- Email: [email protected]
Hepatitis E Is a Rare Finding in Liver Transplant Patients With Chronic Elevated Liver Enzymes and Biopsy-Proven Acute Rejection
Hepatitis E Is a Rare Finding in Liver Transplant Patients With Chronic Elevated Liver Enzymes and Biopsy-Proven Acute Rejection F. Darsteina,*, F. Häuserb, J. Mittlerc, A. Zimmermanna, A. Lautemc, M. Hoppe-Lotichiusc, G. Ottoc, H. Langc, P.R. Gallea, and T. Zimmermanna a First Department of Internal Medicine, Gastroenterology and Hepatology, Universitätsmedizin Mainz, Mainz, Germany; bInstitute for Clinical Chemistry and Laboratory Medicine, Universitätsmedizin Mainz, Mainz, Germany; and cDepartment of Hepatobiliary and Transplantation Surgery, Universitätsmedizin Mainz, Mainz, Germany
ABSTRACT Background. In past decades, liver transplant (LT) patients were not routinely screened for hepatitis E virus (HEV) infection, and thus it might have been misdiagnosed as an acute rejection episode. Our aim was to analyze a real-world cohort of LT patients who presented with at least 1 episode of biopsy-proven acute rejection (BPAR) and suffered from persistent elevated transaminases, to evaluate the frequency of HEV infection misdiagnosed as a rejection episode. Methods. Data from 306 patients transplanted between 1997 and 2017, including 565 liver biopsies, were analyzed. Biopsies from patients suffering from hepatitis C (n ¼ 79; 25.8%) and from patients who presented with a Rejection Activity Index <5 (n ¼ 134; 43.8%) were excluded. A subgroup of 74 patients (with 134 BPAR) with persistently elevated liver enzymes was chosen for further HEV testing. Results. Positive HEV IgG was detectable in 18 of 73 patients (24.7%). Positive HEV RNA was diagnosed in 3 of 73 patients with BPAR (4.1%). Patients with HEV infection showed no difference in etiology of the liver disease, type of immunosuppression, or median Rejection Activity Index. Conclusion. Few HEV infections were misdiagnosed as acute rejection episodes in this real-world cohort. Thus, HEV infection is an infrequent diagnosis in cases with persistent elevated liver enzymes and BPAR after LT.
E
LEVATED liver enzymes following liver transplantation (LT) are a frequent finding. The elevation might be caused by harmless or severe diseases, with possible liver damage or loss of the transplanted liver [1]. A common reason for increased liver enzymes is an episode of biopsy-proven acute cellular rejection (BPAR) [2]. However, many pathologies can be responsible for elevated liver enzymes after LT, including recurrence of the underlying disease, viral infections, vascular or biliary complications, and others [3,4]. In recent years, hepatitis E virus (HEV) infection has been reported more frequently in patients after LT [5]. However, the true incidence of HEV infection in LT recipients is still unclear [6]. A potential risk factor for HEV infection in LT recipients might be transfusion, especially as blood concentrates have not been tested regularly for HEV in the ª 2020 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, XX, 1e6 (2020)
past [7]. It might be difficult to distinguish HEV infection from BPAR in liver biopsies. Therefore, LT recipients with HEV infection might have been misdiagnosed as suffering from acute cellular rejection [8,9]. HEV infection can present as chronic hepatitis responsible for liver cirrhosis and organ loss [10,11]. Limited data are available concerning the true rate of post-LT HEV infections that become chronic and cause recurrent cirrhosis.
*Address correspondence to Felix Darstein, First Department of Internal Medicine, Gastroenterology and Hepatology, University Medical Center Mainz, Langenbeckstraße 1, 55131 Mainz, Germany. Tel: (0049) 06131 170. E-mail: felix.darstein@ unimedizin-mainz.de 0041-1345/20 https://doi.org/10.1016/j.transproceed.2020.01.011
1
2
Ribavirin is an effective treatment option for HEV [12]. As ribavirin has been frequently used to treat LT recipients with hepatitis C virus (HCV) reinfection, HCV-positive patients might have been cured from HEV while treating their HCV infection. In the past, many LT recipients presented with BPAR and persistent elevated liver enzymes despite treatment with steroids (with complications such as organ loss and death). HEV infection could have been the reason for those chronic steroid-refractory hepatic injuries. The aim of our study was to determine the burden of HEV infection in LT patients with BPAR and persistent elevated liver enzymes in a real-world cohort and to determine the rate of HEV infections misdiagnosed as BPAR between 1997 and 2017. PATIENTS AND METHODS All patients were selected from an administrative transplant database, and all data were retrieved from patients’ charts and reports. The selection process is presented in Fig 1. To determine the burden of chronic HEV infection in LT patients with BPAR and persistent elevated liver enzymes, inclusion and exclusion criteria were as follows: BPAR was diagnosed according to the recommendations of the BANFF working group [13]. Clinically relevant liver damage was presumed when patients underwent at least 2 liver biopsies with at least 1 Rejection Activity Index (RAI) score 5 due to persistent elevation of liver enzymes. Patients who did not meet these criteria were excluded from our analysis. Since HCV infection might cause a bias because of the difficult histologic differentiation between BPAR and HCV reinfection and because of the unintended treatment of HEV during ribavirin therapy for HCV infection, HCV-positive patients (n ¼ 79) were excluded [14]. Finally, 74 patients met the inclusion criteria. Blood samples for the detection of HEV (IgM, IgG, and RNA) were initially taken at the time of liver biopsy and were available from 73 of 74 patients (Fig 1). A frequent immunosuppressive regimen consisted of the combination of a calcineurin inhibitor (cyclosporine or tacrolimus) with mycophenolate mofetil. During the first year, target trough levels were 5 to 7 ng/mL for tacrolimus and 3 to 5 ng/mL after 1 year. Cyclosporine target trough levels during the first year were 70 to 90 ng/mL and 50 to 70 ng/mL thereafter. Patients were treated with steroids in the first 3 to 6 months after LT. Methylprednisolone was reduced from 1.5 mg/kg on day 1 and 2 after LT to 1.0 mg/kg on days 3 and 4, 0.5 mg/kg on days 5 to 14, and 0.2 mg/kg from day 15 until 3 to 6 months [15]. Methylprednisolone (500 mg/d intravenously) was given for 3 days whenever a BPAR with an RAI 5 was diagnosed. For HEV, polymerase chain reaction (PCR) viral RNA was extracted from 200 mL of serum using the QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s recommendation. Prior to the elution of the nucleic acid, an additional centrifugation step of 10 minutes at 14,000g was performed to eliminate the entire ethanol-containing buffer. HEV RNA was detected via real-time PCR using the RealStar HEV RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany). PCR was set up according to the manufacturer’s instructions and processed on a LightCycler 480 Instrument II (Roche Diagnostics GmbH, Mannheim, Germany) [16].
