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

European Union Reference Laboratory for Monitoring Bacteriological and Viral Contamination

of Bivalve Molluscs, Cefas, Weymouth

Technical Report for Calendar Year 2012

EURL for monitoring bacteriological and viral contamination of bivalve molluscs annual technical report calendar year 2012

Page 1 of 15

Contents

Legal functions and duties 2

Introduction 2

1. Scientific advice and support 2

2. Co-ordination of activities of NRL network and provision of technical assistance and

training

5

3. Proficiency testing and quality assurance 11

4. Confirmatory testing 15

5. Development of analytical methods 15

List of Annexes

Annex I - Work programme for the EURL for bacteriological and viral contamination of

bivalve molluscs 2012.

Annex II - Resolutions of the 11th

workshop of NRLs for bacteriological and viral

contamination of Bivalve Molluscs, 2012.

Annex III - Agenda of the 11th

workshop of NRLs for bacteriological and viral

contamination of Bivalve Molluscs, 2012.

Annex IV - Confidential participant feedback results from the 11th

workshop of NRLs

for bacteriological and viral contamination of Bivalve Molluscs, 2012.

Annex V - 2nd

International workshop on shellfish area classification and management -

application of sanitary surveys.

Annex VI - Escherichia coli and Salmonella spp EQA - PT 48.

Annex VII - Enumeration of Escherichia coli and the detection of Salmonella spp. in

Pacific oysters (Crassostrea gigas) and Common mussels (Mytilus edulis) - PT45.

Annex VIII - PT46 - norovirus (genogroup I and II) and hepatitis A virus proficiency

testing.


Page 2 of 15

European Union Reference Laboratory for Monitoring Bacteriological and Viral

Contamination of Bivalve Molluscs, Cefas, Weymouth

Technical Report for Calendar Year 2012

Legal functions and duties

The functions and duties of the European Union Reference Laboratory (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 2012 was approved by the European Union (EU) in

December 2011. This report details activities of the EURL according to the work programme 2012

(Annex I), additional tasks described under the resolutions of the 11th

workshop of microbiological

NRLs held in Weymouth (Annex II) and other responsibilities outlined in Commission Regulation

(EC) 882/2004 for the calendar year 2012.

1. Scientific advice and support

To the European Union

The EURL has provided the following advice and support to the EU (DG Sanco) through

participation in expert working groups, provision of briefing documents and guidance, specifically

in 2012 this has comprised:

Participation in the Commission restricted expert working group on bivalve molluscs

(meetings in 2012 in January and March)

Elaboration of the Community Guide on microbiological monitoring of bivalve mollusc

harvesting areas now published on the Europa website.

http://ec.europa.eu/food/food/biosafety/hygienelegislation/good_practice_en.htm.

Contribution to ESFA opinion (mandate EFSA-Q-2010-00926) on ―Scientific opinion on

norovirus (NoV) in oysters: methods, limits and control options‖. EFSA published January

2012 http://www.efsa.europa.eu/en/search/doc/2500.pdf

Final recommendations on harmonisation of E. coli standards for live bivalve molluscs

(LBM) under Codex (CODEX STAN 292-2008) and EU Food Hygiene legislation.

Expert advice to SCFCAH on validated alternative methods for official control analysis of

LBM.

EU/US negotiations on LBM and associated hygiene standards. Including representation at

trade negotiations with US FDA, Washington in December 2012.

http://ec.europa.eu/food/food/biosafety/hygienelegislation/good_practice_en.htm

http://www.efsa.europa.eu/en/search/doc/2500.pdf


Page 3 of 15

Response to FDA comments regarding the EU ‗Community Guide to the Principles of Good

Practice for the Microbiological Monitoring of Bivalve Mollusc Harvesting Areas with

regard to Regulation 854/2004‘.

Preliminary discussions and advice on viruses in LBM, potential future control options.

Provision of advice to DG Sanco on virus (norovirus and hepatitis A virus) methods and

application including sampling plans for foodstuffs of non-animal origin (specifically

strawberries) in response to Regulation (EU) No. 1235/2012.

Advice to other entities and contributions to standardisation activities under ISO or CEN working

groups

The EURL led CEN/TC 275/WG6/TAG4 group developing a horizontal ISO standard for

determination of norovirus and hepatitis A virus in LBM. In 2012 this has included:

Completion of the standard reference methods for determination of norovirus and hepatitis

A virus in foodstuffs, including LBM,

o ISO TS 21516-1, Microbiology of food and animal feed: determination of norovirus

and hepatitis A virus in foodstuffs by PCR, part 1: quantitative determination.

o ISO TS 21516-2, Microbiology of food and animal feed: determination of norovirus

and hepatitis A virus in foodstuffs by PCR, part 2: qualitative determination.

The EURL led the CEN/TC 275/WG6/TAG3 programme on work on food borne pathogenic vibrios

using molecular approaches, including:

Development of real-time PCR method to enable semi-quantitative (and quantitative) in

seafood including LBM (attendance at one meeting in 2012, see also section 2 – Vibrio

expert working group July 2012).

Revision of ISO TS 21872 parts 1 and 2, Microbiology of food and animal feed (part 1),

Detection of pathogenic Vibrio spp., Detection of Vibrio parahaemolyticus and V. cholerae

in foodstuffs and (part 2) Detection of pathogenic Vibrio spp., detection of pathogenic

Vibrio spp. other than Vibrio parahaemolyticus and V. cholerae in foodstuffs.

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 16649-3). This revision is now completed and

will establish the EU E. coli reference method as a full standard thus removing the need to

withdraw the standard.

The EURL has led the ISO/SC9/WG8 expert group on the revision of the ISO 6887-3, sample

preparation and preparation of initial dilutions for LBM. In 2012 the EURL attended two meetings

and completed the revision of the standard – this now is harmonised with EU legislation (Reg (EC)

No. 2073/2005).

The EURL acted as rapporteur at the first expert working group for method verification.

The EURL contributed to the FAO/WHO initiatives for the conduct of national risk assessments for

human pathogenic vibrios in seafoods, http://www.fao.org/food/food-safety-quality/scientific-

advice/jemra/en/

The EURL contributed reports and publications to the joint Canadian/US risk assessment on

norovirus in shellfish in January 2012.

http://www.fao.org/food/food-safety-quality/scientific-advice/jemra/en/

http://www.fao.org/food/food-safety-quality/scientific-advice/jemra/en/


Page 4 of 15

The EURL contributed to the expert working group on the EFSA scientific opinion on the minimum

hygiene criteria to be applied to clean seawater and on the public health risks and hygiene criteria

for bottled sea water intended for domestic use. http://www.efsa.europa.eu/en/search/doc/2613.pdf

Support was provided to FVO in the form of provision of National Experts for LBM audits to South

Korea and Italy. The EURL provided follow-up technical assistance to FVO following audit

responses from New Zealand and Mexico.

Technical reports and advisory documents

In 2012 the EURL authored technical reports and advice notes covering the following topics:

Report of the 11th

workshop of NRLs for monitoring bacteriological and viral contamination

of bivalve molluscs.

NRLs participation and performance assessment in the whole animal distribution for E. coli

and Salmonella (PT41).

NRLs participation and performance assessment in the EURL/HPA shellfish EQA scheme

for E. coli and Salmonella (PT44).

NRLs participation and performance assessment in the EURL/HPA shellfish EQA scheme

for E. coli and Salmonella (PT48)

Supervision of official control laboratories by NRLs.

EURL guidance on the frequency and level of proficiency test participation.

Intra-laboratory trial for generation of data for the revision of ISO TS 21872 series.

Control options for norovirus.

Quantitative detection of norovirus and hepatitis A virus (draft generic protocol)

EURL norovirus and hepatitis A virus proficiency testing (PT) PT43.

Application of sanitary surveys in EU Member States (MS).

EURL recommendations regarding possible harmonisation of EU and Codex standards for

LBM – final version.

Plenary meeting of microbiology committees CEN/TC275 and ISO SC9 Brussels 2012 - in

collaboration with EURLs in the food and feed sector.

Response to FDA comments regarding the EU ‗Community Guide to the Principles of Good

Practice for the Microbiological Monitoring of Bivalve Mollusc Harvesting Areas with

regard to Regulation 854/2004‘.

2nd

International workshop on shellfish area classification and management – applications of

sanitary surveys.

Real-time RT-PCR results for norovirus in oysters; the relationship between CT values and

copies/g digestive tissues.

Copies of all reports are available from the EURL co-ordinator on request or from the information

centre of the EURL website (www.eurlcefas.org)

http://www.efsa.europa.eu/en/search/doc/2613.pdf

http://www.eurlcefas.org/


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2. Co-ordination of activities of NRL network and provision of technical assistance and

training

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 www.eurlcefas.org (Table 1).

Table 1. Designated NRLs in Member States, EFTA and Accession states in 2012.

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

Luxembourg

Scientific Institute of Public Health (IPH), Rue Juliette Wytsmanstraat 14,

1050 Brussels.

Bulgaria National Diagnostic and Research Veterinary Institute, Pencho, Slaveikov,

15 BG - 1606 Sofia

Croatia Croatian Veterninary Institute, Regional Veterinary Laboratory Split,

Poljicka Cesta 33, 21000 Split, Croatia

Cyprus No NRL designated

The Czech

Republic

State Veterinary Institute, Rantirovska 93, Jihlava, CZ – 58605, Jihlava

Denmark Department of Microbiology and Risk Assessment, National Food Institute,

Technical University of Denmark, Morkhoj Bygade 19, DK 2860 Soborg.

Estonia No NRL designated

Finland Finnish Food Safety Authority Evira, Research Department Microbiology

Unit, Mustialankatu 3, FI-00790, Helsinki

France IFREMER, Departement Microbiologie et phycotoxines, Centre de Nantes,

Rue de I'lle de'Yeu, BP 21105, 44311 Nantes Cedex 3.

Germany Federal Institute for Risk Assessment, Diedersdorfer Weg, D-12277,

Berlin.

Greece Institute of Food Hygiene of Athens, Neapoleos 25, 15310 Ag. Paraskevi,

Attiki, Athens.

Hungary Central Agricultural Institiute, Food and Feed Safety Directorate Mester u.

81, H-1095 Budapest.

