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Protecting the health of Europeans by improving methods for the detection of pathogens in drinking water and water used in food preparation

Final Report Summary - AQUAVALENS (Protecting the health of Europeans by improving methods for the detection of pathogens in drinking water and water used in food preparation)

Executive Summary:
Executive Summary
The goal of AQUAVALENS was to reduce the incidence of waterborne and foodborne infectious diseases by developing new techniques for monitoring water quality that are robust and suitable for routine application. The work was divided into four consecutive but overlapping clusters of activities.
In cluster one, we focussed on identifying those genes associated with virulence or host specificity so that we could develop better targets for detection of pathogens of human health significance or for determining sources of contamination. We made several important advances in our understanding of virulence and in the application of microbial source tracking. We developed PCR methods able to detect multiple viruses simultaneously and also give some indication on whether such viruses were or were not still viable. We extended the use of whole genomes sequencing to a range of bacterial pathogens and combined this technique with Whole Genome Enrichment to allow direct genotyping of bacterial pathogens in water, so speeding outbreak investigation. We developed improved methods for the detection of toxoplasma in water supplies and also improved our knowledge of the pathogenicity of Giardia and the evolutionary genetics of host specificity in Cryptosporidium. In Work Package 5 we were able to develop and trial a new approach to microbial source tracking that was able to determine the sources of faecal pollution of water sources with high accuracy.
In cluster two we used the findings of cluster 1 to develop technological platforms that could be used in subsequent field studies. A particularly important advance within the AQUAVALENS project was the development of improved sample preparation. Sample preparation and concentration has traditionally been a time consuming and costly step in sample analysis and different approaches needed to be used for viruses, bacteria or protozoa. By using the Rexeed(TM) filter we were able to dramatically cut the cost of sample concentration, especially as we have demonstrated that this filter can simultaneously concentrate all three domains, viruses, bacteria and protozoa. For detection technologies we expanded the range of available PCR kits and investigated the value of several in line microbiological monitoring systems and developed an automated sampling, preparation and detection platform. All technologies were subject to multi-lab standardisation and validation.
Cluster 3 was concerned with the trialling of the new technologies and platforms in the real world. We concentrated on work in three main areas; large water systems, small water systems and water used in the food industry. The overall conclusions of this cluster were that the technologies and platforms used in field show a great potential for the improvement of microbiological sampling and pathogen detection in terms of time, money and human resources. For small water systems we showed that random sampling for indicator organisms will miss uncommon events that are likely to be the main driver of human health risk. Our in-line monitoring and event sampling showed substantially greater contamination. AQUAVALENS technologies have an important role in improving food safety for those fresh foods reliant on water in their production.
Finally, in cluster 4 we considered the broader implications of the use of these technologies in the impacts on public health, risk assessment, and the economics of water testing. The broad conclusions of this cluster were that the platforms developed within AQUAVALENS have the real potential to improve public health. Principally these benefits come from the use of more cost and time efficient technologies to screen for pathogens that will better inform Water Safety plans. In addition, the development of in line microbiological monitoring will allow more rapid detection of events so enabling control measures to be implemented earlier to protect public health. We also considered how AQUAVLENS technologies could improve our management of emerging infectious diseases and deliberate contamination. Finally the technologies developed were shown to be economically beneficial and in many situations can reduce carbon footprints.

Paul Hunter Coordinator
Project Context and Objectives:
Cluster 1: Platform Targets
Cluster 1 focused on identifying appropriate molecular targets or markers and gene sequences that could be used to provide insights into the presence of waterborne pathogens in water, their viability and virulence or could be used for strain discrimination in microbial typing and source tracking. Because the issues facing microbiologists working with viruses, bacteria and protozoa can be quite different we have chosen to have separate work packages covering viruses (WP2), bacteria (WP3) and protozoa (WP4). In addition WP 5, which is independent of the others in this cluster, was on Microbial Source Tracking [MST] as this could include all three categories of microorganisms and also non-microbiological markers. To achieve the overall goal of cluster 1 the following objectives were defined:
Objective 1: to determine, for a range of significant waterborne pathogens, including viruses, bacteria and protozoa, those genes or other molecular targets that are associated with infectivity or virulence to humans, to serve as targets for identification or in epidemiological investigations and which may be used to make assumptions about viability.
Objective 2: to select and define the best molecular tools for microbial source tracking (MST), based on inductive learning and statistical methods, capable of distinguishing between faecal pollution from more than two different sources that can be used routinely in Europe.

Cluster 2: Platform Development (WP 6 to WP 9)
Cluster 2 focused on developing the targets identified in cluster 1 into viable tools suitable for use in the detection and characterization of pathogens in water or for source tracking. This cluster was broken down in 4 workpackages (WP6 to WP9) related to sample preparation and enrichment, detection technologies, automation of bespoke systems and standardisation of operating procedures.
As one of the most problematic issues in molecular detection of pathogens in water is sample preparation a specific WP6 was dedicated to improving sample preparation technologies.WP7 developed a portfolio of electronic sensors and other detection methods on different platforms suitable for different test beds (surface, ground, tap, processing water), laboratories (high, low tech) and water management systems (on-site, on-line measurement, large, small distribution systems). As we are aware that some of the highest risk drinking water systems in Europe are very small supplies whose owners do not have the skills or resources to undertake complex or costly microbiological examinations, an important part of WP7 was to develop low cost/ easy to use technologies. WP8 had the objective to develop bespoke systems that integrate some of the technologies developed in WP6 and WP7. WP9 had the responsibility of taking the technologies developed in cluster 2 and developing standard operating procedures and microbial standards suitable for use in service laboratories. In addition, WP9 undertook the validation of some of the technologies by designing and managing appropriate external quality assurance schemes and supporting internal quality assurance though the provision of standards.
To achieve the overall goal of cluster 2 the following objectives were defined like this in the DoW:
Objective 3: to develop robust and efficient technologies for recovery of waterborne pathogens from large volumes of different types of water that are compatible with the corresponding platform designated for detection.
Objective 4: to develop a portfolio of detection methods on a range of different platforms suitable for various applications (source water, treated water for drinking and food production surface, ground, tap, processing water), laboratories (high, low tech) and water management systems (on-site, on-line measurement, large, small distribution systems).
Objective 5: to develop selected detection systems for specific applications that are suitable for commercial production fully supported with appropriate hardware and software for effective operation.
Objective 6: to establish procedures for validating and verifying the performance of the newly developed platforms, that includes standardised reference materials that will form an external quality assurance scheme to ensure consistency between different users.

Cluster 3: Field studies in European drinking water systems
A thorough assessment of the performance of the various techniques and technologies under field conditions was essential for demonstrating their benefit for improving the microbiological safety of drinking water. It was important, therefore, that trials were conducted under conditions representative of those found across Europe. In particular, variation in water quality was viewed as a significant factor that would affect their performance.
Recognition was also given to the requirement that not all the techniques and technologies would be appropriate for different types of end users. The work was undertaken in three separate work packages (WP) to provide separate recognition to the needs of (1) large water supply systems (WP10) (2) small water supply systems (WP11) and (3) water used for food production and preparation (WP12).
The outcome would lead to validated methods, expand knowledge on pathogen occurrence and improved understanding of the risk.
Main objective
Objective 7: to implement and evaluate the acceptability and effectiveness of developed platforms under conditions encountered in practice for large and small water supplies, for food processing and bottled mineral water and assess their suitability to inform management decisions and to generate important new information on the presence of pathogens in European drinking water supplies.

Cluster 4: Improving Public Health through safer water
The aim of Cluster 4 has been to look into the results of all the previous work done in the project and examine its implications given the current priorities in potable water management: Water Safety Plans, climate change, emerging pathogens and diseases, full cost benefit analysis, environmental impact, sustainability and the carbon footprint of the proposed method.
WHO has developed comprehensive guidance in their Guidelines for Drinking-water Quality which now promotes a systematic risk assessment and management approach towards ensuring microbial safety, called the “Water Safety Plans” (WSP). These plans provide the basis for effective operation to ensure that numbers of waterborne pathogens present a negligible risk to public health. In the context of the European Union the WSP are expected to be implemented by Member States by October 2017.
The specific objectives are:
Objective 8: To determine the value of the platforms for supporting the implementation of water safety plans (WSPs) either for improved risk assessment, monitoring critical locations to act as an early warning system or to derive preventive management strategies, that would be beneficial to both developed and developing countries.
Objective 9: To better understand the risks to European public health by developing scenario models that enable future risks to be estimated to take account of climate change and the threat within Europe from the emergence of new pathogens and assess the impact of different technological responses to controlling these risks.
Objective 10: To inform the European Commission on the implications of our work for European Food and Water policy through assessment of the potential gains brought about by improved health in Europe, based on a full cost-benefit analysis including their environmental impact using the carbon footprint as an indicator.
Cluster 4 synthesizes the outputs from all the previous clusters to increase our knowledge of the health risks associated with drinking water in Europe, how these risks may be affected by climate change or the emergence of new pathogens and how these risks may be reduced. A particular focus of this cluster have been on assessing the impact of our work on European policy, especially in relation to water safety plans. Also, this cluster was involved in dissemination where there was added value over and above those dissemination activities that would be part of the original WSPs.
In cluster 4 we tested how the technologies can be used to protect human health, through improving the effectiveness of Water Safety Plans, adaptation to climate change, and attempts to control outbreaks of infectious disease. We have also determined the sustainability and potential economic impacts of the developed technologies.

