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hepatitis G virus infection in patients with cryptogenic chronic liver disease and in patients with primary biliary cirrhosis or Wilson’s disease
THE AMERICAN JOURNAL OF GASTROENTEROLOGY Copyright © 1999 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 94, No. 2, 1999 ISSN 0002-9270/99/$20.00 PII S0002-9270(98)00763-1
Prevalence of GB Virus-C/Hepatitis G Virus Infection in Patients With Cryptogenic Chronic Liver Disease and in Patients With Primary Biliary Cirrhosis or Wilson’s Disease Alessandro Tagger, Ph.D, Maria Lisa Ribero, Ph.D., Alberto Larghi, M.D., Francesco Donato, Ph.D., Massimo Zuin, M.D., Roberta Chiesa, M.D. Giampiero Benetti, M.D., Giuliano Ramella, M.D., Mauro Borzio, M.D., and Mauro Podda, M.D. Institute of Virology, Institute of Hygiene, and Institute of Internal Medicine (San Paolo Hospital), University of Milano, Milano; Institute of Hygiene, University of Brescia, Brescia; Liver Unit, 1st Department of Medicine, Melegnano Hospital, Melegnano; and 1st Department of Medicine, Fatebenefratelli Hospital, Milano, Italy
Objective: To assess the role of hepatitis G virus (HGV) in cryptogenic chronic liver disease (CLD), we investigated the prevalence of HGV RNA among patients with cryptogenic CLD, patients with nonviral CLD (primary biliary cirrhosis [PBC] and Wilson’s disease [WD]) and subjects without clinically evident liver disease (controls). Methods: Ninety patients with cryptogenic CLD (43 with chronic hepatitis, 20 with cirrhosis, and 27 with hepatocellular carcinoma [HCC]), 143 patients with PBC, 22 patients with WD, and 134 controls were recruited. HGV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and antibodies against HGV E2 protein (anti-E2) by an immunoassay test. Results: HGV RNA was detected in 7.8% of patients with cryptogenic CLD (chronic hepatitis, 9.3%; cirrhosis, 5.0%; HCC, 7.4%), in 2.4% of patients with PBC or WD, and in 2.2% of controls. As a consequence, a positive association of HGV infection with cryptogenic CLD was found (odds ratio, 3.1; 95% confidence interval [CI], 1.0 –9.7; p 5 0.05). No difference was observed between HGV RNA-positive and -negative patients by age, sex, histology, or liver function tests. Anti-E2 prevalence did not differ between patients with cryptogenic CLD (26.5%), patients with PBC (28.1%), and controls (22.1%). Transfusion history was associated with HGV RNA but not with anti-E2 seropositivity. Conclusions: Although an association was found between cryptogenic CLD and HGV infection, the role of the virus seems far from important, the proportion of cryptogenic CLD attributable to it being only 5.2%. (Am J Gastroenterol 1999;94:484 – 488. © 1999 by Am. Coll. of Gastroenterology)
INTRODUCTION A new hepatitis-associated RNA virus of the Flaviviridae family was recently identified using reverse transcriptionpolymerase chain reaction (RT-PCR) and designated hepatitis G virus (HGV) (1). It is almost identical to another virus associated with human non–A-E hepatitis and named GB virus-C (2), and therefore they are usually referred to as a single agent named GBV-C/HGV (3). HGV can be transmitted parenterally through blood transfusions, blood products, intravenous drug use and, hemodialysis (4 –9), and maybe through sexual (10) and vertical transmission (11). HGV RNA has been found in both patients with acute or chronic hepatic diseases and in subjects without liver diseases (1, 2). A controversial association between HGV and fulminant hepatic failure (12) has been described. Among patients with chronic liver disease of unknown etiology (cryptogenic), HGV RNA seroprevalence has been found to vary from 2% to 39% (13–22), usually higher than the 1–2% found among blood donors and healthy controls. However, few studies have enrolled a sufficiently large number of patients with biopsy-proven cryptogenic CLD and of controls unaffected by liver disease living in the same geographical area. Therefore, the role of HGV in cryptogenic CLD is still controversial. An immunoassay test for the detection of antibodies against HGV E2 protein (anti-E2) has been developed (23–25), and seropositivity has been found to be associated with recovery from the infection (26, 27). Only one study has been so far performed on anti-E2 seroprevalence among patients with cryptogenic CLD (19); thus, it is unknown whether patients with this disease have had higher exposure to the virus than patients with nonviral liver disease or those without clinically evident liver disease. The aim of our study was to evaluate the association between HGV infection and cryptogenic CLD by comparing HGV RNA prevalence in patients with cryptogenic CLD,
Received Jan. 29, 1998; accepted Oct. 1, 1998. 484
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MATERIALS AND METHODS
14.3% of the controls. History of intravenous drug abuse was found in one of the WD patients and in none of the cryptogenic CLD patients, PBC patients, or controls. Informed consent was given by all patients, and the project was approved by local Ethics Committees.