DARSTEIN, HÄUSER, MITTLER ET AL For statistical analysis, IBM SPSS statistics version 22 (SPSS Inc, Chicago, Ill, United States) was used. Continuous variables were expressed as median (with interquartile range) for descriptive analysis. Inequality between groups was compared using the Fisher exact test for 2 categorical variables, and the nonparametric MannWhitney U test was calculated for differences between 2 groups for continuous outcome variables. P values <.05 were considered significant. This research was approved by the local ethics committee of Rhineland-Palatinate and was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent of the patients was obtained during the evaluation process for LT, with general approval to use anonymous data in clinical research.
RESULTS
In our LT recipient cohort, we found 565 cases of BPAR in 306 patients. Characteristics of the patients and the performed therapy are presented in Table 1. Seventy-four patients with at least 2 liver biopsies at different times with at least 1 RAI score 5 (and no HCV infection) were suspected for possible chronic HEVassociated liver inflammation. Characteristics of these patients are presented in Table 1. Blood samples stored at the time of liver biopsy were available for HEV testing in 73 of 74 patients. In 2 patients, an intermediate HEV IgG level was found, and these patients were defined as positive for further analysis. Positive HEV IgG was detected in 18 of 73 patients (24.7%). Differences between HEV IgGepositive and HEV IgGenegative patients are shown in Table 2. No differences in clinical characteristics between patients with and without HEV IgG or patients with and without acute HEV infection were found. An active HEV infection with positive HEV RNA was diagnosed in 3 patients (4.1%). Clinical Course of HEV RNAePositive Patients
In patient 1, liver-kidney transplantation for primary biliary cholangiopathy and kidney failure was simultaneously performed at the age of 63 years. The patient had a complicated postoperative course with ischemic-type biliary lesions. An HEV-positive blood sample was taken on day 375 after LT. The patient died on day 682 after LT from sepsis (HEV as a relevant cofactor cannot be excluded). Patient 2 underwent orthotopic LT at the age of 54 years for primary sclerosing cholangitis with mixed hepato-/cholangiocellular carcinoma, which was diagnosed 2 years before LT and treated by liver resection and radiofrequency ablation. Tumor recurrence occurred 2 years after LT in the transplanted liver. In this patient, HEV infection was diagnosed 10 months after LT. Therapy with ribavirin was initiated 13 months after LT and continued for 3 months. The patient died of tumor progression 1091 days after LT. Patient 3 underwent LT due to hepatocellular carcinoma and hemochromatosis at the age of 66 years. He tested positive for HEV RNA on day 26 after LT. Today, more than 2000 days after LT, the patient is alive and free of symptoms.
HEPATITIS E IN LIVER TRANSPLANT PATIENTS
DISCUSSION
There are several causes of elevated liver enzymes, ranging from harmless findings to pathologies such as BPAR or recurrence of the underlying disease [3,17]. Because of the
3
availability of different treatment options (especially in case of viral hepatitis or acute rejection), it is very important to find the correct diagnosis of the underlying reason for elevated liver enzymes [10]. In contrast to the decreasing
Fig 1. Patient selection. BPAR, biopsy-proven acute rejection; HCV, hepatitis C virus; LT, liver transplantation; RAI, Rejection Activity Index.
4
DARSTEIN, HÄUSER, MITTLER ET AL
number of cases of recurrent hepatitis C, increasing diagnoses of HEV infections after LT have been reported [18]. Because testing for HEV infection was not available in the past, HEV might have been underdiagnosed. As a result, a delay in diagnosis might have resulted in relevant liver damage. The occurrence of chronic hepatitis E is reported to be as high as 65.9% in solid organ transplant recipients after acute infection, and the progression to liver cirrhosis is reported in 14.3% of patients [19]. Since these data were collected from several centers retrospectively, without a standard protocol and from recipients of different organ types, it is not ensured that reported rates are representative of common LT recipient cohorts. The prevalence of antieHEV IgG in the general German adult population is 15.3% for all ages [20]. In our selected cohort of LT recipients, we found an antieHEV IgG seroprevalence of 24.7% (18 of 73 patients). As antieHEV IgG seroprevalence increases with age, the prevalence in LT patients (mean age 55.0 years) is comparable with the seroprevalence in the general population (25.0% for the age group 50-59 years) [20]. Because of a similar seroprevalence, a relevant number of new infections can be assumed for LT recipients each year (seroconversion of 5.2/1000 inhabitants/year according to 417,242 estimated and 1991
