Article Text

Original article
Chronic hepatitis E infection in children with liver transplantation
  1. Ugur Halac1,
  2. Kathie Béland1,
  3. Pascal Lapierre1,
  4. Natacha Patey2,
  5. Pierre Ward3,
  6. Julie Brassard3,
  7. Alain Houde3,
  8. Fernando Alvarez1
  1. 1Division of Gastroenterology, Hepatology and Nutrition, Department of, Pediatrics, CHU Sainte-Justine, Université de Montréal, Quebec, Canada
  2. 2Division of Pathology and Cellular Biology, CHU Sainte-Justine, Université de Montréal, Quebec, Canada
  3. 3Agriculture and Agri-Food Canada, Food Research and Development Centre, Saint-Hyacinthe, Quebec, Canada
  1. Correspondence to Dr Ugur Halac, Division of Gastroenterology, Hepatology and Nutrition, CHU Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, Québec H3T 1C5, Canada; ugur.halac{at}umontreal.ca

Abstract

Objective Chronic hepatitis E virus (HEV) infection has been described in immunosuppressed adult patients. A study was undertaken to establish the presence of HEV infection in children after orthotopic liver transplantation (OLT).

Methods Children undergoing liver transplantation between 1992 and 2010 with available serum were classified into two groups: group 1 (control group, n=66) with normal serum aminotransferases and group 2 (n=14) with persistently increased serum aminotransferases and histological features of chronic hepatitis. Patients were tested for HEV RNA by reverse transcription-polymerase chain reaction (RT-PCR). HEV amplicons were sequenced and compared with published sequences. Antibody titres (IgG and IgM) to 12 HEV immunodominant regions were measured by enzyme-linked immunosorbent assays.

Results In group 1 (control group), 15% of children were anti-HEV IgG-positive during follow-up. No anti-HEV IgM antibodies were detected in any of these children. After OLT, 86% of patients in group 2 had anti-HEV IgG compared with 36% pre-OLT. Thus, two-thirds of children acquired anti-HEV IgG after OLT. Seven anti-HEV IgG-positive patients (58%) were also anti-HEV IgM-positive more than once during follow-up after OLT. Eight years post-OLT, one girl presented with anti-HEV IgG and IgM that remained positive afterwards. In this patient, HEV RNA was found in five different annual samples from 10 years post-OLT, concomitantly with increased serum aminotransferases and cirrhosis development during that period. Phylogenetic analysis revealed two different HEV strains (detected 3 years apart) that were highly similar to swine genotype 3, suggesting a possible case of zoonotic re-infection.

Conclusion The diagnosis of HEV infection is technically challenging and should be made simultaneously with RT-PCR methods, viral load quantification and serological markers. In immunosuppressed children who develop chronic hepatitis, the prevalence of HEV is high and could explain the chronic liver inflammation potentially leading to cirrhosis. Re-infection by different HEV strains from zoonotic transmission can result in progressive liver disease in immunocompromised children.

  • Hepatitis E virus
  • liver transplantation
  • chronic hepatitis
  • immunosuppression

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Significance of this study

What is already known about this subject?

  • Chronic HEV infection is possible in immunosuppressed adults but has not yet been described in children.

  • Diagnosis of HEV infection is difficult.

What are the new findings?

  • Chronic HEV infection is possible in immunosuppressed children.

  • This is the first report of chronic HEV infection in children from North America.

  • Re-infection by two different HEV strains in the same patient is probably possible.

  • Diagnostic techniques are challenging, but improved approaches are described.

How might it impact on clinical practice in the foreseeable future?

  • In the long-term follow-up of immunosuppressed children, HEV infection is one of the main differential diagnoses of liver abnormalities.

  • The prevention of HEV infection in immunosuppressed patients should include new long-term recommendations concerning food consumption habits (avoiding undercooked farm meats, especially pork, and thoroughly washing fruits and vegetables).

