Hepatitis After Liver Transplantation: The Role of the Known and Unknown Viruses

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1 Hepatitis After Liver Transplantation: The Role of the Known and Unknown Viruses Mario G. Pessoa,*00 Norah A. Terrault,*00 Linda D. Ferrell, Jill Detmer, Janice Kolberg, Mark L. Collins, Maurene Viele, John R. Lake, John P. Roberts, Nancy L. Ascher, and Teresa L. Wright*00 This study was designed to determine the cause of posttransplantation hepatitis in patients undergoing transplantation for liver disease of nonviral cause; the role of acquired hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis G virus (HGV) in posttransplantation hepatitis; and the course of posttransplantation hepatitis of unknown cause. Two hundred forty-three patients underwent transplantation for nonviral liver diseases (mean age, 48 years; 103 men, 140 women). Serological and virological assays for HBV and HCV were performed pretransplantation to exclude preexisting infection and posttransplantation to investigate the cause of posttransplantation hepatitis. Histology was graded on all available biopsy specimens; posttransplantation hepatitis was assessable in 150 patients. Posttransplantation hepatitis was present in 29% (44 of 150) of the patients after a median follow-up of 47 months (range, 1 to 101 months). Actuarial survival was significantly lower in patients with posttransplantation hepatitis compared with patients without (71% v 89% at 5-year follow-up; P.03). HCV and HBV were identified posttransplantation in 14% and 9% of patients with hepatitis, respectively. After the exclusion of HCV and HBV infection, 22% (33 of 150) of the patients had posttransplantation hepatitis of unknown cause. HGV was present in 58% of these patients, but HGV was equally prevalent in patients without posttransplantation hepatitis. When patients with HBV and HCV were excluded, there was no difference in survival between patients with posttransplantation hepatitis compared with patients without (P.08, log-rank test). Posttransplantation hepatitis was present in approximately 30% of the patients undergoing transplantation for nonviral diseases, with a median follow-up of 47 months. Known hepatitis viruses (HBV, HCV) were present in one fourth of the patients with posttransplantation hepatitis; 22% (33 of 150) of the patients had hepatitis of unknown cause, suggesting that other, as yet undiscovered, hepatitis viruses may exist. Copyright 1998 by the American Association for the Study of Liver Diseases A significant number of patients develop liver disease after liver transplantation. This may represent a recurrence of the disease process that was present before liver transplantation, as with primary biliary cirrhosis, hepatitis B virus (HBV), and hepatitis C virus (HCV), or a de novo process acquired after transplantation, such as HBV- or HCV-induced liver disease. In a study performed before the routine screening of organ donors and blood products for HCV, our group had shown that 17 of 89 patients (19%) with pretransplantation diagnoses of alcoholic liver disease or cryptogenic cirrhosis acquired HCV infection during liver transplantation. 1 A more recent study addressed the issue of de novo HBV and HCV infection after liver transplantation using serology and viral detection by polymerase chain reaction and found that 2.5% of liver transplant recipients acquired HBV infection and 7.4% acquired HCV infection. 2 The transmission of viruses has been well documented in the transfusion setting. For example, previous studies have shown that HCV infection was responsible for the majority of transfusionassociated hepatitis. 3 However, it is noteworthy that in 10% of transfusion-associated hepatitis, no known viruses have been identified. 4 In a series of 357 blood transfusion recipients, acute posttransfusion hepatitis occurred in 79 patients. HCV infection was shown in 63 patients (80%), but hepatitis remained unexplained in a proportion. 5 From the Departments of *Medicine, Pathology, Laboratory Medicine, and Surgery, 00Veterans Affairs Medical Center and University of California, San Francisco; and the Chiron Corp, Emeryville, CA. T.L.W. is supported by grant no. R29AI32242 from the National Institutes of Health, a Veterans Administration Merit Review, and by grant no. DK from the National Institute of Diabetes and Digestive and Kidney Diseases (Liver Center). N.A.T. is supported by the Hepatitis Foundation International. Address reprint requests to Teresa L. Wright, MD, Gastroenterology Unit, 111B, Veterans Affairs Medical Center, 4150 Clement St, San Francisco, CA Copyright 1998 by the American Association for the Study of Liver Diseases /98/ $3.00/0 Liver Transplantation and Surgery, Vol 4, No 6 (November), 1998: pp

