The risk of transfusion-transmitted hepatitis E virus (HEV) infections by contaminated blood products remains unknown. In the present study, we evaluated and compared different nucleic acid amplification technique (NAT) methods for the detection of HEV in blood components. Minipools of a total of 16,125 individual blood donors were screened for the presence of HEV RNA using the highly sensitive RealStar HEV RT-PCR kit, revealing a minimum detection limit of 4.66 IU/ml. Thirteen donors were HEV RNA positive (0.08%), and of these donors, only three already showed reactive IgM antibody titers. The detected HEV strains all belonged to genotype 3 and were most closely related to German HEV strains from wild boars and pigs as well as from human hepatitis E cases. Furthermore, HEV RNA and HEV-specific IgM and IgG titers were determined in 136 blood donors with elevated alanine aminotransferase (ALT) levels and in 200 donors without pathological findings. HEV RNA was not detectable, but 8.08% (elevated ALT) and 0.5% (nonelevated ALT) of donors showed reactive HEV IgM titers. The overall seroprevalence rate of HEV IgG amounted to 5.94% (elevated ALT, 5.88%; nonelevated ALT, 6.0%). The clinical relevance of transfusionassociated hepatitis E infection still requires further investigation. However, in connection with raising concerns regarding blood safety, our NAT method provides a sensitive possibility for HEV testing.

Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes.

Asymptomatic hepatitis E virus (HEV) infections have been found in blood donors from various European countries, but the natural course is rarely specified. Here, we compared the progression of HEV viraemia, serostatus and liver-specific enzymes in 10 blood donors with clinically asymptomatic genotype 3 HEV infection, measuring HEV RNA concentrations, plasma concentrations of alanine/aspartate aminotransferase, glutamate dehydrogenase and bilirubin and anti-HEV IgA, IgM and IgG antibodies. RNA concentrations ranged from 77.2 to 2.19×105 IU/mL, with viraemia lasting from less than 10 to 52 days. Donors showed a typical progression of a recent HEV infection but differed in the first detection of anti-HEV IgA, IgM and IgG and seropositivity of the antibody classes. The diagnostic window between HEV RNA detection and first occurrence of anti-HEV antibodies ranged from eight to 48 days, depending on the serological assay used. The progression of laboratory parameters of asymptomatic HEV infection was largely comparable to the progression of symptomatic HEV infection, but only four of 10 donors showed elevated liver-specific parameters. Our results help elucidate the risk of transfusion-associated HEV infection and provide a basis for development of screening strategies. The diagnostic window illustrates that infectious blood donors can be efficiently identified only by RNA screening.

summary In many countries, screening of hepatitis B virus (HBV) in blood donors is limited to HBsAg testing. However, if anti-HBc testing and sensitive HBV nucleic acid amplification testing (NAT) for routine screening are not prescribed, HBV viraemia might remain unrecognized. A clinically inconspicuous HBsAg-negative 35-year-old female blood donor was detected with anti-HBc antibodies following the introduction of anti-HBc screening of donors. Based on her history, she had seroconverted to anti-HBs positive (titre >7000 IU/L) after vaccination. Blood donations were routinely tested HBV-DNA negative by minipool NAT. The individual donor samples were reinvestigated by an ultrasensitive NAT with a lower detection limit of 3.8 IU/mL. Intermittent HBV viraemia was detected over a 7-year period from this donor, with a concentration ranging from 8 to 260 IU/mL. In the subsequent donor-directed lookback study, no post-transfusion hepatitis was detected. Low-level HBV viraemia in simultaneous anti-HBc- and anti-HBs-positive blood donors could only be identified with enhanced sensitivity individual polymerase chain reaction assays and is not detectable by pool HBV NAT.

BACKGROUND:The cost-benefit question of general screening of blood products for the hepatitis E virus (HEV) is currently being discussed. One central question is the need for individual nucleic acid amplification techniques (NAT) screening (ID-NAT) versus minipool NAT screening (MP-NAT) approaches to identify all relevant viremias in blood donors. Here, the findings of ID-NAT versus MP-NAT in pools of 96 samples were compared. STUDY DESIGN AND METHODS: From November2017 to January 2018, a total of 10,141 allogenic blood donations from 7650 individual German blood donors were screened for the presence of HEV RNA using MP-NAT (96 samples) (RealStar HEV RT-PCR Kit) compared to ID-NAT (cobas HEV assay) on the fully automated cobas 6800 platform. RESULTS:Parallel screening of MP (n = 122, 96 samples/MP) using both methods detected seven reactive pools. After pool resolution, 8 HEV RNApositive donations were identified by the in-house detection method, whereas 17 HEV RNA-positive donations were identified by ID-NAT with the cobas HEV assay. This resulted in an incidence of 1:1268 donations (0.079%) for MP-NAT screening and 1:597 donations (0.168%) for ID-NAT screening. CONCLUSIONS:The detection frequency of HEV RNA was approximately 50% higher if ID-NAT was used compared to MP-NAT. However, viral loads of ID-NATonly samples were below 25 IU/mL and will often not result in transfusion-transmitted HEV (TT-HEV) infection, taking into account the currently known infectious dose of 5.0E + 04 IU inevitably resulting in TT-HEV infection. The clinical relevance and need for identification of these low-level HEV-positive donors still require further investigation.T he emergence of the hepatitis E virus (HEV) as a potential new candidate pathogen of transfusiontransmitted HEV (TT-HEV) infections introduces procedural questions about the safety of blood products. The cost-benefit question of a general screening of blood products for HEV is being discussed extensively. An important factor influencing the discussion is the required minimum viral load to be detected in the donor blood, which is determined mainly by the currently known lowest infectious dose of a certain blood product triggering an infection in the recipient. Increased importance of TT-HEV has been recognized since 2004, although earlier reports pointed to the risk of infection. 1,2 Several European committees, local blood authorities, and a large number of blood transfusion facilities question the necessity of HEV-NAT screening of blood donors. In the "European Pharmacopeia," Chapter 8.3, the implementation of an HEV run control for screening of human plasma pools was recently demanded. 3 A summarized assessment of the current situation in 11 European countries by Domanovic et al. described the situation as "a shift to screening," 4 but ABBREVIATIONS: ALT = alanine aminotransferase; APC = apheresis-derived platelet concentrates; FFP = fresh frozen plasma; HEV = hepatitis E virus; ID-NAT = individual NAT; MP-NAT = minipool NAT; PPC = pooled platelet concentrate; RBC ...