Introduction

Hepatitis E virus (HEV) appears to be an emerging threat in industrialised countries.1 2 HEV infection usually induces acute but self-limiting hepatitis in immunocompetent individuals,3 but complications such as fulminant hepatic failure and increased mortality rate are frequent, especially in patients with chronic liver diseases and pregnant women.4

Historically, HEV has been considered mainly as a source of acute hepatitis in developing countries with suboptimal sanitary conditions. The anti-HEV seroprevalence is high (17–45%) in Asia, Africa and Latin America5 where transmission is mainly orofaecal by drinking contaminated water. In developed countries, HEV infection was previously thought to be a disease ‘imported’ by worldwide travel, but growing evidence shows some sporadic HEV cases due to autochthonous (locally acquired) viral strains.6

Phylogenetic studies of HEV have produced evidence of four main genotypes identified as 1–4, divided into 24 different subtypes.7 Epidemic presentations are caused by genotypes 1 and 2, considered as primarily ‘human’ genotypes, and typically affect young adults. Genotypes 3 and 4 are associated with zoonotic infections and appear in farm-raised (swine) or wild (boar, deer) animals. Sporadic HEV infection is suspected to be a zoonosis, and transmission could occur by consumption of infected animal meat.5 8 Indeed, swine and human HEVs show extremely close genetic similarities (HEV genotype 3 in particular), suggesting that pigs may serve as reservoirs for human HEV infection.9

Recently, the idea that HEV infection leads to acute hepatitis was reconsidered when chronic HEV infection was found in organ transplant recipients10–15 and patients undergoing chemotherapy.16 These observations led us to consider HEV infection in the management and follow-up of liver transplant recipients. In paediatrics, available data regarding HEV infection are limited to case reports and to a single Spanish study that detected 4.6% IgG seroprevalence in a large cohort of healthy children.17

Unexplained liver anomalies, such as increased serum aminotransferases levels or histological features of chronic hepatitis, are a permanent issue for some patients followed in liver transplant programmes. Because no aetiology has been defined in numerous patients, we studied a cohort of children who had undergone liver transplantation in order to investigate past and current HEV infection as a cause for these abnormalities.

Methods

Patients and serum samples

We studied all children who have undergone liver transplantation in our centre from 1992 to 2010 and for whom enough serum was available. The cohort was divided into two groups based on features observed during follow-up: patients with normal serum aminotransferases (group 1) and those with persistently increased serum aminotransferases levels and histological features of chronic hepatitis (group 2).

Group 1 (control group)

Sixty-six patients who had a liver transplantation from 1992 to 2010 (37 girls, median age 13.7 years (range 1.8–25.5) and biliary atresia (BA) as the most frequent indication of orthotopic liver transplantation (OLT) (31/66)) were included in the control group. None of them showed any sign of chronic liver disease during follow-up. Two hundred and twenty-one serum samples from these 66 patients were tested for anti-HEV immunoglobulin G (IgG) and anti-HEV immunoglobulin M (IgM).

Group 2

Group 2 was composed of 14 patients who underwent liver transplantation (eight girls, median age 17.4 years (range 5.9–19.8)) with past or current abnormal serum aminotransferases levels post-OLT (at least two times the upper limit of normal) and histological features of chronic hepatitis without any defined aetiology despite extensive screening. In our hospital, the upper limit for normal alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are 25–34 U/l and 43–70 U/l, respectively, according to the age of the patients. These children underwent OLT at a median age of 2.5 years (range 10 months–5.6 years). The original organ diseases were tyrosinaemia (7/14), BA (5/14), Amerindian cirrhosis (1/14) and Alagille syndrome (1/14). Current median follow-up duration was 14.6 years (range 5.1–17.6). The chart of each patient was reviewed to study all reported clinical features. The demographic characteristics of all the children are reported in table 1. One hundred and twelve serum samples freeze-stored at −80°C from these 14 OLT children (representing all available blood samples for these patients in our unit's biobank) were studied for HEV RNA detection and for anti-HEV IgG and anti-HEV IgM. These serum samples included pre-OLT samples from 11 of the 14 children who underwent liver transplantation. Sixty-four liver biopsies from all children were reviewed by experienced pathologists and given Metavir scores.18