2 462 Pessoa et al This study examines the presence and severity of posttransplantation liver disease in patients undergoing liver transplantation for chronic liver disease of nonviral cause and investigates the relationship between viruses known to be associated with chronic hepatitis (HBV, HCV) and the clinical course and severity of posttransplantation liver disease. Patients and Methods Study Population Between February 1988 and December 1995, 624 adult patients underwent liver transplantation at the University of California at San Francisco. Two hundred fiftynine patients (41.5%) had a diagnosis of end-stage liver disease related to viral hepatitis; 215 patients (34.4%) with HCV, 33 patients (5.3%) with HBV, 7 patients (1.1%) with HBV/hepatitis D virus (HDV), 2 patients (0.3%) with HBV/HCV, and 2 patients (0.3%) with HBV/HDV/HCV. Fifty-four patients (8.6%) had a diagnosis of fulminant or subfulminant hepatitis. The remaining 311 patients had a diagnosis of cirrhosis unassociated with known viral causes and are the focus of this study. Serum samples were available posttransplantation in 243 of the 311 patients (78.1%). The 68 patients who lacked serum samples to evaluate for the presence of viral infections posttransplantation were excluded. These 68 patients had the following diagnoses: alcoholic liver disease (n 11), primary biliary cirrhosis (n 8), primary sclerosing cholangitis (n 8), cryptogenic cirrhosis (n 8), autoimmune hepatitis (n 5), alpha 1 - antitrypsin (n 2), Budd-Chiari syndrome (n 1), and others (n 25; Table 1). Clinical data were obtained from reviewing the patients charts. The clinical characteristics assessed were age, sex, ethnicity, date of liver transplantation, time to re-transplantation, and time to death or last follow-up. Ethnicity was classified into five categories: white, Hispanic, black, Asian, and other. Biochemical data included alanine (ALT) and aspartate (AST) aminotransferase and total bilirubin levels, measured within 15 days of the liver biopsy. All serum samples were stored at 70 C after being aliquoted into 1.5-mL eppendorf tubes. All patients gave consent under a protocol approved by the Institutional Review Board of the University of California, San Francisco. Serological Assays Hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-hbc), and anti-hcv (enzymelinked immunoassay [EIA]-1 or EIA-2) were tested in all patients before and after liver transplantation using EIAs (Abbott Diagnostics, North Chicago, IL). Virological Methods HCV detection. After extraction with proteinase K/phenol-chloroform from 50 µl of sera, complementary DNA was generated from HCV RNA using methods previously described. 1 All pretransplantation and posttransplantation serum samples were tested for HCV RNA. Hepatitis G virus (HGV) detection and quantitation. Polymerase chain reaction amplification was performed as previously described. 6 The research-based HGV branched DNA assay has a provisional limit of detection of approximately 50,000 Eq/mL, based on dilutions of a 700-nucleotide synthetic HGV RNA transcript. The methods were previously described. 7 Levels of viremia were measured in serum samples collected before and after liver transplantation (mean, 19.3 months after transplantation; range, 1 to 80 months). Histological Assessment Protocol liver biopsies were performed weekly in the early period after transplantation and annually thereafter in the majority of patients. Additional liver biopsies were performed when liver tests became abnormal. Biopsy specimens were stained with hematoxylin and eosin and reviewed by a single pathologist who was blinded to the virological status of the patient. Serial biopsies performed after transplantation were evaluated, and a histological score was assigned to patients with evidence of hepatitis on each biopsy. The severity of posttransplantation hepatitis was evaluated on the last liver biopsy available. Biopsy specimens showing conditions that prevented assessment of hepatitis, such as acute and chronic rejection or moderate to severe fatty change, or those with definitive criteria for other recurrent conditions, such as primary biliary cirrhosis or primary sclerosing cholangitis, were excluded from the assessment of posttransplantation hepatitis. To be classified as having chronic posttransplantation hepatitis, histological hepatitis had to be present on at least two liver biopsies during a 6-month interval. Patients with transient inflammation were not included in the posttransplantation hepatitis group. The amount of portal/periportal activity, lobular activity, fibrosis, and cytopathic swelling was quantified on a scale of 0 to 4 for each criterion (0, absent; 1, minimal; 2, mild; 3, moderate; and 4, severe). 