codon 28 is not a general condition in fulminant hepatitis B and might reflect epidemiological factors.
Key words: Polymerase chain reaction; Direct sequencing
It has been suggested that variants of the HBV genome with mutations in the precore gene may cause severe forms of hepatitis. In particular a high incidence of these mutants has been reported in patients with sporadic HBsAg positive fulminant hepatitis, from the Far East (1) and Southern Mediterranean countries (2). Furthermore, the precore mutations have been also detected in fulminant hepatitis type B in Israel (3) and in Japan after nosocomial (2), interspouse (4) or intrafamilial transmission (2). In these cases, the same mutation was present in both the contaminant and the recipients. Factors other than mutations in the HBV genome, such as coinfection by the delta virus (HDV) and hepatitis C virus (HCV) (5) might be also determinant in the pathogenesis of fulminant hepatitis in HBsAg positive patients. In order to further investigate this issue, the precore gene was sequenced in French patients with fulminant hepatitis, and referred to our :enter for emergency liver transplantation (LT).
Patients Ten patients referred for HBsAg positive FH to the H6pital Beaujon and Villejuif liver transplant center were studied retrospectively. Fulminant hepatitis was diagnosed if patients developed hepatic encephalopathy within 15 days of the onset of jaundice (6) The only criteria for being included in the study were the availability and quality of serum and liver samples. Each patient had circulating hepatitis B surface antigen (HBsAg) and eight had detectable IgM anti-HBc. Hepatitis B virus e antigen (HBeAg) and antibody to HBeAg (anti-HBe) were detected in two and seven cases, respectively. The mean period between the onset of jaundice and encephalopathy was 4 days. Two patients had evidence of acute coinfection (n = 2) by hepatitis delta virus (HDV) and were intravenous drug users. Sexual transmission of infection by HBV was identified in three other patients. None of these 10 patients had
Correspondence to: Cyrille F6ray, M.D., Service de Chirurgie H6pato-biliaire, 14 Avenue Paul Vaillant Couturier, (94) Villejuif France.
120
C. FERAY et al.
received blood transfusions or had taken potentially hepatotoxic drugs. All had been living in France for at least the past year. In addition, there was no serological evidence of acute infection by hepatitis A virus, EpsteinBarr virus, herpes virus hominis, Toxoplasma, cytomegalovirus or human immunodeficiency virus. Serum ceruleoplasmin and alpha-1 antitrypsin were within normal ranges. Immunoserologic studies showed the absence of anti-nuclear and anti-smooth-muscle antibodies. Liver weight was 580 g (S.D. = 210; range 148-940). Histological examination revealed massive necrosis with regeneration in only one subject. There was no evidence of steatosis (7) or of syncitial giant-cell hepatitis (8). The control group included 20 patients with chronic anti-HCV-positive NANB hepatitis (n = 10), primary biliary cirrhosis (n = 7) or primary sclerosing cholangitis (n = 3). Serum samples were obtained at the time of admission. Liver samples were obtained during orthotopic liver transplantation (OLT) from all patients. Serum samples were also available for all patients during a follow-up of 1 year after OLT. The samples were stored under appropriate conditions for RNA analysis at -20°C (sera) or -80°C (liver).
proteins (c33, c100.3 and 511). Sera reacting with just one antigen were considered indeterminate. HB V and HD V markers HBV and HDV serologies were determined by means of enzyme-linked immunosorbent assays (ELISAs) (HBsAg, anti-HBc, HBeAg and anti-HBe, Wellcome diagnostic, Kent, UK; anti-HBs, Hoechst Behring Diagnostic, Marburg, Germany; HDV-Ag, Organon Teknika, Durham, Netherlands and anti-HDV, Diagnostic Pasteur, Marnes La Coquette). HBV DNA detection HBV DNA was extracted as previously described from 160 ttl of serum. HBV DNA PCR was performed as previously described (9). Briefly, 10 #1 of DNA preparation, corresponding to 40/~1 of serum and 1 /zg of liver DNA, was added to a final reaction volume of 50/zl containing 2 U of Taq polymerase, 20 pmol of each primer in PCR mixture previously described. The HBV primer sequences were located within the pre-C/C sequence (Table 1). Sequencing of the pre C region of HB V PCR products obtained with primers MD 40 and MD 31 were directly sequenced using T7 DNA polymerase (Pharmacia, Sweden) and primer MD 31.
Methods
Anti-HCV antibodies The presence of anti-HCV antibodies was determined using a recombinant immunoblot assay (RIBA-2, ORTHO diagnostic) which detects antibodies against epitopes located in the capsid (c22) and non-structural
HCV RNA detection RNA was extracted as described by Chomczynski (10) from 50 mg liver in guanidinium isothiocyanate, phenol/chloroform using a commercially available kit ('RNAZOL', Cinna-Biotecx laboratories, Friendswood, Texas, USA). Serum extraction of HCV RNA, eDNA
TABLE 1 Oligonucleotide sequences used for HCV and HBV primers
'outer' SFI ( - b ) SRI (_c) 'inner' SF2 (--) SR2 (--) probe
5' terminal sequence of HCV a
Length of PCR products
5' 5' 5' 5' 5'
259 bp 212 bp
GCC ATG GGC GTT AGT ATG AG 3' (-259; -239) GG TGC ACG GTC AAC GAG ACC 3' (-21; - I ) GTG CAG CCT CCA GGA CCC CCGA 3' (-236; -216) ACG GGT GAG GTA GTA GAC CC 3' (-24; -44) CCG GAA TTG CCA GGA CCG GGT CCT TTC TTG 3'(-76; -46)
H B V precore region of H B V
MD40 ( - ) 5' GCK} AGG AGA TTA GGT TAA 3' 1746-1764 MD31 (--) 5' CTT AGG AGT GCG AAT CCA 3' 2287-2269 aNucleotide sequences are numbered from initiating codon ATG. b(_) sense. c(_) antisense.
541 bp
PRECORE
MUTATIONS
IN HEPATITIS
B VIRUS DNA
121
synthesis and nested-PCR were performed according to a previously described protocol (11) using primers located in the untranslated part of the HCV RNA (Table 1).
AGCT
AG CT
•;
Specificity and sensitivity controls
',
) ~ t .-.am
To detect carry-over, HCV-PCR was performed with the following controls: (i) serum-free lysis buffer to detect contamination at every step of the procedure, (ii) three negative sera (blood donors) for every 12 test sera, (iii) the PCR mix alone. Prevention of carry-over included complete separation of pre-PCR and post-PCR steps. • It has been shown that most false-positive results with PCR occur during handling of amplification products (12). Results were only considered valid if they were consistent in independent experiments repeated at least twice (including the nucleic acid extraction step).
Results and Discussion
HBV DNA was detected using PCR in the liver of the 10 subjects and in the serum of half of them (5/10) (Table 2). HBV precore gene sequences were available for each patient. In one case, a G to A mutation at nucleotide 83 in the precore region which converted codon 28 for tryptophan (TGG) to a stop mutation (TAG), was detected (Fig. 1). The patient infected with this precore mutant did not have detectable HBV DNA in the serum nor HCV RNA in the serum or in the liver. It is noteworthy that this patient had evidence of chronic HBV infection since anti-HBc of IgM class was not detected. In the remaining nine patients, direct sequencing of HBV DNA did not show any mutations in the precore region but a minor population of mutant HBV
Fig. 1. Direct s e q u e n c i n g o f p r e C r e g i o n p o s i t i v e - f u l m i n a n t hepatitis. P a t i e n t N o . b y a w i l d - t y p e s t r a i n o f H B V . In p a t i e n t p o s i t i o n 1896 c r e a t i n g a s t o p m u t a t i o n
in t w o p a t i e n t s with H B s A g 5 ( o n the right) w a s infected N o . 6 a G to A m u t a t i o n in in p r e C gene w a s d e t e c t e d
(arrow).