reported new infections in the general population each year in Germany) [20,21]. Compared with other countries, a similarly high seroprevalence was found in blood donors in Denmark (20.6%), southwestern England (16%), and the United States (18%), while estimates from Switzerland and the Netherlands are considerably lower [20]. Reasons for different seroprevalence could be due to different exposure to infected food or due to different test systems, for example. In our selected cohort of LT recipients, we found positive HEV RNA (as an indication for acute or chronic HEV infection) in only 3 of 73 LT recipients (4.1%). Because of the estimated high number of new infections (with an antieHEV IgG seroprevalence of 24.7%) and the abovementioned chronification rate of 65.9%, chronic hepatitis E would be expected more frequently [19]. In our selected LT cohort, patients with hepatitis C infection were excluded to avoid an impact on treatment with ribavirin or interferon. Therefore, the most likely reason for the few patients with positive HEV RNA is a rare transition from acute to chronic HEV infection in our LT cohort. Because calcineurin inhibitors are known for stimulating replication of HEV, one explanation for the difference in the reported conversion rates could be a lower target trough level of immunosuppression in our LT recipients compared with
Table 1. Patient Characteristics 306 Pts With 565 Suspected Rejections
Age at time of LT, y, median (min-max) Time to rejection, d, median (min-max) Sex (male/female), % (n) Rejections, median (IQR) Immunosuppression Tacrolimus Cyclosporine Azathioprine Sirolimus/everolimus MMF Methylprednisolone Etiology of liver disease, % (n) HCV HBV HBV/HDV HCC Autoimmune, PBC, PSC Alcohol Other Acute liver failure Therapy, % (n) No therapy Steroid bolus Steroid and change immunosuppression Steroid and other therapy Antibody Change immunosuppression Dose escalation of immunosuppression RAI 5, % (n)
53.50 55.00 62.1 1
(16-74) (4-4173) (190)/37.9 (116) (1)
74 Pts With 134 Suspected Rejections
55.00 60.50 62.2 2
(17-68) (5-3197) (46)/37.8 (28) (1)
57.2 28.1 3.6 6.5 37.9 46.1
(175) (86) (11) (20) (116) (141)
69.0 25.4 7.0 11.3 40.8 54.9
(49) (18) (5) (8) (29) (39)
25.8 6.2 2.6 35.0 11.1 28.4 16.4 9.5
(79) (19) (8) (107) (34) (87) (50) (29)
0 6.8 2.7 26.7 20 41.1 17.6 10.7
(0) (5) (2) (20) (15) (30) (13) (8)
35.3 51.6 10.1 0.7 0.6 1.3 0.3 36.6
(108) (158) (31) (2) (2) (4) (1) (112)
9.5 74.3 10.8 2.7 0 2.7 0 100
(7) (55) (8) (2) (0) (2) (0) (74)
Abbreviations: Autoimmune, autoimmune hepatitis; BPAR, biopsy-proven acute rejection; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; IQR, interquartile range; LT, liver transplantation; MMF, mycophenolate mofetil; PBC, primary biliary cholangiopathy; PSC, primary sclerosing cholangitis; RAI, Rejection Activity Index.
HEPATITIS E IN LIVER TRANSPLANT PATIENTS
5
Table 2. Characteristics of Patients With Recurrent BPAR According to HEV Status
Age at time of LT, y, median (min-max) Time to rejection, d, median (min-max) Sex (male/female), % (n) Immunosuppression, %(n) Tacrolimus Cyclosporine Azathioprine Sirolimus/everolimus MMF Methylprednisolone Etiology of liver disease, % (n) HBV HBV/HDV HCC Autoimmune, PBC, PSC Alcohol Other Acute liver failure Therapy, % (n) No therapy Steroid bolus Steroid and change immunosuppression Steroid and other therapy Change immunosuppression Rejection, median (IQR) RAI, median (IQR)
HEV IgG Positive (n ¼ 18)
HEV IgG Negative (n ¼ 55)
P Value
52.5 (25-68) 35 (5-224) 61.1 (11)/38.9 (7)
56.0 (17-68) 74 (6-3179) 61.8 (35)/38.2 (21)
.44 .35 1.00
72.2 22.2 5.6 11.1 38.9 66.7
(13) (4) (1) (2) (7) (12)
67.9 26.4 7.5 11.3 41.5 50.9
(36) (14) (4) (6) (22) (27)
1.00 1.00 1.00 1.00 1.00 .28
11.1 5.6 27.8 27.8 33.3 16.7 5.6
(2) (1) (5) (5) (6) (3) (1)
5.5 1.8 25.5 18.2 43.6 18.2 12.7
(3) (1) (14) (10) (24) (10) (7)
.59 .44 1.00 .50 .58 1.00 .67
0 88.9 5.6 0.0 5.6 3.0 5.5
(0) (16) (1) (0) (1) (1.25) (1)
12.7 70.9 10.9 3.6 1.8 2.0 5.0
(7) (39) (6) (2) (1) (1) (1)
.18 .21 .67 1.00 .44 .49 .73
Abbreviations: BPAR, biopsy-proven acute rejection; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, hepatitis E virus; IQR, interquartile range; LT, liver transplantation; MMF, mycophenolate mofetil; PBC, primary biliary cholangiopathy; PSC, primary sclerosing cholangitis; RAI, Rejection Activity Index.