Iceland 1 Matis, Matvaelarannsoknir Islands, Skulagotu 4, 101 Reykjavik.

Ireland Marine Institute, Orinville, Oranmore, Galway.

Italy 3 Istituto Zooprofilattico Sperimentale Umbria e Marche, Via Cupa di

Posatora 3, 60131, Ancona.

Dipartmento di Sanita Pubblica Veterinaria e Sicurezza Alimentare, Viale

Regina Elena 299, Roma.

Latvia Institute of Food Safety, Animal Health and Environment (BIOR), Lejupes

str.3, LV-1076 Riga.



Page 6 of 15

Lithuania National Veterinary Laboratory, J.Kairiukscio 10, LT-2021 Vilnius.

Malta No NRL designated

The Netherlands National Institute for Public Health and the Environment (RIVM), PO Box

1 A van, Leeuwenhoeklaan 9, 3720 BA, Bilthoven.

Norway 2 Institute for Food Safety and Infectious Biology, Department for Food

Safety, P.O Box 8146 Dep, 0033 Oslo.

Poland National Veterinary Research Institute, Panstwowy Instytut,

Weterynaryjny, Al.Partyzantów 57, PL - 24-100 Pulawy.

Portugal Instituto de Investigacao das Pescas e do Mar (IPIMAR), Avenida Brasilia,

1449-006 Lisboa.

Romania Institute for Diagnosis and Animal Health, Str. Dr. Staicovici, nr. 63

Sector 5, cod 050557, Bucuresti.

Slovakia State Veterinary and Food Institute, Janoskova 1611/58 Ministry of

Agriculture, SK - 02601 Dolny Kubin.

Slovenia National Veterinary Laboratory, Gerbiceva 60, SI – 1000, Ljubljana.

Spain Centro Nacional de Alimentacion, Agencia Espanola de Seguridad

Alimentaria, E-28220 Majadahonda, Madrid.

Sweden National Food Administration, P.O. Box 622, 751 26 Uppsala.

Turkey 1 No NRL established

United Kingdom Centre for Environment, Fisheries and Aquaculture Science (Cefas),

Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4

8UB. 1 Candidate Country

2 Member of the EFTA

3 2 NRLs established (microbiological monitoring and virology)

EURL website

The EURL website www.eurlcefas.org was used extensively by stakeholders in 2012 and continues

to provide a very useful repository for information for NRLs and other stakeholders. In addition to

the well established sections covering legislation, methods, quality assurance and workshops in

2012, on request of the EU, the site was extended to enable dissemination of information and

protocols for virus analysis of soft fruit to support implementation of Regulation (EU) No.

1235/2012. This illustrated the utility of a centralised method of dissemination and enabled rapid

communication of important technical information to MS and relevant laboratories across the EU.

During 2012 the website has been substantively reviewed and accessibility improved following

feedback from NRLs and other stakeholders. The improved site will be live in 2013.



Page 7 of 15

Figure 1. Hits per month www.eurl.cefas.org in 2011 and 2012

Figure 2. Visitors per month www.eurl.cefas.org in 2011 and 2012

Annual workshop of NRLs

The EURL institute in Weymouth, U.K hosted the 11th

workshop of NRLs on the 24th

, 25th

and 26th

April 2012. Thirty-seven delegates representing 23 EU MS, EFTA countries and accession

countries took part in the workshop. Following a short introductory meeting, the workshop

discussed microbiological monitoring and classification, liaison with ISO and CEN (method

development and standardisation), PT, marine vibrios and viruses. The main focus of this year‘s

workshop was hepatitis A virus in LBM. Professor Albert Bosch attended as an invited expert from

the University of Barcelona, Spain and presented an excellent overview of hepatitis A virus and the

contribution seafood makes to community disease burden. Twenty-two resolutions were agreed by

the workshop. A full report of workshop of NRLs is available from the information centre of the

EURL website (www.eurlcefas.org). As part of the programme of continuing cooperation with the

EURL for biotoxin in Vigo, Spain, Prof Bermudez attended the workshop as an observer. The

Hits are defined as requests from

users to a web server for a file, web

page or document.

Between January and December

2012 users registered an average of

12,249 hits per month representing a

9% increase on 2011.

Visitors are defined as a unique

individual visiting the website.

Between January and December

2012 the EURL website received an

average of 2,280 visitors per month.

This represented an increase of 37%

on 2011.

http://www.eurl.cefas.org/

http://www.eurl.cefas.org/


http://en.wikipedia.org/wiki/Web_server


Page 8 of 15

workshop resolutions are included as Annex II of this report and the workshop agenda as Annex III.

Anonymised feedback was obtained from delegates attending the workshop, this indicated

predominantly good to very good performance, feedback breakdown is provided as Annex IV.

2nd

International Workshop on Molluscan Shellfish Area Classification

The EURL co-organised the 2nd

International Workshop on Molluscan Shellfish Area Classification

to be held in Rhode Island, USA, 24th

- 28th

September 2012. Presentations from speakers from EU

and US covered the main philosophical considerations of the EU and US approaches, and desk

based and practical exercises proved very effective in illustrating the programmes. Fifty-three

delegates from Australia, Canada, Denmark, France, Ireland, Italy, Mexico, The Netherlands, New

Zealand, Peru, United Kingdom and the US attended the 5 day event. A set of recommendations

were agreed in brief, these considered that a best practice approach should be elaborated. In the first

instance FAO/WHO would be approached to elicit support for an international expert working

group to draft a ―best practice‖ guide based upon the pre-existing framework used in the Codex

Ailmentarius: Code of Practice for fish and fisheries products – section 7 - live and raw bivalve

molluscs. The EURL would progress this activity in 2013. The full outputs of the workshop are

provided as Annex V and are available at

http://www.crlcefas.org/InformationCentre/docs/EU_US_workshop_on_shellfish_sanitation_outco

mesFINAL.pdf.

1st international expert practical working group of Vibrio methods, controls and their application for

bivalve molluscs

The EURL organised and hosted the first international expert practical working group of Vibrio

methods, controls and their application for bivalve molluscs in July 2012. The workshop was

attended by 12 representatives from NRLs with a particular interest in pathogenic Vibrio spp.. Prof.

James Oliver from University of North Carolina provided additional expert input.

Additional ad hoc training and study visits

The EURL hosted a study visit for a member of staff from the National Diagnostic and Research

Veterinary Institute (The Bulgarian NRL) - specifically covering test methods for classical and

molecular detection of human pathogenic Vibrio spp. in LBM.

Provision of advice to NRLs and others

In 2012 the EURL provided advice (briefing notes, technical reports, etc) to laboratories within the

network of NRLs and others. Advisory activities requiring input of half a person day or more, or

those with written outputs are included here:

Proficiency testing and statutory determinands

Advisory note to laboratories on the participation and recommended frequency of PT for

statutory determinands.

Trend analyses for ongoing performance in statutory comparative testing for E. coli for all

http://www.crlcefas.org/InformationCentre/docs/EU_US_workshop_on_shellfish_sanitation_outcomesFINAL.pdf

http://www.crlcefas.org/InformationCentre/docs/EU_US_workshop_on_shellfish_sanitation_outcomesFINAL.pdf


Page 9 of 15

NRLs.

Microbiological monitoring and classification

The EURL provided advice to FVO on the technical equivalence of methods following the

Mexican response to the EU audit of LBM.

Advice to FVO on New Zealand response to FVO specifically relating to compositing of

samples for detection of Salmonella spp. in LBM.

Advice to the Danish Food Administration on the origins of US FDA faecal coliform

standards for shellfish growing waters.

Virus

The EURL provided a preliminary dossier to the US Food and Drug Administration (FDA)

and Canadian Food Inspection Agency, Environment Canada, and Fisheries and Oceans

Canada as part of the Quantitative food safety risk assessment on norovirus in bivalve

molluscan shellfish.

Additional technical advice was provided to laboratories via e-mail or telephone on an ad hoc basis.

Other Scientific activities –- participation in international scientific fora

EURL staff presented norovirus methodology development at several international conferences in

2012; Noro2012 - Norovirus and Other Caliciviruses on the Rise, Lubeck, 3rd

Food and

Environmental Virology Conference, Lisbon.

The EURL is a member of the International Steering Committee responsible for planning and

organisation of the next International Conference Molluscan Shellfish Sanitation (ICMSS)

conference in Sydney in March 2013.

Additional outputs

EURL staff have produced a number of peer-review papers in scientific journals, book chapters and

other publications:

EURL Publications

In 2012 the EURL produced the following peer review publications and book chapters:

1. Baker-Austin, C., Campos, C.J.A., Turner, A., Lees, D. Human health risks from the marine

environment. MCCIP Report Card. Submitted.

2. Campos, C.J.A., Kershaw, S.R., Lee, R.J. Environmental influences on faecal indicator

organisms in coastal waters and their accumulation in bivalve shellfish. Estuaries and Coasts.

Submitted.

3. Morrison, S. A. Cain, B. Froelich, T. Williams, C. Taylor, D. Verner-Jeffreys, R. Hartnell, J. D.

Oliver, C. Baker-Austin, and C. Gibas. Pyrosequencing-based comparative genome analysis of


Page 10 of 15

Vibrio vulnificus environmental isolates. 7(5):e37553. PLOS One 2012.

4. Verner-Jeffreys, D., C. Baker-Austin, et al. Zoonotic bacterial pathogens isolated from

imported Doctor fish (Garra rufa) destined for use in UK pedicure spa treatments centers.

Emerging Infectious Disease, 8:1006-1008, 2012.

5. Baker-Austin, C., J. Trinanes, R. Hartnell, N. Taylor, A. Siitonen, and J. Martinez-Urtaza.

Emerging Vibrio risk at high-latitudes in response to ocean warming. Nature Climate Change,

3:73-77. doi:10.1038/nclimate1628.

6. Lowther JA, Gustar NE, Powell AL, Hartnell RE, Lees DN. (2012). Two-year systematic study

to assess norovirus contamination in oysters from commercial harvesting areas in the United

kingdom. Appl Environ Microbiol 78:5812-5817.

7. Baker-Austin C, Lemm E, Hartnell R, Lowther J, Onley R, Amaro C, Oliver JD, Lees D.

(2012). pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of

human health relevance. Food Microbiol. 30:17-23.