might still have been missed due to direct sequencing. However, in previous studies analyzing precore mutations in fulminant hepatitis, the mutants represented the major strain of HBV as demonstrated by cloning and sequencing of the amplified HBV DNA (2). Thus, our study indicates that infection by a wild-type HBV
TABLE 2 Serological d a t a , d e t e c t i o n o f H B V D N A , H C V R N A , a n d p r e c o r e s t o p m u t a t i o n in 10 p a t i e n t s with f u l m i n a n t hepatitis type B Patient No. 1 2
3 4 5 6 7 8 9 10
Sex
Age years
IgM antiHBc
HBeAg/ anti H B e
AgDelta/ anti-Delta
anti-HCV
HBV DNA serum/liver
HCV RNA serum/liver
precore mutation
F
M F F M F M M M F
36
+
+ + + + + + +
-/+ -/+ +/-/+ -/+ -/+ _/+ -/-/+ +/-
-/+/-/-/-/-/_/_ -/-/+/-
_a -
-/+ -+ +/+ +/+ -/+ -/+ _/+ +/+ +/+ +/+
-/+ -/+/+ +/+ --/_/_ -/-/-/-
-
19 20 36 36 37 28 28 39 28
a l n d e t e r m i n a t e results o f R I B A II test.
+ -
122 prevails in F r e n c h patients with H B s A g - p o s i t i v e fulminant hepatitis. In addition, a m o n g the nine patients with wild-type H B V infection, two H D V A g - p o s i t i v e patients h a d acute coinfection by H D V a n d three others were p r o b a b l y acutely coinfected by H C V as assessed by detection o f H C V R N A a n d negative H C V serology (Table 2). The high incidence o f p r e c o r e m u t a t i o n previously r e p o r t e d in patients with f u l m i n a n t hepatitis might therefore p a r t l y reflect an e p i d e m i o l o g i c a l bias. Indeed, patients in these r e p o r t s were f r o m areas with a high prevalence o f p r e c o r e m u t a n t s in c h r o n i c a l l y infected patients a n d there is evidence which suggests precore m u t a n t selection d u r i n g the course o f c h r o n i c HBV infection (13). A direct role o f these m u t a n t s in the pathogenesis o f f u l m i n a n t hepatitis was h o w e v e r supp o r t e d by the finding o f the same m u t a t i o n in the cont a m i n a n t a n d in the recipient (4). O n the o t h e r h a n d , the same m u t a t i o n has been now identified in patients with a s y m p t o m a t i c (14) H B V infection w h o were the source o f c o n t a m i n a t i o n in the transmission o f f u l m i n a n t hepatitis. Thus, in conclusion, these o b s e r v a t i o n s as well as the present study seem to indicate that the H B V m u tant p h e n o t y p e is due to a c o m b i n a t i o n o f o t h e r viral (i.e., m u t a t i o n s in o t h e r regions o f the H B V genome) a n d / o r host factors.
c. FERAY et al.
References i
2 3 4
5 6 7 8
9 10 I1 12
Acknowledgments
13
T h e a u t h o r s t h a n k V. Thiers for technical assistance, D. Castaing, J.P. B e n h a m o u a n d F. Saliba for including patients in the liver t r a n s p l a n t p r o g r a m . C. F 6 r a y h o l d s grants from Assistance P u b l i q u e - H 6 p i t a u x de Paris (No. 92-0302). This w o r k was s u p p o r t e d by I N S E R M U370.
