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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

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.

During the SARS-CoV-2 pandemic, several studies reported a lower incidence of respiratory infections other than COVID-19 compared to previous years, but bias due to changes in uptake and capacity of non-COVID care cannot be excluded. The routine screening of Dutch blood donors remained unchanged during the SARS-CoV-2 pandemic and showed a significant reduction of silent parvovirus B19 (B19V) infections from 4.0/100,000 donations in 2013 to 2019 to 0.6/100,000 donations in 2020 and 0.0/100,000 donations in first 6 months of 2021.

SARS-CoV-2 serosurveys can estimate cumulative incidence for monitoring epidemics but require characterization of employed serological assays performance to inform testing algorithm development and interpretation of results. We conducted a multi-laboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serological assays using blinded panels of 1,000 highly-characterized blood-donor specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%) and precision (IIC 0.55-0.99). Durability of antibody detection in longitudinal samples was dependent on assay format and immunoglobulin target, with anti-spike, direct, or total Ig assays demonstrating more stable, or increasing reactivity over time than anti-nucleocapsid, indirect, or IgG assays. Assays with high sensitivity, specificity and durable antibody detection are ideal for serosurveillance. Less sensitive assays demonstrating waning reactivity are appropriate for other applications, including characterizing antibody responses after infection and vaccination, and detection of anamnestic boosting by reinfections and vaccine breakthrough infections. Assay performance must be evaluated in the context of the intended use.

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.

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.

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.

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.

The world is combating an ongoing COVID-19 pandemic1-4. Health-care systems, society and the economy are impacted in an unprecedented way. It is unclear how many people have contracted the causative coronavirus (SARS-CoV-2) unknowingly. Therefore, reported COVID-19 cases do not reflect the true scale of outbreak5-9. Natural herd immunity has been suggested as a potential exit strategy during COVID-19 outbreaks, which may arise when 50-67% of a community has been infected10. Here we present the prevalence and distribution of antibodies to SARS-CoV-2 in a healthy adult population of a highly affected country using a novel immunoassay, indicating that one month into the outbreak (i) the seroprevalence in the Netherlands is 2.7% with substantial regional variation, (ii) the hardest-hit areas show a seroprevalence of up to 9.5%, (iii) the seroprevalence is sex-independent throughout age groups (18-72 years), (iv) antibodies are significantly more often detected in younger people (18-30 years), and (v) the number of immune individuals in the current epidemic stage is far below the herd immunity threshold. This study provides vital information on the extent of virus spread in a country where social distancing is in place, concluding that herd immunity to SARS-CoV-2 is not a realistic short-term exit strategy option.

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 The incidence of hepatitis E virus (HEV) has increased substantially in Europe recently, thereby threatening blood safety. A cost-effectiveness analysis for HEV screening of blood donations in the Netherlands was performed. STUDY DESIGN AND METHODS A simulation model was developed to mimic the process of donation, infections in the donor population, donation testing, and transmission to transfusion recipients. The variability of viral loads among donors was modeled using observed loads. The number of (incurable) chronic HEV infections among organ and stem cell transplant patients and the costs avoided by implementing blood screening were estimated. RESULTS HEV screening of whole blood donations in pools of 24 would prevent 4.52 of the 4.94 transfusion-associated chronic HEV infections expected annually, at approximately €310,000 per prevented chronic case. Per case not curable by ribavirin prevention, costs are approximately 10 times higher. Selective screening, if logistically feasible, could reduce screening costs by 85%. Sensitivity analyses show that uncertainty in the HEV transmissibility and the frequency of HEV clearing greatly impact the estimated cost-effectiveness. Of all HEV infections nationwide one in 700 is estimated to be due to blood transfusion, while for chronic infections this is one in 3.5. CONCLUSION Despite uncertainties in our estimates, preventing HEV transmission by screening of blood donations appears not excessively expensive compared to other blood-screening measures in the Netherlands. However, the impact on HEV disease burden may be relatively small as only a minority of all HEV cases is transmitted by blood transfusion.

