Epidemiology of Pathogens Listed as Potential Bioterrorism Agents, the Netherlands, 2009‒2019.

We provide incidences (cases/10 million persons) in the Netherlands during 2009-2019 for pathogens listed as potential bioterrorism agents. We included pathogens from the highest categories of the European Medicines Agency or the US Centers for Disease Control and Prevention. Notifiable diseases and recently published data were used to calculate the average annual incidence. Coxiella burnetii had the highest incidence because of a Q fever epidemic during 2007-2010. Incidence then decreased to 10.8 cases/. Pathogens with an incidence >1 were Brucella spp. (2.5 cases), Francisella tularensis (1.3 cases), and Burkholderia pseudomallei (1.1 cases). Pathogens with an incidence <1 were hemorrhagic fever viruses (0.3 cases), Clostridium botulinum (0.2 cases), and Bacillus anthracis (0.1 cases). Variola major and Yersinia pestis were absent. The generally low incidences make it unlikely that ill-meaning persons can isolate these pathogens from natural sources in the Netherlands. However, the pathogens are stored in laboratories, underscoring the need for biosecurity measures.

Surface chemistry-dependent antiviral activity of silver nanoparticles.

The toxicity towards viruses of silver nanoparticles (AgNPs) has been reported to be dependent on several factors such as particle concentration, size, and shape. Although these factors may indeed contribute to the toxicity of AgNPs, the results presented in this work demonstrate that surface chemistry and especially surface charge is a crucial factor governing their antiviral activity. Here, this work investigated the influence of capping agents representing various surface charges ranging from negative to positive. These AgNPs were capped with citrate, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) mercaptoacetic acid (MAA) and (branched polyethyleneimine (BPEI). We show that AgNPs exhibited surface charge-dependent toxicity towards MS2 bacteriophages. Among the capping agents under investigation, BPEI capped AgNPs (Ag/BPEI) exhibited the highest reduction of MS2 resulting in ≥6 log10-units reductions, followed by 4-5 log10-units reductions with PVP and PEG capping's and 3-4 log10-units with MAA and citrate cappings. Bare nanoparticles reported a mere 1-2 log10-units reduction. Electrostatic interaction between the positively charged BPEI-coating and the negatively charged virus surface played a significant role in bringing the MS2 closer to toxic silver ions (Ag+). Further results obtained from TEM showed that Ag/BPEI nanoparticles could directly damage the structure of the MS2 bacteriophages. AgNPs and cationic capping agents' observed synergy can lead to much lower and much more efficient dosing of AgNPs for antiviral applications.

Aichi virus in sewage and surface water, the Netherlands.

Detection of Aichi virus in humans was initially reported in Japan in 1989. To establish a timeline for the prevalence of Aichi virus infection among humans in the Netherlands, we conducted molecular analysis of archival water samples from 1987-2000 and 2009-2012. Aichi virus RNA was detected in 100% (8/8) of sewage samples and 100% (7/7) of surface water samples collected during 1987-2000 and 100% (8/8) of sewage samples and 71% (5/7) of surface water samples collected during 2009-2012. Several genotype A and B Aichi virus lineages were observed over the 25-year period studied, but the time course of viral genetic diversity showed recent expansion of the genotype B population over genotype A. Our results show that Aichi virus has been circulating among the human population in the Netherlands since before its initial detection in humans was reported and that genotype B now predominates in this country.

Biosafety and biosecurity challenges during the COVID-19 pandemic and beyond.

As the world continues to battle the SARS-CoV-2 pandemic, it is a stark reminder of the devastation biological threats can cause. In an unprecedented way the global community saw a massive surge in the demand for diagnostic capacities, which had a substantial impact on biosafety and biosecurity. Laboratories had to cope with a surge in laboratory testing capacity, while resources and training possibilities were limited. In addition, the pandemic highlighted the impact biological threats can have, thereby giving rise to new dialogue about biosecurity and new biological threats. This paper aims to highlight some of the most pressing issues regarding biosafety and biosecurity observed during the COVID-19 pandemic with special focus on low and lower middle-income countries. The authors provide lessons learned, tools and recommendations to improve future biosafety and biosecurity and increase preparedness for the next global health crisis.