recipients of kidney, heart, lung, and other transplants [15,19,22]. Another reason for the low rate of positive HEV RNA could be missing cases due to the exclusion of patients with HCV, without liver biopsy, and with low histologic RAI score. Interestingly, a low rate (3%) of HEV RNAepositive LT recipients is also reported from other German LT centers, which is still much higher than the rate (0.12%) in immunocompetent blood donors [7,23]. There are several limitations of our study. This was a retrospective analysis with a potential bias due to patient selection, as all studied LT recipients were transplanted in only a single center. LT recipients who were not subject to liver biopsy or did not meet the inclusion criteria (no HCV infection, less than 2 suspected rejection episodes and without at least 1 RAI 5) were not included in our evaluation. Another potential bias in our study is the inclusion of patients with suspected BPAR with an RAI 5. Although findings of HEV infection consistent with acute rejection are reported in the literature, the histopathologic finding in liver biopsies from immunocompromised patients is highly variable [9,23,24]. Therefore, cases of hepatitis E in LT recipients without relevant histopathologic signs of rejection could have been missed in our analysis. Despite these limitations, we analyzed a large LT cohort of 306 patients with 565 liver biopsies with BPAR and thus selected the subgroup with the highest risk for persistent liver damage. In this high-risk group, we found a seroprevalence of
antieHEV IgG (24.7%) comparable with that of the general population, but only 4.1% of patients (3 of 73 patients) had positive HEV RNA. In summary, HEV infections seem to be common in LT recipients with persistent elevated liver enzymes and BPAR, but persistent infections resulting in chronic liver damage seem to be rather rare. Therefore, the misdiagnosis of HEV infection as a rejection episode was not a relevant issue in the past, as shown by only 3 of 73 patients in our study with positive HEV RNA. Nevertheless, molecular HEV testing of all LT patients with elevated liver enzymes is recommended, and in positive cases, treatment according to the UK guidelines for HEV and solid organ transplantation and the European Association for the Study of the Liver guidelines should be started [25,26]. In view of a frequent incidence of HEV infection in our LT cohort, further studies investigating the route of transmission, therapy, and natural course after LT are necessary. Raw or undercooked pork products (eg, salami, liver sausage, raw ground meat) should be avoided by patients after LT. We recommend heating food to >72 C for at least 20 minutes [27]. CONCLUSION
In the selected subgroup of LT recipients with BPAR and persistent elevated liver enzymes, the misdiagnosis of HEV infection as BPAR was a rare finding and did not result in
6
relevant organ damage. Because of the rare finding of positive HEV RNA despite high IgG seroprevalence, a rather low rate of HEV chronification in this collection of patients must be assumed. Further studies concerning the impact of HEV infection in LT recipients and the reason for persistent elevated liver enzymes are needed. REFERENCES [1] Puliti Reigada CH, de Ataide EC, de Almeida Prado Mattosinho T, Boin IFSF. Hepatic artery thrombosis after liver transplantation: five-year experience at the State University of Campinas. Transplant Proc 2017;49:867e70. [2] Voigtländer T, Alten TA, Kirstein MM, et al. Clinical impact of liver biopsies in liver transplant recipients. Ann Transplant 2017;22:108e14. [3] Lim J, Curry MP, Sundaram V. Risk factors and outcomes associated with alcohol relapse after liver transplantation. World J Hepatol 2017;9:771e80. [4] Martin-Gandul C, Stampf S, Héquet D, et al. Preventive strategies against cytomegalovirus and incidence of a-herpesvirus infections in solid organ transplant recipients: a nationwide cohort study. Am J Transplant 2017;17:1813e22. [5] Inagaki Y, Oshiro Y, Tanaka T, et al. A nationwide survey of hepatitis E virus infection and chronic hepatitis E in liver transplant recipients in Japan. EBioMedicine 2015;2:1607e12. [6] Behrendt P, Steinmann E, Manns MP, Wedemeyer H. The impact of hepatitis E in the liver transplant setting. J Hepatol 2014;61:1418e29. [7] Westhölter D, Hiller J, Denzer U, et al. HEV-positive blood donations represent a relevant infection risk for immunosuppressed recipients. J Hepatol 2018;69:36e42. [8] Aggarwal A, Perumpail RB, Tummala S, Ahmed A. Hepatitis E virus infection in the liver transplant recipients: clinical presentation and management. World J Hepatol 2016;8:117e22. [9] Allaire M, Bazille C, Selves J, Salamé E, Altieri M. Hepatitis E virus infection mimicking acute graft rejection in a liver transplant recipient. Clin Res Hepatol Gastroenterol 2018;42:e68e71. [10] Mazzola A, Tran Minh M, Charlotte F, et al. Chronic hepatitis E viral infection after liver transplantation: a regression of fibrosis after antiviral therapy. Transplantation 2017;101:2083e7. [11] Kamar N, Selves J, Mansuy J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811e7. [12] Kamar N, Izopet J, Tripon S, et al. Ribavirin for chronic hepatitis E virus infection in transplant recipients. N Engl J Med 2014;370:1111e20. [13] Demetris AJ, Bellamy C, Hübscher SG, et al. 2016 Comprehensive update of the Banff Working Group on Liver
DARSTEIN, HÄUSER, MITTLER ET AL Allograft Pathology: introduction of antibody-mediated rejection. Am J Transplant 2016;16:2816e35. [14] Sreekumar R, Rasmussen DL, Wiesner RH, Charlton MR. Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation. Liver Transplant 2002;8:814e21. [15] Darstein F, König C, Hoppe-Lotichius M, et al. New onset of diabetes after transplantation is associated with improved patient survival after liver transplantation due to confounding factor. Eur J Intern Med 2015;26:439e44. [16] Darstein F, Häuser F, Straub BK, et al. Hepatitis E virus genotype 3 is a common finding in liver-transplanted patients undergoing liver biopsy for elevated liver enzymes with a low De Ritis ratio and suspected acute rejection: a real-world cohort. Clin Transplant 2018;32:e13411. [17] Taner T. Liver transplantation: rejection and tolerance. Liver Transplant 2017;23:S85e8. [18] Koning L, Charlton MR, Pas SD, et al. Prevalence and clinical consequences of Hepatitis E in patients who underwent liver transplantation for chronic Hepatitis C in the United States. BMC Infect Dis 2015;15:371. [19] Kamar N, Garrouste C, Haagsma EB, et al. Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants. Gastroenterology 2011;140:1481e9. [20] Faber MS, Wenzel JJ, Jilg W, Thamm M, Höhle M, Stark K. Hepatitis E virus seroprevalence among adults, Germany. Emerg Infect Dis 2012;18:1654e7. [21] Robert Koch Institut. Infektionsempidemiologisches Jahrbuch meldepflichtiger Erkrankungen für 2016. https://www.rki.de/DE/ Content/Infekt/Jahrbuch/Jahrbuch_2016.pdf?__blob¼publicationFile [Accessed 21.02.2020]. [22] Wang Y, Zhou X, Debing Y, et al. Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus. Gastroenterology 2014;146:1775e83. [23] Protzer U, Böhm F, Longerich T, et al. Molecular detection of hepatitis E virus (HEV) in liver biopsies after liver transplantation. Modern Pathol 2015;28:523e32. [24] Lenggenhager D, Weber A. An update on the clinicopathologic features and pathologic diagnosis of hepatitis e in liver specimens. Adv Anat Pathol 2018;25:273e81. [25] McPherson S, Elsharkawy AM, Ankcorn M, et al. Summary of the British Transplantation Society UK guidelines for hepatitis E and solid organ transplantation. Transplantation 2018;102:15e20. [26] European Association for the Study of the Liver. EASL Clinical Practice Guidelines on hepatitis E virus infection. J Hepatol 2018;68:1256e71. [27] Cook N, Van der Poel, Wim HM. Survival and elimination of hepatitis E virus: a review. Food Environ Virol 2015;7: 189e94.
