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GBV-C infection in liver transplant recipients: antibodies to the viral E2 envelope glycoprotein protect from de novo infection
Journal of Hepatology 1998; 29: 533–540 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
HGV/GBV-C infection in liver transplant recipients: antibodies to the viral E2 envelope glycoprotein protect from de novo infection Enrico Silini2, Luca Belli1, Alberto B. Alberti1, Margherita Asti2, Antonella Cerino2, Morena Bissolati2, Gianfranco Rondinara3, Luciano De Carlis3, Domenico Forti3, Mario U. Mondelli2 and Gaetano Ideo1 2
Department of Pathology and Infectious Diseases, University of Pavia and IRCCS Policlinico S. Matteo, Pavia, and 1Department of Internal Medicine, Center for Liver Diseases and 3Abdominal Organs’ Transplantation Unit, Niguarda Ca’ Granda Hospital, Milan, Italy
Background/Aims: Liver transplantation for endstage liver cirrhosis provides a useful model to investigate the pathogenetic role of hepatotropic viral agents. Recently, a new member of the Flaviviridae family, provisionally named HGV/GBV-C virus, has been associated with acute and chronic non A–E hepatitis. We studied 136 patients with cirrhosis consecutively transplanted at our institution for evidence of hepatitis G virus infection and correlation with the patients’ clinical course. Methods: All patients survived for at least 6 months after transplantation (median follow-up 44 months) and underwent routine liver biopsies. Hepatitis G virus infection was studied using both direct viral RNA identification by RT-PCR and indirect detection of antibodies to the E2 glycoprotein. Results: There was a high frequency of the hepatitis G virus among patients undergoing liver transplantation, with HGV RNA and anti-E2 prevalence rates of 18.4% and 26.5%, respectively. HGV RNA preva-
lences significantly increased after transplantation (47.8%), with 47.3% rate of new infections in susceptible subjects. Anti-E2 antibodies were significantly more prevalent among patients transplanted for HCVrelated cirrhosis and represented a strong protective factor against hepatitis G virus reinfection or recurrent infection. No correlation was found between HGV RNA or anti-E2 prevalences and survival after transplantation or rates of recurrent liver damage. Conclusions: All available evidence suggests that, although liver transplant patients are heavily exposed to hepatitis G virus both before and after transplantation, hepatitis G virus does not induce liver disease in this setting. Most infections appear to be selflimited and induce a protective immunity which is marked by the presence of anti-E2 antibodies.
fulminant hepatic failure (FHF) and cryptogenic cirrhosis are relatively frequent indications for orthotopic liver transplantation (OLT) worldwide: in Italy they account for about 10% of all liver transplants (1). It is presumed that as yet unknown infectious agents may be implicated in the etiology of these conditions (2). This hypothesis is supported by evidence derived from experimental inoculations of primates with human sera from non A–E hepatitis cases, which have
demonstrated the existence of at least two separate filterable agents able to induce liver disease (3). Epidemiological population-based studies also indicate that about 20% of community-acquired non A, non B acute hepatitis (4,5), 3–9% of all chronic hepatitis (2,6,7) and over 90% of fulminant non A, non B hepatitis (8–10) cannot be referred to known causes. Chronic liver damage of unknown etiology can be observed among long-term survivors of liver transplantation (11,12), including over 70% of patients transplanted for seronegative FHF (13), suggesting the possible involvement of new or persistent infections of the liver graft. Recently, a novel member of the Flaviviridae family of positive-strand RNA viruses has been isolated by molecular cloning by two independent groups (14,15). The new virus has beeen provisionally designated
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Received 17 October 1997; revised 22 January; accepted 9 February 1998
Correspondence: Gaetano Ideo. Divisione di Medicina Interna ed Epatologia ‘Crespi’, Ospedale di Niguarda Ca` Granda, Piazza dell’Ospedale Maggiore 3, 20162 Milan, Italy. Tel: 0039-2-64442396. Fax: 0039-2-64442788.
Key words: Anti-E2 antibodies; GBV-C; HGV; Liver transplant; Recurrent hepatitis.
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hepatitis G virus (HGV)/GB-C virus. HGV infection is distributed worldwide, is highly prevalent among individuals at risk for parenterally transmitted infections and is self-limited in most individuals (16,17), although long-term persistence is not infrequent (18). Circulating HGV RNA has occasionally been associated with acute and chronic liver disease (14) and FHF (19,20), but conclusive evidence implicating HGV in the etiology of non A–E hepatitis is still lacking (21– 23). We studied HGV infection in 136 liver transplant patients consecutively treated at our institution between 1990 and 1996, who survived for at least 6 months after transplantation (median follow-up time 44 months, range 7–88). The aims of the study were: (i) to investigate the prevalence of HGV infection using both direct viral identification by RT-PCR and indirect detection by antiE2 antibodies; (ii) to evaluate the incidence of de novo and persistent infection following transplantation; (iii) to correlate HGV infection prevalence with the etiology of cirrhosis; and (iv) to assess the role of HGV infection in the patients’ clinical course.
cal findings were scored and staged semi-quantitatively according to the criteria recommended by Desmet et al. (24). Informed consent to participate in the study was obtained from each patient, and the study protocol was approved by the ethical board of our insitution and conformed to the guidelines of the 1975 Declaration of Helsinki. Serological assays Patients’ sera were tested for anti-HCV by second-generation ELISA, according to the manufacturer’s instructions (Ortho Diagnostics System Inc. Raritan, NJ, USA) and in selected cases by second-generation immunoblot assay (RIBA 2, Chiron Corporation, Emeryville, CA, USA). Anti-HGV was detected in pre-transplant sera of all patients by a two-step sandwich ELISA (mPlate antiHGenv, Boehringer Mannheim, Germany), which uses as antigen a recombinant E2 envelope glycoprotein of HGV expressed in mammalian cells and captured in solid phase with a specific murine monoclonal antibody. Only repeat reactive sera were considered positive.