8. Lowther JA, Gustar NE, Hartnell RE, Lees DN. (2012) Comparison of norovirus RNA levels in

outbreak-related oysters with background environmental levels. J Food Prot. 75:389-393.

9. Hartnell R, Lowther J, Avant J, Dancer D, Lees D, Russell J. (2012). The development of

LENTICULES™ as reference materials for noroviruses. J Appl Microbiol. 112:338-345.

10. C.J.A. Campos, R. Lee, S. Kershaw, O. Morgan, J. Crowther, and D. Kay (2012) Factors

affecting the microbial quality of shellfish. Shellfish News, May 2012.

11. A.D. Turner, M. Rapkova, M. Algoet, B. A. Suarez-Isla, M. Cordova, C. Caceres, C. J. Murphy,

M. Casey and D. N. Lees (2012) Investigations into the matrix components affecting the

performance of the official bioassay reference method for quantitation of paralytic shellfish

poisoning toxins in oysters. Toxicon 59 (2012); p 215-230.

Note. Abstracts of all EURL publications will shortly be available on the EURL website; selected

publications can be obtained directly from the EURL co-ordinator on request.

U333333 3 Provision of methods, proficiency testing, reference materials and quality assurance

Standard methods

EURL generic protocols for reference methods and approved alternative methods were reviewed

according to the annual cycle and maintained on the EURL website. All methods were distributed

on request to NRLs, Official Control Laboratories and third country laboratories.

The virus methods (ISO TS 15216 parts 1 and 2) were agreed at the relevant ISO committee stages

and will be published in early 2013. Therefore a reference method for norovirus and hepatitis A

virus in bivalve shellfish will be available very shortly as an International Standard. In preparation

the EURL made available a generic protocol on the EURL website.

A protocol, sampling plan and additional information for testing laboratories undertaking analysis

of soft fruits for norovirus and hepatitis A virus was produced to support the publication of


Page 11 of 15

Regulation (EU) No. 1235/2012.

Proficiency testing

PT distributions have been organised by the EURL between January and December 2012 these

comprised.

For official control deteminands - E. coli and Salmonella spp. in March, July and November

2012 - Lenticules™ EURL reference PT48.

The annual distribution of whole shellfish for E. coli and Salmonella - October 2012. EURL

reference PT45.

The annual distribution of laboratory constructed and bivalve shellfish for detection of

norovirus and hepatitis A virus – November 2012. EURL reference PT46.

All PT reports were distributed to participants and posted on the EURL website www.eurlcefas.org.

PT reports or intended results are presented as Annexes VI, VII, and VIII of this technical report.

Follow-up procedures were not triggered in 2012 as all laboratories performed satisfactorily in

statutory determinand distributions.

Participation of MS NRLs, accession and third countries in EURL organised PT in 2012 is

tabulated in Table 2 and summarised in this section.



Page 12 of 15

Table 2 - Summary of participation amongst NRLs and others in EURL organised PT in 2012

1 no designated NRL

2 not registered for E. coli/Salmonella EQA scheme

3 more than one laboratory participated in PT 45 distribution

4 more than one laboratory participated in PT 46 distribution

EU

RL

pro

fici

ency

tes

tin

g r

efer

ence

nu

mber

Pro

fici

ency

tes

tin

g d

escr

ipti

on

EU NRLs EFTA Third country laboratories

Au

stri

a

Bel

giu

m a

nd L

uxem

bou

rg3 4

Bulg

aria

2

Cro

atia

Cyp

rus

1

Cze

ch R

epub

lic

2

Den

mar

k 3

Est

onia

1

Fin

lan

d

Fra

nce

4

Ger

man

y 3

4

Gre

ece

3

Hu

ng

ary

Icel

and

2

Irel

and

Ital

y 3

4

Lat

via

2

Lit

hu

ania

2

Mal

ta 1

Net

her

land

s 4

Po

land

Po

rtu

gal

Ro

man

ia 3

Slo

vak

ia 2

Slo

ven

ia

Sp

ain

4

Sw

eden

Un

ited

Kin

gdo

m 4

No

rway

3

Au

stra

lia

Can

ada

4

Chil

e

New

Zea

land

Per

u 4

Sin

gap

ore

So

uth

K

ore

a 4

Tun

isia

Tu

rkey

3

Un

ited

Sta

tes

PT 45 E. coli / Salmonella whole

animal 2012 x x x x x x x x x x x

PT 46 Norovirus / Hepatitis A

whole animal 2012 x x x x x x x x x x x x x x

PT 48 E. coli / Salmonella EQA

2012 x x x x x x x x x x x x x x x x x x x


Page 13 of 15

EURL proficiency testing for E. coli / Salmonella (EURL reference PT 48 and PT 45)

The Shellfish EQA scheme is targeted at analysis of the statutory determinants E. coli and

Salmonella spp. in bivalve molluscs. The scheme uses laboratory constructed Lenticules™ which

are easy to transport and provide simulated bacterial samples representative of the natural flora

found in bivalves from a typical harvesting area.

In addition, to test the initial preparation of bivalve samples i.e. opening bivalves on receipt in the

laboratory and preliminary dilutions a matrix distribution is also provided. NRLs are expected to

participate in at least 1 of 3 Lenticule™ distributions and the matrix PT annually. NRLs are

assigned PT scores for each distribution and a cumulative performance calculated. Laboratories

achieving over 70% of the attainable maximum are considered to be performing satisfactorily. NRL

performance for E. coli is tabulated in Table 3 and Table 4 for Salmonella spp. for 2012.

Table 3. Summary of participants’ performance in the EURL matrix scheme and the EQA

scheme – E.coli

Lab

no.

EURL PT 45 EURL PT48 All distributions

Sample

1

Sample

2 SF0090 SF0091 SF0092 SF0093 SF0094 SF0095

Cumulative

score

Max

score %

121 12 12 12 12 12 12 12 12 96 96 100

391 12 12 12 12 - - 12 12 72 72 100

403 12 12 12 12 12 12 - - 72 72 100

413 12 12 12 12 12 12 4 9 85 96 89

493 12 12 - - - - 12 12 48 48 100

583 12 12 12 12 - - 12 12 72 72 100

596 a - - - - - - 12 12 - - -

597 10 10 12 12 12 12 12 12 92 96 96

601 a - - 12 12 - - 12 12 - - -

649 12 12 12 12 12 12 12 10 94 96 98

651 12 12 12 12 12 12 12 12 96 96 100

653 12 12 12 12 12 12 12 12 96 96 100

658 a 6 12 - - - - - - - - -

660 a 12 12 - - - - - - - - -

662 a - - - - - - - - - - -

701 12 12 12 9 12 12 12 12 93 96 97

703 12 12 12 6 12 12 12 12 90 96 94

715 10 10 - - - - 12 12 44 48 92

718 12 12 12 12 9 12 12 12 93 96 97

720 12 12 12 12 12 12 12 12 96 96 100

744 12 12 12 12 12 12 - - 72 72 100

983 a 12 12 - - - - - - - - -

1498 12 12 12 12 12 12 12 12 96 96 100

1527 12 12 12 12 12 12 12 12 96 96 100

1578 12 10 12 12 12 12 - - 70 72 97

1798 12 12 - - 12 12 10 12 70 72 97 a full performance assessment was not carried as laboratories did not participate in PT 45 and 1 EQA distribution during 2012.


Page 14 of 15

Table 4. Summary of participants’ performance in the EURL matrix scheme and the EQA

scheme - Salmonella

a full performance assessment was not carried as laboratories did not participate in PT 45 and 1 EQA distribution during 2012.

All NRLs that participated in at least two annual distributions for E. coli and Salmonella spp. met

the satisfactory performance indicator of >70%. NRLs Bulgaria, The Czech Republic, Latvia,

Lithuania and Slovakia have not participated sufficiently in 2012. The EURL has contacted these

laboratories to request further information on lack of participation in EURL PT under stage one of

the Commission protocol (Anon 2009).

Full performance reports are provided as Annexes IV and V.

Proficiency testing for non-statutory determinands - viruses

Norovirus and hepatitis A virus proficiency testing (PT 46)

In October 2012 the EURL distributed a PT distribution comprising of both naturally and

artificially contaminated matrix samples (C. gigas and M. edulis), laboratory constructed

Lenticule™ discs and control material. Material was distributed to 42 laboratories (16 NRLs, see

Table 2). The intended results are included as Annex V.

A full report of participant performance is in preparation and will be presented to NRLs at the 11th

workshop in May 2012. In brief, performance of NRLs undertaking the analyses has improved from

Lab

no.

EURL PT 45 EURL PT 48 All distributions

Sample

1

Sample

2 SF0090 SF0091 SF0092 SF0093 SF0094 SF0095

Cumulative

score

Max

score %

121 2 2 2 2 2 2 2 2 16 16 100

391 2 2 2 2 - - 2 2 12 12 100

403 2 2 2 2 2 0 - - 10 12 83

413 2 2 2 2 2 2 0 2 14 16 88

493 2 2 - - - - 2 2 8 8 100

583 2 2 2 2 - - 2 2 12 12 100

596 a - - - - - - 2 2 - - -

597 2 2 2 2 2 2 2 2 16 16 100

601 a - - 2 2 - - 2 2 - - -

649 2 2 2 2 2 2 2 2 16 16 100

651 2 2 2 2 2 2 2 2 16 16 100

653 2 2 2 2 2 2 2 2 16 16 100

658 a 2 2 - - - - - - - - -

660 a 2 2 - - - - - - - - -

662 a - - - - - - - - - - -

701 2 2 2 2 2 2 2 2 16 16 100

703 2 2 2 2 2 2 2 2 16 16 100

715 2 2 - - - - 2 2 8 8 100

718 2 2 2 2 2 2 2 2 16 16 100

720 2 2 2 2 2 2 2 2 16 16 100

744 2 2 2 2 2 2 - - 12 12 100

983 a 2 2 - - - - - - - - -

1498 2 2 2 2 2 2 2 2 16 16 100

1527a 2 2 - - - - - - - - -

1578 2 0 2 2 2 2 - - 10 12 83

1798 2 2 - - 2 2 2 2 12 12 100


Page 15 of 15

2011 with respect to both qualitative and quantitative determination. This illustrates the importance

of ongoing PT and EURL assistance in this method implementation period. However, nine NRLs

currently lack the capacity to participate in virus testing exercises. The EURL has organised a

training course for NRLs with no/limited experience of virus testing methods in early 2013. It is

anticipated that will help to standardise virus testing capacity across EU MS laboratories.

