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 2015 FINAL report (version 3) Contract Reference: Cefas ref (C6472) Document approved by: James Lowther Review date: Not applicable Document checked by: David Lees 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 Comparative testing and ring trials 9 6. Development of analytical methods 15 Annexes 17 Annex I - Work programme for the EURL for bacteriological and viral contamination of bivalve molluscs Annex II - Resolutions of the 14 th workshop of NRLs for bacteriological and viral contamination of Bivalve Molluscs, 2015 Annex III Report of the 14 th workshop of NRLs for bacteriological and viral contamination of Bivalve Molluscs, 2015 Annex IV PT 55; Noroviruses and hepatitis A virus in bivalve molluscan shellfish; final report Annex V PT 58; Detection of V. parahaemolyticus and V. vulnificus; final report Annex VI PT 59; Noroviruses and hepatitis A virus; final report Annex VII - PT 60; Enumeration of Escherichia coli in bivalve molluscan shellfish; final report Annex VIII - PT 63; Escherichia coli and Salmonella spp. EQA; final report EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 1 of 16

4 European Union Reference Laboratory for Monitoring Bacteriological and Viral Contamination of Bivalve Molluscs, Cefas, Weymouth Technical Report for Calendar Year 2015 Legal functions and duties 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 annual work programme for the EURL for 2015 was approved by the European Union in December This report details activities of the EURL according to the work programme 2015 (Annex I), additional tasks described under the resolutions and report of the 14 th workshop of microbiological NRLs held in Nantes, France (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 2015 this has comprised: Provision of discussion papers, participation, and advice to the Commission and Member States in the Commission working group on bivalve molluscs. Three working groups attended in The EURL has provided scientific guidance relating to microbiological monitoring and classification (particularly the pending revision of E.coli requirements under Regulation 2015/2285) and norovirus particularly in relation to options for EU controls. Support to the Commission regarding discussion with EU producer stakeholders on possible norovirus controls including a stakeholder meeting. Support to the Commission regarding EU-US trade agreements on bivalve molluscs. Including technical discussion and negotiation with US FDA, participation with the DG Sante audit of the USA (March 2015), and assistance to EU MS with preparations for the FDA audit of the EU. Working with EFSA on the development of a study plan for a harmonised EU wide baseline survey of viruses in bivalve molluscs. Including working with MS to collect information on oyster production areas and processing establishments, participation in 5 EFSA working group meetings, and progress presentations to MS. The survey plan is now published EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 2 of 16

5 Participation in EFSA working group on alternative heat treatments for bivalve molluscs (5 working group meetings attended in 2015). The opinion was published in December Advice on viruses in non-animal matrices, specifically soft fruit incl. provision of an updated method protocol for application under Commission Implementing Regulation (EU) 2015/1607 amending Annex I to Regulation (EC) No 669/2009 implementing Regulation (EC) No 882/2004 of the European Parliament and of the Council as regards the increased level of official controls on imports of certain feed and food of non-animal origin (including raspberries from Serbia). In addition 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) was updated and maintained. Provision of advice to the Commission on a submission from the competent authority in Peru in relation to the emergency measures suspending imports from Peru of certain bivalve molluscs intended for human consumption. Reconvening the Good Practice Guide Working Group in order to review the content of the EURL Good Practice Guide further to the publication of Commission Regulation (EU) 2015/2285 and to address questions raised by NRLs on the application of the previous issue. The content of the Community Guide has been reviewed alongside these. Revised drafts have been produced for discussion at the 2016 EURL Workshop in Berlin. Further to a resolution taken at the 2015 EURL workshop development of guidance for the estimation of measurement uncertainty in relation to the enumeration of E. coli in bivalve molluscs. This will be presented to NRLs at the 2016 EURL Workshop in Berlin. 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/DIS , Microbiology of food and animal feed: determination of norovirus and hepatitis A virus in foodstuffs by PCR, part 1: quantitative determination. Project leader activities associated with acceptance of the mandate to CEN (M/381) on methods in microbiology to formally validate ISO TS (quantitative determination) through expert laboratory study and interlaboratory study. Attendance at the plenary meeting of CEN/TC275/WG6, 22 24th June 2015, Delft, the Netherlands The EURL led the CEN/TC 275/WG6/TAG15 programme on work on food borne pathogenic vibrios using molecular approaches, including: EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 3 of 16

6 Preparation of a new draft ISO 21782, Microbiology of food and animal feed: Horizontal method for the detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. Project leader activities associated with acceptance of the mandate to CEN (M/381) on methods in microbiology to formally validate ISO TS parts 1 and 2 through interlaboratory study. The EURL led the ISO/SC9/WG14 expert group on the revision of the EU standard reference method for enumeration of E. coli in LBM (ISO TS ). The standard has now been published. The EURL led the ISO/SC9/WG8 expert 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. Standard at the DIS stage. The EURL participated in the second FAO/WHO expert working group meeting on the development of Technical Guidance for the Implementation of Bivalve Mollusc Sanitation Programs and contributed to the development of the guidance. Following invitation, the EURL attended a workshop in Hong Kong (September 2015) to inform the authorities and other stakeholders on EU developments on controls for norovirus in bivalve molluscs and to advise on risk management. Following invitation by the US FDA the EURL attended and presented information on EU norovirus developments at a special session of the International Association of Food Protection Conference in Portland USA in July The EURL worked closely with technical experts from the US FDA, including advanced training sessions and conducting two intensive joint fieldwork exercises, on the application of effluent dilution (dye tracing) techniques to the determination of sewage discharge buffer zones. This knowledge assisted development of EURL guidance and supported advice to MS wishing to export to USA. 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). EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 4 of 16

7 Table 1. Designated NRLs in Member States, EFTA and Accession states in calendar year Member State Laboratory Austria Austrian Agency for Health and Food Safety, Institute for Food Control, AGES-LMU Wien, Abt. Mikrobiologie, Spargelfeldstraße 191, A-1220 Wien. Belgium and Scientific Institute of Public Health (IPH), Rue Juliette Wytsmanstraat 14, Luxembourg 1050 Brussels. Bulgaria National Diagnostic and Research Veterinary Institute, Pencho, Slaveikov, 15 BG Sofia Croatia Croatian Veterninary Institute, Regional Veterinary Laboratory Split, Poljicka Cesta 33, Split, Croatia Cyprus No NRL designated The Czech Republic Denmark Estonia Finland No NRL designated DTU Food, National Food Institute, Technical University of Denmark, Morkhoj Bygade 19, DK 2860 Soborg. No NRL designated Finnish Customs Laboratory, Tekniikantie 13, FI-02150, Espoo. France IFREMER, Departement Microbiologie et phycotoxines, Centre de Nantes, Rue de I'lle de'yeu, BP 21105, Nantes Cedex 3. Germany Bundesinstitut fur Risikobewertung (BfR), Federal Institute for Risk Assessment, Diedersdorfer Weg, D-12277, Berlin. Greece Institute of Food Hygiene of Athens, Neapoleos 25, Ag. Paraskevi, Attiki, Athens. Hungary Central Agricultural Institiute, 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, Orinville, Oranmore, Galway. Italy 2 Istituto Zooprofilattico Sperimentale Umbria e Marche, Via Cupa di Posatora 3, 60100, Ancona. Dipartmento di Sanita Pubblica Veterinaria e Sicurezza Alimentare, Viale Regina Elena 299, 00161, Rome. Latvia Institute of Food Safety, Animal Health and Environment (BIOR), Lejupes str.3, LV Riga. Lithuania National food and Veterinary Risk Assessment Institute, J.Kairiukscio Str. 10, LT , Vilnius. Malta No NRL designated The Netherlands Norway 1 Poland Portugal National Institute for Public Health and the Environment (RIVM), PO Box 1 A van, Leeuwenhoeklaan 9, 3720 BA, Bilthoven. Institute for Food Safety and Infectious Biology, Department for Food Safety, P.O Box 8146 Dep, 0033 Oslo. National Veterinary Research Institute, Panstwowy Instytut, Weterynaryjny, Al.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. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 5 of 16

8 Member State Laboratory Romania 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 National Veterinary Laboratory, Gerbiceva 60, SI 1000, Ljubljana. Spain Centro Nacional de Alimentacion, Agencia Espanola de Seguridad Alimentaria, E Majadahonda, Madrid. Sweden National Food Administration, 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 the EFTA 2 2 NRLs established (one for microbiological monitoring and one for virology) EURL website The EURL website continues to provide a very useful repository for information for NRLs and other stakeholders. The website attracted 15,383 page views from 11,441 unique visits throughout Annual workshop of NRLs The 14 th workshop of NRLs was hosted at IFREMER between May Forty one delegates representing 24 EU Member States, 2 EFTA countries and the EURL alongside invited experts from EFSA, the Food and Consumer Product Safety Authority of The Netherlands, the Centro de Control da Calidade do Medio Marino of Galicia, Spain and the Centre for Disease Control of the United States took part in the workshop. The workshop covered official controls, E.coli and Salmonella, viruses and marine vibrios. 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 good to very good performance, feedback breakdown is provided within the annual workshop report. 3. Provision of technical advice and training Formal training workshops EU VIBRIO PCR METHODS WORKSHOP April 2015 The EURL provided a workshop on methods for the detection and quantitation of pathogenic vibrios at the Croatian NRL (Split), Croatia in May The main objective of the workshop was to provide in depth training to Croatian, Albanian and Italian microbiologists using conventional and real-time PCR for the detection and quantification of major pathogenic vibrios of human health relevance (e.g. Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae). The workshop was a mixture of theory and practical activities, carried out over 5 days (20-24 th April 2015). Additional ad hoc training and study visits EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 6 of 16

9 The EURL hosted a visitor from the Namibian Standards Institute for 4 days training in practical methods for detection of microbiological indicators and pathogens in shellfish, partially funded through the Better Training for Safer Food programme. Other technical assistance Reference and control materials for the virus detection method were provided to laboratories in Spain, Poland, Slovenia and the United Kingdom. Provision of advice to NRLs and others In 2015 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 Italy, Albania, the United Kingdom, Spain, Bulgaria, Ireland, Poland, Canada, Singapore, the Netherlands, France and the United States. Provision of advice to the Moroccan competent authority in relation to the use of indicator species and monitoring of offshore bivalve areas Provision of advice to the Sardinian regional competent authority in relation to the use of indicator species Provision of advice to NRL Greece in relation to the use of indicator species Provision of advice to NRL France in relation to application of the EURL Good Practice Guide Provision of advice to NRL Italy on the quality control of media with respect to the use of ISO for bivalve molluscs Provision of advice to the Regional Authority of Andalusia on sampling plans for bivalve molluscs Additionally ad hoc technical advice was provided to laboratories via or telephone. A register of advice provided by the EURL is available from the EURL co-ordinator on request. Other Scientific activities EURL staff have produced a number of peer-review papers in scientific journals, book chapters, other publications and conference proceedings: Le Roux, F., M. Wegner, C. Baker-Austin, L.Vezzulli, C. R. Osorio, C. Amaro, J. Ritchie, T. Defoirdt, D. Destoumieux-Garzón, M. Blokesch, D. Mazel, A. Jacq, F. Cava, L. Gram, C. Wendling, E. Strauch, A. Kirschner, and S. Huehn. The Emergence of Vibrio pathogens in Europe: Ecology, Evolution and Pathogenesis (Paris, March 2015). Frontiers in Microbiology, 6:830, Campos, C.J.A., J. Avant, N. Gustar, J. Lowther, A. Powell., L. Stockley, and D. N. Lees. Fate of human noroviruses in a shellfish water impacted by frequent sewage pollution events. Water Science & Technology 49(14): , EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 7 of 16

10 Campos, C.J.A., J. Lowther, N. Gustar, J. Avant, L. Stockley, and D. Lees. Improving knowledge of human norovirus in the marine environment to reduce the incidence of shellfishrelated illness. Proceedings of the EFSA s 2nd Scientific Conference Shaping the Future of Food Safety, Together, October 2015, Milan, Italy, EFSA Journal, Supplement 13(10): Martinez-Urtaza J., A. Powell, J. Jansa, J. L. Castro Rey, O. Paz Montero, M. García Campello, M. J. Zamora López, A. Pousa, M. J. Faraldo Valles, J. Trinanes, D. Hervio-Heath, W. Keay, A. Bayley, R. Hartnell and C. Baker-Austin. Epidemiological investigation of a foodborne outbreak in Spain associated with U.S. West Coast genotypes of Vibrio parahaemolyticus. SpringerPlus, (accepted). 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 (accepted). Scientific presentations (international conferences) Baker-Austin C. Vibrio infections acquired in sub-arctic waters. The Emergence of Vibrio pathogens in Europe: Ecology, Evolution and Pathogenesis, Paris, France, March Lees, D.N. New approaches to NoV controls in the EU: method development and application. International Association of Food Protection meeting, Portland, Oregon, United States, July Campos, C.J.A., J. Avant, J. Lowther, D. Till, and D. Lees. Effectiveness of sewage treatment processes in removing human norovirus. Poster presentation. 8th International Symposium on Health-Related Water Microbiology, Lisbon, Portugal, September Campos, C.J.A., G. Goblick., D. Till, R. Lee, and D. N. Lees. Assessing the fate of human norovirus in shellfish waters through microbiological analysis and effluent dilution studies: a case study in the South of England, UK. 8th International Symposium on Health-Related Water Microbiology, Lisbon, Portugal, September 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 2015 version 1 Page 8 of 16

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 2015 is tabulated in Table 2 and summarised in this section. Proficiency testing reports and other documentation are provided as Annexes IV, V, VI, VII and VIII. Proficiency testing for statutory determinands E. coli and Salmonella spp. proficiency testing PT 60 and PT 63 NRLs have previously agreed that participation in a minimum of two annual PT distributions is mandatory with the EURL matrix PT (comprising of bivalve molluscs samples) to be compulsory and at least one other non-matrix 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 participation in PT. All designated NRLs completed the EURL matrix PT with the exception of Denmark and Finland. Bulgaria, Finland, Greece and Latvia did not take part in a second nonmatrix PT distribution. The reasons for lack of participation will be investigated according to the Commission Protocol 1. Overall performance of NRLs are shown in Table 3 and 4. PT 60 - Matrix distribution Matrix distributions enable participants to examine all aspects of the methodology. In December 2015 the EURL distributed two samples of bivalve molluscan shellfish (Common mussels (Mytilus edulis) and Pacific oysters (Crassostrea gigas)). This PT distribution (PT 60) was 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. Thirty-seven laboratories participated in PT 60, including 23 of the 25 nominated NRLs for bacteriology within the network (all designated NRLs except Denmark and Finland). Participants examined each sample for E. coli in duplicate. Reference samples were assessed for homogeneity in accordance with ISO Reported E. coli MPN values for each sample were compared to the median of all participants results. Upper and lower acceptability limits were calculated as the participants median ±3 theoretical standard deviations (SD) and ±3 SD ( 99% and 99.9% confidence intervals respectively). Performance assessment was according to the EURL/PHE EQA scheme for a single distribution, with modifications to reflect replicate analyses of a single sample. Table 3 shows abstracted performance scores from NRLs for PT 60 assessed in combination with all participants. Belgium, France, Ireland, Italy and Poland had points deducted as one or both MPN values reported for sample 1 or sample 2 were outside ±3 SD of the participants median. Further points were deducted from NRLs 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 should be used. A full report is included as Annex VII. PT 63 Non-matrix distributions 1 Commission Protocol for lack of collaboration or underperformance in proficiency testing EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 9 of 16

12 NRLs were offered distributions (SF050, SF051, SF052) in March, July and November 2015 for examination of E. coli and Salmonella in simulated bivalve mollusc matrices. Uptake of the PT was more variable. Tables 3 and 4 show abstracted performance scores from NRLs for PT 63 assessed in comparison with NRL participants. A full report is included as Annex VIII. 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 ISO7218:2007/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 should be used. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 10 of 16

13 Table 2 - Summary of participation amongst NRLs and others in EURL organised proficiency testing for calendar year 2015 EU NRLs EFTA Third country laboratories EURL PT reference number Proficiency testing description Austria 1 Belgium and Luxembourg 1 2 Bulgaria 3 Croatia Cyprus Czech Republic Denmark Estonia Finland 3 France 1 Germany 1 Greece Hungary Ireland Italy 1 Latvia 3 Lithuania Malta Netherlands 1 2 Poland Portugal Romania Slovakia Slovenia Spain 1 2 Sweden 2 United Kingdom 1 2 Iceland Norway 1 2 Australia Canada 1 Chile Serbia Singapore PT 58 PT 59 PT 60 PT 63 Detection of V. parahaemolyticus and V. vulnificus Norovirus and Hepatitis A virus Matrix and LENTICULES Enumeration of E. coli in shellfish E. coli and Salmonella spp. EQA 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 x x x EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 11 of 16

14 Table 3. Performance scores for NRLs in E. coli PT in 2015 Distribution SF050 Distribution SF051 Distribution SF052 PT 60 All distributions NRL Sample Sample Cumulative Max SF0108 SF0109 SF0110 SF0111 SF0112 SF score score % Austria Belgium & Luxembourg 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 2015 version 1 Page 12 of 16

15 Table 4. Performance scores for NRLs in Salmonella spp. PT in 2015 Distribution SF050 Distribution SF051 Distribution SF052 All distributions NRL Cumulative Max SF0108 SF0109 SF0110 SF0111 SF0108 SF0109 score score % Austria Belgium& Luxembourg Bulgaria Croatia Denmark Finland France Germany Greece Hungry Iceland Ireland Italy Latvia Lithuania Netherlands Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden UK Proficiency testing for non-statutory determinands The EURL also offers PT for non-statutory determinands, in 2015 PT distributions were provided for Vibrio spp, 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 55 - Norovirus and hepatitis A virus in matrix material and LENTICULES In November 2014 the EURL distributed 9 samples comprising naturally and artificially contaminated matrix (Crassostrea gigas and M. edulis) and laboratory constructed discs. Outline details, summary tables of results and the intended results sheet were included in the Annual Technical Report for 2014, however the full report (which was not complete at the time) is included in this document as Annex IV. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 13 of 16