Table 1

Demographic characteristics of patients from group 2

Viral RNA extraction and detection of HEV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR)

Viral genomic RNA was extracted from 200 μl freeze-stored serum with the QIAamp Viral RNA Mini Kit (Qiagen, Mississauga, Ontario, Canada) according to the manufacturer's instructions with a final elution of 50 μl. The presence of HEV was based on HEV RNA detection by nested RT-PCR amplification with primers derived from the ORF2 region.19 Three different volumes of prepared RNA (2, 10 and 18 μl) were analysed to ensure high sensitivity. We employed external primers 3156N (forward, 5′-AATTATGCYCAGTAYCGRGTTG-3′; nucleotide positions 5663–5684) and 3157N (reverse, 5′-CCCTTRTCYTGCTGMGCATTCTC-3′; nucleotide positions 6371–6393) to obtain an amplification product of 731 base pairs (bp).19 Amplifications were performed with the Qiagen OneStep RT-PCR Kit (Qiagen) according to the manufacturer's instructions. Nested PCR (PyroStart Fast PCR Master Mix, Fermentas, Burlington, Ontario, Canada) was then performed on the first PCR product with the internal primers 3158N (forward 5′-GTWATGCTYTGCATWCATGGCT-3′; nucleotide positions 5948–5969) and 3159N (reverse 5′- AGCCGACGAAATCAATTCTGTC-3′; nucleotide positions 6274–6295), resulting in a PCR product of 348 bp.19 Nucleotide positions were based on the HEV reference sequence (GenBank accession number NC_001434). The expected nested PCR product size was 348 bp.

The PCR parameters for first-round PCR with primers 3156N and 3157N were: a reverse transcription step at 50°C for 30 min followed by a denaturation step at 94°C for 15 min, then 40 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 50°C, extension for 1 min at 72°C, with a final extension at 72°C for 7 min. The parameters for nested PCR were an initial denaturation step at 94°C for 5 min followed by 39 cycles of denaturation at 94°C for 45 s, an annealing step at 50°C for 1 min and extension at 72°C for 1 min, finishing with a final extension at 72°C for 7 min.

Determination of relative HEV viral load by TaqMan RT-qPCR

The relative HEV viral load was quantified at the Agriculture and Agri-Food Canada Food Research and Development Centre (Quebec, Canada) according to published techniques.20 Feline calicivirus (FCV) served as sample process control to monitor nucleic acid extraction and identify possible amplification inhibitors.20 3.2×103 plaque-forming units of FCV were added to 200 μl serum samples before RNA extraction with the QIAamp Viral RNA Mini Kit. Multiplex TaqMan RT-qPCR assays in a final volume of 25 μl were conducted with Brilliant II QRT-PCR Core Reagent Kit, 1-Step (Stratagene, La Jolla, California, USA) for the concomitant detection of HEV and FCV, as previously published.20 Amplifications were performed with a Stratagene Mx3005P system combined with MxPro4.01 software (Stratagene). The amplification profile was 30 min at 50°C followed by 10 min at 95°C and 45°C cycles of 94°C for 15 s and 60°C for 1 min. A standard curve was generated for HEV with 10-fold serial dilution (108–102 copies) of purified HEV DNA plasmid in a 5 ng/ml salmon sperm DNA solution and amplified in triplicate.20 Standard curves for HEV were included on each plate, allowing a relative quantification of all samples. The lower limit of detection for HEV RNA was 1×103 copies/ml of sample.