Reference materials

Reference and control materials in the form of faecal material containing characterised norovirus,

plasmids with dsDNA for norovirus and hepatitis A virus quantitative and qualitative analysis and

fully characterised strains of human pathogenic Vibrio spp. were supplied on request to NRLs and

third country laboratories. Provision of these materials continues to support laboratories in

implementation and quality assurance of methods. A full distribution list of control material is

available on request.

4. Confirmatory testing

The EURL is accredited by the United Kingdom Accreditation Service (UKAS) schedule number

UKAS 2293. UKAS is a member of the European co-operation for accreditation (EA).

Accreditation to ISO 17025 was retained in 2012 for the following methods and associated standard

operating procedures:

Detection of Salmonella spp. in bivalve molluscan shellfish (ISO 6579).

Enumeration of E. coli in bivalve molluscan shellfish (ISO TS 16649-3).

Detection of V. parahaemolyticus in bivalve molluscan shellfish (ISO TS 21872-1).

Determination of norovirus in bivalve molluscan shellfish (ISO TS 15216-1)

Samples of green-lipped mussels originating in New Zealand were tested as part of an investigation

into a RASFF notification concerning detection of hepatitis A virus.

5. Development of analytical methods

The EURL has progress work according to the 2012 work programme on the development of direct

detection methods for the enumeration of pathogenic strains of V. parahaemolyticus and V.

vulnificus. This work is in support of method development activities through CEN TAG3. It also

compliments the activities of the CEN working group on revision of ISO TS 21872 series (Vibrios).

The EURL has undertaken practical investigations into the performance of the draft standard

norovirus method (ISO TS 15216-1, 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) particularly with respect to limit of detection, limit of quantitation

and linearity in oysters (Crassostrea gigas).

In house verification of the approved alternative methods for the enumeration of E. coli in bivalve

shellfish has also been undertaken in 2012.

EURL, March 2013

Page | 1 EURL monitoring bacteriological and viral contamination of bivalve molluscs v1 26_08_11 work programme

European Union Reference laboratory for

monitoring bacteriological and

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) 1305 206600, Fax +44 (0) 1305 206601

Email: [email protected] http://www.crlcefas.org

WORK PROGRAMME FOR THE EURL FOR BACTERIOLOGICAL AND

VIRAL CONTAMINATION OF BIVALVE MOLLUSCS, 2012

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 30.4.2004).

In the 2012 work programme year 27 Member States and 3 candidate countries (Croatia, Turkey and

Republic of Macedonia) are considered eligible for EURL assistance and invited to participate in

EURL organised training programmes, comparative testing etc. The full integration into the European

Union of recent accession Member States continues to be a priority area, and is facilitated via the

provision of additional advice, training and assistance.

WORK PROGRAMME, 2012

1. Scientific advice and support – (up to 100 days)

1.1. The EURL will provide scientific assistance to DG SANCO in operation and implementation

of European Union food hygiene legislation, and in particular in 2012 the following activities

have been identified:

1.1.1. Provide scientific and statistical assistance with the ongoing equivalency negotiations

between EU and US for live bivalve molluscs (LBM).

1.1.2. Provide final recommendations on harmonisation of standards for class A shellfish

and end products in EU hygiene legislation with CODEX standards for LBM with

respect to E. coli and potential implications for Salmonella.

1.1.3. Publish harmonised protocols for alternative methods validated according to ISO

16140 or equivalent for official control use.

1.1.4. Provision of updated dossier of information on application of sanitary surveys across

MS with LBM production areas.

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.

1.2 Participate in relevant EU and International scientific committees (EFSA, ISO/CEN,

WHO/FAO, ICMSS etc). In 2012 the EURL will:

1.2.1 Co-ordinate the activities of the CEN/TC 275/WG6/TAG4 for the elaboration of the

standard methods for detection of norovirus and hepatitis A in foodstuffs, including

bivalve molluscs. In 2012 this activity will be restricted to responding through

electronic working groups to technical comments arising from the publication process

for the technical specifications for quantitative and qualitative standards.






Science


Barrack Road,

The Nothe,

Weymouth,



1.2.2 Lead and co-ordinate the activities of CEN/TC 275/WG6/TAG3 in the elaboration of

molecular based enumeration methods for pathogenic marine vibrios in bivalve

shellfish, particularly for V. parahaemolyticus. Two missions associated with this

activity are anticipated in 2012.

1.2.3 Lead the revision of the EU reference method for enumeration of E. coli in LBM for

official control (ISO TS 16649-3) to establish the method as a full standard. To be

conducted through electronic working group.

1.2.4 Project leader for the revision of the ISO 6887 series part 3 initial preparation and

dilutions for aspects of microbiology associated with LBM (incl. in fish and fisheries

products). Two missions are envisaged in 2012.

1.2.5 Lead the revision of ISO TS 21872-1 and 2 detection of Vibrio spp. in seafood. One

mission is envisaged in 2012.

1.2.6 To continue to contribute to relevant EFSA expert working groups as required, to

identify potential control options for viruses in LBM, e.g. risk based, quantitative

virus standards, water quality improvements, environmental legislation and

depuration. Up to two missions envisaged in 2012.

1.2.7 Assist DG SANCO with specialist advice in relation to food and veterinary

inspections of Member States, Accession Countries and Third Countries as they arise.

1.2.8 Represent the EURL at the annual plenary meeting of the ISO SC9 and CEN WG6

Microbiology working group meeting. One meeting in Brussels in anticipated in

2012.

2 Co-ordination of activities of NRL network – (up to 180 days)

2.2 Participate in annual EURL Director’s co-ordination meeting and other EURL co-

ordination meetings/workshops as appropriate.

2.3 Organise, host, and participate in the eleventh annual EURL workshop, produce

resolutions and other workshop outputs (April 24th to 26

th 2012, Ljubljana, Slovenia). To

include administrative assistance.

2.4 Undertake EURL activities and commitments agreed in resolutions at annual workshop

above (as posted on www.crlcefas.org).

2.5 Provide specialist training and/or training courses to NRLs, accession country NRLs and

others in relation to analyses of E.coli, Salmonella spp., Vibrio spp., FRNA bacteriophage,

Norovirus, hepatitis A virus and other aspects of bivalve shellfish hygiene as required.

http://www.crlcefas.org)/






Science


Barrack Road,

The Nothe,

Weymouth,



2.6 Conduct a review in consultation with NRLs and other stakeholders of the EURL website

(www.crlcefas.org) to improve contents and ease of accessing information.

3 Provision of technical advice and training - (up to 70 days)

3.1 Organise an expert technical meeting to further develop and harmonise scientific expertise

across the EU with respect to control methods for human pathogenic Vibrio spp. associated

with LBM (particularly raw oysters). It envisaged that a small working group will be

convened with up to 2 international invited experts; the physical working group will be

hosted at the EURL, to enable practical activities and demonstrations to be undertaken.

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.

3.3 To include assistance on implementation of methods, accreditation to IEC ISO17025 and

quality control requirements (see above).

3.4 To provide guidance and review of procedures/data to laboratories wishing to undertake

studies to validate of alternative methods according to ISO 16140.

3.5 Provide specialist training and/or training courses to NRLs, accession country NRLs and

others in relation to analyses of LBM for microbiological contaminants as required.

4 Comparative testing and ring trials - (up to 270 days)

4.2 Organise comparative testing for NRLs for E. coli and Salmonella spp. in bivalve molluscs

via the EURL/HPA shellfish EQA scheme. Analyse results, produce report, advice and

recommendations (by May 2012).

4.3 Organise norovirus and hepatitis A virus comparative testing distribution for quantitative

and qualitative analyses. Analyse results, produce report and recommendations (by May

2012).

4.4 Undertake collaborative trials to test aspects of developmental Vibrio spp. methods in

matrix and laboratory constructed samples. Analyse results, produce report (By December

2012).

4.5 Organise comparative testing amongst NRLs for E. coli and Salmonella spp. in live

bivalve molluscs samples to test aspects of official methods no examined in standard EQA,

i.e. initial dilutions, homogenisation. This item is specifically at the request of the NRL

network to assist in the requirements of accreditation bodies. Analyse results, produce

report, advice and recommendations (by May 2012).

4.6 Distribution of reference materials for all relevant microbiological determinants on request

of NRLs.

http://www.crlcefas.co.uk/






Science


Barrack Road,

The Nothe,

Weymouth,



5 Confirmatory testing and quality assurance – (up to 80 days)

5.1 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 17025 accreditation for quantitative

determination of norovirus in LBM.

5.2 Maintenance of EURL laboratory competence and expertise in analytical methods for

monitoring bacteriological contaminants of bivalve molluscs (E. coli, Salmonella spp.,

marine vibrios). To include maintenance of ISO/IEC 17025 accreditation of enumeration

of E. coli, and the detection of Salmonella spp. and Vibrio parahaemolyticus.

5.3 Contribution to costs of the maintenance of EURL capability to perform analysis for

human pathogenic strains of marine vibrios associated with LBM (e.g. serotyping V.

parahaemolyticus, molecular characterisation of pathogenic strains of V.

parahaemolyticus, V. vulnificus and) non01/0139V. cholerae).

5.4 Performance of above tests on outbreak material or on occasion of disputed test results (on

request of DG SANCO).

6 Development of analytical methods – (up to 110 days)

6.1 In collaboration with JRC/IRMM Geel production of norovirus and hepatitis A virus

reference material using freeze-dried oyster samples.

6.2 Characterisation studies of virus reference materials (homogeneity, stability, shelf life etc).

6.3 Limited interlaboratory studies amongst NRLs to establish reproducibility and precision of

reference materials.

6.4 Practical developmental to support elaboration of standard molecular methods to detect

pathogenic vibrios in foodstuff; including bivalve shellfish (see section 1.2.2).

NOTE. In 2012 it is recommended that a studentship is partially supported by the EURL up to a maximum

value of £5,000 to enable the establishment of a fully characterised EU/global Vibrio strain bank.