Materials and Methods Patients From August 1989 through January 1996, 185 patients with cirrhosis underwent orthotopic liver transplantation (OLT) at our institution. One hundred and fiftysix patients survived for more than 6 months after transplantation; paired pre- and post-transplant frozen sera were available for 136 of them (103 males, median age 49 years, range 23–61). These patients represented the population base of our study. Sixty-four patients had HCV-related liver cirrhosis, 25 had HBV-related cirrhosis, 18 had HDV superinfection, 10 patients were HCV-HBV coinfected, 14 had cryptogenic cirrhosis, 16 were alcoholics and seven had miscellaneous indications for transplantation (primary biliary cirrhosis: three patients, and primary sclerosing cholangitis, Budd-Chiari syndrome, Wilson’s disease and Caroli’s disease: one patient each). Demographic, epidemiological and biochemical data from all patients were retrieved from our files, including the regimen of immunosuppression, the number and type of blood transfusions performed and the detailed post-operative course. Patient follow-up was 7–88 months (median 44). Patients were regularly reviewed every 2 weeks until day 90, monthly until the sixth post-operative month, every 3 months until 1 year, and every 3–6 months thereafter. Percutaneous liver biopsies were performed whenever clinically indicated and at yearly intervals in all patients. Histologi-
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Virological methods Total RNA was extracted from 0.5 ml of serum stored frozen at ª70æC using a modification of the guanidinium acid-phenol method (25). RNA from 100 ml of serum was retrotranscribed and PCR-amplified using a nested-primer protocol. Two sets of 4 primers were used, one based on sequences from the helicase/NS3 gene, which are essentially a modification of previously described primers (15,19) and a second set based on conserved sequences of the 5ø non-coding region: 5ø non-coding, sense primer (1st round) 5ø GGC-CAA-AAG-GTG-GTG-GAT-GG 3ø 5ø non-coding, antisense primer (1st round) 5ø GTG-GGC-GTC-GTT-TGC-CCA-GG 3ø 5ø non-coding, sense primer (2nd round) 5ø TTG-GTA-GCC-ACT-ATA-GGT-GG 3ø 5ø non-coding, antisense primer (2nd round) 5ø GGT-AGG-ACC-AAC-ACC-TGT-GG 3ø. Selected amplicons were cloned in a T-tailed plasmid (TA cloningTM kit, Invitrogen Corp., San Diego, CA, USA) and sequenced using a thermostable DNA polymerase (CircumVentTM kit , New England Biolabs Inc., Beverly, MA, USA). HGV genotypes were determined by phylogenetic analysis of sequences of the 5ø noncoding region in 10 samples and in the remaining cases by a PCR assay which uses a set of type-specific primers of the same genomic region (E. Silini, unpublished results).
HGV infection in liver transplant patients
HCV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR), using conserved primers localized in the 5ø non-coding region of the viral genome (26). HCV genotyping was performed by PCR-amplification of core region sequences with universal and 5 subtype-specific primers according to Okamoto et al. (27,28), with a modified type 2a-specific primer, as previously described (26). HCV type distribution in this cohort has been previously described (29). Statistical methods Actuarial survival rates and rates of recurrent hepatitis were calculated using the Kaplan-Meier method; comparison of actuarial rates was made using the log-rank test. The chi-square test, t-test and analysis of variance were used as appropriate. P-values∞0.05 were considered statistically significant.
Results Three hundred and twenty-three serum samples derived from 136 patients were analyzed for the study. All patients were tested on at least two separate sera: one collected pre-transplantation and the other 6–12 months after grafting. HGV RNA detection was performed by nested RT-PCR of sequences from the helicase-NS3 gene region and confirmed in 85 samples by the use of a second set of primers from the 5ø noncoding region (5øNCR). The results obtained by the two detection systems showed a concordance of 93%. The reliability of HGV RNA detection was further assessed by cloning and sequencing 14 amplicons from the NS3 region and 10 amplicons from the 5øNCR. HGV genotypes were also determined in 50 cases; all HGV RNA sequences were classifiable as type 2a (30), which represents the most frequent HGV variant present in Italy and in the Western world. No differences were found as to the infecting HGV type with respect to other patient categories in the same geographic area
(E. Silini, work in preparation). Anti-E2 seroprevalence was determined in pre-transplantation sera of all patients, using a commercial two-step sandwich ELISA which uses as antigen a recombinant E2 viral glycoprotein expressed in mammalian cells. Prevalences of serum HGV RNA and anti-E2 antibodies in pre-transplantation sera are shown in Table 1. Overall, HGV RNA was found in 18.4% (25/136) of patients and anti-E2 seroreactivity in 26.5% (36/136); among the 36 anti-E2 positive patients, only a single subject was HGV RNA positive, as compared to 24 out of 100 patients in the anti-E2 negative group (p∞0.01). Sex, age at transplantation or previous history of blood transfusion did not differ between patients according to their anti-HGV or serum HGV RNA status. Conversely, significant differences in the prevalence of HGV infection were observed between patient categories (Table 2). Specifically, at transplantation the 64 patients with HCV-induced cirrhosis had higher rates of anti-E2 seropositivity (37.5% (24/64) vs 16.7% (12/72), p∞0.01) and lower rates of serum HGV RNA (12.5% (8/64) vs 23.6% (17/72), pΩns) with respect to the other groups of patients . Detection of serum HGV RNA markedly increased after transplantation among patients of all groups, with an overall prevalence of 47.8% (65/136) (Table 2). The risk of developing post-transplantation HGV viremia was significantly dependent on the anti-E2 status of the patient prior to transplantation, since only 4 of 36 (11%) anti-E2 positive subjects showed viremia compared to 61 out of 100 (61%) anti-E2 negative patients (p∞0.001). These findings are in agreement with previous observations, which also reported the presence of serum HGV RNA in about 10% of anti-E2 positive subjects (16). Accordingly, HCV-infected patients showed significantly lower rates of post-transplan-
TABLE 2 Presence of serum HGV RNA and anti-E2 antibodies in 136 liver transplant patients according to their indication for transplantation
TABLE 1 Presence of HGV RNA and anti-E2 antibodies in the pre- and postoperative sera of 136 liver transplant patients Anti-E2 antibodies
Serum HGV RNA Pre-OLT
Positive Negative Total
Total Post-OLT
Positive
Negative
Positive
Negative
1 24 25
35 76 111
4 61 65
32* 39 76
36 100 136
* Anti-E2 positive patients are significantly protected from the development of post-transplant HGV viremia (p∞0.01 by chi-square test). OLTΩorthotopic liver transplantation.
Indication for transplantation HGV RNA positive* (number of patients) Pre-OLT Post-OLT
Anti-E2 positive*
Cryptogenic cirrhosis (14) Alcoholic cirrhosis (16) HCV cirrhosis (64) HBV cirrhosis (25) HBV/HCV cirrhosis (10) Other (7)
3 (21) 3 (19) 24 (37)æ 4 (16) 2 (20) ª
4 4 8 4 3 2
(29) (25) (12) (16) (30) (29)
10 9 21 13 6 6
(71) (56) (33)æ (52) (60) (86)
* The percent of total in the group is shown in brackets. æ Anti-HCV positive patients show a significantly higher prevalence of anti-HGV (p∞0.001 by chi-square) compared with patients of other groups and, accordingly, they show a reduced incidence of posttransplant HGV viremia (p∞0.01). OLTΩorthotopic liver transplantation.
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Fig. 1. Kaplan-Meier analysis of actuarial survival in 136 liver transplant patients according to their anti-HGV (Fig. 1a) and HGV RNA (Fig. 1b) status. No significant differences in actuarial survival were observed (1a pΩ0.48; 1b pΩ0.74 log-rank test).
tation HGV viremia compared to patients in other groups (32.8% (21/64) vs 61.1% (44/72), p∞0.01). All 24 patients who were HGV RNA positive at transplantation continued to harbor circulating virus during the first year after graft. To assess the duration of posttransplant viremia in the long term, we also examined sera of 20 patients after 3 years of follow-up and we detected HGV RNA in 15 (75%) of them. Thirty-six of 76 (47.4%) subjects who were anti-E2 negative and HGV RNA negative before OLT developed HGV RNA viremia after transplantation. These presumably represent new infections, since no direct (HGV RNA) or indirect (anti-HGV) evidence of HGV was found in these patients. Anti-HCV positive cases had a lower risk of acquiring de novo HGV infections; in fact, 12 of 32 (37.5%) potentially at-risk HCVinfected patients (HGV RNA anti-HGV double negative at transplantation) developed post-OLT HGV viremia compared with 24 of 44 (54.5%) patients from other groups, although this difference did not reach statistical significance (Table 3). To estimate the transfusion-related risk of HGV infection, we evaluated the prevalence of serum HGV
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Fig. 2. Actuarial rates of hepatitis recurrence in 64 antiHCV positive liver transplant patients according to their anti-HGV (Fig. 2a) and HGV RNA (Fig. 2b) status. No significant differences in hepatitis recurrence rate were observed (1a pΩ0.48; 1b pΩ0.33 log-rank test).