Patients Ninety-two patients with cryptogenic CLD were studied. Cryptogenic CLD was defined by exclusion of any known causes of CLD (viral, metabolic, toxic, alcoholic, autoimmune diseases). In addition to the standard serological tests (HBsAg, anti-HBc, anti-HCV), sera were also tested for the presence of HBV DNA and HCV RNA by nested PCR or RT-PCR, respectively. All patients tested HCV RNA-negative and two (both with hepatocellular carcinoma [HCC]) were found seropositive for HBV DNA and thus were excluded from the cryptogenic CLD series. Patients with cryptogenic chronic hepatitis (n 5 43) or cirrhosis (n 5 20) were selected among all subjects with the disease who were admitted to one of three main hospitals in Greater Milan, in Lombardy, northern Italy, in 1988 –1996. Cirrhosis was diagnosed on the basis of histology (n 5 3) or clinical picture (n 5 17), i.e., the presence of ascites, portal hypertension, or gastroesophageal varices. Diagnosis of chronic hepatitis was based on liver histology and, therefore, patients who could not be biopsied, or who refused to undergo biopsy, were excluded. Patients with HCC were enrolled in either of two hospitals in Brescia, a town near Milan, among all those included in an on-going hospital-based, case-control study on the determinants of HCC, some data of which have been already published (28, 29). All of the newly recognized HCC cases, who were admitted to the two main hospitals in the Brescia province between January 1995 and May 1998, were included in the study. Of the 305 HCC patients enrolled in the case-control study, only 27 (8.8%) had no known risk factor for HCC and thus were included in the present study as cryptogenic HCC cases. Of the HCC cases, 79.3% had a diagnosis by histology or cytology or had a-fetoprotein serum levels .500 ng/ml, the rest having been diagnosed by sonography or computed tomography after intra-arterial injection of lipiodol or both. A total of 143 patients with primary biliary cirrhosis (PBC) and 22 patients with Wilson’s disease (WD), both attending a third referral center for these diseases in Milan, were enrolled in this study as CLD cases of nonviral etiology. Diagnosis of PBC or WD was based on current criteria (30 –31). As a control group, we enrolled 134 patients not affected by liver disease or neoplasms and admitted to the same hospitals from which the cryptogenic CLD cases were recruited. None of patients with PBC or WD or the controls had a history of heavy alcohol intake (.60 g of ethanol/day in men and .40 g in women for $5 yr), and all were negative for both HBsAg and HCV antibody. History of blood transfusion was found in 6.2% of the cryptogenic CLD, 8.5% of the patients with PBC or WD and
Laboratory methods All blood samples were collected recently and taken at 280°C until tested for HGV RNA. The presence of HBsAg and other HBV markers was assessed using commercial tests for enzyme-linked immunosorbent assay (ELISA) (Abbott Laboratories, North Chicago, IL), and that of anti-HCV antibodies using third-generation ELISA and RIBA (Abbott Laboratories, North Chicago, IL; Ortho Diagnostic Systems, Raritan, NJ). Total RNA was extracted from 100 ml of serum by the guanidine isothiocianate-phenol-chloroform method. RTPCR assay for detection of HCV RNA was performed using nested primers of the 59 noncoding region (28). Serum HGV RNA was detected by RT-PCR amplification with nested primers from 59 noncoding region. The methods have been described elsewhere (29). Briefly, primer sequences were derived from the published gene sequences after multiple sequence alignment using the Pile Up program of GCG (Genbank accession numbers: U 44402 for HGV PNF 2161, U 45966 for HGV R 10291, U 36380 for GBV-C, U 63715 for GBV-C (EA), and U 59518 to U 59558 for the GBV-C 59-UTR sequences) (32). Specimens that gave positive results were confirmed by RT-PCR analysis using two sets of nested primers derived from the putative NS3 helicase and NS5b regions. In addition to HGV RNA, we have also investigated the presence of antibody against HGV E2 protein (anti-E2) as a marker of previous exposure to the virus. The anti-E2 ELISA test for antibodies against an HGV protein was performed by a microtiter plate assay (Boehringer, Mannheim, Germany) in all cryptogenic CLDs and controls, and in 100 PBC cases. Because no HGV-positive subject was also anti-E2 positive, total prevalence of HGV exposure was computed as the sum of HGV RNA-positive and anti-E2positive individuals. For the detection of HBV DNA, nucleic acid was extracted from 100 ml of serum with the proteinase K-sodium dodecyl sulphate-phenol-chloroform method. Two oligonucleotide primer pairs were used to amplify a sequence of 222 nt of the HBV surface gene, which were derived from the published gene sequences after multiple sequence alignment (Genbank accession numbers: V00867 and X14193 for subtype adr, X04615 for ayr, X024496, X65257, X65258, X65259, and X59795 for ayw, and V00866 for adw). The first-round PCR was performed with primer B261 (sense: 59 GCT GCT ATG CCT CAT CTT CTT G 39) and primer BR526 (antisense: 59 GTT AAC AGA GCC AGG AGA AAC GG 39); and the second-round PCR with primer B282 (sense: 59 GTT GGT TCT TCT GGA CTC TCA AGG 39)
patients with nonviral CLD (primary biliary cirrhosis and Wilson’s disease), and patients hospitalized for nonliver diseases (controls).