ABSTRACT Background. In past decades, liver transplant (LT) patients were not routinely screened for hepatitis E virus (HEV) infection, and thus it might have been misdiagnosed as an acute rejection episode. Our aim was to analyze a real-world cohort of LT patients who presented with at least 1 episode of biopsy-proven acute rejection (BPAR) and suffered from persistent elevated transaminases, to evaluate the frequency of HEV infection misdiagnosed as a rejection episode. Methods. Data from 306 patients transplanted between 1997 and 2017, including 565 liver biopsies, were analyzed. Biopsies from patients suffering from hepatitis C (n ¼ 79; 25.8%) and from patients who presented with a Rejection Activity Index <5 (n ¼ 134; 43.8%) were excluded. A subgroup of 74 patients (with 134 BPAR) with persistently elevated liver enzymes was chosen for further HEV testing. Results. Positive HEV IgG was detectable in 18 of 73 patients (24.7%). Positive HEV RNA was diagnosed in 3 of 73 patients with BPAR (4.1%). Patients with HEV infection showed no difference in etiology of the liver disease, type of immunosuppression, or median Rejection Activity Index. Conclusion. Few HEV infections were misdiagnosed as acute rejection episodes in this real-world cohort. Thus, HEV infection is an infrequent diagnosis in cases with persistent elevated liver enzymes and BPAR after LT.
E
LEVATED liver enzymes following liver transplantation (LT) are a frequent finding. The elevation might be caused by harmless or severe diseases, with possible liver damage or loss of the transplanted liver [1]. A common reason for increased liver enzymes is an episode of biopsy-proven acute cellular rejection (BPAR) [2]. However, many pathologies can be responsible for elevated liver enzymes after LT, including recurrence of the underlying disease, viral infections, vascular or biliary complications, and others [3,4]. In recent years, hepatitis E virus (HEV) infection has been reported more frequently in patients after LT [5]. However, the true incidence of HEV infection in LT recipients is still unclear [6]. A potential risk factor for HEV infection in LT recipients might be transfusion, especially as blood concentrates have not been tested regularly for HEV in the ª 2020 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, XX, 1e6 (2020)
past [7]. It might be difficult to distinguish HEV infection from BPAR in liver biopsies. Therefore, LT recipients with HEV infection might have been misdiagnosed as suffering from acute cellular rejection [8,9]. HEV infection can present as chronic hepatitis responsible for liver cirrhosis and organ loss [10,11]. Limited data are available concerning the true rate of post-LT HEV infections that become chronic and cause recurrent cirrhosis.
*Address correspondence to Felix Darstein, First Department of Internal Medicine, Gastroenterology and Hepatology, University Medical Center Mainz, Langenbeckstraße 1, 55131 Mainz, Germany. Tel: (0049) 06131 170. E-mail: felix.darstein@ unimedizin-mainz.de 0041-1345/20 https://doi.org/10.1016/j.transproceed.2020.01.011
1
2
Ribavirin is an effective treatment option for HEV [12]. As ribavirin has been frequently used to treat LT recipients with hepatitis C virus (HCV) reinfection, HCV-positive patients might have been cured from HEV while treating their HCV infection. In the past, many LT recipients presented with BPAR and persistent elevated liver enzymes despite treatment with steroids (with complications such as organ loss and death). HEV infection could have been the reason for those chronic steroid-refractory hepatic injuries. The aim of our study was to determine the burden of HEV infection in LT patients with BPAR and persistent elevated liver enzymes in a real-world cohort and to determine the rate of HEV infections misdiagnosed as BPAR between 1997 and 2017. PATIENTS AND METHODS All patients were selected from an administrative transplant database, and all data were retrieved from patients’ charts and reports. The selection process is presented in Fig 1. To determine the burden of chronic HEV infection in LT patients with BPAR and persistent elevated liver enzymes, inclusion and exclusion criteria were as follows: BPAR was diagnosed according to the recommendations of the BANFF working group [13]. Clinically relevant liver damage was presumed when patients underwent at least 2 liver biopsies with at least 1 Rejection Activity Index (RAI) score 5 due to persistent elevation of liver enzymes. Patients who did not meet these criteria were excluded from our analysis. Since HCV infection might cause a bias because of the difficult histologic differentiation between BPAR and HCV reinfection and because of the unintended treatment of HEV during ribavirin therapy for HCV infection, HCV-positive patients (n ¼ 79) were excluded [14]. Finally, 74 patients met the inclusion criteria. Blood samples for the detection of HEV (IgM, IgG, and RNA) were initially taken at the time of liver biopsy and were available from 73 of 74 patients (Fig 1). A frequent immunosuppressive regimen consisted of the combination of a calcineurin inhibitor (cyclosporine or tacrolimus) with mycophenolate mofetil. During the first year, target trough levels were 5 to 7 ng/mL for tacrolimus and 3 to 5 ng/mL after 1 year. Cyclosporine target trough levels during the first year were 70 to 90 ng/mL and 50 to 70 ng/mL thereafter. Patients were treated with steroids in the first 3 to 6 months after LT. Methylprednisolone was reduced from 1.5 mg/kg on day 1 and 2 after LT to 1.0 mg/kg on days 3 and 4, 0.5 mg/kg on days 5 to 14, and 0.2 mg/kg from day 15 until 3 to 6 months [15]. Methylprednisolone (500 mg/d intravenously) was given for 3 days whenever a BPAR with an RAI 5 was diagnosed. For HEV, polymerase chain reaction (PCR) viral RNA was extracted from 200 mL of serum using the QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s recommendation. Prior to the elution of the nucleic acid, an additional centrifugation step of 10 minutes at 14,000g was performed to eliminate the entire ethanol-containing buffer. HEV RNA was detected via real-time PCR using the RealStar HEV RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany). PCR was set up according to the manufacturer’s instructions and processed on a LightCycler 480 Instrument II (Roche Diagnostics GmbH, Mannheim, Germany) [16].