14
Kosaka Y, Takase K, Kojima Met al. Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen. Gastroenterology 1991; 100: 1087-94. Carman WF, Fagan EA, Hadziyannis S e t ai. Association of precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 1991; 14: 219-22. Liang TJ, Hasegawa K, Rimon N, Wands JR, Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med 1991; 324: 1705-9. Yotsomuto S, Kojima M, Shoji IS, Yamamoto K, Okamoto H, Mishiro S. Fulminant hepatitis related to transmission of hepatitis B variants with precore mutations between spouses. Hepatology 1992; 16: 31-5. F&ay C, Gigou M, Samuel D et al. Hepatitis C Virus RNA and Hepatitis B virus DNA in fuiminant hepatitis. Gastroenterology (in press). Bernuau J, Rueff B, Benhamou JP. Fulminant and subfulminant liver failure: definition and causes. Semin Liver Dis 1986; 6: 97-106. Meythaler J, Varma R. Reye's syndrome in adults. Archiv Intern Med 1987; 147: 61-4. Phillips J, Blendis L, Poucell Set al. Syncytial giant-cell hepatitis: sporadic hepatitis with distinctive pathological features, a severe clinical course, and paromyxoviral features. N Engl J Med 1991; 324: 455-60. Thiers V, Nakajima E, Kremsdorf D et al. Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet 1988; ii: 1273-6. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guadinium thiocyanate-phenol-chloroform extraction. Ann Biochem 1987; 162: 156-9. F~ray C, Samuel D, Thiers Vet al. Reinfection of liver graft by hepatitis C virus after liver transplantation. J Clin Invest 1992; 89: 1361-5. Kwok S, Higushi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. Thomas H, Carman W. The host immune response may be responsible for selection of envelope and precore/core variants of HBV. J Hepatol 1991; 13: S108. Okamoto H, Yotsumoto S, Akahane Y, et al. Hepatitis B viruses with precore region defects prevail in persistenly infected hosts along with seroconversion to the antibody against e antigen. J Virol 1990; 64: 1298-1303.
1 19
HEPAT 01466
Rapid Publication
Low prevalence of precore mutations in hepatitis B virus DNA in fulminant hepatitis type B in France Cyrille F 6 r a y a'b, Michelle G i g o u a'b, D i d i e r S a m u e l b, J a c q u e s B e r n u a u c, H e n r i B i s m u t h b a n d Christian Br6chot a aHybridotest, Institut Pasteur, Paris, bService de Chirurgie H~pato-biliaire et Transplantation H~patique, H6pital Paul Brousse, Villejuif, cUnit~ d'Hdpatologie, H6pital Beaujon, Clichy and dlNSERM U-370, CHU Necker and Unit~ d'H#patologie, H6pital La#nnec, Paris, France
The presence of mutants of the precore region of the hepatitis B virus genome was investigated in French patients with fulminant hepatitis. Only one of the 10 subjects had a detectable mutation by direct sequencing. On the other hand, 2/10 and 3/10 had evidence of coinfection by hepatitis D virus and hepatitis C virus. These results indicate that the precore stop mutation at codon 28 is not a general condition in fulminant hepatitis B and might reflect epidemiological factors.
Key words: Polymerase chain reaction; Direct sequencing
It has been suggested that variants of the HBV genome with mutations in the precore gene may cause severe forms of hepatitis. In particular a high incidence of these mutants has been reported in patients with sporadic HBsAg positive fulminant hepatitis, from the Far East (1) and Southern Mediterranean countries (2). Furthermore, the precore mutations have been also detected in fulminant hepatitis type B in Israel (3) and in Japan after nosocomial (2), interspouse (4) or intrafamilial transmission (2). In these cases, the same mutation was present in both the contaminant and the recipients. Factors other than mutations in the HBV genome, such as coinfection by the delta virus (HDV) and hepatitis C virus (HCV) (5) might be also determinant in the pathogenesis of fulminant hepatitis in HBsAg positive patients. In order to further investigate this issue, the precore gene was sequenced in French patients with fulminant hepatitis, and referred to our :enter for emergency liver transplantation (LT).