Nucleotide sequencing and phylogenetic analysis

The amplified 348 bp fragments from nested RT-PCR were purified with the QIAquick Gel Extraction Kit (Qiagen). Sequencing was performed on the amplified fragments in an ABI 3730 DNA analyser (Applied Biosystems, Carlsbad, California, USA). All sequences were deposited in GenBank under accession numbers PT11-03: HM772982, PT11-06: HM772983, PT11-07: HM772984, PT11-08: HM772985, PT11-09: HM772986. The HEV genotype of amplicons was determined by sequence alignment with publicly available HEV sequences from all known HEV genotypes using the BLAST program (http://www.ncbi.nlm.nih.gov). HEV viral amplicon sequences were also used to construct a phylogenetic tree with ClustalW2 and the neighbour-joining algorithm (MEGA 5.0). The statistical confidence of phylogenetic relationships was ascertained by bootstrap analysis.

Serological diagnosis of HEV

Independently of PCR results, all serum samples were tested to establish the anti-HEV status. Antibody reactivity (IgM and IgG) to 12 HEV immunodominant regions was measured by ELISA with a chimeric HEV antigen (Hepatitis E Virus Mosaic Recombinant, Feldan Bio Inc, Saint-Augustin, Quebec, Canada), as previously described.21 Briefly, microwell ELISA plates (Corning, New York, New York, USA) were coated with 0.2 μg per well of chimeric HEV antigen. Serum samples were loaded at a dilution of 1:50. The presence of anti-HEV antibodies was revealed by incubation with anti-human IgG or anti-human IgM-alkaline phosphatase-conjugated antibodies (Sigma-Aldrich, Oakville, Ontario, Canada) at a dilution of 1:1000. Alkaline phosphatase was developed by incubation with p-nitrophenyl phosphate and the result read at 405 nm. Antiserum was considered positive if its specific optical density was at least two times higher than the mean optical density of healthy control serum.

Results

In group 1 (control group), 10/66 (15%) children were anti-HEV IgG-positive during follow-up. None of these children showed IgM type anti-HEV antibodies.

In group 2, before OLT, serum samples from four out of 11 children tested (36%) were anti-HEV IgG-positive (table 2). Three of them were French-Canadian and came from semi-rural regions in the centre and north of Quebec, while the fourth child was an Amerindian boy living in a First Nations community under inadequate socioeconomic conditions and who regularly ate wild animal meat. After OLT, the rate of positive anti-HEV IgG patients increased to 86% (12/14, table 2). Thus, at least two-thirds of children acquired anti-HEV IgG after OLT. Nine (75%) out of 12 anti-HEV IgG-positive patients were also anti-HEV IgM-positive after OLT (table 2). Among these nine children, seven had anti-HEV IgM several times in different serum samples during the post-OLT follow-up period. These results indicate a significantly lower seroprevalence in transplanted children without abnormal serum aminotransferases values and histological features of chronic hepatitis (p<0.0001 according to Poisson model with exposure: follow-up time and number of tested serum samples). The retrospective nature of our work could be responsible for some heterogeneity in this group. Serum samples were not obtained at regular intervals after OLT owing to the timing of their follow-up. This has limited our ability to determine precisely the time of seroconversion (see table 1 in online supplement).

Table 2

Hepatitis E virus (HEV) serology and the presence of serum viral RNA in children who have had a liver transplant from group 2

The liver histology and Metavir scores for patients from group 2 (table 3) showed a trend towards deterioration through time post-OLT for disease activity level and fibrosis stage. Stage 2 or higher fibrosis was observed at the first liver biopsy in four out of 13 patients. Fibrosis was progressive or stable but marked (stage 2 or higher) in at least 11 children in subsequent liver biopsies during follow-up. A contact with HEV was suspected in nine of these 11 patients, as they concomitantly had IgG and IgM types anti-HEV antibodies.

