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.