This will provide an extremely valuable resource to European laboratories (NRLs and others) and

will assist in standardisation and harmonisation of Vibrio methods.

Rachel Hartnell

EURL Co-ordinator

August 2011

Resolutions of the 11th workshop of NRLs for monitoring bacteriological and viral contamination of bivalve molluscs v1 26.04.12 1


monitoring bacteriological and viral


The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK

Tel: +44 (0) 1305 206600, Fax +44 (0) 1305 206601 Email: [email protected] http://www.eurlcefas.org

Resolutions of the 11th workshop of Microbiological NRLs for Bivalve Molluscs, 24 - 26th April 2012

Official controls – microbiological monitoring and classification

1. The final recommendations to the Commission concerning harmonisation of Codex Standards (292-2008) and EU hygiene regulations for E. coli criteria were presented to NRLs. Recommendations include adoption of the Codex 3 class plan as an end product microbiological criteria and introduction of the Codex criteria applied ‘over time’ as criteria for classification of A grade production areas. These recommendations were now with Member States (MS) for consideration and the EURL would report progress to NRLs in due course.

2. Issue 3 (incorporating MS comments) of the proposed Community Guide to the Principles of Good Practice for the Microbiological Classification and Monitoring of Bivalve Mollusc Harvesting Areas was presented to NRLs. The guide was now with MS for comment prior to submission to SCFCAH. It was noted that adoption as an official Community guide would confer legal status. The EURL would report progress to NRLs in due course.

3. The EURL thanked NRLs for the updated information on sanitary surveys conducted in their MS. Outstanding information would be supplied within 3 weeks of the workshop. The finalised document would be circulated to NRLs prior to distribution to the Commission and publication on the EURL website (public domain).

4. The EURL presented recommendations for revision of the stability assessment included in the Microbiological Monitoring Good Practice Guide (a reduced frequency of monitoring is acceptable for ‘stable’ areas). The EURL proposed additional work to develop new criteria and, following definition of requirements, will request monitoring datasets to assist evaluation. In the interim a revised revision of the Guide (issue 5) will be distributed to NRLs and published on the EURL website (public domain).

5. The EURL announced the 2nd international workshop on molluscan shellfish area classification on 24th – 28th September 2012 at Newport, Rhode Island, USA jointly organised by the EURL and the US FDA. It was noted that this workshop would focus on comparison of EU and US systems and was regarded as important for informing the ongoing US/EU equivalency negotiations. NRLs and Competent Authorities from MS interested in exporting to the US were encouraged to attend via notification of either the Commission or the EURL.

Official control – Comparative testing

6. The EURL thanked NRLs for the updated information on official control laboratories (OCL) and proficiency testing in their MS. Following supply of outstanding information (within 3 weeks of the workshop) a summary document would be circulated to NRLs prior to distribution to the Commission and publication on the EURL website (public domain).

7. Supervision of the quality of OCL testing through proficiency testing and other measures is a legislative responsibility of NRLs under EC Reg 882/2004. NRLs require access to OCL proficiency test results to discharge this responsibility. Competent Authorities have an obligation to work with NRLs to ensure access to this information.







8. Four annual proficiency testing distributions for E. coli and Salmonella are available (3 HPA/ EURL non-matrix and 1 EURL matrix based distribution). It was agreed that the minimum requirement for satisfactorily (>70% score) performing NRLs was participation in the EURL matrix distribution and at least one other non matrix distribution. This was in accordance with the minimum participation frequency specified in the Good Practice Guide. NRLs with <70% scores should participate in all available distributions.

Marine vibrios

9. NRLs noted that, with the exception of O1/O139 V. cholerae, illness related to Vibrio spp. were not generally notifiable in the EU. Considering the potential impact of climate change on Vibrio spp. occurrence, better quality epidemiological information was vital to inform risk assessment. The EURL would contact the European Centre for Disease Prevention and Control (ECDC) regarding this issue.

10. The EURL announced a Vibrio methods workshop in July 4-5th 2012, NRLs were encouraged to consider attending the workshop. Expressions of interest should be sent to the EURL.

Viruses

11. NRLs considered control limits for hepatitis A virus (HAV) in bivalve molluscs. Given the severity of the infection and the rare occurrence in mollusc production areas in the EU, the workshop recommended a bivalve mollusc microbiological criterion of HAV absence (non detectable) when tested by the ISO CEN standard method.

12. It was agreed that to improve the quality of viral analysis, and hence risk management support available to producers, NRLs should write to commercial laboratories undertaking testing of live bivalve shellfish for norovirus and HAV and recommend the use of virus reference materials (available from the UK HPA) and participation in available, appropriate proficiency testing.

13. Several cross border EU outbreaks related to norovirus in oysters were reported by NRLs. It was noted that traceability of batches related to outbreaks was often problematical. This was particular relevant to trans-shipment of product between MS for further processing prior to final packaging and issue of health marks. The workshop recommended that this issue be brought to the attention of the Commission.

14. Following the recent EFSA reports, and data available from outbreaks and EU production area surveillance, NRLs considered possible control limits for norovirus in bivalve molluscs. NRLs agreed that an absence standard, whilst conservative for public heath, would have a high impact on producers that would not seem justified by available epidemiological evidence.

15. The workshop agreed that for norovirus differential quantitative standards for products intended to be eaten raw, and cooked in the home or restaurant may be appropriate. Data gaps were the protection afforded by home and restaurant cooking, commercial depuration and on norovirus levels in species other than oysters.







16. NRLs were informed that the ISO CEN standard method - ISO TS 15216-1 Microbiology of food and animal feed: Detection of norviruses and hepatitis A virus, part 1: Quantitative determination, would be published in 2012/13. An EURL harmonised protocol for molluscs based upon the standard was distributed and will be available on the EURL website (public domain).

17. The workshop noted encouraging performance in virus proficiency testing, particularly with respect to an increasing number of laboratories applying quantitative methods. NRLs noted that performance of laboratories using methods closely derived from the CEN standard method (ISO TS 15216-1) tended to perform better in proficiency testing than those using alternative methods.

18. Further to the above NRLs noted that further work was still required to improve comparability of quantitative results for norovirus prior to the introduction of quantitative legislative criteria. The EURL agreed to continue to develop quantitative reagents (standards) to help underpin accurate quantitation and to organise further proficiency testing distributions using matrix samples and dsDNA standards.

19. NRLs requested that the EURL provide technical training on quantitative determination of norovirus in bivalve shellfish. A formal training workshop would be proposed for early 2013.

20. NRLs agreed with the EFSA recommendation on pollution reduction strategies (e.g. prohibition zones) and agreed to have a dedicated session on this aspect at the next workshop.

21. NRLs agreed with the EFSA opinion that the current class B criterion of 10% of samples up to 46,000 E. coli MPN/100g was too high and may allow highly contaminated product to be placed on the market. It was agreed that the upper tolerance should be based upon practical performance of the method.

Date and time of next meeting

22. The next workshop would provisionally (subject to confirmation of costs) be held at ISS in Rome, Italy 7-9th May 2013.

WS11/05

- 1 -

AGENDA

11TH WORKSHOP OF MICROBIOLOGICAL NRLS, 24 – 26 APRIL 2012

Venue: EURL Weymouth Cefas Weymouth Laboratory Main reception Tel: +44 (0) 1305 206600 Barrack Road Main reception Fax: +44 (0) 1305 206601 The Nothe Weymouth DORSET DT4 8UB UK Enquiries : Prior to the workshop enquiries should be directed to Rachel Hartnell or

Samantha Arkell. Direct line : +44 (0) 1305 206707 +44 (0) 1305 206698 E-mail : [email protected] or [email protected]

Day 1 - Tuesday 24 April 9:30 - 17:30

1 Introductory meeting

1.1 Welcome, introductions and apologies.

1.2 Domestic arrangements including reclaim of expenses (papers WS11/01, WS11/02).

1.3 Actions arising from the 10th workshop 2010 (paper WS11/04).

1.4 Agreement of the agenda (paper WS11/05).

1.5 EURL work programme 2012 (EURL) (paper WS11/06).

Coffee/tea break (10:30 am)

2 Official Controls - Microbiological monitoring and classification

2.1 Feedback from the Commission Restricted Working Group on Classification and

Monitoring of Bivalve Molluscs (EURL).

2.2 Harmonisation of Codex and EU standards (paper WS11/07) (EURL).

2.3 Community Guide to the Principles of Good Practice for the Microbiological Monitoring

of Bivalve Mollusc Harvesting Areas with regard to Regulation 854/2004 (provisional

paper WS11/08) (EURL).

2.4 Update on application of sanitary surveys across Member States (paper WS11/09, paper

WS11/10) (All – roundtable)

2.5 Proposal for a revision to the stability assessment for monitoring frequency in the good

practice guide (EURL).

2.6 Second International Workshop on Molluscan Shellfish Area Classification (paper

WS11/11, paper WS11/11a) (EURL).

mailto:[email protected]

mailto:[email protected]

WS11/05

- 2 -

2.7 Temporal trends of E. coli level in clams (Chamelea gallina) harvesting areas of the

Marche Region (NRL Italy, Ancona).

Lunch (1:00 pm)

3 Liaison with ISO and CEN

3.1 Update on activities at ISO SC9 and CEN WG6 groups (EURL).

4 Official Controls - Proficiency Testing

4.1 E. coli and Salmonella spp.

4.1.1 Proficiency testing among Spanish Official Control Laboratories for bivalve

molluscs (NRL Spain).

4.1.2 NRLs participation and performance assessment in the EURL/HPA Shellfish

EQA scheme for E. coli and Salmonella (RT 44) (paper WS11/12) (EURL).

4.1.3 NRLs participation and performance assessment in the whole animal

distribution for E. coli and Salmonella (RT 41) (paper WS11/13) (EURL).

4.1.4 Ring trial on fresh shellfish: an overview on several years experience (NRL

France)

4.1.5 Supervision of Official Control Laboratories by NRLs (paper WS11/14) (All –

roundtable)

4.1.6 EURL draft guidance on the level and frequency of proficiency testing

participation (paper WS11/15) (EURL).

