European Union Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs

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1 European Union Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs Annual technical report for calendar year 2016 FINAL report (version 3) Contract Reference: Cefas ref (C6927) Document approved by: James Lowther Review date: Not applicable Document checked by: David Lees, Rachel Hartnell Classification: Official Document prepared by: James Lowther, Louise Stockley Location CRL$

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3 Contents Legal functions and duties 2 Introduction 2 1. Scientific advice and support 2 2. Project management and co-ordination of activities of NRL network 4 3. Provision of technical advice and training 4. Confirmatory testing and quality assurance 5. Comparative testing and ring trials 9 6. Development of analytical methods 13 Annexes 16 Annex I - Work programme for the EURL for bacteriological and viral contamination of bivalve molluscs, Annex II - Resolutions of the 15 th workshop of microbiological NRLs, th May 2016, Berlin, Germany Annex III Report of the 15 th workshop of NRLs for bacteriological and viral contamination of Bivalve Molluscs, 2016 Annex IV PT 61; Noroviruses and hepatitis A virus; final report Annex V PT 64; Enumeration of Escherichia coli and the detection of Salmonella spp. in bivalve molluscan shellfish; final report Annex VI PT 65; Noroviruses and hepatitis A virus; final report Annex VII - PT 67; Escherichia coli and Salmonella spp. EQA; final report 6 8 EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 1 of 15

4 European Union Reference Laboratory for Monitoring Bacteriological and Viral Contamination of Bivalve Molluscs, Cefas, Weymouth Legal functions and duties Technical Report for Calendar Year 2016 The functions and duties of the EURL are specified in Article 32 of Council Regulation (EC) 882/2004 (Official Journal of the European Communities No L165). Introduction The two-year work programme for the EURL for was approved by the European Union in December This report details activities of the EURL according to the work programme (Annex I), additional tasks described under the resolutions and report of the 15 th workshop of microbiological NRLs held in Berlin, Germany (Annexes II and III) and other responsibilities outlined in Commission Regulation (EC) 882/2004 for the calendar year Scientific advice and support To the European Union The EURL has provided the following advice and support to the European Commission (DG Sante, the FVO, and EFSA) through participation in expert working groups, provision of briefing documents, guidance and audits, specifically in 2016 this has comprised: Provision of advice to the Commission and Member States in the Commission working group on bivalve molluscs. Three working groups attended in 2016 (January, March, September). Including preparation for meetings, attendance in Brussels, and post meeting follow up. In particular, during 2016 the Commission were supported at the MS WG with development of Regulation 2015/2285 amending class A classification, with MS input into consequential redrafts of the European guidance, with the development of the EU wide norovirus baseline survey, and with the developing EU-US trade agreement. Support to the Commission regarding EU-US trade: This activity included correspondence and a report to the US FDA regarding the implications of adoption of Regulation 2015/2285 for trade, further statistical analysis of EU and US datasets and a full report. Attendance of 3 EURL staff (including statistical support) at a meeting in Washington DC in September 2016 to present our report to the FDA, to scrutinise the corresponding analysis and presentation from the FDA, and to support the Commission in various other trade aspects under discussion including issue on Vibrio controls. Post meeting finalisation of technical documents, including redrafted Guidance annexes, and publication of material on the EURL website. Working with EFSA on the development of a study plan for a harmonised EU wide baseline survey of viruses in bivalve molluscs, including hosting a meeting of the EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 2 of 15

5 protocol working group and analytical sub-group in Weymouth, th January Further support to the Commission and EFSA on various aspects of the baseline survey including provision of data relevant to financial arrangements. Advice on viruses in non-animal matrices, including maintenance of a database including details of laboratories in EU Member States with capacity for determination of noroviruses and hepatitis A virus in soft fruits (and detailing ISO IEC accreditation status). Redrafting the EURL Good Practice Guide and the EU Principles of Good Practice Guide to update principally sections regarding implementation of Regulation 2015/2285 for classification and monitoring and also amending the annexes regarding EU exports to the USA linked to the trade negotiations. This activity included several rounds of technical discussion with the EURL steering group, tabling and presentation of draft proposals to NRLs, the Commission and MS, and consequential redrafting of the guidance according to comments made. Advice to other entities and contributions to standardisation activities under ISO or CEN working groups The EURL led the CEN/TC 275/WG6/TAG4 programme on work on viruses in foods, including: Preparation of ISO/FDIS , Microbiology of the food chain -- Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR -- Part 1: Method for quantification Attendance at the plenary meetings of ISO/TC34/SC9 and CEN/TC275/WG6, 9-13 th May 2016, Paris, France The EURL led the CEN/TC 275/WG6/TAG15 programme on work on food borne pathogenic vibrios using molecular approaches, including: Preparation of ISO/DIS 21782, Microbiology of the food chain -- Horizontal method for the detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. The EURL led the ISO/SC9/WG14 expert group on the EU standard reference method for enumeration of E. coli in LBM (ISO TS ). In 2016 a technical corrigendum was developed to harmonise incubation controls and performance testing of media with ISO Microbiology of food, animal feed and water -- Preparation, production, storage and performance testing of culture media The EURL led the ISO/SC9/WG8 expert sub-group on the revision of the ISO , sample preparation and preparation of initial dilutions to include harmonisation of the ISO standard with the requirements of EU Regulation (EC (No.) 2073/2005) for live bivalve molluscs. This standard is currently at the FDIS stage. The EURL provided advice to FAO/WHO on the theoretical LOD for the determination of FRNA bacteriophage in LBM. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 3 of 15

6 2. Project management and co-ordination of activities of NRL network The EURL promotes full and active participation by NRLs in the activities of the network as outlined in Article 32 of Commission Regulation (EC) No. 882/2004. To assist in co-ordination activities a list of designated NRLs is updated annually. This information is published on the EURL website (Table 1). Table 1. Designated NRLs in Member States, EFTA and Accession states in calendar year Member State Austria Belgium and Luxembourg Laboratory Austrian Agency for Health and Food Safety, Institute for Food Control, AGES-LMU Wien, Abt. Mikrobiologie, Spargelfeldstraße 191, A-1226 Wien Scientific Service of Food-borne Pathogens, Operational Directorate of Communicable and Infectious Diseases, Juliette Wytsmanstraat 14, 1050 Brussels Bulgaria National Diagnostic and Research Veterinary Institute, Pencho, Slaveikov, 15 BG Sofia Croatia Cyprus Czech Republic Croatian Veterninary Institute, Regional Veterinary Laboratory Split, Poljicka Cesta 33, Split No NRL designated No NRL designated Denmark National Food Institute, The Technical University of Denmark, Mørkhøj Bygade 19, DK Søborg Estonia No NRL designated Finland Finnish Customs Laboratory, Tekniikantie 13, FI-02150, Espoo France Germany Greece IFREMER, Departement Microbiologie et phycotoxines, Centre de Nantes, Rue de I'lle de'yeu, BP 21105, Nantes Cedex 3 Bundesinstitut fur Risikobewertung (BfR), Federal Institute for Risk Assessment, Diedersdorfer Weg, D-12277, Berlin Institute of Food Hygiene of Athens, Neapoleos 25, Ag. Paraskevi, Attiki, Athens Hungary Central Agricultural Office, Food and Feed Safety Directorate, Mester u. 81, H-1095 Budapest Iceland 1 Matís ohf. / Icelandic Food and Biotech R&D. Vínlandsleið 12, 113 Reykjavík Ireland Marine Institute, Rinville, Oranmore, Co. Galway EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 4 of 15

7 Member State Laboratory Italy 2 Istituto Zooprofilattico Sperimentale Umbria e Marche, Via Cupa di Posatora 3, 60100, Ancona (NRL for bacteriology) Latvia Lithuania Malta Netherlands Norway 1,2 Poland Portugal Romania Instituto Superiore di Sanità, Dipartmento di Sanita Pubblica Veterinaria e Sicurezza Alimentare, Viale Regina Elena 299, 00161, Rome (NRL for virology) Institute of Food Safety, Animal Health and Environment (BIOR), Lejupes iela 3, LV Riga National Food and Veterinary Risk Assessment Institute, J.Kairiukscio Str. 10, LT , Vilnius No NRL designated National Institute for Public Health and the Environment (RIVM), PO Box 1 A van Leeuwenhoeklaan 9, 3720 BA, Bilthoven NIFES - The National Institute of Nutrition and Seafood Research, Box 2029 Nordnes, NO-5817, Bergen (NRL for bacteriology) NMBU Campus Adamstuen, Department of Food Safety and Infection Biology, P.O. Box 8146 Dep., 0033 Oslo (NRL for virology) National Veterinary Research Institute, Partyzantów 57, PL Pulawy Portuguese Institute of Sea and Atmosphere, I.P. (IPMA)/Department of Sea and Marine Resources, IPMA-Alges, Avenida de Brasilia, Lisboa Institute for Diagnosis and Animal Health, 63 Dr. Staicovici Street, Sector 5, Code 76202, Bucharest Slovakia State Veterinary and Food Institute, Janoskova 1611/58 Ministry of Agriculture, SK Dolny Kubin Slovenia Institute for Food Hygiene and Bromatology, Veterinary Faculty, Gerbiceva 60, SI 1000, Ljubljana Spain Centro Nacional de Alimentacion, Agencia Española de Seguridad Alimentaria, E Majadahonda, Madrid Sweden National Food Agency, P.O. Box 622, Uppsala United Kingdom Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB 1 Member of EFTA 2 2 NRLs established (one for bacteriology and one for virology) EURL website The EURL website continues to provide a very useful repository for information for NRLs and other stakeholders. Annual workshop of NRLs The 15 th workshop of NRLs was hosted at BfR, Berlin, Germany between May Forty-two delegates representing 23 EU Member States, 2 EFTA countries and the EURL alongside EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 5 of 15

8 invited experts from EFSA, the Lower Saxony State Office for Consumer Protection and Food Safety, Germany, National Laboratory of Schleswig-Holstein, Germany and the Centro de Control da Calidade do Medio Marino of Galicia, Spain and observers from DG SANTE and the FVO. The workshop covered official controls, E. coli and Salmonella, marine vibrios and viruses. Twenty-five resolutions were agreed by the workshop (Annex II). A full report of the workshop of NRLs is included as Annex III of this technical report and is available to download from the EURL website ( Anonymised feedback from delegates attending the workshop, showed predominantly good to very good performance, feedback breakdown is provided within the annual workshop report. EURL Director s meeting The EURL Director designate attended the meeting of Directors of EURLs in the field of Animal Health, Food and Feed in Brussels on Dec 2 nd Provision of technical advice and training Methods, protocols and guidance notes The EURL updated its generic protocols for detection of E. coli and viruses in shellfish during 2016, to reflect the developing ISO standards in these areas. Further assistance for laboratories undertaking virus testing was provided in the form of an updated spreadsheet for quantity calculations, and a guidance note on the determination of limits of detection and quantification. The EURL contributed to the survey protocol for the EFSA European baseline survey of norovirus in oysters, including leading the sub-group responsible for generation of the method specification detailing the analytical methods for use in the survey. Formal training workshops VIRUS METHOD TRAINING FOR BASELINE SURVEY LABS June-July 2016 The EURL provided 3-day training workshops in consecutive weeks (28-30 th June & 5-7 th July 2016) for laboratories designated for analysis in the EFSA European baseline survey of norovirus in oysters covering all elements of the method including analysis of data and calculation of quantities and production of associated control materials. The workshops were attended by representatives of designated labs from Ireland, Sweden, Denmark, the Netherlands, Spain, Portugal, Italy, Croatia and Greece. Additional ad hoc training and study visits The EURL hosted a visitor from the Institute of Public Hygiene and Veterinary Health in Romania for training in practical methods for detection of viruses in November Other technical assistance Reference and/or ready-to-use control materials for the virus detection method were provided to laboratories designated for analysis in the EFSA European baseline survey of norovirus in oysters in Ireland, Norway, Sweden, Denmark, Germany, the Netherlands, France, Spain, Portugal, Italy, Croatia and Greece. Control materials for the method were also separately provided to a laboratory in France. Provision of advice to NRLs and others EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 6 of 15

9 In 2016 the EURL provided advice (briefing notes, technical reports, etc) to laboratories within the network of National Reference Laboratories and others. Advisory activities requiring input of half a person day or more, or those with written outputs are included here: Advice on the implementation of methods for detection of viruses in shellfish (and fresh produce) (ISO 15216) to labs in China, Canada, France and Germany. Presentation of EURL activities to annual French Live Bivalve Mollusc Stakeholders meeting Health, Environment and Microbiology September 2016, Nantes. Advice to NRL Croatia on the length of review periods for interpretation of monitoring data for the purposes of classification of LBM production areas. Advice to NRL Croatia on application of E. coli MPN/100g hygiene standards to LBM placed on the market. Advice to the Competent Authority of the UK devolved administration (Scotland) on the classification review frequency following implementation of Commission Regulation (EU) 2015/2285. Advice to the UK Competent Authority on the rationale for and calculation of 99.9%tile value for rainfall events and the relationship between one in five-year rainfall event when considering anomalous results from LBM classification programmes. Additionally ad hoc technical advice was provided to laboratories via or telephone. Other Scientific activities EURL staff have produced a number of peer-review papers in scientific journals, book chapters, etc.: Church, S., C. Baker-Austin and S. Michell. Vibrio vulnificus Type VI Secretion System 1 contains anti-bacterial properties. PLOS One, 11(10):2016. Baker-Austin, C., J. A. Trinanes, N. Gonzalez-Escalona, and J. Martinez-Urtaza. Non-cholera vibrios the microbial barometer of climate change. Trends in Microbiology, published ahead of print, Nov Olalemi, A., C. Baker-Austin, J. Ebdon and H. Taylor. Bioaccumulation and faecal bacterial and viral indicators in Mytilus edulis and Crassostrea gigas. International Journal of Hygiene and Environmental Health, 219: , Baker-Austin, C., J. A. Trinanes, S. Salmenlinna, M. Löfdahl, A. Siitonen and J. Martinez- Urtaza. Heatwave-associated vibriosis in Sweden and Finland, Emerging Infectious Diseases, 22(7): , Jennings, S., C. Baker-Austin et al. Aquatic food security: insights into challenges and solutions from an analysis of interactions between fisheries, aquaculture, food safety, human health, fish and human welfare, economy and environment. Fish and Fisheries, DOI: /faf Campos, C.J.A., J. Avant, J. Lowther, D. Till and D.N. Lees. Human norovirus in untreated sewage and effluents from primary, secondary and tertiary treatment processes. Water Research 103: , EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 7 of 15

10 Winterbourn J.B., K. Clements, J.A. Lowther, S.K. Malham, J.E. McDonald and D.L. Jones. Use of Mytilus edulis biosentinels to investigate spatial patterns of norovirus and faecal indicator organism contamination around coastal sewage discharges. Water Research 105: , Boxman, I.L., L. Verhoef, H. Vennema, S. Ngui, I.H. Friesema, C. Whiteside, D. Lees and M. Koopmans. International linkage of two food-borne hepatitis A clusters through traceback of mussels, the Netherlands, Eurosurveillance 21:1-9, European Food Safety Authority (Working group and sub-group members including D. Lees and J. Lowther). Technical specifications for a European baseline survey of norovirus in oysters. EFSA Journal 2016;14(3):4414 [62 pp.], Scientific presentations (international conferences) Lowther, J. Methodology for detection of norovirus and hepatitis A virus in foods; current status and future challenges, FSA-EFSA International Workshop on Foodborne Viruses, London, England, February Baker-Austin, C. Vibrio wound infections in Sub-Arctic waters. Vibrio, Roscoff, France, March Lees, D. Improving health controls for viruses in bivalve molluscs. Keynote. Water Microbiology Conference, Chapel Hill, North Carolina, USA, May Lowther, J. A One Year Survey of Norovirus in UK Oysters Collected at the Point of Sale, 5th Food and Environmental Virology conference, Kusatsu, Japan, September Campos, C. Satellite-based HAB and water quality monitoring for shellfish farms to support management decisions. 58th Marine Measurement Forum, Plymouth Marine Laboratory, Plymouth, United Kingdom, September Campos, C. Microbiological pollution in shellfish production areas: environmental risk factors and management options. Marine BioIsle Seminar, Universidade dos Açores, Ponta Delgada, Portugal, October Baker-Austin, C. Pathogenic vibrios the microbial barometer of climate change. University of North Carolina, Institute for Marine Sciences, Morehead city, NC, USA, October Confirmatory testing and quality assurance EURL standard operating procedures (SOPs) for ISO/IEC accredited methods have been reviewed and revised according to the annual cycle. All SOPs are available as generic protocols for statutory (and some non-statutory) methods through the EURL website and on request from the EURL co-ordinator. Accreditation to ISO was retained for the following methods and associated procedures: Detection of Salmonella spp in bivalve molluscan shellfish (ISO 6579). Enumeration of E. coli in bivalve molluscan shellfish (ISO TS ). Detection of V. parahaemolyticus in bivalve molluscan shellfish (ISO TS ). Quantification of norovirus in bivalve molluscan shellfish (ISO TS ). EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 8 of 15

11 The EURL is accredited for these analyses by the United Kingdom Accreditation Service (UKAS) schedule number UKAS UKAS is a member of the European co-operation for accreditation (EA). No confirmatory testing for third parties as a result of disputed analysis was undertaken in Comparative testing and ring trials Participation of Member State NRLs, EFTA and third countries in EURL organised proficiency testing (PT) in 2016 is tabulated in Table 2 and summarised in this section. All PT distributions are open to non-nrl participants on a cost recovery basis. Samples were provided free of charge to NRLs under the agreed annual work programme of the EURL. PT reports and other documentation are provided as Annexes IV, V, VI and VII. Reference samples produced by the EURL were assessed for homogeneity in accordance with ISO Proficiency testing for statutory determinands E. coli and Salmonella spp. proficiency testing PT 64 and PT 67 NRLs have previously agreed that participation in a minimum of two annual PT distributions is mandatory with the EURL matrix PT (comprising of bivalve mollusc samples) to be compulsory and at least one PHE EQA scheme distribution to be examined per year. The minimum requirement for satisfactory performance is a score of greater than 70%. Table 2 describes the level of PT participation. All designated NRLs completed the EURL matrix PT (currently there are no NRLs designated in Malta, Cyprus, the Czech Republic or Estonia). NRLs in Bulgaria, Finland and Norway did not take part in the PHE EQA scheme. The reasons for lack of participation were reviewed at the 2016 annual workshop. To assist NRLs in participating in the mandatory PT distributions, the EURL agreed to pay NRLs participation in a single PHE distribution each year from 2017 onwards (Resolution 8). Tables 3 and 4 show abstracted performance scores for NRLs from PT 64 and PT 67 assessed in comparison with NRL participants. PT 64 - Matrix distribution Matrix distributions enable participants to examine all aspects of the methodology. In November 2016, the EURL distributed three samples of bivalve molluscan shellfish (Pacific oysters - Crassostrea gigas). Thirty-nine laboratories participated, including all 25 nominated NRLs for bacteriology within the network. Participants duplicate E. coli MPN values for each sample were compared to the median of all participants results (Table 3). Upper and lower acceptability limits were calculated as the participants median ±3 theoretical standard deviations (SD) and ±5 SD ( 99% and 99.9% confidence intervals respectively). Performance assessment was determined according to the EURL/PHE EQA scheme for a single distribution, with modifications to reflect replicate analyses of a single sample. NRL Bulgaria, Finland and Poland had points deducted as one or both MPN values reported for sample 1 or sample 3 were outside ±3 SD of the participants median. Further points were deducted from NRLs Germany, Finland and Latvia due to the reporting of tube combinations inconsistent with the guidance given in ISO7218:2007/Amd 1:2013 for interpretation of MPN tables and/or the EURL generic protocol for enumeration of E. coli in bivalve molluscs. For Salmonella spp. analyses, all NRLs correctly detected the presence of Salmonella spp. in sample 3 (Table 4). Due to Salmonella spp. being detected naturally in 4 out of 20 replicates reference samples for sample 2 (un-spiked sample), scores were not allocated to participants. A full report is included as Annex V. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 9 of 15