DARSTEIN, HÄUSER, MITTLER ET AL For statistical analysis, IBM SPSS statistics version 22 (SPSS Inc, Chicago, Ill, United States) was used. Continuous variables were expressed as median (with interquartile range) for descriptive analysis. Inequality between groups was compared using the Fisher exact test for 2 categorical variables, and the nonparametric MannWhitney U test was calculated for differences between 2 groups for continuous outcome variables. P values <.05 were considered significant. This research was approved by the local ethics committee of Rhineland-Palatinate and was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent of the patients was obtained during the evaluation process for LT, with general approval to use anonymous data in clinical research.
RESULTS
In our LT recipient cohort, we found 565 cases of BPAR in 306 patients. Characteristics of the patients and the performed therapy are presented in Table 1. Seventy-four patients with at least 2 liver biopsies at different times with at least 1 RAI score 5 (and no HCV infection) were suspected for possible chronic HEVassociated liver inflammation. Characteristics of these patients are presented in Table 1. Blood samples stored at the time of liver biopsy were available for HEV testing in 73 of 74 patients. In 2 patients, an intermediate HEV IgG level was found, and these patients were defined as positive for further analysis. Positive HEV IgG was detected in 18 of 73 patients (24.7%). Differences between HEV IgGepositive and HEV IgGenegative patients are shown in Table 2. No differences in clinical characteristics between patients with and without HEV IgG or patients with and without acute HEV infection were found. An active HEV infection with positive HEV RNA was diagnosed in 3 patients (4.1%). Clinical Course of HEV RNAePositive Patients
In patient 1, liver-kidney transplantation for primary biliary cholangiopathy and kidney failure was simultaneously performed at the age of 63 years. The patient had a complicated postoperative course with ischemic-type biliary lesions. An HEV-positive blood sample was taken on day 375 after LT. The patient died on day 682 after LT from sepsis (HEV as a relevant cofactor cannot be excluded). Patient 2 underwent orthotopic LT at the age of 54 years for primary sclerosing cholangitis with mixed hepato-/cholangiocellular carcinoma, which was diagnosed 2 years before LT and treated by liver resection and radiofrequency ablation. Tumor recurrence occurred 2 years after LT in the transplanted liver. In this patient, HEV infection was diagnosed 10 months after LT. Therapy with ribavirin was initiated 13 months after LT and continued for 3 months. The patient died of tumor progression 1091 days after LT. Patient 3 underwent LT due to hepatocellular carcinoma and hemochromatosis at the age of 66 years. He tested positive for HEV RNA on day 26 after LT. Today, more than 2000 days after LT, the patient is alive and free of symptoms.
HEPATITIS E IN LIVER TRANSPLANT PATIENTS
DISCUSSION
There are several causes of elevated liver enzymes, ranging from harmless findings to pathologies such as BPAR or recurrence of the underlying disease [3,17]. Because of the
3
availability of different treatment options (especially in case of viral hepatitis or acute rejection), it is very important to find the correct diagnosis of the underlying reason for elevated liver enzymes [10]. In contrast to the decreasing
Fig 1. Patient selection. BPAR, biopsy-proven acute rejection; HCV, hepatitis C virus; LT, liver transplantation; RAI, Rejection Activity Index.
4
DARSTEIN, HÄUSER, MITTLER ET AL
number of cases of recurrent hepatitis C, increasing diagnoses of HEV infections after LT have been reported [18]. Because testing for HEV infection was not available in the past, HEV might have been underdiagnosed. As a result, a delay in diagnosis might have resulted in relevant liver damage. The occurrence of chronic hepatitis E is reported to be as high as 65.9% in solid organ transplant recipients after acute infection, and the progression to liver cirrhosis is reported in 14.3% of patients [19]. Since these data were collected from several centers retrospectively, without a standard protocol and from recipients of different organ types, it is not ensured that reported rates are representative of common LT recipient cohorts. The prevalence of antieHEV IgG in the general German adult population is 15.3% for all ages [20]. In our selected cohort of LT recipients, we found an antieHEV IgG seroprevalence of 24.7% (18 of 73 patients). As antieHEV IgG seroprevalence increases with age, the prevalence in LT patients (mean age 55.0 years) is comparable with the seroprevalence in the general population (25.0% for the age group 50-59 years) [20]. Because of a similar seroprevalence, a relevant number of new infections can be assumed for LT recipients each year (seroconversion of 5.2/1000 inhabitants/year according to 417,242 estimated and 1991
reported new infections in the general population each year in Germany) [20,21]. Compared with other countries, a similarly high seroprevalence was found in blood donors in Denmark (20.6%), southwestern England (16%), and the United States (18%), while estimates from Switzerland and the Netherlands are considerably lower [20]. Reasons for different seroprevalence could be due to different exposure to infected food or due to different test systems, for example. In our selected cohort of LT recipients, we found positive HEV RNA (as an indication for acute or chronic HEV infection) in only 3 of 73 LT recipients (4.1%). Because of the estimated high number of new infections (with an antieHEV IgG seroprevalence of 24.7%) and the abovementioned chronification rate of 65.9%, chronic hepatitis E would be expected more frequently [19]. In our selected LT cohort, patients with hepatitis C infection were excluded to avoid an impact on treatment with ribavirin or interferon. Therefore, the most likely reason for the few patients with positive HEV RNA is a rare transition from acute to chronic HEV infection in our LT cohort. Because calcineurin inhibitors are known for stimulating replication of HEV, one explanation for the difference in the reported conversion rates could be a lower target trough level of immunosuppression in our LT recipients compared with