RNA in 217 active donors from the local blood bank. Prevalences of 2.3% (5/217) and 6% (9/149) were found for serum HGV RNA and anti-E2 antibodies, respectively. The mean number of units of blood or blood derivates transfused per patients in the operative period was: 21 units of packed red cells, 26 units of fresh frozen plasma and 6 units of platelets. Therefore, the estimated cumulative risk of acquiring HGV in the peri-transplantation period was 1–0.97753Ω0.71, which is compatible with the figure actually observed. To evaluate whether HGV infection might affect the
TABLE 3 De novo HGV infection among 76 orthotopic liver transplantation patients who were anti-E2/HGV RNA negative in pre-transplant sera Indication for transplantation (no. of patients)
De novo infection
Exposed subjects
Cryptogenic cirrhosis (14) Alcoholic cirrhosis (16) HCV cirrhosis (64) HBV cirrhosis (25) HBV/HCV cirrhosis (10) Other (7)
5 5 12 8 2 4
8 9 32 17 5 5
HGV infection in liver transplant patients TABLE 4 Biochemical indices of liver damage in 29* patients with cirrhosis and HCV recurrence according to HGV RNA status after transplant Patients (no.)
Peak values AST ALT GGT Bilirubin
and only one had a concurrent post-transplantation HGV viremia.
Discussion
HGV RNA negative (21)
HGV RNA positive (8)
262∫348 (range 23–1400) 292∫250 (range 40–800) 120∫99 (range 12–300) 1.7∫0.9 (range 0.8–7)
146∫116 (range 26–310) 292∫237 (range 29–700) 153∫123 (range 30–370) 1.6∫1.0 (range 0.6–3.3)
At time of the first protocol biopsy AST 149∫128 (range 23–470) ALT 195∫107 (range 40–380) GGT 113∫98 (range 12–300) Bilirubin 1.7∫0.9 (range 0.8–4)
105∫103 (range 26–310) 247∫218 (range 29–632) 130∫124 (range 30–370) 1.4∫0.8 (range 0.6–2.7)
Mean follow-up values AST 145∫183 (range 22–610) ALT 153∫173 (range 14–590) GGT 71∫74 (range 10–240) Bilirubin 1.6∫1.7 (range 0.7–4)
56∫35 (range 26–106) 125∫174 (range 26–515) 81∫48 (range 19–156) 1.2∫0.8 (range 0.6–3)
* Five patients were excluded from the analysis because of intercurrent cholestasis due to mechanical obstruction to bile outflow. ALTΩ alanine aminotransferase; ASTΩaspartate aminotransferase; GGTΩ gamma glutamyltransferase.
outcome of OLT, we performed a survival analysis dividing patients according to their anti-E2 status and the presence of serum HGV RNA before or after transplantation (Fig. 1). No significant differences were observed considering the whole cohort or groups of patients with specific transplant indications (HCV infection, HBV infection, other indications). In particular, none of the 19 patients transplanted for cryptogenic or alcoholic cirrhosis, who were HGV RNA positive after transplantation, has shown so far evidence of recurrent liver damage (median follow-up time 39 months, range 8–74). Thirty-four of 64 (53.1%) subjects transplanted for HCV-related cirrhosis developed recurrent hepatitis C and in these patients HGV coinfection did not influence the frequency, the timing or the severity of HCV recurrence (Fig. 2). In addition, no correlations were found between the presence of HGV RNA after transplantation and the infecting HCV genotype or the mean (or peak) levels of ALT, AST, gGT or bilirubin during the entire follow-up period, at the time of the diagnosis of recurrent hepatitis or when the first protocol liver biopsy was performed (Table 4). Five patients with recurrent hepatitis C were excluded from the analysis of the results due to concurrent biliary complications. These five patients all required repeated biliary stenting to treat extrahepatic biliary strictures. Eight of the 34 patients with recurrent hepatitis C have progressed to stage 3–4 so far; two were anti-E2 positive
Liver transplantation provides an excellent model to investigate the possible pathogenetic relevance of new viral hepatitis agents in humans. It overcomes the limitations of cross-challenge studies in animals; these are often not relevant due to the differences in viral pathogenesis which can exist even between closely related species. Indeed, hepatitis virus infections almost invariably recur after transplantation and sometimes induce a more rapid and overt clinical course than in immunocompetent patients. In addition, in the liver transplant setting the natural history of recurrent infections can be accurately monitored, since dates of infection are known, subjects are followed up very closely, and collection of biological samples and repeated biopsies are readily available. We used the OLT model to study the clinical and pathological correlations of HGV infection in a large group of Italian patients who were transplanted for cirrhosis of different etiologies. Although previous studies have already addressed this issue (12,31–33), our work provides significant and novel information. Thus, to assess the prevalence and the incidence of HGV infection, we looked for both HGV RNA and anti-E2 antibody. This approach allowed us to distinguish between de novo and persistent infections and to evaluate more precisely the real number of HGV-exposed patients, which is largely underestimated by the use of HGV RNA detection alone. We analyzed consecutive patients with different indications for OLT and found significant differences in the distribution of HGV infection among patient categories. The study of subjects with long follow-up times (median 44 months) and the exclusion of patients who survived less than 6 months enabled us to assess more precisely the role of HGV infection in the long-term outcome and in the recurrence of HCV infection. Detailed molecular analysis was performed on paired pre- and post-OLT serum samples of all patients, including study of the possible role of viral heterogeneity in determining the severity of liver disease. Estimates of the prevalence of HGV viremia and anti-E2 positivity in a large number of sera from active blood donors from the same geographical area was also obtained to evaluate the risk of HGV infection associated with blood transfusion. Here we show that HGV infection is highly prevalent among patients undergoing OLT, with 45% cumulative infection rates as assessed by combined serum anti-E2 and HGV RNA detection. This finding is in agreement with all previous studies (12,31–33), although anti-
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HGV seroprevalence has never been reported before in this setting. Simultaneous evaluation of both anti-E2 and HGV RNA indicates that the number of exposures to HGV considerably exceeds that of active infections as assessed by the presence of circulating virus. HCVinfected cirrhotic patients are significantly more exposed to HGV than patients from other categories, and in 24 (75%) of 32 patients our findings indicate a past, probably resolved infection with circulating antiE2 and absence of serum HGV RNA. This finding is in agreement with most epidemiological studies published so far, which show a strong association of HCV and HGV infections in all patient categories. However, these findings also suggest that HGV infection is shortlived since most HCV positive patients had cleared HGV RNA. HGV infection invariably recurs after OLT and most patients both with de novo and persistent infections harbor viral sequences for a long time after transplantation. Furthermore, the risk of being infected by HGV in the peri-operative period is very high, since about half of the potentially at-risk patients (i.e. HGV RNA and anti-E2 double-negative subjects at transplantation), develop serum HGV RNA during the first year of follow-up. The risk of acquiring new HGV infections is significantly dependent on the presence of anti-E2 antibodies at transplantation. Indeed, our study provides the first formal prospective