Chronic Hepatitis E in Heart Transplant Recipients

Authors


Heiner Wedemeyer, Wedemeyer.Heiner@mh-hannover.de

Abstract

Chronic courses of hepatitis E virus (HEV) infections have been described in immunosuppressed patients. We aimed to study the role of HEV infections in heart transplant recipients (HTR). 274 HTR were prospectively screened for HEV infection using an anti-HEV-IgG ELISA and HEV-PCR. In addition, 137 patients undergoing cardiac surgery (non-HTR) and 537 healthy subjects were studied cross-sectionally. The anti-HEV-IgG seroprevalence was 11% in HTR, 7% in non-HTR and 2% in healthy controls (HTR vs. healthy controls p<0.0001; non-HTR vs. healthy controls p<0.01). Anti-HEV tested positive in 4.0% in control cohorts of other immunocompromised patients (n = 474). Four HTR (1.5%) were chronically infected with HEV as shown by HEV-PCR and all four patients had liver transaminases of >200 IU/L and histological or clinical evidence of advanced liver disease. In three patients ribavirin treatment was successful with a sustained biochemical and virological response while treatment failed in one cirrhotic patient after ribavirin dose reduction. Heart transplant recipients and patients undergoing cardiac surgery have an increased risk for HEV infections. Chronic hepatitis E may explain elevated liver enzymes in heart transplant recipients. Treatment of HEV infection with ribavirin is effective but the optimal dose and duration of ribavirin therapy remains to be determined.

Abbreviations: 
ALT

alanine aminotransfrase

AST

aspartate aminotransferase

ELISA

enzyme-linked immunosorbent assay

HEV

hepatitis E virus

HTR

heart transplant recipients

non-HTR

patients undergoing cardiac surgery, without transplantation

PCR

polymerase chain reaction

Introduction

Hepatitis E virus (HEV) infection takes a clinically silent course in a large majority of cases (1–4). Few subjects develop symptomatic acute hepatitis, which may rarely progress to severe hepatic failure, this in particular in pregnant women or patients with underlying chronic liver diseases (1,5,6). In recent years chronic courses of HEV infection have been described in immunocompromised individuals such as liver and kidney transplant recipients (7–10) or HIV-infected patients (11). An association between chronic HEV infections and progressive liver disease leading to fibrosis or in severe cases to life-treating cirrhosis has been established (7–9). An impaired T cell response against HEV is associated with persistent HEV viremia (12) and certain immunosuppressive drugs may be associated with chronicity (13). Treatment options of chronic hepatitis E include reduction of immunosuppression, administration of interferon alpha and monotherapy with ribavirin (4). Chronic HEV infection has recently also been described in a single heart transplant recipient (14). However the frequency of HEV infections or HEV-specific antibodies in heart transplant recipients has not been studied prospectively.

The aims of this study were (i) to investigate the prevalence of HEV-antibodies in heart transplant recipients (HTR) and (ii) to determine the frequency and clinical relevance of chronic hepatitis E in HTR in a low endemic Central European country.

Patients and Methods

We prospectively studied 274 consecutive heart transplant recipients (HTR) for anti-HEV IgG antibodies and HEV RNA. The study was performed between August 2010 and April 2011 and included all heart transplant patients attending the heart transplant outpatient clinic of the Hannover Medical School. HTR were tested for hepatitis E markers after a median of 121 months after transplantation (6–308). Additionally, stored sera of patients testing positive for either anti-HEV or HEV RNA were tested if available to determine the time point of exposure to HEV. A control group of nontransplant patients with heart disease undergoing cardiac surgery was recruited from one of our cardio-thoracic surgery wards (n = 137, non-HTR). In addition, 537 healthy subjects (employees of Hannover Medical School (n = 167) and blood donors (n = 370)) were tested for anti-HEV to determine the overall seroprevalence of HEV in Northern Germany as described previously (15). Other cohorts of immunocompromised patients at Hannover Medical have been studied previously for hepatitis E with identical methods (total number of published cases 474) (9,16–18).

Anti-HEV and HEV RNA testing

Anti-HEV immunoglobuline G antibodies were tested with the MP Diagnostic assay (MP Biomedicals, formerly Genelabs Diagnostics, Singapore) according to the manufacturer's instructions and as reported previously (9).