16 PT 58 - Vibrios In May 2015, the EURL organised a distribution for the detection of V. parahaemolyticus and V. vulnificus. The distribution comprised of 4 shellfish homogenates inoculated with known levels of Vibrio spp.. This distribution was only available to NRLs with 16 requesting to participate in PT 58. NRLs analysed using the classical detection method at both 37 C and 41.5 C with nine providing additional PCR data. Analysis of results indicated that detection of V. vulnificus was more problematic that the detection of V. parahaemolyticus. A questionnaire was included in the distribution to provide additional information on NRLs methodology. On collating the data the favoured reference for Tox R for V. parahaemolyticus strain identification was Kim et al., for the detection of the tdh and trh pathogenic markers was Bej et al. and for the detection of V. vulnificus was Hill et al. A full report is included as Annex V. PT 59 - Noroviruses and hepatitis A virus In June 2015 the EURL distributed 2 laboratory constructed LENTICULE discs containing some combination of norovirus GI, GII and HAV. This PT was 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. Material was distributed to 33 laboratories (15 NRLs), and 30 laboratories returned results. Scores were based upon qualitative measures of relative accuracy, relative specificity and relative sensitivity as determined by comparison with EURL reference results. Abstracted performance of NRLs is shown in Table 5. All participating NRLs produced satisfactory performance scores. A full report is included as Annex VI. Table 5. Performance scores from NRLs for norovirus and hepatitis A virus in LENTICULES 2015 Performance Norovirus GI Norovirus GII Hepatitis A virus NRL score AC SP SE AC SP SE AC SP SE NoV HAV Belgium A A Denmark A A France A A Greece A A Hungary A A Ireland A A Italy A A Norway A A Poland A A Romania NR NR NR NR NR NR NR NR NR - - Slovenia A A Spain A A Sweden A A Netherlands A A UK A A NR = Not returned. Performance scores; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Norovirus (NoV) and HAV scored separately. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 14 of 16

17 6. Development of analytical methods ISO/DIS , the revision of ISO TS , Microbiology of food and animal feed: determination of norovirus and hepatitis A virus in foodstuffs by PCR, part 1: quantitative determination, was completed by the ISO TC275/WG6 TAG4 group, and submitted to ISO/CEN for preparation for technical enquiry. At the technical enquiry stage a positive vote of 15 to 1 was obtained at the CEN level, and a positive vote of 21 to 2 was obtained at the ISO level. A final draft (FDIS) of ISO will be prepared with revisions based on the comments received at the enquiry and submitted for final approval during Ongoing standardisation of ISO Microbiology of the food chain - Horizontal method for the detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus was carried out (under the EU mandate M/381) throughout The standard has been modified by consolidating parts 1 and 2 into a single standard, introducing molecular confirmation methods and simplifying some biochemical tests. Research aimed at developing a real-time PCR based method for quantification of pathogenic strains of V. parahaemolyticus was carried out. The EURL undertook liaison with the US FDA with respect to the conduct of field studies in bivalve production areas to define buffer zones. This was undertaken in order to support advice to NRLs and CAs on the application of buffer zones to meet US export requirements and to assist in the provision of advice on the potential wider application of buffer zones within the EU. EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 15 of 16

18 U Annexes [This page is left blank intentionally] EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2015 version 1 Page 16 of 16

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 WORK PROGRAMME FOR THE EURL FOR BACTERIOLOGICAL AND VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, 2015 (VARIATION JULY 20 th 2015) 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 2015 work programme year 28 Member States, are considered eligible for EURL assistance and invited to participate in EURL organised training programmes, comparative testing etc. Acceding countries and additional laboratories are also invited to participate in comparative testing and training workshops (on a cost recovery basis). The full integration into the European Union of Member States continues to be a priority area, and is facilitated via the provision of additional advice, specific training and assistance. WORK PROGRAMME, 2015 (VARIATION JULY 20 th 2015) 1. Scientific advice and support Expected outputs 1.1. The EURL will provide scientific assistance to DG SANCO in operation of existing, and implementation of new, European Union food hygiene legislation, and in particular in 2015 the following activities have been identified: The provision of expert scientific and technical advice with regard to the setting quantitative limits / criteria for noroviruses in live bivalve mollusc (LBM) production areas and/or products placed on the market a Further to the above, the development of technical guidance (based upon analysis of existing data) describing the precision characteristics associated with the virus reference method (ISO TS ) e.g. level of detection, limit of quantitation, linearity and measurement uncertainty to support the setting of quantitative limits in LBM b The provision of expert scientific and technical advice with regard to a potential criterion for hepatitis A virus in LBM. a Regulation covering inter alia standards for norovirus in LBM improvement in public health b Evidence to support the setting of robust standards for EU legislation The provision of supplementary advice with regard to implementation of 3 class plan (applied over time in harvesting area assessments) for testing E. coli in LBM to harmonise EU legislative requirements with Codex STAN c Provide technical advice to the Commission on issues associated with microbiological monitoring of bivalve molluscs, including but not restricted to decisions after monitoring, implementation of Page 1 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015 c Successful implementation of amendments to EU legislation.

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 prohibition buffer zones around sources of faecal contamination, virus (or other microbiological) outbreaks d. d EU and US trade in LBM To provide support to DG SANCO and priority MS, as required with recommencement of trade between EU and US LBM d Provide scientific advice and assistance to DG SANCO with respect to determination of norovirus and hepatitis A virus in matrices other than bivalve shellfish (eg soft fruits) covered in the ISO TS parts 1 and 2 (the virus reference method) e 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 MS, NRLs and Official Control testing laboratories e. NOTE. The EURL will provide any other additional advice within its area of expertise as required, and undertake supporting expert missions on request of the European Union. e Scientific and technical advice to support EU Reg 1235/ Participate in relevant EU and International scientific committees (EFSA, ISO/CEN, WHO/FAO, ICMSS etc). In 2015 the EURL will: Act as convener for CEN/TC275/ WG6/TAG4 Viruses to assist in the development of CEN methods for the determination of viruses in foodstuffs. In 2015 including, but not restricted, to providing responses to official technical and editorial comments from voting members at ISO SC9 level for the publication of 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 and 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 2: Method for qualitative determination) f Assist as a member of the international steering committee towards the organisation of 11 th International Conference on Molluscan Shellfish Safety providing guidance and comment upon the developing programme as it relates to microbiological safety of bivalve molluscs g.. f Publication of full ISO standard (currently a Technical Specification) as a reference method for viruses in LBM Further to the above up to two members of EURL staff to attend the 11 th International Conference on Molluscan Shellfish Safety. To chair sessions, deliver keynote presentations in the area of viruses in shellfish, future direction of legislative controls and to contribute to EU / US technical and advisory initiatives on the development of best practice in microbiological monitoring to LBM to enhance public safety and trade g. g Enhanced EURL scientific reputation and Lead and co-ordinate the activities of CEN/TC275/WG6/TAG15 in impact, provision of best quality, state of art advice Page 2 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

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 the elaboration methods for the determination of pathogenic marine vibrios in bivalve shellfish, particularly for V. parahaemolyticus and V. vulnificus. Up to 2 missions associated with this activity are anticipated in 2015 h Complete the revision of the EU reference method for enumeration of E. coli in LBM for official control (ISO TS ) to establish the method as a full standard. No physical meetings in 2015 activity restricted to review of technical and editorial comments at ISO SC9 level i 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. No physical meetings in 2015, activity restricted to review of technical and editorial comments at ISO SC9 level j 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). Two missions in to EU h Reference method for determination of total and toxigenic V. parahaemolyticus and V. vulinificus in LBM of particular importance in US traded product i Publication of the EU E. coli reference method as a full ISO standard j Standard harmonised with EU Reg. 2073/2005 with respect to minimum number of animals comprising a sample To contribute to relevant EFSA expert working groups as required g Assist DG SANCO with specialist advice in relation to food and veterinary inspections of Member States, Accession Countries and Third Countries as they arise Represent the EURL at the annual plenary meeting of the ISO SC9 and CEN WG6 Microbiology working group meeting. One mission (Berlin) in 2015 k Contribute towards the FAO/WHO initiative developing best practice guidance for the application of bivalve molluscan shellfish sanitation programmes: global technical application activity restricted to 10 days to support production and review of documentation l Support EFSA in the initial planning and organization of the anticipated baseline survey for viruses in oysters, to include participation in planning discussions and review of documentation. 2 Project management and co-ordination of activities of NRL network k EU representation at CEN and ISO microbiology expert groups l Enhanced EURL scientific reputation and impact m retention of EN ISO 9001 demonstration of project management quality 2.1 Project management of EURL programme according to the requirements of EN ISO 9001 Quality Management Systems m. 2.2 Participate in EURL Director s co-ordination meeting and other EURL n EURL impact, harmonisation across co-ordination meetings/workshops as appropriate. network of EURLs Page 3 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

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 In 2015 the EURL will participate in the EURLs in the food and feed and animal health sectors expert working groups on n ; Development and evaluation of NRL workshops and training programmes, Performance monitoring and peer review of proficiency programmes, Development of best practices amongst the EURL network. 2.4 Organise, host, and participate in the fourteenth annual EURL workshop of NRLs for monitoring bacteriological and viral contamination of bivalve molluscs, produce resolutions and other workshop outputs. In 2015, the workshop will be held at the NRL France at IFREMER, Nantes (May 19 th -21 st 2015) o. 2.5 Further to the above, undertake EURL activities and commitments agreed in resolutions at annual workshop 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. To include in 2015 the launch of online registration for comparative testing services p. 3 Provision of technical advice and training o harmonisation of official controls across network of NRLs p increased accessibility of website, revised content and increase usage q improvements in performance and increased standardisation across laboratories 3.1 Provide specialist training and/or training courses to NRLs, and others in relation to official control analyses (E. coli, Salmonella spp.) and nonstatutory analyses (Vibrio spp., norovirus, hepatitis A virus FRNA bacteriophage,) and other aspects of bivalve shellfish hygiene as required q. 3.2 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 include approved alternative methods for official control analysis q. 3.3 To include assistance on implementation of methods, accreditation to IEC ISO17025 and quality control requirements (see above) q. 3.4 To provide guidance and review of procedures/data to laboratories wishing to undertake studies to validate of alternative methods according to ISO To include formal assessment of validation data as required to support official approval (at SCFCAH) for alternative methods q. 3.5 Provide specialist ad hoc training and/or training courses to NRLs and Page 4 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

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 others in relation to analyses of LBM for microbiological contaminants as required q. 3.6 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 q. 4 Confirmatory testing and quality assurance 4.1 Maintenance of 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 r. r retention of ISO IEC accreditation where relevant at annual external audit demonstration of science quality 4.2 Maintenance of 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 the detection of Salmonella spp. and Vibrio parahaemolyticus r. 4.3 Carry out activities associated with extension of scope of ISO IEC accreditation to include V. vulnificus and V. cholerae r. 4.4 Contribution to costs of the maintenance of EURL capability to perform enumeration analyses in LBM for the sanitary indicator organism FRNA bacteriophage r. 4.5 Contribution to costs of the maintenance of EURL capability to perform further characterisation of human pathogenic strains of marine vibrios associated with LBM (e.g. serotyping and molecular characterisation of pathogenic strains of V. parahaemolyticus, V. vulnificus and) non01/0139 V. cholerae, to include next generation sequencing of strains of clinical relevance) r. 4.6 Maintain competence and expertise in practical analytical methods for determination of norovirus and hepatitis A virus in matrices other than bivalve shellfish (specifically soft fruit including strawberries), to include development of appropriate sampling strategies. In support of Commission Regulation (EU) No. 1235/2012 r. s technical advice to support EU 4.7 Performance of above tests on outbreak material or on occasion of disputed test results (on request) s. 5 Comparative testing and ring trials Page 5 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

24 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 Organise comparative testing for NRLs for statutory determinands (E. coli and Salmonella spp.) in bivalve molluscs. Analyse results, produce report, advice and recommendations (4 distributions in 2015, March, July, October and November). 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 t. 5.2 To support implementation of quality assurance standards for virus testing organise one distribution of norovirus and hepatitis A virus for comparative testing for quantitative and qualitative analyses (provisionally June 2015). Analyse results, produce report and recommendations q. 5.3 Distribution of norovirus and hepatitis A virus reference materials and control materials, and Vibrio spp. EURL reference strains on request of NRLs u. t comparative testing reports published on the EURL website, presented at workshop, identification of satisfactory performance of NRLs and follow-up of unsatisfactory performance u improvements in performance and increased harmonisation across laboratories 5.4 Organise comparative testing for pathogenic Vibrio spp. (specifically in 2015, it is proposed that this comprises analyses for toxigenic strains of V. parahaemolyticus and V. vulnificus in LBM r. 6 Development of analytical methods 6.1 Development of methods for inclusion in ISO TS (Standard method for detection of vibrios) of an enumeration annex to enable enumeration of total / pathogenic V. parahaemolyticus in imported / indigenously produced bivalve shellfish h,s. 6.2 Standardisation of a typing method using nucleic acid hybridisation to speciate the sanitary indicator organism FRNA bacteriophage detected in LBM to enable source attribution (human and animal sources of pollution) u. 6.3 Examination and statistical assessment of norovirus datasets to determine precision characteristics of ISO TS (virus reference method) including measurement uncertainty / confidence intervals etc to support setting of norovirus limits in LBM a, b. 6.4 Further to Recommendation 364 taken at CEN/WG6/TAG4 work with colleagues, including the EU Joint Research Council, Geel, to elaborate control materials to assist in the standardisation of ISO technical specifications for noroviruses and hepatitis A virus o. 6.5 Undertake activities associated with dye-tracing studies to assess size of prohibition buffer zones to assist in development of guidance to support Annex II of the Community Guide to the Principles of Good Practice for the Microbiological Classification and Monitoring of Bivalve Mollusc Production and Relaying Areas with regard to Regulation 854/2004 Page 6 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

25 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 covering additional requirements for production areas from which LBM are harvested for export to the USA s. NOTE. In 2015 it is proposed that a studentship is partially supported by the EURL up to a maximum value of 7,174 to assist with development of analytical methods under 6.1 and 6.2. Page 7 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

26 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 Page 8 EURL monitoring bacteriological and viral contamination of bivalve molluscs v3final work programme2015

27 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 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015 Official controls 1. The EURL briefed the workshop on pending legislative changes regarding classification and end-product criteria for E.coli. The EURL noted that they would amend Community Guidance appropriately following adoption of the Community legislation. NRLs agreed to notify the EURL of any other issues that should be considered in this revision. 2. The workshop noted the recent RASFF alerts regarding norovirus contamination in cooked products imported into the EU. It was agreed that the use of PCR to assess risk in cooked products was not appropriate. The workshop was informed that EFSA had received a mandate from the Commission to investigate the application of Community heat treatment requirements and would report in due course. 3. With respect to the potential application of measurement uncertainty to microbiological results, the EURL noted that this was due to be discussed between the Commission and Member States. The EURL would circulate a previous Commission discussion document on this topic and draft a section covering this issue in Community Guidance. 4. The workshop considered developments in land-based and enclosed production systems for bivalve molluscs, noted the lack of clarity regarding the legal basis for classification, and agreed that this needed to be clarified by Competent Authorities. Following this clarification the EURL agreed to develop monitoring guidance for incorporation within Community Guidance, but also noted that it was the responsibility of FBOs to incorporate risks from such systems into their HACCP plans. 5. Following discussion of approaches applied in Member States to disregard E.coli results associated with high rainfall the EURL agreed to review and further clarify the Good Practice Guidance on this issue. E.coli and Salmonella 6. NRLs identified that there was no appropriate guidance on the use of the MPN Calculator referenced in ISO The EURL undertook to propose to ISO that the MPN calculator be amended to include a bivalve-specific worksheet, to be made more user friendly, and to be accompanied by a user guide. The EURL also agreed to request information from ISO on how the calculator was validated and to provide this information to NRLs. In the short-term, the EURL would amend the generic E.coli protocol to provide guidance on the use of the MPN calculator. 7. The workshop considered a presentation on E.coli levels in echinoderms (Paracentrotus lividus) in the vicinity of a pollution source and noted surprisingly high levels of contamination. The implications for classification of this species were noted. The workshop noted a lack of data and experience on approaches to official control testing and classification for other non-bivalve species covered by EU legislation. Resolutions of the 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015

28 Viruses 8. In the content of virus outbreaks in non-bivalve mollusc foods the workshop briefly discussed whether there were any plans for establishment of a EURL/NRL network for this issue. The importance of retaining virus responsibility within the network of bivalve microbiology NRLs was endorsed by all participants of the workshop. It was agreed that viruses in non-bivalve foods fell outside of the remit of the bivalve mollusc network but the group was interested in developments on viruses in other foods and exchange of information. 9. The workshop considered the performance characteristics of the virus method as determined by the validation of ISO/TS under mandate M/381. It was noted that, for accreditation purposes, laboratories would be required to generate their own limits of detection and quantification. The EURL agreed to produce guidance on approaches for laboratories to determine these characteristics. 10. The results of virus PT distributions 53 and 55 showed continued improvements in qualitative performance and method harmonisation amongst NRLs. However, further improvements to quantitative comparability would be desirable. 11. Further to the above the EURL agreed to assist NRLs with calculation of sample concentrations from Ct values, by provision of worked examples for PT 55 and by placing on the EURL website a calculation spreadsheet for ISO/TS Further to resolution 10 the workshop also agreed that in-house generation of quantification standards could contribute to variability. The EURL agreed that, for the next matrix PT, quantification standards would be supplied and information on in-house generation of standards requested from participants. The EURL would analyse responses in relation to the standards generation methodology utilised, to identify potential sources of variability. 13. The EURL agreed to continue to develop accessible quantification standards to assist laboratories with the application of ISO/TS It was agreed to continue virus PT with 2 distributions per year including one matrix distribution which should include at least one whole animal sample (shucked or unshucked). 15. The workshop considered presentations on recent hepatitis A virus outbreaks attributed to consumption of bivalve shellfish produced in the EU. It was recognised that with the low immune status of hepatitis A virus in the EU, and the low infective dose of this virus, that sporadic community cases of hepatitis A virus in coastal locations could present a high risk for shellfish contamination. 16. Further to the above, the workshop recommended that risk mitigation for hepatitis A virus needs to be improved and this would be achieved by better communication between health authorities and shellfish risk managers. In this context it was noted that laboratory analysis significantly assists risk management. 17. Further to resolution 15 the workshop considered resolutions 11 and 14 of 2012 in relation to risks posed by shellfish contamination with hepatitis A virus and norovirus respectively. The workshop agreed that emerging data confirmed these resolutions and that they continued to be appropriate advice. 18. The workshop considered emerging plans for an EU-wide harmonised survey for viruses in oysters. EFSA presented considerations for the sampling plan. It was agreed that it was important to consider analytical aspects, volumes of shellfish production, logistics and cost in designing the sampling plan. Resolutions of the 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015

