Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater Sequencing, the Netherlands and Belgium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a major global health problem, and public health surveillance is crucial to monitor and prevent virus spread. Wastewater-based epidemiology has been proposed as an addition to disease-based surveillance because virus is shed in the feces of ≈40% of infected persons. We used next-generation sequencing of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level in the Netherlands and Belgium. Phylogenetic analysis revealed the presence of the most prevalent clades (19A, 20A, and 20B) and clustering of sewage samples with clinical samples from the same region. We distinguished multiple clades within a single sewage sample by using low-frequency variant analysis. In addition, several novel mutations in the SARS-CoV-2 genome were detected. Our results illustrate how wastewater can be used to investigate the diversity of SARS-CoV-2 viruses circulating in a community and identify new outbreaks.

Spatial and Temporal Dynamics in Attached and Suspended Bacterial Communities in Three Drinking Water Distribution Systems with Variable Biological Stability.

Microbial presence and regrowth in drinking water distribution systems (DWDSs) is routinely monitored to assess the biological stability of drinking water without a residual disinfectant, but the conventional microbiological culture methods currently used target only a very small fraction of the complete DWDS microbiome. Here, we sequenced 16S rRNA gene amplicons to elucidate the attached and suspended prokaryotic community dynamics within three nonchlorinated DWDSs with variable regrowth conditions distributing similarly treated surface water from the same source. One rural location, with less regrowth related issues, differed most strikingly from the other two urban locations by the exclusive presence of Pseudonocardia (Actinobacteria) in the biofilm and the absence of Limnobacter (Betaproteobacteriales) in the water and loose deposits during summer. There was a dominant seasonal effect on the drinking water microbiomes at all three locations. For one urban location, it was established that the most significant changes in the microbial community composition on a spatial scale occurred shortly after freshly treated water entered the DWDS. However, summerly regrowth of Limnobacter, one of the dominant genera in the distributed drinking water, already occurred in the clean water reservoir at the treatment plant before further distribution. The highlighted bacterial lineages within these highly diverse DWDS communities might be important new indicators for undesirable regrowth conditions affecting the final drinking water quality.

Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences.

Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140-2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43-1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on "in silico" analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.

Pyrosequence analysis of the hsp65 genes of nontuberculous mycobacterium communities in unchlorinated drinking water in the Netherlands.

Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.

Normalisation of SARS-CoV-2 concentrations in wastewater: The use of flow, electrical conductivity and crAssphage.

Over the course of the Corona Virus Disease-19 (COVID-19) pandemic in 2020-2022, monitoring of the severe acute respiratory syndrome coronavirus 2 ribonucleic acid (SARS-CoV-2 RNA) in wastewater has rapidly evolved into a supplementary surveillance instrument for public health. Short term trends (2 weeks) are used as a basis for policy and decision making on measures for dealing with the pandemic. Normalisation is required to account for the dilution rate of the domestic wastewater that can strongly vary due to time- and location-dependent sewer inflow of runoff, industrial discharges and extraneous waters. The standard approach in sewage surveillance is normalisation using flow measurements, although flow based normalisation is not effective in case the wastewater volume sampled does not match the wastewater volume produced. In this paper, two alternative normalisation methods, using electrical conductivity and crAssphage have been studied and compared with the standard approach using flow measurements. For this, a total of 1116 24-h flow-proportional samples have been collected between September 2020 and August 2021 at nine monitoring locations. In addition, 221 stool samples have been analysed to determine the daily crAssphage load per person. Results show that, although crAssphage shedding rates per person vary greatly, on a population-level crAssphage loads per person per day were constant over time and similar for all catchments. Consequently, crAssphage can be used as a quantitative biomarker for populations above 5595 persons. Electrical conductivity is particularly suitable to determine dilution rates relative to dry weather flow concentrations. The overall conclusion is that flow normalisation is necessary to reliably determine short-term trends in virus circulation, and can be enhanced using crAssphage and/or electrical conductivity measurement as a quality check.

Capturing the SARS-CoV-2 infection pyramid within the municipality of Rotterdam using longitudinal sewage surveillance.

Despite high vaccination rates in the Netherlands, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate. Longitudinal sewage surveillance was implemented along with the notification of cases as two parts of the surveillance pyramid to validate the use of sewage for surveillance, as an early warning tool, and to measure the effect of interventions. Sewage samples were collected from nine neighborhoods between September 2020 and November 2021. Comparative analysis and modeling were performed to understand the correlation between wastewater and case trends. Using high resolution sampling, normalization of wastewater SARS-CoV-2 concentrations, and 'normalization' of reported positive tests for testing delay and intensity, the incidence of reported positive tests could be modeled based on sewage data, and trends in both surveillance systems coincided. The high collinearity implied that high levels of viral shedding around the onset of disease largely determined SARS-CoV-2 levels in wastewater, and that the observed relationship was independent of variants of concern and vaccination levels. Sewage surveillance alongside a large-scale testing effort where 58 % of a municipality was tested, indicated a five-fold difference in the number of SARS-CoV-2-positive individuals and reported cases through standard testing. Where trends in reported positive cases were biased due to testing delay and testing behavior, wastewater surveillance can objectively display SARS-CoV-2 dynamics for both small and large locations and is sensitive enough to measure small variations in the number of infected individuals within or between neighborhoods. With the transition to a post-acute phase of the pandemic, sewage surveillance can help to keep track of re-emergence, but continued validation studies are needed to assess the predictive value of sewage surveillance with new variants. Our findings and model aid in interpreting SARS-CoV-2 surveillance data for public health decision-making and show its potential as one of the pillars of future surveillance of (re)emerging viruses.

Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.

Rise and fall of SARS-CoV-2 variants in Rotterdam: Comparison of wastewater and clinical surveillance.

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity.

Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities.

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Surveillance of influenza A and the pandemic influenza A (H1N1) 2009 in sewage and surface water in the Netherlands.

The role of the water cycle in spreading human pathogenic influenza viruses is poorly studied and is not considered to be significant. However, gastrointestinal symptoms developed in a large proportion of influenza A (H1N1) 2009 virus infected people during the pandemic in 2009 and fecal shedding was reported. This fecal route could potentially play a role in the entry of human pathogenic influenza viruses in to the water cycle. Monitoring of influenza viruses in sewage and surface water during the pandemic in 2009 showed that influenza A viruses were detected in sewage and surface water. However, the pandemic influenza A (H1N1) 2009 virus was not detected. These findings imply that the water cycle did not play a relevant role in spreading the pandemic influenza virus during the epidemic in the Netherlands in 2009. Analyses of deliberately contaminated water samples confirmed the ability of quantitative RT-PCR to detect influenza viruses in sewage samples whereas the analysis of large volumes of surface water was strongly hampered by the presence of PCR-inhibiting substances.

Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater.

Wastewater surveillance has shown to be a valuable and efficient tool to obtain information about the trends of COVID-19 in the community. Since the recent emergence of new variants, associated with increased transmissibility and/or antibody escape (variants of concern), there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. In this study, we demonstrate the use of RT-ddPCR on wastewater samples for specific detection of mutation N501Y. This assay enabled simultaneous enumeration of lineage B.1.351 (containing the 501Y mutation) and Wild Type (WT, containing 501N) SARS-CoV-2 RNA. Detection of N501Y was possible in samples with mixtures of WT with low proportions of B.1.351 (0.5%) and could accurately determine the proportion of N501Y and WT in mixtures of SARS-CoV-2 RNA. The application to raw sewage samples from the cities of Amsterdam and Utrecht demonstrated that this method can be applied to wastewater samples. The emergence of N501Y in Amsterdam and Utrecht wastewater aligned with the emergence of B.1.1.7 as causative agent of COVID-19 in the Netherlands, indicating that RT-ddPCR of wastewater samples can be used to monitor the emergence of the N501Y mutation in the community. It also indicates that RT-ddPCR could be used for sensitive and accurate monitoring of current (like K417N, K417T, E484K, L452R) or future mutations present in SARS-CoV-2 variants of concern. Monitoring these mutations can be used to obtain insight in the introduction and spread of VOC and support public health decision-making regarding measures to limit viral spread or allocation of testing or vaccination.

Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and challenges.

Analysing wastewater can be used to track infectious disease agents that are shed via stool and urine. Sewage surveillance of SARS-CoV-2 has been suggested as a tool to determine the extent of COVID-19 in cities and serve as an early warning for (re-)emergence of SARS-CoV-2 circulation in communities. The focus of this review is on the strength of evidence, opportunities and challenges for the application of sewage surveillance to inform public health decision making. Considerations for undertaking sampling programs are reviewed including sampling sites, strategies, sample transport, storage and quantification methods; together with the approach and evidence base for quantifying prevalence of infection from measured wastewater concentration. Published SARS-CoV-2 sewage surveillance studies (11 peer reviewed and 10 preprints) were reviewed to demonstrate the current status of implementation to support public health decisions. Although being very promising, a number of areas were identified requiring additional research to further strengthen this approach and take full advantage of its potential. In particular, design of adequate sampling strategies, spatial and temporal resolution of sampling, sample storage, replicate sampling and analysis, controls for the molecular methods used for the quantification of SARS-CoV-2 RNA in wastewater. The use of appropriate prevalence data and methods to correlate or even translate SARS-CoV-2 concentrations in wastewater to prevalence of virus shedders in the population is discussed.

Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands.