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TABLE 1 Patients With Cryptogenic Chronic Liver Disease (CLD), Patients With Primary Biliary Cirrhosis (PBC) or Wilson’s disease (WD), and Controls, According to Age, Sex, and Seropositivity for HGV RNA
Patients
Cryptogenic CLD (n 5 90) PBC or WD (n 5 165) Controls (n 5 134)
Age (yr)
Male/ Female
HGV RNAPositive
Mean (SD)
Ratio
No. (%)
54.4 (15.2) 58.5 (14.9) 63.4 (10.0)
58/34 24/141 96/38
7 (7.8)* 4 (2.4)* 3 (2.2)*
* p 5 0.08 by the exact test.
and primer BR503 (antisense: 59 GCT GAG GCC CAC TCC CAT AGG 39). Serum transaminases and total bilirubin were measured by the methods commonly used in hospital settings, normal values being ,45 units/ml for ALT, ,35 units/ml for AST, and ,1 mg/dl for bilirubin according to our laboratory reference values. Statistical methods The analysis of proportions was performed according to the methods commonly suggested, and exact methods were used when necessary, because of the small number of patients (33). The odds ratios (OR) and their 95% confidence intervals (95% CI) were computed as relative risk (RR) estimates according to common methods for epidemiological research. Sex and age were included as independent variables in logistic regression models as possible confounders to compute adjusted estimates of the ORs. All of the statistical tests were performed using the alpha value of 0.05 to reject the null hypothesis, although values between 0.05 and 0.1 are reported. All of the analyses were performed using the BMDP/Dynamic (34) and the StatXact computer program (35). RESULTS Age, sex, and HGV RNA prevalence among patients with cryptogenic CLD, PBC, or WD and controls are shown in Table 1. The prevalence of HGV RNA was slightly higher in cryptogenic CLD patients (7.8%) than in patients with PBC or WD (2.4%) or in controls (2.2%) at the limit of statistical significance (p 5 0.08). Demographic and clinical features of cryptogenic CLD patients according to HGV RNA seroprevalence are compared in Table 2. The patients with HGV infection did not differ significantly from those without HGV infection with regard to sex, age, liver function tests, and proportion of transfused subjects. HGV RNA prevalence did not differ according to type of liver disease (9.3% in chronic hepatitis, 5% in cirrhosis, and 7.4% in HCC on cirrhosis, p . 0.1). Anti-E2 antibodies were found in 26.5% of patients with cryptogenic CLD, 28.1% of patients with PBC, and 22.1% of controls (p . 0.1) (Table 3). Among cryptogenic CLD
TABLE 2 Demographic Features, Liver Function Tests and Type of Liver Disease Among the 90 Patients With Cryptogenic CLD According to HGV RNA Status
Features Age (yr): mean (SD)* Male/female ratio Liver function: mean (SD)* Bilirubin (mg/dl) ALT (U/ml) AST (U/ml) Transfusion history Cryptogenic CLD† Chronic hepatitis (n 5 43) Cirrhosis hepatitis (n 5 20) HCC (n 5 27)
HGV RNAPositive (n 5 7)
HGV RNANegative (n 5 83)
53.1 (12.9) 4/3
54.5 (15.4) 53/30
1.1 (0.5) 88.2 (37.0) 53.2 (17.6) 1 (14.3%)
1.1 (1.2) 84.3 (53.2) 48.9 (24.8) 3 (3.6%)
4 (9.3%) 1 (5.0%) 2 (7.4%)
39 19 25
* Mean values of age, liver function tests, and proportions of transfused subjects did not differ between HGV RNA-positive and -negative patients (p . 0.1 for each comparison). †Percentages are computed on the row total (total number of patients with each disease) (comparison of HGV RNA prevalence between chronic hepatitis, cirrhosis, and HCC, p . 0.1 by the exact test). TABLE 3 Patients With Cryptogenic Chronic Liver Disease (CLD), Patients With Primary Biliary Cirrhosis (PBC) or Wilson’s disease (WD), and Controls, According to Seropositivity for Anti-E2 in Subjects Negative for HGV RNA Anti-E2 Subjects
No. Tested
No. Positive
(%)
Patients with cryptogenic CLD Chronic hepatitis Cirrhosis HCC Patients with PBC Controls
83 39 19 25 96 131
22 8 5 9 27 29
(26.5) (20.5) (26.3) (36.0) (28.1) (22.1)
patients, no significant difference in anti-E2 prevalence was observed among patients with chronic hepatitis, cirrhosis, and HCC. The proportion of total HGV exposure (HGV RNA plus anti-E2) was 32.2%, 31.0%, and 23.9% among cryptogenic CLDs, PBC cases, and controls, respectively (p . 0.1). When considering all patients together, history of blood transfusion was claimed by 33.3% of HGV RNA-positive and 7.9% of HGV RNA-negative subjects (p 5 0.01), and by 7.1% of anti-E2-positive and 11.9% of anti-E2-negative subjects (p . 0.1). Transfusion history was not associated with total exposure to HGV (13% among subjects with and 10.1% among those without exposure). After dividing all subjects into patients with (n 5 90) and without (n 5 299) cryptogenic CLD, the odds ratio for having cryptogenic CLD by HGV RNA positivity, adjusted for age and sex, was 3.1 (95% CI, 1.0 –9.7, p 5 0.05) using logistic regression analysis. The OR did not change when
