CONCLUSIONS ON THE WBP ACTION October 2011

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1 CONCLUSIONS ON THE WBP ACTION October 2011 Report written by : Sophie Courtois (Suez Environnement), Nicolas Boudaud and Karine Delabre (Veolia Environnement) 1. Introduction Given the necessity to minimize public health risks, the effective removal or inactivation of pathogenic microorganism from water intended for human consumption becomes a major issue because of urbanization, demographic growth and the reuse of waste water potentially contaminated by viruses. The conclusions of MICRORISK project (2006) have previously shown that source waters were contaminated by various degrees of waterborne pathogens, especially Cryptosporidium, Giardia, Campylobacter, E. coli O157 and Enteroviruses. Their presence and also their persistence in source waters were impacted by a number of parameters, such as survival, transport and control of inputs, particularly depending on the water type. However, the levels of contamination of source waters were strongly influenced by the detection methods used and particularly the detection limit (DL) and quantification limit (QL). To date, no single and/or standardized methods are taking place in order to accurately assess the waterborne pathogenic risk. There is consequently a crucial need to further improve detection methods all over the world. More studies will be necessary to compare detection of different waterborne pathogens in order to determine with relevancy the risk levels of waterborne pathogens (Microrisk, 2006). As a follow up to the GWRC Research Strategy Workshop on Waterborne Pathogens (Paris, December 2005), a specific project on analytical methods, identified as a priority issue related to Waterborne Pathogens, was launched in 2008 by the Global Water Research Coalition (GWRC). The overall objective of the project was to compare and harmonise protocols for the preparation of microbial extracts from water samples for rapid molecular detection of pathogens, and an international evaluation of molecular methods for the detection of WaterBorne Pathogens (WBP) in water. In the GWRC WBP workshop meeting organized in December 2008, two analytical methods based on molecular biology for the quantification of Legionella and Norovirus in water samples were identified as relevant assays for the organization of inter-laboratory round-robin tests. During 2010, although it was unanimously agreed by all the GWRC members that there was a need to assessing the capabilities of the molecular tools to respond to the opportunity to increase our knowledge on emerging pathogens, the number of laboratories that expressed their ability to participate in one of the proposed proficiency tests was too low (around 4 laboratories for each parameter) for the correct organization of such assays. In particular, the current absence of international standard method based on PCR (Polymerase Chain Reaction) could have circumscribed the capability of additional laboratories to join to this international evaluation. To date, CEN and ISO groups are still working on the standardization of these two

2 methods and their publication will doubtless help to harmonize the practices of the laboratories in the near future. Considering the overall potential of each GWRC member to study the occurrence of (emergent) pathogens in water resources, a new orientation of the project has been given. The defined objectives of this new GWRC project were: to collect data on the occurrence of (emergent) waterborne pathogens that are monitored by the GWRC members in the source water using molecular methods; to exchange, between the GWRC network, the protocols and practices of molecular methods; to correlate, if possible, the occurrence of waterborne pathogens with faecal indicators. In particular, the use of viral indicators for faecal contamination could be investigated since the relatively common presence of pathogenic parasites, bacteria and viruses in source waters extend the use of relevant indicators (surrogates) of contamination. 10 GWRC members participated in the project : Wouter Leroux; CSIR (South Africa), Tobas Barnard; Université de Johannesburg (South Africa), Albert Bosch; Université de Barcelone (Spain), Siao Yun Chang; PUB (Singapore), Fout Shay; USEPA (USA), Gertjan Medema; KWR (Netherland), Beate Hambsch; TZW (Germany), Michele Akeroyd; WQRA (Australia), Sophie Courtois; Suez Environment (France), Nicolas Boudaud and Karine Delabre; Veolia Environment (France).