Project Results:
Summary of results of Cluster 1: Platform Targets [Technical abbreviations explained in the glossary added as an attachment.]
WP2 produced robust quantitative molecular systems for detection of health-significant enteric viruses. Methodological modifications enabled better estimation of virus infectivity through molecular assays. Typing tools for these viruses were developed. In WP3 advanced molecular tools and test kits were developed for the identification and quantification of major waterborne bacterial pathogens and successfully validated with water samples representing the drinking water supply chain. WP4 has validated and applied molecular targets for detection and assessment of human infectivity potential of the protozoan parasites Cryptosporidium, Giardia and Toxoplasma, and investigated targets that might be associated with virulence. In WP5 molecular markers for Microbial Source Tracking have been identified by considering up to 4-log units of dilution and up to 300 hours of environmental persistence. All molecular assays based on the identified genetic targets were validated for mass application on the technical platforms of Cluster 2 and in the field campaigns of Cluster 3.
The following major Pathogen targets were identified in Cluster 1:
Viruses: Hepatitis A virus, Norovirus, Adenovirus, Hepatitis E virus, Rotavirus, Cosackievirus
Bacteria: Campylobacter jejuni, Pseudomonas aeruginosa, Salmonella enterica, Campylobacter coli, Vibrio cholerae, pathogenic Escherichia coli, Arcobacter butzleri
Protozoa: Cryptosporidium, Giardia, Toxoplasma

Scientific and technical results of Cluster 1
Work Package 2: Applied Virology – Molecular detection, quantification and typing of infectious waterborne viruses
A set of robust quantitative molecular multiplex systems for the detection of health-significant enteric viruses was established. For example, a quadruplex Real-Time RT-qPCR assay fulfilling the requirements of the method developed by the European Committee on Standardization (CEN) was adapted for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (MgV). Pre-treatments of target viruses in order to provide a better estimation of infectivity through genome copy determination were investigated. Propidium MonoAzide [PMA] treatment before RT-qPCR amplification was optimized in all participating laboratories for the detection and quantification of viruses with intact cohesive undamaged capsids. Overall, our data confirm that capturing of viruses using PGM coated beads, may indeed facilitate selection of viruses with intact surface proteins. Another approach to assess viral infectivity was pursued using specific virus damage through some specific virucidal treatments. One result of these treatments (applying sprayed EOW on stainless steel surfaces with dried murine norovirus (MNV) was that it required 10-20 minutes contact time to reduce MNV by 3-4 log. Finally, within this WP new typing tools for health significant human and animal viral pathogens in clinical and environmental samples were generated. Several genotyping tools have been developed in this WP enabling the application to environmental samples, usually water or food samples, with a low quantity of viruses. Work on the development on NGS-pre-processing has been carried out on a NoV GII.1 positive stool sample adding mengovirus as an index. The use of endonuclease was very important to remove extracellular DNA/RNA, increasing the fraction of reads mapping to viruses from 2 % to 40 %. It was necessary to amplify the extracted RNA/DNA substantially (40 PCR cycles) to reach concentrations suitable for NGS. This method has been applied to African sewage samples, where preliminary data shows the presence of more than 450 different virus species, including NoV, enteroviruses (EV) and RV. Overall, the new genotyping tools for waterborne viruses are ready now for future applications of NGS technologies.
Highlights of WP2
Development of highly reliable singleplex and multiplex molecular assays for detection of waterborne viral pathogen
• Methods for better discrimination of infectivity through determination of genome copies
• Procedures to estimate the inactivation of non-cultivable viruses were developed
• New molecular typing methodologies for characterization of waterborne viruses during outbreaks.

Work Package 3: Applied Bacteriology - Assessment of load and virulence of waterborne bacterial pathogens
Advanced pyro-printing were developed and applied using NGS-technologies to determine the load and identity of all selected bacterial pathogens at the species level. After validation in-silico, in-vitro experiments were performed using targeted reference species to determine specificity and detection limits. Two different Patho-printing approaches were developed: i) pyro-printing based on the Roche 454 technology, and ii) SBS-printing based on the Illumina MiSeq technology. Both NGS approaches were used for in-situ validation with a set of reference water samples to determine their specificity and accuracy. Genus-specific patho-printing was demonstrated for the genus Pseudomonas with high specificity and accuracy for Pseudomonas aeruginosa in highly contaminated river water. All developed NGS methods are now ready for mass application on field samples at the intended level of taxonomic resolution. DNA-based markers for the identification of single strains of the bacterial species were developed and validated. Three different types of molecular markers were explored for the high resolution identification of bacteria to the strain level: i) highly conserved but variable functional genes (gyrB and rpoB), ii) specific virulence genes and iii) whole-genome enrichment (WGE). For the functional genes, gyrB was most promising because it could be demonstrated in-silico and in-vitro that it is possible to differentiate the major subtypes of Salmonella enterica and identify clearly Escherichia coli and Shigella flexneri. In-situ application in a set of contaminated water samples confirmed its high resolution if specific primers were applied in a patho-printing approach. For V. cholerae a novel approach was developed resulting in single strain detection and identification of individual virulence genes using WGE. A field trial with this WGE method in African river water allowed identification of individual virulence genes in low abundance (2000 fold enrichment of the gene target). Molecular methods for assessment of in-situ viability of bacterial populations using viability PCR (vPCR) were developed and validated. A combination of qPCR with propidiummonoazide (PMA) treatment may improve the detection of live cells by exclusion of false positive results due to DNA from dead cells. We investigated the application of PMA-qPCR (vPCR) for rapid detection of viable Escherichia coli in freshwater. Different PMA concentrations, light sources and exposure times were employed. By comparing results obtained with qPCR, vPCR (E. coli primers are reported in SOP) and plate counts, the best working conditions were determined. In-situ validation of the vPCR assay with surface water samples from the Genoa water supply produced satisfactory results. Molecular and experimental methods to assess virulence and infectivity of pathogenic bacteria in freshwater complemented the vPCR approach. V. cholera El Tor N16961 and pathogenic E. coli 7402 induced the activation of the viable but non culturable (VBNC) state after 40 and 80 day incubation at 4°C, respectively. VBNC cells maintained the adhesive potential towards cultured intestinal cells although the efficiency of attachment was reduced by about 40-60% compared to that of the controls. The experimental and molecular data indicated that A. butzleri, E. coli and V. cholera are viable, can activate the VBNC survival strategy and maintain the potential to colonize human intestinal cells during several weeks in freshwater.

• Various NGS-based molecular identification technologies were developed and validated allowing the detection and quantification of the specific bacterial genera
• A novel whole genome enrichment technology for V. cholerae was developed and applied to contaminated river water demonstrating a more than 2000 fold enrichment of the pathogens’ DNA at the genome level
• Molecular test kits for the detection and quantification of all targeted bacterial species were validated in several ring trials with representative selections of water samples and are now commercialized by SMEs.

Work Package 4 Applied parasitology – Detection, virulence assessment and subtyping of major waterborne protozoa
Protozoal reference genomes of Cryptosporidium species were sequenced. Quality parameters for the genome sequences were assessed based on the mapping of reads to the C. hominis and C. parvum reference genomes: high sequence identity and similar guanine and cytosine (GC) content (~30%) to the reference genomes, a high proportion of target DNA compared to background, and ideally 30-50x coverage of the genomes was achieved. The genome data have been deposited on the NCBI database ( Putative virulence markers of C. hominis and C. parvum were differentially expressed based on the sequence information of the newly generated whole genome sequences. This approach has been remarkably successful in identifying several families of putative virulence factors, including extracellular proteases and mucins that are specifically expressed in different lineages. Based on the bio-informatic analysis we have identified several loci as targets for qPCR and fluorescence in-situ hybridization (FISH). Identification and validation of C. parvum and C. hominis subtyping markers was achieved. This has allowed us to work on optimizing PCR fragment sizing using a convenient platform (QiaExcel, Qiagen) compared with DNA sequencing. For Giardia virulence, factors were identified using proteomics. Comparative analysis of assemblage specific proteins as well as conserved secreted proteins were characterized. To this end, two families of secreted proteins, Cathepsins and Tenascins, were identified, which were highlighted as the most abundant secreted proteins in our proteomic analysis. Our analyses suggest these proteins are mediators of pathology and so are the key virulence factors mediating host pathogen interactions. Both are constituents of large multi-gene families but we have succeeded in the design and testing of allele specific probes with which to evaluate isolate variation to discriminate virulent strains. Infectivity markers for Toxoplasma gondii were assessed for high resolution identification. A mouse bioassay has been completed using 3 isolates of Toxoplasma gondii oocysts of different viabilities. The reference material from this experiment was frozen down and RNA extracted followed by PCR targeting the SporoSAG gene. Results indicated that SporoSAG was not a sensitive enough marker for the low quantities of oocysts likely to be present in water samples. The reference material referred to above, including RNA, is available to Cluster 2 for platform development experiments. Selected protozoal markers were integrated in the platform development of Cluster 2. Markers for the detection of: the genus Cryptosporidium, human pathogenic Cryptosporidium spp., and the main human pathogenic Cryptosporidium species, Giardia duodenalis, and human pathogenic assemblages A and B have been identified and validated.
• Development and application of sensitive and specific qPCRs for Cryptosporidium spp, C. parvum, C. hominis, Giardia spp., Giardia duodenalis assemblages A and B., Toxoplasma gondii
• Application of a sensitive and specific quantification assay for detection of Toxoplasma gondii DNA in water samples
• Identification and validation of new C. parvum subtyping markers for multilocus fragment sizing