BACKGROUND Hepatitis E virus (HEV) Genotype 3 (G3) infection is a zoonosis that may be transmitted during the acute phase by transfusion. The aim of this study was to determine the incidence of HEV and seroprevalence among Irish blood donors. STUDY DESIGN AND METHODS Anonymized samples from 1076 donations collected in 2012 were tested for HEV immunoglobulin (Ig)G using the Wantai enzyme-linked immunosorbent assay. A total of 24,985 anonymized donations collected between December 2013 and June 2014 were individually tested for HEV RNA using the Procleix HEV assay; reactive donations were confirmed by an in-house real-time polymerase chain reaction (PCR) test. RESULTS Seroprevalence for anti-IgG was 5.3% (95% confidence interval [CI], 4.0%-6.8%), ranging from 1.1% in the 18- to 29-years age group to 33.3% in males over 60 years. HEV RNA screening of 24,985 samples yielded five PCR-confirmed donations (1:4997, 0.02%; 95% CI, 0.0065%-0.0467%), only one of which was serologically reactive (HEV IgM reactive only). Viral loads ranged from 10 to 44,550 IU/mL. Genotype analysis on three samples identified HEV G3 virus. Four of the five viremic donations were from donors in the 18- to 29-years age group (p = 0.01). CONCLUSION Seroprevalence for anti-HEV IgG was low compared to some European countries, but 1 in 5000 donations was viremic. Viremia was predominantly in younger Irish donors. After Department of Health approval the Irish Blood Transfusion Service implemented individual blood donation HEV RNA screening initially for a 3-year period from January 2016.

BACKGROUND The incidence of hepatitis E virus (HEV) infection in the Netherlands is high. Blood donors are not routinely screened for HEV infection, but since January 2013, donations used for the production of solvent/detergent (S/D)-treated plasma have been screened for HEV RNA. STUDY DESIGN AND METHODS Donations were screened for HEV RNA in pools of 96 and 192 donations. In addition, all donations made between 60 days before and after each HEV RNA-positive donation were tested individually for HEV RNA and anti-HEV immunoglobulin G. RESULTS The screening of 59,474 donations between January 2013 and December 2014 resulted in identification of 45 HEV RNA-positive donations (0.076%) from 41 donors. HEV RNA loads ranged from 80 to 2.3 × 106 IU/mL. The number of positive donations increased significantly over time (p = 0.03). Thirty-three of 90 donations made up to 60 days before or after HEV RNA-positive donations were positive when tested individually, while they had not been detected in the pool screening. The mean duration of HEV viremia in the healthy blood donor is estimated to be 68 days. CONCLUSION The incidence of HEV infection in the Netherlands is high and increased during the study period. In 2013 and 2014, HEV RNA was detected in 1 per 762 donations intended for production of S/D plasma.

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 Hepatitis E virus (HEV) is a nonenveloped emerging virus of increasing worldwide interest. Antibody prevalence, RNA frequencies, and transfusion transmissions have been reported. We investigated the HEV RNA and antibody frequencies in US blood donors. STUDY DESIGN AND METHODS Individual-donation HEV RNA testing was performed on 18,829 donations from six US geographic regions using a CE-marked nucleic acid test (95% limit of detection, 7.9 IU/mL). Repeat-reactive donations were confirmed by in-house, real-time polymerase chain reaction (PCR; 10.3 IU/mL). Total HEV seroprevalence in a randomly selected subset of donations (n = 4499) was assessed by a direct, double-antigen sandwich assay; reactives were further tested for immunoglobulin (Ig)G and IgM. As part of the total antibody confirmatory algorithm, the cutoff was adjusted. RESULTS Two donations tested confirmed-positive for RNA (PCR not quantifiable, IgM/IgG positive; and 14 IU/mL, antibody negative) for a frequency of 1 in 9500 (95% confidence interval [CI], 1:2850-1:56,180) and 99.96% specificity (95% CI, 99.92%-99.98%); both donors were from the Midwest United States. Antibody prevalence was 9.5% (95% CI, 8.7-10.5) before the cutoff adjustment and 7.7% (95% CI, 7.0%-8.5%) after adjustment; 0.58% (95% CI, 0.39%-0.85%) were IgM positive. CONCLUSIONS We confirmed comparatively low rates and low viral loads of HEV RNA in US blood donors indicating the need for individual-donation testing if screening is implemented. Antibody prevalence rates were comparable to those reported by one US study using a different assay, but lower than those reported in another study using yet a third assay. We did not answer the question of whether US blood donation screening is warranted. Selective strategies involving providing HEV-negative blood to severely immunosuppressed patients at risk of developing hepatitis may be considered.