29 Marine vibrios 19. The workshop noted data on variation in norovirus levels between individual oysters and its implications for risk assessment. Furthermore, it was noted that the data reinforced the approach in ISO/TS of analysis of a pooled sample of 10 animals. 20. The workshop considered data on norovirus monitoring in oyster production areas associated with outbreaks. It was noted that outbreaks may reoccur in areas that remain virus contaminated but are reopened. The workshop noted the implications of this for risk management. 21. The workshop noted the application of ISO/TS in diverse Member States and that the method seemed to provide useful risk management information. However the lack of correlation between E.coli and norovirus contamination was noted in several studies. 22. The workshop considered a presentation from CDC Atlanta USA giving a comprehensive overview of vibriosis in the USA. The workshop noted the significant underreporting of this disease in the USA, despite it being a notifiable illness, and considered that it was possible that vibriosis could be significantly underreported in the EU. More structured epidemiological reporting in the EU would improve understanding of the health burden from vibrios associated with shellfish consumption. 23. The workshop considered findings from the validation studies of the Vibrio method and agreed that aspects of the methodology were not yet satisfactory. The EURL agreed to organise a discussion of specialists to agree future strategies for method improvement. To assist this it was agreed that the detailed results of the validation study will be shared with NRLs. Furthermore it was agreed that NRLs would share relevant methods with the discussion group. Future EURL PT would be used to support such methodological improvements. 24. The EURL presented information on international microbiological criteria regarding vibrios in seafood. The workshop agreed that no international consensus had yet emerged and that it was therefore difficult to recommend appropriate microbiological criteria relevant to laboratory testing in the EU. 25. The next workshop will be held on 25 th -27 th May 2016 at the Federal Institute for Risk Assessment, Berlin, Germany. Resolutions of the 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015

30 Resolutions of the 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015

31 Annex III European Union Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs Report of the 14 th workshop of NRLs for monitoring bacteriological and viral contamination of bivalve molluscs. Nantes, France, 20 th 22 nd May, Final report version 3 07/10/2015 Contract Reference: C6472 Document prepared by: Document checked by: Document approved by: James Lowther David Lees Review date: n/a David Lees Classification: Unclassified

32 Contents Foreword 3 Delegate list 4 Agenda of the 14 th workshop 7 List of workshop papers 9 Workshop minutes 10 Workshop actions 18 Workshop resolutions 21 Workshop feedback 24 Workshop declaration 26 2

33 Foreword This document summarises relevant information from the 14 th workshop of National Reference Laboratories for monitoring bacteriological and viral contamination of bivalve molluscs held at IFREMER, France 20 th 22 nd 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) E mail: james.lowther@cefas.co.uk General e mail: fsq@cefas.co.uk EURL Website: 3

34 Delegate List Country Country Status Delegate Specialist area E mail Austria Member State Johann Ladstaetter NRL Representative johann.ladstaetter@ages.at Belgium & Luxembourg Member State Nadine Botteldoorn NRL Representative Nadine.Botteldoorn@wiv isp.be Bulgaria Member State Vanya Chikiova NRL Representative vchikova@abv.bg Croatia Member State Ines Škoko NRL Representative i.skoko.vzs@veinst.hr Denmark Member State Anna Charlotte Schultz NRL Representative acsc@food.dtu.dk Finland Member State Elina Vatunen NRL Representative Elina.Vatunen@tulli.fi Soizick Le Guyader NRL Representative Soizick.Le.Guyader@ifremer.fr Pascal Garry NRL Representative pascal.garry@ifremer.fr France Member State Dominique Hervio Heath NRL Representative dominique.hervio.heath@ifremer.fr Sylvain Parnaudeau NRL Representative sylvain.parnaudeau@ifremer.fr Jean Côme Piquet NRL Representative jean.come.piquet@ifremer.fr Germany Member State Eckhard Strauch NRL Representative Eckhard.Strauch@bfr.bund.de Greece Member State Ntina Vasileiadi NRL Representative iyt@otenet.gr Hungary Member State Zsuzsanna Sreterne Lancz NRL Representative LanczZs@nebih.gov.hu Iceland Member State Franklin Georgsson NRL Representative franklin.georgsson@matis.is 4

35 Country Country Status Delegate Specialist area E mail Ireland Member State Bill Dore NRL Representative bill.dore@marine.ie Elisabetta Suffredini NRL Representative elisabetta.suffredini@iss.it Italy Member State Francesca Leoni NRL Representative f.leoni@pg.izs.it Mario Latini NRL Representative m.latini@izsum.it Latvia Member State Nataļja Ivanova NRL Representative natalja.ivanova@bior.lv Lithuania Member State Audinga Verbickiene NRL Representative averbickiene@vet.lt Netherlands Member State Irene Pol Hofstad NRL Representative irene.pol@rivm.nl Norway Member State Mette Myrmel NRL Representative Mette.Myrmel@nvh.no Poland Member State Ewelina Bigoraj NRL Representative ewelina.bigoraj@piwet.pulawy.pl Portugal Member State Sonia Pedro NRL Representative spedro@ipimar.pt Romania Member State Alina Popescu NRL Representative Popescu.Alina@IDAH.RO Slovakia Member state Zuzana Kubicova NRL Representative zuz.kubicova@gmail.com Slovenia Member state Urška Henigman NRL Representative Urska.Henigman@vf.uni lj.si Spain Member state Cristina Acebal NRL Representative cacebal@msssi.es Sweden Member State Magnus Simonsson NRL Representative magnus.simonsson@slv.se United Kingdom Member State Craig Baker Austin NRL Representative Craig.baker austin@cefas.co.uk 5

36 Country Country Status Delegate Specialist area E mail David Lees EURL Director David.n.lees@cefas.co.uk James Lowther EURL Co ordinator James.lowther@cefas.co.uk EURL EURL Louise Stockley EURL Representative louise.stockley@cefas.co.uk Lisa Cross EURL Representative lisa.cross@cefas.co.uk Ron Lee EURL Representative Ron.lee@cefas.co.uk Italy Federica Barrucci Invited Expert Federica.BARRUCCI@efsa.europa.eu Netherlands Ingeborg Boxman Invited Expert i.l.a.boxman@nvwa.nl Spain Cristina Alvarez Invited Expert calvarez@intecmar.org United States Anna Newton Invited Expert ivz9@cdc.gov France Ainhoa Pare Observer ainhoa.pare@agriculture.gouv.fr 6

37 AGENDA 14 TH WORKSHOP OF MICROBIOLOGICAL NRLS, MAY 2015, FRANCE Venue: IFREMER Rue de l'île d'yeu Nantes France Tel.: Enquiries: Prior to the workshop enquiries should be directed to James Lowther or Samantha Arkell on: Direct line: +44 (0) (0) E mail : james.lowther@cefas.co.uk or samantha.arkell@cefas.co.uk Day 1 Wednesday 20 May 9:00 17:30 1 Introduction 1.1 Welcome to IFREMER (Murielle Millot, IFREMER, France) 1.2 Introductions and apologies (paper WS14/01) 1.3 Domestic arrangements including reclaim of expenses (papers WS14/02, WS14/03) 1.4 Actions arising from the 13 th workshop 2014 (paper WS14/04) 1.5 Agreement of the agenda (paper WS14/05) 1.6 EURL work programme 2015 (EURL) (paper WS14/06) 2 Official controls 2.1 Commission Restricted Working Group and trade negotiations (EURL) (paper WS14/07 ) Coffee/tea break (10:30 11:00) 2.2 Community Guide and Good Practise Guide (EURL) 2.3 Sanitary Survey of Ría de Aldán, special case for export to United States (Cristina Alvarez, INTECMAR) 2.4 The new national database for control of shellfish harvesting areas (NRL Italy) 2.5 Shellfish production in land based systems: how to manage shellfish quality (NRL Netherlands) 2.6 Microbiological monitoring, classification and sanitary surveys in Galicia (Cristina Alvarez, INTECMAR) 2.7 The International guide (EURL) 3 E. coli and Salmonella Lunch break (13:00 14:00) 3.1 E. coli and Salmonella methodology (EURL) 3.2 EURL proficiency testing for E. coli and Salmonella (EURL) (papers WS14/08, WS14/09) 3.3 Detection of E. coli contamination in urchins (NRL France) 3.4 Optimizing the preparative sample for enumeration of E. coli in Venus gallina by ISO/TS (NRL Italy) 3.5 Bivalve related outbreaks of non Vibrio bacterial pathogens (EURL) 4 Viruses Coffee/tea break (15:30 16:00) 4.1 Update on the progress of the validation of ISO TS under the CEN validation (M/381) (EURL) 7

38 4.2 Amendments to the EURL generic protocol (EURL) (paper WS14/10) 4.3 EURL proficiency testing for norovirus and HAV PT 53 and PT 55 (EURL) (paper WS14/11, WS14/12) 4.4 Proficiency Testing for viruses in shellfish in Italy ( ) (NRL Italy) Day 2 Thursday 21 May 9:00 17: International linkage of two food borne HAV clusters in the Netherlands in 2012 through trace back of mussels harvested in a non endemic area (Ingeborg Boxman, Food and Consumer Product Safety Authority, the Netherlands) 4.6 HAV in Naples area: investigation and control measures (NRL Italy) 4.7 Norovirus persists in oysters and may be implicated in an outbreak (NRL France) 4.8 NoV Concentrations in Individual Oysters implications for sampling and consumer exposure (NRL Ireland) Coffee/tea break (10:30 11:00) 4.9 Detection and phylogenetic analysis of noroviruses from bivalve molluscs at production areas in the republic of Croatia (NRL Croatia) 4.10 Norovirus analyses in 2 Dutch production areas with low levels of E.coli (NRL, the Netherlands) 4.11 EU harmonised baseline survey - Opinion of Member States (EURL) (paper WS14/13) - Provisional sampling plan (EFSA) (paper WS14/14) - Analytical considerations including quantification (round table discussion) Lunch break (13:00 14:00) 5 Marine vibrios 5.1 Management of bivalve related Vibriosis in the USA (Anna Newton, CDC, United States) 5.2 Validation of ISO TS under the CEN mandate (M/381) and revision of ISO TS (EURL) 5.3 Update on EURL proficiency testing for vibrios (EURL) Coffee/tea break (15:30 16:00) 5.4 A miniaturized MPN real time PCR method for rapid quantification of total and enteropatogenic Vibrio parahaemolyticus in shellfish (NRL France) 5.5 Overview of microbiological criteria for vibrios (EURL) (paper WS 14/15) Day 3 Friday 22 May 9:00 12:00 6 Any other business 7 Agreement of Workshop resolutions 8 Date and venue for next meeting Coffee/tea break (10:15 10:45) Meeting close After the meeting close lunch will be provided and there will be an opportunity to visit the IFREMER laboratories (optional). 8

39 List of papers for 14th Workshop of Microbiological NRL s WS14/00 WS14/01 WS14/02 WS14/03 WS14/04 WS14/05 List of papers Delegates List EURL Workshop Payment Details Form EURL Workshop Expenses Claim Form Actions from 13th workshop Agenda WS14/06 EURL Work Programme 2015 WS14/07 WS14/08 PT54 Report PT57 Report WS14/09 Method characteristics for detection of viruses in bivalve shellfish using ISO WS14/10 WS14/11 WS14/12 Generic Virus Protocol PT53 Report PT55 Report WS14/13 Blank Questionnaire for EU virus in LBM baseline survey WS14/14 Conclusions from questionnaire for EU virus in LBM baseline survey EURL Feb 2015 WS14/15 WS14/16 WS14/17 Number of Production Areas and Dispatch Centres for each Member State Validation of ISO TS under the CEN mandate Overview of microbiological criteria for vibrios 9

40 Minutes of the 14 th Workshop of Microbiological NRLs for Bivalve Molluscs, 20 th 22 nd May, 2015 Attendees EURL Director David Lees (DNL) (chair) Cefas, Weymouth, UK EURL Coordinator James Lowther (JL) Cefas, Weymouth, UK EURL Louise Stockley (LS) Cefas, Weymouth, UK EURL Ron Lee (RL) Cefas, Weymouth, UK EURL Lisa Cross (LC) Cefas, Weymouth, UK NRL Austria Johann Ladstaetter (JLa) AGES ILMU, Wien NRL Belgium and Luxembourg NRL Bulgaria Nadine Botteldoorn (NB) Vanya Chikiova (VC) Scientific Service of Food borne Pathogens, Brussels National Diagnostic and Research Veterinary Institute, Sofia NRL Croatia Ines Skoko (IS) Croatian Veterinary Institute, Split NRL Denmark Anna Charlotte Schultz (ACS) Institute of Food Safety and Nutrition, Søborg NRL Finland Elina Vatunen (EV) Finnish Customs Laboratory, Espoo Soizick Le Guyader (SG) Pascal Garry (PG) NRL France Dominique Hervio Heath Institut Français de Recherche pour L'Exploitation (DHH) de la Mer (IFREMER), Nantes Sylvain Parnaudeau (SPa) Jean Côme Piquet (JCP) NRL Germany Eckhard Strauch (ES) 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 NRL Italy NRL Latvia NRL Lithuania NRL Netherlands Elisabetta Suffredini (ELS) Francesca Leoni (FL) Mario Latini (ML) Nataļja Ivanova (NI) Audinga Verbickiene (AV) Irene Pol Hofstad (IPH) Istituto Superiore di Sanità (ISS) Rome 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 Ewelina Bigoraj (EB) National Veterinary Research Institute, Pulawy NRL Portugal NRL Romania Sonia Pedro (SPe) Alina Popescu (APo) Portuguese Institute of Sea and Atmosphere (IPMA), Lisbon Institute for Diagnosis and Animal Health, Bucharest NRL Slovakia Zuzana Kubicova (ZK) State Veterinary and Food Institute, Dolny Kubin 10

41 NRL Slovenia NRL Spain Urska Henigman (UH) Cristina Acebal (CA) 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 Anna Newton (AN) Centre for disease control, Atlanta, United States Invited expert Ingeborg Boxman (IB) Invited expert Federica Barrucci (FB) EFSA, Parma, Italy Invited expert Observer Cristina Alvarez Alvarez(CAA) Ainhoa Pare (APa) Apologies Paolo Caricato DG SANTE, European Commission Acronyms Food and Consumer Product Safety Authority, The Netherlands Centro de Control da Calidade do Medio Marino, Pontevedra Ministry of Agriculture, Agrifood, and Forestry, Paris, France CA Competent Authority NRL National Reference Laboratory CG Community Guidance OCL Official Control Laboratory CEN Comité Européen de Normalisation PCR Polymerase Chain Reaction DG SANTE Directorate General for Health and Food Safety PHE Public Health England EFSA European Food Safety Authority PT Proficiency Testing EU European Union RASFF Rapid Alert System for Food and Feed EURL European Union Reference Laboratory RNA Ribose Nucleic Acid FBO Food Business Operator SS Sanitary Survey GPG Good Practice Guide TAG Technical Advisory Group HAV Hepatitis A Virus TS Technical Specification ISO International Standard 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 NoV Norovirus 11

42 1. Introduction 1.1. Welcome to IFREMER Murielle Millot from IFREMER 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 13th workshop All actions identified were either complete or covered separately as agenda items Agreement of the agenda The workshop agenda (WS14/05) was agreed EURL Work Programme 2015 JL presented the EURL work programme (WS14/06) agreed with DG SANTE for the calendar year Official Controls 2.1. Commission Restricted Working Group and trade negotiations DNL gave an overview on the developments within the Restricted working group. In summary, it was agreed to adopt the Codex criterion for E. coli in end product, to make corresponding revisions to the criteria for class A, and to retain the Salmonella criterion for end product testing. Regarding control options for norovirus the Restricted WG had decided to follow the previous recommendation given by EFSA and to undertake a baseline survey for viruses in shellfish in MS, to be coordinated by EFSA with EURL support. Resolutions 1 2 Actions 1 4 DNL also provided an overview of the proposed amendment to Commission Regulation 2073 on microbiological criteria for foodstuffs. The use of heat treatment on imported products and NoV was discussed with reference to RASFF alerts against Vietnamese products. It was noted the interpretation of PCR results on heat treated produce was difficult. DNL stated EFSA have been given a mandate to investigate the application of community heat treatment requirements. DNL finished by reporting that the EU/US trade equivalence negotiations had been finalised and highlighted the latest amendments Community Guide and Good Practice Guide RL presented the latest changes to the CG and GPG including guidance on prohibited zones (based on sewage levels). Some issues in applying the GPG were raised amongst NRLs and some discussion of application of method uncertainty to microbiological results was held. Resolution 3 Actions