demonstration that anti-E2 antibodies are protective against HGV reinfection. This finding may explain the counterintuitive observation that HCV-naive patients are more prone to develop HGV infection following transplantation than HCVinfected patients, who, having been exposed to HGV in the past, have had time to develop a protective immune response. Similar observations were made by previous authors who were not able to offer a plausible explanation for this finding (31–33). Berg and co-authors (32) suggested that HGV infection might have been transmitted through anti-HBsAg immunoglobulin used for prophylaxis of HBV recurrence, but this conclusion seems unwarranted in the light of the present results. It is also interesting to note that the risk of acquiring de novo HGV infection is lower in HCV patients irrespective of their anti-E2 status than in patients from other groups. This might be explained either by the presence of anti-E2 antibodies with different specificity and/or affinity for the E2 protein utilized as antigen in the test or by the existence of protective antibodies directed against other HGV proteins or, possibly, by the interference of HCV in HGV replication. It is reasonable to believe that the high risk of ac-
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quiring HGV infection in the peri-transplant period is due to the heavy exposure of liver transplant patients to blood and blood derivates, the mean number of blood units received by the patients in our cohort in the peri-operative period being 53. A significant, but presumably small risk might also derive from the donor liver (12), but this could not be ascertained in our study. Nonetheless, the risk estimate of acquiring HGV during transplantation, which can be inferred by the frequency of de novo infections (0.47), is comparable to the figures that can be extrapolated from the prevalence of HGV viremia among blood donors (0.71), as also demonstated by other authors (12,33). This indicates that viral infectivity is high, and it also provides further evidence that HGV is easily transmitted by transfusion. Since anti-E2 antibodies were frequently found among blood donors (6–7%), it is highly likely that most patients received these antibodies with transfusions. Passive immunity by transfer of anti-E2, however, seems to play a minor role, if any, in protecting against primary HGV infection, given the high proportion of new infections among HGV-naive patients. This might be explained either by the low titers of the transfused antibodies or by differences in antibody specificity among individuals. In addition, it should be considered that, in analogy with other hepatitis virus models, other anti-viral mechanisms, such as T-cytotoxic responses, might be relevant in the clearance of or in the protection against HGV infection. A possible explanation for the high carrier rate, such as that observed in this and previous studies among otherwise ‘‘healthy subjects’’ in the general population, would be that HGV is efficiently transmitted via non parenteral routes. Evidence in favor of efficient vertical transmission of HGV has already been gathered (34), and there is also evidence suggesting possible sexual transmission (35). HGV infection, both de novo and persistent, is not associated with clinical or pathological signs of damage of the liver graft and does not influence overall survival (Fig. 1). No patient with HGV infection alone has shown recurrence of liver disease up to 6 years post-transplantation. In the larger group of HCV patients, HGV coinfection does not influence the timing, the frequency or the clinical course of recurrent hepatitis C (Fig. 2). These findings are in agreement with all previous studies from different cohorts (12,31– 33,36–38). It has been suggested that HGV infection might be preferentially associated with bile duct damage (39); however, in our 34 patients with recurrent hepatitis C, we did not observe a cholestatic picture associated with HGV coinfection. Finally, HGV se-
HGV infection in liver transplant patients
quence variants present among patients with cirrhosis of different etiologies are similar, indicating that viral heterogeneity plays no role in determining the possible clinical features of the infection (30). In conclusion, all the available evidence suggests that HGV does not induce liver disease in the setting of orthotopic liver transplantation. It is indeed possible that the liver is not the primary target organ of this peculiar human virus.
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Acknowledgements This study was supported in part by grants no. 030RFM93/01 and 240RFM96/01 from the Italian Ministry of Health to IRCCS Policlinico San Matteo. We also gratefully acknowledge the financial support of the Italian Red Cross, Committee of Ponte San Pietro, Bergamo, Italy.
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Detmer J, et al. Hepatitis G virus in patients with cryptogenic liver disease undergoing liver transplantation. Hepatology 1997; 25: 1266–70. Mohamed R, Hubscher SG, Mirza DF, Gunson BK, Mutimer DJ. Posttransplantation chronic hepatitis in fulminant hepatic failure. Hepatology 1997; 25: 1003–7. Linnen J, Wages J, Zhang-Keck Z-Y, Fry KE, Krawczynski KZ, Alter H, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996; 271: 505–8. Simons JN, Leary TP, Dawson GJ, Pilot-Matias TJ, Muerhoff AS, Schlauder GG, et al. Isolation of novel virus-like sequences associated with human hepatitis. Nature Med 1995; 1: 564–9. Tacke M, Kiyosawa K, Stark K, Sclueter V, OfenlochHaehnle B, Hess G, et al. Detection of antibodies to a putative hepatitis G virus envelope protein. Lancet 1997; 349: 318–20. Feucht HH, Zollner B, Polywka S, Knodler B, Schroter M, Nolte H, et al. Distribution of hepatitis G viremia and antibody response to recombinant proteins with special regard to risk factors in 709 patients. Hepatology 1997; 26: 491–4. Masuko K, Mitsui T, Iwano K, Yamazaki C, Okuda K, Meguro T, et al. Infection with hepatitis GB virus C in patients on maintenance hemodialysis. N Engl J Med 1996; 334: 1485–90. Yoshiba M, Okamoto H, Mishiro S. Detection of the GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown aetiology. Lancet 1995; 346: 1131–2. Heringlake S, Osterkamp S, Trautwein C, Tillman HL, Bo¨ker K, Muerhoff S, et al. Association between fulminant hepatic failure and a strain of GBV virus C. Lancet 1996; 348: 1626– 9. Alter HJ, Nakatsuji Y, Melpolder J, Wages J, Welsey R, Shih JW-H, et al. The incidence of transfusion associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 1997; 336: 747–54. Alter MJ, Gallagher M, Moris TT, Moyer LA, Meeks EL, Krawczynski K, et al. for the Sentinel Counties Viral Hepatitis Study Team. Acute non-A–E hepatitis in the United States and the role of hepatitis G virus infection. N Engl J Med 1997; 336: 741–6. Kanda T, Yokosuka O, Ehata T, Maru Y, Imazeki F, Saisho H, et al. Detection of GBV-C RNA in patients with nonA–E fulminant hepatitis by reverse-transcription polymerase chain reaction. Hepatology 1997; 25: 1261–5. Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology 1994; 19: 1513–20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-penol-chloroform extraction. Anal Biochem 1987; 162: 156–9. Silini EM, Bono F, Cividini A, Cerino A, Bruno S, Rossi S, et al. Differential distribution of hepatitis C virus genotypes in patients with and without liver function abnormalities. Hepatology 1995; 21: 285–90. Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73: 673–9. Okamoto H, Tokita H, Sakamoto M, Horikita M, Kojima M, Iizuka H, et al. Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection. J Gen Virol 1993; 74: 2385–90.
