The Elecsys® Anti-SARS-CoV-2 and Elecsys® Anti-SARS-CoV-2 S antibody assays: Differentiating between vaccination and infection, and assessing long-term performance.

INTRODUCTION: Serological surveillance is useful for assessing SARS-CoV-2 immunity in populations. To effectively study the presence and persistence of antibodies, it is necessary to distinguish between persons with past infection, and persons who only received vaccination. Knowledge of the duration of antibody persistence is essential for correct interpretation of surveillance results. METHODS: Starting in April 2020, waning of SARS-CoV-2 antibodies was studied in a longitudinal cohort study of 495 SARS-CoV-2 antibody-positive Dutch blood donors, not pre-selected by PCR testing or disease severity. Additionally, in May 2021, a sample of donors representative for the Dutch population was tested for antibodies against the SARS-CoV-2 spike (S) protein, using the Wantai Ab ELISA and the Elecsys® Anti-SARS-CoV-2 S assay; and for antibodies against the nucleocapsid protein, which indicate past infection, using the Elecsys® Anti-SARS-CoV-2 assay. RESULTS: The anti-S response in donors that were infected in April or May 2020 remained positive in 100% of donors in the Elecsys® Anti-SARS-CoV-2 S assay one year after infection, after which follow up of waning was no longer possible because of large scale vaccination. The anti-nucleocapsid response results were still positive in approximately 80% of donors two years after infection. In May 2021, 51% of the donors showed anti-S reactivity and 16.8% tested positive for anti-nucleocapsid antibodies. CONCLUSION: Infection with SARS-CoV-2 resulted in spike and nucleocapsid antibody levels still detectable in the majority of donors 1-2 years after infection. In May 2021, 51% of donors were vaccinated and 16.8% had had an infection. Thus, both Elecsys® SARS-CoV-2 antibody assays can be used to reliably assess the vaccination and infection status of individuals.

Blood safety markers in Dutch donors after relaxation of deferral for men who have sex with men: re-emergence of syphilis and HIV pre-exposure prophylaxis use.

BACKGROUND: Less discriminatory donor selection policies for men who have sex with men (MSM) may impact transfusion safety in terms of higher residual risks for known transfusion-transmitted infections (TTIs), increased vulnerability toward new TTIs that are also transmitted via sex, and HIV infections masked by pre-exposure prophylaxis (PrEP). STUDY DESIGN AND METHODS: TTI trends in Dutch donors were studied over a 13-year period (2011-2023), characterized by successive relaxations of MSM deferral criteria. Structured posttest counseling was performed to determine risk factors in TTI-positive donors. PrEP drug levels were measured in 9977 donations from male donors living in urban areas and in 67 donors with active or resolved syphilis. RESULTS: HIV incidence (from 5.8 to 1.5 per 1,000,000 donor years (DY)) and HBV incidence (from 12.4 to 4.5 per 1,000,000 DY) in Dutch donors decreased with less stringent MSM deferral criteria, while syphilis prevalence (from 26.4 to 44.1 per 100,000 new donors) and syphilis incidence (from 18.3 to 46.3 per 1,000,000 DY) increased over time. The proportion of MSM-related syphilis rose from 2% to 32% in new donors and from 12% to 27% in repeat donors. PrEP was detected in 2 of 9977 (0.02%) donations from male donors living in urban areas, and in 1 of 39 (2.6%) male donors with syphilis. DISCUSSION: To date, phasing out donor deferral for MSM had no significant impact on transfusion safety in the Netherlands. However, rising syphilis rates and (recent) PrEP use in the blood donor population, albeit rare, suggest an influx of donors with higher sexual risk profiles and requires intensified TTI surveillance in donors.

Assessing West Nile virus (WNV) and Usutu virus (USUV) exposure in bird ringers in the Netherlands: a high-risk group for WNV and USUV infection?