43 2.3. Sanitary Survey of Ría de Aldán, special case for export to United States (Cristina Álvarez Álvarez, INTECMAR, Spain) CAA presented on the SS work undertaken in the Ria de Aldan estuary in preparation for possible exportation to the US of mussels and oysters from 2 specified harvesting areas within the estuary. It was evident from the data presented that a lot of work was involved to complete this SS and a very thorough job had been done. DNL requested an update on the progress of the export negotiations at the next WS. Action The new national database for control of shellfish harvesting areas (NRL Italy) A database for controlling production areas in Italy was presented by ML. OCLs can easily upload their data with each production area having a unique code, map and specified sampling points. At present the information retained is not available in the public domain Shellfish production in land based systems: how to manage shellfish quality (NRL Netherlands) IPH presented to the group the work NRL Netherlands had undertaken regarding the development of land based shellfish production areas. It was noted that there is no guidance written in legislation identifying how classification of a land based production areas should be performed. It was agreed that clarify was needed from the CA Microbiological monitoring, classification and sanitary Surveys in Galicia (Cristina Álvarez Álvarez, INTECMAR, Spain) CAA presented information on management of shellfish monitoring in Galicia. Some discussion of the approach to results associated with anomalous rainfall or sewage events, both in Galicia and across the EU was undertaken. Resolution 4 Actions Resolution 5 Actions The International guide (EURL) RL gave a presentation on the developing International Guide (Technical Guidelines for the Development of Shellfish Sanitation Programmes), and explained that this guide was designed to help third countries with implementation of regulations to enable safe shellfish sanitation systems. 3. E.coli and Salmonella 3.1. E.coli and Salmonella methodology (EURL) RL provided updates on the progress with publication of new versions of the ISO standards relevant to the E.coli and Salmonella methods and confirmed that the EURL generic protocols would need to be updated as a result EURL proficiency testing for E. coli and Salmonella (EURL) LS presented a summary of the performance of the NRLs network in the last two E.coli and Salmonella proficiency testing schemes (PT54 and PT57). Problems arising from incorrect application of the MPN calculator spreadsheet referenced in ISO 7218 were discussed, and a number of actions to improve the situation identified Detection of E.coli contamination in urchins (NRL France) JCP gave a presentation on research into E.coli in sea urchins from an area in southern France. The WS discussed the surprisingly high levels detected and noted the potential implications for classification and human health. Actions Resolution 6 Actions Resolution 7 13

44 3.4. Optimizing the preparative sample for enumeration of E. coli in Venus gallina by ISO/TS (NRL Italy) FL presented an investigation into the use of reduced sample weights (25g cf g) for the clam Venus gallina in the standard E.coli method. Although no statistically significant differences between the sample weights were noted the WS noted some possible non significant effects, possibly requiring extra investigation. The WS then moved onto a discussion of certain technical difficulties surrounding the testing of sea cucumbers for E.coli. 4. Viruses 4.1. Update on the progress of the validation of ISO TS under the CEN validation (M/381) (EURL) JL presented the group with the progress with the virus validation, with particular focus on the bivalve mollusc matrices. The method characteristics determined through the validation exercise, and particularly the approach used to generate LOD and LOQ characteristics were discussed, and it was agreed that guidance for laboratories for determination of in house LOD and LOQ values would be important. The issue of viruses in non bivalve foods was also briefly discussed, and the WS agreed that while this issue fell outside of the scope of the NRLs network, they would be interested in developments in this area. Action 19 Resolutions 8 9 Action Amendments to the EURL generic protocol (EURL) JL detailed the changes made to the virus generic protocol to harmonise with the new draft of ISO EURL proficiency testing for norovirus and HAV PT 53 and PT 55 (EURL) JL gave a presentation of the performance of the NRLs in the last two virus proficiency testing round. Encouraging progress was noted in terms of participation, method harmonisation and qualitative detection. Some variation in the quantitative results was noted, with evidence that some laboratories were making incorrect calculations presented. The WS agreed that provision by the EURL of a calculation spreadsheet would be helpful. In addition it was recognised that in house generation of quantification controls was a potential source of error that could be improved through e.g. development of commercially available standards. Resolutions Actions Proficiency Testing for viruses in shellfish in Italy ( ) (NRL Italy) ELS provided details of the virus proficiency testing schemes in operation in Italy in the last years. Through this initiative improvements in performance across the OCL network in Italy had been effected International linkage of two food borne HAV clusters in the Netherlands in 2012 through trace back of mussels harvested in a nonendemic area (Ingeborg Boxman, Food and Consumer Product Safety Authority, the Netherlands) IB provided an in depth analysis of an investigation into an outbreak of HAV in the Netherlands linked to consumption of mussels harvested in another MS. Sequences from the Dutch clusters matched those from a nonfoodborne cluster in the catchment of the mussel production area, and the timings of the cluster were consistent with a causal relationship mediated 14

45 via contamination of the mussels in the production area. The index case in the production area cluster had a history of travel to an HAV endemic region HAV in Naples area: investigation and control measures (NRL Italy) ELS presented data on an outbreak of HAV in the Naples area with consumption of locally produced shellfish as a risk factor. HAV presence was detected in shellfish samples from local production areas. Sequencing of clinical and shellfish positives indicated a linkage, and ruled out a linkage with a large outbreak of HAV related to consumption of frozen berries. The sequences clustered with those found in an HAV endemic region outside Europe. Following on from this and the previous presentation the WS considered the risks posed by HAV in shellfish in the EU, and discussed possible improvements to risk mitigation practices Norovirus persists in oysters and may be implicated in an outbreak (NRL France) SPa gave a presentation on a case study of outbreaks of norovirus linked to a single oyster production area in France in the years In one case the area was re opened following an outbreak, despite the persistence of norovirus signal in the oysters, and some weeks later a further illness outbreak was linked to the area. The delegates considered the implications of this for risk management NoV Concentrations in Individual Oysters implications for sampling and consumer exposure (NRL Ireland) BD presented a study where norovirus concentration in 30 individual oysters within a single growing bag was assessed. Although variation from oyster tooyster was noted, statistical assessment suggested that pooling 10 oysters as in ISO should give a result that was representative of the levels in the population. Resolutions Resolution 20 Resolution Detection and phylogenetic analysis of noroviruses from bivalve molluscs at production areas in the republic of Croatia (NRL Croatia) IS presented the results of an initial survey into norovirus in production areas in Croatia. Although no NoV illness has yet been linked to consumption of Croatian shellfish, viral RNA was found in ~10% of samples Norovirus analyses in 2 Dutch production areas with low levels of E.coli (NRL, the Netherlands) IPH detailed a small survey of 2 Dutch stable class A sites for norovirus. Although one site was negative for norovirus, high levels were found in the other, highlighting the limitations of E.coli as an indicator of viral risk. The WS discussed this issue and also noted the encouraging use of ISO methods in this and previous presentations EU harmonised baseline survey The WS considered emerging plans for an EU wide harmonised survey for viruses in oysters. Resolution 21 Resolution 18 Action 24 Opinion of Member States (EURL) DNL prepared a summary of responses from the MS CAs to a questionnaire designed to define the scope of the survey. 15

46 Provisional sampling plan (EFSA) APa provided an overview of the responsibilities and activities of EFSA, particularly with regards to their previous organisation of EU baseline surveys, and presented the basics of the draft sampling plan. The WS discussed the survey and agreed that additional considerations e.g. logistics and cost were important and should be taken into account. Analytical considerations including quantification (round table discussion) The WS discussed analytical considerations regarding the baseline survey. 5. Marine vibrios 5.1. Management of bivalve related Vibriosis in the USA (Anna Newton, CDC, United States) AN provided a comprehensive overview of the US system for management of shellfish related Vibriosis including the Cholera and Other Vibrio Illness Surveillance (COVIS) system, and presented the data generated therein. The WS discussed how the lessons of the US system could be used to improve the understanding of shellfish related Vibriosis in the EU Validation of ISO TS under the CEN mandate (M/381) and revision of ISO TS (EURL) CBA presented a summary of the progress of the validation of the vibrio ISO including the results. NRLs that had taken part in the validation noted that they had not seen the full data set and were not able to put their results in context. The EURL agreed to collate and distribute the full data set. The WS discussed the findings of the validation and all agreed that the methods as currently included in the ISO were not sufficiently fit for purpose and needed improvement. DNL suggested that the NRLs network should take a lead on developing improved methodology since the network had the relevant expertise. Some delegates expressed confusion at the current state of vibrio standardisation within the CEN/ISO organisation due to the emergence of a new vibrio group TAG15 with CEN TC275/WG6, while up to that point vibrio standardisation had fallen within the remit of TAG3. The EURL agreed to investigate and clarify the situation Update on EURL proficiency testing for vibrios (EURL) LS provided a brief update on the ongoing vibrio proficiency testing scheme PT 58. The WS agreed that this and future vibrio PT schemes should be used to try to improve methodology. Resolution 22 Action 25 Resolution 23 Actions Action A miniaturized MPN real time PCR method for rapid quantification of total and enteropatogenic Vibrio parahaemolyticus in shellfish (NRL France) DHH gave a presentation on the development of a PCR based method for enumeration of total and pathogenic Vibrio parahaemolyticus, which has been identified by ISO/CEN as a priority for future methods development. The WS noted that this was a welcome development and that it would be helpful to share the protocol within the network to further method development and harmonisation, DHH agreed subject to final revisions of the method. 16

47 5.5. Overview of microbiological criteria for vibrios (EURL) In response to a request from NRL Germany, CBA gave a presentation on international microbiological criteria for vibrios in shellfish and other foods. A wide diversity of approaches was noted and the WS agreed that there was currently no international consensus, and it was therefore difficult to make recommendations for criteria within the EU. 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 at the Federal Institute for Risk Assessment, Berlin, Germany, 25 th 27 th May Resolution 23 17

48 Actions of the 14 th Workshop of Microbiological NRLs for Bivalve Molluscs, Ifremer, Nantes 20 th 22 nd May, 2015 Action Owner Completed Notes Official controls 1. EURL to revise Note 16 (regarding pooled samples) to the proposed amendment to Commission Regulation 2073 on microbiological criteria for foodstuffs 2. EURL to check text of 2073 to see whether stipulation of recording unit as MPN/100g in Table 1.25 of the proposed amendment is appropriate EURL EURL 3. Gastropods to be added to note 5 of the proposed amendment for consistency EURL 4. EURL to update guidance documents following publication of the amended regulations EURL 5. EURL to distribute Commission discussion document on measurement uncertainty (MU) to the NRLs network EURL 6. EURL to include advice on how to determine MU in revised guidance documents EURL 7. NRLs to advise the EURL on any issues they have had when applying the guidance documents so that these issues can be included in the revision NRLs 8. EURL to check that all community guidance has been changed regarding alternative methods for E.coli. EURL 9. Cristina Alvarez Alvarez (CAA) to provide feedback to the next workshop on negotiations/audits relating to export of LBMs from Spain to the USA CAA 10. NRL Netherlands to ask Netherlands Competent Authority to seek clarification on the legislative requirements for classification of land based LBM production systems 11. EURL to consider how to address monitoring of land based and multi trophic systems in the Good Practise Guide 12. CAA to send the Galician classification protocol to the EURL CAA 13. EURL to include further clarification in the Good Practice Guide on waiving results due to high rainfall events EURL E.coli and Salmonella NRL Netherlands EURL 14. EURL to review and, where necessary, update E.coli generic protocol following publication of new version of ISO EURL 15. EURL to review and, where necessary, update Salmonella generic protocol following publication of new EURL 18

49 versions of ISO 6579 part EURL to review and, where necessary, update E.coli and Salmonella generic protocols following publication of new version of ISO EURL to propose to ISO that the MPN calculator referenced in ISO 7218 be amended to include a bivalvespecific worksheet, to be made more user friendly, and to be accompanied by a user guide. The EURL also to request information from ISO on how the calculator was validated and to provide this information to NRLs 18. In the short term, the EURL to amend the generic E.coli protocol to provide guidance on the use of the calculator 19. NRL Iceland to provide clarification on whether production of sea cucumbers in Iceland (for export to China) is subject to classification EURL EURL EURL NRL Iceland Viruses 20. EURL to produce guidance document on approaches for laboratories to determine limit of detection and limit of quantification for in house application of ISO EURL 21. EURL to provide worked examples of quantification calculations for PT 55 EURL 22. EURL to provide a calculation spreadsheet for ISO/TS by e mail to NRLs and also on the website EURL 23. EURL to supply quantification standards with the next matrix PT for viruses. In addition information on inhouse generation of quantification standards to be requested from participants EURL 24. NRLs from producer states to ask Competent Authorities to ask about funding for the EFSA baseline survey at the next restricted working group meeting Marine vibrios 25. EURL to request from Anna Newton (CDC, Atlanta USA) papers demonstrating the effectiveness of new food safety procedures (icing etc.) in reducing shellfish related vibriosis and to inform NRLs 26. EURL to collate results of the CEN mandate vibrio validation study and forward to all study participants EURL 27. EURL to clarify current status with regards to vibrio standardisation (particularly with regards to the new CEN TC275 WG6 TAG15) and inform NRLs 28. EURL to organise a discussion of vibrio specialists from within the NRLs to agree future strategies for method EURL NRLS from producer states EURL EURL 19

50 improvement; NOTE: following clarification of ISO responsibilities post workshop, EURL will progress this objective through TAG NRLs to share potentially useful in house protocols for vibrio detection/quantification with the discussion group; NOTE: see action 28 protocols will be shared with TAG EURL to request methodology used by participants in vibrio PT 58, to analyse the impact of methodology on results, and to include findings in PT report. NRLs EURL 20

51 Resolutions of the 14 th workshop of microbiological NRLs for bivalve molluscs, nd May 2015 Official controls 1. The EURL briefed the workshop on pending legislative changes regarding classification and end product criteria for E.coli. The EURL noted that they would amend Community Guidance appropriately following adoption of the Community legislation. NRLs agreed to notify the EURL of any other issues that should be considered in this revision. 2. The workshop noted the recent RASFF alerts regarding norovirus contamination in cooked products imported into the EU. It was agreed that the use of PCR to assess risk in cooked products was not appropriate. The workshop was informed that EFSA had received a mandate from the Commission to investigate the application of Community heat treatment requirements and would report in due course. 3. With respect to the potential application of measurement uncertainty to microbiological results, the EURL noted that this was due to be discussed between the Commission and Member States. The EURL would circulate a previous Commission discussion document on this topic and draft a section covering this issue in Community Guidance. 4. The workshop considered developments in land based and enclosed production systems for bivalve molluscs, noted the lack of clarity regarding the legal basis for classification, and agreed that this needed to be clarified by Competent Authorities. Following this clarification the EURL agreed to develop monitoring guidance for incorporation within Community Guidance, but also noted that it was the responsibility of FBOs to incorporate risks from such systems into their HACCP plans. 5. Following discussion of approaches applied in Member States to disregard E.coli results associated with high rainfall the EURL agreed to review and further clarify the Good Practice Guidance on this issue. E.coli and Salmonella 6. NRLs identified that there was no appropriate guidance on the use of the MPN Calculator referenced in ISO The EURL undertook to propose to ISO that the MPN calculator be amended to include a bivalve specific worksheet, to be made more user friendly, and to be accompanied by a user guide. The EURL also agreed to request information from ISO on how the calculator was validated and to provide this information to NRLs. In the short term, the EURL would amend the generic E.coli protocol to provide guidance on the use of the MPN calculator. 7. The workshop considered a presentation on E.coli levels in echinoderms (Paracentrotus lividus) in the vicinity of a pollution source and noted surprisingly high levels of contamination. The implications for classification of this species were noted. The workshop noted a lack of data and experience on approaches to official control testing and classification for other non bivalve species covered by EU legislation. 21

52 Viruses 8. In the content of virus outbreaks in non bivalve mollusc foods the workshop briefly discussed whether there were any plans for establishment of a EURL/NRL network for this issue. The importance of retaining virus responsibility within the network of bivalve microbiology NRLs was endorsed by all participants of the workshop. It was agreed that viruses in non bivalve foods fell outside of the remit of the bivalve mollusc network but the group was interested in developments on viruses in other foods and exchange of information. 9. The workshop considered the performance characteristics of the virus method as determined by the validation of ISO/TS under mandate M/381. It was noted that, for accreditation purposes, laboratories would be required to generate their own limits of detection and quantification. The EURL agreed to produce guidance on approaches for laboratories to determine these characteristics. 10. The results of virus PT distributions 53 and 55 showed continued improvements in qualitative performance and method harmonisation amongst NRLs. However, further improvements to quantitative comparability would be desirable. 11. Further to the above the EURL agreed to assist NRLs with calculation of sample concentrations from Ct values, by provision of worked examples for PT 55 and by placing on the EURL website a calculation spreadsheet for ISO/TS Further to resolution 10 the workshop also agreed that in house generation of quantification standards could contribute to variability. The EURL agreed that, for the next matrix PT, quantification standards would be supplied and information on in house generation of standards requested from participants. The EURL would analyse responses in relation to the standards generation methodology utilised, to identify potential sources of variability. 13. The EURL agreed to continue to develop accessible quantification standards to assist laboratories with the application of ISO/TS It was agreed to continue virus PT with 2 distributions per year including one matrix distribution which should include at least one whole animal sample (shucked or unshucked). 15. The workshop considered presentations on recent hepatitis A virus outbreaks attributed to consumption of bivalve shellfish produced in the EU. It was recognised that with the low immune status of hepatitis A virus in the EU, and the low infective dose of this virus, that sporadic community cases of hepatitis A virus in coastal locations could present a high risk for shellfish contamination. 16. Further to the above, the workshop recommended that risk mitigation for hepatitis A virus needs to be improved and this would be achieved by better communication between health authorities and shellfish risk managers. In this context it was noted that laboratory analysis significantly assists risk management. 17. Further to resolution 15 the workshop considered resolutions 11 and 14 of 2012 in relation to risks posed by shellfish contamination with hepatitis A virus and 22