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E. Silini et al. 29. Belli LS, Silini E, Alberti A, Bellati G, Vai C, Minola E, et al. HCV genotypes, hepatitis, and HCV recurrence after liver transplantation. Liver Transpl Surg 1995; 2: 1–7. 30. Muerhoff AS, Smith DB, Leary TP, Erker JC, Desai SM, Mushahwar IK. Identification of GB virus C variants by phylogenetic analysis of 5ø-untranslated and coding region sequences. J Virol 1997; 71: 6501–8. 31. Berenguer M, Terrault NA, Piatak M, Yun A, Kim JP, Lau JYN, et al. Hepatitis G virus infection in patients with hepatitis C virus infection undergoing liver transplantation. Gastroenterology 1996; 111: 1569–75. 32. Berg T, Naumann U, Fukumoto T, Bechstein WO, Neuhaus P, Lobeck H, et al. GB virus C infection in patients with chronic hepatitis B and C before and after liver transplantation. Transplantation 1996; 62: 711–4. 33. Fried MW, Khudyakov YE, Smallwood GA, Cong M, Nichols B, Diaz E, et al. Hepatitis G virus co-infection in liver transplantation recipients with chronic hepatitis C and nonviral chronic liver disease. Hepatology 1997; 25: 1271–5. 34. Viazov S, Riffelmann M, Sarr S, Ballauff A, Meisel H, Roggendorf M. Transmission of GBV-C/HGV from drug-addicted mothers to their babies. J Hepatol 1997; 27: 85–90.
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35. Kao J-H, Chen P-J, Lai M-Y, Chen W, Liu D-P, Wang J-T, et al. GB virus-C/hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease and liver cancer. Gastroenterology 1997; 112: 1265–70. 36. Cotler SJ, Gretch DR, Bronner MP, Tateyama H, Emond MJ, dela Rosa C, et al. Hepatitis G virus co-infection does not alter the course of recurrent hepatiits C virus infection in liver transplantation recipients. Hepatology 1997; 26: 432– 6. 37. Dickson RC, Quian KP, Lau JY. High prevalence of GB virus-C/hepatitis G virus infection in liver transplant recipients. Transplantation 1997; 63: 1695–7. 38. Haagsma ER, Cuypers HTM, Gouw ASH, Sjerps MC, Huizenga JR, Slooff MJH, et al. High prevalence of hepatitis G virus after liver transplantation without apparent influence on long-term graft function. J Hepatol 1997; 26: 921–5. 39. Colombatto P, Randone A, Civitico G, Monti Gorin J, Dolci L, Medaina N, et al. Hepatitis G virus RNA in the serum of patients with elevated gamma glutamyl transpeptidase and alkaline phosphatase: a specific liver disease. J Viral Hepatitis 1996; 3: 301–6.
Copyright C European Association for the Study of the Liver 1998
Journal of Hepatology ISSN 0168-8278
HGV/GBV-C infection in liver transplant recipients: antibodies to the viral E2 envelope glycoprotein protect from de novo infection Enrico Silini2, Luca Belli1, Alberto B. Alberti1, Margherita Asti2, Antonella Cerino2, Morena Bissolati2, Gianfranco Rondinara3, Luciano De Carlis3, Domenico Forti3, Mario U. Mondelli2 and Gaetano Ideo1 2
Department of Pathology and Infectious Diseases, University of Pavia and IRCCS Policlinico S. Matteo, Pavia, and 1Department of Internal Medicine, Center for Liver Diseases and 3Abdominal Organs’ Transplantation Unit, Niguarda Ca’ Granda Hospital, Milan, Italy
Background/Aims: Liver transplantation for endstage liver cirrhosis provides a useful model to investigate the pathogenetic role of hepatotropic viral agents. Recently, a new member of the Flaviviridae family, provisionally named HGV/GBV-C virus, has been associated with acute and chronic non A–E hepatitis. We studied 136 patients with cirrhosis consecutively transplanted at our institution for evidence of hepatitis G virus infection and correlation with the patients’ clinical course. Methods: All patients survived for at least 6 months after transplantation (median follow-up 44 months) and underwent routine liver biopsies. Hepatitis G virus infection was studied using both direct viral RNA identification by RT-PCR and indirect detection of antibodies to the E2 glycoprotein. Results: There was a high frequency of the hepatitis G virus among patients undergoing liver transplantation, with HGV RNA and anti-E2 prevalence rates of 18.4% and 26.5%, respectively. HGV RNA preva-
lences significantly increased after transplantation (47.8%), with 47.3% rate of new infections in susceptible subjects. Anti-E2 antibodies were significantly more prevalent among patients transplanted for HCVrelated cirrhosis and represented a strong protective factor against hepatitis G virus reinfection or recurrent infection. No correlation was found between HGV RNA or anti-E2 prevalences and survival after transplantation or rates of recurrent liver damage. Conclusions: All available evidence suggests that, although liver transplant patients are heavily exposed to hepatitis G virus both before and after transplantation, hepatitis G virus does not induce liver disease in this setting. Most infections appear to be selflimited and induce a protective immunity which is marked by the presence of anti-E2 antibodies.
fulminant hepatic failure (FHF) and cryptogenic cirrhosis are relatively frequent indications for orthotopic liver transplantation (OLT) worldwide: in Italy they account for about 10% of all liver transplants (1). It is presumed that as yet unknown infectious agents may be implicated in the etiology of these conditions (2). This hypothesis is supported by evidence derived from experimental inoculations of primates with human sera from non A–E hepatitis cases, which have
demonstrated the existence of at least two separate filterable agents able to induce liver disease (3). Epidemiological population-based studies also indicate that about 20% of community-acquired non A, non B acute hepatitis (4,5), 3–9% of all chronic hepatitis (2,6,7) and over 90% of fulminant non A, non B hepatitis (8–10) cannot be referred to known causes. Chronic liver damage of unknown etiology can be observed among long-term survivors of liver transplantation (11,12), including over 70% of patients transplanted for seronegative FHF (13), suggesting the possible involvement of new or persistent infections of the liver graft. Recently, a novel member of the Flaviviridae family of positive-strand RNA viruses has been isolated by molecular cloning by two independent groups (14,15). The new virus has beeen provisionally designated
S
Received 17 October 1997; revised 22 January; accepted 9 February 1998
Correspondence: Gaetano Ideo. Divisione di Medicina Interna ed Epatologia ‘Crespi’, Ospedale di Niguarda Ca` Granda, Piazza dell’Ospedale Maggiore 3, 20162 Milan, Italy. Tel: 0039-2-64442396. Fax: 0039-2-64442788.
Key words: Anti-E2 antibodies; GBV-C; HGV; Liver transplant; Recurrent hepatitis.