3 2. Results Following a template sending in 2011 March 31, a data collection has been harvested on the occurrence of waterborne pathogens and microbial indicators that are monitored in source waters by the GWRC members using conventional and molecular methods, including: Resource type, Characterization of sampling points (name of sampling points, location, date ), Physicochemical properties of sampling points in terms of turbidity, conductivity, ph, Waterborne pathogens monitored and analysed (volume analysed, type of concentration and detection methods used (included standardized and conventional procedures), expression of result (Unit), detection limit (DL) and quantification limit (QL), Indicators (surrogates) monitored and analysed (same parameters as waterborne pathogens). To date, the data analysis has been carried out by using the pool of data provided by 6/10 partners. Using the data received, it was possible to only elaborate correlations and differences between either GWRC partners in terms of detection methods used for the waterborne pathogens. A global synthesis has been described on Table 1 and Table 2. Firstly, Table 1 describes the methodologies applied for the detection of waterborne pathogens and microbial indicators in water resources. The physical characteristics and date of sampling points are also been mentioned.

4 Table 1. Global synthesis of The type of data provided by each partner Resource type / Characteristics Drinking water Surface water Groundwater data Physicochemical (P-C) parameters: ph, turbidity, T C, total organic carbon (TOC), conductivity Drinking water Surface water Feed water data P-C parameters: ph, turbidity, T C, conductivity, salinity, dissolved O 2 Surface water data Waterborne pathogens & indicators analysed GWRC Partner 1 Anaerobic Sulfate-Reducing Bacteria (ASRB), Enterococci, Campylobacter spp., Legionella spp., Clostridium spp. Total coliforms, viable bacteria at 22 & 37 C, E. coli Detection method used Parasites: Cryptosporidium, Giardia NF T Viruses: Norovirus GI & GII, Rotavirus, Astrovirus, Adenovirus, Hepatitis A Virus (HAV), Enterovirus, Somatic phages, F- specific RNA phages GWRC Partner 2 Clostridium perfringens, Aeromonas spp., Campylobacter spp., Legionella spp., Salmonella spp., Shigella spp., Vibrio cholerae, Pseudomonas aeruginosa, Helicobacter pylori, Mycobacterium avium Enterococcus spp., Bacteroides spp. Parasites: Entamoeba histolytica, Cryptosporidium, Giardia Amibes / viruses: Naegleria fowleri (amibe), Astrovirus, Adenovirus, Enterovirus, Norovirus Clostridium perfringens,, Legionella spp. Coliforms, E. coli, Enterococci Parasites: non indicated P-C parameters: : conductivity (non indicated for the others Viruses : Norovirus GI & GII, Sapovirus, HAV, Rotavirus Surface water Wastewater Swimming pond data P-C parameters: : non indicated Groundwater ; & 2010 data GWRC Partner 3 GWRC Partner 4 Pseudomonas aeruginosa, L. Pneumophila, C. jejuni Total coliforms, E. coli, Parasites: Cryptosporidium, Giardia Viruses: non indicated GWRC Partner 5 C. perfringens Coliforms, E. coli, faecal Enterococci, Bacteroides spp. Parasites: Cryptosporidium ISO (Coliforms), ISO 6222 (viable bacteria), NF EN (ASRB), ISO (Enterococci), NF T (E. coli), ISO/DIS (Campylobacter), ISO (Legionella) Culture for cultivable viruses Specific real-time PCR for uncultivable viruses and phagic surrogates Culture NF EN ISO (somatic phages), NF EN ISO (F- RNA phages) Real-time PCR, standardized procedure inside laboratory (Enterococcus spp., Bacteroides spp.) Conventional PCR, construction of representative biomarker (rdna) population gene clone library, DNA sequencing Conventional PCR, real-time PCR, standardized procedure inside laboratory Conventional PCR, clone library, sequencing Colilert-18 method (Coliforms & E. coli), ISO (Enterococci), ISO (C. Perfringens) Non indicated Specific real-time PCR (methodologies described by Da silva et al., 2007; Kageyama et al., 2003; Kageyama et al., 2004; Kojima et al., 2002; Loisy et al., 2005; Sano et al., 2011) Cultural technique (chromoculture coliform agar for Coliforms and E. coli, ISO for P. aeruginosa), FISH Non indicated Non indicated Conventionnal and standardized methods Non indicated