53 Marine vibrios norovirus respectively. The workshop agreed that emerging data confirmed these resolutions and that they continued to be appropriate advice. 18. The workshop considered emerging plans for an EU wide harmonised survey for viruses in oysters. EFSA presented considerations for the sampling plan. It was agreed that it was important to consider analytical aspects, volumes of shellfish production, logistics and cost in designing the sampling plan. 19. The workshop noted data on variation in norovirus levels between individual oysters and its implications for risk assessment. Furthermore, it was noted that the data reinforced the approach in ISO/TS of analysis of a pooled sample of 10 animals. 20. The workshop considered data on norovirus monitoring in oyster production areas associated with outbreaks. It was noted that outbreaks may reoccur in areas that remain virus contaminated but are reopened. The workshop noted the implications of this for risk management. 21. The workshop noted the application of ISO/TS in diverse Member States and that the method seemed to provide useful risk management information. However the lack of correlation between E.coli and norovirus contamination was noted in several studies. 22. The workshop considered a presentation from CDC Atlanta USA giving a comprehensive overview of vibriosis in the USA. The workshop noted the significant underreporting of this disease in the USA, despite it being a notifiable illness, and considered that it was possible that vibriosis could be significantly underreported in the EU. More structured epidemiological reporting in the EU would improve understanding of the health burden from vibrios associated with shellfish consumption. 23. The workshop considered findings from the validation studies of the Vibrio method and agreed that aspects of the methodology were not yet satisfactory. The EURL agreed to organise a discussion of specialists to agree future strategies for method improvement. To assist this it was agreed that the detailed results of the validation study will be shared with NRLs. Furthermore it was agreed that NRLs would share relevant methods with the discussion group. Future EURL PT would be used to support such methodological improvements. 24. The EURL presented information on international microbiological criteria regarding vibrios in seafood. The workshop agreed that no international consensus had yet emerged and that it was therefore difficult to recommend appropriate microbiological criteria relevant to laboratory testing in the EU. 25. The next workshop will be held on 25 th 27 th May 2016 at the Federal Institute for Risk Assessment, Berlin, Germany. 23

54 EURL Workshop 20 th to 22 nd May 2015, Nantes Confidential Participant Feedback Results 24

55 Comments : 1. a. We were really out of the agenda. It is important to keep it. 2. a. More time for discussion. b. More participation of more laboratory. Comments from an observer 3. a. Very interesting (almost) on Wednesday (unfortunately I could not attend the end of the workshop). b. Too much topics and not enough discussion because we have not all presentations* to prepare the discussion. Suggestion 1) put apart laboratories topics (i.e. 3.1, 3.2, 3.4, 4.1 of agenda) 2) Please invite competent authorities to discuss about monitoring, management, guidance (at least 2 days twice a year!) (i.e. 2.2, 2.3, 2.5, 2.6, 2.7, 3.3 of the agenda +4.6, 4.7, , 5.5 ) *These documents should contain Presentation (of the case on local situation) Proposition for harmonising : through EU legislation or through guidance 25

56 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 Nantes 20th 22nd May Dr David Lees EURL Director 7 th October 2015 Dr James Lowther EURL Co ordinator 7 th October

57 Annex IV European Union Reference Laboratory (EURL) Proficiency Testing Scheme Noroviruses and hepatitis A virus in bivalve molluscan shellfish EURL PT reference number: PT55 Final report version pages Contract Reference: Cefas ref (C6095) Document approved by: C6095 Project Manager Rachel Hartnell Review date: 30 h April 2015 Document checked by: David Lees Classification: Official Document prepared by: Louise Stockley and James Lowther Location PT CRL$

58 Contents Page number Samples 2 Results 3 Conclusion and discussion 7 References 7 Appendices 8 Proficiency testing 55 Final 1 Page 1 of 21

59 Samples Materials dispatched consisted of bioaccumulated Pacific oysters (Crassostrea gigas) and common mussels (Mytilus edulis) (Samples 1, 2, 3 and 4), laboratory constructed test sample LENTICULES, dsdna LENTICULES and dsdna control solutions for quantification (1x10 5 copies/µl) for each target virus. The reference results for each shellfish sample and test sample LENTICULE are shown in Table 2. Sample preparation Shellfish Sample 1 Approximately 500 Pacific oysters (Crassostrea gigas) from a UK commercial harvesting area were collected following commercial purification. Testing of the sample indicated the shellfish were negative for HAV, GI and GII norovirus. The oysters were shucked, then randomly sorted into samples of 10 animals and placed in a 250ml sample bottle. The sample was held at <-15 C until required for quality control testing, dispatch or reference analysis. Shellfish Sample 2 Approximately 500 Pacific oysters (Crassostrea gigas) were placed in trays and re-immersed in 500 litres of recirculating natural seawater at 16±1 C. The shellfish were left for 24 hours to acclimatise before 50ml of shellfish food containing known levels of genogroup I (GI) norovirus was added to the tank. After approximately 16 hours the shellfish were removed from the tank, the oysters were shucked, then sorted randomly into samples of 10 animals and placed in 250ml sample bottles. The samples were held at <-15 C until required for quality control testing, dispatch or reference analysis. Shellfish Sample 3 Approximately 500 Pacific oysters (Crassostrea gigas) were placed in trays and re-immersed in 500 litres of recirculating natural seawater at 16±1 C. The shellfish were left for 24 hours to acclimatise before 50ml of shellfish food containing known levels of genogroup I and II (GI and GII) norovirus from human faeces and hepatitis A virus (HAV) cell culture supernatant was added to the tank. After approximately 16 hours the shellfish were removed from the tank, the oysters were shucked, then sorted randomly into samples of 10 animals and placed in 250ml sample bottles. The samples were held at <-15 C until required for quality control testing, dispatch or reference analysis. Shellfish Sample 4 Approximately 500 common mussels (Mytilus edulis) were placed in trays and re-immersed in 500 litres of recirculating natural seawater at 16±1 C. The shellfish were left for 24 hours to acclimatise before 50ml of shellfish food containing known levels of GI and GII norovirus from human faeces and HAV cell culture supernatant was added to the tank. After approximately 16 hours the shellfish were removed from the tank, the oysters were shucked, then sorted randomly into samples of 10 animals and placed in 250ml sample bottles. The samples were held at <- 15 C until required for quality control testing, dispatch or reference analysis. 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 mix prepared for LENTICULE 2 included known levels of GI and GII norovirus from human faeces and HAV cell culture supernatant. Table 1 shows details of the stock viruses used in the preparation of LENTICULE 2. 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) Proficiency testing 55 Final 1 Page 2 of 21

60 Sample distribution Samples were dispatched on dry ice in accordance with IATA packing instructions 650 for UN3373 Diagnostic Specimens on 17 th November 2014 or shortly thereafter to 48 participating laboratories. Participants were requested to analyse the test 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 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 samples and test sample LENTICULES 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. Note that the subsamples tested for Shellfish Sample 4 provided both positive and negative results for norovirus GII; for this reason participant results for this sample and genogroup are not considered in performance scoring. Table 2: Reference results for PT 55 proficiency testing material Norovirus Sample GI GII HAV Shellfish sample 1 a Shellfish sample 2 a - + (2.67 x x 10 4 ) - Shellfish sample 3 a + (1.92 x x 10 3 ) + (1.30 x x 10 3 ) + (1.61 x x 10 2 ) Shellfish sample 4 a + (7.69 x x 10 3 ) + (1.49 x x 10 1 ) c + (5.12 x x 10 3 ) Test sample LENTICULE 1 b Test sample LENTICULE 2 b + (4.60 x x 10 2 ) + (7.38 x x 10 3 ) + (1.33 x x 10 4 ) a Copies / g; b Copies / LENTICULE. c some subsamples provided negative results participant results for this sample and genogroup are 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. The dsdna LENTICULES provided were designed to contain 1x10 8 copies/lenticule. Participants results Performance assessment was assessed as percentage relative accuracy, specificity and sensitivity for each determinant according to the calculations described in Appendix II. As this proficiency testing distribution included both shellfish matrices and test sample LENTICULE discs, an overall performance assessment was performed to assess each laboratory s performance for all samples (Table 3) as well as assessing the performance on shellfish matrices (Table 4) and LENTICULE discs only (Table 5). Note: Participants results were expressed as percentage concordance with intended results generated by the EURL. In this assessment presence/absence data was used and no consideration of quantitative measurements (Ct values) were made. Proficiency testing 55 Final 1 Page 3 of 21

61 Table 3: Participants results for all dispatched material Lab ID GI GII HAV Performance score No. AC SP SE AC SP SE AC SP SE NoV HAV B B 3* A A 7* A A 9* NR NR NR NR NR NR NR NR NR * C A 13* A A C C 17* A A 19* A A A A 21* A A A A 25* A A 27* A A 32* A A 33* A A NE NE NE C - 35* B A 39* A B 41* A A 43* NR NR NR NR NR NR NR NR NR * A B C C A A B A NE NE NE A - 90* A A A A A A A A A A 102* A A A C 147* C C A A A A A A A A 186 NE NE NE NE NE NE A A B A A A A A A A A 218 NE NE NE NE NE NE A NE NE NE A A A NE NE NE C - * - Designated NRL, NE= Determinand not examined, NR = Results not returned. AC - Relative accuracy, SP Relative specificity, SE Relative sensitivity. Performance scoring; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Norovirus (NoV) and HAV scored separately. Note 29/46 laboratories that returned results tested all determinands and scored 100% overall accuracy. 4/46 laboratories scored 100% overall accuracy but did not test all determinands. Proficiency testing 55 Final 1 Page 4 of 21

62 Table 4: Participants results for all shellfish material (Samples 1, 2, 3 and 4) Lab ID GI GII HAV No. AC SP SE AC SP SE AC SP SE * * * NR NR NR NR NR NR NR NR NR 10* * * * * * * * * NE NE NE 35* * * * NR NR NR NR NR NR NR NR NR 47* NE NE NE 90* * * NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE * - Designated NRL, NE= Determinand not examined, NR = Results not returned. AC - Relative accuracy, SP Relative specificity, SE Relative sensitivity. Note 29/46 laboratories that returned results tested all determinands and scored 100% overall accuracy for shellfish samples. 4/46 laboratories scored 100% overall accuracy for shellfish samples but did not test all determinands. Proficiency testing 55 Final 1 Page 5 of 21

63 Table 5: Participants results for test sample LENTICULES (L1 L2) Lab ID GI GII HAV No. AC SP SE AC SP SE AC SP SE * * * NR NR NR NR NR NR NR NR NR 10* * * * * * * * * NE NE NE 35* * * * NR NR NR NR NR NR NR NR NR 47* NE NE NE 90* * * NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE * - Designated NRL, NE= Determinand not examined, NR = Results not returned. AC - Relative accuracy, SP Relative specificity, SE Relative sensitivity. Note 38/46 laboratories that returned results tested all determinands and scored 100% overall accuracy for test sample LENTICULES. 5/46 laboratories scored 100% overall accuracy for test sample LENTICULES but did not test all determinands. Proficiency testing 55 Final 1 Page 6 of 21

64 Conclusion and discussion General comments Forty-eight laboratories (20 NRLs and 28 other laboratories) received samples. Laboratories 9 and 43 did not return results. Laboratories 186 and 218 did not examine for GI or GII norovirus and laboratories 34, 72, 222 and 231 did not examine for HAV. Results reported to the EURL are shown in Appendices III - VII. Discussion Thirty-three (72%) of the laboratories which returned results obtained the intended results (as determined by EURL reference designations) for all samples, for all determinands that they tested. The overall accuracies across all laboratories were 94%, 96% and 95% for GI, GII and HAV respectively. The false positive reporting rates for GI, GII and HAV were 2%, 0% and 0% respectively. The false negative reporting rates for GI, GII and HAV were 10%, 7% and 10% respectively. The overall accuracies for LENTICULES were 98%, 98% and 100% for GI, GII and HAV respectively and for shellfish samples they were 93%, 95% and 93% for GI, GII and HAV respectively. The false positive reporting rates for LENTICULES were 2%, 0% and 0% for GI, GII and HAV respectively and in shellfish samples the false positive reporting rates were 1%, 0% and 0% respectively. The false negative reporting rates for GI, GII and HAV were 5%, 5% and 0% for LENTICULES and 10%, 8% and 14% for shellfish samples respectively. Forty four laboratories (96%) returned data expressed as Ct values for at least one test sample or the dsdna material (Appendices III, IV and V). Thirty one laboratories (67%) returned quantitative data using their own routine method and/or using the standard materials provided expressed as detectable copies per g or copies per LENTICULE for at least one sample (Appendix VI). Quantitative results from individual labs alongside reference results are shown graphically in Appendix VII. Reference results were not corrected using extraction efficiency data. Results (Ct values +/- quantities) for the dsdna LENTICULES and control solutions are shown as Appendix V. Methods used by participants to analyse the shellfish matrix, with the labs listed according to their overall accuracy score for the shellfish samples, are shown in Appendix VIII. References Codd AA, Richardson IR, Andrews N Lenticules for the control of quantitative methods in food microbiology. J Appl Microbiol. 85(5): Anonymous 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. Proficiency testing 55 Final 1 Page 7 of 21

65 Appendix I: EURL reference results displayed as box and whisker plots (log scale) of detectable genome copies per gram or 25μl LENTICULE. Shellfish sample 2 Shellfish sample 3 SF2 - GII SF3 copies per gram copies per gram GI GII HAV Shellfish sample 4 Proficiency testing 55 Final 1 Page 8 of 21

66 LENTICULE 2 Proficiency testing 55 Final 1 Page 9 of 21

67 Appendix II: Percentage relative sensitivity: Relative sensitivity (SE) = TP (TP+FN) Percentage relative specificity: Relative specificity (SP) = TN (TN+FP) Percentage relative accuracy: Relative accuracy (AC) = TP+TN N Where TP = true positives FN = false negatives FP = false positives TN = true negatives N = total number of tests x 100% x 100% Note: Participants results were expressed as percentage concordance with intended results generated by the EURL. In this assessment presence/absence data was used and no consideration of quantitative measurements (Ct values) was made. Proficiency testing 55 Final 1 Page 10 of 21

68 Appendix III: Participants results and Ct values for shellfish samples Shellfish sample 1 Shellfish sample 2 Shellfish sample 3 Shellfish sample 4 Lab ID No. GI GII HAV GI GII HAV GI GII HAV GI GII HAV - Ct - Ct - Ct - Ct + Ct - Ct + Ct + Ct + Ct + Ct +/- a Ct + Ct NR b NR b NR b 3* * * NR NR NR NR NR NR NR NR NR NR NR NR 10* * * * * * * * * NE c NE c NE - - NE 35* * * * NR NR NR NR NR NR NR NR NR NR NR NR 47* c c NE NE NE NE 90* * * NE NE - NE NE - NE NE NE NE d d d d NE NE - NE NE - NE NE + NE NE NE NE NE NE NE - - NE - - NE - - NE *= Designated EU member state NRL, NE= determinand not examined, NR= results not returned, dark red denotes false negative, dark blue denotes false positive results a; some reference subsamples provided negative results participant results for this sample and genogroup are not considered in performance scoring, negative and positive participant results highlighted in light red and light blue respectively for information. b; participant lab did not return results for SF sample 4, results are scored as incorrect. c; Ct values recorded considered too high by participant lab and results reported as negative. d; Ct values derived from 1/10 diluted RNA. Proficiency testing 55 Final 1 Page 11 of 21

69 Appendix IV: Participants results and Ct values for test sample LENTICULES Test sample LENTICULE 1 Test sample LENTICULE 2 Lab ID No. GI GII HAV GI GII HAV - Ct - Ct - Ct + Ct + Ct + Ct * * * NR NR NR NR NR NR 10* * * * * * * * * NE NE 35* * * * NR NR NR NR NR NR 47* NE NE 90* * * NE NE - NE NE a a NE NE - NE NE NE NE NE - - NE *= Designated EU member state NRL, NE= determinand not examined, NR= results not returned, dark red denotes false negative, dark blue denotes false positive results a; Ct values derived from 1/10 diluted RNA. Proficiency testing 55 Final 1 Page 12 of 21

70 Appendix V: Participants results and Ct values for dsdna control solutions and dsdna LENTICULES Lab ID No. GI GII HAV a b c d a b c d a b c d E E E E+08 3* E E E E E E+08 7* E E E E E E+07 10* E E E E E E+08 13* * E E E E E E+08 19* E E E+07 21* E E E E E E E+08 25* E E E E E E+07 27* E E E E * E E E+08 33* E E E E E+05 39* E E E+04 41* E E E E E E+07 47* E E E E E E E E E E E E E E E E+08 90* E E E E E E E E E E E E E E E * E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E a (Average) Ct value for undiluted dsdna control solution (1x10 5 copies/µl); b Ct value for dsdna LENTICULE (1x10 8 copies/lenticule; 1x10 5 copies/µl in solution following preparation according to instruction sheet); c - Quantity for dsdna LENTICULE in copies/lenticule determined using routine method; d - Quantity for dsdna LENTICULE in copies/lenticule determined using standard material provided. Labs which did not report any Ct values or quantities for the dsdna control materials and LENTICULES not included. Proficiency testing 55 Final 1 Page 13 of 21

71 Appendix VI: Participants reported quantities for each target (copies/g (shellfish) or copies/lenticule) Lab ID No. Shellfish sample 2 Shellfish sample 3 Shellfish sample 4 Test sample LENTICULE 2 GII GI GII HAV GI GII a HAV GI GII HAV a b a b a b a b a b a b a b a b a b a b E E E E E E E E E E+03 3* 1.55E E E E E E E E E E E E E E E E E E+03 7* 3.10E E E E E E E E E E E E E E E E E E+04 10* 9.66E E E E E E E E E E+02 <1.00E+02 <1.00E E E E E E E E E+03 13* 1.60E E E E E E E E E E E E E E E E E E+02 17* 2.33E E E E E E E E E E E E E E E E E+04 19* 6.68E E E E E E E E E+04 21* 6.48E E E E E E E E E E E E E E E E E E E E E E E E E E+03 25* 1.80E E E E E E E E E E E E E E E E E E E E+03 27* 1.02E E E E E E E E E E E E E E E E E E E E+03 32* 4.18E E E E E E E E E+04 33* 5.30E E E E E E E E E E-01 b 3.70E-01 b 3.10E E+00 39* <1.00E+02 <1.00E+02 <1.00E+02 <1.00E+02 <1.00E E E E+03 41* 1.40E E E E E E E E E E E E E E E E E E E E+04 47* 9.70E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E+02 <2.00E E+03 <2.00E+02 <2.00E+02 <2.00E+02 90* 6.91E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E+02 a - Quantity determined using routine method, b - Quantity determined using standard material provided. Labs which did not report any quantities not included. Proficiency testing 55 Final 1 Page 14 of 21

72 Appendix VII: Participants and reference quantities for each sample. Shellfish sample 2 GII Shellfish sample 3 GI Proficiency testing 55 Final 1 Page 15 of 21