Work Package 5: Microbial Source Tracking [MST] - Combining cultivation-dependent and molecular techniques
The feasibility of novel MST indicators across several countries of the European Union was evaluated based on pre-selected methods and microbiological parameters related to MST. Partners agreed to use the International standard (ISO, CEN) protocols when available. Other protocols of new indicators were written up and added to those listed in a booklet of standard operating procedures (SOPs) for internal use. The verification test for traditional microbial parameters was performed among all participants. Two raw urban sewage samples (high and low faecal concentration) were sent around. All the partners analysed these “blind samples” on the same day for E. coli enterococci, Cl. perfringens, somatic coliphages and Total and fermenting-sorbitol bifidobacteria, following the agreed SOP. Numerical results were sent to the University of Barcelona for statistical comparison of results. No significant differences were observed between the results of partners, though some higher variance was observed for certain parameters. A final selection of indicators was done and predictive MST models were developed by inductive learning methods. The selection of MST markers was done twice: considering all the studied indicators and MST markers (culture dependent and culture independent) and only molecular indicators and MST markers (culture independent). The selected set of MST markers providing models with highest accuracy were identified and suggested to the partners of Cluster 2 as a preliminary selection for integration in platform development. The developed MST models were tested and validated at European level. A list of 15 indicators and MST markers to be analysed during the main sampling campaign was selected from the obtained models and agreed by all participants. Additionally, WP5 partners decided that 8 culture-dependent indicators and MST markers would be also included. The test set of data from the sampling campaign constituted of a total of 52 samples grouped in two data matrices: 38 samples faecally polluted from a unique source and 14 water samples experimentally prepared by mixtures of several faecal sources. These data matrices allowed confirming the usefulness of the molecular MST markers. The selected indicators were integrated in platform development of Cluster 2. Only a fraction of the parameters (molecular MST markers) should be used. The use of all parameters (culture dependent and molecular targets) was considered for comparison with the use of only molecular MST markers using the previously defined predictive models. Analyses were performed concerning two different decision situations: Human versus non-human and distinguishing among four main faecal sources (human, porcine, bovine, poultry). The confirmation of the set of molecular MST markers was done considering only diluted and aged faecal polluted water samples. A list of molecular MST targets was obtained using random forest predictive models based on the Ichnaea® software (University of Barcelona). These selected molecular MST markers are providing practical solutions to two different scenarios for MST: distinguishing Human versus Non-human faecal sources, and distinguishing 4 main faecal sources (human, bovine, porcine, poultry) in Europe.
• To distinguish Human from Non-human faecal sources only 9 parameters are needed to be included in a technical platform
• To distinguish 4 faecal sources (human, bovine, porcine, poultry) 14 parameters are needed in a technical platform, finally 10 derived ratios are requested for the best prediction with random forest models

Summary of results of Cluster 2: Platform Development.
In WP6, improved and novel methods to concentrate waterborne pathogens have been developed, characterized and validated. Guidelines were created for end users regarding the suitability of the sampling technology with respect to three kingdoms (viruses, bacteria and parasites) and water type (treated water, ground water, raw water, etc.) to sample. In WP7, Companies like GPS(Trademark), CEERAM (Biomerieux), Vermicon and MicroLAN developed successfully and validated kits, in collaboration with WP9, able to detect viruses, bacterial pathogens such as coliform bacteria and E. coli. Rapid qPCR protocols were also created by GPS allowing the reduction of detection from 45 minutes down to 8 minutes. In WP8, new techniques using megasonic assisted elution of filters and membranes were demonstrated to either enhance the recovery rate of protozoa such as Cryptosporidium Parvum and Giardia without reduction of their viability, or to enable cost savings in equipment and manpower in the sampling protocol. In WP9, standardised controls were designed, developed, tested and produced with the help of CEERAM and GPSTM for accurate and highly sensitive detection using PCR of pathogens or markers in environmental samples. Rounds of validation were carried out amongst the relevant laboratories for comparative studies and an automated sampling system created in WP8 was validated. The validated molecular methods of detections were provided to members of Cluster 3.

Scientific and technical results of Cluster 2
Work Package 6: Sample Preparation – Robust and rapid sample preparation technologies
The ultrafiltration protocol, which efficiently concentrates bacteria, viruses and parasites, has been successfully used in an outbreak of gastroenteritis in Southern Sweden where a small ground water treatment plant was identified as the likely source. Eight 10-L samples were collected from day 2 of the outbreak and analysed using the in-house protocols and two samples were found to contain norovirus Genotype 1 [GI].
The Monolithic Affinity Filtration technique developed in WP2 was used successfully in (a) an assessment of airborne bacteria and noroviruses in air emission from a new highly-advanced hospital wastewater treatment plant, where NoV GII [Genotype II or 2] was detected in the wastewater at inlet ((2.4±1.4) ×103 GC/L) and outlet (below quantifiable range) and (b) in water distributed in a pipe installation below ground at a party at Svendborg harbor, Denmark where NoV GII.2/p2 was detected.
The first report ever was given on virus detection in bottled water using glass powder and PEG precipitation whereby NoV GI and GII were detected in office water-coolers associated with the outbreak. Between 11 and 25 April 2016, a total of 4,136 cases of gastrointestinal illness were reported to the official health authorities in the Barcelona and Tarragona provinces of Catalonia (Spain).
A study was conducted to test the different concentration methods developed to compare the ease of handling of the filters and obtained recovery efficiencies on different water types. Three methods 1) hollow fibre ultra-filters, 2) MAF-filters (Monolithic Affinity Filters) and 3) glass wool filters were tested by all participating laboratories. Nucleic acids were extracted using the protocol developed by WP6 partners. Organisms were detected using qPCR-kits from Ceeram and GPS as well as in-house qPCR protocols. Waters were seeded with Salmonella enteritidis, Listeria monocytogenes, Campylobacter jejuni, Cryptosporidium parvum, norovirus GGII.4 murine norovirus and Mengovirus. The results showed that all filters could be used to recover microorganisms by staff in all laboratories, even in a laboratory where the staff had no previous experience with the protocols developed in the project. The study showed that no single filter performed better than the others for simultaneous detection of all organism groups, although variations were seen between techniques and among recovered microorganisms. The MAF-filter showed great promise, especially for bacteria in drinking water and due to ease of handling.

Highlights of WP6:
• First report on virus detection in bottled water using the developed glass wool concentration method which confirmed a norovirus disease outbreak.
• Demonstration of validity of the Monolithic Affinity Filtration (MAF) and glass wool concentration procedures for metagenome sequencing of viruses in sewage water.
• Hollow fibre ultra-filtration, MAF and glass wool filtration demonstrated for:
o implementation by laboratory personnel with no previous experience in the techniques
o simultaneous concentration of bacteria, viruses and parasites
o processing of both drinking water and river water
o positive detection when used in surveillance studies of different water types with hollow fibre ultrafiltration for treated drinking and river water, MAF for sewage inlet, secondary and tertiary treated outlet water, and glass wool for ground and surface water.

Work Package 7: Detection – Assessment and development of a portfolio of pathogen detection methods
A food and water viral quadroplex panel was successfully validated for the simultaneous detection of norovirus genogroup I and II, hepatitis A virus and mengovirus internal control. AQUAVALENS SMEs GPS and Ceeram (Biomerieux) developed, in collaboration with WP9, validated kits and results were highly satisfactory with no false positives, few false negatives and insignificant variation between labs. The developed GPS MONODOSE format simplifies automation and minimises possible cross contamination and deterioration of enzyme/fluorophores by freezing/thawing. Fast-Cycling protocol developed by GPS allowed qPCR reaction time to be reduced from 45 to 8 min (total PCR run depends of ramping time of instrument). Kits are available for the detection of more than 200 genes provided, including all prioritised targets from Cluster 1.
An on-site chip detection platform for HAdV was developed as a viral indicator for human wastewater. The lab on a chip (LOC) consists of a 1x1x0.1 cm prototype polymeric chip with an injection moulding insert of hard aluminium using a micro-milling system. The chip with a 12 μL chamber volume was replicated in the high refractive index thermal plastics, COC Topas 5013L-10 using an injection moulding system Victory 80/45 Tech. On-chip PCR detection of virus was carried out using a flatbed thermocycler. The size of amplified products was visualized and confirmed on ethidium bromide stained agarose gels under UV light. Overall, this resulted in similar or even better detection efficiencies as obtained with the qPCR; as approximately 1 GC/reaction of both HAdV-41 DNA constructs and HAdV-35 DNA extracted from whole cell cultured viruses could be detected with no signs of inhibiting effect during amplification. The LOC system allowed also detection of HAdV DNA in extracts from two 10 L tap water samples spiked with HAdV-35 after concentration by either monolithic affinity filtration or glass wool filtration (WP6).
The BACTcontrol system from microLAN is an automated system for semi-continuous measurements of the bacterial indicators, coliform bacteria and Escherichia coli. The detection of these bacteria is based on enzymatic hydrolysis of fluorogenic substrates for β-d-galactosidase (GAL) and β-d-glucuronidase (GLUC). WP7 did set the detection limit of the BACTcontrol system for coliform bacteria and E. coli. Water samples from different sources (river water, karst water) were analysed over a period of time to determine the detection limit for these natural water samples. Altogether, the obtained results indicate that the BACTcontrol online-device could be used for monitoring of coliform bacteria and E. coli in water. Cell numbers of 102 were found to be detected reliably in natural water samples. However, some discrepancies were observed between detected enzymatic activities and cell numbers, which is why cultural methods should be used in addition to the BACTcontrol system.
A ready-to-use kit was developed by Vermicon and others for the quantitative detection of Campylobacter spp. with the FISH technology. In addition, a Catalysed Reporter Deposition (CARD)-FISH protocol was established by the partner MUW in order to use FISH in combination with a solid phase cytometer for quantification of Campylobacter jejuni and thermotolerant Campylobacter spp. and pathogenic Vibrio spp. Furthermore, two alternative semi quantitative methods for the cultural detection of Campylobacter spp. were established by TZW to improve and facilitate cultural detection of Campylobacter spp., which is important for the validation and confirmation of the developed FISH protocols. These cultural methods were validated on natural water samples. Finally, a ready-to-use FISH kit for thermotolerant Campylobacter was developed by the partner Vermicon, which was tested for its usability and specificity. Altogether, the FISH methodology is a fast and sensitive method for monitoring of Campylobacter spp. in water samples. The established FISH protocol and other FISH kits developed by Vermicon were tested in field studies and large drinking water systems of Cluster 3.
The established protocol based on the use of Easystain (BTF Biomerieux) and Dynabeads GC Combo (Idexx) in combination with the ChemScan solid phase cytometer (AES Chemunex Biomerieux) was used to quantify Cryptosporidium spp. in different water matrices. Tenfold dilutions of known concentrations of Cryptosporidium parvum oocysts were spiked into sterile water and numbers of less than 10 oocysts could be reliably quantified with the kit in the SPC. The protocol, applied for groundwater column experiments and wastewater studies, was suitable for reliable detection and quantification of low concentrations of Cryptosporidium spp. However, this protocol did not separate between human pathogenic and non-pathogenic Cryptosporidium species. A new approach was then adapted combining the amplification of a specific virulence factor within individual Cryptosporidium parvum oocysts via PCR and subsequent FISH-detection of the amplified DNA products. A protocol was successfully developed that gave satisfying results for detection of the oocysts with epifluorescence microscopy. However, staining was weak and concentration techniques of large sample volumes (100 to 10,000 L) studied in WP6 that are compatible with subsequent immunomagnetic separation and solid phase cytometry analysis, should be pursued.
Highlights of WP7:
From the seven different platforms, four have been developed for use in field studies for the rapid detection of faecal pollution, waterborne pathogens and virulence genes:
• AQUAVALENS SMEs GPS and Ceeram have developed validated kits for more than 200 pathogens, including all prioritised targets from cluster 1, enabling the first broad European study on waterborne pathogens providing comparable results on a real-time PCR platform.
• Creation by GPS of the MONODOSE format which simplifies automation, minimises possible cross contamination, avoids deterioration of enzyme/fluorophores by freezing/thawing and allows simultaneous detection of different pathogens.
• ATP biosensor for the on-line detection of bacterial activity in drinking water distribution networks in collaboration with WP8.
• MicroLAN fully automated platform BACTcontrol for detection of faecal pollution in source water in less than an hour.
• Vermicon kits for FISH detection of thermophilic Campylobacter and other bacterial pathogens.