Background In the Netherlands, universal antibody to hepatitis B core antigen (anti-HBc) donor screening was introduced in July 2011 to intercept potentially infectious donations slipping through hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA minipool screening (HBV DNA MP6). Study Design and Methods The yield and donor loss were evaluated after the first 2 years of universal anti-HBc donor screening. A total of 382,173 donors were tested for anti-HBc and, if positive, for antibody to HBsAg (anti-HBs). Anti-HBc–reactive donors with anti-HBs of less than 200 IU/L were deferred, but repeat donors were allowed retesting after 6 months if anti-HBs was less than 10 IU/mL. Anti-HBc false positivity was estimated using the crude anti-HBc signal, family name–based ethnicity scoring, and donor follow-up. Results Anti-HBc screening identified 13 confirmed or potential HBsAg- and HBV DNA MP6–negative recent HBV infections. In addition, 820 anti-HBc–reactive donors with low anti-HBs titers (

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 The Netherlands experienced major Q fever outbreaks from 2007 through 2009. An increasing number of human chronic Q fever cases has been reported in the affected area. Blood donors unaware of chronic Coxiella burnetii infection might be infectious for transfusion recipients. Local blood donations were screened for serologic signs of chronic Coxiella infection. Study Design and Methods From August 2012 through January 2013, a total of 2490 serum samples were collected from all consenting blood donors in the most affected Q fever outbreak area and screened for Phase II anti-Coxiella immunoglobulin G antibodies using an enzyme-linked immunosorbent assay (ELISA). (Phase II antibodies are considered indicative for resolved or ongoing Coxiella infection.) Reactive samples were confirmed by quantitative immunofluorescent Phase I and II antibody testing. A Phase I antibody titer of at least 1024 was considered indicative for chronic Coxiella infection. For 179 donors archived samples from 2009 and 2010 were available to study the long-term course of Coxiella antibodies. Results A total of 110 of 2490 donors were confirmed positive for Phase II Coxiella antibodies (4.4%; 95% confidence interval, 3.7%-5.3%), of which 79 were reactive for Phase I antibodies, with a maximum titer of 256. In 15 of 24 donors (62.5%), testing positive for Phase II antibodies in 2009 and 2010, ELISA reactivity had declined to negativity in 2012 and 2013. Conclusion After large Q fever outbreaks in the Netherlands, no sign of potentially infectious chronic Coxiella infection was found among blood donors in the most affected area. Using an ELISA for detection, Coxiella antibodies in previously exposed donors waned quickly.

Background Recent studies show that endemic hepatitis E virus (HEV) infection occurs frequently in some developed countries. In the Netherlands in 2013, the routine screening of 35,220 plasma donations for HEV RNA showed 20 donors to be viremic (1:1761), which seems to contradict reports of declining HEV seroprevalence in the recent past. Study Design and Methods To asses HEV infection pressure changes over time, archived samples from Dutch blood donations collected in 1988 and 2000 were tested for anti-HEV immunoglobulin (Ig)G. The findings were compared to the HEV seroprevalence among donors in 2011. Results The age-adjusted prevalence of anti-HEV IgG for Dutch donors aged 18 to 64 declined from 46.6% in 1988 to 27.3% in 2000 and to 20.9% in 2011. The reduction of seroprevalence was apparent for all age groups between 1988 and 2000, and for donors older than 40 between 2000 and 2011, but the seroprevalence among donors aged 18 to 29 increased between 2000 and 2011. Recent changes in HEV infection pressure are more apparent in the youngest donors, who to a lesser extent reflect cumulative exposure to HEV in the past. Donors aged 18 to 21 showed decreasing HEV seroprevalence from 19.8% in 1988 to 7.0% in 1995 and to 4.3% in 2000, followed by an increase to 12.7% in 2011. Conclusion HEV antibody patterns in young and old Dutch donors, in 1988 to 2011, suggest that decades ago, HEV was ubiquitous and most persons acquired infection. Subsequently HEV incidence was low during a prolonged period, to increase again in recent years.

The in vitro perfused rectal gland of the dogfish shark ( Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

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.