12 EURL PT reference number Proficiency testing description Austria Bulgaria Croatia Czech Republic Estonia Finland Hungary Ireland Latvia Lithuania Malta Slovakia Slovenia Sweden Iceland Norway Chile New Zealand Singapore Switzerland South Korea Vietnam Table 2 - Summary of participation amongst NRLs and others in EURL organised proficiency testing for calendar year 2016 National Reference Laboratories EFTA Third countries PT 61 PT 64 PT 65 PT 67 Norovirus and Hepatitis A virus in LENTICULES E. coli and Salmonella spp. in shellfish matrix Norovirus and Hepatitis A virus shellfish matrix and LENTICULES E. coli and Salmonella spp. - EQA Belgium and Luxembourg Cyprus 3 Denmark France 1 3 Germany Greece 2 3 Italy 2 3 Netherlands 1 Poland 3 Portugal 1 3 Romania 2 3 Spain United Kingdom 2 3 Canada 3 Peru 1 x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x X X X X X X X X X X X X X X X No designated NRL 1 more than one laboratory participated in PT more than one laboratory participated in PT more than one laboratory participated in PT 66. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 10 of 15

13 PT 67 Non-matrix distributions NRLs were offered distributions (SF053, SF054, SF055) in February, June and October 2016 for examination of E. coli and Salmonella spp. in simulated bivalve mollusc matrices. Uptake to the PHE/EQA scheme was variable (Table 3 and 4). A full report is included as Annex VII. Overall NRL performance of proficiency testing for statutory determinands Overall performance assessments for participating NRLs indicated generally satisfactory performance over the annual cycle. Point deductions were due to reported tube combinations being inconsistent with the guidance given in ISO 7218:2007/Amd 1:2013 for interpretation of 5 x 3 MPN tables and/or the EURL generic protocol for enumeration of E. coli in bivalve molluscs. Table 3. Performance scores for NRLs in E. coli PT in 2016 Lab no. a Distribution Distribution Distribution PT 64 All distributions SF053 SF054 SF055 Cumulative Max S - 1 S - 3 SF0114 SF0115 SF0116 SF0117 SF0118 SF0119 % score score Austria Belgium Bulgaria Croatia Denmark Finland France Germany Greece Hungary Iceland Ireland Italy Latvia Lithuania Netherlands Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden UK laboratories submitted results from reference and alternative methods EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 11 of 15

14 Table 4. Performance scores for NRLs in Salmonella spp. PT in 2016 Lab no. a PT 64 Distribution SF053 Proficiency testing for non-statutory determinands The EURL also offers PT for non-statutory determinands, in 2016 PT distributions were provided for norovirus (genogroups I and II) and for hepatitis A virus. Non-statutory PT helps laboratories in the implementation and accreditation of new methods, and can demonstrate continuous improvements. This is particularly important for virus methods for which there is a need for capacity building across the network of NRLs to support potential future food hygiene legislation. PT 61 - Norovirus and hepatitis A virus in matrix material and LENTICULES In September 2016 the EURL distributed 6 samples comprising of naturally contaminated and bioaccumulated Pacific oysters (Crassostrea gigas) and laboratory constructed discs. Double-stranded DNA control material for each target was also included with the sample material to assist laboratories with quantification. Material was distributed to 47 laboratories (19 NRLs). Of those laboratories receiving material, 45 returned results, with 27 (60%) obtaining the intended presence/absence result (as determined by EURL reference designations) for all determinands tested. Thirty-one laboratories reported quantitative data for at least one sample/target virus combination with 14 reporting results within an acceptable range defined as the participants median ±2δ median absolute deviation (MAD) for all sample/target virus combinations tested. A full report is included as Annex IV. PT 65 - Noroviruses and hepatitis A virus Distribution SF054 Distribution SF055 All distributions S - 1 S - 3 SF0114 SF0115 SF0116 SF0117 SF0118 SF0119 Cumulative Max score score % Austria Belgium Bulgaria Croatia Denmark Finland France Germany Greece Hungary Iceland Ireland Italy Latvia Lithuania Netherlands Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden UK EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 12 of 15

15 In July 2016 the EURL distributed 2 laboratory constructed LENTICULE discs containing a combination of norovirus GI, GII and HAV. Material was distributed to 43 laboratories (18 NRLs), with 39 laboratories returned results. Thirty-six laboratories correctly reported presence/absence results (as determined by the EURL reference samples) for all determinands tested. Twenty-one laboratories returned quantification data with 12 and 14 laboratories obtained a satisfactory performance score for NoV and HAV positive samples respectively. A full report is included as Annex VI. 6. Development of analytical methods A meeting of CEN/TC275/WG6/TAG4 was convened in Brussels on 2 nd -3 rd February Based on the results of the votes (and associated comments) on ISO/DIS , the revision of ISO/TS :2013, Microbiology of food and animal feed: determination of norovirus and hepatitis A virus in foodstuffs by PCR, Part 1: Quantitative determination, a series of revisions was agreed and a new final draft of the standard ISO/FDIS was prepared. The final vote (8 weeks duration) on the revised standard was launched on 25 th November Assuming a positive outcome of the vote publication will be anticipated in the first quarter of Following successful conclusion of a research collaboration with the EU Joint Research Council, Geel, to elaborate control materials to assist in the standardisation of ISO methods for noroviruses, a teleconference between the EURL and representatives of JRC was held on 28 th October 2016, to identify further opportunities for collaboration in this area. Further research into methods for the determination of the infectivity of noroviruses detected in bivalve shellfish by PCR was carried out, and progress with the use of FRNA bacteriophage as an indicator of infectivity was made. The initial technical enquiry on ISO/DIS 21872, the draft document amalgamating and revising ISO/TS :2007, Microbiology of food and animal feeding stuffs: Horizontal method for the detection of potentially enteropathogenic Vibrio spp., Part 1: Detection of Vibrio parahaemolyticus and Vibrio cholerae and ISO/TS :2007, Part 2: Detection of species other than Vibrio parahaemolyticus and Vibrio cholerae, was carried out from 17 th March to 17 th June The DIS was approved with 23 positive votes and no negative votes. Following this the EURL convened a meeting of CEN/TC275/WG6/TAG15 to discuss the vote and the comments received and a final draft revision ISO/FDIS was prepared and forwarded to the CEN/ISO secretariat in preparation for the final vote due February Assuming a positive outcome of the vote publication will be anticipated in the second quarter of A teleconference meeting of CEN/TC275/WG6/TAG15 was convened on 19 th May 2016 to discuss the first steps necessary for development of methods to enable enumeration of total / pathogenic Vibrio parahaemolyticus. Further meetings are scheduled for the early part of The EURL has worked with statistical experts from the Bundesanstalt für Materialforschung und - prüfung, Berlin, Germany (Prof. Cordula Wilrich), to develop a bivalve-specific E. coli MPN calculator, to assist NRLs and OCLs and to standardise the approach to MPN calculations derived from positive and negative tube combinations EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 13 of 15

16 The EURL has worked with the relevant expert from the US FDA (Dr Greg Goblick) to work up data resulting from practical dye tracing studies carried out in A joint peer reviewed paper submission, with the US FDA, is in late draft. This work includes establishing experience with dye dilution studies used by the US FDA to establish buffer zones in complex situations. The EURL is in a good position to advise MS, in support of the trade annexes in the EU guidance, should MS need to establish buffer zones in production areas targeted for exports to the US. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 14 of 15

17 U Annexes [This page is left blank intentionally] EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2016 v2 Page 15 of 15

18 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk WORK PROGRAMME FOR THE EURL FOR BACTERIOLOGICAL AND VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, LEGAL FUNCTIONS AND DUTIES The functions and duties of the EURL are specified in Article 32 of Regulation (EC) No 882/2004 (Official Journal of the European Communities No L 165 of ). In the work programme 28 Member States and 2 EFTA Member States (Iceland and Norway) are considered eligible for EURL assistance and invited to participate in EURL organised training programmes, comparative testing etc. Third country and other MS laboratories are also invited to participate as appropriate in comparative testing and training workshops on a cost recovery basis. WORK PROGRAMME, Scientific advice and support Expected outputs 1.1. The EURL will provide scientific assistance to DG SANTE in operation of existing, and implementation of new, European Union food hygiene legislation, and in particular in the following activities have been identified: Attendance at the meetings of the EU Member States restricted working group on bivalve molluscs Provision of expert scientific and technical advice with regard to criteria for noroviruses and hepatitis A virus in live bivalve mollusc (LBM) production areas and/or products placed on the market. In particular sampling plans, analytical methods, and reporting for an EU wide baseline survey (with EFSA) a. a Regulation covering inter alia standards for viruses in LBM improvement in public health Revision of Community Guidance with regard to pending revision of the criteria for E.coli in end-products and class A harvest areas Provision of technical advice and any necessary revision to Community Guidance on microbiological monitoring of bivalve molluscs, mitigation of faecal contamination (e.g. buffer zones), virus (or other microbiological) contamination or outbreaks Provision of support to DG SANTE and priority MS, as required with recommencement of trade of LBM between EU and US b Provision of ad-hoc scientific advice and assistance to DG SANTE with respect to determination of norovirus and hepatitis A virus in matrices other than bivalve shellfish (eg soft fruits) covered in ISO (the virus reference method) c. b EU and US trade in LBM c Scientific and technical advice to support EU Regulations on viruses in non-bivalve foods. Page 1 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme

19 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk Assistance to DG SANTE FVO with audits of MS and third countries as requested and as other EURL priorities allow (mission costs funded by FVO). Specialist technical advice and support to FVO in support of their remit as requested The EURL will participate in relevant EU and International scientific committees (EFSA, ISO/CEN, WHO/FAO, ICMSS etc). In the EURL will: Act as convener for CEN/TC275/WG6/TAG4 Viruses in food to assist in the development of EN methods for the determination of viruses in foodstuffs. In including, but not restricted, to providing responses to official technical and editorial comments from voting members at ISO SC9 level for the publication of EN/ISO , Microbiology of food and animal feed Horizontal method for detection of hepatitis A virus and norovirus in food using real-time RT-PCR Part 1: Method for quantitative determination (due for publication by end of 2016) and drafting of a new revised DIS ISO/TS , Microbiology of food and animal feed Horizontal method for detection of hepatitis A virus and norovirus in food using real-time RT-PCR Part 2: Method for detection to harmonise with the DIS of part 1 d Lead and co-ordinate the activities of CEN/TC275/WG6/TAG15 Vibrios in the elaboration of methods for the determination of pathogenic marine vibrios in bivalve shellfish, particularly for V. parahaemolyticus and V. vulnificus e Act as project leader for the revision of the ISO 6887 series part 3 initial preparation and dilutions for aspects of microbiology to harmonise ISO standard with Commission Regulation (EC) No 2073/2005 f Act as sub-project leader for the revision of the ISO 7218 General rules for microbiology, covering revision of sections on MPN tables (with relevance to the EU reference method for E. coli) and molecular detection methods (viruses and vibrios). d Publication of full ISO standard (currently a Technical Specification) as a reference method for quantification of viruses in LBM and other foods. Publication of an updated TS for detection of viruses in LBM and other foods including fresh produce. e Reference method for determination of total and toxigenic V. parahaemolyticus and V. vulinificus in LBM of particular importance in US traded product f Standard harmonised with EU Reg. 2073/2005 with respect to minimum number of animals comprising a sample Contribute to the project for revision of ISO on organisation of proficiency testing Contribute to relevant EFSA expert working groups, including the WG on heat treatment of bivalve molluscs, and the WG to provide scientific and technical assistance on the baseline survey on Norovirus in oysters, and additional groups as required Represent the EURL at the annual plenary meeting of the CEN WG6 and ISO SC9 microbiology working groups g Contribute towards the FAO/WHO initiative developing best practice guidance for the application of bivalve molluscan shellfish sanitation Page 2 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme g EU representation at CEN and ISO microbiology expert groups

20 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk programmes h. 2. Project management and co-ordination of activities of NRL network h Enhanced EURL scientific reputation and impact The EURL will: Conduct project management of the EURL programme according to the requirements of EN ISO 9001 Quality Management Systems j Participate in the EURL Director s co-ordination meeting and other EURL co-ordination meetings/workshops as appropriate Organise, host, and participate in the annual EURL workshop of NRLs for monitoring bacteriological and viral contamination of bivalve molluscs, produce resolutions and other workshop outputs. In 2016, the workshop will be held at the NRL Germany at the Federal Institute for Risk Assessment, Berlin, (25 th -27 th May 2016). A venue and date for the 2017 workshop will be agreed at the 2016 meeting k. j retention of EN ISO 9001 demonstration of project management quality k harmonisation of official controls across network of NRLs 2.4. Further to the above, undertake EURL activities and commitments agreed in resolutions at the annual workshops above (as posted on Maintain and continue to improve the EURL website to improve relevance and accessibility of contents and development strategies to increase usage of the website services by NRLs and other stakeholders l. l increased accessibility of website, revised content and increase usage 3. Provision of technical advice and training The EURL will: Develop and maintain technical guidance documents describing the generation and calculation of precision characteristics associated with the virus reference method (ISO TS ) e.g. limit of detection, limit of quantitation, linearity and measurement uncertainty to support the setting of quantitative limits in LBM m. m Evidence to support the setting of robust standards for EU legislation Provide technical advice on request on ISO (the virus reference method) for matrices other than bivalve molluscs (e.g. soft fruits) to MS, NRLs and Official Control testing laboratories c Provide specialist training to NRLs, and others in relation to official control analyses (E. coli, Salmonella spp.) and non-statutory analyses (Vibrio spp., norovirus, hepatitis A virus, FRNA bacteriophage,) and other aspects of bivalve shellfish hygiene as required n. n improvements in performance and increased standardisation across laboratories 3.4. Supply technical advice on bacteriological and viral methods to NRLs, Official Control testing laboratories, and third county laboratories in the form of EURL harmonised protocols, standard operating procedures etc, to Page 3 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme

21 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk include approved alternative methods for official control analysis. These will be available in the public domain on the EURL website n Provide assistance on implementation of methods, accreditation to IEC ISO17025 and quality control requirements (see above) n Generate and distribute norovirus and hepatitis A virus reference materials and control materials, and Vibrio spp. EURL reference strains on request of NRLs n Provide guidance and review of procedures/data to laboratories wishing to undertake studies to validate alternative methods according to ISO To include formal assessment of validation data as required to support official approval (at SCFCAH) for alternative methods n Provide specialist ad hoc training to NRLs and others in relation to analyses of LBM for microbiological contaminants as required n Supply technical advice on request on ISO TS parts 1 and 2 (the virus reference method) for matrices other than bivalve molluscs (e.g. soft fruits) to NRLs and Official Control testing laboratories. To include advice on sampling plans, sample transport and result interpretation n Provide specialist training in application of the standard method for quantification of viruses (ISO ), to NRLs and Member State OCLs, designated by MS competent authorities to participate in the laboratory testing element of the EFSA baseline survey on Norovirus in oysters. This training to be provided through dedicated workshops hosted at the EURL p. p ensuring the EFSA baseline survey is as robust as possible Further to the above, the EURL will provide assistance to DG SANTE, EFSA, MS competent authorities, NRLs and OCLs in support of the EFSA baseline survey, through provision of protocols and reagents p Provide advice to NRLs and competent authorities regarding the implementation of official controls. 4. Confirmatory testing and quality assurance The EURL will: Maintain EURL laboratory competence and expertise in analytical methods for monitoring virological contaminants of bivalve molluscs (norovirus and hepatitis A virus). To include maintenance of requirements for ISO IEC accreditation for quantitative determination of norovirus in LBM q. q retention of ISO IEC accreditation where relevant at annual external audit demonstration of science quality 4.2. Maintain EURL laboratory competence and expertise in analytical methods for monitoring bacteriological contaminants of bivalve molluscs (E. coli, Salmonella spp., marine vibrios) using reference methods. To include maintenance of ISO IEC accreditation of enumeration of E. coli, and Page 4 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme

22 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk the detection of Salmonella spp. and Vibrio parahaemolyticus and attainment and/or maintenance of ISO IEC accreditation for detection of Vibrio vulnificus and V. cholerae q Perform the above-named tests on outbreak material or on occasion of disputed test results (on request) r. r technical advice to support EU 5. Comparative testing and ring trials The EURL will: Organise comparative testing for NRLs for statutory determinands (E. coli and Salmonella spp.) in bivalve molluscs. To include organisation of one whole-animal distribution per calendar year, and co-ordination of NRL participation in one or more of the three non-matrix External Quality Assessment (EQA) schemes organised in collaboration with Public Health England (PHE) per calendar year. For both EURL- and EURL/PHEorganised schemes, the EURL will analyse results and produce a report, plus additional advice and recommendations. To improve quality assurance in test results for EU controls comparative tests organised by the EURL will be available to laboratories outside of the NRLs network on a cost recovery basis s. s comparative testing reports published on the EURL website, presented at workshop, identification of satisfactory performance of NRLs and follow-up of unsatisfactory performance 5.2. Support implementation of quality assurance standards for virus testing by organising two distributions of samples containing norovirus and hepatitis A virus in each calendar year for comparative testing for quantitative and qualitative analyses. The EURL will analyse results, and produce a report and recommendations. To improve quality assurance in test results for EU controls comparative tests will be available to laboratories outside of the NRLs network on a cost recovery basis s Further to the above, the EURL will include non-nrl Member State OCLs, designated by MS competent authorities to participate in the laboratory testing element of the EFSA baseline survey for Norovirus in oysters, in one distribution of samples containing norovirus and hepatitis A virus in 2016 for comparative testing for quantitative analyses. Analyse results, produce report and recommendations specifically to support the survey p Organise comparative testing for pathogenic Vibrio spp. (one distribution per calendar year) to support methodological improvements. Analyse results, produce report and recommendations s. 6. Development of analytical methods The EURL will: Develop methods for inclusion in ISO TS (detection of vibrios), particularly to enable enumeration of total / pathogenic V. parahaemolyticus in imported / indigenously produced bivalve shellfish e,. Page 5 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme

23 Annex I European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) fsq@cefas.co.uk Work with colleagues, including the EU Joint Research Council, Geel, to elaborate control materials to assist in the standardisation of ISO methods for noroviruses and hepatitis A virus n Work with the NRLs network to identify means to improve the harmonisation of quantification between laboratories using ISO , the standard method for detection of viruses in foods n Develop methods to improve determination of the potential infectivity of noroviruses detected in bivalve shellfish t. t improved understanding of the public health significance of norovirus test results 6.5. Undertake analysis of dye-tracing studies carried out in 2015, and using the expertise developed during those studies, provide support to Member States wishing to apply that approach in support of satisfying additional requirements for production areas from which LBM are harvested for export to the USA b. NOTE: to support development of analytical methods under 6.1 and 6.4 it is proposed that a studentship(s) is supported by the EURL up to a maximum value of 10,000 per calendar year. Page 6 EURL for monitoring bacteriological and viral contamination of bivalve molluscs work programme

24 Annex II European Union Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) RESOLUTIONS OF THE 15 th WORKSHOP OF MICROBIOLOGICAL NRLs, TH MAY 2016, BERLIN, GERMANY Official controls 1. The commission reported to the workshop the emerging situation with tetrodotoxin contamination of LBMs. The possible connection with Vibrio sp. was noted and the workshop supported the need for further collaborative research with algal toxin scientists. 2. The commission informed the workshop that consequential on trade discussions with the US FDA, an audit of additional Member States would not be required prior to approval of that Member State for export. The EURL reminded the workshop of the need for Member States seeking approval to comply with the Good Practice Guide including Annex 4 on export to the USA. 3. The EURL presented draft version 6 of the Good Practice Guide and draft issue 3 of the Community Guide. The EURL further agreed to address comments on holding areas and alert levels for class A areas, and to recirculate the updated drafts amongst NRLs by June 17 th NRLs agreed to provide any additional comments by July 15 th Further to the above, the EURL informed the workshop of the complications with respect to seasonal classification and proposed to further consider this within the expert Working Group and report back at the next workshop. E.coli and Salmonella 5. The EURL reiterated the requirement to test 10 animals for E.coli analysis and acknowledged the current discrepancy within ISO ;2003 (cross-referenced in regulation 2015/2285) and informed the workshop that this will be resolved with the forthcoming publication of the revised ISO (currently at FDIS stage). 6. The EURL agreed to progress the development of a bivalve-specific MPN calculator (including reporting values of <18, >18,000 and >180,000 E.coli MPN/100g) and to endeavour to incorporate reference to this calculator within the forthcoming revision of ISO The EURL agreed to review ISO and undertake an evaluation of any available validation data for proprietary Salmonella detection systems to determine whether such methods can be used within the scope of the ISO as a screen. 8. The EURL informed the workshop that for the February 2017 EURL/Public Health England E.coli and Salmonella External Quality Assessment distribution, and for future annual distributions, the EURL would meet the costs of NRLs, and that participation would therefore be mandatory.