Thermal Inactivation of Hepatitis E Virus in Pork Products Estimated with a Semiquantitative Infectivity Assay.

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.

Complementarity of International Instruments in the Field of Biosecurity.

The COVID-19 pandemic has demonstrated the devastating impact of infectious disease outbreaks and the threat of emerging and re-emerging dangerous pathogens, independent of their origin. Natural, accidental, and deliberate disease outbreaks all need systems in place for an effective public health response. The best known international instrument in the field of public health is the WHO International Health Regulations (2005). Although the International Health Regulations are mainly focused on natural disease outbreaks, the actions to take to comply with them also contribute to biosecurity and non-proliferation. This paper examines in case of full implementation of the International Health Regulations, what other actions states should take to comply with international biosecurity instruments, including the Biological and Toxin Weapons Convention and United Nations Security Council Resolution 1540, to effectively prevent and defend against intentional biological threats. An overview of international instruments from different disciplines regarding biosecurity is presented. Furthermore, this paper clarifies the similarities between the international biosecurity instruments and addresses the additional requirements that instruments stipulate. From a detailed comparison between the instruments it can be concluded that, to adhere to all legally-binding international biosecurity instruments, specific non-proliferation and export control measures are necessary in addition to full implementation of the International Health Regulations. Additionally, an overview of non-legally binding instruments in the field of biosecurity is presented and practical implementation examples are highlighted. Compliance with legally binding instruments can be improved by precise guidance provided by non-legally binding instruments that are clear and attuned to the situation on the ground. To improve understanding of the existing international instruments, this paper aims to provide an overview of the international legal biosecurity framework to biosecurity experts, policymakers, civil servants, and practitioners. It offers possible practical applications for the politico-legal context and accommodates the enhancement of full employment of biosecurity resources for an improved multidisciplinary capacity to prevent, detect, and respond to infectious disease outbreaks.

Dual-Use Quickscan: A Web-Based Tool to Assess the Dual-Use Potential of Life Science Research.

Research on pathogenic organisms is crucial for medical, biological and agricultural developments. However, biological agents as well as associated knowledge and techniques, can also be misused, for example for the development of biological weapons. Potential malicious use of well-intended research, referred to as "dual-use research", poses a threat to public health and the environment. There are various international resources providing frameworks to assess dual-use potential of the research concerned. However, concrete instructions for researchers on how to perform a dual-use risk assessment is largely lacking. The international need for practical dual-use monitoring and risk assessment instructions, in addition to the need to raise awareness among scientists about potential dual-use aspects of their research has been identified over the last years by the Netherlands Biosecurity Office, through consulting national and international biorisk stakeholders. We identified that Biorisk Management Advisors and researchers need a practical tool to facilitate a dual-use assessment on their specific research. Therefore, the Netherlands Biosecurity Office developed a web-based Dual-Use Quickscan (www.dualusequickscan.com), that can be used periodically by researchers working with microorganisms to assess potential dual-use risks of their research by answering a set of fifteen yes/no questions. The questions for the tool were extracted from existing international open resources, and categorized into three themes: characteristics of the biological agent, knowledge and technology about the biological agent, and consequences of misuse. The results of the Quickscan provide the researcher with an indication of the dual-use potential of the research and can be used as a basis for further discussions with a Biorisk Management Advisor. The Dual-Use Quickscan can be embedded in a broader system of biosafety and biosecurity that includes dual-use monitoring and awareness within organizations. Increased international attention to examine pathogens with pandemic potential has been enhanced by the current COVID-19 pandemic, hence monitoring of dual-use potential urgently needs to be encouraged.

Systematic approach towards establishing a National Inventory of Dangerous Pathogens.