Table 3

Histopathological evolution (Metavir score) after OLT in patients from group 2

Anti-HEV IgG, anti-HEV IgM and HEV RNA were present simultaneously in one female patient (patient 11). No pre-OLT serum sample was available for this patient. Currently 20 years old, she was transplanted at the age of 2.7 years for tyrosinaemia. Eight years after OLT, anti-HEV IgG and IgM were detected in her serum and were continuously positive afterwards. HEV viral RNA was found in five different annual samples from 10th years after OLT (from 2003 to 2009, respectively, at 10–16 years post-OLT), concomitantly with abnormal serum ALT and AST levels. Viral load was quantified in four out of five HEV RNA-positive samples and revealed a rate of 106–108 copies/ml serum (6.8×104 to 1.1×106 copies/μl of isolated RNA). Her serum aminotransferases activity was significantly and chronically abnormal, despite some slight fluctuations (figure 1). No definitive aetiology was found that could explain these anomalies after extensive screening. Histological examination showed portal inflammation and interface hepatitis which developed into progressive fibrosis and finally cirrhosis (table 3 and figure 1). Phylogenetic analysis of HEV sequences identified them as belonging to HEV genotype 3a (figure 2 and supplemental figure 1). The close similarity of HEV sequences detected from 2006 to 2009 in this patient (13–16 years post-OLT) strongly indicated that she was infected with the same HEV isolate during that period. However, phylogenetic analysis showed that the HEV RNA sequence from the 2003 sample (10 years post-OLT) was different (figure 2). Isolated HEV sequences were compared with all publicly available HEV sequences. The HEV isolate from year 2003 was 93% homologous to an HEV swine sequence found in Quebec (swSTHY51, GenBank accession number DQ860009) , while the sequences from 2006 to 2009 were 96% homologous to another swine sequence also isolated in Quebec (swSTHY7, GenBank accession number DQ859990). Among all HEV sequences compared, these swine sequences showed the highest homology.

Figure 1

Liver disease activity and hepatitis E virus (HEV) infection. Course of HEV infection and markers of liver disease activity in patient 11 between 2 months and 16.2 years after orthotopic liver transplantation (OLT). Major increases in serum aminotransferase levels and histological deterioration were concomitant with positivity of HEV IgG, IgM and viral RNA. ALT, alanine aminotransferase; AST, aspartate aminotransferase; IgG, immunoglobulin G; IgM, immunoglobulin M; A, activity (Metavir score); F, fibrosis (Metavir score).

Figure 2

Phylogenetic tree analysis of isolated hepatitis E virus (HEV) sequences. Phylogenetic analysis was performed with sequences obtained from patient 11 and the genome sequence of each HEV genotype. HEV sequences isolated in 2003 (hHEV_Pt11_03) are from a different HEV strain than those obtained from 2006 to 2009 (hHEV_Pt11_06 to hHEV_Pt11_09). All HEV sequences from patient 11 belong to HEV genotype 3. Bootstrap values are reported in the tree.

This girl was on calcineurin inhibitors (tacrolimus) at dose levels between 0.06 and 0.08 mg/kg/day for 7 years after OLT. From 7 to 13 years post-OLT, mycophenolate mofetil (MMF) at 25 mg/kg/day was added because of the presumptive diagnosis of chronic rejection. Rapamune replaced MMF from 13 years after OLT at a dose level of 0.04 mg/kg/day for a 2-year period. After pejorative biological evolution and histological suspicion of severe chronic rejection, corticosteroid recycling was instituted. Rapamune was stopped and MMF in enteric-coated form (Myfortic®) was started from 2008 until today.