8 The maximum score was 16. In the presence of chronic hepatitis of unknown cause, special stains for HBsAg and HBC antigen in the liver were performed. Statistical Analyses Values are expressed as median, mean, percentage, and range, as appropriate. The chi-squared test was used to compare the presence of posttransplantation hepatitis between the different groups. The Mann-Whitney U test was used to compare groups on continuous variables,

3 Hepatitis After OLT 463 Table 1. Posttransplantation Hepatitis in Liver Transplant Recipients of Nonviral Causes Pre-OLT Diagnosis No. No. Excluded for Lack of Serum No. Excluded for F/U 30 Days No. Included Histological Post-OLT Diagnosis Excluded Histological Post-OLT Diagnosis Included ALD Acute rejection 9 NL 6 Biliary obstruction 2 NS 19 Cholestasis 2 CH 5 CMV 1 Fatty change 10 PBC Acute rejection 4 NL 4 Chronic rejection 3 NS 13 CMV 1 CH 7 Fatty change 2 Recurrent PBC 12 CC Acute rejection 2 NL 5 Chronic rejection 2 NS 17 Fatty change 2 CH 15 FK 506 toxicity 1 LPD 1 PSC Acute rejection 3 NL 5 Biliary obstruction 1 NS 9 Chronic rejection 1 CH 6 Fatty change 2 FK 506 toxicity 1 Recurrent PSC 3 AICH Acute rejection 6 NL 4 Fatty change 1 NS 6 Recurrent AICH 1 CH 5 Hemoch Fatty change 3 NL 0 NS 2 CH 1 A1AT Fatty change 1 NL 1 NS 3 CH 1 BD-CH NL 1 NS 2 CH 0 Others Acute rejection 1 NL 2 Chronic rejection 2 NS 7 CH 4 Total Abbreviations: PSC, primary sclerosing cholangitis; AICH, autoimmune chronic hepatitis; Hemoch, hemochromatosis; A1AT, 1 -antitrypsin; BD-CH, Budd-Chiari syndrome; LPD, lymphoproliferative disease; NL, normal; NS, nonspecific; CH, chronic hepatitis; OLT, orthotopic liver transplantation; F/U, follow-up; ALD, alcoholic liver disease; PBC, primary biliary cirrhosis; CC, cryptogenic cirrhosis; CMV, cytomegalovirus. including ALT, AST, and total bilirubin levels; histological score; duration of follow-up; and duration of histological follow-up. The log-rank test was used to compare actuarial survival of patients with and without posttransplantation hepatitis. P less than.05 was considered statistically significant. Results Characteristics of the Study Population Two hundred forty-three patients with cirrhosis of nonviral cause referred for liver transplantation were followed up for a median of 47 months

4 464 Pessoa et al (range, 1 to 101 months). The diagnoses before liver transplantation were: 58 patients (23.8%), alcoholic liver disease; 47 patients (19.3%), primary biliary cirrhosis; 45 patients (18.5%), cryptogenic cirrhosis; 33 patients (13.6%), primary sclerosing cholangitis; 24 patients (9.9%), autoimmune chronic hepatitis; and 36 patients (14.8%) classified as other. The diagnoses in this miscellaneous category were hemochromatosis (n 7), alpha 1 - antitrypsin (n 6), Budd-Chiari syndrome (n 5), Wilson s disease (n 3), secondary biliary cirrhosis (n 3), amyloidosis (n 3), fibrolamellar carcinoma (n 1), cholangiocarcinoma (n 1), hepatocellular carcinoma (n 1), glycogen storage disease (n 1), polycystic liver (n 1), abetalipoproteinemia (n 1), oxalosis (n 1), portal thrombosis with hepatopulmonary syndrome (n 1), and liver abscess (n 1). There were 103 men (42.4%) and 140 women (57.6%), with a mean age at the time of transplantation of 48 years (range, 13 to 71 years). The majority of patients were white (72%); 15% were Hispanic, 4% were black, 4% were Asian, and 5% were other ethnicities. Prevalence of Posttransplantation Hepatitis To evaluate the presence and cause of posttransplantation hepatitis in patients who underwent transplantation for nonviral causes, serial biopsies from 230 patients with a follow-up of at least 1 month were reviewed. In 80 patients, the presence of specific nonhepatitis histological conditions in the last follow-up biopsy prevented assessment for posttransplantation hepatitis (Table 1). In the remaining 150 patients, 44 patients (29.3%) met the histological criteria for chronic hepatitis (Table 1). Among the patients with posttransplantation hepatitis, 3 patients were