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also including transfusion history in the model as a possible confounder (OR, 2.9; 95% CI, 0.8 –10.9; p . 0.1). Based on this OR estimate and on the prevalence rate of HGV infection among cryptogenic CLD cases, the attributable risk for HGV RNA positivity was 5.2%. DISCUSSION In Italy, the main causes of CLD are hepatitis B, hepatitis C, and alcohol intake (36 –38), and a small proportion of CLD cannot be attributed to known agents. Unknown hepatitis viruses have been hypothesized to cause cryptogenic CLD, and the newly discovered HGV has been seen as a possible candidate for this role. To assess the actual role of HGV, we enrolled a large series of cryptogenic CLDs diagnosed by excluding any known causes of liver disease. The prevalence of HGV infection among them (7.8%) is in line with the 2–22% value found in previous studies among non-B, non-C CLDs (13–21), whereas a higher prevalence (39%) was found in one study based on a small number of patients (n 5 18) (22). HGV prevalence among patients with nonviral CLD and controls is in agreement with the 1–2% usually reported among healthy subjects and blood donors around the world (1, 4, 5, 20 –22). Therefore, we found a higher prevalence of HGV RNA in cryptogenic CLD patients than in patients with PBC or WD and in controls, with an OR estimate of 3.1. This finding is in line with previous studies showing a higher HGV RNA prevalence in cryptogenic CLD than in healthy subjects (13, 18, 21), and it suggests that HGV infection may represent a risk factor for developing cryptogenic CLD. However, no differences were found in liver function tests and type of liver disease according to HGV infection, in agreement with studies that found no association between HGV infection and severity of cryptogenic (15) or of HBV- or HCV-related chronic hepatitis (13, 21, 39 – 41). Furthermore, HGV RNA was found in many patients after liver transplantation but was not associated with the presence of posttransplantation liver disease (14, 42). In fact, HGV replication was not found in the liver of patients seropositive for HGV RNA who had end-stage liver disease that was of nonviral etiology or related to HCV infection (43, 44). Although subjects with HGV infection may develop a chronic carrier state, the duration of this state has been found to vary, and spontaneous clearance of the virus with seroconversion to anti-HGV status seems to occur in many subjects after some months or some years (26, 27). In our study, prevalence of anti-E2 among cryptogenic CLD was 26.5%, similar to that found among PBC cases and among controls, in agreement with another study that found a similar proportion of anti-E2 positivity (16%) in both non-B, non-C CLDs and in healthy controls (19). These findings suggest that patients with cryptogenic CLD did not experience a higher HGV infection rate than subjects with
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nonviral liver disease or those without clinically evident liver disease. We found a higher prevalence of transfusion history among HGV RNA-positive than -negative subjects, in agreement with other studies (18, 21), suggesting that blood transfusion is an important way of acquiring HGV infection in this area. On the opposite side, no association was evident between anti-E2 positivity and transfusion. These findings suggest that long lasting HGV infection may occur more often after transfusion-transmitted infection than infection transmitted through other routes, such as sexual intercourse. An alternative explanation for the association found between cryptogenic CLD and HGV prevalence is that other viral agents, possibly transmitted by the same route as HGV, may be involved in the pathogenesis of cryptogenic CLD. The recent findings of posttransplantation liver disease, in the absence of known viruses (A-G), suggests that other, yet unidentified, viruses may play a role (14). However, should HGV have caused some cases of chronic liver disease of unknown etiology, its role seems far from being important, as the prevalence of HGV among cryptogenic CLD is much lower than that of HBV and HCV infections among subjects with CLD in Italy (37). In fact, based on the proportion of cases with the presence of HGV in serum, and based on the OR estimate of 3.1, the proportion of cryptogenic CLD eventually attributable to HGV is 5.2%. ACKNOWLEDGMENTS The recruitment of the HCC cases and controls was performed within the Brescia HCC Study, thanks to the precious collaboration of the following: U. Gelatti and A. Albertini (Cattedra di Igiene), M. Favret (Cattedra di Anatomia Patologica), N. Portolani (Cattedra di Chirurgia Generale II) and M. Ghirardi (Cattedra di Chirurgia Generale) of the University of Brescia; L. Bettini and M. G. De Tavonatti (I Medicina), G. Pelizzari (II Medicina), E. Radaeli and L. Biasi (III Medicina), R. Farfaglia (II Chirurgia), M. Puoti (II Malattie Infettive), F. Bonetti (I Anatomia Patologica) and C. Leali (Dermatologia) at the Spedali Civili di Brescia; A. Salmi and M. Graffeo (Medicina), M. Valli (Anatomia Patologica), and M. Garatti (Chirurgia) at the Ospedale S. Orsola di Brescia. Reprint requests and correspondence: Alessandro Tagger, Ph.D., Institute of Virology, University of Milano, Via C. Pascal, 38-20133 Milano, Italy.