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hepatitis G virus (HGV)/GB-C virus. HGV infection is distributed worldwide, is highly prevalent among individuals at risk for parenterally transmitted infections and is self-limited in most individuals (16,17), although long-term persistence is not infrequent (18). Circulating HGV RNA has occasionally been associated with acute and chronic liver disease (14) and FHF (19,20), but conclusive evidence implicating HGV in the etiology of non A–E hepatitis is still lacking (21– 23). We studied HGV infection in 136 liver transplant patients consecutively treated at our institution between 1990 and 1996, who survived for at least 6 months after transplantation (median follow-up time 44 months, range 7–88). The aims of the study were: (i) to investigate the prevalence of HGV infection using both direct viral identification by RT-PCR and indirect detection by antiE2 antibodies; (ii) to evaluate the incidence of de novo and persistent infection following transplantation; (iii) to correlate HGV infection prevalence with the etiology of cirrhosis; and (iv) to assess the role of HGV infection in the patients’ clinical course.
cal findings were scored and staged semi-quantitatively according to the criteria recommended by Desmet et al. (24). Informed consent to participate in the study was obtained from each patient, and the study protocol was approved by the ethical board of our insitution and conformed to the guidelines of the 1975 Declaration of Helsinki. Serological assays Patients’ sera were tested for anti-HCV by second-generation ELISA, according to the manufacturer’s instructions (Ortho Diagnostics System Inc. Raritan, NJ, USA) and in selected cases by second-generation immunoblot assay (RIBA 2, Chiron Corporation, Emeryville, CA, USA). Anti-HGV was detected in pre-transplant sera of all patients by a two-step sandwich ELISA (mPlate antiHGenv, Boehringer Mannheim, Germany), which uses as antigen a recombinant E2 envelope glycoprotein of HGV expressed in mammalian cells and captured in solid phase with a specific murine monoclonal antibody. Only repeat reactive sera were considered positive.
Materials and Methods Patients From August 1989 through January 1996, 185 patients with cirrhosis underwent orthotopic liver transplantation (OLT) at our institution. One hundred and fiftysix patients survived for more than 6 months after transplantation; paired pre- and post-transplant frozen sera were available for 136 of them (103 males, median age 49 years, range 23–61). These patients represented the population base of our study. Sixty-four patients had HCV-related liver cirrhosis, 25 had HBV-related cirrhosis, 18 had HDV superinfection, 10 patients were HCV-HBV coinfected, 14 had cryptogenic cirrhosis, 16 were alcoholics and seven had miscellaneous indications for transplantation (primary biliary cirrhosis: three patients, and primary sclerosing cholangitis, Budd-Chiari syndrome, Wilson’s disease and Caroli’s disease: one patient each). Demographic, epidemiological and biochemical data from all patients were retrieved from our files, including the regimen of immunosuppression, the number and type of blood transfusions performed and the detailed post-operative course. Patient follow-up was 7–88 months (median 44). Patients were regularly reviewed every 2 weeks until day 90, monthly until the sixth post-operative month, every 3 months until 1 year, and every 3–6 months thereafter. Percutaneous liver biopsies were performed whenever clinically indicated and at yearly intervals in all patients. Histologi-
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Virological methods Total RNA was extracted from 0.5 ml of serum stored frozen at ª70æC using a modification of the guanidinium acid-phenol method (25). RNA from 100 ml of serum was retrotranscribed and PCR-amplified using a nested-primer protocol. Two sets of 4 primers were used, one based on sequences from the helicase/NS3 gene, which are essentially a modification of previously described primers (15,19) and a second set based on conserved sequences of the 5ø non-coding region: 5ø non-coding, sense primer (1st round) 5ø GGC-CAA-AAG-GTG-GTG-GAT-GG 3ø 5ø non-coding, antisense primer (1st round) 5ø GTG-GGC-GTC-GTT-TGC-CCA-GG 3ø 5ø non-coding, sense primer (2nd round) 5ø TTG-GTA-GCC-ACT-ATA-GGT-GG 3ø 5ø non-coding, antisense primer (2nd round) 5ø GGT-AGG-ACC-AAC-ACC-TGT-GG 3ø. Selected amplicons were cloned in a T-tailed plasmid (TA cloningTM kit, Invitrogen Corp., San Diego, CA, USA) and sequenced using a thermostable DNA polymerase (CircumVentTM kit , New England Biolabs Inc., Beverly, MA, USA). HGV genotypes were determined by phylogenetic analysis of sequences of the 5ø noncoding region in 10 samples and in the remaining cases by a PCR assay which uses a set of type-specific primers of the same genomic region (E. Silini, unpublished results).
HGV infection in liver transplant patients
HCV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR), using conserved primers localized in the 5ø non-coding region of the viral genome (26). HCV genotyping was performed by PCR-amplification of core region sequences with universal and 5 subtype-specific primers according to Okamoto et al. (27,28), with a modified type 2a-specific primer, as previously described (26). HCV type distribution in this cohort has been previously described (29). Statistical methods Actuarial survival rates and rates of recurrent hepatitis were calculated using the Kaplan-Meier method; comparison of actuarial rates was made using the log-rank test. The chi-square test, t-test and analysis of variance were used as appropriate. P-values∞0.05 were considered statistically significant.
Results Three hundred and twenty-three serum samples derived from 136 patients were analyzed for the study. All patients were tested on at least two separate sera: one collected pre-transplantation and the other 6–12 months after grafting. HGV RNA detection was performed by nested RT-PCR of sequences from the helicase-NS3 gene region and confirmed in 85 samples by the use of a second set of primers from the 5ø noncoding region (5øNCR). The results obtained by the two detection systems showed a concordance of 93%. The reliability of HGV RNA detection was further assessed by cloning and sequencing 14 amplicons from the NS3 region and 10 amplicons from the 5øNCR. HGV genotypes were also determined in 50 cases; all HGV RNA sequences were classifiable as type 2a (30), which represents the most frequent HGV variant present in Italy and in the Western world. No differences were found as to the infecting HGV type with respect to other patient categories in the same geographic area
(E. Silini, work in preparation). Anti-E2 seroprevalence was determined in pre-transplantation sera of all patients, using a commercial two-step sandwich ELISA which uses as antigen a recombinant E2 viral glycoprotein expressed in mammalian cells. Prevalences of serum HGV RNA and anti-E2 antibodies in pre-transplantation sera are shown in Table 1. Overall, HGV RNA was found in 18.4% (25/136) of patients and anti-E2 seroreactivity in 26.5% (36/136); among the 36 anti-E2 positive patients, only a single subject was HGV RNA positive, as compared to 24 out of 100 patients in the anti-E2 negative group (p∞0.01). Sex, age at transplantation or previous history of blood transfusion did not differ between patients according to their anti-HGV or serum HGV RNA status. Conversely, significant differences in the prevalence of HGV infection were observed between patient categories (Table 2). Specifically, at transplantation the 64 patients with HCV-induced cirrhosis had higher rates of anti-E2 seropositivity (37.5% (24/64) vs 16.7% (12/72), p∞0.01) and lower rates of serum HGV RNA (12.5% (8/64) vs 23.6% (17/72), pΩns) with respect to the other groups of patients . Detection of serum HGV RNA markedly increased after transplantation among patients of all groups, with an overall prevalence of 47.8% (65/136) (Table 2). The risk of developing post-transplantation HGV viremia was significantly dependent on the anti-E2 status of the patient prior to transplantation, since only 4 of 36 (11%) anti-E2 positive subjects showed viremia compared to 61 out of 100 (61%) anti-E2 negative patients (p∞0.001). These findings are in agreement with previous observations, which also reported the presence of serum HGV RNA in about 10% of anti-E2 positive subjects (16). Accordingly, HCV-infected patients showed significantly lower rates of post-transplan-
TABLE 2 Presence of serum HGV RNA and anti-E2 antibodies in 136 liver transplant patients according to their indication for transplantation
TABLE 1 Presence of HGV RNA and anti-E2 antibodies in the pre- and postoperative sera of 136 liver transplant patients Anti-E2 antibodies
Serum HGV RNA Pre-OLT
Positive Negative Total
Total Post-OLT
Positive
Negative
Positive
Negative
1 24 25
35 76 111
4 61 65
32* 39 76
36 100 136
* Anti-E2 positive patients are significantly protected from the development of post-transplant HGV viremia (p∞0.01 by chi-square test). OLTΩorthotopic liver transplantation.