HEV RNA testing was performed by nested PCR in all patients irrespective of the presence of anti-HEV IgG. The following primer sequences were used:

1. PCRHE 361FGCR GTG GTT TCT GGG GTG AC
 HE 364RCTG GGM YTG GTC DCG CCA AG
2. PCRHE 366FGYT GAT TCT CAG CCC TTC GC
 HE 363RGMY TGG TCD CGC CAA GHG GA

In viremic patients a quantitative real-time PCR was performed using the Cobas Taqman platform similarly to our previous studies in hepatitis D virus infection (19).

Statistics

Data for the different patient groups were presented in means and standard deviations. A comparison of continuous and categorical data between groups was performed using the chi-square test. Comparison of quantitative data between groups was performed using the Mann–Whitney test. A p-value < 0.05 was considered significant.

Ethics

This study has been approved by the Ethics Committee of Hannover Medical School.

Results

The majority of patients included in this prospective study were male (76% of all patients, 80% of HTR and 69% of non-HTR). The mean age of HTR was 57 years (range 18–80, SD 13 years) and 66 years (range 27–83 years, SD 11 years) in non-HTR, respectively (p<0.001). Elevated ALT levels were detected in 28 HTR (10%) and 20 non-HTR (15%) with mean ALT levels of 30 and 38 in the two groups (p = 0.002).

Prevalence of HEV-specific antibodies in heart transplant recipients

Anti-HEV antibodies were detected in 31 of the 274 heart transplant recipients (11.3%; Table 1). Anti-HEV antibodies were more frequent in heart transplant recipients than in healthy individuals (p < 0.001) and or in other cohorts of immunocomprimised individuals (4.0%, total number 474; p < 0.001) including liver transplant recipients, patients after allogenic stem cell transplantation, patients with common variable immunodeficiency syndrome and HIV-infected individuals (Table 1).

Table 1.  Prevalence of HEV antibodies and HEV-RNA in different patient groups in Germany
GroupsNumber of patientsAnti-HEV-IgG positive nHEV-RNA positive np-Value; seroprevalence rate in comparison to seroprevalence rate in HTR (chi-square)
  1. *Healthy controls were only tested for HEV-RNA if they tested positive for IgG.

Heart transplant recipients27431 (11.3%)4 (1.5%) 
Healthy controls (15)53711 (2.0%)0 (0%)*<0.001  
Non transplanted cardiac patients13710 (7.3%)0 (0%)ns
Liver transplant recipients (9)22610 (4.4%)2 (<1%)0.005
Non transplant liver patients (9)1294 (3.1%)0 (0%)0.006
HIV-infected patients (16)1236 (4.9%)0 (0%)0.04 
Stem cell transplant recipients with strongly elevated transaminases (17) 523 (5.8%)0 (0%)ns
Patients with common variable immunodeficiency (CVID) (18) 730 (0%)0 (0%)0.003

Eleven of the anti-HEV positive HTR (4.0%) were already anti-HEV-IgG-positive prior to heart transplantation, while 19 patients underwent seroconversion after heart transplantation. For one patient no pretransplant serum was available. Anti-HEV-IgG-positive and -negative transplanted patients did not differ in age (57 ± 10 years vs. 57 ± 14 years, p = 0.313 Mann–Whitney test) or sex (81% male vs. 80% male, p = 0.255 chi-square test). ALT levels were significantly higher in IgG positive versus negative heart transplant recipients (54 ± 71 U/ mL vs. 27 ± 14 U/mL, p = 0.020 Mann–Whitney test). Dilatative cardiomyopathy and ischemic heart disease were the underlying causes of transplantation in 6/11 and 4/11 HTR, respectively, who were anti-HEV positive at the time of transplantation (Figure 1). Anti-HEV negative HTR were transplanted due to dilatative cardiomyopathy in 130/243 cases (54%) and due to ischemic heart disease in 82/243 patients (34%).

Figure 1.

Distribution of underlying disease in transplant patients testing positive for anti-HEV antibodies (for one patient the information on the time point of seroconversion is missing). Only 4 out of 19 patients who had contact with HEV after transplantation developed chronic hepatitis E and tested positive for HEV RNA.

Prevalence of HEV RNA in heart transplant recipients

All patients were tested for HEV RNA irrespective of the presence of anti HEV antibodies. HEV RNA was detected in four HTR but in none of the non-HTR (Table 1). All four HEV RNA positive patients had ALT levels above 200 IU/L (range 211–285 IU/L) while none of the 270 HEV RNA negative subjects showed ALT values above 100 IU/L (mean 27 IU/L, range 8–95 IU/L). The four viremic patients had been anti-HEV-IgG and HEV RNA negative at the time of transplantation and thus became infected after transplantation.