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Vol. 94, No. 2, 1999 ISSN 0002-9270/99/$20.00 PII S0002-9270(98)00763-1
Prevalence of GB Virus-C/Hepatitis G Virus Infection in Patients With Cryptogenic Chronic Liver Disease and in Patients With Primary Biliary Cirrhosis or Wilson’s Disease Alessandro Tagger, Ph.D, Maria Lisa Ribero, Ph.D., Alberto Larghi, M.D., Francesco Donato, Ph.D., Massimo Zuin, M.D., Roberta Chiesa, M.D. Giampiero Benetti, M.D., Giuliano Ramella, M.D., Mauro Borzio, M.D., and Mauro Podda, M.D. Institute of Virology, Institute of Hygiene, and Institute of Internal Medicine (San Paolo Hospital), University of Milano, Milano; Institute of Hygiene, University of Brescia, Brescia; Liver Unit, 1st Department of Medicine, Melegnano Hospital, Melegnano; and 1st Department of Medicine, Fatebenefratelli Hospital, Milano, Italy
Objective: To assess the role of hepatitis G virus (HGV) in cryptogenic chronic liver disease (CLD), we investigated the prevalence of HGV RNA among patients with cryptogenic CLD, patients with nonviral CLD (primary biliary cirrhosis [PBC] and Wilson’s disease [WD]) and subjects without clinically evident liver disease (controls). Methods: Ninety patients with cryptogenic CLD (43 with chronic hepatitis, 20 with cirrhosis, and 27 with hepatocellular carcinoma [HCC]), 143 patients with PBC, 22 patients with WD, and 134 controls were recruited. HGV RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and antibodies against HGV E2 protein (anti-E2) by an immunoassay test. Results: HGV RNA was detected in 7.8% of patients with cryptogenic CLD (chronic hepatitis, 9.3%; cirrhosis, 5.0%; HCC, 7.4%), in 2.4% of patients with PBC or WD, and in 2.2% of controls. As a consequence, a positive association of HGV infection with cryptogenic CLD was found (odds ratio, 3.1; 95% confidence interval [CI], 1.0 –9.7; p 5 0.05). No difference was observed between HGV RNA-positive and -negative patients by age, sex, histology, or liver function tests. Anti-E2 prevalence did not differ between patients with cryptogenic CLD (26.5%), patients with PBC (28.1%), and controls (22.1%). Transfusion history was associated with HGV RNA but not with anti-E2 seropositivity. Conclusions: Although an association was found between cryptogenic CLD and HGV infection, the role of the virus seems far from important, the proportion of cryptogenic CLD attributable to it being only 5.2%. (Am J Gastroenterol 1999;94:484 – 488. © 1999 by Am. Coll. of Gastroenterology)
INTRODUCTION A new hepatitis-associated RNA virus of the Flaviviridae family was recently identified using reverse transcriptionpolymerase chain reaction (RT-PCR) and designated hepatitis G virus (HGV) (1). It is almost identical to another virus associated with human non–A-E hepatitis and named GB virus-C (2), and therefore they are usually referred to as a single agent named GBV-C/HGV (3). HGV can be transmitted parenterally through blood transfusions, blood products, intravenous drug use and, hemodialysis (4 –9), and maybe through sexual (10) and vertical transmission (11). HGV RNA has been found in both patients with acute or chronic hepatic diseases and in subjects without liver diseases (1, 2). A controversial association between HGV and fulminant hepatic failure (12) has been described. Among patients with chronic liver disease of unknown etiology (cryptogenic), HGV RNA seroprevalence has been found to vary from 2% to 39% (13–22), usually higher than the 1–2% found among blood donors and healthy controls. However, few studies have enrolled a sufficiently large number of patients with biopsy-proven cryptogenic CLD and of controls unaffected by liver disease living in the same geographical area. Therefore, the role of HGV in cryptogenic CLD is still controversial. An immunoassay test for the detection of antibodies against HGV E2 protein (anti-E2) has been developed (23–25), and seropositivity has been found to be associated with recovery from the infection (26, 27). Only one study has been so far performed on anti-E2 seroprevalence among patients with cryptogenic CLD (19); thus, it is unknown whether patients with this disease have had higher exposure to the virus than patients with nonviral liver disease or those without clinically evident liver disease. The aim of our study was to evaluate the association between HGV infection and cryptogenic CLD by comparing HGV RNA prevalence in patients with cryptogenic CLD,
Received Jan. 29, 1998; accepted Oct. 1, 1998. 484
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MATERIALS AND METHODS
14.3% of the controls. History of intravenous drug abuse was found in one of the WD patients and in none of the cryptogenic CLD patients, PBC patients, or controls. Informed consent was given by all patients, and the project was approved by local Ethics Committees.