International regulations stipulate that countries need to organize their biosafety and biosecurity systems to minimize the risk of accidental (biosafety) or malicious intentional (biosecurity) release of dangerous pathogens. International Health Regulations (IHR) benchmarks from the WHO state that even for a level of limited capacity countries need to 'Identify and document human and animal health facilities that store/maintain dangerous pathogens and toxins in the relevant sectors and health professionals responsible for them'. This study provides a stepwise, systematic approach and best practices for countries to initiate a national inventory of dangerous pathogens. With a national inventory of dangerous pathogens a country can identify and document information in a dedicated electronic database on institutes that store or maintain dangerous pathogens. The systematic approach for the implementation of a national inventory of dangerous pathogens consists of four stages; identification, preparation, implementation, and maintenance and evaluation. In the identification phase, commitment of the relevant national ministries is to be established, and a responsible government entity needs to be identified. In the preparatory phase, a list of pathogens to be incorporated in the inventory, as well as a list of institutes to include, is to be agreed upon. In the implementation phase, the institutes are contacted, and the collected data is stored safely and securely in a electronical database. Finally, in the maintenance and evaluation phase meaningful insights are derived and reported to the relevant government authorities. Also, preparations for updates and modifications are undertaken, such as modifications of pathogen lists or institute lists. The approach and database, which is available from the authors, have been tested for the implementation of a national inventory of dangerous pathogens in multiple East-African countries. A national inventory of dangerous pathogens helps countries in strengthening national biosafety and biosecurity as well as in their compliance to IHR.

Pathogenic Vibrio species in dutch shellfish destined for direct human consumption.

Vibrio parahaemolyticus is a common cause of shellfish-related gastroenteritis all over the world. V. parahaemolyticus and Vibrio alginolyticus have previously been detected in water samples from the Oosterschelde, a large inlet on the North Sea, which is used for both recreational purposes and shellfish production. In 2006, oysters (Crassostrea gigas) from a noncommercial oyster bed in the Oosterschelde and oysters bought in Dutch fish shops were tested for the presence of pathogenic Vibrio species; in 2007 and 2008, oysters (C. gigas) and mussels (Mytilus edulis) from Oosterschelde production areas were examined. Total Vibrio numbers were related to water temperatures to study joint patterns. Vibrio was found in oysters and mussels from the production areas, and levels ranged from 6 to 622 most probable number (MPN) per g in oysters and 6 to 62 MPN/g in mussels. Vibrio levels in oysters from fish shops were 231 to >333 MPN/g, whereas levels in noncommercial oysters ranged from 231 to >2,398 MPN/g. About 80% of the isolated strains were V. alginolyticus, and approximately 10% were identified as V. parahaemolyticus. Vibrio counts in shellfish samples increased with increasing water temperature and declined when water temperatures dropped; Vibrio was not detected when water temperatures declined to <13.5 degrees C. Based on the obtained results and the known high V. parahaemolyticus dose (>10(4) cells per serving of oysters) required for infection, it is concluded that the risk of gastrointestinal infections with V. parahaemolyticus through consumption of shellfish from the Oosterschelde production sites is presumably low.

Sources of hepatitis E virus genotype 3 in The Netherlands.

Non-travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans.

The course of hepatitis E virus infection in pigs after contact-infection and intravenous inoculation.

BACKGROUND: Worldwide, hepatitis E virus (HEV) genotype 3 is observed in pigs and transmission to humans is implied. To be able to estimate public health risks from e.g. contact with pigs or consumption of pork products, the transmission routes and dynamics of infection should be identified. Hence, the course of HEV-infection in naturally infected pigs should be studied. RESULTS: To resemble natural transmission, 24 HEV-susceptible pigs were infected either by one-to-one exposure to intravenously inoculated pigs (C1-pigs; n = 10), by one-to-one exposure to contact-infected pigs (C2-pigs: n = 7; C3-pigs: n = 5) or due to an unknown non-intravenous infection route (one C2-pig and one C3-pig). The course of HEV-infection for contact-infected pigs was characterized by: faecal HEV RNA excretion that started at day 7 (95% confidence interval: 5-10) postexposure and lasted 23 (19-28) days; viremia that started after 13 (8-17) days of faecal HEV RNA excretion and lasted 11 (8-13) days; antibody development that was detected after 13 (10-16) days of faecal HEV RNA excretion. The time until onset of faecal HEV RNA excretion and onset of viremia was significantly shorter for iv-pigs compared to contact-infected pigs, whereas the duration of faecal HEV RNA excretion was significantly longer. At 28 days postinfection HEV RNA was detected less frequently in organs of contact-infected pigs compared to iv-pigs. For contact-infected pigs, HEV RNA was detected in 20 of 39 muscle samples that were proxies for pork at retail and in 4 of 7 urine samples. CONCLUSION: The course of infection differed between infection routes, suggesting that contact-infection could be a better model for natural transmission than iv inoculation. Urine and meat were identified as possible HEV-sources for pig-to-pig and pig-to-human HEV transmission.