25 9. The EURL agreed to include both E.coli and Salmonella analysis in future whole animal proficiency testing distributions to assist NRLs in their accreditation requirements. 10. Following analysis of responses to an EURL questionnaire, the workshop noted that all EU Official Control Laboratories performing analysis for E.coli and Salmonella in Member States with production areas participated in proficiency testing for those determinands, under the supervision of their NRL, with the exception of Official Control Laboratories in Bulgaria and Norway. 11. The workshop noted the guidance on determination of measurement uncertainty for MPN methods provided by the EURL. The importance of sub-sampling prior to the homogenisation stage, for tests where replicate sample test measurements are required, was reinforced. Vibrios 12. The workshop noted the need for a clearer definition of fresh or frozen seafood within ISO in relation to the selection of the initial incubation temperatures. NRLs were encouraged to submit comments through their National Standardisation Bodies to facilitate this. 13. The TAG 15 group leader informed the workshop that the primary objective of TAG 15 was to establish robust quantitative methods for pathogenic Vibrio parahaemolyticus. The workshop agreed that the primary focus of this methodological development should be an MPN format with sufficient precision to enable robust quantification, confirmed using molecular methods. 14. The TAG 15 group leader encouraged NRLs with expertise in vibrios to join TAG 15 through nomination by their National Standardisation Bodies. In addition NRLs were encouraged to propose other relevant experts in their Member States to seek nomination. 15. The workshop agreed that EURL ring trials for vibrios organised during would focus on assisting method development and standardisation in collaboration with TAG 15 rather than assessment of laboratory competence. 16. In relation to EU/US trade, the workshop noted the increasing risk of shellfish-related vibriosis in the USA however data indicates that recently-introduced cold-chain measures may mitigate this risk. 17. The workshop agreed the need to share information regarding vibriosis associated with bivalve mollusc consumption, including any associated with US imports, in order to inform future review of risk. Viruses 18. The workshop commended the excellent work conducted by the European Food Safety Authority regarding alternative heat treatments for bivalve molluscs and noted the further steps necessary to revise the criteria if appropriate. 19. Regarding the EU-wide baseline survey for norovirus in oysters, the workshop noted an inconsistency within the European Food Safety Authority protocol between the body text (section 2.3.3) and the method specification (Appendix B) regarding use of archived materials for EU-wide and national studies and sought clarification from the European Food Safety Authority on this issue. The workshop recommended that Member State Competent Authorities should be free to decide the most appropriate use

26 of archived samples collected in their Member State without the need for consultation of the Standing Committee on Plants, Animals, Food and Feed. 20. Further to the above, the workshop agreed the importance of analysis of the baseline survey samples for HAV as a priority, considering the severity of this pathogen and the lack of data on the prevalence of HAV in oysters in the EU. 21. The workshop noted the growing awareness and importance of Hepatitis E virus as a foodborne pathogen and acknowledged the lack of data regarding the potential role of bivalve molluscs as a transmission vehicle for this virus within the EU. The workshop agreed the possibility to address this data gap through analysis of the archive from the baseline survey. 22. Following discussion, the EURL agreed to circulate the proposal for competence scoring for use in the virus proficiency testing prior to the baseline survey by the end of June 2016 for comment by NRLs by end of July Following discussion on the non-mandatory EURL guidance on determination of a Limit of Detection (LOD) and Limit of Quantification (LOQ) for the norovirus method, the EURL agreed to incorporate comments on the rationale for the assigned maximum standard deviation value, and to incorporate a provision for user laboratories to use appropriate methods for interpolation of LOQ between tested levels. A revised draft would be circulated by end of June 2016 for comment by NRLs by end of July Based on information supplied by NRLs, the workshop noted that in most Member States samples for the baseline survey were planned to be collected by official samplers for both production areas and dispatch centres. 25. The next workshop will be held 3 rd -5 th May 2017, in Split, Croatia (subject to confirmation by the EURL).

27 Annex III European Union Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs Report of the 15 th workshop of NRLs for monitoring bacteriological and viral contamination of bivalve molluscs. Berlin, Germany, 25 th 27 th May, Final report version 3 03/10/2016 Contract Reference: C6927 Document prepared by: Document checked by: Document approved by: James Lowther David Lees Review date: n/a David Lees Classification: Unclassified

28 Contents Foreword 3 Delegate list 4 Agenda of the 15 th workshop 7 List of workshop papers 9 Workshop minutes 10 Workshop actions 18 Workshop resolutions 21 Workshop feedback 24 Workshop declaration 26 2

29 Foreword This document summarises relevant information from the 15 th workshop of National Reference Laboratories for monitoring bacteriological and viral contamination of bivalve molluscs held at BFR, Berlin, Germany 25 th 27 th May It includes the workshop agenda, delegate contact information, workshop minutes, lists of associated papers, and the resolutions agreed by the meeting. Supplementary supporting information identified in this report can be accessed on the website of the EURL or may be supplied on request by the EURL. All requests should be made to the EURL co-ordinator. Dr James Lowther EURL Co-ordinator Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom Telephone: +44 (0) Direct line: +44 (0) Fax: +44 (0) General EURL Website: 3

30 Delegate List Country/Organisation Country Status Delegate Specialist area Austria Member State Johann Ladstaetter NRL Representative Belgium & Luxembourg Member State Nadine Botteldoorn NRL Representative Bulgaria Member State Gergana Krumova-Valcheva NRL Representative Croatia Member State Irena Listes NRL Representative Eddy Listes NRL Representative Denmark Member State Anna Charlotte Schultz NRL Representative France Germany Member State Member State Soizick Le Guyader NRL Representative Pascal Garry NRL Representative Eckhard Strauch NRL Representative Reimar Johne NRL Representative Greece Member State Ntina Vasileiadi NRL Representative Hungary Member State Zsuzsanna Sreterne Lancz NRL Representative Iceland EFTA Franklin Georgsson NRL Representative Ireland Member State Bill Dore NRL Representative Elisabetta Suffredini NRL Representative Italy Member State Francesca Leoni NRL Representative Mario Latini NRL Representative 4

31 Country/Organisation Country Status Delegate Specialist area Latvia Member State Nataļja Ivanova NRL Representative Lithuania Member State Audinga Verbickiene NRL Representative Netherlands Member State Irene Pol-Hofstad NRL Representative Norway EFTA Mette Myrmel NRL Representative Poland Member State Artur Rzezutka NRL Representative Magdalena Lopatek NRL Representative Portugal Member State Sonia Pedro NRL Representative Romania Member State Alina Popescu NRL Representative Slovakia Member State Julia Jurovcikova NRL Representative Slovenia Member State Urška Henigman NRL Representative Spain Member State Juan Olmedo NRL Representative Sweden Member State Magnus Simonsson NRL Representative United Kingdom Member State Craig Baker-Austin NRL Representative David Lees EURL Director EURL James Lowther EURL Co-ordinator Samantha Arkell EURL Administrator 5

32 Country/Organisation Country Status Delegate Specialist area Rachel Hartnell EURL Representative EURL Louise Stockley EURL Representative Lisa Cross EURL Representative Spain Cristina Alvarez Invited Expert Germany Thorsten Trumpf Invited Expert Germany Edda Bartelt Invited Expert EFSA Jane Richardson Invited Expert FVO Julia Uriol Observer DG SANTE Paolo Caricato Observer 6

33 AGENDA 15 th Workshop of Microbiological NRLs, MAY 2016, GERMANY Venue: Room D146 Bundesinstitut für Risikobewertung (BfR) Diedersdorfer Weg Berlin-Marienfelde Germany Tel.: +49- (0) Enquiries: Prior to the workshop enquiries should be directed to James Lowther or Samantha Arkell on: Direct line: +44 (0) (0) james.lowther@cefas.co.uk or samantha.arkell@cefas.co.uk Day 1 - Wednesday 25 May 9:00-17:30 1 Introduction 1.1 Welcome to BfR 1.2 Introductions and apologies (paper WS 15/01) 1.3 Domestic arrangements including reclaim of expenses (papers WS15/02, WS15/03) 1.4 Actions arising from the 14 th workshop 2015 (paper WS15/04) 1.5 Agreement of the agenda (paper WS15/05) 1.6 EURL work programme (EURL) (paper WS15/06) 2 Official controls 2.1 Update on the Commission Restricted Working Group (EURL) Coffee/tea break (10:30-11:00) 2.2 EU/US trade negotiations - FVO audit of the US (EURL) - Equivalence of microbiological standards (EURL) (paper WS15/07) - FDA audit: The Netherlands (NRL Netherlands) - FDA audit: Galicia (Cristina Alvarez, INTECMAR, Spain) 2.3 Update on the Good Practise Guide (EURL) (papers WS15/08, WS15/09) 2.4 Mussel and oyster production in Germany: Production areas, Monitoring and Future Developments (Edda Bartelt, Lower Saxony State Office for Consumer Protection and Food Safety, Germany & Torsten Trumpf, Landeslabor Schleswig-Holstein, Germany) 3 E.coli and Salmonella Lunch break (13:00 14:00) 3.1 E.coli and Salmonella methodology update (EURL) (paper WS15/10) 3.2 EURL proficiency testing for E. coli and Salmonella (EURL) (papers WS15/11, WS15/12) 3.3 Update on discussions regarding the ISO MPN calculator (EURL) 3.4 Method uncertainty (EURL) (paper WS15/13) 3.5 Summary of NRL organised PT schemes for Member State OCLs (EURL) (paper WS15/14) 3.6 Elevated E. coli results from a UK official control laboratory, July 2015 (NRL United Kingdom) 7

34 Coffee/tea break (15:30-16:00) 4 Marine vibrios 4.1 Validation of ISO TS under the CEN mandate (M/381) and revision of ISO (EURL) (paper WS15/15) 4.2 Update on activities of CEN/TC 275/WG6/TAG15 (vibrios) (EURL) 4.3 Vibrio proficiency testing (EURL) (paper WS15/16) Day 2 - Thursday 26 May 9:00-17: Trh (tdh /trh+) gene analysis of clinical, environmental and food isolates of Vibrio parahaemolyticus (NRL Italy) 4.5 Cold chain measures to reduce Vibrio incidence from the USA (EURL) 5 Viruses 5.1 EFSA Scientific Opinion on alternative heat treatment profiles for processing of live bivalve molluscs (Winy Messens, EFSA) (paper WS15/17) Coffee/tea break (10:30-11:00) 5.2 Update on the progress of ISO (EURL) 5.3 EURL proficiency testing for norovirus and HAV PT 59 (EURL) (paper WS15/18) 5.4 European baseline survey of norovirus in oysters - The sampling plan (Jane Richardson, EFSA) (paper WS15/19) Lunch break (13:00 14:00) - The method specification (EURL) - Training and proficiency testing (EURL) - Determination of LOD and LOQ (EURL) (paper WS15/20) - Approaches to sampling in Member States (round-table discussion) Coffee/tea break (15:30-16:00) 5.5 Optimisation of digital PCR and application to naturally contaminated oysters (NRL France) 5.6 The experience of the Belgian NRL for BMS on Norovirus detection, especially during outbreak investigations (NRL Belgium) 5.7 Hepatitis E virus; an overview (NRL Germany) 6 Any other business 7 Agreement of Workshop resolutions 8 Date and venue for next meeting Day 3 - Friday 27 May 9:00-12:00 Coffee/tea break (10:15-10:45) Meeting close 8

35 List of papers for 15th Workshop of Microbiological NRL s WS15/00 WS15/01 WS15/02 WS15/03 WS15/04 WS15/05 List of papers Delegates List EURL Workshop Payment Details Form EURL Workshop Expenses Claim Form Actions from 14th workshop Agenda WS15/06 EURL Microbiological Contamination of Bivalve Molluscs Work Programme WS15/07 WS15/08 Comparison of Current and Proposed European Union Classification Criteria for Category A Shellfish Harvesting Waters Community Guide Microbiological Monitoring Bivalve Mollusc Harvesting Areas Issue 3 draft revision WS15/09 Commission Regulation (EU) WS15/10 WS15/11 WS15/12 WS15/13 WS15/14 Issue 12 EURL Generic SOP E-coli Proficiency testing 60 E-coli Proficiency testing 63 - EQA Determining Uncertainty of Measurement draft v2 Summary of NRL organised PT schemes for Member State OCLs WS15/15 Validation of ISO TS under the CEN mandate (M 381) and revision of ISO WS15/16 WS15/17 WS15/18 WS15/19 WS15/20 Proficiency testing 58 - Vibrio EFSA Scientific Opinion on alternative heat treatment profiles Proficiency testing 59 - Virus Baseline survey sampling plan LOD and LOQ guidance note v1 Draft 9

36 Minutes of the 15 th Workshop of Microbiological NRLs for Bivalve Molluscs, 25 th 27 th May, 2016 Attendees EURL Director David Lees (DNL) (chair) Cefas, Weymouth, UK EURL Coordinator James Lowther (JL) Cefas, Weymouth, UK EURL Administrator Samantha Arkell (SA) Cefas, Weymouth, UK EURL Rachel Hartnell (RH) Cefas, Weymouth, UK EURL Louise Stockley (LS) Cefas, Weymouth, UK EURL Lisa Cross (LC) Cefas, Weymouth, UK NRL Austria Johann Ladstaetter (JLa) AGES ILMU, Wien NRL Belgium and Luxembourg NRL Bulgaria NRL Croatia NRL Denmark NRL France NRL Germany Nadine Botteldoorn (NB) Gergana Krumova- Valcheva (GKV) Eddy Listes (EL) Irena Listes (IL) Anna Charlotte Schultz (ACS) Soizick Le Guyader (SLG) Pascal Garry (PG) Eckhard Strauch (ES) Reimar Johne (RJ) Scientific Service of Food-borne Pathogens, Brussels National Diagnostic and Research Veterinary Institute, Sofia Croatian Veterinary Institute, Split Institute of Food Safety and Nutrition, Søborg Institut Français de Recherche pour L'Exploitation de la Mer (IFREMER), Nantes Federal Institute for Risk Assessment, Berlin NRL Greece Ntina Vasileiadi (NV) Institute of Food Hygiene of Athens, Athens NRL Hungary Zsuzsanna Sreterne Lancz (ZL) NRL Iceland Franklin Georgsson (FG) Matis, Reykjavik Central Agricultural Office, Food & Feed Directorate, Budapest NRL Ireland Bill Dore (BD) Marine institute, Galway Elisabetta Suffredini (ELS) Istituto Superiore di Sanità (ISS) Rome NRL Italy NRL Latvia NRL Lithuania NRL Netherlands Francesca Leoni (FL) Mario Latini (ML) Nataļja Ivanova (NI) Audinga Verbickiene (AV) Irene Pol-Hofstad (IPH) Istituto Zooprofilattico Sperimentale, dell'umbria e delle Marche, Ancona National Diagnostic Centre of Food & Veterinary Service (FVS), Riga National Food and Veterinary Risk Assessment Institute, Lithuania National Institute of Public Health and the Environment (RIVM), Bilthoven NRL Norway Mette Myrmel (MM) Norwegian School of Veterinary Science, Oslo NRL Poland Artur Rzezutka (AR) Magdalena Lopatek (MLo) National Veterinary Research Institute, Pulawy NRL Portugal Sonia Pedro (SPe) Portuguese Institute of Sea and Atmosphere (IPMA), Lisbon NRL Romania Alina Popescu (APo) Institute for Diagnosis and Animal Health, Bucharest NRL Slovakia Julia Jurovcikova (JJ) State Veterinary and Food Institute, Dolny Kubin 10

37 NRL Slovenia NRL Spain Urska Henigman (UH) Juan Olmedo (JO) Institute for Food Hygiene and Bromatology, Ljubljana Agencia Espanola de Seguridad Alimentaria, Majadahonda, Madrid NRL Sweden Magnus Simonsson (MS) National Food Administration, Uppsala NRL UK Craig Baker-Austin (CBA) Cefas, Weymouth Invited expert Invited expert Cristina Alvarez Alvarez (CAA) Edda Bartelt (EB) Centro de Control da Calidade do Medio Marino, Pontevedra Lower Saxony State Office for Consumer Protection and Food Safety, Cuxhaven Invited expert Thorsten Trumpf (TT) Landeslabor Schleswig-Holstein, Neumünster Invited expert Jane Richardson (JR) EFSA Observer Julia Uriol (JU) FVO Observer Paolo Caricato (PC) DG SANTE Apologies Elina Vatunen, NRL Finland Acronyms CEN Comité Européen de Normalisation MU Method Uncertainty DG SANTE Directorate-General for Health and Food Safety NoV EQA External Quality Assessment NSSP Norovirus US National Shellfish Sanitation Program EFSA European Food Safety Authority NRL National Reference Laboratory EU European Union OCL Official Control Laboratory EURL European Union Reference Laboratory PCR Polymerase Chain Reaction FDA US Food and Drug Administration PHE Public Health England FVO Food and Veterinary Office PT Proficiency Testing HAV Hepatitis A Virus TAG Technical Advisory Group HEV Hepatitis E Virus TS Technical Specification ISO International Standards Organisation UK United Kingdom LBM Live Bivalve Molluscs US/USA United States of America MPN Most Probable Number WG Working Group MS Member State WS Workshop 11

38 1. Introduction 1.1. Welcome to BFR Prof. Dr. Reiner Wittkowski from the Federal Institute for Risk Assessment, Berlin welcomed the delegates to the institute Introduction and apologies DNL opened the meeting, followed by round table introductions Domestic arrangements including reclaim form Delegates were given instructions on electronic submission of forms and supporting information to enable payment of expenses Actions arising from the 14th workshop All actions identified were either complete or covered separately as agenda items Agreement of the agenda The workshop agenda (WS15/05) was agreed EURL Work Programme 2015 JL presented the EURL work programme (WS15/06) agreed with DG SANTE for the calendar year Official Controls 2.1 Update on the Commission Restricted Working Group DNL gave an overview on the developments within the Restricted Working Group particularly with regards to Regulation (EU) No. 2015/2285, the EU baseline survey for norovirus, method uncertainty, and classification and monitoring. JU clarified the position of the Commission with regards to closing/reclassification of non-compliant class A areas, whereby they would seek a consensus between MS, rather than specific legal advice on the matter. There was some discussion between the EURL/NRLs and PC and JU from the Commission as to whether the definition of production areas in EU legislation excludes artificial ponds on land. The issues of accumulation of tetrodotoxin and parasites (pathogenic to humans) within LBM were raised and it was agreed that further collaborations with relevant experts were desirable. 2.2 EU/US trade negotiations a. FVO audit of the US DNL provided an update on the findings of the FVO audit of the US, following the publication of the audit report. It was clarified that the audit was to check compliance with US rules, not EU rules. It was noted, in particular, that the US control programmes based on the NSSP were comprehensive, and that the US states visited implemented them adequately. There were gaps identified in certain areas regarding compliance with the NSSP and the report included recommendations for rectification. Resolution 1 Actions 1&4 Resolution 2 b. Equivalence of microbiological standards RH presented a study looking at the equivalence of US approved and EU class A standards, in light of the forthcoming change to a 3 class standard (Regulation (EU) No. 2015/2285). No significant difference in equivalence of 12