5 P-C parameters: ph, turbidity, T C, conductivity Surface water Well water Groundwater data P-C parameters: ph, turbidity, T C,TOC, conductivity Viruses: Enterovirus, Reovirus, Rotavirus, HAV, Norwalk virus, Poliovirus, Norovirus, somatic phages, male phages F-specific coliphages GWRC Partner 6 E. coli O157:H7; Salmonella spp., Pseudomonas aeruginos, Legionella pneumophila, Legionella spp. E. coli, total coliforms Parasites: Cryptosporidium parvus & lamblia Viruses: Enteroviruses, HAV, Noroviruses Culture for cultivable viruses Conventional RT-PCR (Fout, 2003), real time RT-PCR Colilert IDEXX (total coliforms, E. coli), ISO (P. aeruginosa), SBG selective media (Salmonella spp.), ISO (Legionella) Specific real-time RT-PCR DNA chip-based multi-detection method, specific real-time RT-PCR Table 2 summarizes the methodological characteristics provided by the different GWRC partners, in relation to the detection of bacteria, parasites, viruses and microbial indicators. For waterborne pathogens and microbial indicators, expression of results (unit), volume analysed, type of concentration, detection limit and quantification limit have been described based on the data collected.

6 Table 2. Specifications of methodologies used for the detection of waterborne microorganisms in terms of expression of result (unit), volume analysed, type of concentration, detection (DL) and quantification (QL) limits Microorganism Unit Volume analysed Type of concentration DL/QL GWRC Partner 1 Bacteria CFU/100mL 500mL 5L Filtration DL/QL depending ISO methods applied Parasite n/100ml 20L 100L Filtration DL ~0.2-5 n/100ml Virus Bacteria P/A, PFU/L, genomic copies/l P/A, CFU/100mL, cells/100ml 100mL 100L Filtration DL ~30 gc/l GWRC Partner 2 Non indicated Centrifugation / filtration 9 CFU/100mL Parasite n/100ml Non indicated Centrifugation / filtration 2-5 cells/100ml Amibe P/A Non indicated Centrifugation / filtration P/A GWRC Partner 3 Bacteria CFU/100mL 500mL 5L Filtration DL/QL : 1-5 CFU/100 ml Coliforms / E. Coli DL Legionella : 300 gc/l; see ISO norms for Enterococci / C. perfringens Parasite Non indicated Non indicated Non indicated Non indicated Virus Genomic copies/l 500mL 1L Filtration QL < 50 gc/l for NoV and < 100 gc/l for Sapovirus GWRC Partner 4 Bacteria CFU/100mL, µcolonies/100ml Techneau D Filtration See Techneau D Parasite Non indicated Non indicated Non indicated Non indicated Virus Non indicated Non indicated Non indicated Non indicated GWRC Partner 5 Bacteria CFU/100mL L Filtration Not carefully calculated Parasite Non indicated Non indicated Non indicated Non indicated Virus P/A, PFU/L; genomic copies/l L Filtration P/A, DL: PFU/L; QL: 0.01 PFU/L (infectious unit); DL: 0.13 gc/l ; QL: 0.4 gc/l for uncultivable viruses GWRC Partner 6 Bacteria CFU/L 250mL-1L Filtration DL: 12 (Salmonella) to 25 (E. coli O157:H7) CFU/L Parasite Cyst/L 30L Filtration DL: cyst/l Virus P/A/, genomic copies/l N: number;p/a: Presence/Absence; gc: genomic copies; NoV: Norovirus; HAV : Hepatitis Virus 30L Filtration DL: 23 gc/l for Enteroviruses, < 7700 gc/l for HAV, <3440 gc/l for NoV