Table 1. Patient Characteristics 306 Pts With 565 Suspected Rejections
Age at time of LT, y, median (min-max) Time to rejection, d, median (min-max) Sex (male/female), % (n) Rejections, median (IQR) Immunosuppression Tacrolimus Cyclosporine Azathioprine Sirolimus/everolimus MMF Methylprednisolone Etiology of liver disease, % (n) HCV HBV HBV/HDV HCC Autoimmune, PBC, PSC Alcohol Other Acute liver failure Therapy, % (n) No therapy Steroid bolus Steroid and change immunosuppression Steroid and other therapy Antibody Change immunosuppression Dose escalation of immunosuppression RAI 5, % (n)
53.50 55.00 62.1 1
(16-74) (4-4173) (190)/37.9 (116) (1)
74 Pts With 134 Suspected Rejections
55.00 60.50 62.2 2
(17-68) (5-3197) (46)/37.8 (28) (1)
57.2 28.1 3.6 6.5 37.9 46.1
(175) (86) (11) (20) (116) (141)
69.0 25.4 7.0 11.3 40.8 54.9
(49) (18) (5) (8) (29) (39)
25.8 6.2 2.6 35.0 11.1 28.4 16.4 9.5
(79) (19) (8) (107) (34) (87) (50) (29)
0 6.8 2.7 26.7 20 41.1 17.6 10.7
(0) (5) (2) (20) (15) (30) (13) (8)
35.3 51.6 10.1 0.7 0.6 1.3 0.3 36.6
(108) (158) (31) (2) (2) (4) (1) (112)
9.5 74.3 10.8 2.7 0 2.7 0 100
(7) (55) (8) (2) (0) (2) (0) (74)
Abbreviations: Autoimmune, autoimmune hepatitis; BPAR, biopsy-proven acute rejection; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; IQR, interquartile range; LT, liver transplantation; MMF, mycophenolate mofetil; PBC, primary biliary cholangiopathy; PSC, primary sclerosing cholangitis; RAI, Rejection Activity Index.
HEPATITIS E IN LIVER TRANSPLANT PATIENTS
5
Table 2. Characteristics of Patients With Recurrent BPAR According to HEV Status
Age at time of LT, y, median (min-max) Time to rejection, d, median (min-max) Sex (male/female), % (n) Immunosuppression, %(n) Tacrolimus Cyclosporine Azathioprine Sirolimus/everolimus MMF Methylprednisolone Etiology of liver disease, % (n) HBV HBV/HDV HCC Autoimmune, PBC, PSC Alcohol Other Acute liver failure Therapy, % (n) No therapy Steroid bolus Steroid and change immunosuppression Steroid and other therapy Change immunosuppression Rejection, median (IQR) RAI, median (IQR)
HEV IgG Positive (n ¼ 18)
HEV IgG Negative (n ¼ 55)
P Value
52.5 (25-68) 35 (5-224) 61.1 (11)/38.9 (7)
56.0 (17-68) 74 (6-3179) 61.8 (35)/38.2 (21)
.44 .35 1.00
72.2 22.2 5.6 11.1 38.9 66.7
(13) (4) (1) (2) (7) (12)
67.9 26.4 7.5 11.3 41.5 50.9
(36) (14) (4) (6) (22) (27)
1.00 1.00 1.00 1.00 1.00 .28
11.1 5.6 27.8 27.8 33.3 16.7 5.6
(2) (1) (5) (5) (6) (3) (1)
5.5 1.8 25.5 18.2 43.6 18.2 12.7
(3) (1) (14) (10) (24) (10) (7)
.59 .44 1.00 .50 .58 1.00 .67
0 88.9 5.6 0.0 5.6 3.0 5.5
(0) (16) (1) (0) (1) (1.25) (1)
12.7 70.9 10.9 3.6 1.8 2.0 5.0
(7) (39) (6) (2) (1) (1) (1)
.18 .21 .67 1.00 .44 .49 .73
Abbreviations: BPAR, biopsy-proven acute rejection; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; HEV, hepatitis E virus; IQR, interquartile range; LT, liver transplantation; MMF, mycophenolate mofetil; PBC, primary biliary cholangiopathy; PSC, primary sclerosing cholangitis; RAI, Rejection Activity Index.
recipients of kidney, heart, lung, and other transplants [15,19,22]. Another reason for the low rate of positive HEV RNA could be missing cases due to the exclusion of patients with HCV, without liver biopsy, and with low histologic RAI score. Interestingly, a low rate (3%) of HEV RNAepositive LT recipients is also reported from other German LT centers, which is still much higher than the rate (0.12%) in immunocompetent blood donors [7,23]. There are several limitations of our study. This was a retrospective analysis with a potential bias due to patient selection, as all studied LT recipients were transplanted in only a single center. LT recipients who were not subject to liver biopsy or did not meet the inclusion criteria (no HCV infection, less than 2 suspected rejection episodes and without at least 1 RAI 5) were not included in our evaluation. Another potential bias in our study is the inclusion of patients with suspected BPAR with an RAI 5. Although findings of HEV infection consistent with acute rejection are reported in the literature, the histopathologic finding in liver biopsies from immunocompromised patients is highly variable [9,23,24]. Therefore, cases of hepatitis E in LT recipients without relevant histopathologic signs of rejection could have been missed in our analysis. Despite these limitations, we analyzed a large LT cohort of 306 patients with 565 liver biopsies with BPAR and thus selected the subgroup with the highest risk for persistent liver damage. In this high-risk group, we found a seroprevalence of
antieHEV IgG (24.7%) comparable with that of the general population, but only 4.1% of patients (3 of 73 patients) had positive HEV RNA. In summary, HEV infections seem to be common in LT recipients with persistent elevated liver enzymes and BPAR, but persistent infections resulting in chronic liver damage seem to be rather rare. Therefore, the misdiagnosis of HEV infection as a rejection episode was not a relevant issue in the past, as shown by only 3 of 73 patients in our study with positive HEV RNA. Nevertheless, molecular HEV testing of all LT patients with elevated liver enzymes is recommended, and in positive cases, treatment according to the UK guidelines for HEV and solid organ transplantation and the European Association for the Study of the Liver guidelines should be started [25,26]. In view of a frequent incidence of HEV infection in our LT cohort, further studies investigating the route of transmission, therapy, and natural course after LT are necessary. Raw or undercooked pork products (eg, salami, liver sausage, raw ground meat) should be avoided by patients after LT. We recommend heating food to >72 C for at least 20 minutes [27]. CONCLUSION