39 the existing and new EU standards compared to the US standard was noted. However, there was some discussion on the number of discordant classifications under the EU and US systems amongst the sites in the data set. Further analysis of the agreed EU US dataset was underway prior to a physical technical meeting planned later in the year. c. FDA audit: The Netherlands IPH provided a comprehensive overview of the audit carried out by the US FDA on mussel and oyster production (6 areas) in the Netherlands. The audit generally found a high level of compliance with EU legislation and additional good practise, however a recommendation around discharge of human waste from commercial vessels was made by FDA, in addition to a request for sight of the validation data for the E. coli method used in the Netherlands. d. FDA audit: Galicia CAA provided a comprehensive overview of the audit carried out by the US FDA on bivalve production in the Xunta de Galicia, Spain. The audit generally found a high level of compliance with EU legislation and additional good practise, however recommendations on reopening following sewage discharges into growing waters, discharge of human waste from commercial vessels, co-purification of shellfish from different classifications and the performance of private laboratories were made by the FDA. Following the audit presentations, PC confirmed that any additional areas within the EU (and the US) will not be subject to formal audits prior to approval for transatlantic trade. 2.3 Update on the Good Practice Guide RH provided an update on the revision of the Good Practice Guide, identifying areas of the document that have undergone changes, and highlighting outstanding areas for discussion, particularly with regards to seasonal classification. The WS discussed the treatment of holding areas within the guidance and PC reiterated that it was important that the documents do not exceed what is included in EU legislation, nor modify the definitions therein. Additional required changes to the class A alert level were identified by the WS. Resolutions 3-4 Actions Mussel and oyster production in Germany EB gave a presentation on bivalve production in the states of Lower Saxony and Schleswig-Holstein in Germany, detailing production areas and volumes, and the classification monitoring programmes, and presenting the results of microbial testing, including for non-regulatory determinands. 3. E. coli and Salmonella 3.1. E. coli and Salmonella methodology LS updated the WS on the revisions of ISO standards , and , and detailed the current status of the EURL generic protocols for E. coli and Salmonella in terms of harmonisation with the standards. The WS discussed the potential discrepancy in the number of animals comprising a sample in the period between the application of Regulation (EU) No. 2015/2285 on 1 st January 2017, and the publication of the new revision of ISO The EURL reiterated that in such an event the number of animals comprising a sample remained 10, as in currently enacted legislation. Resolutions 5&7 Actions 6,7&11 13

40 SPe noted a discrepancy between the loop size in ISO and the EURL generic protocol; the EURL agreed to amend the generic protocol to remove the discrepancy. The WS discussed whether the use of proprietary Salmonella detection systems was permissible according to ISO The EURL indicated that it would investigate EURL proficiency testing for E. coli and Salmonella LS presented the results of PT 60 and the EURL/PHE EQA scheme, with particular emphasis on the performance of NRLs and follow-up actions resulting from underperformance. The EURL informed the WS that from 2017, it would cover the costs of participation for all NRLs in one EQA distribution annually (in February). It would therefore be mandatory for all NRLs to participate. The WS agreed that this was a positive development. NRLs emphasised the importance of inclusion of Salmonella analysis in all PT distributions the EURL agreed and promised to include this in all future distributions Update on discussions regarding the ISO MPN calculator RH provided the WS with an update on issues around the MPN calculator, including the production of guidance documents, and discussions with ISO TC34 SC9 WG2 (Food Microbiology - Statistics) to produce a bivalve specific worksheet incorporating <18, >18,000 scores etc., and to incorporate reference to the worksheet in ISO NRLs welcomed the developments and reiterated the importance of resolving this issue, as accredited labs are currently risking problems due to the discrepancy between ISO 7218 and the EURL guidance documents Method uncertainty RH presented an update on the EURL guidance on determination of method uncertainty (MU) for E. coli analysis. Following questions from NRLs, the EURL confirmed that although MU is not referenced in ISO , national accreditation bodies generally require labs to calculate MU in order to approve accreditation to ISO Summary of NRL organised PT schemes for Member State OCLs LS presented findings from a questionnaire distributed to NRLs to determine both their participation in PT schemes (both organised by the EURL and by others) and their oversight of the participation of OCLs within their MS in such schemes. Following a round-table discussion between the EURL and NRLs from producer countries, it was noted that in two countries (Bulgaria and Norway), OCLs did not participate in PT schemes under the supervision of the NRL. The EURL reiterated that it was a responsibility of NRLs to ensure that labs in their OCL network participated in PT schemes. Resolutions 8-9 Action 8 Resolution 6 Action 5 Resolution 11 Action 10 Resolution 10 Action Elevated E. coli results from a UK official control laboratory, July 2015 CBA gave a presentation on a series of high E. coli results reported by a UK OCL during a short period in The WS discussed this interesting case and some NRLs reported some similar unusual E. coli results, but on a less significant scale. 14

41 4. Marine Vibrios 4.1 Validation of ISO TS under the CEN mandate (M/381) and revision of ISO (M/381) (EURL) RH gave a comprehensive overview of the CEN project to validate the vibrio ISO 21872, including presentation of the method characteristics determined. Following questions from NRLs, RH confirmed that under ISO 21872, there is a choice of conventional, real-time or biochemical confirmation tests. The WS suggested some modifications to improve the new draft of 21872, including the clarification of definitions of fresh and frozen seafood, and the inclusion of information regarding the strains used for contamination. 4.2 Update on activities of CEN/TC 275/WG6/TAG15 (vibrios) CBA updated the WS on the activities of TAG15 relating to the development of new PCR based methods for the detection and quantification of pathogenic vibrios. It was noted that a focus of work would be directed at quantitative methods for V. parahaemolyticus. The call for relevant national experts to participate in, and share methods with TAG15 was reiterated, particularly from countries where vibrio testing is currently widespread and routine. 4.3 Vibrio proficiency testing (EURL) LS presented the findings of PT 58 distribution for vibrios. The WS discussed and agreed the need for future EURL vibrio PT rounds to focus on method development, to assist the activities of TAG Trh (tdh /trh+) gene analysis of clinical, environmental and food isolates of Vibrio parahaemolyticus FL updated the WS with the results of genome sequencing efforts for V. parahaemolyticus isolates carried out by NRL Italy, highlighting in particular the possible use of trh gene characterisation for assessment of potential pathogenicity. The EURL agreed to share relevant strains with NRL Italy to assist in further research in this area. 4.5 Cold chain measures to reduce Vibrio incidence from the USA CBA presented epidemiological data supplied by colleagues in the US on the spread of Vibrio parahaemolyticus cases to new States and on the effectiveness of new cold chain measures (particularly the use of ice slurries) to reduce shellfish-related vibriosis. The WS acknowledged that the epidemiological data suggested these measures were potentially effective however there were possible problems (mortality, organoleptic issues etc.). CBA suggested that more epidemiological data was required to fully determine the efficacy of these control measures, and further noted that given the potential risk of Vibrio contamination of oysters in the US, and the new approval for importing oysters from approved areas within the US, that efforts should be taken to collate information on shellfish-related vibriosis cases within the EU. It was further agreed that it was important to share all information on this issue whether linked to EU-US trade or not, and mechanisms for capturing the information were discussed. The WS was informed that the EU and US had agreed to keep bivalve shellfish related vibriosis, potentially associated with traded products, under review and to revisit trade agreements if necessary. Resolution 12 Action 15 Resolutions 13&14 Actions 12&17 Resolution 15 Action 16 Resolutions 16&17 Actions 13&14 15

42 5. Viruses 5.1 EFSA Scientific Opinion on alternative heat treatment profiles for processing of live bivalve molluscs Winy Messens of EFSA gave a presentation by telephone detailing the preparation and findings of the scientific opinion of alternative heat treatments. The WS discussed the significance of the conclusions with regards to the existing legal heat treatment criteria, and agreed that the experimental evidence broadly supported the existing criteria, and that it would be difficult to recommend heat treatment at <90 C, due to the wide confidence intervals on the appropriate length of time at lower temperatures. DNL, who was part of the EFSA WG formed to address this question, further emphasised that considerations of organoleptic qualities were important. 5.2 Update on the progress of ISO (EURL) JL provided a brief update on the status of both parts of the virus ISO; (quantification) had passed the first vote and a final draft based on the comments received had been prepared. Publication was anticipated by the end of Further, the CEN parent committee had requested that a new revision of (detection) was prepared to harmonise with the revised part 1, and to incorporate method characteristics. 5.3 EURL proficiency testing for norovirus and HAV PT 59 (EURL) LS provided an overview of PT 59 for viruses in LENTICULE samples, with particular emphasis on the participation and performance of NRLs. 5.4 European baseline survey of norovirus in oysters a. The sampling plan JR gave a presentation on the development of the sampling plan for the baseline survey, detailing the input parameters used in the model, and presenting the total samples per MS required. Resolution 18 Actions 22&23 Action 24 Resolutions & Actions b. The method specification (EURL) JL presented the baseline survey method specification to the WS, explaining the development of the document and its relationship to ISO The additional technical support in terms of control materials offered by the EURL was also detailed. The WS engaged in considerable discussion on the use of archived test sample materials for additional purposes e.g. HAV or HEV analysis. It was noted that there were some discrepancies in the baseline survey protocol as to how priority for use of the archive was decided, and the WS agreed that clarification on this point would be important. c. Training and proficiency testing (EURL) LC presented the EURL plans for two training workshops for laboratories designated for analysis in the baseline survey, and in addition presented a first draft of a proposal for competence scoring of labs, following the planned PT distribution, prior to approval to take part in the baseline survey. d. Determination of LOD and LOQ (EURL) JL gave an overview of an EURL guidance note for determination of LOD and LOQ for the quantification of viruses in LBM. It was reiterated that although the method included in the guidance note was not mandatory, it was necessary for all designated laboratories undertaking analysis for the 16

43 baseline survey to report LOQ values to EFSA. e. Approaches to sampling in Member States (round-table discussion) NRLs from those MS involved in the baseline survey presented what they knew of their national plans to collect samples for the survey. In the majority of cases sampling was due to be carried out by Competent Authority officials. 5.5 Optimisation of digital PCR and application to naturally contaminated oysters SLG gave a presentation on application of the digital PCR quantification method to oysters. The WS discussed the positive aspects and limitations of this technology compared with real-time PCR. 5.6 The experience of the Belgian NRL for BMS on Norovirus detection, especially during outbreak investigations NB gave an overview of food and waterborne outbreaks of norovirus in Belgium, with diverse sources and epidemiologies described. 5.7 Hepatitis E virus; an overview RJ provided a comprehensive overview on HEV, covering its virology, epidemiology, occurrence in LBM and other foods, and methods for its culture and detection. The WS discussed the growing emergence of this pathogen and agreed that it should be a priority for shellfish hygiene research in the short and medium term. The WS identified that archived samples from the norovirus baseline survey presented a good opportunity to gather information on the occurrence of HEV in molluscs. Resolution Any other business No other business was raised. 7. Agreement of Workshop resolutions The workshop resolutions were agreed by the delegates. 8. Date and venue for next meeting The workshop agreed with the proposal that the next meeting would be held on 3 rd - 5 th May 2017, in Split, Croatia (subject to confirmation by the EURL). Resolution 25 17

44 Actions of the 15 th Workshop of Microbiological NRLs for Bivalve Molluscs, Bundesinstitut für Risikobewertung (BfR), Berlin 25 th 27th May, 2016 Action Owner Completed Notes Official controls 1. EURL to investigate the possibility of a collaboration with the EURL on marine biotoxins on tetrodotoxin. 2. EURL to update guidance documents to address comments on holding areas and alert levels for class A areas, then to circulate to NRLs for comment prior to presentation at the meeting of the Commission Restricted Working Group. 3. EURL to consider the complications with respect to seasonal classification within the expert Working Group and report back at the next workshop. 4. EURL to write to relevant contacts at EURL Parasites to ask whether they have any data on the occurrence of parasites (Giardia, Cryptosporidium etc.) in bivalve shellfish, particularly with respect to human illness. EURL to report at next WS. E.coli and Salmonella 5. EURL to contact the relevant expert (Cordelia Wilrich) to progress the bivalve specific MPN calculator and the convenor of ISO/TC 34/SC 9/WG 7 General requirements and guidance for microbiological examinations to discuss inclusion of the calculator within the revision of ISO EURL EURL EURL EURL EURL 6. EURL to consider the scope of ISO , to determine if it is possible to use proprietary Salmonella detection systems as a screen. 7. Subject to the findings from the previous action, EURL to undertake an evaluation of any available validation data relating to bivalves/seafood. 8. EURL to liaise with PHE to make necessary arrangements and determine details re: registration and payment etc. for participation of NRLs in the February 2017 EURL/PHE E. coli and Salmonella External Quality Assessment distribution at the expense of the EURL. EURL EURL EURL 18

45 9. EURL to finalise the report on the responses to the questionnaire on proficiency testing and distribute to NRLs, to assess the findings and decide on further actions, and to report back at the next WS. 10. EURL to complete a final version of the guidance on Method Uncertainty and to publish on the website. Final version to incorporate comments received, in addition to the EURL in-house MU value. 11. EURL to amend E. coli generic protocol to change recommended loop size in body text to 10µl to align with ISO , and to add a footnote allowing the use of other loop sizes (e.g. 1µl) subject to appropriate verification. Marine vibrios 12. All NRL members with expertise in vibrios to consider joining TAG 15 through nomination by their National Standardisation Bodies and in addition to propose other relevant experts in their Member States to seek nomination. 13. EURL to develop a simple database, to be held on the restricted area of the website, to capture details of shellfish-related vibriosis across the EU. 14. NRLs to share any data that they may have on shellfish-related vibriosis, through the mechanism of the database mentioned in the previous action. EURL EURL EURL NRLs EURL NRLs 15. EURL, as project leader for validation of ISO 21872, to add relevant Vibrio strain details to the forthcoming FDIS. 16. EURL to send trh+ Vibrio strains to IZS to facilitate research. EURL 17. EURL to clarify whether Vibrio methods that are currently in routine use for large number of samples in the US, e.g. Washington State, have been shared with TAG15. Viruses 18. EURL to contact EFSA with a view to producing a joint note clarifying the approach regarding use of archived materials from the baseline survey for EU-wide and national studies for distribution to NRLs and to MS survey coordinators. EURL EURL EURL 19

46 19. EURL to contact EFSA to ask them to consider adding a reporting parameter on whether a sanitary survey has been carried out on the production area for samples collected in the baseline survey 20. EURL to circulate the proposal for competence scoring for use in the virus proficiency testing prior to the baseline survey to NRLs for comment. 21. EURL to finalise the non-mandatory EURL guidance on determination of a Limit of Detection (LOD) and Limit of Quantification (LOQ) for the norovirus method and distribute to NRLs. 22. EURL to update generic protocol for quantification of viruses to harmonise with final draft of ISO EURL to clarify with CEN TC 275 WG6 secretariat whether it is allowed to share precision characteristics from the validation of ISO with NRLs prior to publication of the new version of the ISO 24. EURL to circulate a lenticule-specific calculation spreadsheet to participants in PT 65 EURL EURL EURL EURL EURL EURL 20

47 RESOLUTIONS OF THE 15 th WORKSHOP OF MICROBIOLOGICAL NRLs, TH MAY 2016, BERLIN, GERMANY Official controls 1. The commission reported to the workshop the emerging situation with tetrodotoxin contamination of LBMs. The possible connection with Vibrio sp. was noted and the workshop supported the need for further collaborative research with algal toxin scientists. 2. The commission informed the workshop that consequential on trade discussions with the US FDA, an audit of additional MS would not be required prior to approval of that MS for export. The EURL reminded the workshop of the need for MS seeking approval to comply with the Good Practice Guide including Annex 4 on export to the USA. 3. The EURL presented draft version 6 of the Good Practice Guide and draft issue 3 of the Community Guide. The EURL further agreed to address comments on holding areas and alert levels for class A areas, and to recirculate the updated drafts amongst NRLs by June 17 th NRLs agreed to provide any additional comments by July 15 th Further to the above, the EURL informed the workshop of the complications with respect to seasonal classification and proposed to further consider this within the expert Working Group and report back at the next workshop. E.coli and Salmonella 5. The EURL reiterated the requirement to test 10 animals for E.coli analysis and acknowledged the current discrepancy within ISO ;2003 (cross-referenced in regulation 2015/2285) and informed the workshop that this will be resolved with the forthcoming publication of the revised ISO (currently at FDIS stage). 6. The EURL agreed to progress the development of a bivalve-specific MPN calculator (including reporting values of <18, >18,000 and >180,000 E.coli MPN/100g) and to endeavour to incorporate reference to this calculator within the forthcoming revision of ISO The EURL agreed to review ISO and undertake an evaluation of any available validation data for proprietary Salmonella detection systems to determine whether such methods can be used within the scope of the ISO as a screen. 8. The EURL informed the workshop that for the February 2017 EURL/Public Health England E.coli and Salmonella External Quality Assessment distribution, and for future annual distributions, the EURL would meet the costs of NRLs, and that participation would therefore be mandatory. 9. The EURL agreed to include both E.coli and Salmonella analysis in future whole animal proficiency testing distributions to assist NRLs in their accreditation requirements. 10. Following analysis of responses to an EURL questionnaire, the workshop noted that all EU Official Control Laboratories performing analysis for E.coli and Salmonella in Member States with production areas participated in proficiency testing for those determinands, under the supervision of their NRL, with the exception of Official Control Laboratories in Bulgaria and Norway. 11. The workshop noted the guidance on determination of measurement uncertainty for MPN methods provided by the EURL. The importance of sub-sampling prior to the homogenisation stage, for tests where replicate sample test measurements are required, was reinforced. 21

