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Enterically transmitted human hepatitis E virus (HEV) has long been known as a major cause of acute hepatitis E in developing countries, with occasional travel-related cases in developed countries.1 Where studied, HEV infections have been associated with self-limiting hepatitis. Recently, chronic HEV infection has been described in immunosuppressed patients after organ transplantation.2–5 We reported on the development of chronic hepatitis E to cirrhosis and subsequent graft failure in 2 liver transplant recipients.4 These observations raise important questions about the prevalence and course of HEV infection in liver transplant recipients.
All organ transplant patients reported with chronic HEV infection were infected with HEV of genotype 3.2–5 This HEV genotype has been recognized in the past decade as a highly prevalent infection in pigs across the world1, 6 and has occasionally been detected as a cause of illness in humans.7–11 The sources of HEV infection in humans still remain unknown, although the high genetic similarity between human and porcine HEV strains suggests some relationship, but the exposure of both hosts from an as yet unknown third reservoir is possible as well. Transmission of HEV via food, direct contact, water, and blood transfusion has been described.1, 8 The observation that organ transplant recipients may be carriers of HEV points at a critical data gap for risk assessments needed to advise on the public health impact of the presence of HEV in pigs and in the environment.
Therefore, we studied a large cohort of adult liver transplant recipients for evidence of incident or past HEV infection.
All 331 patients presently alive (as of January 2008) and transplanted at an adult age between March 1979 and January 2007 at the University Medical Center Groningen were considered for the present study. The presence of freeze-stored sera in a biobank allowed for screening of sera for evidence of incident or past HEV infection. Most sera were collected during annual controls. We selected the most recent serum samples for immunoglobulin M (IgM) and immunoglobulin G (IgG) screening using an enzyme-linked immunosorbent assay (ELISA) technique and confirmatory western immunoblot analysis. Also, samples were analyzed with real-time polymerase chain reaction. Serum samples were considered to be indicative of a past or present HEV infection when positive for HEV RNA and/or positive for IgM or IgG by both ELISA and confirmatory immunoblotting.
In these cases, older serum samples were taken from the bank and tested by the same methods. Charts from patients highly suspected for past or present HEV infection were reviewed for details of the medical history.
Diagnostic Workup for HEV
The serological diagnosis of HEV was made by assays of sera for the presence of anti-HEV IgM and IgG with commercially available methods (Genelabs Diagnostics, Inc., Redwood City, CA) with confirmatory testing by immunoblotting (Mikrogen GmbH, Neuried, Germany). Cutoff values for the assays were defined for testing in a low-endemic setting as described.12 A combination of ELISA screening and confirmatory Western immunoblotting gave specificities of 96% and 99% for IgM and IgG, respectively. The sensitivity was 100% for IgG detection but somewhat lower (88%) for IgM following genotype 3 infection in the combined test regimen.12
In addition, serum samples were tested for the presence of viral RNA. For detection, a real-time reverse-transcription polymerase chain reaction method with a TaqMan probe was used as described.13
Recent serum samples were available for 285 of the eligible 331 patients (86%).
Patient characteristics are listed in Table 1. The serum samples were obtained in 2006 or 2007. Serological signs of past or present infection were found in 11 of the 285 patients (3.9%).
Samples from 274 patients (96.1%) tested negative for all HEV parameters. This included a patient described in an earlier article.4 She experienced chronic HEV infection (genotype 3) with hepatitis after her first liver transplant, but the HEV infection was cleared after retransplantation, and the antibody levels decreased below the detection limit over the course of 1 year.
One patient was found positive for HEV RNA without HEV antibodies. This patient was already known to us and was described earlier.4 Briefly, after her first transplant, she developed chronic HEV hepatitis (genotype 3) with subsequent cirrhosis. After retransplantation, chronic HEV hepatitis recurred.
Sera from 9 patients tested positive for HEV IgG without HEV IgM or HEV RNA 1, 5, 5, 5, 6, 7, 12, 13, or 18 years after orthotopic liver transplantation (OLT; median, 6; range, 1-18).
A retrospective analysis pointed to an HEV infection in the past, well before transplantation, in 6 of these 9 patients. Five of these 6 patients were found to be HEV IgG–positive from the pretransplant sample onward up to the most recent samples from 1, 5, 5, 12, or 13 years after OLT. In these patients, in-between samples were determined every 2 years on average. One of the 6 patients tested positive before transplantation, but HEV IgG was negative in the first 4 years after OLT and reappeared in the sample of year 5.
A posttransplant episode of HEV infection was suspected in retrospect in 1 of the 9 patients presently positive for HEV IgG. This patient underwent transplantation in January 2000. The case is illustrated in Fig. 1. Before transplantation and 1 year after OLT, the patient tested negative for HEV antibodies and HEV RNA. At 2 years after OLT, HEV IgM and IgG were positive in combination with the presence of HEV RNA. The serum from the fourth year showed IgG reactivity and HEV RNA. In the seventh year, only IgG was detected, and HEV RNA was absent. The medical charts revealed normal liver tests and normal liver histology 1 year after OLT. A mild increase in aminotransferases of unknown origin was seen between February 2002 and September 2004. Liver histology in March 2004 showed only mild nonspecific changes. In retrospect, we now conclude that this patient suffered from mild hepatitis caused by HEV infection for almost 3 years, after which the virus was successfully cleared. With respect to possible risk factors for HEV, the patient is known to use a diet that includes pig meat, and she did not travel abroad the year before she acquired the HEV infection.