Patients Ninety-two patients with cryptogenic CLD were studied. Cryptogenic CLD was defined by exclusion of any known causes of CLD (viral, metabolic, toxic, alcoholic, autoimmune diseases). In addition to the standard serological tests (HBsAg, anti-HBc, anti-HCV), sera were also tested for the presence of HBV DNA and HCV RNA by nested PCR or RT-PCR, respectively. All patients tested HCV RNA-negative and two (both with hepatocellular carcinoma [HCC]) were found seropositive for HBV DNA and thus were excluded from the cryptogenic CLD series. Patients with cryptogenic chronic hepatitis (n 5 43) or cirrhosis (n 5 20) were selected among all subjects with the disease who were admitted to one of three main hospitals in Greater Milan, in Lombardy, northern Italy, in 1988 –1996. Cirrhosis was diagnosed on the basis of histology (n 5 3) or clinical picture (n 5 17), i.e., the presence of ascites, portal hypertension, or gastroesophageal varices. Diagnosis of chronic hepatitis was based on liver histology and, therefore, patients who could not be biopsied, or who refused to undergo biopsy, were excluded. Patients with HCC were enrolled in either of two hospitals in Brescia, a town near Milan, among all those included in an on-going hospital-based, case-control study on the determinants of HCC, some data of which have been already published (28, 29). All of the newly recognized HCC cases, who were admitted to the two main hospitals in the Brescia province between January 1995 and May 1998, were included in the study. Of the 305 HCC patients enrolled in the case-control study, only 27 (8.8%) had no known risk factor for HCC and thus were included in the present study as cryptogenic HCC cases. Of the HCC cases, 79.3% had a diagnosis by histology or cytology or had a-fetoprotein serum levels .500 ng/ml, the rest having been diagnosed by sonography or computed tomography after intra-arterial injection of lipiodol or both. A total of 143 patients with primary biliary cirrhosis (PBC) and 22 patients with Wilson’s disease (WD), both attending a third referral center for these diseases in Milan, were enrolled in this study as CLD cases of nonviral etiology. Diagnosis of PBC or WD was based on current criteria (30 –31). As a control group, we enrolled 134 patients not affected by liver disease or neoplasms and admitted to the same hospitals from which the cryptogenic CLD cases were recruited. None of patients with PBC or WD or the controls had a history of heavy alcohol intake (.60 g of ethanol/day in men and .40 g in women for $5 yr), and all were negative for both HBsAg and HCV antibody. History of blood transfusion was found in 6.2% of the cryptogenic CLD, 8.5% of the patients with PBC or WD and
Laboratory methods All blood samples were collected recently and taken at 280°C until tested for HGV RNA. The presence of HBsAg and other HBV markers was assessed using commercial tests for enzyme-linked immunosorbent assay (ELISA) (Abbott Laboratories, North Chicago, IL), and that of anti-HCV antibodies using third-generation ELISA and RIBA (Abbott Laboratories, North Chicago, IL; Ortho Diagnostic Systems, Raritan, NJ). Total RNA was extracted from 100 ml of serum by the guanidine isothiocianate-phenol-chloroform method. RTPCR assay for detection of HCV RNA was performed using nested primers of the 59 noncoding region (28). Serum HGV RNA was detected by RT-PCR amplification with nested primers from 59 noncoding region. The methods have been described elsewhere (29). Briefly, primer sequences were derived from the published gene sequences after multiple sequence alignment using the Pile Up program of GCG (Genbank accession numbers: U 44402 for HGV PNF 2161, U 45966 for HGV R 10291, U 36380 for GBV-C, U 63715 for GBV-C (EA), and U 59518 to U 59558 for the GBV-C 59-UTR sequences) (32). Specimens that gave positive results were confirmed by RT-PCR analysis using two sets of nested primers derived from the putative NS3 helicase and NS5b regions. In addition to HGV RNA, we have also investigated the presence of antibody against HGV E2 protein (anti-E2) as a marker of previous exposure to the virus. The anti-E2 ELISA test for antibodies against an HGV protein was performed by a microtiter plate assay (Boehringer, Mannheim, Germany) in all cryptogenic CLDs and controls, and in 100 PBC cases. Because no HGV-positive subject was also anti-E2 positive, total prevalence of HGV exposure was computed as the sum of HGV RNA-positive and anti-E2positive individuals. For the detection of HBV DNA, nucleic acid was extracted from 100 ml of serum with the proteinase K-sodium dodecyl sulphate-phenol-chloroform method. Two oligonucleotide primer pairs were used to amplify a sequence of 222 nt of the HBV surface gene, which were derived from the published gene sequences after multiple sequence alignment (Genbank accession numbers: V00867 and X14193 for subtype adr, X04615 for ayr, X024496, X65257, X65258, X65259, and X59795 for ayw, and V00866 for adw). The first-round PCR was performed with primer B261 (sense: 59 GCT GCT ATG CCT CAT CTT CTT G 39) and primer BR526 (antisense: 59 GTT AAC AGA GCC AGG AGA AAC GG 39); and the second-round PCR with primer B282 (sense: 59 GTT GGT TCT TCT GGA CTC TCA AGG 39)
patients with nonviral CLD (primary biliary cirrhosis and Wilson’s disease), and patients hospitalized for nonliver diseases (controls).
