Towards a research agenda for water, sanitation and antimicrobial resistance.

Clinically relevant antimicrobial resistant bacteria, genetic resistance elements, and antibiotic residues (so-called AMR) from human and animal waste are abundantly present in environmental samples. This presence could lead to human exposure to AMR. In 2015, the World Health Organization (WHO) developed a Global Action Plan for Antimicrobial Resistance with one of its strategic objectives being to strengthen knowledge through surveillance and research. With respect to a strategic research agenda on water, sanitation and hygiene and AMR, WHO organized a workshop to solicit input by scientists and other stakeholders. The workshop resulted in three main conclusions. The first conclusion was that guidance is needed on how to reduce the spread of AMR to humans via the environment and to introduce effective intervention measures. Second, human exposure to AMR via water and its health impact should be investigated and quantified, in order to compare with other human exposure routes, such as direct transmission or via food consumption. Finally, a uniform and global surveillance strategy that complements existing strategies and includes analytical methods that can be used in low-income countries too, is needed to monitor the magnitude and dissemination of AMR.

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.

Evaluation of water quality guidelines for public swimming ponds.

Swimming ponds are artificial ecosystems for bathing in which people imitate the conditions of natural waters. Swimming in natural water may pose health risks if the water quality is microbiologically poor. Swimming ponds are small water bodies that may be used by relatively large groups of people, moreover, the water is not disinfected, e.g. by using chlorine. The draft new swimming pool legislation in the Netherlands includes water quality requirements for swimming ponds. This study focused on the examination and evaluation of the new microbiological water quality requirements, including Escherichia coli, intestinal enterococci, Pseudomonas aeruginosa and Staphylococcus aureus, in thirteen public swimming pools. In eight of thirteen swimming ponds the water quality met the requirements for fecal indicators; 93-95% of the samples met the requirement for E. coli (≤100/100 ml) and intestinal enterococci (≤50/100 ml). The requirement for P. aeruginosa (≤10/100 ml) was met in eleven of thirteen swimming ponds (99% of the samples). In 68% of the samples the requirement for S. aureus (<1/100 ml) was met. A linear mixed effect analysis showed that E. coli and intestinal enterococci concentrations were significantly dependent on the log10 of turbidity. P. aeruginosa concentrations were significantly dependent on water temperature. 31-45% of the variation between swimming ponds was explained by considering 'pond' as a random effect in the analysis. The monitoring of microbiological parameters in swimming pond water needs selective analytical methods, such as those used in this study, due to large numbers of background bacteria. The draft new Dutch swimming pool legislation provides proper guidance to ensure the microbiological safety of swimming pond water; it would benefit from inclusion of turbidity as an extra parameter. S. aureus is a relevant parameter for non-fecal shedding, although scientific literature does not provide evidence for a norm value based on a dose-response relation for exposure to S. aureus in water.

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.

Establishment of a National Inventory of Dangerous Pathogens in the Republic of Uganda.

One of the challenges of global biosecurity is to protect and control dangerous pathogens from unauthorized access and intentional release. A practical and feasible option to protect life science institutes against theft and sabotage, and secure their biological materials against misuse, is to establish a national electronic database with a comprehensive overview of the locations of all controlled dangerous pathogens in a country. This national database could be used as an instrument to secure and account for dangerous pathogens in a country, but it could also assist in establishing a biosecurity assessing and monitoring system for laboratories that work with these controlled biological agents. The Republic of Uganda is one of the first countries, prompted by the World Health Organization's (WHO's) Joint External Evaluation (JEE), to implement a national electronic database that assembles information collected from relevant Ugandan laboratories. This Ugandan Inventory of Dangerous Pathogens is different from an institute-specific pathogen inventory system, as it is intended to store the information collected from laboratories in the country working with dangerous pathogens in 1 centralized secure location. The Uganda National Council for Science and Technology (UNCST) has coordinated the implementation of the Ugandan national inventory. The inventory was recognized by the WHO JEE as contributing to Uganda's developed capacities regarding biosafety and biosecurity. This article describes the steps in implementing Uganda's National Inventory of Dangerous Pathogens. In addition, it presents a straightforward approach that can be adapted by other countries that aim to enhance their biosecurity capacities.

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.

Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, The Netherlands.

The intestinal parasites Cryptosporidium and Giardia cause gastro-enteritis in humans and can be transmitted via contaminated water. Oysters are filter feeders that have been demonstrated to accumulate pathogens such as Salmonella, Vibrio, norovirus and Cryptosporidium from contaminated water and cause foodborne infections. Oysters are economically important shellfish that are generally consumed raw. Commercial and non-commercial oysters (Crassostrea gigas) and oyster culture water from the Oosterschelde, The Netherlands, were examined for the presence of Cryptosporidium oocysts and Giardia cysts. Nine of 133 (6.7%) oysters from two non-commercial harvesting sites contained Cryptosporidium, Giardia or both. Six of 46 (13.0%) commercial oysters harboured Cryptosporidium or Giardia in their intestines. Data on the viability of (oo)cysts recovered from Oosterschelde oysters were not obtained, however viable (oo)cysts were detected in surface waters that enter the Oosterschelde oyster harvesting areas. The detection of Cryptosporidium and Giardia in oysters destined for human consumption has implications for public health only when human pathogenic (oo)cysts that have preserved infectivity during their stay in a marine environment are present. Our data suggest that consumption of raw oysters from the Oosterschelde may occasionally lead to cases of gastro-intestinal illness.

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.

Determination of the recovery efficiency of cryptosporidium oocysts and giardia cysts from seeded bivalve mollusks.

The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.

A mathematical model for removal of human pathogenic viruses and bacteria by slow sand filtration under variable operational conditions.

Slow sand filtration (SSF) in drinking water production removes pathogenic microorganisms, but detection limits and variable operational conditions complicate assessment of removal efficiency. Therefore, a model was developed to predict removal of human pathogenic viruses and bacteria as a function of the operational conditions. Pilot plant experiments were conducted, in which bacteriophage MS2 and Escherichia coli WR1 were seeded as model microorganisms for pathogenic viruses and bacteria onto the filters under various temperatures, flow rates, grain sizes and ages of the Schmutzdecke. Removal of MS2 was 0.082-3.3 log10 and that of E. coli WR1 0.94-4.5 log10 by attachment to the sand grains and additionally by processes in the Schmutzdecke. The contribution of the Schmutzdecke to the removal of MS2 and E. coli WR1 increased with its ageing, with sticking efficiency and temperature, decreased with grain size, and was modelled as a logistic growth function with scale factor f0 and rate coefficient f1. Sticking efficiencies were found to be microorganism and filter specific, but the values of f0 and f1 were independent of microorganism and filter. Cross-validation showed that the model can be used to predict log removal of MS2 and ECWR1 within ±0.6 log. Within the range of operational conditions, the model shows that removal of microorganisms is most sensitive to changes in temperature and age of the Schmutzdecke.

Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification.

Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.