Patients Ten patients referred for HBsAg positive FH to the H6pital Beaujon and Villejuif liver transplant center were studied retrospectively. Fulminant hepatitis was diagnosed if patients developed hepatic encephalopathy within 15 days of the onset of jaundice (6) The only criteria for being included in the study were the availability and quality of serum and liver samples. Each patient had circulating hepatitis B surface antigen (HBsAg) and eight had detectable IgM anti-HBc. Hepatitis B virus e antigen (HBeAg) and antibody to HBeAg (anti-HBe) were detected in two and seven cases, respectively. The mean period between the onset of jaundice and encephalopathy was 4 days. Two patients had evidence of acute coinfection (n = 2) by hepatitis delta virus (HDV) and were intravenous drug users. Sexual transmission of infection by HBV was identified in three other patients. None of these 10 patients had
Correspondence to: Cyrille F6ray, M.D., Service de Chirurgie H6pato-biliaire, 14 Avenue Paul Vaillant Couturier, (94) Villejuif France.
120
C. FERAY et al.
received blood transfusions or had taken potentially hepatotoxic drugs. All had been living in France for at least the past year. In addition, there was no serological evidence of acute infection by hepatitis A virus, EpsteinBarr virus, herpes virus hominis, Toxoplasma, cytomegalovirus or human immunodeficiency virus. Serum ceruleoplasmin and alpha-1 antitrypsin were within normal ranges. Immunoserologic studies showed the absence of anti-nuclear and anti-smooth-muscle antibodies. Liver weight was 580 g (S.D. = 210; range 148-940). Histological examination revealed massive necrosis with regeneration in only one subject. There was no evidence of steatosis (7) or of syncitial giant-cell hepatitis (8). The control group included 20 patients with chronic anti-HCV-positive NANB hepatitis (n = 10), primary biliary cirrhosis (n = 7) or primary sclerosing cholangitis (n = 3). Serum samples were obtained at the time of admission. Liver samples were obtained during orthotopic liver transplantation (OLT) from all patients. Serum samples were also available for all patients during a follow-up of 1 year after OLT. The samples were stored under appropriate conditions for RNA analysis at -20°C (sera) or -80°C (liver).
proteins (c33, c100.3 and 511). Sera reacting with just one antigen were considered indeterminate. HB V and HD V markers HBV and HDV serologies were determined by means of enzyme-linked immunosorbent assays (ELISAs) (HBsAg, anti-HBc, HBeAg and anti-HBe, Wellcome diagnostic, Kent, UK; anti-HBs, Hoechst Behring Diagnostic, Marburg, Germany; HDV-Ag, Organon Teknika, Durham, Netherlands and anti-HDV, Diagnostic Pasteur, Marnes La Coquette). HBV DNA detection HBV DNA was extracted as previously described from 160 ttl of serum. HBV DNA PCR was performed as previously described (9). Briefly, 10 #1 of DNA preparation, corresponding to 40/~1 of serum and 1 /zg of liver DNA, was added to a final reaction volume of 50/zl containing 2 U of Taq polymerase, 20 pmol of each primer in PCR mixture previously described. The HBV primer sequences were located within the pre-C/C sequence (Table 1). Sequencing of the pre C region of HB V PCR products obtained with primers MD 40 and MD 31 were directly sequenced using T7 DNA polymerase (Pharmacia, Sweden) and primer MD 31.
Methods
Anti-HCV antibodies The presence of anti-HCV antibodies was determined using a recombinant immunoblot assay (RIBA-2, ORTHO diagnostic) which detects antibodies against epitopes located in the capsid (c22) and non-structural
HCV RNA detection RNA was extracted as described by Chomczynski (10) from 50 mg liver in guanidinium isothiocyanate, phenol/chloroform using a commercially available kit ('RNAZOL', Cinna-Biotecx laboratories, Friendswood, Texas, USA). Serum extraction of HCV RNA, eDNA
TABLE 1 Oligonucleotide sequences used for HCV and HBV primers
'outer' SFI ( - b ) SRI (_c) 'inner' SF2 (--) SR2 (--) probe
5' terminal sequence of HCV a
Length of PCR products
5' 5' 5' 5' 5'
259 bp 212 bp
GCC ATG GGC GTT AGT ATG AG 3' (-259; -239) GG TGC ACG GTC AAC GAG ACC 3' (-21; - I ) GTG CAG CCT CCA GGA CCC CCGA 3' (-236; -216) ACG GGT GAG GTA GTA GAC CC 3' (-24; -44) CCG GAA TTG CCA GGA CCG GGT CCT TTC TTG 3'(-76; -46)
H B V precore region of H B V
MD40 ( - ) 5' GCK} AGG AGA TTA GGT TAA 3' 1746-1764 MD31 (--) 5' CTT AGG AGT GCG AAT CCA 3' 2287-2269 aNucleotide sequences are numbered from initiating codon ATG. b(_) sense. c(_) antisense.