Substantial developments have been achieved with regard to:
• Prototype Lab on a Chip developed for on-site detection of food- and waterborne adenoviruses with comparable performance to traditional qPCR.
• Solid phase Cytometry (SPC) developed for quantitative detection of: 1. Bacterial pathogens using CARD-FISH, 2. Cryptosporidium spp. oocysts using immunofluorescence kits and 3. Pathogenic Cryptosporidium parvum using in-situ PCR.
• VOCMA developed for a real time PCR platform to the extent that enables take-up for biotechnology companies, mainly within the sector of clinical microbiology, for the simultaneous detection of gastro-intestinal pathogens and antimicrobial resistance genes.

Work Package 8: Integration – Integrated and automated platforms for effective pathogen detection
Modern pattern recognition techniques were developed to enhance the functionality of an automated optical imaging system commercialized by Vermicon used in the quantification of fluorescent bacterial cells. The imaging system consists of a microscope, a camera, and an XYZ table, which are automatically controlled via software. The software automatically detects and counts the FISH-stained fluorescent bacterial cells. The techniques automatically learned to distinguish cells from the background by using a large number of examples of correct cells provided by the experts. Microscopic systems for the automatic detection of FISH-stained fluorescent bacterial cells were, up to now, not available with automatic focus, with 66 hours per membrane in term of scanning time. This made FISH techniques for bacteria detection more time-consuming than conventional culture-based methods. The system overcame these issues and achieved the objectives of this task by taking only 3 hours per scan. Another useful feature of the system is that the user can reposition the microscope after a scan, at the same exact location where cells have been detected, and manually validate the cell counting. The SME Vermicon intends to commercialise the technology developed.
Tests to remove Cryptosporidium oocysts from filters and membranes of IDEXX systems using for water sampling demonstrated the same recovery rate of protozoa using megasonic assisted agitation as the standard EPA method, 1623.1 . The tests were extended to Giardia and demonstrated again a high recovery rate and increased viability of the oocysts compared to manual rubbing of the membranes. Megasonic elution was demonstrated to replace the need for centrifugation, resulting in saving in centrifugation equipment and skilled manpower. This elution technique has the potential for integration within a fully automated, stand-alone detection system and could be extended to the enrichment of other pathogens.
A microfluidic-based system for the detection of living bacteria was developed by measuring the optical signal from the chemiluminescent reaction between ATP and luciferin in the presence of the luciferase enzyme ATP (Adenosine TriPhosphate). The system was automated with the potential of becoming a robust standalone piece of equipment that can be installed at the waterworks and in the distribution system. Total and free ATP were measured on a series of standard solutions in the 2.5-500 pg/mL range by adding extraction reagent or replacing it with ATP-free water respectively. The sensitivity was estimated to be better than 2.5 pg/mL for both total and free ATP. Tap water samples and tap water spiked with untreated wastewater and showed a higher level of total ATP than free ATP as expected.

Highlights of WP8:
• Successful validation in collaboration with WP9 for viruses and bacteria of an integrated platform using hollow-fibre ultrafiltration filters.
• Demonstrated increased recovery and viability rate of protozoa Giardia and Cryptosporidium from elution of membrane and IDEXX filters using megasonic transducers.
• Demonstrated reduction of costs in capital equipment and manpower for the EPA method by avoiding the use of centrifugation step.
• Reduction of scanning time from 64h to around 3h of the Vermicon FISH scanning system.
• Validation of a microfluidic-based ATP sensor for detection of bacterial pathogen in collaboration with WP7.
• Validation with WP9 of an automated dead-end ultrafiltration system for viruses and bacteria.

Work Package 9: Standardisation and validation
Standardised controls have been designed, developed, tested, and produced in a large quantity (Ceeram, GPS) given the susceptibility of a sensitive PCR to cross-contamination (false positives) and also matrix interferences (false negatives). The controls included a preparation control (enrichment, extraction), an internal positive control, a negative (blank) and an external positive control. Guidelines, written as a technical aid in a report, were also provided for the preparation of standard operation protocols (SOPs).
Samples in the validation were characterized by specific analyte/matrix/concentration combinations. The main results were:
• No false positives for blank samples (tested in the General Test) in case of qPCR for HAV, E. coli, C. jejuni, and C. parvum. In case of NoV GI and NoV GII one (out of six) and two (out of six) labs reported very low gene copy numbers, respectively.
• In all tests, higher concentrations of spiked nucleic acids corresponded to higher analysed gene copy numbers.
• In particular the RNA samples (viruses) gave results close to the expected values, i.e. the spiked number of gene copies.
• In particular the DNA samples (bacteria and C. parvum) in pure water gave lower results than expected. Most probably, a decrease of targets occurred during shipping and storage.
• In the inhibition test, a humic acid concentration of 5 ng/µL had negligible effects. However, a concentration of 27 ng/µL in some tests led to decreased copy numbers.
• For the complex matrix samples, remarkably reproducible results were obtained for extracts of surface water no 1. Results within 1 Log deviation were reported for viruses, bacteria and Cryptosporidium parvum from all participating labs. However, larger deviations were obtained for spiked extracts of surface water no 2.

Different qPCR kits from Ceeram and GPS for the detection of bacterial (Campylobacter jejuni, Escherichia coli) and viral targets (adenovirus, mengovirus) were tested with spiked water samples. The investigated commercial PCR detection kits proved to be suitable for detection of bacteria such as C. jejuni and E. coli, and viruses such as adenovirus and mengovirus.
Highlights of WP9:
• Validation experiments using PCR methods for the detection of bacterial target DNA resulted in:
o No false positive results for blank samples
o In all tests, higher concentrations of spiked nucleic acids corresponded to higher analysed gene copy numbers.
o Obtained results are close to the expected values
o Specificity of the qPCR assays using the kit systems.
o Negligible effects of humic acid on dtec qPCR kits
• PCR kits developed suitable for detection of bacteria such as C. jejuni and E. coli. The results of the molecular kit assays were consistent with expected values.
Validation using model bacteria and model viruses (E. coli phiX174 and MS2) of a concentration system based on dead-end ultra-filtration in collaboration with WP 8.

Summary of results of Cluster 3: Field Studies in European Drinking water systems.

Description of progress
Surveys of pathogen occurrence in large and small water supplies were conducted monthly over a period of one year at several locations. Pathogen monitoring in food production and food preparation facilities was undertaken to coincide with the growing season for the particular crop.

Work package 10 – Large systems
Sites for the evaluation of the Aquavalens’ technologies were located in the United Kingdom (UK), Spain (ES), Germany (DE) and Denmark (DK) that were carefully selected to provide a suitable range of water types and representing different catchment activities. Additionally, each of their corresponding water treatment works provided a range of processes for comparing their ability to remove pathogens.

Monitoring was conducted monthly over one year at each site for a selected range of viral, bacterial and protozoan parasites with E. coli also included because of its application as an indicator organism for the presence of faecal contamination. The dead-end hollow fibre ultrafiltration procedure developed by Cluster 2 proved an effective technique for concentrating large volumes of the different source waters and permitted organisms from all three kingdoms to be sampled in a single step. The subsequent secondary concentration procedure varied between laboratories depending on the availability of specific apparatus and comprised methods such as centrifugation or PEG (polyethylene glycol) precipitation, which was followed by nucleic acid extraction.