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TABLE 1 Patients With Cryptogenic Chronic Liver Disease (CLD), Patients With Primary Biliary Cirrhosis (PBC) or Wilson’s disease (WD), and Controls, According to Age, Sex, and Seropositivity for HGV RNA
Patients
Cryptogenic CLD (n 5 90) PBC or WD (n 5 165) Controls (n 5 134)
Age (yr)
Male/ Female
HGV RNAPositive
Mean (SD)
Ratio
No. (%)
54.4 (15.2) 58.5 (14.9) 63.4 (10.0)
58/34 24/141 96/38
7 (7.8)* 4 (2.4)* 3 (2.2)*
* p 5 0.08 by the exact test.
and primer BR503 (antisense: 59 GCT GAG GCC CAC TCC CAT AGG 39). Serum transaminases and total bilirubin were measured by the methods commonly used in hospital settings, normal values being ,45 units/ml for ALT, ,35 units/ml for AST, and ,1 mg/dl for bilirubin according to our laboratory reference values. Statistical methods The analysis of proportions was performed according to the methods commonly suggested, and exact methods were used when necessary, because of the small number of patients (33). The odds ratios (OR) and their 95% confidence intervals (95% CI) were computed as relative risk (RR) estimates according to common methods for epidemiological research. Sex and age were included as independent variables in logistic regression models as possible confounders to compute adjusted estimates of the ORs. All of the statistical tests were performed using the alpha value of 0.05 to reject the null hypothesis, although values between 0.05 and 0.1 are reported. All of the analyses were performed using the BMDP/Dynamic (34) and the StatXact computer program (35). RESULTS Age, sex, and HGV RNA prevalence among patients with cryptogenic CLD, PBC, or WD and controls are shown in Table 1. The prevalence of HGV RNA was slightly higher in cryptogenic CLD patients (7.8%) than in patients with PBC or WD (2.4%) or in controls (2.2%) at the limit of statistical significance (p 5 0.08). Demographic and clinical features of cryptogenic CLD patients according to HGV RNA seroprevalence are compared in Table 2. The patients with HGV infection did not differ significantly from those without HGV infection with regard to sex, age, liver function tests, and proportion of transfused subjects. HGV RNA prevalence did not differ according to type of liver disease (9.3% in chronic hepatitis, 5% in cirrhosis, and 7.4% in HCC on cirrhosis, p . 0.1). Anti-E2 antibodies were found in 26.5% of patients with cryptogenic CLD, 28.1% of patients with PBC, and 22.1% of controls (p . 0.1) (Table 3). Among cryptogenic CLD
TABLE 2 Demographic Features, Liver Function Tests and Type of Liver Disease Among the 90 Patients With Cryptogenic CLD According to HGV RNA Status
Features Age (yr): mean (SD)* Male/female ratio Liver function: mean (SD)* Bilirubin (mg/dl) ALT (U/ml) AST (U/ml) Transfusion history Cryptogenic CLD† Chronic hepatitis (n 5 43) Cirrhosis hepatitis (n 5 20) HCC (n 5 27)
HGV RNAPositive (n 5 7)
HGV RNANegative (n 5 83)
53.1 (12.9) 4/3
54.5 (15.4) 53/30
1.1 (0.5) 88.2 (37.0) 53.2 (17.6) 1 (14.3%)
1.1 (1.2) 84.3 (53.2) 48.9 (24.8) 3 (3.6%)
4 (9.3%) 1 (5.0%) 2 (7.4%)
39 19 25
* Mean values of age, liver function tests, and proportions of transfused subjects did not differ between HGV RNA-positive and -negative patients (p . 0.1 for each comparison). †Percentages are computed on the row total (total number of patients with each disease) (comparison of HGV RNA prevalence between chronic hepatitis, cirrhosis, and HCC, p . 0.1 by the exact test). TABLE 3 Patients With Cryptogenic Chronic Liver Disease (CLD), Patients With Primary Biliary Cirrhosis (PBC) or Wilson’s disease (WD), and Controls, According to Seropositivity for Anti-E2 in Subjects Negative for HGV RNA Anti-E2 Subjects
No. Tested
No. Positive
(%)
Patients with cryptogenic CLD Chronic hepatitis Cirrhosis HCC Patients with PBC Controls
83 39 19 25 96 131
22 8 5 9 27 29
(26.5) (20.5) (26.3) (36.0) (28.1) (22.1)
patients, no significant difference in anti-E2 prevalence was observed among patients with chronic hepatitis, cirrhosis, and HCC. The proportion of total HGV exposure (HGV RNA plus anti-E2) was 32.2%, 31.0%, and 23.9% among cryptogenic CLDs, PBC cases, and controls, respectively (p . 0.1). When considering all patients together, history of blood transfusion was claimed by 33.3% of HGV RNA-positive and 7.9% of HGV RNA-negative subjects (p 5 0.01), and by 7.1% of anti-E2-positive and 11.9% of anti-E2-negative subjects (p . 0.1). Transfusion history was not associated with total exposure to HGV (13% among subjects with and 10.1% among those without exposure). After dividing all subjects into patients with (n 5 90) and without (n 5 299) cryptogenic CLD, the odds ratio for having cryptogenic CLD by HGV RNA positivity, adjusted for age and sex, was 3.1 (95% CI, 1.0 –9.7, p 5 0.05) using logistic regression analysis. The OR did not change when