HCV RNA positive, 1 patient was HBsAg and HCV RNA positive, 19 patients were HGV RNA positive, 3 patients were HGV RNA and HCV RNA positive, and 4 patients were HGV RNA and HBsAg positive. Fourteen patients had negative tests for all three viruses (designated as Non A through G [NA-G] hepatitis; Table 2). Among the 106 patients without posttransplantation hepatitis, 2 patients were HCV RNA positive, 1 patient was HBsAg positive, 1 patient was HCV RNA and HGV RNA positive, 50 patients were HGV RNA positive, and 53 patients were negative for all three viruses. Posttransplantation hepatitis was significantly associated with HBV or HCV infection (P.001) but not with HGV infection (P.17). HGV Infection in Patients Who Underwent Transplantation for Nonviral Causes The prevalence of HGV RNA after liver transplantation was 52.3% (127 of 243 patients). Thirty-three patients had histological findings of chronic hepatitis not related to HBV or HCV. The proportion of patients with hepatitis who were HGV positive (24%; 19 of 78 patients) was not significantly different from the proportion of NA-G patients (19%; 14 of 72 patients) with hepatitis. The median histological scores were also similar in HGVpositive compared with NA-G patients (4.0 [range, 2 to 14] and 3.5 [range, 2 to 8], respectively; P.47). Overall, the total histological scores were low. There was no difference in ALT, AST, or bilirubin levels in HGV-positive compared with NA-G patients. The duration of follow-up was Table 2. Characteristics of Patients With Posttransplantation Hepatitis NA-G HGV Alone HGV, HCV HCV Alone HGV, HBV HBV, HCV PTH (no.) HGV viremia (Eq/mL, median) Histological score Median Range Duration of histological follow-up (mo) ALT ( ULN) Total bilirubin ( ULN) Abbreviations: PTH, patients with posttransplantation hepatitis; ULN, upper limits of normal.

5 Hepatitis After OLT 465 similar in the HGV-positive versus the NA-G group (47 months and 44 months, respectively). Graft and patient survival were not statistically different between HGV-positive and NA-G patients (82% v 89%; P.26; Table 3). Clinical Characteristics of Patients With Posttransplantation Hepatitis Because patients with HGV did not differ in any significant way from patients with NA-G hepatitis, further clinical analysis of posttransplantation hepatitis of unknown cause combined the HGV-positive and NA-G groups into a single group designated hepatitis NA-E. Thirty-three patients with chronic hepatitis after liver transplantation were in the NA-E group and 11 patients had acquired HBV or HCV. The NA-E patients had a median of nine posttransplantation liver biopsies performed (range, 3 to 25). The acquired HBV/HCV patients had a median of 10 posttransplantation liver biopsies (range, 8 to 19). The median histological follow-up was 33 months (range, 3.5 to 87 months) in the NA-E group compared with a median follow-up of 27.5 months (range, 3 to 89 months) in the acquired HBV/HCV group (P.82). Sixteen patients (48%) in the NA-E group had mild chronic hepatitis at their last biopsy, 3 of them with a prominent lobular component. Thirteen patients (39%) had moderate chronic hepatitis without bridging fibrosis, and 4 patients (12%) had chronic active hepatitis with bridging fibrosis. The median time from transplantation to first diagnosis of hepatitis was 12 months (range, 2 to 81 months) in the NA-E group, and 21.5 months (range, 4 to 36 months) in the group with acquired HBV or HCV (P.65). Elevated liver enzyme levels during posttransplantation follow-up prompted the performance of the liver biopsies in all patients. Figures 1 and 2 compare median ALT and total bilirubin values of patients with NA-E hepatitis, patients with acquired HBV/HCV, and patients without hepatitis. Only those patients with acquired HBV or HCV had median ALT values that were persistently greater than the normal range. Patients with NA-E hepatitis had median ALT values that were within the normal range for the first 60 months of follow-up, but with abnormal values after this time. Total bilirubin levels were greater than the normal range in the acquired HBV/HCV group during the first 12 months posttransplantation but decreased to within the normal range in subsequent follow-up. Elevations in total bilirubin levels in patients with acquired HBV/HCV and NA-E hepatitis occurred in the few patients with extended periods of follow-up ( 84 months). Actuarial survival was significantly lower in patients with posttransplantation hepatitis compared with patients