In the current COVID-19 pandemic, a significant proportion of cases shed SARS-Coronavirus-2 (SARS-CoV-2) with their faeces. To determine if SARS-CoV-2 RNA was present in sewage during the emergence of COVID-19 in The Netherlands, sewage samples of six cities and the airport were tested using four qRT-PCR assays, three targeting the nucleocapsid gene (N1-N3) and one the envelope gene (E). No SARS-CoV-2 RNA was detected on February 6, 3 weeks before the first Dutch case was reported. On March 4/5, one or more gene fragments were detected in sewage of three sites, in concentrations of 2.6-30 gene copies per mL. In Amersfoort, N3 was detected in sewage 6 days before the first cases were reported. As the prevalence of COVID-19 in these cities increased in March, the RNA signal detected by each qRT-PCR assay increased, for N1-N3 up to 790-2200 gene copies per mL. This increase correlated significantly with the increase in reported COVID-19 prevalence. The detection of the virus RNA in sewage, even when the COVID-19 prevalence is low, and the correlation between concentration in sewage and reported prevalence of COVID-19, indicate that sewage surveillance could be a sensitive tool to monitor the circulation of the virus in the population.

Mobility and redox transformation of arsenic during treatment of artificially recharged groundwater for drinking water production.

In this study we investigate opportunities for reducing arsenic (As) to low levels, below 1 µg/L in produced drinking water from artificially infiltrated groundwater. We observe that rapid sand filtration is the most important treatment step for the oxidation and removal of As at water treatment plants which use artificially recharged groundwater as source. Removal of As is mainly due to As co-precipitation with Fe(III)(oxyhydr)oxides, which shows higher efficiency in rapid sand filter beds compared to aeration and supernatant storage. This is due to an accelerated oxidation of As(III) to As(V) in the filter bed which may be caused by the manganese oxides and/or As(III) oxidizing bacteria, as both are found in the coating of rapid sand filter media grains by chemical analysis and taxonomic profiling of the bacterial communities. Arsenic removal does not take place in treatment steps such as granular activated carbon filtration, ultrafiltration or slow sand filtration, due to a lack of hydrolyzing iron in their influent and a lack of adsorption affinity between As and the filtration surfaces. Further, we found that As reduction to below 1 µg/L can be effectively achieved at water treatment plants either by treating the influent of rapid sand filters by dosing potassium permanganate in combination with ferric chloride or by treating the effluent of rapid sand filters with ferric chloride dosing only. Finally, we observe that reducing the pH is an effective measure for increasing As co-precipitation with Fe(III)(oxyhydr)oxides, but only when the oxidized arsenic, As(V), is the predominant species in water.

Multiple Sources of the Outbreak of Legionnaires' Disease in Genesee County, Michigan, in 2014 and 2015.

BACKGROUND: A community-wide outbreak of Legionnaires' disease (LD) occurred in Genesee County, Michigan, in 2014 and 2015. Previous reports about the outbreak are conflicting and have associated the outbreak with a change of water source in the city of Flint and, alternatively, to a Flint hospital. OBJECTIVE: The objective of this investigation was to independently identify relevant sources of Legionella pneumophila that likely resulted in the outbreak. METHODS: An independent, retrospective investigation of the outbreak was conducted, making use of public health, health care, and environmental data and whole-genome multilocus sequence typing (wgMLST) of clinical and environmental isolates. RESULTS: Strong evidence was found for a hospital-associated outbreak in both 2014 and 2015: a) 49% of cases had prior exposure to Flint hospital A, significantly higher than expected from Medicare admissions; b) hospital plumbing contained high levels of L. pneumophila; c) Legionella control measures in hospital plumbing aligned with subsidence of hospital A-associated cases; and d) wgMLST showed Legionella isolates from cases exposed to hospital A and from hospital plumbing to be highly similar. Multivariate analysis showed an increased risk of LD in 2014 for people residing in a home that received Flint water or was located in proximity to several Flint cooling towers. DISCUSSION: This is the first LD outbreak in the United States with evidence for three sources (in 2014): a) exposure to hospital A, b) receiving Flint water at home, and c) residential proximity to cooling towers; however, for 2015, evidence points to hospital A only. Each source could be associated with only a proportion of cases. A focus on a single source may have delayed recognition and remediation of other significant sources of L. pneumophila. https://doi.org/10.1289/EHP5663.

Quantitative detection of E. coli, E. coli O157 and other shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR.

Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species.

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.

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.

Method for rapid detection of viable Escherichia coli in water using real-time NASBA.

A rapid real-time NASBA method was developed for detection of Escherichia coli in water samples. In this method, a fragment of the clpB-mRNA is amplified and a specific molecular beacon probe is used to detect the amplified mRNA fragment during the NASBA reaction. The method was shown to be specific and sensitive (1 viable E. coli in 100ml) and can be performed within 3-4h. Different inactivation processes (starvation, heat, UV-irradiation and chlorine) were employed to study the relationship between culturability and the ability to detect E. coli using NASBA. Detection of clpB-mRNA correlated with culturability after starvation or chlorine treatment. After UV-irradiation or heat-inactivation, detection of the increase in production of clpB-mRNA in viable E. coli cells after heat-shock induction correlated with culturability. Application of the NASBA method on tap water, treated sewage and surface water samples showed that culture and NASBA yielded comparable results in these different matrices. This study demonstrates that the NASBA method has high potential as a rapid test for microbiological water quality monitoring.