The DK-NRL activities according to Council Decision 99/313/EC

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1 Microbiological analysis of bivalve molluscs in Denmarks Anna Charlotte Schultz DK-NRL for monitoring bacteriological and viral contamination of bivalve molluscs, National Food Institute, Technical University of Denmark, DTU. Phone: Workshop om ostronkvalitet på Tjärnö den 26 april 2007

2 The DK-NRL activities according to Council Decision 99/313/EC Bacteria and viral analysis Accreditation status of NRL and national laboratories (NL s) Proficiency testing (PT) programmes Supervision to the Competent Authority (CA) Risk assessment of harvest areas CEN Tag4: EU standardisation of Virus detection in food and bottled water Ring trials Viral screening of DK shellfish harvesting areas Training of NL and technology transfer Research projects Nordic FoodVirus - Nordic network (NKJ: ) Foodborne virus - application for a Nordic research project (NKJ: ) European Cost 929 a European network for food-borne viruses ( ) Biotracer a EU research project ( ) DK-NRL Anna Charlotte Schultz, FOOD.DTU

3 Accreditation status of NRL and NL s NRL is not accredited and do not carry out any official control analyses on bivalve molluscs. Statutory E. coli analyses are conducted by four NL s Accreditation by the Danish Accreditation Body (DANAK) Live bivalve molluscs (BM) E. coli (ISO ) Salmonella (NMKL 71) Cooked and frozen BM E. coli (NMKL 125*) Pathogenic Vibrio s (NMKL 156/AOAC /DK STD*) Proficiency tests / programs HPA EQA Shellfish Scheme Swedish Livsmedelverket (E. coli* and Vibrio s)

4 PT programmes NRL participate in HPA EQA Shellfish Scheme Via a private NL Written contract between CA/NRL and the private laboratory. Res.11, 2nd NRL meeting NRL supervises in-country PT in testing laboratories HPA EQA Shellfish Scheme (since August 2005) PT performance assessment The scoring system of HPA has been adapted PT results from NL s to HPA are copied to NRL who catch poor performance as soon as possible take action if necessary An excel sheet is updated for each PT including Comments of NL s individual performance Explanation behind poor performance (if any) Action taken by the NL (if any) The CA and DANAK receives yearly a PT performance assessment document.

5 Example of PT performance assessment Opsamling og vurdering af NL's PT performance november 2006, SF025 Laboratorie resultater af præstationsprøvning SF025 Lab nr E. coli /100g Salmonella pr 25g SF0058 SF0058 SF0059 SF0059 SF0058 SF0059 A B A B Lab a a ip pos Lab b b ip pos Lab c c ip pos Lab d d ip pos Lab median 2.0E+2 7.5E+3 Endeligt resultat (HPA/lab median ± 3SD) E+3 1.4E+3-4.4E+4 HPA/lab median ± 5SD > E+3 3.8E+3-1.5E+5 SF023 SF024 SF025 Vurdering af sidste tre Kummulativ score* Max. mulig score Procent score Lab nr April 06 Juli 06 November 06 præstationer SF0054 SF0055 SF0056 SF0057 SF0058 SF0059 % E. coli Salmonella E. coli Salmonella E. coli Salmonella Lab a a 12 / 2 12 / 2 12 / 2 12 / 2 12 / 2 12 / Lab b b 12 / 2 6 / 2 12 / 2 12 / 2 12 / 2 12 / Lab c c 12 / 2 6 / 2 9 / 2 9 / 2 12 / 2 12 / Lab d d 12 / 2 12 / 2 9 / 2 12 / 2 12 / 2 12 / E. coli analyseres kvantitativt jvnf. ISO Alle værdier er MPN værdier og angiver koncentrationen af mikroorganismer / 100 g. < : opfattes som ikke påvist. Salmonella analyseres kvalitativt efter NMKL 71. pos: positiv. ip: Ikke påvist. NR : Resultater ikke returneret til ringtest udbyder. SD: standardafvigelse. HPA / lab median: middelværdi af resultater fra samtlige deltagende laboratorier. * Kummulativ score er baseret på (op til) de sidste tre deltagelser i HPA EQA shellfish scheme. Vurdering af resultater: Alle fire laboratorier deltog i ringtesten og reporterede tilbage med korrekte værdier indenfor både E. coli og Salmonella. Alle opnåede maximal score. ACS 09. jan 2007.

6 Supervision to the Competent Authority - 1 Risk assessment of harvest areas based on historical and incoming data from statutory E. coli analysis. Re-classification to long termed classified areas where possible Introducing fixed sample point where appropriate Member of CEN TC275/WG6/Tag4: EU standardisation of Virus detection in food and bottled water Aim: Validated standard method for detection of food-borne viruses 2012 Agents: norovirus and hepatitis A Matrices: soft fruits, salad, vegetables, bottled water, hard surfaces and bivalve molluscan shellfish Status: First draft standard Current work: Preparation for method validation trials including quality assurance Participation in yearly ring trials provided by CRL (CEFAS) since 2002 Agents: norovirus and hepatitis A Matrices: Faeces, cell culture and bivalve molluscs

7 Supervision to the Competent Authority - 2 Viral screening of DK shellfish harvesting areas using earlier in-house methods -> using proposed quantitative and quality assured CEN methods Training of NL and method technology transfer In connection with viral analyses a guest from NL will visit NRL and conduct the analyses Aim: Method transfer to NL in 2008 Viral analyses of bivalve moluscs (etc.) associated in outbreaks DK-NRL Anna Charlotte Schultz, FOOD.DTU

8 Viral screening of Danish bivalve molluscs Samples: 300 samples collected in the period of March December 2005 Oysters (Orstrea edulis) and Mussels (Mytilus edulis) Aim of harvest: commercial use Harvest areas: natural and aqua culture establishments located in The Liimfiord, The East coast of Jutland and the South of Funen and Isefjord Methods: RNA extraction: Le Guyader et al mod. NoV detection Screening: RT-realtime PCR (Loisy et al. 2005, Vinjé 2005). Typing: RT-nested PCR (De Medici et at 2004 mod.) and Reverse Line Blot (Vinjé et al. 2000) or sequencing HAV detection Screening: RT-realtime PCR (Bosch et al. 2005) Typing: RT-nested PCR (Pina et al and De Medici et al. 2001) DK-NRL Anna Charlotte Schultz, FOOD.DTU

9 Preliminary results So far 120 samples are analysed All HAV negative All NoV GI negative One sample confirmed positive for NoV GII (Melksham) Due to old samples the action has been to evaluate results from samples harvested in the area of interest prior and after the positive finding. re-evaluate the classification of the harvest area Evaluate the propose of harvest and sale (e.g. direct consumption or post-treatment)

10 Nordic collaboration FoodVirus A Nordic network for validation and standardization of methods for detection of Food and waterborne Viruses. Funding: Nordic Agriculture Research, NKJ Partners: two food/waterborne virus colleagues pr. Nordic country Webpage: 3rd and last workshop was held in Stockholm March 2007 Foodborne virus Application on its way to (NKJ: ) Proposal for a Nordic research project Partners based on the FoodVirus - network