7 3. Discussion The specific and representative evaluation of the levels of waterborne pathogen contamination is actually critical for drinking water (wastewater) suppliers and regulatory agencies. Currently, an urgent need appears in order to standardize the detection of waterborne pathogen, and consequently better appreciate the effective pathogenic risk in water resources used for drinking water production and distribution. Detection of microorganisms (especially virus) means remain limited because of difficulties related to extraction, culture and implementation of standardized methods. Despite the fact that GWRC data collection was not fully completed, this study highlights the characteristics of molecular methods used for the detection of waterborne pathogens in order to establish the degree of diversity (and homologies) inside the GWRC members. Based on the synthetic consolidation previously described in Table 1 and Table 2, the analysis of molecular methods used for the detection of waterborne pathogen inside the GWRC consortium can potentially show: Relative homogeneity in terms of physicochemical characteristics of water samples analysed, such as turbidity, conductivity, temperature, ph Large heterogeneity in terms of waterborne pathogens analysed. This observation is clearly related to the potential vulnerability/risk identified in water resources used. It was not markedly demonstrated but we can suppose that most of waterborne pathogens are analysed by the GWRC members, according to research needs, instead of regulatory purposes. Relative good homogeneity in terms of microbial indicators monitored. To date, the scientific community has well admitted the relevance of the purpose of microbial surrogates to anticipate and evaluate the pathogenic risk (or fecal risk) in resource waters (total coliforms and E.coli for pathogenic bacterial/parasitical risk, bacteriophages for viral risk, for example). Wide range of cultural and molecular methods applied by the GWRC members. Even if some GWRC partners used globally the same expression units (CFU/100mL for bacterial detection, genomic copies/l for virus detection, for example), further investigations should be carried out to develop standardized detection method, following inter-laboratory comparisons. Consequently the definition of normative expression units, detection limits and quantification limits could potentially be anchored, and therefore facilitate the validation by regulatory authorities. Importance of standard methods of sampling and sample processing to ensure comparable results. Since source water pathogen concentrations may be very low, concentration/enrichment of large volumes of water may be necessary for an accurate detection. Thus, it is important to collect adequate and standardized sample volumes. The relevance of filtration for sample concentration before analyses. The literature data underlines the strengths of filtration methods for the conservation of microbial integrity/viability during waterborne pathogen detection and/or quantification.

8 To date, detection of waterborne pathogens is clearly not optimal and standardized. There are a number of limitations especially due to the sensitivity of analytical tools and the lack of knowledge regarding the virus properties. According to the GWRC study based on internal evaluation of molecular methods used for the detection of waterborne pathogens, the assessment of human risk of infection requires accurate determinations of microbial occurrence, concentration, viability, infectivity and human dose response data (Le Chevallier et al., 2003). In agreement with Microrisk project (2006) regarding the definition of relevant and standardized pool of analytical methods, it is initially essential during the screening to balance the strengths and weaknesses of each in terms of the required output. But the main fundamental topic to carry out this ambitious project is that the selected methods ensure comparable results. The good sensitivity and specificity of real-time PCR methodology supports its status as the best methodology since few years. To correlate genomic copies obtained by real-time PCR and viable character of microorganisms, the use of viability markers could discriminate between viable and non-viable microorganisms (PMA-PCR for example). The rapid melting curve output provides an unambiguous result, without the need for hazardous and time consuming gel electrophoresis. Savings in labour costs and an increased water samples throughput should offset the increased costs of running the real-time assay. Laboratories should therefore provide their quality control on the performance characteristics of detection methods used, including statistical interpretation of results obtained. 4. Recommendations for future GWRC actions The latest GWRC actions on the standardization and international evaluation of molecular methods used for detection of WBP showed that the diversity of microbial targets that can be assessed by such molecular tools, (leading to a highly diversity of laboratory practices) hampered the attempt to standardize and validate the molecular tools in their wholeness. In particular, the choice of microbial targets are mainly driven by the potential vulnerability/risk of water resources identified locally. Nevertheless, as highlighted in this report, an important need remains to establish standardized practices for water analysis by molecular tools. As important works are being made through national and international actions (US EPA, National Agencies for Standardisation, CEN and ISO), GWRC can play a central role in promoting scientific exchange through the GWRC experts network. Once every two years, GWRC workshops could be organized in order to : o communicate on method developments, research project results and international standard methods, o centralize the state-of-the art methods for microbial targets identified as priorities o establish certain GWRC internal guidelines for developing and validating new methods for detecting WBP in water samples (for example in quality assurance and quality controls) o fill the gaps in the map of knowledge described in the GWRC / IWA book on waterborne pathogens (Loret and Guillot, 2009).