without posttransplantation hepatitis (71% v 89% at 5 years, respectively; P.03; Fig. 3). However, when patients with HBV- and HCV-associated hepatitis were removed from the posttransplantation hepatitis group (n 13), survival in patients with and without hepatitis was not significantly different (P.08; Fig. 4). Discussion Infection of the grafted liver with HBV or HCV infection occurs almost universally within the first weeks after transplantation in patients with pretransplantation infection in the absence of prophylactic therapy. De novo acquisition of viral infec- Table 3. Characteristics of HGV RNA Positive and HGV RNA Negative Patients HGV RNA Positive HGV RNA Negative P Total tested (n 150) Total with hepatitis,* no. (%) 19 (24) 14 (19).14 Total histological score (range) 4.0 (2-14) 3.5 (2-8).47 Median duration follow-up (mo) Median donor exposure Actuarial 5-year survival (%) *HCV and HBV co-infected patients excluded. Chi-squared test. Possible histological score, 0 to 16. Mann-Whitney U test.

6 466 Pessoa et al Figure 1. Median ALT levels in patients with NA-E hepatitis ( z ), patients with acquired HBV/ HCV ( s ), and patients without hepatitis ( ). tion, although much less common, may also occur. Both situations are frequently associated with serious liver disease in the graft. This study highlights the frequency of posttransplantation hepatitis in patients undergoing liver transplantation for nonviral causes. With a median follow-up of almost 4 years, we observed that 29% of the patients had hepatitis after transplantation. Acquired HBV or HCV infection was present in 7% of the patients, whereas the remaining 22% had hepatitis of unclear cause. An initial series from our center found a rate of HCV acquisition of 35%. 1 This was drawn from a population of patients who underwent transplantation between 1988 and 1991, thus including many patients who underwent transplantation before the availability of anti-hcv screening procedures for blood and organs. By contrast, the rate of HCV acquisition in this study that included patients who underwent transplantation between 1988 and 1995 was only 4%. Among the 7 patients in our current series with posttransplantation-acquired HCV infec- Figure 2. Median total bilirubin levels in patients with NA-E hepatitis ( z ), patients with acquired HBV/HCV ( s ), and patients without hepatitis ( ).

7 Hepatitis After OLT 467 Figure 3. Patient survival (in months) was significantly lower in patients with posttransplantation hepatitis (n 44; ) than in patients without posttransplantation hepatitis (n 106; ; P.03, log-rank test). tion, 6 of 7 patients underwent transplantation between 1988 and 1991, and only 1 of 7 patients underwent transplantation between 1992 and The substantial decrease in the frequency of HCV acquisition after transplantation since 1991 reflects the effectiveness of donor-screening procedures. To date, no consistent relationship between HGV and acute or chronic liver disease has been shown. 5,9 Although HGV was prevalent among transplant recipients (52% were HGV RNA positive after transplantation), it was not associated with the presence of posttransplantation liver disease. This is consistent with other reports of HGV in transplant recipients. 6,7,10 Additionally, among the patients with chronic posttransplantation hepatitis of unknown cause (non-b, non-c), the histological disease was generally mild. However, 12% of these patients developed cirrhosis in as few as 37 months. Thus, other viruses yet to be identified may be responsible for the 33 patients with posttransplantation hepatitis of unknown cause, including the 19 patients with HGV infection. Previous studies have shown that nonviral liver diseases, such as primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis, may recur after transplantation Early lesions related to these recurrent diseases may be relatively nonspecific and their histological features overlap with those of viral hepatitis. In our study, 5 patients with pretransplantation diagnoses of primary biliary cirrhosis and primary sclerosing cholangitis had a histological picture more consistent with posttransplantation hepatitis than with recurrence of the pretransplantation condition in their initial Figure 4. When patients with HBV and HCV infection are excluded, patient survival (in months) in patients with posttransplantation hepatitis (n 34; ) was not significantly different from patients without posttransplantation hepatitis (n 103; ; P.08, log-rank test).