Coffee/tea (3:30 pm)

5 Marine vibrios

5.1 Characterisation of Vibrio cholerae isolated in Italy (NRL Italy, Ancona).

5.2 Outcomes of Vibrio ring trial (RT38) and ongoing revision of ISO TS 21872 (paper

WS11/16) (EURL)

5.3 Progress at CEN TAG3 – direct quantitative determination of Vibrio spp. and proposals

for practical methods workshop July 4-5th 2012 (EURL).

5.4 The research consortium VibrioNet (NRL Germany).

5.5 Proposals for a COST initiative (EURL).

Dinner – Sense Restaurant and Wine Bar for 7.30pm

WS11/05

- 3 -

Day 2 - Wednesday 25 April 9:15 - 17:30

6 Impact of chronic microbial pollution on shellfish – (EURL).

7 Viruses - Hepatitis A virus

7.1 Hepatitis A virus, a somewhat neglected shellfish-borne pathogen (Prof. Albert Bosch-

University of Barcelona).

7.2 Looking for HAV in a shellfish production area over 2 years (NRL France).

7.3 Any more contributions on HAV? Please

Coffee/tea (10:30am)

8 Viruses - noroviruses

8.1 Tracing norovirus in oysters from Ireland to Denmark through France (NRLs Denmark and

France).

8.2 Norovirus surveillance in the UK – prevalence and levels of norovirus in Crassostrea gigas

and Ostrea edulis in England, Wales and Scotland (UK NRL).

9 Viruses - Towards virus controls

9.1 EFSA Panel on Biological Hazards (BIOHAZ) - Scientific Opinion on Norovirus (NoV) in

oysters: methods, limits and control options (paper WS11/17) (NRL Ireland)

9.2 Discussion – Control options for norovirus (paper WS11/18)(all)

Lunch 12:30 pm

10 Viruses - Proficiency testing for norovirus and hepatitis A virus

10.1 The first ring trial for the detection of HAV and NoV (GI and GII) in bivalve mussels

organized in Italy (NRL Italy, Roma).

10.2 EURL norovirus and hepatitis A virus proficiency testing – PT43 (EURL) (Paper WS11/19).

10.3 Proposals for:

10.3.1 Proficiency testing,

10.3.2 EURL harmonised protocol (Paper WS11/20),

10.3.3 Training requirements.

10.4 Discussion (all)

10.5 Update on the progress of CEN validation (M/381) (EURL).

Coffee/tea (3:30 pm)

WS11/05

- 4 -

Day 3 - Thursday 26 April 9:30 - 12:00

11 Agreement of Workshop resolutions.

12 Any other business

13 Date and venue for next meeting.

Meeting close

EU-RL Workshop 24th to 26th April 2012, Weymouth Confidential Participant Feedback Results

84%

16%

Invited Speaker(s)

Very Good

Good

Satisfactory

Poor

76%

20%

4%

Administration

Very Good

Good

Satisfactory

Poor

36%

56%

8%

Presentations

Very Good

Good

Satisfactory

Poor

28%

52%

16%

4%

Accommodation

Very Good

Good

Satisfactory

Poor

N/A

40%

48%

12%

Workshop Content

Very Good

Good

Satisfactory

Poor

50% 39%

11%

Overall Performance

Very Good

Good

Satisfactory

Poor

Comments:

1. The support in advance by EU-RL of funding travelling and accommodation is very helpful.

2. Many interesting discussions. However, for few there is no outcome on decision made.

3. a. The first day content was somewhat too long.

b. Is it possible to have the workshop on 2 days only?

4. a. Lower part of sheets are not well visible from seats in the back.

b. Discussions are very long, with loose ends.

c. Lot of extra time needed every day perhaps extend program?

5. Better weather!

Report of the 2nd International workshop on shellfish area classification and management 31/01/13 FINAL Page 1

European Union Reference Laboratory for



2nd

INTERNATIONAL WORKSHOP ON SHELLFISH AREA CLASSIFICATION

AND MANAGEMENT – APPLICATIONS OF SANITARY SURVEYS

A. PHILOSOPHICAL CONSIDERATIONS - U.S. AND EU SYSTEMS

U.S. and EU systems each provide a framework of controls for the safe production of

bivalve molluscan shellfish. Whilst the two systems differ in a number of aspects, the

overarching aim of both is to protect shellfish consuming public from fecally borne and

autochthonous pathogens - in this they are largely successful.

Comparability and equivalency initiatives between the FDA and EU have been underway

for some time and are considered likely to reach a conclusion shortly. The tentative result

of discussions to date has been the recognition that EU Class A and US Approved area

shellfish are very similar with respect to risk whereas shellfish from EU Class B and

Restricted areas appear to differ by degrees, in part dependent on the quality of the Class

B area. Technical and microbiological basis of these, and other similarities (and

differences) were further explored by delegates at this workshop1.

Sections below summarize workshop findings.

1. Sanitary Surveys

Sanitary surveys can be considered to be akin to risk assessments

Reports- Components and outputs of NSSP and EU survey reports are similar;

however, outcomes (uses) are different –NSSP surveys inform decisions on area

classification and management (including monitoring), whereas EU surveys

enable the establishment of sampling plans for ongoing monitoring.

Initial survey and periodicity of review – EU legislation requires that all new

(from 2006) and reclassified areas require a sanitary survey. EU guidance further

recommends Member States put systems in place to establish sanitary surveys for

all areas by 2015, with updates if significant changes occur or within 6 years.

NSSP shellfish area classification requires detailed sanitary surveys to be

completed for all shellfish areas, with annual updates, triennial updates, complete

reassessments every 12 years, and a watershed approach.

1 Biotoxin management strategies were considered beyond the scope of the workshop and were excluded

from these considerations





NSSP pollution source assessments quantify or estimate contamination loads and

impacts from sources on shellfish areas.

2. Classification and harvesting

NSSP classifications aim to reflect the enduring and predictable sanitary quality

of shellfish growing areas, with consideration of contiguous waters and based

upon the outcomes of the sanitary surveys and periodic review. Verification is by

regular water sampling and testing for faecal coliforms – classification refers to a

body of water, not shellfish species.

In the EU classified areas consist of discrete, delineated portions of an estuary or

shoreline from which shellfish are harvested, regular monitoring data informs the

classification (with monitoring points selected on the basis of the outcomes of the

sanitary survey) – classification refers generally to a shellfish species, not body

of water.

The NSSP requires establishment of prohibited areas around sewage sources and

forbids any direct fecal discharges into areas classified as Approved. EU

legislation does not explicitly require the establishment of buffer zones, or

minimum distances from pollution sources. Guidance in this respect is however

provided in the EU Guide to Good Practice.

Harvesting under the NSSP is managed, when prescribed under a shellfish area

management plan. This is achieved by officially closing and re-opening areas

according to predefined criteria (such as rainfall), and is controlled by law

enforcement officials. EU legislation does not prescribe management plans.

However, the EU system does require that areas are closed „where the results of

sampling show that the health standards for molluscs are exceeded, or where there

may otherwise be a risk to human health.

3. Flesh versus Water

The EU requires collection and testing for Escherichia coli from at least 8

shellfish flesh samples per year, results are reviewed annually (taking into account

the last three years of monitoring data), classification is awarded on the basis of

the assessment. Water testing may be carried out as part of the sanitary survey but





is not collected routinely as part of the monitoring programme. Methods are

prescribed and required to meet ISO/IEC 17025 quality standards. Flesh not

water. E. coli not fecal coliforms.

The NSSP requires water sample locations be established to assess impacts from

pollution sources and verify shellfish area water quality, with designated locations

in the shellfish area sampled and tested for fecal coliforms at least 5 or 6 times per

year, and results of the most recent 15 or 30 samples are reviewed annually.

Water not flesh. Fecal coliforms not E. coli.

Under the NSSP water sample locations may be established anywhere and up to

any maximum number to characterize sanitary quality of shellfish harvest areas.

Under the EU system flesh sample locations (sampling points) are established,

according to the sanitary survey, to characterize the sanitary quality of shellfish

harvested.

4. Management of harvesting

NSSP Approved and EU Class A areas are designated for the production of

shellfish for direct marketing and raw consumption – no further post harvest

processing is required.

NSSP Restricted and Class B areas are designated to produce shellfish for post-

harvest purification processing by depuration or relay before marketing for raw

consumption. Restricted area harvesting permitted with special permit and

supervised by Authority only.

EU Class C areas are designated to produce shellfish for purification by relay

before marketing for raw consumption.

“Unclassified” areas are managed under the NSSP as prohibited. Harvesting from

“Prohibited” areas is not permitted under the EU system.

Emergency closures are introduced for WWTP failures, flooding, major storms,

spills, or other adverse events under both systems.

Most US States tend to manage harvest of all shellfish versus a species-by-species

approach. With some exceptions EU Member States separately classify, monitor

and manage on species-by-species basis.

5. End-Product Compliance





Testing is not required by the NSSP for Approved area and long-term relayed shellfish.

EU shellfish testing and method is stipulated in HACCP plan requirements and regulatory

end product compliance with an E. coli criterion.

6. Pathogen Monitoring

Testing for pathogens is not required by NSSP for shellfish, unless implicated by

reported illness or as stipulated for State management plan or hazard control plan.

Pathogen testing is not required by EU legislation although is foreshadowed in

legislative text when methods (for viruses and Vibrio parahaemolyticus) are

sufficiently developed.

7. Training and supervision

NSSP standardized training provided to State shellfish program officials by FDA

annually or as needed. EU training and supervision of official control laboratories

is the responsibility of the EU and National Reference laboratories. EU reference

methods are prescribed and are required to accredited to ISO IEC 17025.

8. Audits

The FDA conducts annual audits in all NSSP States and biennial audits in NSSP-

participating countries. In the EU the Member State Competent Authority is

required to audit a food business operators HACCP plan. The EU‟s Food and

Veterinary Office carries out audit of shellfish systems every 3 – 5 years on a risk

assessment basis.

9. Law Enforcement

Law enforcement officers are routinely required to enforce harvest restrictions

and control shellfish harvest in the U.S. Competent Authorities are tasked with

enforcement across the EU.