Indication for transplantation HGV RNA positive* (number of patients) Pre-OLT Post-OLT
Anti-E2 positive*
Cryptogenic cirrhosis (14) Alcoholic cirrhosis (16) HCV cirrhosis (64) HBV cirrhosis (25) HBV/HCV cirrhosis (10) Other (7)
3 (21) 3 (19) 24 (37)æ 4 (16) 2 (20) ª
4 4 8 4 3 2
(29) (25) (12) (16) (30) (29)
10 9 21 13 6 6
(71) (56) (33)æ (52) (60) (86)
* The percent of total in the group is shown in brackets. æ Anti-HCV positive patients show a significantly higher prevalence of anti-HGV (p∞0.001 by chi-square) compared with patients of other groups and, accordingly, they show a reduced incidence of posttransplant HGV viremia (p∞0.01). OLTΩorthotopic liver transplantation.
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Fig. 1. Kaplan-Meier analysis of actuarial survival in 136 liver transplant patients according to their anti-HGV (Fig. 1a) and HGV RNA (Fig. 1b) status. No significant differences in actuarial survival were observed (1a pΩ0.48; 1b pΩ0.74 log-rank test).
tation HGV viremia compared to patients in other groups (32.8% (21/64) vs 61.1% (44/72), p∞0.01). All 24 patients who were HGV RNA positive at transplantation continued to harbor circulating virus during the first year after graft. To assess the duration of posttransplant viremia in the long term, we also examined sera of 20 patients after 3 years of follow-up and we detected HGV RNA in 15 (75%) of them. Thirty-six of 76 (47.4%) subjects who were anti-E2 negative and HGV RNA negative before OLT developed HGV RNA viremia after transplantation. These presumably represent new infections, since no direct (HGV RNA) or indirect (anti-HGV) evidence of HGV was found in these patients. Anti-HCV positive cases had a lower risk of acquiring de novo HGV infections; in fact, 12 of 32 (37.5%) potentially at-risk HCVinfected patients (HGV RNA anti-HGV double negative at transplantation) developed post-OLT HGV viremia compared with 24 of 44 (54.5%) patients from other groups, although this difference did not reach statistical significance (Table 3). To estimate the transfusion-related risk of HGV infection, we evaluated the prevalence of serum HGV
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Fig. 2. Actuarial rates of hepatitis recurrence in 64 antiHCV positive liver transplant patients according to their anti-HGV (Fig. 2a) and HGV RNA (Fig. 2b) status. No significant differences in hepatitis recurrence rate were observed (1a pΩ0.48; 1b pΩ0.33 log-rank test).
RNA in 217 active donors from the local blood bank. Prevalences of 2.3% (5/217) and 6% (9/149) were found for serum HGV RNA and anti-E2 antibodies, respectively. The mean number of units of blood or blood derivates transfused per patients in the operative period was: 21 units of packed red cells, 26 units of fresh frozen plasma and 6 units of platelets. Therefore, the estimated cumulative risk of acquiring HGV in the peri-transplantation period was 1–0.97753Ω0.71, which is compatible with the figure actually observed. To evaluate whether HGV infection might affect the
TABLE 3 De novo HGV infection among 76 orthotopic liver transplantation patients who were anti-E2/HGV RNA negative in pre-transplant sera Indication for transplantation (no. of patients)
De novo infection
Exposed subjects
Cryptogenic cirrhosis (14) Alcoholic cirrhosis (16) HCV cirrhosis (64) HBV cirrhosis (25) HBV/HCV cirrhosis (10) Other (7)
5 5 12 8 2 4
8 9 32 17 5 5
HGV infection in liver transplant patients TABLE 4 Biochemical indices of liver damage in 29* patients with cirrhosis and HCV recurrence according to HGV RNA status after transplant Patients (no.)
Peak values AST ALT GGT Bilirubin
and only one had a concurrent post-transplantation HGV viremia.
Discussion
HGV RNA negative (21)
HGV RNA positive (8)
262∫348 (range 23–1400) 292∫250 (range 40–800) 120∫99 (range 12–300) 1.7∫0.9 (range 0.8–7)
146∫116 (range 26–310) 292∫237 (range 29–700) 153∫123 (range 30–370) 1.6∫1.0 (range 0.6–3.3)
At time of the first protocol biopsy AST 149∫128 (range 23–470) ALT 195∫107 (range 40–380) GGT 113∫98 (range 12–300) Bilirubin 1.7∫0.9 (range 0.8–4)
105∫103 (range 26–310) 247∫218 (range 29–632) 130∫124 (range 30–370) 1.4∫0.8 (range 0.6–2.7)
Mean follow-up values AST 145∫183 (range 22–610) ALT 153∫173 (range 14–590) GGT 71∫74 (range 10–240) Bilirubin 1.6∫1.7 (range 0.7–4)
56∫35 (range 26–106) 125∫174 (range 26–515) 81∫48 (range 19–156) 1.2∫0.8 (range 0.6–3)
* Five patients were excluded from the analysis because of intercurrent cholestasis due to mechanical obstruction to bile outflow. ALTΩ alanine aminotransferase; ASTΩaspartate aminotransferase; GGTΩ gamma glutamyltransferase.