BACKGROUND: Since 2007, large outbreaks of Q fever occurred in the Netherlands. The unprecedented number of Q fever infections resulted in the need for the Dutch blood transfusion service to evaluate the risk of transmission of Coxiella burnetii via blood. STUDY DESIGN AND METHODS: A lookback procedure (recipient tracing) was performed for transfused blood products of whole blood donors with confirmed C. burnetii infection within 3 weeks before to 3 weeks after blood donation. Repository samples of index donations were tested with real-time polymerase chain reaction (PCR) for C. burnetii DNA. Hospitals were asked to review the medical records of recipients and—if considered necessary—to test the recipients for infection with C. burnetii. RESULTS: From 2007 through 2011, a total of 33 blood donors notified the blood bank of infection with C. burnetii. Thirteen donations fulfilled the criteria for a lookback procedure (18 blood products). C. burnetii PCR was positive in 1 of 13 repository samples of index donations. Blood products were transfused to 18 recipients. Information was retrieved from 12 of them; seven were tested for C. burnetii. Two recipients showed positive serology. However, transmission of C. burnetii via transfusion was unlikely, especially since most recipients lived in the same Q fever–affected area as the donors. CONCLUSION: Blood donors who have clinical Q fever around the time of blood donation are unlikely to test positive for C. burnetii by PCR in repository samples. Transmission of C. burnetii via transfusion of blood products could not be demonstrated in a lookback exercise.

Exposure to hepatitis E virus (HEV) is common in the United States, but there are few data on prevalence of HEV/human immunodeficiency virus (HIV) coinfection in U.S. populations. We tested 2,919 plasma samples collected from HIV-infected (HIV+) women and men enrolled in U.S. cohort studies for HEV viremia using a high-throughput nucleic acid testing (NAT) platform. NAT+ samples were confirmed by real-time polymerase chain reaction. Samples were selected for testing primarily on the basis of biomarkers of liver disease and immune suppression. Prevalence of HEV viremia was 3 of 2,606 and 0 of 313 in tested plasma samples collected from HIV+ women and men, respectively. All HEV isolates were genotype 3a. Based on follow-up testing of stored samples, 1 woman had chronic HEV infection for >4 years whereas 2 women had acute HEV detectable at only a single study visit. Conclusions: To our knowledge, this is the first reported case of chronic HEV infection in an HIV+ U.S. individual. We also confirm that chronic HEV infection can persist despite a CD4+ count >200 cells/mm3. Overall, though, these data suggest that HEV infection is rare in the HIV+ U.S. population. (Hepatology 2016;63:712–720)

BACKGROUND: In 2007, 2008, and 2009 outbreaks of Q-fever occurred in the Netherlands with increasing magnitude. The 2009 outbreak with 2354 reported cases is the largest human Q-fever outbreak ever recorded. To assess the extent of infection and the safety of donated blood, we tested local blood donations for presence of Coxiella burnetii antibodies and DNA. STUDY DESIGN AND METHODS: Starting May 2009, more than 40,000 serum samples were collected from all consenting blood donors in the areas with high Q-fever incidence. The 1004 samples from the areas with the highest number of reported cases were tested for C. burnetii DNA by polymerase chain reaction; seroprevalence and incidence were determined using enzyme-linked immunosorbent assay and immunofluorescence assays (IFAs) in the subset of 543 donors of whom a follow-up sample was available. RESULTS: A total of 6 of 1004 donor samples tested reactive for C. burnetii DNA. Confirmatory testing (IFA) on the index and follow-up samples demonstrated seroconversion in two donors, high-level preexisting antibodies in one donor, and no seroconversion in three donors. Immunoglobulin (Ig)G testing of the 543 serum pairs showed that 66 were reactive in the latest sample, of which 10 represented seroconversions. CONCLUSION: In the area with highest incidence during a large Q-fever outbreak, 3 of 1004 blood donations contained C. burnetii DNA (0.3%; 95% confidence interval, 0.1%-1.0%). A total of 66 of 543 (12.2%) donors tested positive for anti-Coxiella IgG. Ten seroconversions were detected, resulting in an incidence of 5.7% per year, which is more than 10-fold higher than the local number of reported clinical cases (0.47% per year). [Correction added after online publication 14-Jul-11: high-level existing antibodies has been updated to highlevel preexisting antibodies.]