Analysis of 19 HTR who had contact with HEV after heart transplantation did not show an association between immunosuppressive drugs used and chronicity of hepatitis E. All but two of the 19 patients were on triple therapy consisting of steroids plus cyclosporine (n = 15) or tacrolimus (n = 4) and azathioprine (n = 2), everolimus (n = 6) or mycophenolate (n = 9). However, clearance of HEV infection was significantly associated with the use of MMF (9/15 vs. 0/4; p = 0.03).

In the 15 patients who seroconverted to anti-HEV antibodies after transplantation but cleared the infection without development of chronic hepatitis E, ALT levels were normal or only moderately elevated (range 11–59 IU/L). 13 of the 15 spontaneous HEV clearers were male and the age ranged from 40 to 74 years (mean 57 years ± 10 years).

Clinical courses of chronic hepatitis E (Figure 2)

Figure 2.

Course of ALT in patients with chronic HEV infection. A = patient 1, B = patient 2, C = patient 3, D = patient 4

Patient HTR-1 was transplanted in 1998, tested HEV negative in April 2008 and became HEV RNA and IgG positive in May 2010. The liver biopsy showed portal-lobular lymphocytic infiltration with strong interface activity (Figure 3). After successful surgical treatment of renal carcinoma without additional chemotherapy, treatment with ribavirin (800 mg/day) was initiated. He rapidly responded to antiviral treatment with normalization of ALT levels and clearance of HEV RNA within 4 weeks of treatment. Treatment was stopped after 5 months and no relapse occurred. Patient HTR-2 tested HEV negative 1 month before transplantation in December 2007 and HEV RNA was detected for the first time in January 2009. Liver histology taken after diagnosis of HEV infection demonstrated chronic hepatitis with mild to moderate necro-inflammatory activity and focal fibrosis with few septa. Seroconversion to anti-HEV could not be documented before September 2010. This patient also responded rapidly to ribavirin treatment (800 mg/day) and he tested HEV RNA negative in plasma after 4 weeks of treatment. Treatment was continued for a total of 5 months and a sustained virological response was achieved. Similarly a sustained response to 5 months of ribavirin therapy was also observed in patient HTR-3 (transplanted in 2008, first diagnosis of hepatitis E in September 2010), even though the dose of ribavirin had to be reduced from 800 mg/day to 600 mg/day due to anemia. This patient tested negative for HEV RNA in a sample taken after 6 weeks of treatment. Liver histology in this patient showed chronic hepatitis with mild to moderate periportal activity and few lobular apoptotic bodies.

Figure 3.

Histology of the liver biopsy of patient HTR 1 (hematoxylin and eosin stain, original magnification 100×).

Patient HTR-4 underwent heart transplantation in May 1998 due to ischemic cardiomyopathy. He first tested anti-HEV and HEV RNA positive in April 2008 (the last HEV negative sample was taken in January 2006). The patient lost HEV antibodies during further follow-up and was anti-HEV-IgG negative in October 2010. Ribavirin therapy (600 mg/day) was started in October 2010. ALT levels rapidly improved and HEV RNA declined by about 3 logs during the first 3 months of therapy (CT value of 25.90 before treatment and a CT value of 34.11 at month 2). However, HTR-4 never became HEV RNA negative and even the HEV RNA levels (CT value 28.60) increased paralleled by a biochemical relapse once the ribavirin dose had been reduced to 200 mg/day due to severe anemia. Transient reincrease of the ribavirin dose up to 600 mg/day supported by frequent blood transfusions had no effects on HEV RNA levels and the patient remained HEV RNA positive throughout follow-up. The patient showed clinical evidence of liver cirrhosis with presence of esophageal varices, hepatic decompensation and development of ascites precluding percutaneous liver biopsy.

Prevalence of HEV-specific antibodies in nonheart transplanted cardiac patients

HEV antibodies were surprisingly frequent in HTR, posing the question whether patients undergoing cardiac surgery per se have an increased risk for HEV infections. Indeed, 10 (7.3%) of 137 non-HTR were anti-HEV positive (p<0.01 versus healthy controls, Table 1), while there was no difference between HTR and non-HTR (p = 0.2). Notably, HEV RNA was undetectable in all non-HTR including the 10 anti-HEV positive patients.