8 468 Pessoa et al biopsies. However, continued follow-up of these patients will be required to exclude evolution of these early hepatitis-like lesions into other histological conditions. Overall, patients with posttransplantation hepatitis had a lower survival than patients without posttransplantation hepatitis, although this was in large part accounted for by the presence of HBV and/or HCV infection. The survival of patients with posttransplantation HBV infection may be improved with the availability of specific nucleoside analogues, such as lamivudine. 14 However, no similar agents are available for the treatment of posttransplantation HCV infection. For the group of patients with posttransplantation hepatitis unassociated with HBV and HCV, the long-term survival was not significantly different from patients without posttransplantation hepatitis, although a trend (P.06) toward decreased survival was seen. This finding suggests that if new viruses are implicated in this syndrome, their pathogenicity is low. Further longitudinal study of these patients will be necessary to determine the full consequences of posttransplantation hepatitis. References 1. Wright T, Donegan E, Hsu H, Ferrell L, Lake J, Kim M, et al. Recurrent and acquired hepatitis C viral infection in liver transplant recipients. Gastroenterology 1992;103: Cavallari A, De Raffele E, Bellusci R, Miniero R, Vivarelli M, Galli S, et al. De novo hepatitis B and C viral infection after liver transplantation. World J Surg 1997;21: Alter H, Purcell R, Shih J, Melpolder J, Houghton M, Choo Q, et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-a, non-b hepatitis. N Engl J Med 1989;321: Alter H, Jett B, Polito A. Analysis of the role of hepatitis C virus in transfusion-associated hepatitis. In: Viral hepatitis and liver disease. Hollinger FB, Morgolis H (eds). Baltimore: Williams & Wilkins, 1991: Alter H, Nakatsuji Y, Melpolder J, Wages J, Wesley R, Shih J, et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. N Engl J Med 1997;336: Berenguer M, Terrault NA, Piatak M, Yun A, Kim JP, Lau J, et al. Hepatitis G virus infection in patients with hepatitis C virus infection undergoing liver transplantation. Gastroenterology 1996;111: Pessoa MG, Terrault NA, Ferrell LD, Kim JP, Kolberg J, Detmer J, et al. Hepatitis G virus (HGV) in patients with cryptogenic liver disease undergoing liver transplantation. Hepatology 1997;25: Scheuer P. Classification of chronic viral hepatitis: A need for reassessment. J Hepatol 1991;13: Alter M, Gallagher M, Morris T, Moyer L, Meeks E, Krawczynski K, et al. Acute non A-E hepatitis in the United States and the role of hepatitis G virus infection. N Engl J Med 1997;336: Hoofnagle J, Lombardero M, Wei Y, Everhart J, Wiesner R, Zetterman R, et al. Hepatitis G virus infection before and after liver transplantation. Liver Transplant Surg 1997;3:S78-S Hubscher S, Elias E, Buckels J, Mayer A, McMaster P, Neuberger J. Primary biliary cirrhosis. Histological evidence of disease recurrence after liver transplantation. J Hepatol 1993;18: Harrison R, Davies M, Neuberger J, Hubscher S. Fibrous and obliterative cholangitis in liver grafts: Evidence of recurrent primary sclerosing cholangitis. Hepatology 1994;20: Pappo O, Ramos H, Starzl T, Demetris A. Structural integrity and identification of causes of liver allograft dysfunction occurring more than 5 years after transplantation. Surg Pathol 1995;19: Perrillo R, Rakela J, Martin P, Levy G, Schiff E, Wright T, et al. Lamivudine for hepatitis B following liver transplantation. Hepatology 1996;24:A223.