Background & Aims Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na + /H + hydrogen exchanger NHE3 and is increased by Na + -glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood. Methods Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 ( NHERF2−/− mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy. Results The nonmetabolized SGLT1 substrate α-methyl-D-Glu (α-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. α-MD-G normalized NHE3 activity after cholera toxin–induced diarrhea. α-MD-G–stimulated jejunal NHE3 activity was defective in NHERF2−/− mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin–binding domain-dependent increase in co-precipitation of ezrin with NHE3. Conclusions SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin–induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

BACKGROUND: Emerging infectious diseases can compromise the safety of tissues for transplantations. A recent outbreak of Q fever, a zoonosis caused by the bacterium Coxiella burnetii, in the Netherlands compelled the Dutch tissue banks to assess the risk of Q fever transmission through tissue transplantation in order to maintain optimal safety. MATHODS: This article describes the systematic approach that was followed in the Netherlands. This approach included a review of the literature, a qualitative risk assessment, expert opinion gathering and investigations for specific strategies that can help to maintain the balance between tissue safety and availability. RESULTS: This resulted in a specific donor selection policy and in development of further research to fill in gaps in knowledge about Q fever in tissue transplantation. CONCLUSION: The strategy described in this article may be useful for tissue bankers facing similar outbreaks of emerging infections or may be useful for development of future guidelines or assessment strategies for tissue banking.

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 1) inhibited NHE3 V(max) activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca(2+)](i) by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](i); 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca(2+).

Urine electrolyte, mineral, and protein excretion in NHERF- 2 and NHERF-1 null mice. Am J Physiol Renal Physiol 294: F1001-F1007, 2008. First published February 6, 2008; doi:10.1152/ajprenal.00504.2007. -The adaptor proteins sodium/hydrogen exchanger regulatory factor (NHERF)-1 and NHERF-2 have overlapping tissue distribution in renal cells and overlapping specificity in their binding to renal transporters and other proteins. To compare the kidney-specific differences in the function of these adaptor proteins, NHERF-1 and NHERF 2 null mice were compared with wild-type control mice. In NHERF-2 null mice, the renal proximal tubule abundance and distribution of NHERF-1 and NHERF-3 were not different from those in wild-type animals. The glomerular expression of podocalyxin and ZO-1 also did not differ. NHERF-1 null mice had increased urinary excretion of phosphate, calcium, and uric acid compared with wild-type control and NHERF-2 null mice. Because of the association between NHERF-2 and podocalyxin in glomeruli and ClC-5 in the renal proximal tubule, the urinary excretion of protein was determined. There were no differences in the urinary excretion of protein or low-molecular-weight proteins between wild-type control, NHERF-1(-/-), and NHERF-2(-/-) mice. These studies indicate that the increased urinary excretion of phosphate and uric acid are specific to NHERF-1 null mice and highlight the fact that predictions about the role of adaptor proteins such as the NHERF proteins obtained from studies of model cell systems must be confirmed in whole animals.

The first detailed description of the proteome of the mouse jejunal brush border membrane vesicle is presented here. This was obtained by a combination of purification via divalent (Mg2+) cation precipitation starting with isolated cells plus strong cation exchange chromatography LC−MS/MS. Five-hundred seventy proteins were identified including 45 transport proteins. Among the latter, 18 had either not been identified in the intestine in the past or there was a single unconfirmed report of their presence. Validation was accomplished by a combination of immunoblotting and immunofluorescence using mouse jejunum and previously described antibodies. The validated BB proteins were aquaporin 7, Glut 9b, Na+I- symporter (NIS), and non-gastric H+/K+-ATPase. This study helps to further define the brush border membrane vesicle, a preparation which has been widely used to identify transport function of the small intestine. Keywords: murine small intestine • brush border • transport protein • proteome • LC−MS/MS

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the "new permeation pathway" for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF.

Succinate semialdehyde dehydrogenase (SSADH; ALDH5A1) deficiency, a rare metabolic disorder that disrupts the normal degradation of GABA, gives rise to a highly heterogeneous neurological phenotype ranging from mild to very severe. The nature of the mutation has so far been reported in patients from six families world wide and eight different mutations were described. Here we report the mutational spectrum in 48 additional unrelated families of different geographic origin. We detected 27 novel mutations at the cDNA level, of which 26 could be attributed to changes at the genomic level. Furthermore, six mutations were detected that did not strongly affect SSADH activity when expressed in HEK 293 cells and are considered nonpathogenic allelic variants. Twenty of the mutations were only found in one family. The spectrum of disease-causing mutations from all patients sequenced thus far consists of 25 point mutations, four small insertions, and five small deletions. Seven of these mutations affect splice junctions, seven are nonsense mutations, and 12 are missense mutations. Although there were no mutational hotspots or prevalent mutations responsible for a significant number of cases, 14 out of 37 (38%) of the missense alleles were present in exon 4 or 5. With one exception, the missense mutations we consider to be causative of SSADH deficiency reduced the SSADH activity to less than 5% of the normal activity in our in vitro expression system. This indicates that residual expression is not likely to be an important factor contributing to the large phenotypic differences observed among different families and even among siblings, suggesting that other modifying factors are of great importance in disease pathology.