The Vulnerability Scan, a Web Tool to Increase Institutional Biosecurity Resilience.

The importance of vigilance within organizations working with high-risk biological material receives increasing attention. However, an in-depth and comprehensive tool, dedicated to increase awareness of potential risks and to assess an organization's current biosecurity vulnerabilities, has not been available yet. We developed the "Biosecurity Vulnerability Scan," a web tool that identifies biosecurity gaps in an organization based on eight biosecurity pillars of good practice. Although the tool aims primarily to assist biosafety and biosecurity officers, it can also be useful to researchers working with dangerous pathogens, their principal investigators, management, or those responsible for security issues in the life sciences. Results are only stored locally and are provided in an "overview report," which includes information on relevant risks and control measures. This can support well-substantiated decision-making on strengthening biosecurity measures within a specific organization. With this article, we aim to support institutes to increase their overall security resilience and to improve institutional biosecurity in particular by providing practical recommendations. The Biosecurity Vulnerability Scan is available at www.biosecurityvulnerabilityscan.nl.

Norovirus Outbreak Associated with Swimming in a Recreational Lake Not Influenced by External Human Fecal Sources in The Netherlands, August 2012.

Swimming in fecally contaminated recreational water may lead to gastrointestinal illness. A recreational water-associated outbreak of norovirus (NoV) infections affecting at least 100 people in The Netherlands occurred in August 2012. Questionnaire responses from patients indicated swimming in recreational lake Zeumeren as the most likely cause of illness. Most patients visited the lake during the weekend of 18⁻19 August, during which the weather was exceptionally warm (maximum temperatures 32⁻33 °C), and visitor numbers elevated. Patients, mostly children, became ill with gastroenteritis 1⁻6 days (median 2 days) after exposure. Four stool samples from patients were NoV GI positive. Subsurface sandy soil from one of the beaches where most patients swam was NoV GI positive; the water sample was negative. The epidemiological curve and the timeline of investigation based on reported symptoms demonstrate the difficulty in discovering the source in recreational water outbreaks. A NoV outbreak in a recreational lake that is not subjected to external fecal contamination sources shows the need for active communication about human shedding of viruses during and after diarrheal episodes and the advice to refrain from swimming, even a few weeks after the symptoms have resolved.

Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RT-PCR.

Pigs have been suggested to be a potential reservoir for locally acquired human hepatitis E virus (HEV) infections in the Netherlands. To study possible trends in HEV prevalence in the Dutch pig population, 97 pig farms have been screened for the presence of HEV in stools. The prevalence rate of HEV was estimated at 55% (53/97) in 2005, indicating a significant increase as compared to the prevalence rate of 22% (25/115) as was reported in 1999. The current data suggest that this increase is due to the inclusion of appropriate quality assurance controls such as internal amplification controls for RT-PCR. The abundant presence of pigs excreting HEV raises concerns on potential zoonotic transmission of the virus, either by exposure through the environment or by consumption of contaminated pork products. Moreover, one of the detected strains belonged to a European cluster which was not detected in the Netherlands before, suggesting that HEV strains spread through European countries. These data demonstrate the need to include appropriate controls in diagnostic assays, especially in complex matrices such as feces which are known to contain PCR inhibitory substances.

Hepatitis E virus RNA in commercial porcine livers in The Netherlands.

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain approximately 65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (10(4) PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers ( approximately 20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended.

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks.

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.

Detection of noroviruses in foods: a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis.

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation-RNeasy-Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.

Induction of insolubility by herpes simplex virus VP22 precludes intercellular trafficking of N-terminal Apoptin-VP22 fusion proteins.