outcome of OLT, we performed a survival analysis dividing patients according to their anti-E2 status and the presence of serum HGV RNA before or after transplantation (Fig. 1). No significant differences were observed considering the whole cohort or groups of patients with specific transplant indications (HCV infection, HBV infection, other indications). In particular, none of the 19 patients transplanted for cryptogenic or alcoholic cirrhosis, who were HGV RNA positive after transplantation, has shown so far evidence of recurrent liver damage (median follow-up time 39 months, range 8–74). Thirty-four of 64 (53.1%) subjects transplanted for HCV-related cirrhosis developed recurrent hepatitis C and in these patients HGV coinfection did not influence the frequency, the timing or the severity of HCV recurrence (Fig. 2). In addition, no correlations were found between the presence of HGV RNA after transplantation and the infecting HCV genotype or the mean (or peak) levels of ALT, AST, gGT or bilirubin during the entire follow-up period, at the time of the diagnosis of recurrent hepatitis or when the first protocol liver biopsy was performed (Table 4). Five patients with recurrent hepatitis C were excluded from the analysis of the results due to concurrent biliary complications. These five patients all required repeated biliary stenting to treat extrahepatic biliary strictures. Eight of the 34 patients with recurrent hepatitis C have progressed to stage 3–4 so far; two were anti-E2 positive
Liver transplantation provides an excellent model to investigate the possible pathogenetic relevance of new viral hepatitis agents in humans. It overcomes the limitations of cross-challenge studies in animals; these are often not relevant due to the differences in viral pathogenesis which can exist even between closely related species. Indeed, hepatitis virus infections almost invariably recur after transplantation and sometimes induce a more rapid and overt clinical course than in immunocompetent patients. In addition, in the liver transplant setting the natural history of recurrent infections can be accurately monitored, since dates of infection are known, subjects are followed up very closely, and collection of biological samples and repeated biopsies are readily available. We used the OLT model to study the clinical and pathological correlations of HGV infection in a large group of Italian patients who were transplanted for cirrhosis of different etiologies. Although previous studies have already addressed this issue (12,31–33), our work provides significant and novel information. Thus, to assess the prevalence and the incidence of HGV infection, we looked for both HGV RNA and anti-E2 antibody. This approach allowed us to distinguish between de novo and persistent infections and to evaluate more precisely the real number of HGV-exposed patients, which is largely underestimated by the use of HGV RNA detection alone. We analyzed consecutive patients with different indications for OLT and found significant differences in the distribution of HGV infection among patient categories. The study of subjects with long follow-up times (median 44 months) and the exclusion of patients who survived less than 6 months enabled us to assess more precisely the role of HGV infection in the long-term outcome and in the recurrence of HCV infection. Detailed molecular analysis was performed on paired pre- and post-OLT serum samples of all patients, including study of the possible role of viral heterogeneity in determining the severity of liver disease. Estimates of the prevalence of HGV viremia and anti-E2 positivity in a large number of sera from active blood donors from the same geographical area was also obtained to evaluate the risk of HGV infection associated with blood transfusion. Here we show that HGV infection is highly prevalent among patients undergoing OLT, with 45% cumulative infection rates as assessed by combined serum anti-E2 and HGV RNA detection. This finding is in agreement with all previous studies (12,31–33), although anti-
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HGV seroprevalence has never been reported before in this setting. Simultaneous evaluation of both anti-E2 and HGV RNA indicates that the number of exposures to HGV considerably exceeds that of active infections as assessed by the presence of circulating virus. HCVinfected cirrhotic patients are significantly more exposed to HGV than patients from other categories, and in 24 (75%) of 32 patients our findings indicate a past, probably resolved infection with circulating antiE2 and absence of serum HGV RNA. This finding is in agreement with most epidemiological studies published so far, which show a strong association of HCV and HGV infections in all patient categories. However, these findings also suggest that HGV infection is shortlived since most HCV positive patients had cleared HGV RNA. HGV infection invariably recurs after OLT and most patients both with de novo and persistent infections harbor viral sequences for a long time after transplantation. Furthermore, the risk of being infected by HGV in the peri-operative period is very high, since about half of the potentially at-risk patients (i.e. HGV RNA and anti-E2 double-negative subjects at transplantation), develop serum HGV RNA during the first year of follow-up. The risk of acquiring new HGV infections is significantly dependent on the presence of anti-E2 antibodies at transplantation. Indeed, our study provides the first formal prospective demonstration that anti-E2 antibodies are protective against HGV reinfection. This finding may explain the counterintuitive observation that HCV-naive patients are more prone to develop HGV infection following transplantation than HCVinfected patients, who, having been exposed to HGV in the past, have had time to develop a protective immune response. Similar observations were made by previous authors who were not able to offer a plausible explanation for this finding (31–33). Berg and co-authors (32) suggested that HGV infection might have been transmitted through anti-HBsAg immunoglobulin used for prophylaxis of HBV recurrence, but this conclusion seems unwarranted in the light of the present results. It is also interesting to note that the risk of acquiring de novo HGV infection is lower in HCV patients irrespective of their anti-E2 status than in patients from other groups. This might be explained either by the presence of anti-E2 antibodies with different specificity and/or affinity for the E2 protein utilized as antigen in the test or by the existence of protective antibodies directed against other HGV proteins or, possibly, by the interference of HCV in HGV replication. It is reasonable to believe that the high risk of ac-
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quiring HGV infection in the peri-transplant period is due to the heavy exposure of liver transplant patients to blood and blood derivates, the mean number of blood units received by the patients in our cohort in the peri-operative period being 53. A significant, but presumably small risk might also derive from the donor liver (12), but this could not be ascertained in our study. Nonetheless, the risk estimate of acquiring HGV during transplantation, which can be inferred by the frequency of de novo infections (0.47), is comparable to the figures that can be extrapolated from the prevalence of HGV viremia among blood donors (0.71), as also demonstated by other authors (12,33). This indicates that viral infectivity is high, and it also provides further evidence that HGV is easily transmitted by transfusion. Since anti-E2 antibodies were frequently found among blood donors (6–7%), it is highly likely that most patients received these antibodies with transfusions. Passive immunity by transfer of anti-E2, however, seems to play a minor role, if any, in protecting against primary HGV infection, given the high proportion of new infections among HGV-naive patients. This might be explained either by the low titers of the transfused antibodies or by differences in antibody specificity among individuals. In addition, it should be considered that, in analogy with other hepatitis virus models, other anti-viral mechanisms, such as T-cytotoxic responses, might be relevant in the clearance of or in the protection against HGV infection. A possible explanation for the high carrier rate, such as that observed in this and previous studies among otherwise ‘‘healthy subjects’’ in the general population, would be that HGV is efficiently transmitted via non parenteral routes. Evidence in favor of efficient vertical transmission of HGV has already been gathered (34), and there is also evidence suggesting possible sexual transmission (35). HGV infection, both de novo and persistent, is not associated with clinical or pathological signs of damage of the liver graft and does not influence overall survival (Fig. 1). No patient with HGV infection alone has shown recurrence of liver disease up to 6 years post-transplantation. In the larger group of HCV patients, HGV coinfection does not influence the timing, the frequency or the clinical course of recurrent hepatitis C (Fig. 2). These findings are in agreement with all previous studies from different cohorts (12,31– 33,36–38). It has been suggested that HGV infection might be preferentially associated with bile duct damage (39); however, in our 34 patients with recurrent hepatitis C, we did not observe a cholestatic picture associated with HGV coinfection. Finally, HGV se-
HGV infection in liver transplant patients
quence variants present among patients with cirrhosis of different etiologies are similar, indicating that viral heterogeneity plays no role in determining the possible clinical features of the infection (30). In conclusion, all the available evidence suggests that HGV does not induce liver disease in the setting of orthotopic liver transplantation. It is indeed possible that the liver is not the primary target organ of this peculiar human virus.
13.
14.
15.
Acknowledgements This study was supported in part by grants no. 030RFM93/01 and 240RFM96/01 from the Italian Ministry of Health to IRCCS Policlinico San Matteo. We also gratefully acknowledge the financial support of the Italian Red Cross, Committee of Ponte San Pietro, Bergamo, Italy.
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