One of the 9 patients tested positive for HEV IgG from the first year after OLT onward. Before transplantation, serology was negative, and samples analyzed in the first year never revealed HEV RNA, HEV IgM, or HEV IgG. The last of the 9 patients tested positive for HEV IgG from 3 to 18 years after OLT, but earlier samples were lacking.
One patient tested positive for HEV IgM 10 to 12 years after transplantation. The charts do not show a rise in liver tests during this time. Earlier samples were negative, and IgG and HEV RNA were never detected.
In general, HEV infection is known to run a self-limiting course with a more or less severe episode of hepatitis.1 Recently, chronic HEV infection has been described in several immunosuppressed patients after organ transplantation.2–5 In the 2 cases reported previously by us, chronic HEV hepatitis developed gradually into cirrhosis, after which retransplantation of the liver was needed.4 It was postulated that HEV may be one of the hepatotropic viruses responsible for the recently recognized entity of unexplained chronic hepatitis after liver transplantation, which has been reported to occur in about 10% of patients.14, 15 Therefore, the present study was focused on signs of HEV infection in an adult population at a median of 9 years after liver transplantation.
Ninety-six percent of the available sera tested negative for HEV RNA and/or HEV antibodies. Therefore, the prevalence of past or present HEV infection in liver transplant recipients seems low. However, this low prevalence may be an underestimation as HEV antibodies may disappear after clearance of the virus, especially under immunosuppression. Examples of this are the absence of antibodies in the patient previously diagnosed with chronic HEV hepatitis after the first transplant, which was cleared after retransplantation,4 and the patient analyzed in this article in whom HEV IgG was present pre-transplant but disappeared after transplantation and subsequently reappeared in the fifth year after transplantation.
Six patients (2.1%) were found to have HEV IgG antibodies in retrospect related to HEV infection at some time pre-transplant as they also tested positive in a pretransplant serum sample. Most of these patients with past HEV infection were native Dutch citizens. Pretransplant seropositivity most likely implies a lifelong immunity against reinfection. Again, the low seropositivity rate before transplantation may be an underestimation, as we did not test pretransplant sera of all study patients and the patients are now under immunosuppression. Still, the figure seems much lower than that seen for hepatitis A virus infection in our country, the prevalence of antibodies to hepatitis A virus being 20% to 36% in native Dutch citizens.16, 17
One presently seropositive patient suffered in retrospect from a chronic HEV infection with mild hepatitis between 2 and 5 years after liver transplantation (Fig. 1). In contrast to the previously published cases from our center, the hepatitis was only mild without long-term sequelae. Seroconversion occurred spontaneously after 3 years. These are important observations because it shows that chronic HEV infection after liver transplantation may be active and of long duration, leading to cirrhosis,3–5 or may be mild with an adequate immune response even after a relatively long time. The successful clearance of the virus in this patient compared to the others might well be related to a different and lower immunosuppressive regimen. The previously reported patients from our center4 used a regimen including prednisolone, whereas the present patient was on monotherapy with tacrolimus.
Overall, 3 patients of the present study population have or had a documented HEV infection with hepatitis acquired after transplantation. This means a 1% prevalence of de novo HEV hepatitis after liver transplantation. This might be higher (2%) if we include the 3 patients in whom we detected for the first time HEV antibodies after transplantation but in whom we could not detect with the use of available sera in retrospect an episode of HEV RNA positivity with hepatitis.
On the basis of this low prevalence, one may conclude that, although each individual patient with chronic HEV infection is a potential reservoir for spreading the infection, on a population scale, transplanted, immunosuppressed patients are not a major source for endemic HEV infection. One should consider this while keeping in mind the potential underascertainment due to the seroreversion of cases to a seronegative state. The currently available serological methods are not specific for genotype 3 HEV infections, thereby potentially being suboptimal for measuring immunity to these viruses. As soon as validated type-specific assays become available, we will recommend reassessing whether patients that became antibody-negative with the current assay will show the same pattern of reactivity.
It is worrisome that if indeed the prevalence of seropositivity for HEV is around 2% in liver transplant candidates at an adult age, another 1% to 2% will acquire the virus within several years after transplantation. So far, the genotype has been shown to be genotype 3 in all studies,2–5 and so we should consider pigs and pig meat as a source of infection. The transplanted patient may run an increased risk because of the use of immunosuppression.
For more insight, prospective studies are needed, with the determination of HEV parameters on a regular basis from the pretransplantation period onward and more often during episodes of liver enzyme abnormalities. For the time being, we recommend determining the HEV serological status during the evaluation phase for liver transplantation and considering the possibility of HEV hepatitis in the case of liver enzyme abnormalities of unknown origin. It is probably too early to advise patients to refrain from pig meat.