Pathogens were detected in the source water at most sites and their presence correlated with the observations from the sanitary surveys conducted for the respective Drinking Water Safety Plans. At one of the sites, it was also able to semi-quantify the viruses recovered, which was not possible with current culture methods. The absence of viruses from two of the four sites was not unexpected as both had little or no inputs of faecal contamination of human origin. A certain amount of variability in the subsequent detection of the different organisms, however, was found between the sites.

Significant discrepancy between PCR and verification results regarding protozoa was observed, which required further investigation. The detection of E. coli by the qPCR method also was found to be highly dependent on the specific type of water.

The work also demonstrated the value of a semi-continuous instrument (BACTcontrol) for online monitoring of indicator bacteria and total bacterial activity. During routine operation, the instrument showed consistent correlation with certain microbial parameters and provided new knowledge about relationship between microorganisms and various treatment processes. During extreme events affecting the water treatment works, the monitor was able to detect change in total bacterial activity providing operators with an early warning of potential deterioration in water quality.
A detection method, that applied Fluorescence in-situ hybridisation (FISH) for the examination of source and treated drinking water, enabled automatic detection and counting Campylobacter and E. coli in the various water types. It was, however, prone to interference from particulate matter in raw waters and required a certain amount of effort to obtain good performance.

In comparison with conventional techniques, reasonable agreement was found with the molecular techniques for detecting the selected pathogens and indicator organisms. Some discrepancies were observed with the detection of the protozoan parasites, Giardia and Cryptosporidium, which was attributed to the methodology applied to extract the genetic material.

Work package 11 – Small systems
Small water systems generally have been viewed as being more vulnerable to poor water quality and are responsible for a greater proportion of infectious intestinal illnesses. Consequently, it was clear that the challenges for improving water quality were greater but that approaches developed in Aquavalens had to be compatible with constraints imposed by their location and the capabilities of their operators.
Several locations were identified in Portugal, Scotland and Serbia, through consultations with local authorities and water providers, for monitoring the status of the microbiological quality of small water supplies. Databases were created on the occurrence of viruses, bacteria and protozoa that provide a useful insight on pathogen distribution in different source waters and to provide a source of data for subsequent risk assessment undertaken by Cluster 4.
The Aquavalens’ techniques were found to be more sensitive than the conventional techniques. A greater proportion of samples were positive for E. coli using large volume filtration and molecular detection compared to conventional culture based techniques that examine a volume of 100mL. Clearly, improved detection of indicator organisms will provide better surveillance of water quality but would require corroboration with data obtained from existing monitoring.
Sanitary surveys of these water supply systems had identified a number of deficiencies that were recognised as risk factors for adversely affecting microbiological safe drinking water.
Significant improvements in water quality were achieved with the development and installation of water treatment stations on selected supplies serving between 40 and 60 inhabitants. The treatment systems were fitted within shipping containers for ease of transport, installation on site and to provide a secure facility for operation. Each station provided removal of iron and manganese by oxidation and filtration to prepare the water for disinfection with free chlorine. The stations were designed for a peak consumption of 0.15 m3 of water per hour for each inhabitant and to operate with minimum intervention. These stations will remain with each of the respective municipalities after the completion of the project to ensure the provision of safe drinking water for their communities.
The ability to detect events that may compromise water quality was addressed by the development of an automatic sampling system. Turbidity, as an indicator of disturbance to the water supply, was continuously monitored and the sampling system was programmed to collect a large volume of water in response to turbidity exceeding a pre-set limit. An alarm alerted the operator to respond to the event.
A number of dissemination activities took place during the course of the work to transfer knowledge and expertise on the principles and practices of pathogen recovery and detection in water systems to a selected range of stakeholders. In particular, various end-users were successfully trained to undertake molecular detection techniques in their own laboratories.
Surveys of various stakeholders revealed a perceived reluctance by end-users to adopt new tools and techniques. Consumers were generally found to appreciate the benefits provided by the Aquavalens’ technologies, several were wary of the additional burden that may be imposed as a consequence of additional monitoring requirements. In some locations, increased operational costs were cited by respondents as an additional barrier to implementation.

Work package 12 – Water for food production
The provision of microbiologically safe drinking water is critical for drinking water used for food production and water retailed in bottles. Various stages in the preparation and processing of these products are vulnerable to contamination of faecal origin and several outbreaks have been reported from across Europe. A review of the current situation identified particular areas of concern associated with leafy green vegetables, sprouted seeds and cultivation of soft fruits.
A systematic monitoring programme was implemented at sites in Ireland, Portugal, Scotland and Serbia to assess the microbiological quality of samples of irrigation water, food products and bottled water. Cryptosporidium, was the only pathogen identified and only on a single occasion, indicating the generally good quality of these particular food and bottled water operations. A number of samples were positive for E. coli as an indicator organism, especially in irrigation water.
Reasonable agreement was found between conventional detection methods and the molecular methods developed within the Aquavalens project. Some discrepancy was observed regarding the detection of E. coli between the two methods. Molecular methods, which detect both living and dead bacteria, tended to give higher detection rates than conventional culture methods. Of more concern, however, was the occurrence of positive detection by the molecular methods for the negative controls where the target organism would be absent. Investigation of the procedural steps identified reagent contamination as the cause and highlighted the need for inclusion of appropriate sterility checks and process controls.
A major dissemination event was organised by Desing (Partner 24) for food producers in Serbia and the surrounding countries. Representatives from WP12 delivered a series of lectures on the microbiological safety of drinking water used for food production. Wider dissemination was undertaken through a presentation at the Society for Applied Microbiology Annual Conference by Teagasc (Partner 12).
Throughout the study, the partners have been aware of the positive engagement by food producers through their interest in the work being undertaken and their willingness to provide support. It was viewed, therefore, that small, local events were probably a better approach for future dissemination.
Successful application of a single large volume procedure for the recovery of all three pathogens types from source water, process water and treated drinking water for a range of water types. The procedure was also found to be of benefit for assessing water quality in the distribution network providing rapid insights into the nature of these events.
The molecular detection techniques improved the ability to detect and, in some circumstances, quantify, pathogen numbers. In comparison with conventional methods, the response was quicker giving operators more timely information on the impact of events on the microbiological safety of drinking water.
Online instrumentation provided the ability to track changes in the microbial contamination especially in source water and provide an early indication of adverse deterioration in water quality.
The development of transportable water treatment stations provided an effective and low cost solution to improving the microbiological safety of drinking water in rural locations.
A number of dissemination activities have taken place during the course of the project. The presentations have attracted interest from researchers and practitioners resulting in greater awareness of the value of the Aquavalens project.
Potential socio-impacts
The principal aim of the activities of Cluster 3’s work was to provide information and data relevant for the work of Cluster 4. There are, however, certain impacts that are directly relevant to the work of Cluster 3.
Many of the tools and techniques developed in the project are now being implemented by Aigües de Barcelona (ES) for managing the microbiological safety of drinking water.
This work has proved to be of significant interest to the water industry in the UK. Their research organisation, UKWIR intends to commission further studies to increase the application and uptake of these methods for improving quantitative microbial risk assessments and Drinking Water Safety Plans.
Our work has increased the knowledge available on the occurrence of waterborne pathogens in different water supplies across Europe. This information is of direct relevance to water utilities and food producers, and it will enable water utilities to improve their risk assessments.
The findings on pathogen occurrence demonstrated that large systems have effective treatment to manage the risk hence providing reassurance to utilities over the safety of their operations.
A lasting legacy from the project is the successful deployment of portable water treatment systems that have been donated to the municipalities that participated in this particular programme of work. These treatment stations will remain in place for the benefit of the respective communities.
Overall, the knowledge obtained from the Aquavalens’ technology platforms, has identified new pathways for process optimisation as well as directions for further scientific research projects.
Summary of results: Cluster 4. Improving Public Health through safer water.
Description of work performed and main results 4-5 pages max.
WP 13
The methods used to reach the objectives of WP13 was through gathering information from the test sites (twenty small and large drinking water supply systems from different EU Member States) with specific questionnaires. In addition the results derived from the monitoring with the new AQV techniques in relation with the AQV verification control with conventional cultural methods and the data from the regular surveillance monitoring were analyzed and compared. This information was used to derive the possible impact on water safety and preventive management tools like Water Safety Plan (WSP) using the WHO’s WSP manuals.
Task 13.1 Develop and administer a questionnaire.
Two set of questionnaires were developed to gather information from each supply that participated in the trial on risk factors and operational management that could be improved with the AQV platforms. One questionnaire for large supplies and one shorter survey for small supplies were prepared and sent out in spring 2016 and another set was sent out in June/July 2017. The first set of questionnaires were first tested in the field in Iceland and Spain at four water supplies (2 large and 2 small supplies) before sending them out to the test sites.
The first set of questionnaire provided information on general subjects, on infrastructure, on the existence of a WSP and/or other preventive management approaches as well as recognized potential risk factors to water safety. Many questions were aiming at revealing risk factors. The main outcome of the first questionnaire was to find out the status of the water systems tested and their main challenges to water safety. In general the main recognized challenges were the existence of old infrastructures and water quality. Fecal contamination, agriculture, farm waste, residential area and traffic were the main challenges of the catchment areas at the twenty water supplies participating in trialing the AQV techniques/platforms.
The second set of questionnaires asked qualitative questions on the experience of using the AQV techniques/platforms. This questionnaire was sent to the people responsible for the system monitoring and for using the AQV techniques. The general conclusion is that the AQV platforms enable improved knowledge on pathogens present or confirm high quality drinking water. New knowledge on risk factors emerged, new hazards were identified and existing surveillance indicators were verified, as for instance total coliform and E.coli. The AQV platforms showed they have the potential to be used for the microbiological control in water treatment processes and management and thus in risk reduction.
Task 13.2 Develop indicators to measure performance of WSP.
In order to be able to evaluate the benefits from the new AQV platforms a set of indicators was developed. The method used to identify potential impact on WSP was to analyze each WSP module and evaluate possible influences of the AQV technique on the performance of the WSP. The focal point for the evaluation was the WSP manuals published by WHO. The principles in preventive management are more or less the same in all management systems so this should apply to other forms of preventive management systems based on the principle of the quality control circle and applies to HACCP and ISO 22000, as well as to other similar systems. Impact on WSP is also assessed based on responses to the first questionnaire on expected impact from AQV platforms.
From the fifteen possible indicators for improved WSP and water safety with AQV platform four showed to be especially relevant. This main four indicators are; 1) water quality, both with operational monitoring, and regular surveillance monitoring as improved monitoring e.g. finding pathogens will assist in identifying water quality problems; 2) pipe breaks and pressure losses indicating integrity of the system, supporting the improvement plan and prioritising maintenance; 3) number of incidents that should decrease with improved water quality; and 4) treatment performance data as pathogenic detection after treatment is an extra barrier for water contamination.