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also including transfusion history in the model as a possible confounder (OR, 2.9; 95% CI, 0.8 –10.9; p . 0.1). Based on this OR estimate and on the prevalence rate of HGV infection among cryptogenic CLD cases, the attributable risk for HGV RNA positivity was 5.2%. DISCUSSION In Italy, the main causes of CLD are hepatitis B, hepatitis C, and alcohol intake (36 –38), and a small proportion of CLD cannot be attributed to known agents. Unknown hepatitis viruses have been hypothesized to cause cryptogenic CLD, and the newly discovered HGV has been seen as a possible candidate for this role. To assess the actual role of HGV, we enrolled a large series of cryptogenic CLDs diagnosed by excluding any known causes of liver disease. The prevalence of HGV infection among them (7.8%) is in line with the 2–22% value found in previous studies among non-B, non-C CLDs (13–21), whereas a higher prevalence (39%) was found in one study based on a small number of patients (n 5 18) (22). HGV prevalence among patients with nonviral CLD and controls is in agreement with the 1–2% usually reported among healthy subjects and blood donors around the world (1, 4, 5, 20 –22). Therefore, we found a higher prevalence of HGV RNA in cryptogenic CLD patients than in patients with PBC or WD and in controls, with an OR estimate of 3.1. This finding is in line with previous studies showing a higher HGV RNA prevalence in cryptogenic CLD than in healthy subjects (13, 18, 21), and it suggests that HGV infection may represent a risk factor for developing cryptogenic CLD. However, no differences were found in liver function tests and type of liver disease according to HGV infection, in agreement with studies that found no association between HGV infection and severity of cryptogenic (15) or of HBV- or HCV-related chronic hepatitis (13, 21, 39 – 41). Furthermore, HGV RNA was found in many patients after liver transplantation but was not associated with the presence of posttransplantation liver disease (14, 42). In fact, HGV replication was not found in the liver of patients seropositive for HGV RNA who had end-stage liver disease that was of nonviral etiology or related to HCV infection (43, 44). Although subjects with HGV infection may develop a chronic carrier state, the duration of this state has been found to vary, and spontaneous clearance of the virus with seroconversion to anti-HGV status seems to occur in many subjects after some months or some years (26, 27). In our study, prevalence of anti-E2 among cryptogenic CLD was 26.5%, similar to that found among PBC cases and among controls, in agreement with another study that found a similar proportion of anti-E2 positivity (16%) in both non-B, non-C CLDs and in healthy controls (19). These findings suggest that patients with cryptogenic CLD did not experience a higher HGV infection rate than subjects with
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nonviral liver disease or those without clinically evident liver disease. We found a higher prevalence of transfusion history among HGV RNA-positive than -negative subjects, in agreement with other studies (18, 21), suggesting that blood transfusion is an important way of acquiring HGV infection in this area. On the opposite side, no association was evident between anti-E2 positivity and transfusion. These findings suggest that long lasting HGV infection may occur more often after transfusion-transmitted infection than infection transmitted through other routes, such as sexual intercourse. An alternative explanation for the association found between cryptogenic CLD and HGV prevalence is that other viral agents, possibly transmitted by the same route as HGV, may be involved in the pathogenesis of cryptogenic CLD. The recent findings of posttransplantation liver disease, in the absence of known viruses (A-G), suggests that other, yet unidentified, viruses may play a role (14). However, should HGV have caused some cases of chronic liver disease of unknown etiology, its role seems far from being important, as the prevalence of HGV among cryptogenic CLD is much lower than that of HBV and HCV infections among subjects with CLD in Italy (37). In fact, based on the proportion of cases with the presence of HGV in serum, and based on the OR estimate of 3.1, the proportion of cryptogenic CLD eventually attributable to HGV is 5.2%. ACKNOWLEDGMENTS The recruitment of the HCC cases and controls was performed within the Brescia HCC Study, thanks to the precious collaboration of the following: U. Gelatti and A. Albertini (Cattedra di Igiene), M. Favret (Cattedra di Anatomia Patologica), N. Portolani (Cattedra di Chirurgia Generale II) and M. Ghirardi (Cattedra di Chirurgia Generale) of the University of Brescia; L. Bettini and M. G. De Tavonatti (I Medicina), G. Pelizzari (II Medicina), E. Radaeli and L. Biasi (III Medicina), R. Farfaglia (II Chirurgia), M. Puoti (II Malattie Infettive), F. Bonetti (I Anatomia Patologica) and C. Leali (Dermatologia) at the Spedali Civili di Brescia; A. Salmi and M. Graffeo (Medicina), M. Valli (Anatomia Patologica), and M. Garatti (Chirurgia) at the Ospedale S. Orsola di Brescia. Reprint requests and correspondence: Alessandro Tagger, Ph.D., Institute of Virology, University of Milano, Via C. Pascal, 38-20133 Milano, Italy.
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