73 Shellfish sample 3 GII Shellfish Sample 3 HAV Proficiency testing 55 Final 1 Page 16 of 21

74 Shellfish Sample 4 GI Shellfish Sample 4 GII Proficiency testing 55 Final 1 Page 17 of 21

75 Shellfish Sample 4 - HAV Test sample LENTICULE 2- GI Proficiency testing 55 Final 1 Page 18 of 21

76 Test sample LENTICULE 2 - GII LENTICULE 2 HAV Proficiency testing 55 Final 1 Page 19 of 21

77 Appendix VIII: Results and methods used for shellfish samples. (For key to method codes see page 21) SF1 SF2 SF3 SF4 Virus extraction LAB ID ACC SF a GI GII HAV GI GII HAV GI GII HAV GI GII b HAV GI GII HAV 3* 100% A D M O BB AA AA 7* 100% A D M O AA c AA c AA c 13* 100% A D M O AA AA AA 17* 100% A D M O AA AA AA 19* 100% A D M O AA AA AA % A D M P CC CC CC 21* 100% A D M O AA AA AA % A E N Q DD EE AA 25* 100% A D M O AA AA AA 27* 100% A D M O BB AA AA 32* 100% A D M O BB AA AA 33* 100% A D M O AA AA AA 41* 100% A D M O AA AA AA % A D M O AA AA AA % ne + ne + + ne + ne A D M R BB AA 90* 100% A D M S AA AA AA % A D M P CC CC CC % A F N d T EE EE FF % A D M O AA AA AA % A D M nr nr nr nr 102* 100% A F M U AA AA CC % A D M P CC CC CC % A D M O AA AA AA % A D M P CC CC CC % A D M O AA AA AA % ne ne ne ne ne ne + ne ne + A D M P CC % A G M V BB AA AA % A D M P CC CC CC % A H M O BB AA AA % A F N W BB AA AA % ne ne ne ne ne ne + ne ne + A I M O AA % ne + ne + + ne + ne A J M X GG e GG e % A D M P CC CC CC 10* 91% A D M O BB AA AA 35* 91% A D M S EE AA AA 39* 91% A D M O EE EE AA 47* 91% A D M S AA e AA e AA 57 91% A D M O AA AA AA % A D M P CC CC CC % A D M Y AA AA CC 2 82% nr f nr nr f A D M g Z EE EE HH 48 64% + + A D M O BB AA AA 147* 64% + + A F M P CC CC CC 16 45% B F M S JJ JJ AA 34 43% ne ne ne ne nr K M S AA AA % ne ne ne ne C L M P CC CC * = Designated EU member state NRL, ne = determinand not examined, nr = information not returned, dark red = false negative, dark blue = false positive, dark grey = ISO/TS (or very similar) method; light grey = ISO/TS method with modifications; a = ACC SF = accuracy for shellfish samples; b = some reference subsamples provided negative results participant results for this sample and genogroup are not considered in performance scoring, negative and positive participant results highlighted in light red and light blue respectively for information; c = IAC assay run as multiplex with target assays; d = conventional two-step used for HAV only; e = norovirus analysis carried out as multiplex; f = participant lab did not return results for SF sample 4, results are scored as incorrect; g = conventional one-step used for HAV only. RNA extraction RT PCR method RT PCR reagents Primers Proficiency testing 55 Final 1 Page 20 of 21

78 Key to method codes Virus extraction methods A Proteinase K method with secondary incubation B Proteinase K digestion with no secondary incubation C Ceeram virus extraction kit (Proteinase K digestion with chloroform/butanol extraction) RNA extraction methods D NucliSens Magnetic extraction reagents (BioMerieux) E High Pure Viral Nucleic Acid Kit (Roche) F QIAamp/Rneasy kits (Qiagen) G Nucleo spin (Macherey Nagel) H 5X MagMax Pathogen RNA / DNA KIT (Lifescience) with the automatic extractor MegMax Express 96 I Viral NA/Carrier RNA Mini Kit (SYNGEN) J foodproof Virus Sample Preparation Kit (BIOTECON Diagnostics GmbH) K MagJET Viral DNA and RNA Kit (Thermo Scientific) L Ceeram Virus extraction kit RT PCR methods M Real time one step N Real time two step RT PCR reagents O RNA Ultrasense (Invitrogen) P ceeram Tools Q Superscript III (RT) & Platinum qpcr SuperMix UDG (Invitrogen) R RNA to Ct One step kit (Applied Biosystems) S Quantitect/Quantifast (Qiagen) T Real time: Taqman Universal Mastermix (Abi); Conventional: High Capacity cdna RT Kit (Abi) U Norovirus: AgPath ID One step real time PCR kit (Life Tech); HAV: Ceeram tools V Platinum quantitative RT PCR Thermoscript One step system (Invitrogen) W RT: High Capacity cdna Reverse Transcription Kit (Applied Biosystems); Real time PCR: LightCycler 480 Probes Master (Roche) X foodproof NoV Detection Kit (BIOTECON) Y GI&GII: RNA Ultrasense (Invitrogen); HAV: Ceeram Tools Z GI&GII: Brilliant II QRT PCR Core Reagent Kit, 1 step (Agilent); HAV: One Step RT PCR Kit (Qiagen) Primers/probes AA ISO/TS Annex C BB Primers; ISO/TS Annex C, Probe; Hohne and Schreier, (2006) CC Ceeram Tools (sequences as ISO/TS Annex C) DD Wolf et al, (2010) EE Kageyama et al, (2003) FF FDA/BAM Chapter 26 Detection and Quantification of Hepatitis A virus in Shellfish by the Polymerase Chain Reaction GG foodproof NoV Detection Kit (BIOTECON) HH Guevremont et al, (2006) JJ Hohne and Schreier, (2004) Proficiency testing 55 Final 1 Page 21 of 21

79 Annex V European Union Reference Laboratory (EURL) Proficiency Testing Scheme Detection of V. parahaemolyticus and V. vulnificus EURL PT reference number: PT 58 Final report version pages Contract Reference: Cefas ref (C6472A1) Document approved by: C6472A1 Project Manager James Lowther Review date: n/a Document checked by: Craig Baker-Austin Classification: Official Document prepared by: Louise Stockley Location

80 Contents Page number Sample preparation 2 Reference results 2 Participant s results PT 58 samples Discussion 7 Methodology 7 Conclusion 8 References 8 Appendix 1: Reference results 9 PT 58 Final - Page 1 of 9

81 Samples Sample preparation Five samples from the EURL Vibrio spp. strain bank (Table 1) were streaked onto non selective marine agar (MA) and incubated for hrs at 30±2 C. Following visual purity checks, Polymerise chain reaction (PCR) was performed on each selected strain to confirm strain identification and pathogenic markers. Between 2 5 colony forming units (cfu) were isolated from the MA purity plates and inoculated into 100 ml of alkaline salt peptone water (ASPW). Each inoculated ASPW was incubated for hrs at 30±2 C. After incubation samples were centrifuged at 2000 rpm for 20 min. The supernatant was discarded and the pellets were re suspended in 15 ml distilled water. Table 1: Source of Vibrio spp. strains Shellfish homogenate Vibrio species cfu/100g ToxR tdh trh VVH Sample 1 V. vulnificus 3.7 x Sample 2 V. parahaemolyticus 2.9 x Sample 2 V. alginolyticus 3.3 x 10 2 Sample 3 V. alginolyticus 5.1 x 10 2 Sample 4 V. parahaemolyticus 4.4 x Approximately 500 Pacific oysters (Crassostrea gigas) were collected from a UK commercial harvesting area. Each shellfish was shucked and homogenised before being dispensed in plastic containers and frozen at < 80 C for 4 days. A proportion of frozen homogenate was analysed to confirm the absence of Vibrio spp.. Defrosted homogenate was aliquoted in 90ml volumes into sterile bottles before being placed at < 20 C for 1 week. Prior to dispatch, frozen homogenate was removed from the freezer and allowed to defrost before being spiked with known levels of selected Vibrio spp. (Table 1). Sample distribution Samples were dispatched in accordance with IATA packing instructions 650 for UN3373 Diagnostic Specimens on the 5 th May 2015 to 16 participating laboratories. On receipt, participants were requested to examine the samples examine the samples using their routine method. Results Reference results Reference analyses were performed on 10 replicates for each sample distributed. Each replicate was tested at 37±1 C and 41.5±1 C. Biochemical tests were carried out on individual colonies isolated from each selective agar plate showing Vibrio spp. characteristics at both incubation temperatures. Where V. parahaemolyticus and V. vulnificus were detected, the presence of pathogenic markers were also determined. Reference results are summarised in Appendix 1. PT 58 Final - Page 2 of 9

82 Sample 1 Contents V. vulnificus (V11/016) Table 2. Participants results for sample 1 Biochemical tests PCR Primers Lab ID 37 C 41.5 C ToxR tdh trh Vvh 7 NG VND C VV V. vulnificus 10 VND VND VP V. parahaemolyticus 13 NE VND VA V. alginolyticus 17 NR NR VND Vibrio spp. not detected 19 VND NE NG No growth 21 NE VND NE Not examined 22 NG NG NR Not returned 32 VV VV + b NE a Enterobacter cloacae detected 35 NG NG b Tox R gene of V. vulnificus detected 39 VND VND C E. coli detected 42 NG NG 44 NE NG 68 NE NE 90 VP VND NE VND 147 NE VND a Results Sample 1 One NRL reported the presence of V. vulnificus at 37 C and 41.5 C using biochemical identification and PCR V. vulnificus tox R gene. One NRL reported the presence of V. parahaemolyticus at 37 C and confirmed using PCR. Eight NRLs reported the absence of Vibrio spp. at 37 C and/ or 41.5 C using biochemical identification. Key PT 58 Final - Page 3 of 9

83 Sample 2 Contents V. parahaemolyticus (V05/030) (Tox R +ve, tdh ve, trh +ve) and V. alginolyticus (V05/007) Table 3. Participants results for sample 2 Biochemical tests PCR Primers Lab ID 37 C 41.5 C ToxR tdh trh Vvh 7 VP VP + + VV V. vulnificus 10 VP & VA VP & VA + + VP V. parahaemolyticus 13 NE VP VA V. alginolyticus 17 NR NR VND Vibrio not detected 19 VP NE NG No growth 21 NE VP NE Not examined 22 VP VP NR Not returned 32 VP VP + + NE a Sample not pure 35 VV VP VP VP + 42 NG VP NE VP 68 VP NE + + NE 90 VP VP NE VP & VA 147 NE VP a + + Results Sample 2 Eight NRLs reported the presence of V. parahaemolyticus at 37 C and 13 reported the presence at 41.5 C using biochemical identification. Two NRLs reported the presence of V. alginolyticus at 37 C and/or 41.5 C using biochemical identification. Nine NRLs confirmed V. parahaemolyticus presence using PCR with all reporting the detecting of Tox R. For the detection of the pathogenic markers all NRLs correctly reported the absence of tdh and 7 correctly reported the presence of trh. Key PT 58 Final - Page 4 of 9

84 Sample 3 Contents V. alginolyticus (V11/008) Table 4. Participants results for sample 3 Biochemical tests PCR Primers Lab ID Key 37 C 41.5 C ToxR tdh trh Vvh 7 VA VA VV V. vulnificus 10 VA VA VP V. parahaemolyticus 13 NE VC VA V. alginolyticus 17 NR NR VM V. mimicus 19 VA NE VC V. cholerae 21 NE VND a VND Vibrio not detected 22 VV VV NG No growth 32 VA VA NE NE Not examined 35 VA VC NR Not returned 39 VA VA a Enterobacter spp. detected 42 VA VM 44 NE VND 68 NE NE 90 VA VA 102 NE VND 147 NE VA Results Sample 3 Eight NRLs reported the presence of V. alginolyticus at 37 C and 6 NRLs reported V. alginolyticus presence at 41.5 C using biochemical identification. Three NRLs incorrectly reported the presence of V. cholerae and/or V. mimicus at 41.5 C and 1 NRL reported the presence of V. vulnificus at 37 C and 41.5 C using biochemical identification. PT 58 Final - Page 5 of 9

85 Sample 4 Contents V. parahaemolyticus (V05/014) (Tox R +ve, tdh +ve, trh +ve) Table 5. Participants results for sample 4 Lab Biochemical tests PCR Primers ID 37 C 41.5 C ToxR tdh trh Vvh Key 7 VP VP VV V. vulnificus 10 VP VP VP V. parahaemolyticus 13 NE VP VA V. alginolyticus 17 NR NR VND Vibrio not detected 19 VP NE NG No growth 21 NE VV NE Not examined 22 VP VP NR Not returned 32 VP VP NE 35 VP VP VP VP VP VP NE VND 68 VP NE NE 90 VP VP NE VV 147 NE VP Results Sample 4 Ten NRLs reported the presence of V. parahaemolyticus at 37 C and 10 NRLs reported V. parahaemolyticus presence at 41.5 C using biochemical identification. Two NRLs incorrectly reported the presence of V. vulnificus 41.5 C using biochemical identification. Nine NRLs reported results for PCR with all NRLs reporting the presence of Tox R, tdh and trh. PT 58 Final - Page 6 of 9

86 Discussion Sixteen NRLs participated in PT 58 distribution with 1 NRLs not returning the results form. From the results reported by NRLs (6 NRLs 40%) using only biochemical tests; for sample 1 100% of NRLs were unable to detect the presence of V. vulnificus; for sample 2 100% of NRL reported the presence of V. parahaemolyticus and 1 NRL (16%) reported the presence of V. alginolyticus. For sample 3, one NRL reported the presence of V. alginolyticus. For this sample V. vulnificus and V. cholerae were incorrectly detected by 2 NRLs. For sample 4, 3 NRLs (50%) correctly reported the presence of V. parahaemolyticus and 2 NRL reported the presence of V. vulnificus. On comparing the results reported by NRLs using both biochemical tests and PCR (9 NRLs 60%), 1 NRL detected the presence of V. vulnificus in sample 1 using biochemical tests and reported the presence of the V. vulnificus tox R gene using PCR. For samples 2 and 4, all 9 NRLs (100%) detected the presence of V. parahaemolyticus using both processes. A summary of the correct reporting of results for both biochemical tests and PCR for each sample is given in table 6. Table 6: Summary of reported results * percentage based on results reported for Tox R only. Methodology A questionnaire was distributed to all participating NRLs to identify procedures adopted within Member States across Europe and to help in the development of a harmonised method. In total 11 NRLs providing information. For the biochemical tests 86% of NRLs stated they used an oxidase strip for genus identification and a selection of halotolerance tests (0% NaCl, 2% NaCl, 6% NaCl, 8% NaCl, 10% NaCl) to determine species identification. It was noted that there was no significant similarity with the biochemical tests used amounst NRLs with a total of 21 different biochemical test being referenced. Table 7: PCR and RT PCR references PT 58 Final - Page 7 of 9

87 Seven NRLs provided information on the PCR and RT PCR assays used to detect the identity of the strain and any associated pathogenic markers using PCR or RT PCR. Table 7 shows the references cited for each target. On comparing the results reported using PCR 1 false positive for Tox R was reported for sample 1 using PCR assay reference Bauer et al.. Two NRLs did not detect the presence of trh in sample 2 using the Bej et al. primer set. Conclusion From the results reported for this distribution and reviewing previous PT distributions it was noted that NRLs within Member States across Europe are able to detect the presence of V. parahaemolyticus in a variety of samples matrix. The detection of V. vulnificus was shown to be more problematic. This has also been evident in previous PT distributions with only around 50 % of NRLs being able to detect V. vulnificus in the samples. For this distribution the reason only 1 NRL was able to detect the V. vulnificus may be due to the low inoculation level. From the information provided by NRLs on PCR assay detection it was evident the favoured references are Tox R for V. parahaemolyticus strain identification is Kim et al., for the detection of the tdh and trh pathogenic markers is Bej et al. and for the detection of V. vulnificus is Hill et al.. References Bej,A.K.; Patterson,D.P.; Brasher,C.W.; Vickery,M.C.L.; Jones,D.D.; Kaysner,C.A., Detection of total and hemolysin producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh and trh. Journal of Microbiological Methods 36, Chun,J.; Huq,A.; Colwell,R.R., Analysis of 16S 23S rrna intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Applied & Environmental Microbiology 65, Hill,W.E.; Keasler,S.P.; Trucksess,M.W.; Feng,P.; Kaysner,C.A.; Lampel,K.A., Polymerase chain reaction identification of vibrio vulnificus in artificially contaminated oysters. Applied & Environmental Microbiology 57, Kim,Y.B.; Okuda,J.; Matsumoto,C.; Takahashi,N.; Hashimoto,S.; Nishibuchi,M., Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxr gene. Journal of Clinical Microbiology 37, Lee,C.Y.; Pan,S.F., Rapid and specific detection of the TDH gene in Vibrio parahaemolyticus by the polymerase chain reaction. Journal of General Microbiology 139, Suthienkul,O.; Ishibashi,M.; Iida,T.; Nettip,N.; Supavej,S.; Eampokalap,B.; Makino,M.; Honda,T., Urease production correlates with possession of the trh gene in Vibrio parahaemolyticus strains isolated in Thailand. Journal of infectious diseases 172, Tarr,C.L.; Patel,J.S.; Puhr,N.D.; Sowera,E.G.; Bopp,C.A.; Strockbine,N.A., Identification of Vibrio Isolates by a multiplex PCR assay and rpob sequence determination. Journal of Clinical Microbiology 45, PT 58 Final - Page 8 of 9

88 Appendix 1. Reference results Sample number Colony growth on selective agar (no. of positive samples) 37 C 41.5 C TCBS VID TCBS VID Biochemical tests 37 C 41.5 C API 20 E Vibrio strain and number positive PCR Primers Percentage ToxR tdh trh Vvh Sample 1 Green (1/10) Blue (7/10) Green (3/10) Blue (6/10) VV VV V. vulnificus (13/13) 99.3 % + Sample 2 Green (10/10) Yellow (10/10) Pink (10/10) Cream (9/10) Green (10/10) Yellow (10/10) Pink (10/10) Cream (7/10) VP VA VP VA V. parahaemolyticus (21/21) V. alginolyticus (10/10) % % Sample 3 Yellow (10/10) Cream (10/10) Yellow (10/10) Cream (10/10) VA VA V. alginolyticus (3/5) * % Sample 4 Green (10/10) Pink (10/10) Green (10/10) Pink (10/10) VP VP V. parahaemolyticus (2/19) ** 98.8 % * VP detected 66.6 % and 96.4 % ** VM detected 88.9 % TCBS Thiosulphate Citrate Bile Sucrose agar VID ChromID Vibrio (Biomerieux) PT 58 Final - Page 9 of 9