Task 13.3 Establish the effectiveness of the developed AQV platforms as a part of preventive management.
The timeframe for this project was shown to be not long enough to get reliable results to measure the impact from the new AQV platforms on WSP with the developed indicators. Those indicators were evaluated before and after using the techniques/ platforms, but the trial time is only one year and finishes near the end of this project. The main indicator used on performance of AQV techniques/platform was the results from the water quality from the performed tests and the regular results obtained with the surveillance monitoring, if available.
Improved knowledge on water quality and on the presence of pathogens in water tested with AQV platform, will have impact on water safety management as WSP in many ways. Results from monitoring with the AQV platform shows presence of a number of pathogenic bacteria, viruses and parasites in samples taken from the raw and processed water from both large and small water supplies sites and also from treated and untreated tap water at some of the small water supplies. When comparing AQV results with results from regular surveillance monitoring of the indicator for faecal contamination i.e. E.coli results from AQV were shown to be more sensitive.
Task 13.4 Development and application
There is little preventive safety planning or assessment of potential risks in the current European drinking water directive (DWD). In light of the importance of drinking water quality there is a need for extensive control and efficiency of the services provided by water supplies with improved monitoring of pathogenic risk and including new early warning instruments. The results from this project are an important input into this process.
Safe drinking water is essential for public health and well-being and has been recognized by the UN Resolution 64/292 as a fundamental human right and the UN Sustainable Development Goal 6 that availability and sustainable management of water should be ensured for all. It has been shown that small water supplies are more at risk of non-compliance to regulation and most waterborne outbreaks occur at the small supplies. To ensure this preventive management as WSP should be included into the requirement in the directive as well as regular surveillance of the small supplies. According to results from this project, the DWD should include action to prevent and reduce leakage and encourage the renewing of old infrastructure. These actions will then be included into national legislation as mandatory requirements to ensure safe water for all the citizen of Europe.

WP 14
The specific objectives of this work package are the following:
Task 14.1. This task has been carried out. However the number of [positive] samples collected in WP10, 11 and 12 was less than foreseen when the Description of Work was written. So scientists at UEA could examine the results of the testing undertaken in all three Cluster 3 work packages but there were not really enough observations to say a great deal about the impact of climate change. We would have needed many hundreds or thousands of samples to say anything useful about the type or presence of pathogens in the samples and how that might be altering as a result of climate change. The obvious and welcome conclusion is that most of the samples taken revealed no human pathogen and thus that safe water was being delivered by large water suppliers, small water suppliers and those using water in food production in most cases. Del 14.1 explains this in detail.
Task 14.2 We conducted in collaboration with WP11 a prospective epidemiological study of the health of people reliant on small supplies and its relationship to the presence of microbial markers in their water supply. The study suffered from the small number of samples taken in WP11, the relatively small number of positive samples, a low survey response rate in many places and the inconsistency of the results in different countries. Consequently the results have not really altered the previously held perception that normally rural users are at higher risk in small rural water supplies.
In task 14.3 we demonstrated that existing risk assessment approaches used for drinking water supplies in Europe may be subject to systematic biases. In particular for small water supplies we showed that exposure assessments based on random microbiological sampling is likely to under-estimate risk because of the low probabilities of detecting extreme events that occur very infrequently but when they do occur are associated with a substantial increase in risk of illness. We also showed that current methods of estimating annual risk from daily risks are flawed and either systematically under-estimate or over-estimate risk. This is because exposure is affected by variables some of which vary from day to day in an individual whilst other variables are fairly constant in an individual but vary between individuals. Instead we developed and presented a microsimulation approach to quantitative microbial risk assessment which gives a more realistic estimation of risk. Microsimulation also allows exposures from multiple pathways to be integrated into a single combined risk assessment.
Within Task 14.4 we have developed a scenario based approach for planning for unexpected risks to water supplies such as from emerging infectious diseases or deliberate attacks. We then went onto use this approach in a workshop in Barcelona attended by people from the water industry and public health agencies. This generated plausible scenarios that support the planning process and Water Safety plans.

WP 15
Task 15.1 Evaluation of the environmental impact and the carbon footprint (URV)
Life Cycle Assessment (LCA) was the methodology used within WP15 for the evaluation of the environmental impacts in the form of carbon footprint (CFP) (or carbon dioxide equivalent emissions) of the manufacturing and application of the different platforms (i.e. methods of pathogens detection in water) developed under the project. The CFP calculation procedure followed the standard norm ISO 14044:2006 and implied the collaboration with Cluster 2 partners that were involved in the manufacturing of the platforms and Cluster 3 partners that tested the platforms in the field.
Specific questionnaires were designed to collect the data about materials, consumables, use of equipment, transport and waste management related to the platforms production. Carbon footprint scores were computed for the production of two platforms based on quantitative real-time Polymerase Chain Reaction (qPCR kits) of partners GPS and Ceeram. Values of CFP between 0.001 and 0.009 kg of CO2-eq/reaction were obtained depending of the presentation format of the kit. These values are comparable to the CFP generated by production of one steel screw. The CFP was lower for those formats that allowed higher number of reactions per presentation. The CFP of the production of the on-line monitoring device for faecal indicator measurement developed by the partner MicroLAN was also calculated. The emissions obtained were 0.013 kg CO2-equivalents/analysis, assuming the most favourable scenario, with continuous working during the life span of the device and 2 hours spent per analysis. In this case the CFP values were comparable to production of one egg.
To evaluate the CFP derived from the use of the methods, new specific questionnaires for the data provision from Cluster 3 partners were developed to obtain information about the materials and energy flows with special attention to sampling, transport, laboratory practices and waste management. The results indicated that the platform use phase was the most environmentally damaging, the CFP being three orders of magnitude higher than for the manufacturing phase in the case of the qPCR kits. The CFP evaluated for the whole processing of a water sample using qPCR kits was around 2 kg CO2-eq/analysis (equivalent to the CFP produced by a fridge working during one day), for which the transport of the sampling accounted for more than 50% of the impact. The CFP calculated for the online monitoring device was around 0.08 kg of CO2eq/analysis (comparable to 10 minutes of use of a 60W light bulb), considerably lower than for qPCR kits since it produces rapid analyses in-situ, reducing materials consumption, time and transport from sampling site to the laboratory.
For the comparative analysis of the CFP generated by the AQV developed and applied technologies in relation to the conventional analytical methods, the CFP for the analysis of a set of microorganisms from the three kingdoms (viruses, bacteria and parasites) was evaluated. The total score of CFP for the analysis of the set of microorganisms was 12.8 kg CO2-eq/sampling point for AQV methods, whereas the conventional methods yielded 37.6 kg CO2-eq/sampling point. The reasons of this improvement in the AQV methods were the reduction in the travelling associated with sampling, the lower energy consumption compared to the cultivation techniques, and the reduction of the amount of materials because the stages of sampling, first and second concentration and DNA extraction in the AQV procedures were common for the analysis of the different pathogens.
Task 15.2 Cost-benefit analysis and determination of economic gains (URV, UEA, UI)
Since the accomplishment of the tasks in 15.1 implied an exhaustive characterization of the AQV and conventional methods for their comparison, this information was also used to carry out the economic analysis. The CFP of the platforms included the identification of the main materials and energy consumption, so the main sources of expenditures were established using the same basis. In this sense the filter used in the first concentration of the sample and the sampling transport were identified as the main cost contributors. As explained in the previous section, the travels for sampling transport and the amount of materials needed for the AQV methods were significantly lower than for the conventional methods, this implied a 50% reduction in the costs.
The methodology used to calculate the economic gains derived from the implementation of the AQV methods was based in the estimation of two parameters: number of cases of disease avoided and cost of disease to the public health system. Firstly the reduction of number of disease cases was estimated considering different scenarios using one of the AQV methods, particularly the early warning online monitoring device as indicator of waterborne pathogen presence, as this could help avoid disease outbreaks in the population. Secondly different publications and public health system databases were consulted to establish an average cost per person of diseases typically related to waterborne pathogen exposure.
Task 15.3 Applying specific technologies and/or platforms in developing countries (URV, UEA)
Three water specialists from Uganda, East Africa and Egypt attended our Lisbon workshop. They said there could be interest in water tests for large water suppliers which helped overcome the lack of trained personnel and the lack of chemicals / facilities. Two wanted to help train staff in suitable molecular testing methods and one said the online testing machine [BACTcontrol] could be part of more effective quality control testing. However all three African representatives claimed that the water companies only want to achieve the WHO recommended standard for bacterial decontamina- tion at the treatment centre, while conscious that the [clean] water will often be re-contaminated after leaving the plant by breaks and illegal connections in the distribution system. One representative said there appeared to be interest among large water providers in African for items such as chlorine sensors. This might form part of a GCRF African UK funding bid.
Task 15.4. Strengthen Europe’s global leadership on the health water related risk (URV, UEA, UI) Deliverable 15-4 has demonstrated a number of ways that Europe can strengthen it’s leadership in health related water risk. Partners from the Aquavalens consortium have explained how technological developments have made it quicker, simpler and cheaper to use molecular testing to monitor and improve water quality in the brochure described in Del 15-4. One obvious application is to involve scientists in the next amendment of Drinking Water Directive to crystalize some of these benefits. Another popular proposal was a regional training programme to provide Water Safety Planning training to small water system organisers / managers. A third exciting proposal was the suggestion that large water users adopt the BACTcontrol machine as an early warning tool to help safeguard their source water quality. There are many more such suggestions and proposals covered in D15-4 and we plan to disseminate them in a follow-on COST Action bid.