48 Vibrios 12. The workshop noted the need for a clearer definition of fresh or frozen seafood within ISO in relation to the selection of the initial incubation temperatures. NRLs were encouraged to submit comments through their National Standardisation Bodies to facilitate this. 13. The TAG 15 group leader informed the workshop that the primary objective of TAG 15 was to establish robust quantitative methods for pathogenic Vibrio parahaemolyticus. The workshop agreed that the primary focus of this methodological development should be an MPN format with sufficient precision to enable robust quantification, confirmed using molecular methods. 14. The TAG 15 group leader encouraged NRLs with expertise in vibrios to join TAG 15 through nomination by their National Standardisation Bodies. In addition NRLs were encouraged to propose other relevant experts in their Member States to seek nomination. 15. The workshop agreed that EURL ring trials for vibrios organised during would focus on assisting method development and standardisation in collaboration with TAG 15 rather than assessment of laboratory competence. 16. In relation to EU/US trade, the workshop noted the increasing risk of shellfish-related vibriosis in the USA however data indicates that recently-introduced cold-chain measures may mitigate this risk. 17. The workshop agreed the need to share information regarding vibriosis associated with bivalve mollusc consumption, including any associated with US imports, in order to inform future review of risk. Viruses 18. The workshop commended the excellent work conducted by European Food Safety Authority regarding alternative heat treatments for bivalve molluscs and noted the further steps necessary to revise the criteria if appropriate. 19. Regarding the EU-wide baseline survey for norovirus in oysters, the workshop noted an inconsistency within the European Food Safety Authority protocol between the body text (section 2.3.3) and the method specification (Appendix B) regarding use of archived materials for EU-wide and national studies and sought clarification from European Food Safety Authority on this issue. The workshop recommended that Member State Competent Authorities should be free to decide the most appropriate use of archived samples collected in their Member State without the need for consultation of Standing Committee on Plants, Animals, Food and Feed. 20. Further to the above, the workshop agreed the importance of analysis of the baseline survey samples for HAV as a priority, considering the severity of this pathogen and the lack of data on the prevalence of HAV in oysters in the EU. 21. The workshop noted the growing awareness and importance of Hepatitis E virus as a foodborne pathogen and acknowledged the lack of data regarding the potential role of bivalve molluscs as a transmission vehicle for this virus within the EU. The workshop agreed the possibility to address this data gap through analysis of the archive from the baseline survey. 22. Following discussion, the EURL agreed to circulate the proposal for competence scoring for use in the virus proficiency testing prior to the baseline survey by the end of June 2016 for comment by NRLs by end of July

49 23. Following discussion on the non-mandatory EURL guidance on determination of Limit of Detection (LOD) and Limit of Quantification (LOQ) for the norovirus method, the EURL agreed to incorporate comments on the rationale for the assigned maximum standard deviation value, and to incorporate a provision for user laboratories to use appropriate methods for interpolation of LOQ between tested levels. A revised draft would be circulated by end of June 2016 for comment by NRLs by end of July Based on information supplied by NRLs, the workshop noted that in most Member States samples for the baseline survey were planned to be collected by official samplers for both production areas and dispatch centres. 25. The next workshop will be held 3 rd -5 th May 2017, in Split, Croatia (subject to confirmation by the EURL). 23

50 EURL Workshop 25 th to 27 th May 2016, Berlin Confidential Participant Feedback Results 24

51 Comments :- 1. When you have no production area s you are out of the scope of this workshop. 2. First day too long. 3. Organised beautifully. Thank you! 4. Excellent content, could do with moving or suggesting that questions and discussions are continued during tea and lunch breaks to help keep to agenda. 25

52 Workshop declaration This technical report is submitted in accordance with the requirements of Commission Implementing Regulation (EC) No 926/2011 laying down detailed rules for the granting of Community financial assistance to Community reference laboratories for feed and food and the animal health sector, following the workshop of National Reference Laboratories for bacteriological and viral contamination of bivalve molluscs held in Berlin 25th-27th May Dr David Lees 06/09/2016 EURL Director Dr James Lowther 06/09/2016 EURL Co-ordinator 26

53 Annex IV European Union Reference Laboratory (EURL) Proficiency Testing Scheme Noroviruses and hepatitis A virus EURL PT reference number: PT 61 Final report version pages Contract Reference: Cefas ref (C6927A) Document approved by: C6472A1 Project Manager James Lowther Review date: n/a Document checked by: James Lowther Classification: Official Document prepared by: Louise Stockley Location

54 Contents Page number Samples 2 Results 3 Discussion 7 References 8 Appendices 9 Proficiency testing 61 Final 1 Page 1 of 21

55 Samples Materials dispatched consisted of naturally contaminated and bioaccumulated Pacific oysters (Crassostrea gigas) (Samples 1, 2, 3 and 4), laboratory constructed LENTICULES and dsdna control solutions for quantification (1 x 10 5 copies/µl) for each target virus. Reference results for each shellfish sample and LENTICULE sample are shown in Table 2. Sample preparation Shellfish Sample 1 Approximately 3000 Pacific oysters (C. gigas) were taken from a UK commercial harvesting area and relayed in an area subject to high levels of sewage pollution (commercial harvesting of bivalves prohibited at this site) to bioaccumulate for approximately 3 weeks before being re-collected. The oysters were then randomly sorted into samples of 10 animals and placed in sample bags. The samples were held at <-15 C until required for quality control testing, dispatch and/or reference analysis. Shellfish Sample 2 Approximately 3000 Pacific oysters (C. gigas) were collected from a UK commercial harvesting area. The oysters were tested to indicate the batch of shellfish was negative for norovirus (NoV) genogroups I and II (GI and GII) and hepatitis A virus (HAV). Following testing, the shellfish were shucked and the digestive glands removed. The digestive glands were pooled together before being blended to form a homogenous mixture and then split into 2 g aliquots. The samples were held at <-15 C until required for quality control testing, dispatch and/or reference analysis. Highly contaminated blends used for preparation of Shellfish Samples 3 and 4 Approximately 1000 Pacific oysters (C. gigas) collected from a UK commercial harvesting area and previously tested to demonstrate they were negative for NoV GI, NoV GII and HAV, were placed in trays and immersed in 500 litres of re-circulating natural seawater at 16 ± 1 C. The shellfish were left for approximately 24 hours to acclimatise before 50 ml of shellfish food containing high levels of NoV GI from human faeces was added to the tank. After approximately 16 hours to allow bioaccumulation the shellfish were removed from the tank, shucked and the digestive glands removed. The digestive glands were pooled together before being blended to form a homogenous mixture. Separately this procedure was repeated with NoV GII from human faeces and HAV cell culture supernatant to produce three highly contaminated blends, each contaminated with a single target virus. Shellfish Sample 3 Blended negative glands (prepared as for Shellfish Sample 2) were mixed with the highly contaminated blends containing NoV GII and HAV (as described above) to obtain the desired target levels before being split into 2 g aliquots. The samples were held at <-15 C until required for quality control testing, dispatch and/or reference analysis. Shellfish Sample 4 Blended negative glands (prepared as for Shellfish Sample 2) were mixed with the highly contaminated blends containing NoV GI and HAV (as described above) to obtain the desired target levels before being split into 2 g aliquots. The samples were held at <-15 C until required for quality control testing, dispatch and/or reference analysis. NOTE: reference analyses for this sample indicated the presence of extremely low concentrations of NoV GII RNA. For this reason GII results for Shellfish Sample 4 have not been considered for performance scoring. Proficiency testing 61 Final 1 Page 2 of 21

56 Test sample LENTICULES 1 and 2 Two batches of laboratory constructed LENTICULES were prepared following the method of Codd et al (1998) with minor modifications. The mixture prepared for LENTICULE 1 included known levels of GI and GII NoV from human faeces and HAV cell culture supernatant. Table 1 shows details of the stock viruses used in the preparation of Shellfish Samples 3 and 4 and LENTICULE 1. Table 1: Origin and strain/genotype of viruses used for shellfish bioaccumulation and preparation of test sample LENTICULES Description Source Strain ID/genotype Hepatitis A virus Cell culture supernatant HM175/43c Norovirus genogroup I Faecal material GI.7 (based on capsid sequence) Norovirus genogroup II Faecal material GII.4 (based on capsid sequence) Sample distribution Samples were dispatched on dry ice in accordance with IATA packing instructions 650 for UN3373 Diagnostic Specimens on 5 th September 2016 or shortly thereafter to 47 participating laboratories. Participants were requested to analyse the test samples using their routine method. Those laboratories using quantitative real-time RT-PCR were requested to calculate the quantity of target virus in each sample using both their own standard material and using the dsdna control solutions provided with this PT distribution. Results Reference results Reference analyses were performed by the EURL on samples stored at <-15 C. For shellfish and LENTICULE samples; six randomly selected samples from each sample type were extracted in duplicate and qrt-pcr (TaqMan ) was carried out using triplicate PCR reactions for each RNA extract and each target. Reference results for each sample are shown in Table 2, with box and whisker plots included in Appendix I. Table 2: Reference results for PT 61 proficiency testing material Sample Norovirus HAV GI GII Shellfish Sample 1 a + (2.99 x x 10 3 ) + (7.17 x x 10 3 ) - Shellfish Sample 2 a Shellfish Sample 3 a - + (6.69 x x 10 3 ) + (7.29 x x 10 3 ) Shellfish Sample 4 a + (1.66 x x 10 3 ) Not attributed c + (4.78 x x 10 3 ) LENTICULE 1 b + (2.57x x 10 3 ) + (6.41 x x 10 2 ) + (1.04 x x 10 4 ) LENTICULE 2 b a Copies / g; b Copies / LENTICULE; c Reference analyses indicated the potential presence of Norovirus GII RNA at extremely low concentrations in this sample. Results were therefore not considered in performance scoring. Note: Ranges based on a 95% confidence limit determined as 2 geometric standard deviations above and below the geometric mean. Proficiency testing 61 Final 1 Page 3 of 21

57 Participants results Participant s results are tabulated in Appendices II, III and IV, and quantitative results are shown in graphical form alongside the reference values in Appendix V. Performance scoring Presence/absence Performance scoring was undertaken on participant s presence/absence results for Shellfish Samples and test sample LENTICULES separately (Tables 3 and 4). A single score for NoV GI and GII analysis was determined, but HAV was considered separately. Scores were assigned as follows: A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Quantification For those participants reporting quantitative data, an additional performance assessment was carried out using an approach based on ISO/TS (Anon 2010; detailed in Appendix VI). Performance scoring was undertaken on quantitative results for Shellfish Samples and test sample LENTICULES separately (Tables 3 and 4), only for sample/target virus combinations where the intended result was positive. For maximum information, separate scores were calculated for all three target viruses separately (NoV GI, NoV GII, HAV). Graphs in Appendix V include lines showing the boundaries of the acceptable and questionable ranges for each sample/target matrix combination. Proficiency testing 61 Final 1 Page 4 of 21

58 Table 3: Performance scoring for shellfish samples Lab ID No. Presence / Absence Quantification Lab ID Presence / Absence Quantification NoV HAV NoV GI NoV GII HAV No. NoV HAV NoV GI NoV GII HAV 3 * A A A A A 94 A A A A A 7 * A A A C A 95 B A 9 * A NE A A 96 A A A A A 10 * A A A A A 98 NR NR 17 * A A A A A 113 C C 19 * B A C A A 147 * A A 20 A A 158 A A 21 * A A A A A 161 C NE 22 * C NE A A 176 A A 24 A A A A 177 A A 25 * A A A A C 183 B A 27 * A A A A A 186 NE A A 32 * A A A A A 190 A A A A A 33 * A A A A A 193 C A C C A 35 * C A 203 A A A A A 39 * A A A A A 209 A A A A C 41 * A A A A A 214 C A 42 * C NE 225 B A C A A 47 * A A B A A 227 A A A A A 48 A A A A A 232 A A A A C 53 A A 239 A A A A A 57 A A A A A 245 B A A C A 72 NR NR 246 NE NE 90 * A A A A A * = Designated NRL, NE= Not examined, NR = Results not returned. Norovirus (NoV) and HAV scored separately for presence/absence. NoV GI, NoV GII and HAV scored separately for quantification. Presence/absence performance scoring; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Quantification performance scoring; A = satisfactory (all results fall within ±2 δmad of participants median), B = questionable (no more than one result falls outside ±2 δmad of participants median, and no result falls outside ±2.58δMAD of participants median or reported as negative), C = unsatisfactory (two or more results fall outside ±2 δmad of participants median, or one or more result falls outside ±2.58 δmad of participants median or reported as negative). Proficiency testing 61 Final 1 Page 5 of 21

59 Table 4: Performance scoring for LENTICULE TM samples Lab ID No. Presence / Absence Quantification Lab ID Presence / Absence Quantification NoV HAV NoV GI NoV GII HAV No. NoV HAV NoV GI NoV GII HAV 3 * A A A A A 94 A A A B A 7 * A A A C A 95 B A 9 * A NE A A 96 A A A A A 10 * A A A C A 98 NR NR 17 * A A A A A 113 NE NE 19 * A A A A A 147 * A A 20 A A 158 A A 21 * A A A A A 161 A NE 22 * B NE A A 176 A A 24 A B A A 177 B A 25 * B A A A A 183 A A 27 * A A C B A 186 NE A A 32 * A A C C A 190 A A A A A 33 * A A A A A 193 B A A A A 35 * A A 203 A A A A A 39 * A A A A A 209 A A B A A 41 * A A B A A 214 A A 42 * B NE 225 A A A A A 47 * A A A B A 227 A A A A A 48 A A A C A 232 A B A A C 53 A A 239 B A A C A 57 A A A A A 245 B A A A A 72 NR NR 246 NE A 90 * B A B A A * = Designated NRL, NE= Not examined, NR = Results not returned. Norovirus (NoV) and HAV scored separately for presence/absence. NoV GI, NoV GII and HAV scored separately for quantification. Presence / absence performance scoring; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Quantification performance scoring; A = satisfactory (all results fall within ±2 δmad of participants median), B = questionable (no more than one result falls outside ±2 δmad of participants median, and no result falls outside ±2.58δMAD of participants median or reported as negative), C = unsatisfactory (two or more results fall outside ±2 δmad of participants median, or one or more result falls outside ±2.58 δmad of participants median or reported as negative). Proficiency testing 61 Final 1 Page 6 of 21

60 Discussion Forty-seven laboratories (19 NRLs and 28 other labs) received samples. Two labs (72 and 98) did not return results. Amongst the 45 labs that returned results, Labs 9, 22, 42 and 161 did not examine for HAV and Labs 186 and 246 did not examine for NoV. Lab 113 tested only the shellfish samples while Lab 246 only examined the LENTICULE TM samples (for HAV only). Methods used by participants to analyse the test samples are shown in Appendix VII, while brief details of the types of materials used as quantification standards are included as Appendix VII. Presence/absence determination Twenty-seven laboratories (60% of those returning results) obtained the intended presence/absence result (as determined by EURL reference designations) for all sample/target virus combinations that they tested. Of these, 24 labs tested all samples for all target viruses. Shellfish samples Thirty-two laboratories (73% of those testing shellfish samples) obtained the intended presence/absence result (as determined by the EURL reference samples) for each shellfish sample/target virus combination tested. For NoV (GI and GII combined), 31 labs (72% of those testing shellfish samples for NoV) achieved a satisfactory performance score for presence/absence determination, with 5 and 7 laboratories obtaining questionable and unsatisfactory performance scores respectively. A total of 23 false negative results were reported by 11 different laboratories (overall false negative reporting rate was 13%); three laboratories (42, 113, 161) reported false negative results for all sample/target virus combinations intended as positive. A total of 3 false positive results were reported by two different laboratories (overall false positive reporting rate was 2%). For HAV, 39 labs (98% of those testing shellfish samples for HAV) achieved a satisfactory performance score for presence/absence determination with only one lab obtaining an unsatisfactory performance score due to two false negative results (equivalent to an overall false negative reporting rate of 3%). No false positive results for HAV were reported. LENTICULE TM samples Thirty-three laboratories (75% of those testing LENTICULE TM samples) obtained the intended presence/absence result (as determined by the EURL reference samples) for each LENTICULE TM sample/target virus combination tested. For NoV (GI and GII combined), 33 labs (79% of those testing LENTICULE TM samples for NoV) achieved a satisfactory performance score for presence/absence determination, with 9 laboratories obtaining a questionable performance score. Two labs reported a single false negative result while a 7 labs reported a single false positive result (overall false negative and false positive reporting rates were 2% and 8% respectively). For HAV, 38 labs (95% of those testing LENTICULE TM samples for HAV) achieved a satisfactory performance score for presence/absence determination with two labs obtaining a questionable score due to a false negative result (equivalent to an overall false negative reporting rate of 5%). No false positive results for HAV were reported. Proficiency testing 61 Final 1 Page 7 of 21

61 Quantification A total of thirty-one labs (69% of labs returning results) reported quantitative data for at least one sample/target virus combination. 14 labs reported results in the acceptable range determined according to the procedure in Appendix VI for all sample/target virus combinations that they tested, including one lab that reported false positive results for negative samples (these samples were not considered for quantification performance scoring). Six laboratories that had each achieved all satisfactory performance scores for presence/absence of target viruses reported results in the acceptable range for all shellfish sample/target virus combinations, but reported at least one LENTICULE TM sample result outside the acceptable range (resulting in a questionable or unsatisfactory score for one target virus in LENTICULE TM samples). References Codd AA, Richardson IR, Andrews N Lenticules for the control of quantitative methods in food microbiology. J Appl Microbiol. 85(5): Anon 2013 ISO/TS :2013 Microbiology of food and animal feed -- Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR -- Part 1: Method for quantification. Anon 2010 ISO/TS Microbiology of food and animal feeding stuffs Specific requirements and guidance for proficiency testing by interlaboratory comparison. Proficiency testing 61 Final 1 Page 8 of 21

62 Copies / g Copies / LENTICULE Copies / g Copies / g Appendix I: EURL reference results displayed as box and whisker plots (log scale) of detectable genome copies per gram or 25μl LENTICULE. Shellfish sample 1 Shellfish sample GI GII GII HAV Shellfish sample 4 LENTICULE TM sample 1 Boxplot of GI, HAV GI HAV GI GII HAV Proficiency testing 61 Final 1 Page 9 of 21

63 Appendix II: Participants results and C t values for shellfish samples a = results for this sample/target virus combination not attributed, reference analyses indicated the presence of extremely low concentrations of NoV GII RNA *= Designated EU member state/efta NRL, NE= sample/target virus combination not examined, NR= results not returned, red shading denotes false positive, yellow shading denotes false negative results Proficiency testing 61 Final 1 Page 10 of 21

64 Appendix III: Participants results and C t values for test sample LENTICULES TM *= Designated EU member state/efta NRL, NE= sample/target virus combination not examined, NR= results not returned, red shading denotes false positive, yellow shading denotes false negative results Proficiency testing 61 Final 1 Page 11 of 21

65 Appendix IV: Participants reported quantities for each target (copies/g [shellfish]) or copies/lenticule TM ) *= Designated EU member state/efta NRL, A = Quantity determined using laboratory s own standard materials B = Quantity determined using standard material provided by EURL. Labs which did not report any quantities are not included. Proficiency testing 61 Final 1 Page 12 of 21

66 Appendix V: Participants and reference quantities for each sample. NOTE: where quantities are reported using both the laboratory s own quantification standards, and those provided by the EURL, only those using the lab s own standards are considered for performance scoring. Shellfish sample 1 GI Shellfish sample 1 GII Proficiency testing 61 Final 1 Page 13 of 21

67 Shellfish sample 3 GII Shellfish Sample 3 HAV Proficiency testing 61 Final 1 Page 14 of 21

68 Shellfish Sample 4 GI Shellfish Sample 4 HAV Proficiency testing 61 Final 1 Page 15 of 21

69 LENTICULE 1 - GI LENTICULE 1 - GII Proficiency testing 61 Final 1 Page 16 of 21

70 LENTICULE 1 HAV Proficiency testing 61 Final 1 Page 17 of 21

71 Appendix VI : Performance scoring for quantification data Performance scoring was undertaken following the median absolute deviation from the median (MAD) approach described in ISO/TS Microbiology of food and animal feeding stuffs specific requirements and guidance for proficiency testing by interlaboratory comparison (ISO 2010). The MAD approach is recommended for assessment of proficiency testing data where less than 50 participants return quantitative results and/or for new proficiency assessment. Where laboratories submitted quantitative results determined using both their own quantification standards, and those provided by the EURL with the other PT materials, only the results using their own standards were considered for performance scoring; however, where laboratories submitted quantitative results using the EURL standards only, these were considered. For each sample/target virus combination where the intended result was positive, a statistically robust acceptability range was determined by calculation of the median absolute deviation (MAD) of each participant s result from the median of all participant s results. This figure was then multiplied by a constant (1.4826) to obtain a robust estimate of the standard deviation (δ MAD). For each individual result, its absolute deviation from the participant s median was compared with the calculated δ MAD to determine its acceptability as follows:- Difference between result and participants median <2 δ MAD = result within acceptable range Difference between result and participants median >2 δ MAD and <2.58 δ MAD = result within questionable range Difference between result and participants median >2.58 δ MAD = result outside acceptable/questionable range Result reported as negative = result outside acceptable/questionable range For each set of results assessed, if all results were within the acceptable range, the laboratory s overall score was recorded as satisfactory (A), if no more than one result was within the questionable range and no results were outside the acceptable/questionable range, it was recorded as questionable (B) and if two or more results were within the questionable range, or one or more results were outside the acceptable/questionable range, it was recorded as unsatisfactory (C). Proficiency testing 61 Final 1 Page 18 of 21