9 5. References ALBINANA-GIMENEZ N, CLEMENTE-CASARES P, CALGUA B, HUGUET JM, COURTOIS S & GIRONES R, Comparison of methods for concentrating human adenoviruses, polyomavirus JC and noroviruses in source waters and drinking water using quantitative PCR. J Virol Methods, 2009, 158: DA SILVA, A.K., LE SAUX,J.C., PARNAUDEAU,S., POMMEPUY,M., ELIMELECH,M., AND LE GUYADER,F.S., Evaluation of Removal of Noroviruses during Wastewater Treatment, Using Real-Time Reverse Transcription-PCR: Different Behaviors of Genogroups I and II. Appl Environ Microbiol, 2007, 73: FOUT, G.S., B.C. MARTINSON, M.W.N. MOYER, AND D.R. DAHLING., A multiplex reverse transcription-pcr method for detection of human enteric viruses in groundwater. Appl. Environ. Microbiol. 2003, 69(6): GUILLOT E., LORET J-F., Waterborne Pathogens: Review for the Drinking-Water Industry; IWA Publishing, pages KAGEYAMA,T., KOJIMA,S., SHINOHARA,M., UCHIDA,K., FUKUSHI,S., HOSHINO,F.B., Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR. J Clin Microbiol, 2003, 41: KAGEYAMA,T., SHINOHARA,M., UCHIDA,K., FUKUSHI,S., HOSHINO,F.B., KOJIMA,S., Coexistence of Multiple Genotypes, Including Newly Identified Genotypes, in Outbreaks of Gastroenteritis Due to Norovirus in Japan. J Clin Microbiol, 2004, 42: KOJIMA,S., KAGEYAMA,T., FUKUSHI,S., HOSHINO,F.B., SHINOHARA,M., UCHIDA,K., Genogroup-specific PCR primers for detection of Norwalk-like viruses. J Virol Methods, 2002, 100: LE CHEVALLIER, M.W., DI GIOVANNI, G.D., CLANCY, J.L., Bukhari, Z., BUKHARI, S., ROSEN, J.S., SOBRINO, J., and FREY, M.M., Comparison of method 1623 and cell culture-pcr for the detection of Cryptosporidium spp. in water sources. Appl. Env. Microbiol. 2003, 69: LOISY,F., ATMAR,R.L., GUILLON,P., LE CANN,P., POMMEPUY,M., AND LE GUYADER,F.S., Real-time RT-PCR for norovirus screening in shellfish. J Virol Methods, 2005, 123: 1-7. MICRORISK, Microbiological risk assessment: a scientific basis for managing drinking water safety from source to tap. Quantitative Microbial Risk Assessment in the Water Safety Plan. EVK1-CT , SANO,D., PEREZ-SAUTU,U., GUIX,S., PINTO,R.M., MIURA,T., OKABE,S., AND BOSCH,A., Quantification and Genotyping of Human Sapoviruses in the Llobregat River Catchment, Spain. Appl Environ Microbiol, 2011, 77: WHO (2004). Guidelines for Drinking-water Quality. Volume 1 Recommendations. Third Edition. WHO, Geneva.

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