Inherited succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) is one of the few neurogenetic disorders of GABA metabolism, and one in which tonic-clonic seizures associate with increased central nervous system GABA and gamma-hydroxybutyrate (GHB). To explore pathomechanisms and develop new preclinical treatment approaches, we developed a murine knockout model of SSADH deficiency. In the absence of intervention, SSADH(-/-) mice suffer 100% mortality at week 3 to 4 of life from generalized tonic-clonic seizures. In this report, we summarize earlier studies indicating disruption of the GABA/glutamine axis in SSADH(-/-) mouse brain, effective pharmacotherapeutic approaches, preliminary gene-therapy results, and electrophysiological analyses of mutant mice. We also present new evidence for oxidative stress in SSADH(-/-) mice, significant alterations of dopamine metabolism, and abnormal neurosteroid levels in brain, potentially implicating the GABA(A) receptor in pathogenesis. In SSADH deficiency, the accumulation of two neuroactive species, GABA and GHB, is significant because GABA is one of the earliest transmitters expressed in mammals, with key roles in synaptogenesis and myelination, whereas GHB displays a vast array of pharmacological actions. The SSADH(-/-) mouse may represent a useful model in which to explore the effect of GABA and GHB accumulation on central nervous system development and function.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1–/–) deficient mice previously revealed elevated γ-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified β-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of β-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

Objectives The current surveillance system in The Netherlands cannot differentiate recent HIV infections from established infections, which is crucial for estimating the HIV incidence; this information is needed for assessing trends of the HIV epidemic and the impact of prevention interventions. We determined the proportion of recent HIV infections (RI) and estimated HIV incidence using a recent infection testing algorithm (RITA) among men who have sex with men (MSM) newly diagnosed as having HIV attending sexually transmitted infection (STI) clinics. Methods Plasma samples collected between 2009 and 2011 were tested for RI with the Architect HIV Ag/Ab Combo immunoassay. Data on viral load, CD4 count and previous HIV testing were incorporated into the RITA. HIV incidence and 95% CIs were estimated. Logistic regression was used to identify factors associated with RI. Results Of the 251 samples tested for RI, 78/251 (31%) infections were determined as recent by the RITA. No significant change over time was observed. The estimated HIV incidence in this high-risk MSM population was 3.3 per 100 person-years (95% CI 2.5 to 4.1). The only factor associated with RI in the multivariable model was being tested for HIV ≥3 times in the past (aOR=7.4; 95% CI 2.0 to 27.8). Conclusions The proportion of RIs was comparable to studies in similar settings in Europe. Implementation of the RITA for routine surveillance in The Netherlands to assess trends in RIs over time, to study the infections in other groups and to inform public health actions, is being planned.

Background: Several methods have been published for measuring γ-aminobutyric acid transaminase (GABA-T) activity, but these methods are either impracticable because of the use of radioisotopes or insufficiently sensitive to determine small enzyme activities in leukocyte extracts. We developed a direct and sensitive enzyme method. Methods: We developed a stable-isotope dilution method for the measurement of [15N]glutamic acid derived from [15N]GABA and α-ketoglutaric acid, catalyzed by GABA-T. The method for analysis of [15N]glutamic acid comprised a solid-phase extraction procedure to isolate this analyte from incubation samples. After derivatization, [15N]glutamic acid was quantified by gas chromatography–mass spectrometry relative to its 2H5-labeled internal standard. In addition to [15N]GABA, [15N]β-alanine was a cosubstrate. Results: GABA-T-deficient lymphoblasts showed diminished enzyme activity, with both [15N]GABA and [15N]β-alanine as substrate. Vigabatrin inhibited the enzyme activity for both substrates. Conclusions: The activity of GABA-T can be accurately determined by our procedure using 15N-labeled substrate, measuring the formation of [15N]glutamic acid. Our results with [15N]β-alanine indicate that GABA and β-alanine transaminases are identical.