72 Appendix VII: Results and methods used for shellfish samples. (For key to method codes see page 20) LAB ID SF1 SF2 SF3 SF4 Extraction RT-PCR Primers GI GII HAV GI GII HAV GI GII HAV GI GII a HAV Virus RNA method reagents GI GII HAV 3* A B H K AA-1 AA AA 7* A B H K AA-2 b AA b AA b 9* + + NE - - NE - + NE + - NE A B H K AA-1 AA - 10* A B H K AA-1 AA AA 17* A B H K AA-1 AA AA 19* A B H K AA-2 AA AA A B H L BB BB BB 21* A B H K AA-2 AA AA 22* + + NE + - NE + + NE + - NE A B H K AA-1 AA A C J d M CC DD FF 25* A B H K AA-2 AA AA 27* A B H N AA-1 AA AA 32* A B H K AA-1 AA AA 33* A B H K AA-2 AA AA 35* A B H O AA-2 AA AA 39* A B H K DD DD AA 41* A B H K AA-2 AA AA 42* - - NE - - NE - - NE - - NE A D H P AA-1 AA - 47* A B H O AA-1 AA AA A B H K AA-1 AA AA A B H K AA-2 AA AA A B H K AA-2 AA AA 90* A B H O AA-2 AA AA A B H L BB BB BB A E J c Q DD DD GG A B H K AA-2 AA AA A B H R AA-2 AA BB 147* A B H S EE EE BB A B H L BB BB BB NE - - NE - - NE - - NE A F H O AA-2 AA A B H L BB BB BB A B H K AA-2 AA AA A E H L BB BB BB 186 NE NE - NE NE - NE NE + NE NE + A B H L - - BB A B H L BB BB BB A B H L BB BB BB A B H T AA-1 AA AA A E J U AA-1 AA AA A H L BB BB BB A C H V AA-2 AA AA A B H L BB BB BB A B H W AA-2 AA AA A G H L BB BB BB A B H K AA-1 AA AA 246 NE NE NE NE NE NE NE NE NE NE NE NE - B H K - - AA *= Designated EU member state/ EFTA NRL, NE = sample/target virus combination not examined, red shading denotes false positive, yellow shading denotes false negative results. Grey shading of method elements indicates method is the same as or very similar to methods in the normative and informative parts of ISO/FDIS a b c d results for this sample/target virus combination not attributed, reference analyses indicated the presence of extremely low concentrations of NoV GII RNA IAC assay run as multiplex with target assays conventional two-step used for HAV only both real-time and conventional two-step used for HAV Proficiency testing 61 Final 1 Page 19 of 21

73 Key to method codes Virus extraction methods A Proteinase K digestion RNA extraction methods B C D E F G NucliSens Magnetic extraction reagents (BioMerieux) High Pure Viral RNA Kit (Roche) PureLink Viral RNA/DNA mini Kits (Invitrogen) QIAamp/Rneasy kits (Qiagen) MagJET Viral DNA and RNA Kit (Thermo Scientific) MagMAX isolation kit (Thermofisher) RT-PCR methods H J Real-time one-step Real-time two-step RT-PCR reagents K L M N O P Q R S T U V W RNA Ultrasense (Invitrogen) ceeram Tools Superscript III (RT) & Platinum qpcr SuperMix-UDG (Invitrogen) TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems) Quantitect/Quantifast RT-PCR kits (Qiagen) AgPath-ID One-Step RT-PCR Reagents (Applied biosystems) High Capacity cdna RT Kit & Taqman Universal Mastermix (Applied Biosystems) NoV; LightCycler 480 RNA Master Hydrolysis Probes (Roche). HAV; Ceeram Tools NoV; SureFast Norovirus PLUS Kit (R-biopharm) for detection, Ceeram Tools for genogroup identification. HAV; Ceeram Tools Platinum quantitative RT-PCR Thermoscript One-step system (Invitrogen) High-Capacity cdna Reverse Transcription Kit (Applied Biosystems) & LightCycler 480 Master Hydrolysis Probes (Roche) GoTaq Probe 1-Step RT-qPCR System (Promega) NoV; ID Gene Norovirus Duplex (IDvet Genetics). HAV; Ceeram Tools Primers/probes AA BB ISO/FDIS ; 1) with TM9 probe for nov GI; 2) with NVGG1p probe for nov GI Ceeram Tools (sequences as AA-2) CC Wolf et al, (2010) DD Kageyama et al, (2003) EE FF GG SureFast Norovirus PLUS Kit & Ceeram Tools (sequences as AA-2) ISO/FDIS (real-time); HAVnet (conventional) FDA/BAM Chapter 26 Detection and Quantification of Hepatitis A virus in Shellfish by the Polymerase Chain Reaction Proficiency testing 61 Final 1 Page 20 of 21

74 Appendix VIII: Details of laboratory's own quantification standards (preparation and quantification) LAB ID 3* linearised TAG4 plasmid DNA, quantified using fluorimetry 7* no details provided 9* EURL standards; PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry 17* linearised TAG4 plasmid DNA, quantified using A260 spectrophotometry 19* EURL standards; PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry 24 circular plasmid, quantified using fluorimetry 32* EURL standards; PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry 41* synthetic linear dsdna, quantified by supplier, confirmed by fluorimetry and digital PCR 47* NoV; EURL standards - PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry. HAV; circular TAG4 plasmid 57 circular plasmid, quantified using A260 spectrophotometry 90* EURL standards; PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry 94 Ceeram standards; linear dsdna quantified using fluorimetry 96 linearised TAG4 plasmids, quantified using A260 spectrophotometry and fluorimetry 190 Ceeram standards; linear dsdna quantified using fluorimetry 203 EURL standards; PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry 209 PCR product amplified from faecal sample, quantified using A260 spectrophotometry 225 RNA, quantified by A260 spectrophotometry 227 Ceeram standards; linear dsdna quantified using fluorimetry 232 circular plasmid DNA quantified using fluorimetry 245 NoV; EURL standards - PCR product amplified from TAG4 plasmid, quantified using A260 spectrophotometry. HAV; standards provided by a 3rd party laboratory Proficiency testing 61 Final 1 Page 21 of 21

75 Annex V European Union Reference Laboratory (EURL) Proficiency Testing Scheme Enumeration of Escherichia coli and the detection of Salmonella spp. in bivalve molluscan shellfish EURL PT reference number: PT 64 Final report version pages Contract Reference: Cefas ref (C6397) Document approved by: C6397 Project Manager James Lowther Review date: Not applicable Document checked by: James Lowther Classification: Official Document prepared by: Louise Stockley Location PT CRL$

76 -Contents Page number Sample preparation 2 Results 2 General comments 6 References 7 Result charts 8 Appendices 10 This scheme is intended to provide proficiency testing samples for laboratories undertaking examination of live bivalve molluscs from production areas in accordance with Regulation (EC) No. 854/2004 and from throughout the production chain in accordance with Regulation (EC) No. 2073/2005. The scheme is organised by the European Union Reference laboratory (EURL) for monitoring bacteriological and viral contamination of bivalve molluscs. The EURL is designated by the European Union in accordance with Regulation (EC) No. 882/2004. The scheme is intended to compliment the EURL/PHE Shellfish Scheme through examination of aspects of the methods not covered under the Shellfish Scheme (initial sample preparation and preparation of initial dilutions) ( encytesting/eqaptforfoodwaterandenvironmentalmicrobiology/shellfishscheme/ and to provide additional data for laboratories for ISO accreditation purposes. The EU stipulated reference method for enumeration of E. coli in live bivalve molluscs in ISO TS , Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β- glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4- chloro-3-indolyl-β-d-glucuronide (Anon 2005). The EU reference method for detection of Salmonella spp. in live bivalve molluscs is ISO 6579, Microbiology of food and animal feeding stuffs Horizontal method for the detection of Salmonella spp. (Anon 2002). A scoring system is used to help assess participants performance. Details of this system are included as Appendix I of this report. The purpose of scoring is to help the EURL, NRLs and other participating laboratories identify incorrect or outlying results. Further information on the use of scoring in proficiency testing and on recommended procedures for following up poor performance can be accessed via the EURL website ( or obtained by contacting the EURL. The European Union has produced a protocol for management of underperformance in comparative testing and/or lack of collaboration of NRLs with EURLs activities. If you are experiencing problems with any aspects of these distributions please contact the EURL (contact details below), or alternately refer to the troubleshooting guide included as Appendix II of this report. Further advice on microbiological testing of bivalve mollluscan shellfish can be obtained via the EURL website ( Due to the nature of this scheme repeat samples are not available. Proficiency testing 64 Final Page 1 of 12

77 Sample preparation Samples 1 and 2 A single batch of approximately 1500 Pacific oysters (Crassostrea gigas) was collected from a UK commercial harvesting area at 8:30am on the 21 st November 2016 and was placed in a large sterile container before being thoroughly mixed. Sample 1 and sample 2 provided to participating laboratories comprised of 15 randomly selected Pacific oysters from this bulk material. Sample 3 A single batch of approximately 400 Pacific oysters (Crassostrea gigas) was collected from a UK commercial harvesting area on the 17 th November On arrival, the Pacific oysters were shucked and homogenised before being pooled together to form one homogenate. Sample 3 was aliquoted in 100 ml volumes on the 20 th November. Prior to distribution sample 3 was spiked with estimated levels of E. coli and Salmonella spp.. Sample distribution and examination Each individual sample was packed in accordance with the Cefas protocol for packaging shellfish for transportation. Samples were collected at 11:00am on the 21 st November 2016 to 39 participating laboratories. Participants were requested to analyse the samples immediately on receipt using their routine methods. Sample 1 and 3 were examined for E. coli and samples 2 and 3 were examined for Salmonella spp. Sample temperature Participants were requested to record the internal sample temperature on arrival. Temperatures recorded by participants are shown in Appendix I. Results Reference results - E. coli Ten randomly selected samples were analysed in duplicate on 2 consecutive days ( and ) for E. coli using EURL SOP No (Table 1). Sample homogeneity was assessed following the procedure described in ISO The sample material was considered sufficiently homogenous. Table 1: E. coli MPN/100g reference results Sample and type Sample 1 - Oysters Date of Analysis Range Median GM Median ±3*SD T x x x x x x x x x x x x 10 2 Sample x x x x x x 10 4 Homogenate x x x x x x 10 3 GM - geometric mean, SDT - theoretical standard deviation (0.24 log10) Reference results Salmonella spp. Ten randomly selected samples were analysed on 2 consecutive days ( and ) for Salmonella spp. using EURL SOP No (Table 2). Note: Regulation (EC) No. 2073/2005 requires presence/absence testing for Salmonella spp. in live bivalve molluscs Proficiency testing 64 Final Page 2 of 12

78 Table 2: Reference results Sample and type Date of Analysis Salmonella spp. No. of replicates giving the expected result Sample 2 - Oysters Absent in 25g Absent in 25g 7 1 Sample Present in 25g 10 Homogenate Present in 25g 10 1 Salmonella spp. was detected in a proportion of the reference samples examined. Participants results Performance assessment was according to the procedures described in the EURL/PHE EQA shellfish scheme for a single distribution, with minor modifications (Appendix II). Participants results and scores allocated for PT 64 are shown in Tables 3, 4, 5 and Figure 1. Note: The median and upper and lower limits (±3 SD and ±5 SD) were calculated from participants results. SDT calculations were based on the inherent variability of the 5 x 3 MPN method (0.24 log10). Reference values were excluded from the calculation of the participants median. Table 3: Participants results GM - geometric mean, SDT theoretical standard deviation (0.24) Sample and type E. coli MPN/100g Range Median GM Median±3*SD T Sample 1 - Oysters <2.0 x x x x x x 10 2 Sample 3 - Homogenate 1.7 x x x x x x 10 4 Table 4: Summary statistics of participants results E. coli Sample 1 - Sample 3 - Oysters Homogenate Participants reporting duplicate results for E. coli MPN Participants reporting a single MPN result 0 0 Participants reporting MPN results within the expected range for both replicates Participants reporting MPN results outside the expected range for one replicate 3 0 Participants reporting MPN results outside the expected range for both replicates 2 1 Participants reporting MPN results as censored results for one replicate 1 0 Participants reporting MPN results as censored results for both replicates 1 0 Participants reporting tube combination and / or MPN results inconsistent with ISO 7218 (Anon 2007) expected range = participants median ± theoretical 3SD. 2 points deducted from participants returning results inconsistent with ISO Proficiency testing 64 Final Page 3 of 12

79 Table 5: Participants results and allocated scores for whole animal samples Sample 1 Sample 2 a Sample 1 Sample 2 a Lab Lab E. coli MPN/100g Sal. spp. in 25g E. coli MPN/100g Sal. spp. in 25g ID ID Rep 1 Rep 2 Score Rep 1 Score Rep 1 Rep 2 Score Rep 1 Score 3 * Not Detected - 54 b Not Detected - 7 * Not Detected - 68 * Present - 9 * Not Detected - 69 * Not Detected - 10 * Not Detected Not Detected - 13 * Not Detected - 83 * Not Detected - 17 * Not Detected - 90 * Not Detected - 19 * Not Detected Not Detected - 22 * Not Detected - 98 b Not Detected - 23 * Not Detected Not Detected - 27 * Not Detected * b Not Detected - 32 * b Not Detected Not Detected - 33 * Present * Not Detected - 35 * Not Detected * d <200 <200 8 NE - 39 * Not Detected Not Detected - 41 * Not Detected * Not Detected - 42 * Not Detected Not Detected - 43 * Not Detected * d NE - 44 * b Not Detected b c 130 <20 3 Not Detected - 47 * Not Detected * Not Detected Not Detected - * Designated NRL s, a No scores allocated as Salmonella spp. was detected 4/20 replicate reference samples. b Scores deducted as tube combination inconsistent with rules specified in ISO c Score deducted as incorrect MPN value given for recorded tube combination. d MPN tube combination is not required for this method, the maximum overall score is reduced to reflect this (8). NE Not examined. Proficiency testing 64 Final Page 4 of 12

80 Table 6: Participants results and allocated scores for homogenate sample Sample 3 Sample 3 Lab Lab E. coli MPN/100g Sal. spp. in 25g E. coli MPN/100g Sal. spp. in 25g ID ID Rep 1 Rep 2 Score Rep 1 Score Rep 1 Rep 2 Score Rep 1 Score 3 * Present 2 54 a Present 2 7 * Present 2 68 * Present 2 9 * Present 2 69 * Not Detected 0 10 * Present Present 2 13 * Present 2 83 * Present 2 17 * Present 2 90 * Present 2 19 * Present 2 96 b Present 2 22 * Present 2 98 a Present 2 23 * Present d NE NE - NE - 27 * Present * a b Present 2 32 * Present Present 2 33 * Present * Present 2 35 * Present * c NE - 39 * Present Present 2 41 * Present * Present 2 42 * Present Present 2 43 * Present * c NE - 44 * a Present a Present 2 47 * Present * Present Present 2 * Designated NRL s, a Scores deducted as tube combination inconsistent with rules specified in ISO b Score deducted as incorrect MPN value given for recorded tube combination. c MPN tube combination is not required for this method, the overall maximum score is reduced to reflect this (8). d On receipt of the laboratory results, it was noted that no results were recorded for Sample 3. The EURL contacted the laboratory and was informed that sample 3 was not included in the box. NE Not examined. Proficiency testing 64 Final Page 5 of 12

81 General comments Thirty-nine laboratories (29 NRLs and 10 other laboratories) were sent material with all laboratories returning results. Information provided by laboratories on the arrival time of the material showed that 90% (35) of laboratories received the material the day after dispatch ( ), with 51% (18) of these laboratories analysing the material on arrival. All laboratories received material within 48 hours of dispatch. In total 56% (22) of laboratories analysed the material on the same day as receiving the material, with 44% (17) analysing the material one day after receipt. Arrival temperatures were recorded in the range of C. All temperature data, arrival and analysis dates and times recorded by participants are shown in Appendix I. Sample analyses Thirty-nine laboratories returned the report form for this PT distribution. Laboratory 100 did not examine sample 3 for E. coli or Salmonella spp. because the sample was not included in the dispatched box. For this reason, no score was given for this sample. The EURL recommends that all laboratories read the accompanying note before performing any analysis to ensure the correct samples and paperwork have been included in the dispatched box. Sample 1 Oysters (E. coli only) Thirty-four laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants median with 31 laboratories (91%) obtaining full marks. Laboratories 7 and 42 reported 1 replicate and Laboratory 54 reported both replicate results between ±3 and ±5 SD of the participants median. Laboratory 235 reported 1 replicate result outside ±5 SD of the participants median. Laboratory 102 reported 1 replicate falling between ±3 and ±5 SD and the other falling outside ±5 SD of the participants median. Six laboratories (laboratories 32, 44, 54, 98, 102 and 235) were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables or for the reporting of an incorrect MPN value for the reported tube combination. Participants are reminded that for official control testing of live bivalve molluscs in the EU, the 5 x 3 MPN tables or MPN calculator in ISO7218:2007/Amd1:2013 and the EURL generic protocol for enumeration of E. coli in bivalve molluscs (Issue 13) should be used. Sample 2 Oysters (Salmonella spp. only) Ten replicate samples were analysed on consecutive days during the testing period. It was noted that presumptive colonies of Salmonella spp. were detected in 4 of the 20 replicate samples. Further identification using whole genome sequencing and multi locus sequence typing was carried out on the isolates and it was confirmed that the Salmonella strains isolated had originated naturally in the sample. Previous studies at the UK NRL estimated that the limit of detection of ISO 6579 in seeded bivalve shellfish matrix is 4 CFU/25g. However, these data were not established using naturally contaminated samples and the potential effects of sample transit were not considered. For this reason, participants Salmonella results were not allocated a score for this sample. Sample 3 Homogenate (E. coli and Salmonella spp. only) Thirty-seven laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants median with 32 laboratories (86%) obtaining full marks. Laboratory 102 reported both replicate results between ±3 and ±5 SD of the participants median. Six laboratories (laboratories 44, 54, 96, 98, 102 and 235) were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables and the reporting of Proficiency testing 64 Final Page 6 of 12