The Period 4 report described the recent work on our website and the videos we made available there. In September 2017 UEA completed the production of a 48 page brochure containing 14 stories of results and achievements created by this Aquavalens project. We attempted to describe the achievements in laypersons English and 3000 paper copies were distributed. We also had a high resolution copy available on the website. Deliverable 16.6 focused on the events that have been chosen by the consortium for dissemination via a team of representatives. A good deal of effort went into the scientific journal publications and the associated presentations at conferences or business meetings, covered in the periodic reports to the FP7 programme. The “List of publications” in the “End of Project” report and contains more than sixty academic articles and books. The “Dissemination Activity list” in the same report includes more than 250 activities that took place over the five year period. The joint presentations reported on here are all important but are only a small part of our dissemination work. These organised themed presentations by the Aquavalens consortium aimed to enable key partners to present to a suitable audience, key sections of our results and achievements. Here they are.
1. Watermicro2015: Health Related Water and Microbiology Conference [2015]
This conference accepted presentations by seventeen of our scientists from Cluster 1.
2. Rapid Methods Europe [2016]. We organised eight presentations by our engineers and test developers from Cluster 2 who focussed on water sampling, concentration and testing.
3. Health Related Water and Microbiology Conference, Chapel Hill, USA [2107]. Sixteen AQV staff presented a large variety of topics and results, another themed session of talks.
4. Society for Applied Microbiology Annual Conference at Gateshead, [July 2017]. Some of the food research results were available. Some of the food research partners also chose to disseminate locally as that met their needs better. Representatives from Aquavalens also visited FEMS in July in Valencia and the large Tampa IAFP conference.
5. S2Small conference 23-25 October [2017], Nantes, France. This is conference that focusses on small water systems and sewage disposal as well. It appeared to be one of the best conferences in Europe for small water system technology in the second half of 2017.
As some or our participants could not present at a suitable conference late in 2017, we organised an additional meeting in Lisbon in January 2018 and brief coverage of that event is provided here too as part of item 5.

Potential Impact:
Description of the potential impact [including socio-economic impact] and the wider societal implications of the project so far, and the main dissemination and exploitation of results.
Impact and Exploitation of Cluster 1: Platform Targets
We developed and validated more sensitive and quantitative molecular detection technologies for all defined waterborne pathogens (viruses, bacteria, protozoa) with high taxonomic resolution, accuracy, and analytical mass capacity. Diagnostic and surveillance tools for waterborne pathogens have been improved in terms of specificity and sensitivity. Based on these technologies molecular quantification kits and genome standards for all targeted species were validated and are now commercially available from SMEs. The following detailed impact and exploitation of the Project can be expected:
1. Development of a new, uniform, Europe-wide approach regarding drinking water safety management plans
Advanced molecular assays were developed for quantification of the major waterborne pathogens and validated for source and drinking water. In addition, viability PCR (vPCR) was applied to indicator bacteria and experimental assessment of infectivity of several pathogens was performed to verify the validity of the molecular assays. These novel molecular tools are available for mass application in small and large water supplies and could contribute, together with the results of the Europe-wide MST analyses, to a science-based approach for drinking water safety in the EU. Additionally, a booklet of SOPs for MST indicators is available facilitating the standardization of protocols between European laboratories.
2. Give support to the Drinking Water Directive (98/83/EC)
Identifying faecal sources in catchments is contributing to the prevention and correction criteria indicated by the Drinking Water Directive 98/83/EC: Quote “28. Whereas, should such remedial action be necessary to restore the quality of water intended for human consumption, in accordance with Article 130r(2) of the Treaty, priority should be given to action which rectifies the problem at source”
3. Give support to the EU Innovation Union
Molecular test kits for all waterborne pathogens relevant to drinking water were developed and validated. These test kits, including certified molecular reference material, are now produced and commercialized by two SME of the AQUAVALENS consortium and can be viewed as a significant contribution to the bio-economy of the EU.
4. Contribute to public health and to climate change preparedness
By identifying molecular markers for detection of waterborne human-pathogenic viruses, bacteria and protozoa the Project has contributed to the development of assays to improve water quality monitoring for these pathogens and enabled monitoring in the absence of any standard method for Toxoplasma. The assays for the detection of human pathogenic strains and identification of putative virulence factors will help in improving public health risk assessments. This will assist in providing baseline and change data during climate change investigations. The development of subtyping tools for the pathogens will improve outbreak investigations by enabling tracking of contamination and infections.
7. Strengthen relations between researchers and industry, and further disseminate results of advanced research towards practical applications in water quality analysis
An important aspect of the development and optimisation of the Toxoplasma qPCR has been the strengthening of the relationship with Scottish Water, the main industry provider of drinking water in Scotland. We have further developed this work as the same water supply has a history of Cryptosporidium infection which has resulted in illness of the village residents and visitors. In conjunction with the land owners, the Cairngorm National Park, the OpenNESS project and Scottish Water, we have completed a whole catchment study of the area. These results have been used to inform the land managers, who along with Scottish Water, have instigated appropriate catchment management strategies, such as appropriate fencing and water trough installation. It is envisaged that this will have multiple benefits including an improvement in drinking water quality and human health to the Scottish people.
9. Added value of a European approach
The MST experimental approach and the MST field trails done in different EU countries will support further EU regulations on total maximum daily loads, the definition of standard methods for determining faecal pollution sources and consequently risk assessment of waterborne pathogens.

Cluster 2 Impact and Exploitation of Cluster 2: Platform Development
We developed a taxonomy of the most relevant filtration and enrichment methods and demonstrated their applicability in terms of kingdoms to be sampled and suitability for the types of water. We also applied successfully these filtration methods in real case studies of contamination outbreak in Denmark and Spain. We also developed commercial kits for molecular detection that cover all pathogens targeted in Cluster 1. We increased or validated the performance of commercial automated systems used in the detection of bacterial pathogens and deployed some of these systems in companies specialised the treatment of water. We demonstrated the use of megasonic assisted agitation in the cost-effective elution of pathogens from filters and membranes. We set in place validation procedures and protocols for the molecular detection kits and an automated filtration system. By so doing we demonstrated the effective and efficient recovery of pathogens in the automated filtration system.

Based on the work achieved across the four work-packages of this cluster, the following impact and exploitation of the Project can be expected:
1. Development of a report providing guidelines for the effective use of filtration and enrichment methods
Very comprehensive guidelines were prepared for the best use of filters depending on the type of water to be filtered as well as the type of pathogens requested to be sampled. This work carried out in WP6 should be expected to be broadcast to the wider scientific and industrial community. Moreover, the glass wool method could be introduced in developing countries due to the low cost of the method. This would require however the commercialisation of the glass wool by more than one company.

2. Enhancement of commercial bacterial pathogens detection systems
The Vermicon (Germany) and MicroLAN (Netherlands) and Genetic PCR Solutions [Trademark] commercial systems have benefited from the expertise of the Aquavalens Consortium in terms of enhancement of performance or validation of the detection method for certain types of water. For the Vermicon system, this performance increase was with regards to the reduction of the scanning time for the detection the pathogens as well as the versatility of the new scanning method. GPS™ has developed PCR kit formats that facilitate the use and transfer of this technology, with protocols allowing the test of many different pathogens simultaneously. It is to be expected that the Companies involved should be able to increase their commercial footprint in the market place as a consequence of this work.

3. Commercialisation of the microfluidic based ATP sensor
The use of microfluidic technology has allowed the ATP sensor system developed at the DTU to become portable and automated. Continuation of the work should be encouraged in order to bring this technology closer to market. The input from the company Epigem was crucial in providing a microfluidic solution to the ATP sensor system which allowed the system to become portable.

4. Dissemination of results related to megasonic assisted elution of filters and membranes for the EPA method number 1623.1.
The demonstration of the advantages of the megasonic assisted elution of filters and membranes for the EPA method should be disseminated to the wider scientific and end users’ communities. The use of such transducers avoids the use of centrifugation systems and reduces the amount of skilled manpower, whilst increasing the viability of oocysts and maintaining the recovery rate of the pathogens. It is expected that the results obtained and CAD layouts as well as programming code be provided as open source documents for all users to benefit from.

Impact and Exploitation of Cluster 3: Field Studies in European Drinking Water Systems

The primary aims of the work undertaken in Cluster 3 were to evaluate the performance of the various techniques under a range of conditions encountered in practice and to provide information to support the activities of Cluster 4.