Discussion

HEV infection has recently been recognised as a possible chronic disease in immunosuppressed adult patients after organ transplantation.10–12 15 HEV data on paediatric populations under immunosuppression are very limited and, interestingly, there are few studies from North America where HEV is, however, a well-known problem in animal herds.19 22 23 In this regard, the present study is an extended analysis of HEV status in paediatric organ recipients. We investigated all liver-transplanted children in our transplant programme to gather evidence of HEV infection. We divided them into two groups based on the presence or absence of previous or current serum aminotransferases elevation (at least two times the upper limit of normal) without any defined aetiology and histological features of chronic hepatitis. As in adult populations,10–12 15 our results confirm that HEV infection can become chronic in immunosuppressed paediatric patients. These data have important implications in the pre-OLT assessment of candidates and in the follow-up of immunosuppressed children, especially for infection prevention and/or treatment.

The origin of ‘locally acquired’ HEV infection in developed countries is not clear. However, in recent years, several studies on farm animals worldwide, including the USA and Canada, have reported significant seroprevalence of HEV, especially in swine herds.19 22 23 In addition, the presence of anti-HEV IgG in pig handlers and farm veterinarians2 24 emphasises the suspicion of zoonotic transmission. Two other factors support the possibility of zoonosis: reports of HEV transmission from pigs to humans and vice versa9 25 and high genomic similarities between swine and human HEV strains.26 Together, these reported findings strongly suggest that HEV may be a zoonotic infection with animals such as pigs acting as viral reservoirs. Patient 11 had not travelled to HEV endemic regions. She has been living in a semi-rural region of central Quebec without specific lifestyle or nutrition restrictions. She therefore acquired the HEV infection in Canada. Established genotype 3a HEV strains in this patient showed high similarities with the genotype 3a strains in swine, particularly two strains found in Quebec. These observations strongly suggest that zoonotic transmission of HEV occurred in this young patient.

The presence of IgG and mainly anti-HEV IgM were significantly more frequent in patients with abnormal serum aminotransferases levels and histopathological features of chronic inflammation after OLT than in the control group. These results are indirect evidence for the role of HEV in the development of chronic hepatitis in immunosuppressed children. Indeed, 12 out of the 14 patients studied showed anti-HEV IgG after transplantation while only four were positive before OLT. Anti-HEV IgM was positive in several serum samples during follow-up in seven patients after OLT, hinting at a possible re-infection or eventual re-activation of HEV. Despite the lack of direct evidence to confirm re-infection or re-activation (except for patient 11), these findings are compatible with the observed features of chronic hepatitis present in several liver biopsies and the elevated levels of ALT and AST present in our patients. The fact that all serum samples from the control group were negative for anti-HEV IgM also supports the assertion that persistent anti-HEV IgM positivity is associated with an ongoing fluctuant viral replication, as was described in patients with chronic hepatitis B infection.27 Transplanted children usually receive firm recommendations concerning potential food restrictions (to avoid raw or undercooked meat and unpeeled fruits) and exposure to animals. Most patients in this cohort were living in semi-rural regions of Quebec with a potential risk of contamination from farm-raised animals. Recently, our group isolated HEV strains in strawberries from southern Quebec, possibly due to contaminated irrigation systems and/or fields fertilised with infected swine manure. This HEV isolate was sequenced (strain CC2009-1047, GenBank accession number HQ415969) and phylogenetic analysis revealed a high similarity with sequences isolated from patient 11 and from previously reported swine HEV sequences from Quebec22 (data not shown). The possibility of this contamination pathway has never been reported for immunosuppressed patients but could represent another potential source of HEV infection for children after OLT. We believe that HEV could also be present in other fruits and vegetables grown in fields close to swine farms.

Our study highlights the importance of viral load quantification to corroborate the final HEV diagnosis. The viral load found in our positive patient is relatively high. However, detection of the viral genome by PCR is technically challenging. Indeed, even in investigations on farm-raised animals (such as swine) where HEV infection is widespread, the threshold of HEV RNA detection is around 103to 104 copies per ml of serum or per gram of faeces.28 29 Manipulation of blood samples, conditions of serum conservation for many years in some cases, and repeated freeze-thaw cycles of samples, are possible causes of HEV RNA degradation. HEV PCR-negative serum samples therefore have to be considered cautiously, particularly when compatible medical history, abnormal ALT/AST values and chronic hepatitis histology are present without any other defined aetiology. Indeed, HEV RNA could be undetectable due to a very low viral load or RNA degradation. Standardisation of the methods used for the detection and quantification of HEV in humans would be beneficial and would ensure accurate diagnosis of HEV infections.

Interestingly, we found two different HEV strains (both genotype 3) at two different time points in the same patient. This may suggest three possibilities: (1) a case of re-infection by a different HEV strain; (2) the presence of both strains in the same serum sample but without the ability to detect them simultaneously; or (3) a different clustering of the same strain due to the viral evolution. The ladder could be verified by clonal analysis and deep sequencing but, unfortunately, due to a lack of serum samples, this could not be performed. Except in case of a very low viral load, we do not believe the second option (a ‘false negative’ result). Despite the technical challenge of the PCR procedure, our technique showed reliable detection results. In addition, the progressive liver deterioration from 2006 to 2009 was different compared with the status in 2003. A higher abnormal serum aminotransferase level and the occurrence of cirrhosis concomitant with an increased viral load suggests a ‘new’ viral episode. To date, no case of re-infection with different phylogenetically-established HEV strains has been reported in humans at different follow-up times. Therefore, in immunosuppressed children, repeated exposure to HEV, even years after transplantation, could induce re-infection by different strains, leading to chronic hepatitis as seen in liver biopsies and eventually to progressive fibrosis and cirrhosis as seen in our patient. This observation could have important consequences, especially in therapeutic and/or vaccination practices. Currently, no therapeutic options are available for HEV infection. Several case reports and studies in adult patients recently delivered encouraging results for HEV clearance with ribavirin and pegylated interferon α.30–32 However, additional studies with larger numbers of patients and longer follow-up are required. At present, when a diagnosis of HEV infection has been established after careful screening with other classical differential diagnoses, reducing immunosuppressive therapy targeting T cells still remains a reasonable first-line option in children.

In the long-term follow-up of immunosuppressed children with sustained increases in serum aminotransferases of unknown aetiology and histological features of chronic hepatitis, HEV infection is one of the main differential diagnoses from chronic graft rejection directed against periportal hepatocytes. Pre-OLT assessment of HEV status should therefore be methodically established for each transplant candidate and HEV infection should be systematically ruled out in patients with abnormal biochemical or histological liver status after OLT. This is particularly relevant to physicians caring for patients who have undergone liver transplantation because the treatment (reducing immunosuppression therapy) for HEV infections is the opposite of that proposed for the treatment of chronic rejection. We also believe that, after transplantation, immunosuppressed children should be careful in their meat consumption habits and avoid undercooked farm meats (especially pork), and should thoroughly wash fruit and vegetables.

In conclusion, the diagnosis of HEV infection is technically challenging and should be based on simultaneous exploration by PCR methods, viral load quantification and serological markers. A high prevalence of HEV is found in paediatric organ recipients who develop chronic hepatitis, and this could explain the chronic liver inflammation potentially leading to cirrhosis. Re-infection by different HEV strains, from probable zoonotic transmission could contribute to progression of the liver disease in immunocompromised children.

Acknowledgments

We thank Dr Dorothée Bouron-Dal Soglio from the Division of Pathology and Cellular Biology (CHU Sainte-Justine, Université de Montréal, Montréal, Québec, Canada) for her helpful comments and valuable assistance with the histopathological analyses. We also thank Lubomir Alexandrov (Applied Clinical Research Unit at Sainte-Justine Hospital) for his valuable help in statistical analyses and Mrs Morgane Stoyanov for her technical assistance in some RT-PCR and ELISA experiments.

References

Supplementary materials

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Footnotes

  • Competing interests None.

  • Ethics approval Ethics committee approval was obtained from Sainte-Justine Hospital.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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