89 Annex VI European Union Reference Laboratory (EURL) Proficiency Testing Scheme Noroviruses and hepatitis A virus EURL PT reference number: PT 59 Final report version pages Contract Reference: Cefas ref (C6472A1) 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

90 Contents Page number Samples 2 Results 2 Conclusion and discussion 4 References 4 Appendices 5 PT 59 Final version 2; Page 1 of 11

91 Samples Material dispatched consisted of laboratory constructed LENTICULES (Vials 1 and 2). Table 1 shows details of the stock viruses used in the preparation of LENTICULE 2. 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.7 (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 2 included known levels of GI and 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 22 nd June 2015 to 33 participating laboratories. Laboratory 78 received multiple sets of samples. 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 24 th July 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 53 proficiency testing material Sample Norovirus HAV GI GII LENTICULE 1 LENTICULE 2 + (2.03 x x 10 2 ) + (2.53 x x 10 2 ) + (9.45 x x 10 3 ) 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 assessed as percentage relative accuracy, specificity and sensitivity for each determinand (Table 3) according to the calculations described in Appendix II. Note: Participants results were expressed as percentage concordance with intended results generated by the EURL. In this assessment presence/absence data was used and no consideration of quantitative measurements or Ct values were made. PT 59 Final version 2; Page 2 of 11

92 Table 3: Participants results for all LENTICUE TM samples Lab ID GI GII HAV Performance score No. AC SP SE AC SP SE AC SP SE NoV HAV 3* A A 7* A A 10* A A 13* A A 17* A A 19* A A A A 21* A A 22* A A 25* A A 27* A A 39* A A 41* A A 43* NR NR NR NR NR NR NR NR NR 47* A A A A B A NE NE NE A 78 a A A 78 b A A A A B B A A 113 NR NR NR NR NR NR NR NR NR 122 NR NR NR NR NR NR NR NR NR A A 147* A A A A a 100 a 100 a n/a n/a n/a A A A B A A A A a 100 a 100 a n/a n/a n/a A B a 100 a 100 a n/a n/a n/a A A * = Designated NRL, NE= Not examined, NR = Results not returned, a = non genogroup discriminating method used for norovirus, n/a = not applicable, AC =Relative accuracy, SP = Relative specificity, SE = Relative sensitivity, Performance scoring; A = satisfactory (100% accuracy), B = questionable (one incorrect result), C = unsatisfactory (two or more incorrect results). Norovirus (NoV) and HAV scored separately. Note Lab 78 received multiple sets of samples and returned two sets of results. Note 26/30 laboratories that returned results scored 100% overall accuracy for all determinands tested. PT 59 Final version 2; Page 3 of 11

93 Conclusion and discussion General comments Thirty three laboratories (15 NRLs and 18 other laboratories) received samples. Laboratories 43, 113 and 122 did not return results. Laboratory 72 did not examine for HAV. Laboratory 78 received multiple sample sets and returned two sets of results. For purposes of calculation of overall laboratory performance, results from a single set only (78 a) are considered. Laboratories 198, 228 and 250 used a non genogroup discriminating method for norovirus. Results for these laboratories are not considered for purposes of calculation of overall laboratory performance for GI and GII. Results reported to the EURL are shown in Appendices III and IV. Discussion 26/30 (86%) of the laboratories that returned results obtained the intended results (as determined by EURL reference designations) for all the sample/determinand combinations which they tested. The overall accuracies across all laboratories were 100%, 96% and 95% for GI, GII and HAV respectively. The false positive reporting rates for GI, GII and HAV were 0%, 0% and 3% respectively. The false negative reporting rates for GI, GII and HAV were 0%, 7% and 7% respectively. Seventeen laboratories (57%) returned data expressed as both C t values and quantities for at least one sample/determinand combination (Appendix III). 12 laboratories (40%) returned data expressed as C t values only. One laboratory (3%) returned results as quantities only. Quantitative results from individual labs alongside reference results are shown in Appendix IV. Reference results were not corrected using extraction efficiency data. Methods used by participants to analyse the test samples are shown in Appendix V. References Codd AA, Richardson IR, Andrews N Lenticules for the control of quantitative methods in food microbiology. J Appl Microbiol. 85(5): PT 59 Final version 2; Page 4 of 11

94 Appendix I: EURL reference results displayed as box and whisker plots of detectable genome copies per LENTICULE. LENTICULE Copies per LENTICULE GI GII HAV PT 59 Final version 2; Page 5 of 11

95 Appendix II: Percentage relative sensitivity: Relative sensitivity (SE) = TP (TP+FN) Percentage relative specificity: Relative specificity (SP) = TN (TN+FP) Percentage relative accuracy: Relative accuracy (AC) = TP+TN N x 100% x 100% Where TP = true positives FN = false negatives FP = false positives TN = true negatives N = total number of tests Note: Participants results were expressed as percentage concordance with intended results generated by the EURL. In this assessment presence/absence data was used and no consideration of quantitative measurements (C t values) was made. Appendix III: Participants results PT 59 Final version 2; Page 6 of 11

96 Lab ID No. GI GII HAV GI GII HAV + CT copies/ LENTICULE + CT copies/ LENTICULE + CT copies/ LENTICULE 3* E E * E E E+02 10* E E E+02 13* * E E E+03 19* E E E * E E E+02 22* E E E+04 25* E E E+02 27* E E E+02 39* E E E+02 41* E E E+03 43* NR NR NR NR NR NR NR NR NR NR NR NR 47* E E E E E E E+02 NE 78 a E E E b E E E E E E NR NR NR NR NR NR NR NR NR NR NR NR 122 NR NR NR NR NR NR NR NR NR NR NR NR 132 NE * E E E a n/a + a 36 a n/a n/a n/a E E E a n/a + a a n/a n/a n/a 250 a n/a + a n/a n/a n/a * = Designated NRL, NE= determinand not examined, NR= results not returned, a = non genogroup discriminating method used for norovirus, n/a = not applicable, Yellow shading denotes false positives, Red shading denotes false negatives. PT 59 Final version 2; Page 7 of 11

97 Appendix IV: Participants and reference quantities for each sample. LENTICULE 2 GI LENTICULE 2 GII PT 59 Final version 2; Page 8 of 11

98 LENTICULE 2 HAV PT 59 Final version 2; Page 9 of 11

99 Appendix V: Results and methods used for test samples. (For key to method codes see page 11) LENTICULE 1 LENTICULE 2 RNA RT PCR RT PCR Primers LAB ID GI GII HAV GI GII HAV extraction method reagents GI GII HAV 3* A J M AA 1 AA AA 7* A J M AA 2 a AA a AA a 10* A J M AA 1 AA AA 13* A J M AA 2 AA AA 17* A J M AA 2 AA AA 19* A J M AA 2 AA AA A J N BB BB BB 21* A J M AA 2 AA AA 22* B J M AA 2 AA AA 25* A j M AA 2 AA AA 27* A J O AA 1 AA AA 39* A J M CC CC AA 41* A J M AA 2 AA AA 47* A J P AA 2 b AA b AA B J M AA 1 AA AA C J O AA 1 AA KK 72 NE + + NE A J Q AA 1 AA 78 a B K c R DD DD LL 78 b B K c R DD DD LL A J N BB BB BB 95 + B L d S CC CC MM D J T EE EE GG A J U AA 1 AA AA B J V FF FF BB A J M AA 2 AA AA 198 e n/a + e n/a + nr nr W GG e n/a GG A J M AA 2 AA AA E J X AA 1 AA AA F J N BB BB AA 228 e n/a + e n/a G J Y HH e n/a AA 250 e n/a + e n/a + H K f Z JJ e n/a AA * = Designated NRL, Red = false negative results; 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 PCR with sequencing confirmation used as detection method, followed by droplet digital RT PCR for quantification; d = conventional two step used for HAV only; e = non genogroup discriminating method used for NoV; f = realtime one step used for HAV only PT 59 Final version 2; Page 10 of 11

100 Key to method codes RNA extraction methods A NucliSens Magnetic extraction reagents (BioMerieux) B QIAamp/Rneasy kits (Qiagen) C Dynabeads MyOne SILANE (LifeTech) /Rneasy kit (QIAGEN) D Column extraction (r Biopharm) E NucleoSpin RNA Virus (Macherey Nagel) F foodproof Virus Sample Preparation Kit (Biotecon) G High Pure Viral RNA Kit (Roche) H iprep Virus Kit (Invitrogen) RT PCR methods J Real time one step K Conventional one step L Real time two step RT PCR reagents M RNA Ultrasense (Invitrogen) N ceeram Tools O TaqMan Fast Virus 1 Step Master Mix (Applied Biosystems) P Quantitect/Quantifast RT PCR kits (Qiagen) Q RNA to Ct One step kit (Applied Biosystems) R One Step RT PCR kit (Qiagen)/One Step RT ddpcr kit for Probes (Bio Rad) S High Capacity cdna RT Kit & Taqman Universal Mastermix (Applied Biosystems) T Surefood kit (r Biopharm)/foodproof Detection Kit (Biotecon) U One step qrt PCR System (Invitrogen) V NoV; Ridagene Norovirus detection kit (r Biopharm)/CeeramTools for genogroup determination. HAV; CeeramTools W Surefood kit (Congen) X SuperScript III Platinum One Step qrt PCR System (Invitrogen) Y NoV; virellanoro real time RT PCR Kit (Gerbion). HAV; Quantitect Probe RT PCR Kit (Qiagen) Z NoV; SuperScript III Platinum One Step qrt PCR System (Invitrogen). HAV; RNA Ultrasense (Invitrogen) Primers/probes AA Primre/probe sets from 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 Kojima et al, (2003)/Kageyama et al., (2003)/Vinje et al., (2011) EE Surefood kit (r Biopharm)/foodproof Detection Kit (Biotecon) FF Ridagene Norovirus detection kit (r Biopharm)/CeeramTools for genogroup determination GG Surefood kit (Congen/r Biopharm) HH virellanoro real time RT PCR Kit (Gerbion) JJ Vennema et al., (2002) KK Guevremont et al, (2006), Houde et al, (2007) LL Guevremont et al, (2006) MM FDA/BAM Chapter 26 Detection and Quantification of Hepatitis A virus in Shellfish by the Polymerase Chain Reaction PT 59 Final version 2; Page 11 of 11

101 Annex VII European Union Reference Laboratory (EURL) Proficiency Testing Scheme Enumeration of Escherichia coli in bivalve molluscan shellfish EURL PT reference number: PT 60 Final report version pages Contract Reference: Cefas ref (C6472A1) 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

102 Contents Page number Sample preparation 2 Results 2 General comments 5 References 6 Result charts 7 Appendices 9 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 ( g/eqaptforfoodwaterandenvironmentalmicrobiology/shellfishscheme/ through examination of aspects of the methods not covered under the Shellfish Scheme (initial sample preparation and preparation of initial dilutions) 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 glucuronidasepositive Escherichia coli Part 3: Most probable number technique using 5 bromo 4 chloro 3 indolyl β Dglucuronide (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. Proficiency testing 60 Final Page 1 of 11

103 Sample preparation Sample 1 Approximately 1,500 common mussels (Mytilus edulis) comprising a single batch were collected from a UK commercial harvesting area. Sample 1 provided to participating laboratories comprised of 25 randomly selected Manila clams from this bulk material. Sample 2 Approximately 1,000 Pacific oysters (Crassostrea gigas) comprising a single batch were collected from a UK commercial harvesting area. Sample 2 provided to participating laboratories comprised of 15 randomly selected common mussels from this bulk material. Sample distribution and examination Each batch of shellfish were mixed thoroughly in a large container before being subsampled and packed in accordance with Cefas protocol for packaging shellfish for transportation and distributed at 12:30 on the 30 th November 2015 to 37 participating laboratories. Participants were requested to analyse the material in duplicate immediately on receipt using their routine laboratory procedures for the enumeration of E. coli. 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 Table 1: E. coli MPN/100g reference results Sample type Analyses dates Range Median GM Median ±3*SD T Sample x x x x x x 10 3 Mussels x x x x x x 10 3 Sample <1.8 x x x x 10 1 <1.8 x x 10 2 Oysters <1.8 x x x x 10 1 <1.8 x x 10 2 GM geometric mean, SD T theoretical standard deviation (0.24) 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 60 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. SD T calculations were based on the inherent variability of the 5 x 3 MPN method (0.24 log 10). Reference values were excluded from the calculation of participants median. Proficiency testing 60 Final Page 2 of 11

104 Table 3: Participants results Sample type E. coli MPN/100g Range Median GM Median±3*SD T Sample 1 Mussels <1.8 x x x x x x 10 3 Sample 2 Oysters <1.8 x x x x x x 10 2 GM geometric mean, SD T theoretical standard deviation (0.24) Table 4: Summary statistics of participants results E. coli Sample 1 Sample 2 Mussels Oysters Participants reporting duplicate results for E. coli MPN Participants reporting a single MPN result 1 0 Participants reporting MPN results within the expected range for both replicates Participants reporting MPN results outside the expected range for one replicate 1 3 Participants reporting MPN results outside the expected range for both replicates 2 2 Participants reporting MPN results as censored results for one replicate 0 0 Participants reporting MPN results as censored results for both replicates 1 8 Participants reporting MPN results inconsistent with ISO 7218 (Anon 2007) Participants not returning results 0 0 Participants not receiving material due to problems at customs expected range = participants median ± theoretical 3SD. 2 points deducted from participants returning results inconsistent with ISO Proficiency testing 60 Final Page 3 of 11

105 Table 5: Participants results and allocated scores Lab ID Sample 1 Sample 2 Lab Sample 1 Sample 2 Rep 1 Rep 2 Score Rep 1 Rep 2 Score ID Rep 1 Rep 2 Score Rep 1 Rep 2 Score 3 * * * a <18 < * NE NE * a NE NE 10 * < * * * 330 Void * c * * * a <18 < * a b c a * * < * 20 < * a * <200 < <200 < * <18 < * * * <67 < * a < * a * a * 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 Scores were not deducted on this occasion as further discussion is required regarding the value given by the MPN calculator reported for tube combinations of NE Not examined. Proficiency testing 60 Final Page 4 of 11

106 General comments Thirty seven laboratories (26 NRL and 11 other laboratories) were sent material with all laboratories returning results. Information provided by laboratories on the arrival time of the material showed that 76% (28) of laboratories received the material the day after dispatch ( ), with 41% (15) of these laboratories analysing the material on arrival. Sixteen percent (6) of laboratories received material within 48 hours of dispatch, with the remaining 8% (3) of laboratories receiving the material within 72 hours after dispatch. Forty one percent (15) of laboratories analysed the material the day after receiving the material with exception of laboratory 23 who analysed their material 42 hours after receipting the material. Temperature record range of C. All temperature data, arrival and analysis dates and times recorded by participants are shown in Appendix I. Sample analyses Thirty seven laboratories returned the report form for this PT distribution. Laboratory 9 and 68 did not examine one of the samples due to laboratory error. Sample 1 Mussels Thirty two laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants median with 24 laboratories obtaining a score of 12. Laboratory 212 reported 1 replicate result between ±3 and ±5 SD of the participants median. Laboratory 7 reported both replicate results between ±3 and ±5 SD of the participants median. Laboratory 147 both replicate results outside ±3 SD of the participants median with 1 replicate falling between ±3 and ±5 SD and the other falling outside ±5 SD of the participants median. Nine laboratories (laboratories 23, 33, 42, 44, 50, 68, 96, 98 and 245) were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables. 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 11) should be used. Sample 2 Oysters Thirty one laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants median with 26 laboratories obtaining a score of 12. Laboratories 3, 17 and 41 reported one replicate result within ±3 SD of the participants median. Laboratories 19 and 23 reported both replicate results as 0. Scores were not deducted on this occasion as further discussion is required regarding the value given by the MPN calculator reported for tube combinations of is 0 previous discussions at an EURL workshop identified that this could be confusing (a zero is given whatever dilution set is used). The tables in the EURL generic protocol give the value for the standard dilution series as <18/ 100g, as agreed at that workshop. Eight laboratories (laboratories 23, 33, 42, 44, 50, 96, 98 and 245) were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables. Laboratory 245 reported MPN values inconsistent with the tube combination provided. 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 11) should be used. Summary Twenty one laboratories (57%) 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 incurred a deduction (7 laboratories) in scores were due to the advice given in ISO7218:2007/Amd1:2013 and/or the EURL generic protocol for calculating the MPN value for E. coli was not followed. Laboratories are requested to note in Proficiency testing 60 Final Page 5 of 11

107 ISO ISO7218:2007/Amd1:2013 it states 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) 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 2013 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 2010 ISO 22117:2010 Microbiology of food and animal feeding stuffs Specific requirements and guidance for proficiency testing by interlaboratory comparison. Geneva, Switzerland. Proficiency testing 60 Final Page 6 of 11