82 incorrect MPN value for the reported tube combination. Participants are reminded that for official control testing of live bivalve molluscs in the EU, the 5 x 3 MPN tables or MPN calculator in ISO7218:2007/Amd1:2013 and the EURL generic protocol for enumeration of E. coli in bivalve molluscs (Issue 13) should be used. Thirty-six laboratories returned results for Salmonella spp. with 35 correctly reporting the presence of Salmonella spp. in sample 3 and received a score of 2. Laboratory 69 did not detect Salmonella spp. in the sample and received a score of 0. Summary Twenty-nine laboratories (74%) achieved full marks for both samples tested for the enumeration of E. coli. For this distribution the EURL recommended participants to analyse the sample with 4 dilutions. Laboratories who did not follow the advice given in ISO7218:2007/Amd1:2013 and/or the EURL generic protocol for calculating the MPN value for E. coli incurred a deduction (7 laboratories). Laboratories are requested to note from ISO ISO7218:2007/Amd1:2013 that In any circumstance when more than three dilutions are made, it is essential that all measured data values be used. It is not scientifically correct to "select" any combination of values on the premise that these values are more "correct" than other combinations. The results from all possible combinations of positive tubes should be recorded and the MPN calculator ( used to derive MPN values. Those laboratories who achieved <40% of the maximum possible score in this distribution for E. coli enumeration (<5 out of the maximum 12 score) and / or Salmonella spp. detection (score of 0) should review their laboratory procedures. In the first instance refer to the troubleshooting guide included as Appendix III. However, further guidance is available from the EURL. References Anon ISO TS Microbiology of food and animal feeding chain - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5- bromo-4-chloro-3-indolyl-β-d-glucuronide. Geneva, Switzerland. Anon ISO Microbiology of food and animal feeding stuffs Horizontal method for the detection of Salmonella spp. Geneva, Switzerland. Anon ISO 7218:2007/FDAM 1:2013. Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations - Amendment 1. International Organization for Standardization, Geneva. Anon ISO TS Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 2: Colony-count technique at 44 C using 5-bromo- 4-chloro-3-indolyl-β-D-glucuronide. Geneva, Switzerland. Anon ISO 22117:2010. Microbiology of food and animal feeding stuffs Specific requirements and guidance for proficiency testing by interlaboratory comparison. Geneva, Switzerland. Proficiency testing 64 Final Page 7 of 12

83 Figure 1: Results chart sample 1 Pacific oysters Proficiency testing 64 Final Page 8 of 12

84 Figure 2: Results chart sample 3 shellfish homogenate Proficiency testing 64 Final Page 9 of 12

85 Appendix I Sample arrival and temperature Lab ID Date arrived Time of arrival Sample ( C) Storage ( C) Date analysed 3 * 23/11/ : /11/ * 22/11/ : ± 2 23/11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : ± 2 23/11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : ± 2 22/11/ * 22/11/ : /11/ * 22/11/ : /11/ * 22/11/ : ± 2 23/11/ * 22/11/ : /11/ /11/ : /11/ /11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ /11/ : /11/ * 23/11/ : /11/ * 23/11/ : /11/ /11/ : ± 2 23/11/ /11/ : /11/ /11/ : ± 2 23/11/ * 22/11/ : /11/ /11/ : /11/ * 22/11/ : /11/ * 22/11/ : /11/ /11/ : /11/ * 22/11/ :25 2 ± 3 2 ± 3 23/11/ /11/ : /11/ * 22/11/ : /11/ /11/ : /11/ * 22/11/ : /11/2016 * Designated NRLs Proficiency testing 64 Final Page 10 of 12

86 Appendix II: E. coli MPN scores allocated to participants returning 2 replicate results Returning Score allocated Result of results Replicate 1 Replicate 2 Total score Both replicate MPN results are within the expected range One replicate MPN result is outside the expected range and falls between the median ±3SD and median ±5SD values Both replicates MPN results are outside the expected range and fall between the median ±3SD and median ±5SD values One replicate MPN result is outside the median ±5SD value Both replicates MPN results are outside the expected range. The first falls between the median ±3SD and median ±5SD value and the second falls outside the median ±5SD values. Both replicates MPN results reported is outside the median ±5SD value E. coli MPN scores allocated to participants returning 1 single replicate result Result Returning of results Score allocated Total score Single replicate MPN result is within the expected range Single replicate MPN result is outside the expected range and falls between the median ±3SD and median ±5SD values Single replicate MPN result reported is outside the median ±5SD value E. coli score deductions Result Tube combination inconsistent with MPN reported and / or tube combination selected not consistent with rules given in ISO 7218:2007/Amd 1:2013 or MPN tables provided by the NRL Score deducted Replicate 1 Replicate High censored result (e.g. MPN = >18000 per 100g) 2 2 Sample not examined or results returned late - no explanation received 12 Salmonella spp. scoring Result Score allocated Fully correct results 2 Misleading result, e.g. failure to isolate Salmonella 0 Proficiency testing 64 Final Page 11 of 12

87 Appendix III: Troubleshooting advice 1. Methods Ensure that the method used is appropriate for the examination of the sample. a. Ensure that any dilutions have been calculated correctly. b. Ensure that the dilutions analysed are as specified on the report form. c. Ensure that MPN tables (if used) are interpreted correctly. Interpretation of MPN tables Where three dilutions have been tested for a sample, record the number of TBGA/TBX positives for each dilution to give a three figure tube combination number. Use the MPN tables included in ISO 7218 and the EURL generic E. coli protocol. Only category 1 or 2 tube combinations are included in the tables and should be reported. Where more than three dilutions have been tested for a sample, use the Excel spreadsheet MPN calculator ( to determine the MPN from all the dilutions tested. Combinations that do not appear in the tables or obtained from the Excel calculator as category 3 are not acceptable and should not be used. If the tube combination result is an unacceptable combination, the result is reported as void. 2. Culture media - Check the quality control data for media to ensure that they are within specifications and performing adequately. 3. Equipment - Check that the equipment used for the procedures (incubators, refrigerators, measuring instruments) are calibrated and performing adequately. 4. Staff training - Check that the staff performing the tests are fully trained and familiar with all the procedural steps. 5. Clerical procedures - Check that the sample labeling, laboratory numbering and clerical procedures are adequate and that you have procedures for ensuring that test results are reported accurately and on time. 6. Accreditation- Check that quality procedures are documented and adhered to at all times. 7. Internal quality controls (IQC) Ensure adequate controls are in place and follow-up procedures are in place to deal with IQC failures. Further advice can be obtained from the EURL on request. Proficiency testing 64 Final Page 12 of 12

88 Annex VI European Union Reference Laboratory (EURL) Proficiency Testing Scheme Noroviruses and hepatitis A virus EURL PT reference number: PT 65 Final report version pages Contract Reference: Cefas ref (C6927A) Document approved by: C6472A1 Project Manager James Lowther Review date: n/a Document checked by: James Lowther Classification: Official Document prepared by: Louise Stockley Location

89 Contents Page number Samples 2 Results 2 Conclusion and discussion 4 References 4 Appendices 5 PT 65 Final report version 1 Page 1 of 14

90 Samples Material dispatched consisted of two laboratory constructed LENTICULES. Table 1 shows details of the stock viruses used in the preparation of the LENTICULES. Table 1: Origin and strain/genotype of viruses used for preparation of LENTICULE sample Description Source Strain ID/genotype Hepatitis A virus Cell culture supernatant HM175/43c Norovirus genogroup I Faecal material GI.4 (based on capsid sequence) Norovirus genogroup II Faecal material GII.4 (based on capsid sequence) Sample preparation Two batches of laboratory constructed LENTICULES were prepared following the method of Codd et al (1998) with minor modifications. The mix prepared for LENTICULE 1 included known levels of GI norovirus from human faeces, while the mix for LENTICULE 2 included known levels of GII norovirus from human faeces and HAV cell culture supernatant. Sample distribution Samples were dispatched in accordance with IATA packing instructions 650 for UN3373 Diagnostic Specimens on 4 th July 2016 to 43 participating laboratories. All participants were requested to examine the samples using their routine method. Those laboratories using quantitative real-time PCR were requested to calculate the quantity of target virus in each sample using their standard quantification methods. Laboratories were requested to submit their results by 12 th August Results Reference results Reference analyses were performed by the EURL on samples stored at <-15 C. Six randomly selected LENTIULES TM from each sample were extracted in duplicate and qrt-pcr (TaqMan ) was carried out using triplicate PCR reactions for each RNA extract and each target. Reference results for each sample are shown in Table 2, with box and whisker plots included in Appendix I. Table 2: Reference results for PT 65 proficiency testing material Sample Norovirus HAV GI GII LENTICULE 1 + (1.60 x x 10 4 ) - - LENTICULE (1.72 x x 10 3 ) + (3.01 x x 10 4 ) Results expressed as copies/lenticule. Ranges based on a 95% confidence limit determined as 2 geometric standard deviations above and below the geometric mean. Participants results Performance assessment was undertaken on participant s presence / absence data for each parameter. Scores were assigned as follows: A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). For those participants reporting quantitative data, an additional assessment of the quantities ascribed to positive samples based on the approach described in ISO/TS (Anon 2010) was adopted according to the calculations described in Appendix II. Allocated scores for NoV (GI and GII) and HAV are shown in table 3. PT 65 Final report version 1 Page 2 of 14

91 Table 3: Participants results for all LENTICUE TM samples Presence / Absence Quantification Presence / Absence Quantification ID No. NoV NoV ID No. NoV NoV HAV HAV HAV (GI and GII) (GI and GII) (GI and GII) (GI and GII) HAV 2 A A A A 96 A A A A 3 * A A A - 98 A A * A A B A 102 * A A * C NE B A A A C 10 * A A A A 147 * A A * A A A A 161 A NE * A A A A 163 A A B A 20 A A A A C B 22 * NR NR A A * A A A B 177 A A * A A A A 183 A A * A A A A * A A C A 193 NR NR * A A A A 203 * A A A A 42 * A NE A A * B A B A 218 A A A A A A A A 49 A A C A 229 A A B NE C A A * A A C B 245 NR NR NR NR NE A A A - - * = Designated NRL, NE= Not examined, NR = Results not returned. Norovirus (NoV) and HAV scored separately. Presence / absence performance scoring; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Quantification performance scoring; A = satisfactory (all results fall within ±2 δmad of participants median), B = questionable (no more than one result falls outside ±2 δmad of participants median, and no result falls outside ±2.58δMAD of participants median), C = unsatisfactory (two or more results fall outside ±2 δmad of participants median, or one or more result falls outside ±2.58 δmad of participants median). PT 65 Final report version 1 Page 3 of 14

92 Conclusion and discussion General comments Forty-three laboratories (18 NRLs and 25 other laboratories) received samples. Laboratories 22, 94, 193 and 245 did not return results. Laboratory 250 did not examine for norovirus and laboratories 9, 42, 72 and 161 did not examine for HAV. Results reported to the EURL are shown in Appendix III. Discussion 36/39 (93%) of laboratories reported correct presence/absence results (as determined by the EURL reference samples) for all determinands that they tested. For NoV (GI and GII) 35 laboratories achieved a satisfactory performance score for presence/absence determination, with 2 (47 and 72) and 1 (9) laboratories obtaining questionable and unsatisfactory performance scores respectively. In all cases questionable and unsatisfactory scores related to false positive results (no false negative results were reported for NoV). For HAV 35 laboratories achieved a satisfactory performance score. Twenty-one laboratories (54%) returned data expressed as both C t values and quantities for at least one sample/determinand combination (Appendix IV; all 21 laboratories reported quantities for NoV and 18 did so for HAV). Twelve laboratories (31%) returned data expressed as C t values but no quantities. Of the 21 laboratories providing quantitative data, 12 laboratories obtained a satisfactory performance score for quantification of positive samples for NoV with 4 and 5 laboratories obtaining questionable and unsatisfactory performance scores respectively. For HAV 14 laboratories achieved a satisfactory performance score for quantification with 3 and 1 obtaining questionable and unsatisfactory performance scores respectively. Quantitative results and scoring are shown in Appendix V. Methods used by participants to analyse the test samples are shown in Appendix VI. References Codd AA, Richardson IR, Andrews N Lenticules for the control of quantitative methods in food microbiology. J Appl Microbiol. 85(5): Anon 2010 ISO/TS Microbiology of food and animal feeding stuffs Specific requirements and guidance for proficiency testing by interlaboratory comparison. PT 65 Final report version 1 Page 4 of 14

93 Appendix I : EURL reference results displayed as box and whisker plots of detectable genome copies per LENTICULE LENTICULE 1 LENTICULE 2 PT 65 Final report version 1 Page 5 of 14

94 Appendix II : Competency assessment of quantification data Competency assessment was undertaken following the approach described in ISO/TS Microbiology of food and animal feeding stuffs specific requirements and guidance for proficiency testing by interlaboratory comparison (ISO 2010). The MAD approach was used to assess proficiency testing data where less than 50 participants return quantitative results and/or for new proficiency assessment. 1. Where the intended result is positive Assessment was performed by evaluation of the median absolute deviation from the median (MAD) value for each laboratories individual samples. The MAD approach produces a statistically robust acceptability range by calculation of the median difference from the median for every participant s result multiplied by a constant (1.4826) to obtain a robust estimate of the standard deviation (MAD value or δ MAD). It is proposed that the individual results from laboratories will be interpreted as follows:- Difference between result and participants median <2 δ MAD = satisfactory Difference between result and participants median >2 δ MAD and <2.58 δ MAD = questionable Difference between result and participants median >2.58 δ MAD = unsatisfactory Result reported as negative = unsatisfactory 2. Where the intended result is negative Assessment of results from samples with sample results intended as negative will be as follows:- Result reported as negative = satisfactory Result reported as positive = unsatisfactory PT 65 Final report version 1 Page 6 of 14

95 Appendix III : Participants results ID No. L1 L2 GI GII HAV GI GII HAV + CT Copies / Copies / CT LENTICULE LENTICULE + CT Copies / LENTICULE E E E+05 3 * , E , E , NQ 7 * E E E+04 9 * , E , E * E E E * , E , E , E * E E E NQ NQ NQ 22 * NR NR NR NR NR NR 25 * E E E * E E E * * E E E * E E E * + - NE - + NE 47 * E E E , NQ , NQ , NQ E E E E E * E E E NR NR NR NR NR NR NQ NQ , E , E , E NQ NQ NQ 102 * NQ NQ NQ E E E * NQ NQ NQ NQ - NE NQ NE E E E E E E NQ NQ NQ NQ NQ NQ NQ NQ + 26 NQ 193 NR NR NR NR NR NR 203 * E E E NQ NQ NQ E E E NQ NQ NQ 245 NR NR NR NR NR NR 250 NE NE - NE NE + * = Designated NRL, NE= determinand not examined, NR= results not returned, NQ = Not quantified, Yellow shading denotes false positives. PT 65 Final report version 1 Page 7 of 14

96 Appendix IV : Participants and reference quantities for each sample. LENTICULE 1 GI LENTICULE 2 GII PT 65 Final report version 1 Page 8 of 14

97 LENTICULE 2 HAV PT 65 Final report version 1 Page 9 of 14

98 Distance from participants' median (σmad) Appendix V : Participants quantification data and allocated performance scores LENTICULE 1 - GI quantification data Within ± 2 σmad of ID Copies / the participants No. LENTICULE median Within ± 2.58 σmad of the participants median Outside ± 2.58 σmad of the participants median E+03 YES E+03 YES E+03 YES E+02 YES E+04 YES E+03 YES E+04 YES E+03 YES E+03 YES E+03 YES E+03 YES E+02 NO YES E+05 NO YES E+05 NO YES E+01 NO YES E+04 YES E+03 YES E+03 YES E+05 NO YES E+04 YES E+03 YES PT 65 Final report version 1 Page 10 of 14

99 Distance from participants' median (σmad) LENTICULE 2 - GII quantification data Within ± 2 σmad ID Copies / of the participants No. LENTICULE median Within ± 2.58 σmad of the participants median Outside ± 2.58 σmad of the participants median E+03 YES E+03 YES E+02 NO YES E+02 NO YES E+03 YES E+03 YES E+03 YES E+03 YES E+02 YES E+01 NO YES E+03 YES E+03 YES E+03 NO YES E+03 YES E+01 NO YES E+03 YES E+02 YES E+02 NO YES E+03 NO YES E+03 YES E+02 YES PT 65 Final report version 1 Page 11 of 14

100 distance from participants' median (σmad) LENTICULE 2 - HAV quantification data Within ± 2 σmad ID Copies / of the participants No. LENTICULE median Within ± 2.58 σmad of the participants median Outside ± 2.58 σmad of the participants median E+05 YES E+04 YES E+04 YES E+04 YES E+04 YES E+03 NO YES E+04 YES E+05 YES E+05 YES E+03 YES E+05 YES E+03 NO YES E+05 YES E+06 NO YES E+04 YES E+06 NO YES E+05 YES E+04 YES PT 65 Final report version 1 Page 12 of 14

101 Appendix VI: Results and methods used for test samples. (For key to method codes see page 13) LAB LENTICULE 1 LENTICULE 2 RNA RT-PCR RT-PCR Primers ID GI GII HAV GI GII HAV extraction method reagents GI GII HAV A K N CC CC GG 3* A K O AA-1 AA AA 7* A K O AA-2 a AA a AA a 9* + + NE + + NE A K O AA-1 AA - 10* A K O AA-1 AA AA 17* A K O AA-1 AA AA 19* A K O AA-2 AA AA A K P BB BB BB 25* A K O AA-2 AA AA 27* A K Q AA-1 AA AA 35* A K R AA-2 AA AA 39* A K O CC CC AA 41* A K O AA-2 AA AA 42* + - NE - + NE B K S AA-1 AA - 47* A K R AA-2 b AA b AA A K O AA-1 AA AA A K T AA-1 AA HH NE + + NE A K O AA-1 AA - 90* A K R AA-2 AA AA C L c U CC CC JJ A K O AA-2 AA AA D K V DD DD DD 102* C K W AA-2 AA BB A K P BB BB BB 147* A K X EE EE BB NE - + NE E K R AA-2 AA A K O AA-2 AA AA A K O AA-2 AA AA A K P BB BB BB A K O AA-2 AA AA C K P BB BB BB F K V DD DD DD G K Y AA-1 AA AA A K O AA-2 AA AA H K O AA-2 AA AA A K P BB BB BB C M d Z FF FF KK I K V DD DD DD 250 NE NE - NE NE + J K O - - AA * = Designated NRL, Yellow = false positive results; Grey = method as in annexes of ISO/DIS draft standard (or very similar); a = IAC assay run as multiplex with target assays; b = norovirus analysis carried out as multiplex; c = conventional two-step used for HAV only; d = real-time one-step used for HAV only. PT 65 Final report version 1 Page 13 of 14

102 Key to method codes RNA extraction methods A NucliSens Magnetic extraction reagents (BioMerieux) B C D E F G H I PureLink Viral RNA/DNA mini Kits (Invitrogen) QIAamp/Rneasy kits (Qiagen) Column extraction MagJET Viral DNA and RNA Kit (Thermo Scientific) SureFast PREP DNA/RNA Virus (CONGEN) NucleoSpin RNA Virus (Macherey-Nagel) Viral Carrier RNA Kit (SYNGEN) High Pure Viral RNA Kit (Roche) & OneStep PCR Inhibitor Removal Kit (Zymo Research) J iprep Virus Kit (Invitrogen) RT-PCR methods K Real-time one-step L Real-time two-step M Conventional one-step RT-PCR reagents N NoV: Brilliant II QRT-PCR Core Reagent Kit (Agilent), HAV: TAQMAN Fast 1-Step RT-QPCR Kit (Thermofisher) O P Q R S T U V W X RNA Ultrasense (Invitrogen) ceeram Tools TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems) Quantitect/Quantifast RT-PCR kits (Qiagen) AgPath-ID One-Step RT-PCR Reagents (Applied biosystems) GI & HAV; TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems). GII; RNA Ultrasense (Invitrogen) High Capacity cdna RT Kit & Taqman Universal Mastermix (Applied Biosystems) SureFast reagents (CONGEN) NoV; AgPath-ID One-Step RT-PCR Reagents (Applied Biosystems). HAV; Ceeram Tools NoV; SureFast reagents (CONGEN) & Ridagene Norovirus detection kit (R-biopharm). HAV; CeeramTools Y Platinum quantitative RT-PCR Thermoscript One-step system (Invitrogen) NoV; PrimeScript One Step RT-PCR Kit and Taq Hot Start kit (TaKaRa). HAV; One Step PrimeScript RT-PCR Kit - Perfect Z Real Time (TaKaRa) Primers/probes AA ISO/DIS ; 1) with TM9 probe for nov GI; 2) with NVGG1p probe for nov GI BB Ceeram Tools (sequences as AA-2) CC Kageyama et al, (2003) DD EE SureFast reagents (CONGEN) SureFast reagents (CONGEN) & Ridagene Norovirus detection kit (R-biopharm) FF Kojima et al, (2003) GG Health Canada method OPFLP-07 HH Guevremont et al, (2006), Houde et al, (2007) JJ FDA/BAM Chapter 26 Detection and Quantification of Hepatitis A virus in Shellfish by the Polymerase Chain Reaction KK dipasquale et al, 2010 PT 65 Final report version 1 Page 14 of 14