541 bp
PRECORE
MUTATIONS
IN HEPATITIS
B VIRUS DNA
121
synthesis and nested-PCR were performed according to a previously described protocol (11) using primers located in the untranslated part of the HCV RNA (Table 1).
AGCT
AG CT
•;
Specificity and sensitivity controls
',
) ~ t .-.am
To detect carry-over, HCV-PCR was performed with the following controls: (i) serum-free lysis buffer to detect contamination at every step of the procedure, (ii) three negative sera (blood donors) for every 12 test sera, (iii) the PCR mix alone. Prevention of carry-over included complete separation of pre-PCR and post-PCR steps. • It has been shown that most false-positive results with PCR occur during handling of amplification products (12). Results were only considered valid if they were consistent in independent experiments repeated at least twice (including the nucleic acid extraction step).
Results and Discussion
HBV DNA was detected using PCR in the liver of the 10 subjects and in the serum of half of them (5/10) (Table 2). HBV precore gene sequences were available for each patient. In one case, a G to A mutation at nucleotide 83 in the precore region which converted codon 28 for tryptophan (TGG) to a stop mutation (TAG), was detected (Fig. 1). The patient infected with this precore mutant did not have detectable HBV DNA in the serum nor HCV RNA in the serum or in the liver. It is noteworthy that this patient had evidence of chronic HBV infection since anti-HBc of IgM class was not detected. In the remaining nine patients, direct sequencing of HBV DNA did not show any mutations in the precore region but a minor population of mutant HBV
Fig. 1. Direct s e q u e n c i n g o f p r e C r e g i o n p o s i t i v e - f u l m i n a n t hepatitis. P a t i e n t N o . b y a w i l d - t y p e s t r a i n o f H B V . In p a t i e n t p o s i t i o n 1896 c r e a t i n g a s t o p m u t a t i o n
in t w o p a t i e n t s with H B s A g 5 ( o n the right) w a s infected N o . 6 a G to A m u t a t i o n in in p r e C gene w a s d e t e c t e d
(arrow).
might still have been missed due to direct sequencing. However, in previous studies analyzing precore mutations in fulminant hepatitis, the mutants represented the major strain of HBV as demonstrated by cloning and sequencing of the amplified HBV DNA (2). Thus, our study indicates that infection by a wild-type HBV
TABLE 2 Serological d a t a , d e t e c t i o n o f H B V D N A , H C V R N A , a n d p r e c o r e s t o p m u t a t i o n in 10 p a t i e n t s with f u l m i n a n t hepatitis type B Patient No. 1 2
3 4 5 6 7 8 9 10
Sex
Age years
IgM antiHBc
HBeAg/ anti H B e
AgDelta/ anti-Delta
anti-HCV
HBV DNA serum/liver
HCV RNA serum/liver
precore mutation
F
M F F M F M M M F
36
+
+ + + + + + +
-/+ -/+ +/-/+ -/+ -/+ _/+ -/-/+ +/-
-/+/-/-/-/-/_/_ -/-/+/-
_a -
-/+ -+ +/+ +/+ -/+ -/+ _/+ +/+ +/+ +/+
-/+ -/+/+ +/+ --/_/_ -/-/-/-
-
19 20 36 36 37 28 28 39 28
a l n d e t e r m i n a t e results o f R I B A II test.