Large outbreaks of Q fever in the Netherlands from 2007 to 2009 were monitored using notification data of acute clinical Q fever. However, the notification system provides no information on infections that remain subclinical or for which no medical attention is sought. The present study was carried out immediately after the peak of the 2009 outbreak to estimate the ratio between Coxiella burnetii infections and Q fever notifications. In 23 postcode areas in the high-incidence area, notification rates were compared with seroconversion rates in blood donors from whom serial samples were available. This resulted in a ratio of one Q fever notification to 12.6 incident infections of C. burnetii. This ratio is time and place specific and is based on a small number of seroconversions, but is the best available factor for estimating the total number of infections. In addition, as subclinical C. burnetii infection may lead to chronic Q fever, the ratio can be used to estimate the expected number of chronic Q fever patients in the coming years and as input for cost-benefit analyses of screening options.

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 & Aims Diarrhea results from reduced net fluid and salt absorption caused by an imbalance in intestinal absorption and secretion. The bulk of sodium and water absorption in the intestine is mediated by Na + /H + exchanger 3 (NHE3), located in the luminal membrane of enterocytes. We investigated the effect of lysophosphatidic acid (LPA) on Na + /H + exchanger activity and Na + -dependent fluid absorption in the intestine. Methods We analyzed the effects of LPA on fluid absorption in intestines of wild-type mice and mice deficient in Na + /H + exchanger regulatory factor 2 (NHERF2; Nherf2 −/− ) or LPA 2 ( Lpa 2 −/− ). Roles of LPA 5 and NHERF2 were determined by analysis of heterologous expression. Results Under basal conditions, LPA increased fluid absorption in an NHE3-dependent manner and restored the net fluid loss in a mouse model of acute diarrhea. Expression of the LPA receptor LPA 5 was necessary for LPA-induced stimulation of NHE3 activity in colonic epithelial cells. Stimulation of NHE3 by the LPA-LPA 5 signaling required coexpression of NHERF2, which interacted with LPA 5 . LPA-mediated intestinal fluid absorption was impaired in Nherf2 −/− mice, demonstrating the requirement for NHERF2 in LPA 5 activity. However, fluid absorption was unaltered in Lpa 2 −/− mice. LPA stimulated NHE3 and fluid absorption in part by increasing NHE3 protein abundance at the brush border membrane of intestinal epithelial cells. Conclusions LPA is a potent stimulant of NHE3 and fluid absorption in the intestine, signaling through LPA 5 . Regulation by LPA 5 depends on its interaction with NHERF2. LPA might be useful in the treatment of certain diarrheal diseases.

Binding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to the Na(+)/H(+) exchanger 3 regulatory factor 1 (NHERF-1) and NHERF-2 scaffolding proteins has been shown to affect its localization and activation. We have for the first time studied the physiological role of these proteins in CFTR regulation in native tissue by determining CFTR-dependent chloride current in NHERF-1- and NHERF-2-deficient mice. The cAMP- and cGMP-activated chloride current and the basal chloride current in basolaterally permeabilized jejunum were reduced by approximately 30% in NHERF-1-deficient mice but not in NHERF-2-deficient mice. The duodenal bicarbonate secretion was affected in a similar way, whereas no significant differences in CFTR activity were observed in ileum. CFTR abundance as determined by Western blotting was unaltered in jejunal epithelial cells and brush border membranes of NHERF-1 and NHERF-2 mutant mice. However, semi-quantitative detection of CFTR by confocal microscopy showed that the level of apically localized CFTR in jejunal crypts was reduced by approximately 35% in NHERF-1-deficient and NHERF-1/2 double deficient mice but not in NHERF-2 null mice. Together our results indicate that NHERF-1 is required for full activation of CFTR in murine duodenal and jejunal mucosa and that NHERF-1 affects the local distribution of CFTR in or near the plasma membrane. These studies provide the first evidence in native intestinal epithelium that NHERF-1 but not NHERF-2 is involved in the formation of CFTR-containing functional complexes that serve to position CFTR in the crypt apical membrane and/or to optimize its function as a cAMP- and cGMP-regulated anion channel.