B. VISIONS OF A UNIFIED SHELLFISH SAFETY SYSTEM

No two molluscan shellfish growing areas are exactly alike; each has a unique set of

characteristics. The microbiological hazards potentially transmitted by shellfish and the





consequent risks to consumers can vary significantly by world region, country, state and

season. Additionally, raw shellfish present a higher risk relative to most other food that

are consumed after cooking. It is not feasible to test for every potential or actual hazard

associated with shellfish routinely thus controls based upon proxy indicators of risk have

evolved since the 1900‟s. Preventing illnesses and intoxications among shellfish

consumers is the prime objective of any shellfish safety programme irrespective of the

approach; when applied effectively these can help to assure a relatively safe product.

Following presentation of technical aspects of both programmes and discussion the

workshop identified that:

For compliance with the EU system, the NSSP and U.S. States would need to

enhance the legal basis of the NSSP, conduct E. coli flesh testing, ensure

analytical labs were certified and employ equivalent, validated methods of

analysis.

For compliance with the NSSP system, the EU Member States need to establish

minimum [management] requirements, consider shellfish area classifications in

light of the sanitary quality in waters including contiguous areas and conduct

comprehensive, regular audits.

At present, it was considered feasible to conduct equivalency assessments at

specific pre-qualified sites using an area-by-area approach.

The workshop further considered building a prospective “best practice” components of a

shellfish safety programme. A best practice approach included:

Classification of shellfish areas based on sanitary quality – considered the most

effective approach for preventing fecal-borne hazards being transmitted by

shellfish.

Sanitary surveys – characterizing a shellfish area is considered to be fundamental

in safe shellfish production.

A documented risk assessment – possible approach for every shellfish area,

feasible and could be relied upon to prevent unsafe shellfish.

Major components of a “documented risk assessment” might include:

a. A Sanitary Survey Report that includes:





(1) Watershed survey results;

(2) Shoreline survey results;

(3) Pollution source assessments;

(4) Hydrographic factors;

(5) Bacteriological survey results, and future viral and biotoxin results;

(6) Impacts from meteorological and environmental factors;

(7) Analyses of all information and data available;

(8) Pollution source assessments and mitigations;

(9) Identification of areas unsuitable for shellfish harvesting;

(10) Identification and delineation of shellfish area, boundaries, and

designated classification

b. A Comprehensive Management Strategy that includes:

(1) Targeted monitoring plans;

(2) Established critical limits (criteria) for designated classifications;

(3) Identification of suitable post-harvest processing control options;

(4) Closure and length of closure criteria;

(5) Definable reopening criteria;

(6) Harvester, processor and boater education programmes;

(7) Regulator training and certification programme;

(8) Control of harvest plan;

(9) Vibrio control plan;





(10) Unified definitive closure criteria and length of closure criteria

needed;

(11) Emergency situation advanced planning.

Labeling is a vital component of maintaining safe shellfish production.

The workshop considered that:

a. Unified, unambiguous product labeling was essential.

b. Trace-back to harvest location was essential.

Pathogens

Noroviruses2 are considered a universal, persistent hazard - approaches to prevent them

are required, these included:

a. Unified testing system – reference methods, internationally recognized quality

assurance programmes.

b. Established critical regulatory limits.

c. Identification of sources of human sewage, which may transmit viral

pathogens.

d. Requirements that shellfish areas are located sufficiently far from polluting

sources (dilutions of sources or distances).

Post harvest processing

a. Defined and verified criteria for depuration;

b. Defined and verified criteria for relay;

c. Defined and verified criteria for other post harvest processing strategies.

Compliance – the workshop recognized that compliance with requirements was

essential.

2 Other fecal borne pathogens should also be considered e.g. hepatitis A virus, Giardia, Salmonella spp.





a. Routine, comprehensive system audits by Competent Authority;

b. Occasional targeted or comprehensive audits by outside competent authority.

Epidemiological surveillance and communication systems - should be active,

not passive, in order to readily and clearly identify illness or intoxication cases

and outbreaks caused by shellfish.

Consumer advisories should be available

a. Standardized language;

b. Unified messages;

c. Wholesale and retail levels.

Comments noted but not fully discussed due to lack of time are as footnotes3,4,5,6,7,8,9,10,11

C. RESEARCH AND DATA GAPS

1. Significance of environmental impacts - influence of seasonal, temperature, and

related environmental factors.

2. Adequacy of management strategies:

a. Verification of appropriate trigger levels, lengths of closures, and re-

opening criteria under different conditions.

b. Adequacy of post-harvest treatment processes (depuration and relaying) to

control viruses in shellfish from Class B and Restricted areas.

3 Sanitary surveys are a critical, fundamental cornerstone for identifying risks 4 Shellfish from pristine areas are readily managed safely 5 Routine monitoring for specific naturally-occurring hazards that historically have never occurred in a region is

unnecessary 6 Even for secondary sewage treatment sources, a standard for a protective buffer zone is needed 7 The variety of wastewater treatment operations necessitates developing options for managing shellfish resources that

are variable and fit for purpose 8 Verify the appropriate trigger levels, lengths of closures, and re-opening criteria for rainfall events with non-point

sources versus those with activated sewage point sources 9 Routine monitoring for intermittent pathogens conveyed by fecal contamination is not essential or cost-beneficial. 10

After closure and remediation, re-opening criteria should be verified by water and meat testing. 11

E. coli may offer a more accurate assessment of fecal inputs than generic fecal colifoms.





c. Definition of the acceptable level of dilution from source to shellfish

resource considering site specific variations. Consideration of differential

survival differences for bacteria, bacteriophage and enteric viruses.

3. Adequacy of post-harvest treatment processes (depuration and relaying) to control

viruses in shellfish from Class B and Restricted areas.

4. Research efforts on noroviruses are recommended.

a. Periodic retail shellfish surveys.

b. Enhanced epidemiological surveillance and reporting.

c. Illness outbreak investigations on root causes and remedies.

d. Pollution source and impact studies.

i. Wastewater treatment plant and collection systems.

ii. Individual subsurface disposal (septic) systems.

iii. Marine sanitation devices and vessel discharges.

e. Infectious dose calculations.

5. Development of a global process for characterizing and managing shellfish areas,

and mandatory actions to take; decision trees etc.

D. WORKSHOP RECOMMENDATIONS

1. It was agreed a consensus approach on best practice should be reached with a

assessment tool box providing fit for purpose strategies according to inter alia the

characteristics of the area, species, pathogen risks, etc.

2. Further to the above, FAO/WHO should be approached to support an international

expert working group to draft a “best practice” guide based upon the preexisting

framework used in the Codex Ailmentarius: Code of Practice for fish and fisheries

products – section 7- live and raw bivalve molluscs12

3. The outcomes from the proposed working group should have the objectives of:

a. Providing a flexible framework for:

i. the technical application of current shellfish sanitation programs

ii. the development of new programs by countries that do not

currently have such health controls

12

http://www.fao.org/docrep/011/a1553e/a1553e00.htm

http://www.fao.org/docrep/011/a1553e/a1553e00.htm





b. Enabling the broader application of shellfish sanitation programmes in

order to:

i. Protect a larger proportion of the world‟s population from

shellfish-associated infections

ii. Assist a wider range of countries to trade safe shellfish

4. Technical consensus on recommended guidance should ultimately lead to a

review of policy requirements to allow its application to established programs.

European Union Reference Laboratory (EURL) and Health Protection Agency (HPA) EQA Shellfish Scheme Escherichia coli and Salmonella spp. EQA EURL ring trial reference number: PT 48 Sample distributions: SF041, SF042 and SF043

PT 48 Page 1 of 20

Contents Page number Samples 2 Results 2 Distribution results SF041 to SF043 3 Performance assessment 9 Summary 10 References 10 Charts 11 Appendices 16

Distribution period: March – November 2013

Report date: 5th March 2013

Report compiled by: Louise Stockley

Authorisation by: Rachel Hartnell

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 Health Protection Agency (HPA) ( Hhttp://www.hpa.org.uk/ProductsServices/InfectiousDiseases/ExternalQualityAssessmentProficiencyTesting/EQAPTForFoodWaterAndEnvironmentalMicrobiology/ShellfishScheme/). The EU reference method for enumeration of E. coli in raw bivalve molluscs is ISO 6649-3, 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. In 2011 EU Standing Committee on Food Chain and Animal Health (SCoFCAH) approved the use of two alternative methods for the enumeration of E. coli. These 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 www.eurl.cefas.org 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 (contact details below), or alternately refer to the troubleshooting guide included as Appendix I of this report. Further advice on microbiological testing of bivalve mollluscan shellfish can be obtained via the EURL website www.eurlcefas.org

http://www.hpa.org.uk/ProductsServices/InfectiousDiseases/ExternalQualityAssessmentProficiencyTesting/EQAPTForFoodWaterAndEnvironmentalMicrobiology/ShellfishScheme/�

http://www.hpa.org.uk/ProductsServices/InfectiousDiseases/ExternalQualityAssessmentProficiencyTesting/EQAPTForFoodWaterAndEnvironmentalMicrobiology/ShellfishScheme/�

http://www.eurl.cefas.org/�

http://www.eurlcefas.org/�

PT 48 Page 2 of 20

Samples Sample preparation 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 preparation instructions and report forms. Participation In total 22 laboratories participated in one, two or all of the distributions reported here, comprising 19 Member State NRLs and 1 EFTA country. All participants were requested to examine the samples using official control methods i.e. those methods routinely used for official control analysis of live bivalve molluscs. NRLs in Bulgaria, The Czech Republic, Iceland, Latvia, Lithuania and Slovakia did not participant in the mandatory number of EQA distributions per year agreed in Resolution 8 of the NRLs annual workshop 2012. Results Reference results For each distribution 10 reference samples were examined by the organising laboratory. Reference analyses were performed using ISO TS 16649-3 for the enumeration of E. coli and ISO 6579 for the detection of Salmonella spp..

Participants’ analysis and scoring system Results reported by NRLs for E. coli and Salmonella spp. examinations are shown in Tables 1, 3, 5, 7, 9, 10 and 11. Reported E. coli MPN values were compared to the median MPN from all participants’ results, reference results were omitted from the calculation. In previous years the acceptable limits were calculated as the participants’ median ±3 standard deviation (SD) and ±5 SD above and below the participants’ medium. On reviewing a two year EQA data set the SD values have been re-calculated to 2.68 and 4 times the inherent variability. This reduction is in line with the notable reduction in variability of participants’ results and is also closer to the variability inherent in the five tube 3 dilution MPN method. The result charts were compiled using log10 transformed MPN values (Figures 1 - 5). Performance assessment was according to the algorithm in Appendix II.