The herpes simplex virus protein VP22 has the intriguing ability to deliver proteins from an expressing cell to neighboring cells. Fusion of VP22 to Apoptin, a protein that induces apoptosis in tumor cells but not in normal cells, might enhance the delivery of Apoptin. To analyze this hypothesis two fusion proteins of VP22 and full-length Apoptin were constructed, namely VP22-VP3 and VP3-VP22, and their apoptosis-inducing ability and intercellular spreading behavior were analyzed by transfection in tumor cells. While both of the Apoptin-VP22 fusion proteins retained the capacity to kill tumor cells, neither of them showed intercellular trafficking. To determine whether the presence of a nuclear localization signal in the C-terminus of Apoptin caused nuclear retention of the fusion protein and the subsequent lack of intercellular spreading, VP22 was fused to the biologically active N-terminal part (residues 1-69) of Apoptin (VP3n), which lacks the nuclear localization signal. However, analysis of the VP3n-VP22 fusion constructs gave no evidence of intercellular transport. A more careful inspection of different fractions of cell lysates expressing Apoptin with or without fusion to VP22 revealed that both the full-length Apoptin protein and its fusion with VP22 are insoluble. Despite the fact that VP3n was found to be soluble on its own, which could make it amenable to transport by VP22, the VP3n-VP22 fusion proteins were present exclusively in the insoluble fraction. We hypothesize that the N-terminal multimerization domain of Apoptin cooperates with VP22 to facilitate aggregation with cellular proteins, thereby inducing insolubility. From these results we conclude that, depending on the fusion partner, VP22 can have a negative effect on the solubility of fusion proteins, which consequently precludes intercellular trafficking. Such properties should be taken into account when establishing new VP22-mediated protein transduction systems.

The tumor-selective viral protein apoptin effectively kills human biliary tract cancer cells.

Biliary tract cancer, or cholangiocarcinoma, has a poor prognosis. Resection is the only curative treatment, but only a minority of patients are eligible. Chemotherapy and gamma-irradiation are merely palliative, as they are unable to remove the malignancy completely. The chicken anemia virus-derived protein apoptin induces apoptosis in a wide range of human tumor cells and is not hindered by mutations inactivating p53 or by overexpression of Bcl-2, changes known to frustrate chemotherapy and radiation therapy. We examined whether apoptin kills human biliary tract cancer cells. Expression of apoptin by means of plasmids caused extensive cell death in three independent cholangiocarcinoma cell lines, CC-LP, CC-SW, and Mz-ChA-1, regardless of their oncogenic mutations, which included inactivated p16 and p53 and the disruption of the transforming growth factor beta signaling pathway. In vitro delivery of apoptin by an adenoviral vector completely eradicated cholangiocarcinoma cells. Moreover, coexpression of the broad-spectrum caspase inhibitor p35 with apoptin only delayed the induced cell death. Changes in nuclear morphology still occurred early after transfection, and nuclei eventually disintegrated, suggesting that apoptin-induced cell death in these cells is not blocked by mutations in either the initiation or execution phase of apoptosis. The efficient induction of cell death by apoptin in cholangiocarcinoma cell lines makes apoptin an attractive candidate for molecular therapy of biliary tract cancer.

Escherichia coli O157:H7 in drinking water from private water supplies in the Netherlands.

The microbiological quality of drinking water from 144 private water supplies in the Netherlands was tested and additionally the occurrence of Escherichia coli O157 was examined. Faecal indicators were enumerated by using standard membrane filtration methods. The presence of E. coli O157 was determined using a specific enrichment method. Eleven percent of the samples contained faecal indicators whereas E. coli O157:H7 was isolated from 2.7% of the samples that otherwise met the drinking water standards. The E. coli O157 positive water supplies were located on camp-sites in agricultural areas with large grazer densities. Pulsed field gel electrophoresis (PFGE) analysis suggested that cattle might have been the cause of contamination. Our results indicate that compliance with microbiological quality standards obtained in routine monitoring does not always guarantee the absence of pathogens. The presence of pathogens such as E. coli O157 may suggest possible health consequences; however, a risk assessment process should be performed as the monitoring of both faecal indicator parameters and pathogens do not predict the effect of microbial contamination of drinking water on a population.