3.1.1. Development of a new, uniform, Europe-wide approach regarding drinking water safety management plans.
Fundamental to developing uniform drinking water safety plans is having reliable and robust techniques for undertaking the risk assessment. A key component of this process is an effective sanitary survey to provide knowledge of the pathogen burden in different sources of drinking water to establish that the corresponding water treatment works is adequate to produce microbiologically safe water. The adoption of fully developed and validated protocols for pathogen detection developed in the Aquavalens project will enable consistency to be achieved for monitoring the microbiological quality of drinking water systems.
Additionally, evaluation of the molecular techniques in Cluster 3 has demonstrated their ability to detect and quantify a wider range of pathogens than was possible with conventional methods. For example, certain viruses, such as noroviruses, can only be detected by these techniques and so, Aquavalens has provided valuable data on their occurrence in different types of source water across Europe.
In the future, adopting consistent recovery and detection methods across Europe will ensure that findings are comparable and the results can be extrapolated to other similar water types. Sharing of information in this way will enable the findings to be available to a wider number of water utilities and food producers and so improve the accuracy of risk assessments.
A direct consequence of our work in Cluster 3 has been to increase knowledge on the occurrence of pathogens in different types of water. This information can be used by water utilities and food producers to improve their risk assessments.
In a wider context, the findings from the field work have demonstrated the generally good quality of drinking water produced by large supplies. Smaller supplies and food producers were identified as being at greater risk and our work has raised awareness of the need to focus on these two areas for improving public health across Europe.
3.1.2. Give support to the Drinking Water Directive (98/83/EC)
Over time, the increased knowledge gained from adopting the tools and techniques developed in the Aquavalens project will provide a better understanding of the risks posed by waterborne pathogens to the microbiological safety of drinking water. This information will support the European Commission’s activities direct towards the adoption of risk assessments for assessing the microbiological safety of drinking water.
3.1.5. Integrated sensor-molecular techniques have applications potential for early warning systems, source tracking and rapid retrospective outbreak analysis for water-borne and foodborne infections.
The development of a fully integrated recovery and molecular detection system did not prove successful within the Aquavalens project although the work led to a number of alternative techniques that were likely to be of more benefit for end-users.
The work on microfluidics emerged as promising technique for pathogen concentration during the project. Whilst proof of concept was established, it was clear that further work would be required to make the method suitable for use under conditions encountered in practice. In particular, the method appeared to useful for separating target organisms from interfering substances.
The molecular methods developed in this project are able to provide better insights into identifying sources of contamination. The molecular source tracking methods, in particular, have considerable value for discriminating between different animal faecal sources and so improve the accuracy of sanitary surveys for understanding catchment based risks.

The techniques developed for organism identification have permitted individual strains to be profiled. This information will be of immense value to outbreak investigations where it is necessary to be able to confirm its nature and extent to enable remedial measures to be targeted more rapidly and more effectively.

3.1.6. More comprehensive, faster assessment of human health risks from surface and ground water will be beneficial to the industry (commercialisation of technologies, new markets), water companies and laboratories dealing with water quality control.

Prior to this project, reliance was placed on individual techniques for pathogen detection in drinking water supplies. The development in Aquavalens of an integrated method for simultaneous recovery of all pathogen types in a single assay will greatly assist the ability to determine pathogen burden. This knowledge will prove useful to operators to ensure their risk assessments are more comprehensive and ensure proper protection of public health.

The development of molecular techniques has been of great interest to the water industries in a number of states. For example, A de B in Spain have now put the Aquavalens methods into practice as part of their strategy for protecting the safety of their drinking water. The water industry in the UK provides another example, where their research organisation, UKWIR, has actively supported one partner in the Aquavalens project. UKWIR are now keen to establish how these techniques will be value.

The online system for determination of number of indicator bacteria will prove useful for water utilities to improve their risk assessment and risk management plans. They can provide an early warning of changes that will enable corrective action to be taken sooner and reduce the impact of any events that would impair the safety and quality of drinking water. The SMEs in Aquavalens responsible for their development have been provided with evidence of the capability of these technologies that can be used to support their commercialisation activities.

The portable water treatment stations for treating small water supplies were a key development in the project. This technology is well suited to exploitation for emergency situations where there is a rapid need for the availability of clean drinking water. For example, the provision of safe drinking water is an absolute necessity in the event of humanitarian disasters that result in large numbers of people being displaced to refugee camps.

Cluster 4: Impact and Exploitation of Cluster 4 Improvement of Public Health
Cluster 4 synthesizes the outputs from all the previous clusters to increase our knowledge of the health risks associated with drinking water in Europe, how these risks may be affected by climate change or the emergence of new pathogens and how these risks may be reduced. A particular focus of this cluster will be on assessing the impact of our work on European policy, especially in relation to water safety plans. The following detailed impact and exploitation can be expected:

1. Development of a new, uniform, Europe-wide approach regarding drinking water safety management plans
According to World Health Organization (Sixty-fourth World Health Assembly A64/24, 2011), an adequate protection of public health is provided by proactive approaches to Drinking Water Quality management. These proactive efforts have to rely on primary prevention measures based on the use of Water Safety Plans (WSP) approach. WP13 has synthesized lessons from the evaluation and implementation of AQV platforms into the existing WSP framework though the identification of challenges in the provision of safe drinking water that the European water supplies must face. This information was used to derive the possible impact on water safety and preventive management tools like Water Safety Plan (WSP).

2. Give support to the Drinking Water Directive (98/83/EC)
According to the results of WP13, a requirement of preventive management, a WSP should be included into the EU drinking water directive. The goal should be to have improved monitoring and regular surveillance of all drinking water, including the small supplies. The directive should include action to prevent and reduce leakage and encourage the renewing of old infrastructure. These actions shall then be included into national legislation as mandatory requirements to ensure safe water for all the citizens of Europe.

3. Give support to the EU Innovation Union
The Innovation Union focuses on major areas of concern for citizens such as climate change, energy efficiency and healthy living. According to the eco-innovation action plan built on the Innovation Union, WP15 measures the environmental and economic gains associated with the developed products (AQV platforms). This information allowed the identification of potential challenges and opportunities for achieving environmental objectives through eco-innovation, pursuing the balance for technological innovation and environmental protection. Besides, the results provided an added value to the novel products in the market.

4. Contribute to public health and to climate change preparedness
WP14 outcomes increased our knowledge of how the risks associated with drinking water may be affected by climate change or the emergence of new pathogens and how these risks may be reduced by using some of the technologies developed within the project. In particular, the consequences of extreme rainfall and drought events are analysed for both large and small water systems, and how the developed platforms improved the early warning of contamination events associated with unusual climate events.

7. Strengthen relations between researchers and industry, and further disseminate results of advanced research towards practical applications in water quality analysis
Outcomes from WP13 provide the formulation of guidelines that should be included in the water safety plans of large and small drinking water companies. This could eventually produce a market for the platforms developed in Aquavalens. Besides, the close cooperation between academic partners and SMEs producers in WP15 enabled future collaboration between industry and academia to strengthen the competitiveness of our SMEs to develop environmentally friendly solutions.

8. Lead to a greater integration of research actors and activities from across the European Union, and the candidate countries.
Especially in WP13 and WP15, through intense cooperation with several partners, particularly from Cluster 2 and 3, we have contributed to this integration goal in the three dimensions: geographically, since partners from up to 13 different countries around Europe were contacted to provide information about the trialled AQV platforms; interdisciplinary, since researchers from a variety of areas (microbiologist, engineers, environmental scientists, epidemiologists) worked together; and integration between industry and academia, through the collaboration with SMEs and water management companies.

9. Added value of a European approach
The conclusions provided by WP13 set criteria for the revision of the Drinking Water Directive. The knowledge generated was based on the research done over multiple European locations, this ensures the representativeness and generalization of the results to any European member state.
On the other hand, the carbon footprint reduction derived from the application of the AQV platforms, calculated in WP15, represent a direct indicator of the impact that the developed technologies may have regarding the environmental objective to be reached through the 2020 European strategy (climate and energy).

10. Potential areas and markets of application over and above current technology
Developed technologies are applicable to a wide range of markets and different sectors. In addition of water control, most methods may be applied to pathogens involved in food production, the environment, and veterinary or clinical diagnosis. The partner Genetic PCR Solutions™ has developed qPCR kits for detection of more than 250 pathogens of interest in most of the markets above. Commercial systems are now provided as additional alternatives to the current phenotypic methods extensively used for species identification and detection. Drawbacks of technologies together with the major hopes from the microbiologists (end-user point of view), were explored by the SMEs. The major needs found in applied markets for microbial controls were simplicity, robustness, suitability, and competitiveness. They are all intrinsic to a concept as desired as it is little used, which we call "pragmatic".

List of Websites:
1. Website address for project website:

2. Address details for Coordinator at UEA.
Professor Paul Hunter, Room 47/01.01A BMRC Building, Norwich Medical School, University of East Anglia, NR4 7TJ, England.

3. Attached - 48 page Aquavalens brochure produced by UEA and all partners in August 2017. Low res PDF copy

4. Attached - Project PERT diagram of outline of project structure and sequence. (P. 25 in part 2 of DoW). PERT Diagram.

5. Attached - List of six project peer-reviewed publications we were unable to load in FP7 software. [Requested but not accepted as journals]

6. Attached - 4 page list of all abbreviations and acronyms used in the project to help readers. Repeated at end of Period 4 report.

7. Attached - Update on the AQV ethics approval as requested in the EOP report, provided by University of Surrey.