Discussion

Elevated liver enzymes in heart transplant recipients are a frequent finding. Our study reveals that chronic hepatitis E should be considered in the differential diagnosis of elevated liver enzymes in heart transplant recipients. Notably, all four HTR with ALT levels above 200 IU/L showed persistent HEV-infections. HEV infection was associated with progressive liver disease and significant fibrosis or cirrhosis in three of the four patients. One of four HTR with chronic hepatitis E developed hepatic decompensation within few years after initially detectable HEV viremia.

The possibility of chronic evolution of HEV infection in immunosuppressed individuals has been well documented in recent years (4,10). The diagnosis of hepatitis E has important clinical consequences. Firstly, HEV-infected HTR may be infectious as HEV is secreted in various bodily fluids. Indeed, HEV RNA was detectable in the stool of one patient after the virus had already been undetectable in plasma. Patient-to-patient transmission of HEV in hospitalized patients with impaired immunity has been described (20). Thus, we strongly recommend to strictly applying hygienic precautions for patients with chronic hepatitis E to prevent further spreading of HEV. Secondly, chronic hepatitis E is a treatable condition. Interferon alpha is contraindicated in heart transplant recipients due to the risk of inducing rejections. However, ribavirin was effective in HTR with persistent HEV infection, this being in line with previous reports on the treatment of chronic hepatitis E with ribavirin (21,22). In all four patients liver transaminases rapidly normalized and three HTR cleared HEV. We used a 5-month course of ribavirin therapy which was slightly longer compared to the French reports (21,22). However, the optimal dose and duration of ribavirin therapy of chronic hepatitis E remain to be determined. Our observation in HTR-4 showing an increase in both HEV RNA and ALT levels once ribavirin had to be reduced due to anemia suggests that lowering the ribavirin dose may lead to treatment failure. We would therefore suggest avoiding doses of ribavirin of less than 600mg/ day until more studies defining the optimal dose of ribavirin have become available.

The overall prevalence of chronic hepatitis E in heart transplant recipients in this prospective study performed in a nonendemic country was low with only 4 out of 274 HTR being HEV RNA positive. These data are well in line with the only two other systematic HEV-screening studies in solid organ transplant recipients. These studies that were performed in the Netherlands and Germany showed HEV RNA frequencies of each 1% in liver transplant recipients (8,9).

Clearance of HEV infection after heart transplantation was more frequent in patients with immunosuppressive treatments containing MMF (p = 0.03). The reason for this finding is unclear, and the number of cases is only very small; thus this finding should not be overestimated and further studies are required to confirm this result. Previous studies did only identify the use of tacrolimus being associated with chronicity (13). Still, one could speculate that MMF, an IMPDH inhibitor, displays some antiviral activity against HEV. MMF increases interferon-stimulated gene expression and has been shown to inhibit hepatitis C virus infection in vitro and in vivo (23). It will be interesting to investigate if a similar mechanism could explain the absence of chronicity in hepatitis E in MMF treated HTR in this study.

A surprising finding of this study was the high prevalence of HEV antibodies both in heart transplant recipients as well as in nontransplanted patients undergoing cardiac surgery being four- to fivefold higher compared to healthy individuals. In previous studies investigating different cohorts of immunosuppressed patients under risk for HEV infection the anti-HEV seroprevalence rates were significantly lower with less than 6% in all studies (9,15–17) and 4.0% overall (Table 1). The reason for the particularly high prevalence of anti-HEV antibodies in patients undergoing cardiac surgery remains unknown. One may speculate that patients with cardiac diseases receive blood products more frequently and thus have a higher risk for exposure to HEV. Plasma donations tested HEV RNA positive in 1:4525 and 1:7986 in Germany and Sweden (24). Overall, plasma pools may be contaminated with HEV in up to 10% of cases (25).

In conclusion, chronic hepatitis E may explain elevated liver enzymes in heart transplant recipients. Treatment of HEV infection with ribavirin is effective but the optimal dose and duration of ribavirin therapy remains to be determined. The prevalence of anti-HEV antibodies is higher in heart transplant recipients and nontransplant cardiac patients undergoing cardiac surgery. The underlying cause for this observation remains to be determined.

Acknowledgments

This work was supported by a grant from the German Federal Ministry of Education and Research (reference number: 01EO0802) and by a grant from the Robert Koch Institute on hepatitis E to Sven Pischke and Heiner Wedemeyer (reference number 1362-1097).

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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