+ -
122 prevails in F r e n c h patients with H B s A g - p o s i t i v e fulminant hepatitis. In addition, a m o n g the nine patients with wild-type H B V infection, two H D V A g - p o s i t i v e patients h a d acute coinfection by H D V a n d three others were p r o b a b l y acutely coinfected by H C V as assessed by detection o f H C V R N A a n d negative H C V serology (Table 2). The high incidence o f p r e c o r e m u t a t i o n previously r e p o r t e d in patients with f u l m i n a n t hepatitis might therefore p a r t l y reflect an e p i d e m i o l o g i c a l bias. Indeed, patients in these r e p o r t s were f r o m areas with a high prevalence o f p r e c o r e m u t a n t s in c h r o n i c a l l y infected patients a n d there is evidence which suggests precore m u t a n t selection d u r i n g the course o f c h r o n i c HBV infection (13). A direct role o f these m u t a n t s in the pathogenesis o f f u l m i n a n t hepatitis was h o w e v e r supp o r t e d by the finding o f the same m u t a t i o n in the cont a m i n a n t a n d in the recipient (4). O n the o t h e r h a n d , the same m u t a t i o n has been now identified in patients with a s y m p t o m a t i c (14) H B V infection w h o were the source o f c o n t a m i n a t i o n in the transmission o f f u l m i n a n t hepatitis. Thus, in conclusion, these o b s e r v a t i o n s as well as the present study seem to indicate that the H B V m u tant p h e n o t y p e is due to a c o m b i n a t i o n o f o t h e r viral (i.e., m u t a t i o n s in o t h e r regions o f the H B V genome) a n d / o r host factors.
c. FERAY et al.
References i
2 3 4
5 6 7 8
9 10 I1 12
Acknowledgments
13
T h e a u t h o r s t h a n k V. Thiers for technical assistance, D. Castaing, J.P. B e n h a m o u a n d F. Saliba for including patients in the liver t r a n s p l a n t p r o g r a m . C. F 6 r a y h o l d s grants from Assistance P u b l i q u e - H 6 p i t a u x de Paris (No. 92-0302). This w o r k was s u p p o r t e d by I N S E R M U370.
14
Kosaka Y, Takase K, Kojima Met al. Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen. Gastroenterology 1991; 100: 1087-94. Carman WF, Fagan EA, Hadziyannis S e t ai. Association of precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 1991; 14: 219-22. Liang TJ, Hasegawa K, Rimon N, Wands JR, Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med 1991; 324: 1705-9. Yotsomuto S, Kojima M, Shoji IS, Yamamoto K, Okamoto H, Mishiro S. Fulminant hepatitis related to transmission of hepatitis B variants with precore mutations between spouses. Hepatology 1992; 16: 31-5. F&ay C, Gigou M, Samuel D et al. Hepatitis C Virus RNA and Hepatitis B virus DNA in fuiminant hepatitis. Gastroenterology (in press). Bernuau J, Rueff B, Benhamou JP. Fulminant and subfulminant liver failure: definition and causes. Semin Liver Dis 1986; 6: 97-106. Meythaler J, Varma R. Reye's syndrome in adults. Archiv Intern Med 1987; 147: 61-4. Phillips J, Blendis L, Poucell Set al. Syncytial giant-cell hepatitis: sporadic hepatitis with distinctive pathological features, a severe clinical course, and paromyxoviral features. N Engl J Med 1991; 324: 455-60. Thiers V, Nakajima E, Kremsdorf D et al. Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet 1988; ii: 1273-6. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guadinium thiocyanate-phenol-chloroform extraction. Ann Biochem 1987; 162: 156-9. F~ray C, Samuel D, Thiers Vet al. Reinfection of liver graft by hepatitis C virus after liver transplantation. J Clin Invest 1992; 89: 1361-5. Kwok S, Higushi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. Thomas H, Carman W. The host immune response may be responsible for selection of envelope and precore/core variants of HBV. J Hepatol 1991; 13: S108. Okamoto H, Yotsumoto S, Akahane Y, et al. Hepatitis B viruses with precore region defects prevail in persistenly infected hosts along with seroconversion to the antibody against e antigen. J Virol 1990; 64: 1298-1303.