103 Annex VII European Union Reference Laboratory (EURL) and Public Health England (PHE) EQA Shellfish Scheme Escherichia coli and Salmonella spp. EQA EURL PT reference number: PT 67 Final Report pages Contract Reference: Cefas ref (C6472A1) Document approved by: C6095 Project Manager James Lowther Review date: Not applicable Document checked by: James Lowther Classification: Official Document prepared by: Louise Stockley Location EURL drive

104 Contents Page number Methodology 2 Reference results 2 Participants analysis and scoring system 2 Participation in statutory determinands 2 Performance assessments 3 Reference 4 Appendix 1 - Distribution SF053: Sample SF0114 and SF Appendix 2 - Distribution SF054: Sample SF0116 and SF Appendix 3 - Distribution SF055: Sample SF0118 and SF Appendix 4 - EURL PT 64 - NRL results and allocated scores 17 Appendix 5 - Scoring for the PHE/EQA and EURL matrix scheme 18 Appendix 6 - Troubleshooting advice 19 Article 32 of Regulation (EC) 882/2004 sets out the organisational responsibilities for EU Reference Laboratories (EURL) with respect to comparative proficiency testing (PT). This PT scheme is intended to provide comparative test samples for laboratories undertaking examination of live bivalve molluscs from production areas in accordance with Regulation (EC) No. 854/2004 and products placed on the market in accordance with Regulation (EC) No. 2073/2005. The scheme is organised in collaboration with the Public Health England (PHE) (Hhttp:// APTForFoodWaterAndEnvironmentalMicrobiology/ShellfishScheme/). The EU reference method for enumeration of E. coli in raw bivalve molluscs is ISO TS , Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide. EU approved alternative methods for the enumeration of E. coli are Enumeration of Escherichia coli in live bivalve molluscan shellfish by the direct impedance technique using the BacTrac 4300 series analyser and Enumeration of Escherichia coli in bivalve molluscan shellfish by the colony-count technique. Protocols for the application of these methods are available at The EU reference method for the detection of Salmonella spp. in live bivalve molluscs is ISO 6579, Microbiology of food and animal feeding stuffs Horizontal method for the detection of Salmonella spp. (Anon 2002). These methods must be used for official control testing of live bivalve molluscs for compliance with EU Regulations. Performance assessments are valuable tools to help laboratories identify any ongoing problems with their procedures or analyses. Scores are given for each distribution to assess participants performance and to highlight any incorrect or outlying results. If you are experiencing problems please contact the EURL, or alternately refer to the troubleshooting guide included as Appendix VI of this report. Further advice on microbiological testing of bivalve mollluscan shellfish can be obtained via the EURL website Proficiency testing 67 Final Page 1 of 20

105 PHE EQA distributions Country Austria Belgium and Luxembourg Bulgaria Croatia Denmark Finland France Germany Greece Hungary Iceland Ireland Italy Latvia Lithuania Netherlands Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden United Kingdom Methodology Sample preparation - PHE EQA Samples comprising of LENTICULE discs containing fully characterised bacterial isolates were distributed during February, June and October The proportions and types of organisms were designed to mirror those found in freshly harvested bivalve molluscs. Samples were packaged according to IATA regulations and distributed with report forms. Sample preparation EURL PT Three shellfish samples comprising of Pacific oysters (Crassostrea gigas) originating from a UK commercial harvesting area were distributed in November Samples were packaged according to IATA regulations and distributed with instructions and report forms. Reference results For each distribution 10 reference samples were examined by the organising laboratory. Reference analyses were performed using ISO for the enumeration of E. coli and ISO 6579 for the detection of Salmonella spp.. Participants analysis and scoring system Reported E. coli MPN values were compared to the median MPN from all participants results, reference results were omitted from the calculation. The acceptable limits were calculated as the participants median ±2.68 standard deviation (SD) and ±4 SD above and below the participants median. Reported MPN values were log 10 transformed before being compiled into charts as shown in Appendix 1 to 3. Performance assessment was carried out according to the algorithm in Appendix 5. Participation in statutory determinands All samples were analysed using participants official control methods i.e. those methods routinely used for official control analysis of live bivalve molluscs. Table 1 shows the participation of NRLs for 2016; all 25 NRLs in the network participated in at least one distribution in 2016 (currently there are no NRLs designated in Malta, Cyprus, the Czech Republic or Estonia). Of those participating labs, 88% participated in both the EURL matrix distribution and 1 or more PHE EQA distributions as agreed in Resolution 8 of the NRLs annual workshop NRLs in Bulgaria, Finland and Norway did not participate in the required number of distributions. However, to assist NRLs to participate in the required number of distributions it was agreed at the 2016 annual workshop that the EURL would pay for NRL participation in a single PHE distribution per year (Resolution 8). Table 1: Participation by NRLs in 2016 for E. coli and Salmonella spp. determinands. EURL PT Proficiency testing 67 Final Page 2 of 20

106 Performance Assessment A cumulative performance assessment was undertaken on participants results for both E. coli and Salmonella spp. from the EURL matrix distribution (PT 64) (Appendix 4) and 3 EQA distributions (March to November 2016). The allocated scores are summarised in Tables 2 and 3 respectively. Good performance is identified where a cumulative score of >70% is achieved. Participants who achieved <70% for E. coli enumeration and/or Salmonella spp. detection should in the first instance refer to the troubleshooting guide included as Appendix 6. NRLs Bulgaria, Finland and Norway did not participate in the mandatory number of EQA distributions (1) per year agreed in Resolution 8 of the NRLs annual workshop 2012 and were given a score of 0% as a result. E. coli MPN assessment Twenty-two laboratories participated in the EURL matrix scheme (Appendix 4) and 1 or more EQA distributions in 2016 and were therefore subject to a full performance assessment. Of these, one laboratory participated using both the reference method and an approved alternative method for both EURL and PHE/EQA distributions. One laboratory participated using both the reference method and an approved alternative in the EURL distribution only (PHE/EQA LENTICULES are not suitable for analyses using this alternative method). In both cases different lab ID numbers are provided for each individual method used. Twenty-one laboratories achieved a cumulative total of >70% for the 2 or more distributions analysed (the lab using both the reference and an alternative method scored >70% using both methods). Laboratory 983 achieved a cumulative total of <70%. Salmonella spp. assessment Twenty-two laboratories participated in the EURL matrix scheme and 1 or more EQA distributions in 2016 and were therefore subject to a full performance assessment. All laboratories achieved a cumulative total of >70% for the distributions analysed. Proficiency testing 67 Final Page 3 of 20

107 Table 2: Summary of participants performance in the EURL matrix scheme and the EQA scheme - E. coli PT 64 Distribution SF053 Distribution SF054 Distribution SF055 All distributions Lab no. a Cumulative Max S - 1 S - 3 SF0114 SF0115 SF0116 SF0117 SF0118 SF0119 score score % 121 [19] [9] [35] [22] [32] [41] [68] [43] [39] [23] [10] [3] [7] [90] [47] [44] [27] [170] b [13] [147] [83] [245] [33] [69] c [42] c [102] c [212] d a NRL ID number from PHE EQA scheme [ID number from EURL PT scheme in brackets]. b The reporting of MPN tube combinations is not required for the alternative method used by this laboratory, the maximum overall score is reduced to reflect this (8). c Full performance assessment was not carried out as the NRL did not register to the PHE EQA scheme during d EQA material cannot be analysed using the alternative method used by this laboratory, therefore a full assessment cannot be completed. Proficiency testing 67 Final Page 4 of 20

108 Table 3: Summary of participants performance in the EQA scheme Salmonella spp. PT 64 b Distribution SF053 Distribution SF054 Distribution SF055 All distributions Lab no. a Cumulative Max S - 1 S - 3 SF0114 SF0115 SF0116 SF0117 SF0118 SF0119 score score % 121 [19] [9] [35] [22] [32] [41] [68] [43] [39] [23] [10] [3] [7] [90] [47] [44] [27] [170] c [13] [147] [83] [245] [33] [69] d [42] d [102] d [212] c a NRL ID number from PHE EQA scheme [ID number from EURL PT scheme in brackets]. b No scores were allocated for sample 1 as Salmonella spp. was detected 4/20 replicate reference samples c Lab ID number is used only for E. coli analysis using an alternative method. d Full performance assessment was not carried out as the NRL did not register to the PHE EQA scheme during References Anon ISO :2015. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo- 4-chloro-3-indolyl-β-D-glucuronide. Geneva, Switzerland. Anon ISO 6579:2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Geneva, Switzerland. Anon 2013 ISO 7218:2007/Amd 1:2013, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations - Amendment 1. International Organization for Standardization, Geneva. Proficiency testing 67 Final Page 5 of 20

109 European Communities Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules. Off. J. Eur. Communities L 165, : European Communities Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Off. J. Eur. Communities L 226, : European Communities Commission Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Off. J. Eur. Communities L338, : ISO/IEC 17043:2010, Conformity assessment General requirements for proficiency testing Proficiency testing 67 Final Page 6 of 20

110 Appendix 1 Distribution SF053 Sample SF0114 contents E. coli (1.3x10²-1.7x10³) (wild strain), Salmonella Livingstone 6,7,14:d:l,w (1.1x10²) (wild strain), Aerococcus viridans (2.0x10³) (NCTC 8251), Staphylococcus epidermidis (9.4x10³) (wild strain) Reference results - E. coli MPN per 100g. Salmonella spp. Detected in 25g. Analysed February / March 2016 Eighteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 493, 658, 983, 1498 and 1892 did not participate in this distribution. Table 4: Participants and reference results median, median 2.68 and 4 SD - SF0114 Median MPN/100g Median -2.68SD MPN/100g Median -4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results Participants results Participants results - SF01114 (Figure 1) Table 5: Results reported by participants and scores allocated - SF0114 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected Detected Detected DNP DNP - DNP Detected Detected Detected Detected Detected DNP DNP - DNP Detected NE Detected Detected Detected DNP DNP - DNP DNP DNP - DNP * NE <18 <18 4 Detected Detected Detected DNP DNP - DNP Detected 2 E. coli MPN Fifteen laboratories reported replicate results within ±2.68 SD of the participants median with all receiving a maximum score. Laboratory 121 and 597 reported one replicate result within ±2.68 SD of the participants median and received a score of 9 and laboratory 1578 reported both replicates outside ±4 SD of the participants median and received an overall score of 4. Salmonella spp. All 16 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 7 of 20

111 Figure 1. Distribution SF053: Sample SF0114 Proficiency testing 67 Final Page 8 of 20

112 Distribution SF053 Sample SF0115 contents E. coli (1.7x10²-5.4x10³) (wild strain), Salmonella Enteritidis 1,9,12:g,m (60) (wild strain), Serratia liquefaciens (1.9x10⁴) (wild strain), Streptococcus bovis (1.9x10³) (NCTC 8177) Analysed February / March 2016 Eighteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 493, 658, 983, 1498 and 1892 did not participate in this distribution. Reference results - E. coli MPN per 100g. Salmonella spp. Detected in 25g. Table 6: Participants and reference results median, median 2.68 and 4 SD - SF0115 Median MPN/100g Median -2.68SD MPN/100g Median -4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results Participants results Participants results - SF0115 (Figure 2) Table 7: Results reported by participants and scores allocated - SF0115 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected Detected Detected DNP DNP - DNP Detected Detected Detected Detected Detected DNP DNP - DNP Detected NE Detected Detected Detected DNP DNP - DNP DNP DNP - DNP * NE <18 <18 4 Detected Detected Detected DNP DNP - DNP Detected 2 E. coli MPN Seventeen laboratories reported replicate results within ±2.68 SD of the participants median with all receiving a maximum score. Laboratory 1578 reported both replicate results outside ±4 SD of the participants median and received an overall score of 4. Salmonella spp. All 16 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 9 of 20

113 Figure 2. Distribution SF053: Sample SF0115 Proficiency testing 67 Final Page 10 of 20

114 Appendix 2 Distribution SF054 Sample SF0116 contents - Salmonella Agona 1,4,[5],12:f,g,s: [1,2] [z₂₇],[z₄₅] (93) (wild strain), Bacillus circulans (<2.0x10²) (wild strain), Enterococcus faecium (1.2x10⁵) (wild strain) Analysed June / July 2016 Fifteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 493, 583, 649, 658, 744, 1578, 1859 and 2118 did not participate in this distribution. Reference results - E. coli MPN <18 per 100g. Salmonella spp. Detected in 25g. Participants results - SF0116 Table 8: Results reported by participants and scores allocated - SF0116 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score 121 <18 <18 12 NE <18 <18 12 Detected <18 <18 12 Detected DNP DNP - DNP DNP DNP - DNP <18 <18 12 Detected DNP DNP - DNP <18 <18 12 Detected <18 <18 12 Detected DNP DNP - DNP <18 <18 12 Detected <18 <18 12 NE <18 <18 12 Detected <18 <18 12 Detected DNP DNP - DNP <1 <1 8 Detected <18 <18 12 Detected <200 <200 8 * NE DNP DNP - DNP <18 <18 12 Detected DNP DNP - DNP <18 <18 12 Detected DNP DNP - DNP - E. coli MPN Fourteen laboratories reported the absence of E. coli in this sample and received a maximum score. Laboratory 983 had points deducted as the tube combinations reported for both replicates were inconsistent with the guidance given in ISO 7218:2007/Amd 1:2013 and received an overall score of 8. Salmonella spp. All 12 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 11 of 20

115 Distribution SF054 Sample SF0117 contents E. coli (4.6x10²- 5.4x10³) (wild strain), Salmonella Anatum 3,{10}{15}{15,34}:e,h:1,6 [z₆₄] (26) (wild strain), Aeromonas hydrophila (1.6x10²) (wild strain), Enterococcus faecium (6.4x10³) (wild strain) Analysed June / July 2016 Fifteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 493, 583, 649, 658, 744, 1578, 1859 and 2118 did not participate in this distribution. Reference results - E. coli MPN per 100g. Salmonella spp. - Detected in 25g. Table 9: Participants and reference results median, median 2.68 and 4 SD - SF0117 Median MPN/100g Median -2.68SD MPN/100g Median -4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results Participants results Participants results - SF0117 (Figure 3) Table 10: Results reported by participants and scores allocated - SF0117 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score NE Detected Detected DNP DNP - DNP DNP DNP - DNP Detected DNP DNP - DNP Detected Detected DNP DNP - DNP Detected NE Detected Detected DNP DNP - DNP Detected Detected * NE DNP DNP - DNP Detected DNP DNP - DNP Detected DNP DNP - DNP - E. coli MPN Thirteen laboratories reported replicate results within ±2.68 SD of the participants median with 12 receiving a maximum score. Laboratories 651 and 1498 reported one replicate result between ±2.68 and ±4SD of the participants median and received a score of 9. Laboratory 983 had points deducted as the tube combinations reported for both replicates were inconsistent with the guidance given in ISO 7218:2007/Amd 1:2013 and received an overall score of 8. Salmonella spp. All 12 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 12 of 20

116 Figure 3. Distribution SF054: Sample SF0117 Proficiency testing 67 Final Page 13 of 20

117 Appendix 3 Distribution SF055 Sample SF0118 contents E. coli (1.3x10³-1.6x10⁴) (wild strain), Pseudomonas putida (1.5x10⁴) (wild strain), Streptococcus bovis (2.6x10³) (NCTC 8177) Analysed November / December 2016 Twenty laboratories received material for examination with 17 laboratories returning results to be included in the assessment. Laboratories 413, 597 and 720 did not return results for this distribution. Laboratories 121 and 1527 did not examine the sample for Salmonella spp.. Laboratories 983, 1498 and 1892 did not participate in this distribution. Reference results - E. coli MPN per 100g. Salmonella spp. Not detected in 25g. Table 11: Participants and reference results median, median 2.68 and 4 SD - SF0118 Median MPN/100g Median -2.68SD MPN/100g Median -4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results Participants results Participants results - SF0118 (Figure 4) Table 12: Results reported by participants and scores allocated - SF0118 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score NE Not detected NE NE - NE Not detected Not detected NR NR 0 NR Not detected Not detected Not detected Not detected Not detected Not detected Not detected NE NE - NE Not detected DNP DNP - DNP DNP DNP - DNP * NE Not detected Not detected Not detected DNP DNP - DNP Not detected 2 E. coli MPN Seventeen laboratories reported replicate results within ±2.68 SD of the participants median with all receiving a maximum score. Laboratory 597 did not provide a reason for not examining this distribution and scored 0. Salmonella spp. All 15 laboratories that reported a result for Salmonella spp. correctly reported the absence of Salmonella spp. and received a maximum score of 2. Laboratory 597 did not provide a reason for not examining this distribution and scored 0. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined NR Not returned * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 14 of 20

118 Figure 4. Distribution SF055: Sample SF0118 Proficiency testing 67 Final Page 15 of 20

119 Distribution SF055 Sample SF0119 contents E. coli (3.1x10²-1.3x10³) (wild strain), Salmonella Pensacola 1,9,12:m,t:[1,2] (73 disc) (wild strain), Bacillus pumilus (1.4x10³) (wild strain), Pseudomonas putida (3.3x10³) (wild strain) Analysed November / December 2016 Twenty laboratories received material for examination with 17 laboratories returning results to be included in the assessment. Laboratories 413, 597 and 720 did not return results for this distribution. Laboratories 121 and 1527 did not examine the sample for Salmonella spp.. Laboratories 983, 1498 and 1892 did not participate in this distribution. Reference results - E. coli MPN per 100g. Salmonella spp. Detected in 25g. Table 13: Participants and reference results median, median 2.68 and 4 SD - SF0119 Median MPN/100g Median -2.68SD MPN/100g Median -4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results Participants results Participants results - SF0119 Table 14: Results reported by participants and scores allocated - SF0119 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score NE Detected NE NE - NE Detected Detected NR NR 0 NR Detected Detected Detected Detected Detected Detected Detected NE NE - NE Detected DNP DNP - DNP DNP DNP - DNP NE Detected Detected Detected DNP DNP - DNP Detected 2 E. coli MPN Sixteen laboratories reported replicate results within ±2.68 SD of the participants median with 15 receiving a maximum score. Laboratory 649 reported one replicate result between ±2.68 and ±4SD of the participants median and received an overall scored 9. Laboratory 658 had points deducted as the tube combination reported for one replicate was inconsistent with the guidance given in ISO 7218:2007/Amd 1:2013 and received an overall score of 10. Laboratory 597 did not provide a reason for not examining this distribution and scored 0. Salmonella spp. All 15 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. Laboratory 597 did not provide a reason for not examining this distribution and scored 0. DNP NRL registered for EQA scheme but did not participate in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported using the alternative method used by this NRL. Proficiency testing 67 Final Page 16 of 20

120 Figure 5. Distribution SF055: Sample SF0119 Proficiency testing 67 Final Page 17 of 20