In the selected subgroup of LT recipients with BPAR and persistent elevated liver enzymes, the misdiagnosis of HEV infection as BPAR was a rare finding and did not result in
6
relevant organ damage. Because of the rare finding of positive HEV RNA despite high IgG seroprevalence, a rather low rate of HEV chronification in this collection of patients must be assumed. Further studies concerning the impact of HEV infection in LT recipients and the reason for persistent elevated liver enzymes are needed. REFERENCES [1] Puliti Reigada CH, de Ataide EC, de Almeida Prado Mattosinho T, Boin IFSF. Hepatic artery thrombosis after liver transplantation: five-year experience at the State University of Campinas. Transplant Proc 2017;49:867e70. [2] Voigtländer T, Alten TA, Kirstein MM, et al. Clinical impact of liver biopsies in liver transplant recipients. Ann Transplant 2017;22:108e14. [3] Lim J, Curry MP, Sundaram V. Risk factors and outcomes associated with alcohol relapse after liver transplantation. World J Hepatol 2017;9:771e80. [4] Martin-Gandul C, Stampf S, Héquet D, et al. Preventive strategies against cytomegalovirus and incidence of a-herpesvirus infections in solid organ transplant recipients: a nationwide cohort study. Am J Transplant 2017;17:1813e22. [5] Inagaki Y, Oshiro Y, Tanaka T, et al. A nationwide survey of hepatitis E virus infection and chronic hepatitis E in liver transplant recipients in Japan. EBioMedicine 2015;2:1607e12. [6] Behrendt P, Steinmann E, Manns MP, Wedemeyer H. The impact of hepatitis E in the liver transplant setting. J Hepatol 2014;61:1418e29. [7] Westhölter D, Hiller J, Denzer U, et al. HEV-positive blood donations represent a relevant infection risk for immunosuppressed recipients. J Hepatol 2018;69:36e42. [8] Aggarwal A, Perumpail RB, Tummala S, Ahmed A. Hepatitis E virus infection in the liver transplant recipients: clinical presentation and management. World J Hepatol 2016;8:117e22. [9] Allaire M, Bazille C, Selves J, Salamé E, Altieri M. Hepatitis E virus infection mimicking acute graft rejection in a liver transplant recipient. Clin Res Hepatol Gastroenterol 2018;42:e68e71. [10] Mazzola A, Tran Minh M, Charlotte F, et al. Chronic hepatitis E viral infection after liver transplantation: a regression of fibrosis after antiviral therapy. Transplantation 2017;101:2083e7. [11] Kamar N, Selves J, Mansuy J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811e7. [12] Kamar N, Izopet J, Tripon S, et al. Ribavirin for chronic hepatitis E virus infection in transplant recipients. N Engl J Med 2014;370:1111e20. [13] Demetris AJ, Bellamy C, Hübscher SG, et al. 2016 Comprehensive update of the Banff Working Group on Liver
DARSTEIN, HÄUSER, MITTLER ET AL Allograft Pathology: introduction of antibody-mediated rejection. Am J Transplant 2016;16:2816e35. [14] Sreekumar R, Rasmussen DL, Wiesner RH, Charlton MR. Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation. Liver Transplant 2002;8:814e21. [15] Darstein F, König C, Hoppe-Lotichius M, et al. New onset of diabetes after transplantation is associated with improved patient survival after liver transplantation due to confounding factor. Eur J Intern Med 2015;26:439e44. [16] Darstein F, Häuser F, Straub BK, et al. Hepatitis E virus genotype 3 is a common finding in liver-transplanted patients undergoing liver biopsy for elevated liver enzymes with a low De Ritis ratio and suspected acute rejection: a real-world cohort. Clin Transplant 2018;32:e13411. [17] Taner T. Liver transplantation: rejection and tolerance. Liver Transplant 2017;23:S85e8. [18] Koning L, Charlton MR, Pas SD, et al. Prevalence and clinical consequences of Hepatitis E in patients who underwent liver transplantation for chronic Hepatitis C in the United States. BMC Infect Dis 2015;15:371. [19] Kamar N, Garrouste C, Haagsma EB, et al. Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants. Gastroenterology 2011;140:1481e9. [20] Faber MS, Wenzel JJ, Jilg W, Thamm M, Höhle M, Stark K. Hepatitis E virus seroprevalence among adults, Germany. Emerg Infect Dis 2012;18:1654e7. [21] Robert Koch Institut. Infektionsempidemiologisches Jahrbuch meldepflichtiger Erkrankungen für 2016. https://www.rki.de/DE/ Content/Infekt/Jahrbuch/Jahrbuch_2016.pdf?__blob¼publicationFile [Accessed 21.02.2020]. [22] Wang Y, Zhou X, Debing Y, et al. Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus. Gastroenterology 2014;146:1775e83. [23] Protzer U, Böhm F, Longerich T, et al. Molecular detection of hepatitis E virus (HEV) in liver biopsies after liver transplantation. Modern Pathol 2015;28:523e32. [24] Lenggenhager D, Weber A. An update on the clinicopathologic features and pathologic diagnosis of hepatitis e in liver specimens. Adv Anat Pathol 2018;25:273e81. [25] McPherson S, Elsharkawy AM, Ankcorn M, et al. Summary of the British Transplantation Society UK guidelines for hepatitis E and solid organ transplantation. Transplantation 2018;102:15e20. [26] European Association for the Study of the Liver. EASL Clinical Practice Guidelines on hepatitis E virus infection. J Hepatol 2018;68:1256e71. [27] Cook N, Van der Poel, Wim HM. Survival and elimination of hepatitis E virus: a review. Food Environ Virol 2015;7: 189e94.