108 Figure 1: Results chart sample 1 Mussels Proficiency testing 60 Final Page 7 of 11

109 Figure 2: Results chart sample 2 Pacific oysters Proficiency testing 60 Final Page 8 of 11

110 Appendix I Sample arrival and temperature Lab ID Date arrived Time of Sample ( C) Storage ( C) Date Time of arrival analysed analysis 3 * 01/12/ :15: /12/ :00:00 7 * 01/12/ :50: /12/ :30:00 9 * 01/12/ :30: /12/ :30:00 10 * 01/12/ :30: /12/ :00:00 13 * 01/12/ :15: /12/ :00:00 17 * 01/12/ :00: /12/ :00:00 19 * 02/12/ :30: /12/ :00:00 21 * 01/12/ :55: /12/ :30:00 22 * 01/12/ :00: /12/ :10:00 23 * 01/12/ :35: /12/ :30:00 27 * 01/12/ :20: /12/ :00:00 32 * 01/12/ :00: /12/ * 01/12/ :15: /12/ :00:00 35 * 01/12/ :15: /12/ :45:00 39 * 01/12/ :00: /12/ :00:00 41 * 01/12/ :00: ±2 01/12/ :00:00 42 * 01/12/ :45: /12/ :00:00 43 * 03/12/ :50: /12/ :00:00 44 * 01/12/ :30:00 2 3±2 02/12/ :00:00 47 * 01/12/ :15: /12/ :00: /12/ :20: /12/ :30:00 68 * 02/12/ :30: ±2 03/12/ :30: /12/ :30: /12/ :00: /12/ :10: /12/ :30:00 83 * 03/12/ :20: /12/ :40:00 90 * 03/12/ :36: /12/ /12/ :00: /12/ :40: /12/ :00: /12/ :30: /12/ :45: /12/ :00: /12/ :45:00 6 3±2 02/12/ :00: * 01/12/ :30:00 4 3±2 02/12/ * 02/12/ :30: /12/ :00: /12/ :15: /12/ :00: /12/ :50: /12/ :00: * 01/12/ :00: /12/ :45:00 3±2 01/12/ /12/ :00: /12/ :45:00 * Designated NRL s Proficiency testing 60 Final Page 9 of 11

111 Appendix II: E. coli MPN scores allocated to participants returning 2 replicate results Reported results Returning 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 values and the second falls outside the median ±5SD values Both replicates MPN results is outside the median ±5SD value E. coli MPN scores allocated to participants returning 1 single replicate results Returning Result of results Score allocated 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 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/FDAM1:2013 or MPN tables provided by the EURL. Total score Score deducted Replicate Replicate High censored result (e.g. MPN = >18000 per 100g) 2 2 Sample not examined or results returned late no explanation received 12 Proficiency testing 60 Final Page 10 of 11

112 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 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 have you 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 60 Final Page 11 of 11

113 Annex VIII European Union Reference Laboratory (EURL) and Public Health England (PHE) EQA Shellfish Scheme Escherichia coli and Salmonella spp. EQA EURL PT reference number: PT 63 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

114 Contents Page number Methodology 2 Reference results 2 Participants analysis and scoring system 2 Participation in statutory determinands 2 Performance assessments 3 Reference 5 Appendix 1 Distribution SF050: Sample SF0108 and SF Appendix 2 Distribution SF051: Sample SF0110 and SF Appendix 3 Distribution SF052: Sample SF0112 and SF Appendix 4 EURL PT 60 NRL results and allocated scores 22 Appendix 5 Scoring for the PHE/EQA and EURL matrix scheme 23 Appendix 6 Troubleshooting advice 24 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 testing 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:// TForFoodWaterAndEnvironmentalMicrobiology/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 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 63 Final Page 1 of 24

115 Methodology Sample preparation PHE EQA Samples distributed consisted of LENTICULE discs containing fully characterised bacterial isolates. 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 Two shellfish samples comprising of common mussels (M. edulis) and Pacific oysters (C. gigas) both 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 TS 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 medium. Reported MPN values were log 10 transformed before being compiled into charts are shown in Appendix 1 to 3. Performance assessment was 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 2015 with 84% participating in the EURL matrix distribution and 1 or more EQA distributions. Currently there is no active participation from a designated NRL in Malta, Cyprus, The Czech Republic or Estonia. NRLs in Bulgaria, Denmark, Finland, Greece and Latvia did not participant in the mandatory number of EQA distributions and the EURL matrix distribution per year agreed in Resolution 8 of the NRLs annual workshop The EURL recommends that NRLs not registered to the EQA scheme should join this scheme. Table 1: Participation by NRLs in 2015 for E. coli and Salmonella spp. determinands. Country EURL PT 60 PHE EQA distributions 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 Proficiency testing 63 Final Page 2 of 24

116 Performance Assessment A cumulative performance assessment was undertaken on participant s results for both E. coli and Salmonella spp. from the EURL matrix distribution (PT 60) (Appendix 4) and 3 EQA distributions (March to November 2015). 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. E. coli MPN assessment Twenty one laboratories participated in the EURL matrix scheme (Appendix 4) and 1 or more EQA distributions in 2015 and were therefore subject to a full performance assessment. All 21 laboratories that were subjected to the full assessment achieved a cumulative total of >70% for the two or more distributions analysed. NRLs Bulgaria, Denmark, Finland, Greece and Latvia did not participate in the mandatory number of EQA distributions (1) and the EURL matrix distribution per year agreed in Resolution 8 of the NRLs annual workshop Table 2: Summary of participants performance in the EURL matrix scheme and the EQA scheme E. coli PT 60 Distribution SF050 Distribution SF051 Distribution SF052 All distributions Lab no. a Sample Sample Cumulative Max SF0108 SF0109 SF0110 SF0111 SF0112 SF score score % 121 [19] [9] [86] b [35] [22] b [32] [41] [68] [43] [39] [23] [10] [3] [21] [7] [90] [47] [44] b [27] [170] c 8 c 8 c 8 c 8 c 8 c [13] [147] [83] [33] [42] b [102] b 0 [212] d 9 12 a NRL Lab number from PHE EQA scheme. ID number in [x] taken from EURL PT scheme. b Full performance assessment was not carried out as NRL did not participate in PT 60 and 1 EQA during c The reporting of MPN tube combinations is not required for this method, the overall score is reduced to reflect this (8). d EQA material cannot be analysed using this method, therefore a full assessment is not completed. Proficiency testing 63 Final Page 3 of 24

117 Salmonella spp. assessment Twenty one laboratories participated in 1 or more EQA distributions in 2015 and were therefore subject to a full performance assessment. All laboratories that were subjected to the full assessment achieved a cumulative total of >70% for the one or more distributions analysed. NRLs Bulgaria, Finland, Greece and Latvia did not participate in the mandatory number of EQA distributions (1) per year agreed in Resolution 8 of the NRLs annual workshop Table 3: Summary of participants performance in the EQA scheme Salmonella spp. Distribution SF050 Distribution SF051 Distribution SF052 All distributions Lab no. a Cumulative Max SF0108 SF0109 SF0110 SF0111 SF0112 SF0113 score score % 121 [19] [9] [86] [35] [22] b [32] [41] [68] [43] [39] [23] [10] [3] [21] [7] [90] [47] [44] b [27] [170] c 1578 [13] [147] [83] [33] [42] b 0 [102] b 0 [212] C a NRL Lab number from PHE EQA scheme. ID number in [x] taken from EURL PT scheme. b Full performance assessment was not carried out as NRL did not participate in 1 EQA during c Salmonella spp. analyses is not performed by these NRLs. Proficiency testing 63 Final Page 4 of 24

118 References Anon ISO TS :2005. 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. 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, : Proficiency testing 63 Final Page 5 of 24

119 Appendix 1 Distribution SF050 Sample SF0108 contents E. coli (10³ 10⁴) (wild strain), S. Indiana 1,4,12:z:1,7 (10²) (wild strain), B. pumilus (10²) (wild strain), E. cloacae (10⁵) (wild strain). Reference results E. coli MPN 3.47 x 10³ 8.33 x 10⁴ per 100g. Salmonella spp. Detected in 25g. Analysed February / March 2015 Seventeen 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 403, 658, 715, 1498 and 1859 did not participate in this distribution. Table 4: Participants and reference results median, median 2.68 and 4 SD SF0108 Median MPN/100g Median 2.68SD MPN/100g Median 4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results 1.3 x x x x x 10 5 Participants results 1.7 x x x x x 10 5 Participants results SF0108 Table 5: Results reported by participants and scores allocated SF0108 (Figure 1) Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected Detected DNP DNP DNP Detected DNR DNR DNR Detected Detected Detected Detected Detected DNP DNP DNP Detected NE 715 DNP DNP DNP Detected Detected Detected DNR DNR DNR 1498 DNP DNP DNP * NE Detected Detected DNP DNP DNP Detected 2 DNR NRL did not register for the scheme 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 this method. Proficiency testing 63 Final Page 6 of 24

120 E. coli MPN Sixteen laboratories reported replicate results within ±2.68 SD of the participants medium with 14 receiving a maximum score. Laboratory 583 reported one replicate result within ±2.68 SD of the participants medium and received a maximum score of 9. Laboratories 651 and 653 were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables. 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. Proficiency testing 63 Final Page 7 of 24

121 Figure 1. Distribution SF050: Sample SF0108 Proficiency testing 63 Final Page 8 of 24

122 Distribution SF050 Sample SF0109 contents E. coli (10 10²), S. Typhimurium 1,4,[5],12:i:1,2 (10²) (wild strain), K. oxytoca (10⁴) (wild strain), E. faecalis (10) (wild strain). Analysed February / March 2015 Seventeen 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 403, 658, 715, 1498 and 1859 did not participate in this distribution. Reference results E. coli MPN < x 10² per 100g. Salmonella spp. Detected in 25g. Table 6: Participants and reference results median, median 2.68 and 4 SD SF0109 Median MPN/100g Median 2.68SD MPN/100g Median 4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results 7.8 x x x x x 10 2 Participants results 7.8 x x x x x 10 2 Participants results SF0109 (Figure 2) Table 7: Results reported by participants and scores allocated SF0109 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected Detected DNP DNP DNP Detected DNR DNR DNR Detected Detected Detected Detected Detected DNP DNP DNP Detected NE 715 DNP DNP DNP Detected Detected Detected DNR DNR DNR 1498 DNP DNP DNP 1527 <200 <200 8 * NE Detected Detected DNP DNP DNP Detected 2 DNR NRL did not register for the scheme 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 this method. Proficiency testing 63 Final Page 9 of 24

123 E. coli MPN Sixteen laboratories reported replicate results within ±2.68 SD of the participants medium with 13 received a maximum score. Laboratory 718 reported one replicate result between ±2.68 and ±4SD of the participants median and reported tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables and received an overall score of 5. Laboratories 649, 653 and 703 were deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables. 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. Proficiency testing 63 Final Page 10 of 24

124 Figure 2. Distribution SF050: Sample SF0109 Proficiency testing 63 Final Page 11 of 24

125 Appendix 2 Distribution SF051 Sample SF0110 contents E. coli (10³ 10⁴) (wild strain), S. London 3, {10}{15}:l,v:1,6 (30) (wild strain), K. oxytoca (10⁵) (wild strain), P. agglomerans (10⁵) (wild strain). Analysed June / July 2015 Twelve laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratories 391, 583, 649, 658, 744, 1498, 1578, 1798, 1859 and 2118 did not participate in this distribution. Reference results E. coli MPN 4.49 x 10³ 1.08 x 10⁵ per 100g. Salmonella spp. Detected in 25g. Table 8: Participants and reference results median, median 2.68 and 4 SD SF0110 Median MPN/100g Median 2.68SD MPN/100g Median 4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results 3.5 x x x x x 10 5 Participants results 1.9 x x x x x 10 5 Participants results SF0110 (Figure 3) Table 9: Results reported by participants and scores allocated SF0110 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected DNP DNP DNP Detected Detected DNR DNR DNR 583 DNP DNP DNP Detected DNP DNP DNP Detected Detected DNP DNP DNP Detected Detected Detected Detected Detected DNP DNP DNP 983 DNR DNR DNR 1498 DNP DNP DNP * NE 1578 DNP DNP DNP 1798 DNP DNP DNP 1859 DNP DNP DNP 2118 DNP DNP DNP DNR NRL did not register for the scheme 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 this method. Proficiency testing 63 Final Page 12 of 24

126 E. coli MPN Ten laboratories reported replicate results within ±2.68 SD of the participants medium with 10 receiving a maximum score. Laboratory 653 reported one replicate result between ±2.68 and ±4SD of the participants median and received a score of 9. Laboratory 715 reported one replicate result between ±2.68 and ±4SD of the participants median and reported both tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables and received an overall score of 5. Salmonella spp. All 11 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. Proficiency testing 63 Final Page 13 of 24

127 Figure 3. Distribution SF051: Sample SF0110 Proficiency testing 63 Final Page 14 of 24

128 Distribution SF051 Sample SF0111 contents E. coli (10² 10³) (wild strain), S. Anatum 3, {10}{15}{15,34} e,h:1,6 [z₆₄] (25) (wild strain), A. hydrophila (<10) (wild strain), E. faecium (10³) (wild strain). Analysed June / July 2015 Twelve laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratories 391, 583, 649, 658, 744, 1498, 1578, 1798, 1859 and 2118 did not participate in this distribution. Reference results E. coli MPN 4.49 x 10² 1.08 x 10⁴ per 100g. Salmonella spp. Detected in 25g. Table 10: Participants and reference results median, median 2.68 and 4 SD SF0111 Median MPN/100g Median 2.68SD MPN/100g Median 4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results 2.2 x x x x x 10 4 Participants results 1.7 x x x x x 10 4 Participants results SF0111 (Figure 4) Table 11: Results reported by participants and scores allocated SF0111 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected DNP DNP DNP Detected Detected DNR DNR DNR 583 DNP DNP DNP Detected DNP DNP DNP Detected Detected DNP DNP DNP Detected Detected Detected Detected Detected DNP DNP DNP 983 DNR DNR DNR 1498 DNP DNP DNP * NE 1578 DNP DNP DNP 1798 DNP DNP DNP 1859 DNP DNP DNP 2118 DNP DNP DNP DNR NRL did not register for the scheme 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 this method. Proficiency testing 63 Final Page 15 of 24

129 E. coli MPN Twelve laboratories reported replicate results within ±2.68 SD of the participants medium with 11 receiving a maximum score. Laboratory 121 was deducted points for reporting tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables. Salmonella spp. All 11 laboratories that reported a result for Salmonella spp. correctly reported the detection of Salmonella spp. and received a maximum score of 2. Proficiency testing 63 Final Page 16 of 24

130 Figure 4. Distribution SF051: Sample SF0111 Proficiency testing 63 Final Page 17 of 24

131 Appendix 3 Distribution SF052 Sample SF0112 contents E. gallinarum (3.6 x 10 4 ) (wild strain), M. varians (3.8 x 10 3 ) (wild strain). Analysed November / December 2015 Eighteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratory 403 reported 1 replicate result for E. coli. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 597, 720, 1578 and 2118 did not participate in this distribution. Reference results E. coli MPN <18 per 100g. Salmonella spp. Not detected in 25g. Participants results SF0112 Table 12: Results reported by participants and scores allocated SF0112 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Not Detected <18 <18 12 Not Detected <18 NE 7 Not Detected Not Detected DNR DNR DNR 583 <18 <18 12 Not Detected DNP DNP DNP 649 <18 <18 12 Not Detected <18 <18 12 Not Detected <18 <18 12 Not Detected Not Detected <18 <18 12 Not Detected <18 <18 12 NE 715 <18 <18 12 Not Detected <18 <18 12 Not Detected DNP DNP DNP 744 <18 <18 12 Not Detected DNR DNR DNR 1498 <18 <18 12 Not Detected <200 <200 8 * NE 1578 DNP DNP DNP 1798 <18 <18 12 Not Detected <18 <18 12 Not Detected DNP DNP DNP DNR NRL did not register for the scheme 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 this method. E. coli MPN Seventeen laboratories reported the absence of E. coli in this sample and received a maximum score. Laboratory 403 reported the absence of E. coli in one replicate result and received a maximum score of 7. Salmonella spp. All 16 laboratories that reported a result for Salmonella spp. correctly reported that Salmonella spp. was not detected and received a maximum score of 2. Proficiency testing 63 Final Page 18 of 24

132 Distribution SF052 Sample SF0113 contents E. coli (1 x x 10 3 ) (wild strain), S. Pensacola 1,9,12:m,t:[1,2] (70) (wild strain), B. pumilus (1.4 x 10 4 ) (wild strain), P. putida (3.3 x 10 3 ) (wild strain) Analysed November / December 2015 Eighteen laboratories received material for examination with all laboratories returning results to be included in the assessment. Laboratory 403 reported 1 replicate result for E. coli. Laboratories 703 and 1527 did not examine the sample for Salmonella spp.. Laboratories 597, 720, 1578 and 1859 did not participate in this distribution. Reference results E. coli MPN 1.59 x 10² 3.82 x 10³ per 100g. Salmonella spp. Detected in 25g. Table 13: Participants and reference results median, median 2.68 and 4 SD SF0113 Median MPN/100g Median 2.68SD MPN/100g Median 4SD MPN/100g Median +2.68SD MPN/100g Median +4SD MPN/100g Reference results 4.9 x x x x x 10 3 Participants results 7.8 x x x x x 10 3 Participants results SF0113 Table 14: Results reported by participants and scores allocated SF0113 Lab E. coli (per 100g) Salmonella spp. (per 25g) No. Replicate 1 Replicate 2 Score Salmonella spp. Score Detected Detected NE 2 Detected Detected DNR DNR DNR Detected DNP DNP DNP Detected Detected Detected Detected Detected NE Detected Detected DNP DNP DNP Detected DNR DNR DNR Detected * NE 1578 DNP DNP DNP Detected Detected DNP DNP DNP DNR NRL did not register for the scheme DNP NRL registered for EQA scheme but did not in this distribution NE Not examined * Score calculated out of 8 rather than 12 as tube combination not reported for this method. Proficiency testing 63 Final Page 19 of 24

133 E. coli MPN Sixteen laboratories replicate results within ±2.68 SD of the participants medium with 15 receiving a maximum score. Laboratory 403 reported a single replicate result which fall outside ±4SD of the participants median and scored 2. Laboratory 121 reported one replicate result between ±2.68 and ±4SD of the participants median and reported both tube combinations inconsistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables and received an overall scored 5. Laboratory 718 had scores deducted as the tube combinations reported were inconsistent with the guidance given in ISO 7218:2007/Amd 1:2013. 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. Proficiency testing 63 Final Page 20 of 24

134 Figure 5. Distribution SF052: Sample SF0113 Proficiency testing 63 Final Page 21 of 24