Introduction: In 2020, the first Dutch West Nile virus (WNV) infected birds were detected through risk-targeted surveillance of songbirds. Retrospective testing of patients with unexplained neurological disease revealed human WNV infections in July and August 2020. Bird ringers are highly exposed to mosquito bites and possibly avian excrements during ringing activities. This study therefore investigates whether bird ringers are at higher risk of exposure to WNV and Usutu virus (USUV). Methods: Dutch bird ringers were asked to provide a single serum sample (May - September 2021) and to fill out a survey. Sera were screened by protein microarray for presence of specific IgG against WNV and USUV non-structural protein 1 (NS1), followed by focus reduction virus neutralization tests (FRNT). Healthcare workers (2009-2010), the national immunity cohort (2016-2017) and blood donors (2021) were used as control groups without this occupational exposure. Results: The majority of the 157 participating bird ringers was male (132/157, 84%) and the median age was 62 years. Thirty-seven participants (37/157, 23.6%) showed WNV and USUV IgG microarray signals above background, compared to 6.4% (6/94) in the community cohort and 2.1% (2/96) in blood donors (p < 0.01). Two seroreactive bird ringers were confirmed WNV or USUV positive by FRNT. The majority of seroreactive bird ringers travelled to EU countries with reported WNV human cases (30/37, 81%) (p = 0.07). No difference was observed between bird ringers with and without previous yellow fever vaccination. Discussion: The higher frequency of WNV and/or USUV IgG reactive bird ringers indicates increased flavivirus exposure compared to the general population, suggesting that individuals with high-exposure professions may be considered to complement existing surveillance systems. However, the complexity of serological interpretation in relation to location-specific exposure (including travel), and antibody cross-reactivity, remain a challenge when performing surveillance of emerging flaviviruses in low-prevalence settings.

External quality assessment of SARS-CoV-2 serology in European expert laboratories, April 2021.

BackgroundCountries worldwide are focusing to mitigate the ongoing SARS-CoV-2 pandemic by employing public health measures. Laboratories have a key role in the control of SARS-CoV-2 transmission. Serology for SARS-CoV-2 is of critical importance to support diagnosis, define the epidemiological framework and evaluate immune responses to natural infection and vaccine administration.AimThe aim of this study was the assessment of the actual capability among laboratories involved in sero-epidemiological studies on COVID-19 in EU/EEA and EU enlargement countries to detect SARS-CoV-2 antibodies through an external quality assessment (EQA) based on proficiency testing.MethodsThe EQA panels were composed of eight different, pooled human serum samples (all collected in 2020 before the vaccine roll-out), addressing sensitivity and specificity of detection. The panels and two EU human SARS-CoV-2 serological standards were sent to 56 laboratories in 30 countries.ResultsThe overall performance of laboratories within this EQA indicated a robust ability to establish past SARS-CoV-2 infections via detection of anti-SARS-CoV-2 antibodies, with 53 of 55 laboratories using at least one test that characterised all EQA samples correctly. IgM-specific test methods provided most incorrect sample characterisations (24/208), while test methods detecting total immunoglobulin (0/119) and neutralising antibodies (2/230) performed the best. The semiquantitative assays used by the EQA participants also showed a robust performance in relation to the standards.ConclusionOur EQA showed a high capability across European reference laboratories for reliable diagnostics for SARS-CoV-2 antibody responses. Serological tests that provide robust and reliable detection of anti-SARS-CoV-2 antibodies are available.

A computational model for prediction of ferritin and haemoglobin levels in blood donors.

Blood donors are at risk of iron deficiency anaemia. While this risk is decreased through ferritin-based deferral, ideally ferritin monitoring should also aid in optimising donation frequencies. We extended an existing model of haemoglobin (Hb) synthesis with iron homeostasis and validated the model on a cohort of 300 new donors whose ferritin levels were measured from stored blood samples collected over a 2-year period. We then used the donor's gender, body weight, height, and baseline Hb and ferritin levels to predict subsequent Hb and ferritin levels. The prediction error was within measurement variability in 88% of Hb level predictions and 64% of ferritin level predictions. A sensitivity analysis of the model revealed that baseline ferritin level was the most important in predicting future ferritin levels. Finally, we used the model to calculate the annual donation frequency at which donors would keep their ferritin level >15 ng/ml when measured after donating for 2 years. The mean annual donation frequency would then be 1.9 for women and 4.1 for men. The computational model, requiring baseline values only, can predict future Hb and ferritin levels remarkably well. This enables determination of optimal donation frequencies for individual donors at the start of their donation career.

Ferritin Trajectories over Repeated Whole Blood Donations: Results from the FIND+ Study.

BACKGROUND: Depending on post-donation erythropoiesis, available iron stores, and iron absorption rates, optimal donation intervals may differ between donors. This project aims to define subpopulations of donors with different ferritin trajectories over repeated donations. METHODS: Ferritin levels of 300 new whole blood donors were measured from stored (lookback) samples from each donation over two years in an observational cohort study. Latent classes of ferritin level trajectories were investigated separately using growth mixture models for male and female donors. General linear mixed models assessed associations of ferritin levels with subsequent iron deficiency and/or low hemoglobin. RESULTS: Two groups of donors were identified using group-based trajectory modeling in both genders. Ferritin levels showed rather linear reductions among 42.9% of male donors and 87.7% of female donors. For the remaining groups of donors, steeper declines in ferritin levels were observed. Ferritin levels at baseline and the end of follow-up varied greatly between groups. CONCLUSIONS: Repeated ferritin measurements show depleting iron stores in all-new whole blood donors, the level at which mainly depends on baseline ferritin levels. Tailored, less intensive donation strategies might help to prevent low iron in donors, and could be supported with ferritin monitoring and/or iron supplementation.

Associations Between Symptoms, Donor Characteristics and IgG Antibody Response in 2082 COVID-19 Convalescent Plasma Donors.

Many studies already reported on the association between patient characteristics on the severity of COVID-19 disease outcome, but the relation with SARS-CoV-2 antibody levels is less clear. To investigate this in more detail, we performed a retrospective observational study in which we used the IgG antibody response from 11,118 longitudinal antibody measurements of 2,082 unique COVID convalescent plasma donors. COVID-19 symptoms and donor characteristics were obtained by a questionnaire. Antibody responses were modelled using a linear mixed-effects model. Our study confirms that the SARS-CoV-2 antibody response is associated with patient characteristics like body mass index and age. Antibody decay was faster in male than in female donors (average half-life of 62 versus 72 days). Most interestingly, we also found that three symptoms (headache, anosmia, nasal cold) were associated with lower peak IgG, while six other symptoms (dry cough, fatigue, diarrhoea, fever, dyspnoea, muscle weakness) were associated with higher IgG concentrations.

Evaluation of Commercially Available High-Throughput SARS-CoV-2 Serologic Assays for Serosurveillance and Related Applications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys can estimate cumulative incidence for monitoring epidemics, requiring assessment of serologic assays to inform testing algorithm development and interpretation of results. We conducted a multilaboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serologic assays using blinded panels of 1,000 highly characterized specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%), and precision (intraclass correlation coefficient 0.55-0.99). Durability of antibody detection was dependent on antigen and immunoglobulin targets; antispike and total Ig assays demonstrated more stable longitudinal reactivity than antinucleocapsid and IgG assays. Assays with high sensitivity, specificity, and durable antibody detection are ideal for serosurveillance, but assays demonstrating waning reactivity are appropriate for other applications, including correlation with neutralizing activity and detection of anamnestic boosting by reinfections. Assay performance must be evaluated in context of intended use, particularly in the context of widespread vaccination and circulation of SARS-CoV-2 variants.

Comparison of Hepatitis E Virus Sequences from Humans and Swine, the Netherlands, 1998-2015.

Pigs are suspected to be a major source of zoonotic hepatitis E virus (HEV) infection in industrialized countries, but the transmission route(s) from pigs to humans are ill-defined. Sequence comparison of HEV isolates from pigs with those from blood donors and patients in 372 samples collected in the Netherlands in 1998 and 1999 and between 2008 and 2015 showed that all sequences were genotype 3 except for six patients (with travel history). Subgenotype 3c (gt3c) was the most common subtype. While the proportion of gt3c increased significantly between 1998 and 2008, it remained constant between 2008 and 2015. Among the few circulating HEV subtypes, there was no difference observed between the human and the pig isolates. Hepatitis E viruses in humans are very likely to originate from pigs, but it is unclear why HEV gt3c has become the predominant subtype in the Netherlands.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

OBJECTIVES: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. METHODS: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. RESULTS: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. CONCLUSION: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.

Blood donor screening in the Netherlands: Universal anti-HBc screening in combination with HBV nucleic acid amplification testing may allow discontinuation of hepatitis B virus antigen testing.

BACKGROUND: In the Netherlands, blood donor screening for hepatitis B virus (HBV) consists of HBsAg screening since the 1970s, HBV DNA minipool testing (MP-NAT) since 2008, and anti-HBc screening since 2011. Anti-HBc reactivity causes deferral only if anti-HBs titers are <200 IU/mL, or when anti-HBc was acquired during follow-up. STUDY DESIGN AND METHODS: Over 5.5 million donations from 582,459 Dutch donors were screened for HBV DNA, HBsAg, anti-HBc, and, if anti-HBc positive, also for anti-HBs. The added value, expressed as the yield of (potentially) infectious and/or recent HBV infections versus unnecessary donor loss, was evaluated for each of the three HBV screening tests. RESULTS: HBV donor screening identified 89 HBV-infected donors with at least two reactive HBV markers (MP-NAT, HBsAg and/or anti-HBc). Single HBV-marker yield was: 5 MP-NAT-only, 0 HBsAg-only, and 20 anti-HBc-only donors. In addition, anti-HBc screening yielded 1,067 potentially infectious donors at risk for occult HBV infection (OBI). In total, 4,126 (0.71%) donors were anti-HBc-reactive at first-time screening, and 1,098 (0.19%) seroconverted during follow-up. Anti-HBc-related donor loss was limited to 2,627 (0.45%) donors using anti-HBs titers and two-strike programs. Donor loss due to MP-NAT and HBsAg screening was extremely low: 0 and 128 donors, respectively. CONCLUSION: HBV donor screening could be limited to MP-NAT and anti-HBc screening. MP-NAT and anti-HBc improved blood safety by intercepting infectious donations from donors with recent infection or OBI, while HBsAg did not. Unnecessary donor loss related to anti-HBc screening is substantial but does not endanger the continuity of the blood supply.

Low awareness of past SARS-CoV-2 infection in healthy plasma donors.

Awareness of infection with SARS-CoV-2 is crucial for the effectiveness of COVID-19 control measures. Here, we investigate awareness of infection and symptoms in relation to antibodies against SARS-CoV-2 in healthy plasma donors. We asked individuals donating plasma across the Netherlands between May 11th and 18th 2020 to report COVID-19-related symptoms, and we tested for antibodies indicative of a past infection with SARS-CoV-2. Among 3,676 with antibodies, and from questionnaire data, 239 (6.5%) are positive for SARS-CoV-2 antibodies. Of those, 48% suspect no COVID-19, despite the majority reporting symptoms; 11% of seropositive individuals report no symptoms and 27% very mild symptoms at any time during the first peak of the epidemic. Anosmia/ageusia and fever are most strongly associated with seropositivity. Almost half of seropositive individuals do not suspect SARS-CoV-2 infection. Improved recognition of COVID-19 symptoms, in particular, anosmia/ageusia and fever, is needed to reduce widespread SARS-CoV-2 transmission.

Low SARS-CoV-2 seroprevalence in blood donors in the early COVID-19 epidemic in the Netherlands.

The world is combating an ongoing COVID-19 pandemic with health-care systems, society and economies impacted in an unprecedented way. It is unclear how many people have contracted the causative coronavirus (SARS-CoV-2) unknowingly and are asymptomatic. Therefore, reported COVID-19 cases do not reflect the true scale of outbreak. Here we present the prevalence and distribution of antibodies to SARS-CoV-2 in a healthy adult population of the Netherlands, which is a highly affected country, using a high-performance immunoassay. Our results indicate that one month into the outbreak (i) the seroprevalence in the Netherlands was 2.7% with substantial regional variation, (ii) the hardest-hit areas showed a seroprevalence of up to 9.5%, (iii) the seroprevalence was sex-independent throughout age groups (18-72 years), and (iv) antibodies were significantly more often present in younger people (18-30 years). Our study provides vital information on the extent of exposure to SARS-CoV-2 in a country where social distancing is in place.

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

Epidemiology of hepatitis E virus infection in a cohort of 4023 immunocompromised patients.

OBJECTIVES: The prevalence of active, chronic, and former hepatitis E virus (HEV) infections was investigated in a cohort of immunocompromised patients. The association with transfusion transmitted HEV was evaluated, and the HEV seroprevalence was compared with that in healthy blood donors. STUDY DESIGN AND METHODS: Serum samples from 4023 immunocompromised patients at Rigshospitalet, Denmark were retrospectively tested for HEV RNA and anti-HEV IgG. HEV RNA-positive patients were followed up by HEV testing, clinical symptoms, and transfusion history. Factors associated with anti-HEV were explored by multivariable logistic regression analysis. Samples from 1226 blood donors were retrospectively tested for anti-HEV IgG. RESULTS: HEV RNA was detected in six patients (0.15%) with no indications of chronic HEV infection. HEV RNA prevalence rates among recipients of allogeneic haematopoietic stem cell transplantation (allo-HSCT) and solid organ transplantation (SOT) were 0.58% and 0.21%, respectively. Transfusion transmitted infections were refuted, and transfusion history was not associated with anti-HEV positivity. The difference in HEV seroprevalence between patients (22.0%) and blood donors (10.9%) decreased when adjusting for age and sex (odds ratio 1.20, 95% confidence interval 0.97-1.48). CONCLUSIONS: HEV viremia among allo-HSCT and SOT recipients suggests that clinicians should be aware of this diagnosis. The lack of association of blood transfusion with anti-HEV positivity supports food-borne transmission as the main transmission route of HEV common to both patients and blood donors.

Hepatitis E Virus Infection in Blood Donors and Risk to Patients in the United States and Canada.

Hepatitis E virus (HEV) is the most common cause of acute hepatitis worldwide including large water-borne outbreaks, zoonotic infections and transfusion transmissions. Several countries have initiated or are considering blood donor screening in response to high HEV-RNA donation prevalence leading to transfusion-transmission risk. Because HEV transmission is more common through food sources, the efficacy of blood donor screening alone may be limited. HEV-nucleic acids in 101 489 blood donations in the United States and Canada were studied. A risk-based decision-making framework was used to evaluate the quantitative risks and cost-benefit of HEV-blood donation screening in Canada comparing three scenarios: no screening, screening blood for all transfused patients or screening blood for only those at greatest risk. HEV-RNA prevalence in the United States was one per 16 908 (95% confidence interval [CI], 1:5786-1:81987), whereas Canadian HEV-RNA prevalence was one per 4615 (95% CI, 1:2579-1:9244). Although 4-fold greater, Canadian HEV-RNA prevalence was not significantly higher than in the United States. Viral loads ranged from 20 to 3080 international units per mL; all successfully typed infections were genotype 3. No HEV-RNA false-positive donations were identified for 100 percent specificity. Without donation screening, heart and lung transplant recipients had the greatest HEV-infection risk (1:366962) versus kidney transplant recipients with the lowest (1:2.8 million) at costs of $225 546 to $561 810 per quality-adjusted life-year (QALY) gained for partial or universal screening, respectively. Higher cost per QALY would be expected in the United States. Thus, HEV prevalence in North America is lower than in countries performing blood donation screening, and if implemented, is projected to be costly under any scenario.

Seroprevalence and risk factors of recent infection with hepatitis E virus during an acute outbreak in an urban setting in Chad, 2017.

BACKGROUND: From September 2016-April 2017, Am Timan, Chad, experienced a large HEV outbreak in an urban setting with a limited impact in terms of morbidity and mortality. To better understand HEV epidemiology in this context, we estimated the seroprevalence of anti-HEV antibodies (IgM and IgG) and assessed the risk factors for recent HEV infections (positive anti-HEV IgM) during this outbreak. METHODS: A serological survey using simple random sampling was implemented in Am Timan at the tail-end of the outbreak (sample size aim = 384 household). Household members provided us with blood samples and household heads answered questions around water, sanitation and hygiene practices and animal ownership. Blood samples were tested for HEV IgG and IgM antibodies using Enzyme-Immune-Assay (EIA). We calculated weighted prevalence estimates and prevalence ratios (PRs) for possible risk factors for recent infection using multivariate Cox regression. RESULTS: We included 241 households (1529 participants). IgM prevalence decreased with age: 12.6% (< 5 years) to 4.3% (> 15 years). IgG prevalence increased with age: 23.5% (< 5 years) to 75.9% (> 15 years). Risk factors for recent HEV infections included: sharing the sanitation facility with other HHs (PR 1.72; 95%CI: 1.08-2.73), not systematically using soap for HW (PR 1.85; 95%CI: 1.30-2.63) and having animals sleeping inside the compound (PR 1.69; 95%CI: 1.15-2.50). CONCLUSIONS: Evidence suggests that Am Timan was already highly endemic for HEV before the outbreak, potentially explaining the limited extent of the outbreak. Recent infection with HEV was linked to household level exposures. Future HEV outbreak response must include ensuring access to safe water, and reducing household level transmission through active hygiene and sanitation promotion activities.

Risk factors for hepatitis E virus seropositivity in Dutch blood donors.

BACKGROUND: A marked increase of hepatitis E cases has recently been observed in the Netherlands. Causes of the (re-)emergence of hepatitis E virus (HEV) and exact sources and routes of transmission of HEV infection are currently unknown. We aimed to identify risk factors for HEV seropositivity. METHODS: Using the Wantai EIA, 2100 plasma samples of blood donors from all over the Netherlands aged 18-70 years were tested for anti-HEV IgG antibodies. A questionnaire on socio-demographic characteristics, health, and potential risk factors for HEV exposure was sent to these participants. RESULTS: The overall IgG-seroprevalence was 31% (648/2100) and increased with age. Several food products were independently associated with IgG-seropositivity in a multivariate analysis adjusting for age and gender among 1562 participants who completed the questionnaire: traditional Dutch dry raw sausages called "cervelaat", "fijnkost", "salami" and "salametti" which are generally made from raw pork and beef (aOR 1.5; 95%CI 1.2-1.9), frequent consumption of bovine steak (aOR 1.3; 95%CI 1.0-1.7), and frequent consumption of smoked beef (aOR 1.3 95%CI 1.0-1.7). Although not frequently reported, contact with contaminated water was also a risk factor for seropositivity (aOR 2.5; 95%CI 1.5-4.4). Lower seroprevalence was associated with eating raspberries, going out for dinner, and contact with wild animals and dogs. CONCLUSION: Several pork food products, mainly dry raw sausages, and contact with contaminated water were associated with past HEV infection in the Netherlands. Further investigation is needed into the prevalence and infectivity of HEV in these risk factor food products, as well as investigation of the production methods and possible origin of HEV-contamination within these sausages, e.g. very small amounts of pork liver, pig-derived blood products as food additive, or the pork muscle tissue.