Material and Methods
Samples
The study was performed on 100 pretested HEV IgG-positive, 53 HEV
IgM-positive, and 49 samples in which HEV IgG and HEV IgM were
undetectable. In addition, 17 samples with serological evidence of acute
Epstein-Barr virus (EBV) infection and two samples with acute human
cytomegalovirus (CMV) infection were included to investigate possible
cross-reactivity. All sera were residual samples which, with the
exception of the 19 samples mentioned above, were sent to the laboratory
Dr. Krause und Kollegen MVZ GmbH Kiel for serodiagnosis of HEV
infection. Information on clinical symptoms as well as liver function
was not available to us.
HEV assays
Initial HEV antibody status was determined manually using therecom Well HEV IgG or HEV IgM ELISA (Mikrogen GmbH, Neuried,
Germany) on a BEP2000 system (Siemens Healthineers AG, Erlangen,
Germany); this assay served as a reference here. Sera in which HEV IgG
or IgM were detected in this test were immunoblotted (recom Line
HEV IgG/IgM on a Dynablot Plus system, Mikrogen). The strips were
automatically scored (BLOTrix Reader and recom Scan software,
Mikrogen). The combination of both tests recommended by the manufacturer
served as a second reference here.
Subsequently, the sera were re-tested with the fully automated
chemiluminescence immunoassay (CLIA) LIAISON® Murex anti-HEV IgM
(qualitative) and LIAISON® Murex anti-HEV IgG (quantitative) (DiaSorin
Italia S.p.A., Saluggia, Italy).
Sera with evidence of acute EBV infection (N=17; i.e., presence of
anti-viral capsid antigen (VCA) IgG/IgM and absence of
anti-Epstein–Barr nuclear antigen (EBNA)-1 IgG; Alinity i™ EBV VCA IgG,
EBV VCA IgM and EBV EBNA-1 IgG Reagent Kits, Abbott, Wiesbaden, Germany)
or acute/reactivated CMV infection (N=2; i.e., presence of CMV IgM;
Abbott Alinity i™ CMV IgM/IgG Reagent Kit, Abbott) were also analyzed
with the HEV antibody assays from Mikrogen and DiaSorin.
Samples with discrepant results were followed up with the WANTAI HEV-IgM
and WANTAI HEV-IgG ELISAs (Beijing Wantai Biological Pharmacy Enterprise
Co., Ltd, Beijing, China), which are known to have particularly high
assay sensitivity and specificity 10-12. It was
assumed that the results obtained with two of the three assays were
correct. Samples with discrepant results were also tested for the
presence of HEV RNA using the RealStar® HEV RT-PCR Kit 2.0 (altona
Diagnostics GmbH, Hamburg, Germany).
All assays are CE-certified for HEV diagnostics and were used according
to the manufacturer’s instructions.
The sensitivity and specificity of the DiaSorin tests were determined by
help of a four-field table in comparison with the reference method. The
95% confidence interval (CI) was calculated using the freely available
software Graph-Pad (https://www.graphpad.com/quickcalcs/) applying the
adjusted Wald method 13.