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Immunocompromised patients are at risk of chronic hepatitis E which can be acquired by blood transfusions. Currently, screening of blood donors (BDs) for HEV RNA with a limit of detection (LOD) of 2,000 IU/ml is required in Germany. However, this may result in up to 440,000 IU of HEV RNA in blood products depending on their plasma volume. We studied the residual risk of transfusion-transmitted (tt) HEV infection when an LOD of 2,000 IU/ml is applied.
undertook a study wherein they performed individual donor nucleic acid testing (ID-NAT) for hepatitis E virus (HEV) in Germany using the Procleix HEV assay (Grifols, Barcelona, Spain). Using a similar approach, between January 4th, 2016 and March 17th, 2017, 172,277 blood donations were screened for HEV by the Irish Blood Transfusion Service. Screening was performed by ID-NAT using the Procleix HEV assay on the automated Procleix Panther System (Grifols). Irish blood donors consented for their samples to be used for the purposes of anonymous research. Forty-three confirmed HEV RNA-positive blood donations were identified, resulting in an overall incidence of 1:4,006. The HEV viral loads ranged from ∼0.5-4.3 log10 IU/ml. Twenty-seven donations were window period donations; one was anti-HEV IgM positive; 4 anti-IgG positive; the remaining donations were positive for both IgM and IgG (Fortress Diagnostics, Antrim, Northern Ireland).
performed molecular typing of the HEV-positive donations with subtype 3c predominating, with a further virus identified as subtype 3f consistent with the prevalence of these subtypes in Germany. Sequence analysis was performed on the HEV RNA-positive Irish donor samples using a combination of methods targeting the methytransferase (Met) and RNA-dependent (directed) RNA polymerase (RdRp) in open reading frame 1 (ORF1).
who reported that they failed to identify rabbit HEV (HEV-3ra) in their cohort, one Irish donor virus (IE-568) was clearly related to HEV-3ra isolates. The Met sequence for IE-568 shares 87.6% nucleotide identity with a HEV-3ra from Japan (LC535077) whilst the RpRp sequence for IE-568 shares 92.58% identity with HEV 3ra sequences obtained from rabbits in China and Mongolia (KX227751 and AB740222, respectively).
Two further unclassified HEV isolates (IE-112 and IE-224) were found to share 88% and 89% nucleotide identity with each other for the Met and RdRp sequences, respectively; however, they were more distantly related to other HEV isolates in the GenBank database. Phylogenetic analysis of concatenated Met and RdRp sequence fragments (525 bp in total) demonstrated that IE-568 lies firmly within the HEV 3ra clade,
and IE-112 and IE-224 form a distinct clade clustering outside of HEV-3ra and basal to most HEV-3 sequences (Fig. 1A). In order to improve resolution of this new phylogenetic clade, it was possible to perform next generation sequencing for IE-112 using an HEV-sequence enriched library approach.
The full-length sequence shared, at best, only ∼77-80% nucleotide identity with HEV-3ra and genotype 3 sequences in GenBank. Phylogenetic analysis of the full-length IE-112 sequence confirmed a clustering outside the HEV-3ra clade and basal to other HEV-3 clades, but distinct from the non-HEV-3 Orthohepevirus A genotypes (Fig. 1B); separate analysis of ORF1, ORF2 and ORF3 of IE-112 showed overall similar phylogeny for the novel sequence (not shown). Using several different approaches, there was no evidence of recombination in the IE-112 sequence, including the hypervariable region in ORF1 (Fig. S1-S3). Although IE-112 does not contain the 90/93 bp insert, within the ORF1 X region, characteristic of HEV-3ra viruses, neither does a recently identified virus (rab81) isolated from a wild rabbit in Germany
(Fig. S4). Both IE-II2 and IE-224 form a distinct and novel HEV clade distantly related to HEV-3ra.
Re-testing of IE-568, IE-112 and IE-224 using the altona Diagnostics realStar® HEV RT-PCR kit (altona Diagnostics, Hamburg, Germany) yielded viral loads of 1.1, 4.3 and 2.8 log10 IU/ml, respectively. Using the Cobas® HEV/Cobas® 6800 System (Roche Diagnostics, Mannheim, Germany) IE-112 and IE-224 had viral loads of 4.4 and 2.5 log10 IU/ml compared to the World Health Organization International Standard (6329/10), similar to the Altona Diagnostics assay values; IE-568 was not detected since the viral load was below the 95% cut-off of the Cobas® HEV assay (18.6 IU/ml). Nevertheless, we have previously observed reduced sensitivity (∼2 log10 IU/ml) for one HEV-3ra isolate (MG211750)
with the Cobas® HEV assay, but not the Procleix assay, although it is not known if this is the case for other HEV-3ra viruses.
All 3 donors ate pork with donor IE-112 reported to have consumed “black pudding” (pork/beef blood sausage) as well as cured meats. Whilst donor IE-224 reported eating medium/rare venison, the other donors denied eating game meat including rabbit. One donor (IE-112) owned a dog, the other donors had no pets. No donors reported contact with farm animals; donor IE-568 reported contact with a koala in an Australian zoo ∼4 months prior to donation. Although donors IE-112 and IE-224 donated within 1 week of each other, one lived in Dublin whilst the other lived in a rural area on the West coast. None of the donors travelled abroad in the 9 weeks prior to donation suggesting autochthonous transmission of HEV. No post-donation illnesses were reported. HEV-3ra sequences have mainly been identified in immunosuppressed individuals
and the detection of IE-568 in a healthy blood donor is noteworthy. With the identification of the novel isolates IE-112 and IE-224, forming a new clade and potentially a tentative new genotype, further investigations are required to understand potential animal origins associated with these Irish isolates and potential routes of transmission which, in the case of HEV-3ra, where it has been identified in humans, remain elusive.
The Irish Department of Health provided financial support for HEV blood donor screening by the Irish Blood Transfusion Service. In addition, this work was funded in part by core funding by the German Federal Ministry of Health (Bundesministerium für Gesundheit) (S.A.B.) and a grant ZMVI1-2518FSB705 (V.M.C).
S.A.B - analysis of HEV-positive donor samples, writing and review of manuscript; N.O’F - HEV screening, review of manuscript; L.B. - HEV screening, review of manuscript; B.H. - analysis of HEV-positive samples, review of manuscript; VMC - analysis of HEV-positive donor samples, writing and review of manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Please refer to the accompanying ICMJE disclosure forms for further details.
We thank Michel Molier and Roswitha Kleiber for excellent technical assistance and Julia Schneider, Jörn Beheim-Schwarzbach and Tobias Bleicker for help with sequencing.
The following are the supplementary data to this article: