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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

BackgroundWhole blood donors lose approximately 200-250 mg of iron per donation. 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.MethodsFerritin levels of 300 new whole blood donors were measured from stored (lookback) samples from each donation over a two-year period in an observational cohort study. Latent classes of ferritin level trajectories were investigated using growth mixture models for male and female donors, separately. Associations of ferritin levels with subsequent iron deficiency and/or low haemoglobin were assessed with generalized linear mixed models.ResultsIn both genders two groups of donors were identified using group-based trajectory modelling. 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.ConclusionRepeated 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.

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. This article is protected by copyright. All rights reserved.

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 ask individuals donating plasma across the Netherlands between May 11th and 18th 2020 to report COVID-19 related symptoms and we test for antibodies indicative of a past infection with SARS-CoV-2. Among 3,676 with antibody and 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, 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., Graphical Abstract, Van den Hurk et al. investigate awareness of SARS-CoV-2 infection and symptoms in relation to antibody status in healthy plasma donors. Forty-eight percent of seropositive individuals do not suspect SARS-CoV-2 infection, 11% are asymptomatic. Improved recognition of COVID-19 symptoms, in particular anosmia/ageusia and fever, is needed to reduce SARS-CoV-2 transmission.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic challenges governments worldwide to balance appropriate virus control measures and their societal and economic consequences. These control measures include the identification, isolation and testing of potentially infected individuals. As this relies on an individual’s awareness of infection, we investigated the extent to which healthy adults suspected having had COVID-19, and how COVID-19 suspicion and symptoms relate to antibodies indicative of a past infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Methods and findingsFor this cross-sectional study, individuals donating plasma anywhere in the Netherlands between May 11th and 18th were screened for total SARS-CoV-2 antibodies using ELISA and invited to participate in an online questionnaire about COVID-19-related symptoms and awareness. Antibody and questionnaire data were complete for 3,676 individuals, including 239 (6.5%) that tested positive for SARS-CoV-2 antibodies. Here, we show that a 38% of the individuals that tested positive for SARS-CoV-2 antibodies reported having had no or only very mild symptoms at any time during the peak of the epidemic. The loss of taste and/or smell in particular was significantly associated with seropositivity, independent of age and sex. Forty-eight percent of antibody-positive persons did not suspect having had COVID-19, in spite of most of them reporting symptoms.ConclusionsAwareness of infection was low among individuals that tested positive for SARS-CoV-2 antibodies, even at the peak of the epidemic. Improved awareness and recognition of COVID-19 symptoms and tracing of asymptomatic contacts is crucial to halting SARS-CoV-2 transmission.

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. Keywords: Hepatitis E, Epidemiology, Immunocompromised, Transfusion transmission, HEV RNA, Anti-HEV IgG, Chronic HEV infection

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., The Netherlands is a country highly affected by the COVID-19 pandemic. In this study, Slot, Hogema and colleagues report a low SARS-CoV-2 seroprevalence one month into the outbreak and provide insights into virus exposure by region and age group when widespread non-pharmaceutical interventions are in place.

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.

Background After a large Q fever outbreak in the Netherlands in the period from 2007 to 2010, the risk of Q fever transmission through tissue and cell transplantation from undiagnosed chronic Q fever cases became a potential issue. Aim: We aimed to evaluate the risk of Q fever transmission through tissue and cell transplantation. Methods: We performed a retrospective observational cohort study among 15,133 Dutch donors of tissues and stem cells from 2010 to 2015 to assess seroprevalence of Coxiella burnetii antibodies, to identify factors associated with presence of C. burnetii antibodies, and to assess the proportion of undiagnosed chronic Q fever cases. Results: The study population consisted of 9,478 (63%) femoral head donors, 5,090 (34%) post-mortal tissue donors and 565 (4%) cord blood donors. Seroprevalence of C. burnetii antibodies gradually decreased after the outbreak, from 2.1% in 2010 to 1.4% in 2015, with a significant trend in time (p Conclusion: This study shows that seroprevalence of C. burnetii antibodies among donors of tissues and cells in the Netherlands after 2014 was similar to pre-outbreak levels in the general population. The proportion of newly detected chronic Q fever patients among donors of tissues and cells was smaller than 0.1%. This study may prompt discussion on when to terminate the screening programme for chronic Q fever in donors of tissues and cells in the Netherlands.

Background The incidence of autochthonous hepatitis E virus genotype 3 (HEV gt3) infections in Western Europe is high. Although pigs are a major reservoir of the virus, the exact sources and transmission route(s) of HEV gt3 to humans remain unclear. Methods To determine the role of meat consumption at a population level, the seroprevalence of anti-HEV IgG antibodies was compared between Dutch blood donors with a vegetarian lifestyle and donors who consume meat on a daily basis. Results The age-weighted anti-HEV IgG seroprevalence among donors not eating meat was significantly lower than among meat-eating donors (12.4% vs 20.5%, p = 0.002). For both groups the prevalence strongly increased with age and the difference in prevalence was apparent for all age groups. Conclusions Compared with meat-eating donors, the incidence of HEV infection is significantly lower among donors not eating meat, indicating that meat consumption is a major risk factor for HEV infection.

Background and objective In several Western countries, silent endemic hepatitis E virus (HEV) infection is common among blood donors. Immunocompromised persons may develop chronic hepatitis E, but the relevance of endemic HEV for immunocompetent persons remains largely unknown. We investigated the immune status and travel history in cases of hepatitis E in the Netherlands. Study design Between January 2009 and May 2014, physicians throughout the Netherlands submitted samples from 4067 hepatitis patients to Sanquin Diagnostic Services for HEV antibody testing. For the 144 patients testing positive for HEV IgM and HEV RNA, travel behavior and immune status were assessed. Complete information was obtained for 81 patients. Results Surprisingly, the majority of patients (52/81, 64%) were immunocompetent and did not travel outside Europe. HEV genotyping was obtained for 47 non-traveling patients, all concerned HEV genotype 3. Discussion Our findings suggest that currently in Western countries the impact of hepatitis E for non-traveling, immunocompetent persons is underestimated. Historically cases of hepatitis A, B and C, but not cases of hepatitis E, are notifiable and warrant preventive measures. However, in parts of Western Europe HEV may have become the most important source of viral hepatitis, in immunocompetent and in immunosuppressed persons. Pending measures against the ongoing transmission of HEV genotype 3 in parts of Europe, physicians should consider hepatitis E in dealing with new hepatitis patients.

The indirect immunofluorescence assay (IFA) is considered the reference method for diagnosing Q fever, but serology is also performed by complement fixation assay (CFA) or enzyme-linked immunosorbent assay (ELISA). However, comparability between these assays is not clear, and therefore a quality assessment was performed. A total of 25 serum samples from negative controls, Q fever patients, and a serial diluted high-positive sample were analyzed in 10 Dutch laboratories. Six laboratories performed CFA, 5 performed IFA, and 5 performed ELISAs. Three international reference laboratories from Australia, France, and the USA also participated in this study. Qualitative values between laboratories using the same methods were within close range, and all 3 methods correctly identified acute Q fever patients. The IFA, ELISA, and CFA are all suitable serodiagnostic assays to diagnose acute Q fever, but the IFA remains an important tool in the follow-up of patients and in identifying patients at risk for developing chronic Q fever.

BACKGROUND: Hepatitis E virus genotype-3 (HEV-gt-3) causes autochthonous infections in western countries, with a primary reservoir in animals, especially pigs. HEV transfusion transmission has been reported, and HEV-gt-3 prevalence is high in some European countries. The prevalence of HEV RNA was investigated among Danish blood donors, and the prevalence of HEV transfusion-transmitted infection (TTI) was investigated among recipients.STUDY DESIGN AND METHODS: Samples from 25,637 consenting donors collected during 1 month in 2015 were screened retrospectively using an individual-donation HEV RNA nucleic acid test with a 95% detection probability of 7.9 IU/mL. HEV-positive samples were quantified by real-time polymerase chain reaction and genotyped. Transmission was evaluated among recipients of HEV RNA-positive blood components. Phylogenetic analyses compared HEV sequences from blood donors, symptomatic patients, and swine.RESULTS: Eleven donations (0.04%) were confirmed as positive for HEV RNA (median HEV RNA level, 13 IU/mL). Two donations were successfully genotyped as HEV-gt-3. Only one donor had a travel history outside Europe. Nine of 11 donors were male, but the gender ratio was nonsignificant compared with the total donor population. Seven available recipients tested negative for HEV RNA and anti-HEV immunoglobulin M in follow-up samples. One recipient was HEV RNA-negative but anti-HEV immunoglobulin G-positive. HEV TTI was considered unlikely, but a transfusion-induced secondary immune response could not be excluded. Phylogenetic analysis showed relatively large sequence differences between HEV from donors, symptomatic patients, and swine.CONCLUSIONS: Despite an HEV RNA prevalence of 0.04% in Danish blood donations, all HEV-positive donations carried low viral loads, and no evidence of TTI was found.

Na+/H+exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na+/H+exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na+absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca2+and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca2+-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca2+-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.

Background After the largest outbreaks of Q fever ever recorded in history occurred in the Netherlands, concern arose that Coxiella may be transmitted via donated tissues of latent or chronically infected donors. The Dutch Health Council recently advised to screen tissue donors, donating high risk tissues, for Coxiella infection. Methods After validation of an enzyme immunoassay (EIA) test for IgG antibodies against phase 2 of C. burnetii for use on post-mortem samples, serum samples of 1033 consecutive Dutch post-mortem tissue donors were tested for IgG antibodies against phase 2 of C. burnetii. Confirmation of reactive results was done by immunofluorescence assay (IFA). All available tissues (corneas, heart valves, skin and bone marrow) from donors with IgG reactivity were tested for presence of Coxiella DNA by PCR. Risk factors for IgG reactivity were investigated. Results After validation of the tests for use on post-mortem samples, 50/1033 donors (4.8%) screened positive for phase 2 anti-Coxiella IgG by EIA, and 31 were confirmed by IFA (3.0%). One donor showed a serological profile compatible with chronic infection. All tested tissues (25 corneas, 6 heart valves, 4 skin and 3 bone marrow) from donors with IgG reactivity tested negative for the presence of Coxiella DNA. Except for living in a postal code area with a high number of Q fever notifications, no risk factors for IgG reactivity were found. Conclusions The strong correlation between notifications and seroprevalence confirms that the used assays are sufficiently specific for use on post-mortem samples, although one has to be aware of differences between batches. Thus, this study provides a validated method for screening tissue donors for infection with Coxiella burnetii that can be used in future outbreaks.

We investigated the role of the Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) on intestinal salt and water absorption, brush border membrane (BBM) morphology, and on the NHE3 mRNA expression, protein abundance, and transport activity in the murine intestine. NHERF1-deficient mice displayed reduced jejunal fluid absorption in vivo, as well as an attenuated in vitro Na(+) absorption in isolated jejunal and colonic, but not of ileal, mucosa. However, cAMP-mediated inhibition of both parameters remained intact. Acid-activated NHE3 transport rate was reduced in surface colonocytes, while its inhibition by cAMP and cGMP was normal. Immunodetection of NHE3 revealed normal NHE3 localization in the BBM of NHERF1 null mice, but NHE3 abundance, as measured by Western blot, was significantly reduced in isolated BBM from the small and large intestines. Furthermore, the microvilli in the proximal colon, but not in the small intestine, were significantly shorter in NHERF1 null mice. Additional knockout of PDZK1 (NHERF3), another member of the NHERF family of adaptor proteins, which binds to both NHE3 and NHERF1, further reduced basal NHE3 activity and caused complete loss of cAMP-mediated NHE3 inhibition. An activator of the exchange protein activated by cAMP (EPAC) had no effect on jejunal fluid absorption in vivo, but slightly inhibited NHE3 activity in surface colonocytes in vitro. In conclusion, NHERF1 has segment-specific effects on intestinal salt absorption, NHE3 transport rates, and NHE3 membrane abundance without affecting mRNA levels. However, unlike PDZK1, NHERF1 is not required for NHE3 regulation by cyclic nucleotides.

Background Hepatitis B virus (HBV) is divided into 8 definite (A-H) and 2 putative (I, J) genotypes that show a geographical distribution. HBV genotype G, however, is an aberrant genotype of unknown origin that demonstrates severe replication deficiencies and very little genetic variation. It is often found in co-infections with another HBV genotype and infection has been associated with certain risk groups such as intravenous drug users and men having sex with men (MSM). We aimed to estimate the prevalence of HBV-G in the Netherlands by analysing samples from HBV-positive patients visiting the Academic Medical Center in Amsterdam. Methods Ninety-six HBV-infected patients, genotyped as HBV-A or HBV-G infected, were retrieved from the clinical database. Blood plasma samples were analysed with a newly-developed real-time PCR assay that detects HBV-A and HBV-G. For three patients, the HBV plasma viral load (pVL) of both genotypes was followed longitudinally. In addition, three complete genomes of HBV-G were sequenced to determine their relationship to global HBV-G strains. Results Ten HBV-G infections were found in the selected Dutch patients. All concerned HIV-1 infected males with HBV-A co-infection. Dutch HBV-G strains were phylogenetically closely related to reference HBV-G strains. Conclusions In this study, HBV-G infection in the Netherlands is found exclusively in HIV-1 infected men as co-infection with HBV-A. A considerable percentage (37%) of men infected with HBV and HIV-1 are actually co- infected with two HBV genotypes.