PT 48 Page 3 of 20

Distribution SF041: Sample SF0090 Contents – Salmonella Typhimurium 1,4,[5],12:I;1,2 (10), Enterococcus faecalis (102) and Pseudomonas aeruginosa (102). Reference results E. coli MPN – <20 per 100g. Salmonella spp. – Detected in 25g. Participants results - SF0090 (Figure 1) Table 1 Results reported by participants and scores allocated - SF0090

DNP – NRL registered for EQA scheme but did not participate in this distribution DNR – NRL did not register for the scheme

Discussion – SF0090 Participation – Eighteen NRLs received material for examination. All laboratories returned results and were included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratories 493, 596, 715 and 1798 did not take part in this distribution. E. coli MPN – All laboratories reported replicate results within the expected range and received a maximum score of 12. Laboratory 1578 reported both replicate MPN result as <18. This laboratory is reminded the MPN values is not consistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables or those previously supplied by the EURL. Salmonella spp. – All laboratories that reported results for Salmonella spp. correctly reported Salmonella spp. as detected and received a maximum score of 2.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 < 20 < 20 12 Detected 2 391 <20 <20 12 Detected 2 403 <20 <20 12 Detected 2 413 <200 <200 12 Detected 2 493 DNP DNP - DNP - 583 <20 <20 12 Detected 2 596 DNP DNP - DNP - 597 <18 <18 12 Detected 2 601 <20 <20 12 Detected 2 649 <20 <20 12 Detected 2 651 <20 <20 12 Detected 2 653 <20 <20 12 Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 <20 <20 12 Detected 2 703 <20 <20 12 Detected 2 715 DNP DNP - DNP - 718 <20 <20 12 Detected 2 720 <20 <20 12 Detected 2 744 <20 <20 12 Detected 2 983 DNR DNR - DNR - 1498 <20 <20 12 Detected 2 1527 <200 <200 12 Not Examined - 1578 <18 <18 12 Detected 2 1798 DNP DNP - DNP -

PT 48 Page 4 of 20

Distribution SF041: Sample SF0091 Contents – Escherichia coli (103), Salmonella Essen 4,12:g,m; (10), Citrobacter freundii (104) and Enterobacter aerugenes (104). Reference results E. coli MPN – 5.5 x 102 – 1.9 x 104 per 100g. Salmonella spp. – Detected in 25g. Table 2 Participants and reference results median, median ±2.68 and ±4 SD - SF0091

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 103 7.0 x 102 3.2 x 102 1.7 x 104 3.8 x 104 Participants results 3.5 x 103 7.0 x 102 3.2 x 102 1.7 x 104 3.8 x 104

Participants results - SF0091 (Figure 1) Table 3 Results reported by participants and scores allocated - SF0091

DNP – NRL registered for EQA scheme but did not participate in this distribution DNR – Did not register

Discussion – SF0091 Participation – Eighteen NRLs received material for examination. All laboratories returned results and were included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratory 493, 596, 715 and 1798 did not take part in this distribution. E. coli MPN – Sixteen laboratories reported replicate results within the expected range and received a maximum score of 12. Laboratory 701 reported one replicate result between ±2.68 and ±4SD of the participants’ median and received a scored of 9. Laboratory 703 reported one replicate result between ±2.68 and ±4SD of the participants’ median and one replicate result outside ±4SD of the participants’ median and received a score of 6. Salmonella spp. – All laboratories that reported results for Salmonella spp. correctly reported Salmonella spp. as detected and received a maximum score of 2.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 2400 16000 12 Detected 2 391 3500 5400 12 Detected 2 403 3500 5400 12 Detected 2 413 3300 3300 12 Detected 2 493 DNP DNP - DNP - 583 5400 5400 12 Detected 2 596 DNP DNP - DNP - 597 2200 5400 12 Detected 2 601 3300 4900 12 Detected 2 649 3500 2400 12 Detected 2 651 3100 9200 12 Detected 2 653 5400 3500 12 Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 490 2400 9 Detected 2 703 230 330 6 Detected 2 715 DNP DNP - DNP - 718 4900 4900 12 Detected 2 720 3500 2200 12 Detected 2 744 9200 5400 12 Detected 2 983 DNR DNR - DNR - 1498 3500 3500 12 Detected 2 1527 2120 1880 12 Not Examined - 1578 7900 4900 12 Detected 2 1798 DNP DNP - DNP -

PT 48 Page 5 of 20

Distribution SF042: Sample SF0092 Contents – Escherichia coli (10), Bacillus pumilus (102) and Pseudomonas fluorescens (102). Reference results E. coli MPN – <20 – 2.4 x 102 per 100g. Salmonella spp. - Not detected in 25g. Table 4 Participants and reference results median, median ±2.68 and ±4 SD - SF0092

Median MPN/100g







DNP – NRL registered for EQA scheme but did not participate in this distribution DNR – Did not register NR – No results returned

Discussion – SF0092 Participation - Sixteen NRLs and 1 EFTA country received material for examination. All laboratories except 715 returned results and were included in the assessment. Laboratories 1527 did not examine the sample for Salmonella spp.. Laboratories 391, 493, 583, 596 and 601 did not take part in this distribution. E. coli MPN – Fifteen laboratories reported replicate results within the expected range and received a maximum score of 12. Laboratory 718 reported one replicate result between ±2.68 and ±4SD of the participants’ median and received a scored of 9. Laboratory 597 reported one replicate MPN result as <18. This laboratory is reminded the MPN values is not consistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables or those previously supplied by the EURL. Salmonella spp. – All laboratories that reported results for Salmonella spp. correctly reported Salmonella spp. as not detected and received a maximum score of 2.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 70 <20 12 Not Detected 2 391 DNP DNP - DNP - 403 20 70 12 Not Detected 2 413 20 20 12 Not Detected 2 493 DNP DNP - DNP - 583 DNP DNP - DNP - 596 DNP DNP - DNP - 597 78 < 18 12 Not Detected 2 601 DNP DNP - DNP - 649 <20 <20 12 Not Detected 2 651 50 50 12 Not Detected 2 653 20 < 20 12 Not Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 20 20 12 Not Detected 2 703 <20 <20 12 Not Detected 2 715 NR NR - NR - 718 80 140 9 Not Detected 2 720 20 50 12 Not Detected 2 744 20 20 12 Not Detected 2 983 DNR DNR - DNR - 1498 20 50 12 Not Detected 2 1527 <200 <200 12 Not Examined - 1578 70 20 12 Not Detected 2 1798 <20 <20 12 Not Detected 2

PT 48 Page 6 of 20

Distribution SF042: Sample SF0093 Contents - Escherichia coli (102 – 103), Salmonella Wentworth 11:z10:1,2 (10), Klebsiella oxytoca (104 – 105) and Pantoea agglomerans (104 – 105). Reference results E. coli MPN – 1.3 x 102 – 4.7 x 103 per 100g. Salmonella spp. - Detected in 25g. Table 6 Participants and reference results median, median ±2.68 and ±4 SD - SF0093

Median MPN/100g








Discussion – SF0093 Participation – Sixteen NRLs and 1 EFTA country received material for examination. All laboratories except 715 returned results and were included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratories 391, 493, 583, 596, and 601 did not take part in this distribution. E. coli MPN – All laboratories reported replicate results within the expected range and received a maximum score of 12. Salmonella spp. – Fourteen laboratories reported results for Salmonella spp correctly reported Salmonella spp. as detected and received a maximum score of 2. Laboratory 403 incorrectly reported Salmonella spp. as not detected and received a score of 0.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 940 490 12 Detected 2 391 DNP DNP - DNP - 403 2400 1300 12 Not Detected 0 413 790 790 12 Detected 2 493 DNP DNP - DNP - 583 DNP DNP - DNP - 596 DNP DNP - DNP - 597 790 330 12 Detected 2 601 DNP DNP - DNP - 649 1100 1400 12 Detected 2 651 790 1300 12 Detected 2 653 700 460 12 Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 700 1300 12 Detected 2 703 330 330 12 Detected 2 715 NR NR - NR - 718 490 790 12 Detected 2 720 700 790 12 Detected 2 744 790 900 12 Detected 2 983 DNR DNR - DNR - 1498 490 700 12 Detected 2 1527 980 560 12 Not Examined - 1578 460 790 12 Detected 2 1798 790 230 12 Detected 2

PT 48 Page 7 of 20

Distribution SF044: Sample SF0094 Contents – Escherichia coli (104 - 105), Salmonella Liverpool 1,3,19:d,e,n,z15 (<10), Enterococcus faecalis (103) and Serratia liquefaciens (105). Reference results E. coli MPN – 2.9 x 104 – 6.9 x 105 per 100g. Salmonella spp. – Detected in 25g. Table 8 Participants and reference results median, median ±2.68 and ±4 SD - SF0094

Median MPN/100g








Discussion – SF0094 Participation – Nineteen NRLs and 1 EFTA NRLreceived material for examination. All laboratories except laboratory 744 returned results and were included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratory 413 and 1578 did not take part in this distribution. E. coli MPN – Seventeen laboratories reported replicate results within the expected range and received a maximum score of 12. Laboratory 413 reported both replicate results outside ±4SD of the participants’ median and received a scored of 4. Laboratory 1798 reported one replicate MPN result inconsistent with the reported tube combination and received a score of 10. Salmonella spp. – Seventeen laboratories that reported results for Salmonella spp. correctly reported Salmonella spp. as detected and received a maximum score of 2. Laboratory 403 incorrectly reported Salmonella spp. as not detected and received a score of 0.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 240000 110000 12 Detected 2 391 160000 92000 12 Detected 2 403 DNP DNP - DNP - 413 5400 9200 4 Not Detected 0 493 220000 110000 12 Detected 2 583 140000 350000 12 Detected 2 596 350000 350000 12 Detected 2 597 92000 92000 12 Detected 2 601 240000 140000 12 Detected 2 649 350000 240000 12 Detected 2 651 79000 170000 12 Detected 2 653 79000 130000 12 Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 92000 160000 12 Detected 2 703 54000 92000 12 Detected 2 715 220000 170000 12 Detected 2 718 160000 110000 12 Detected 2 720 240000 92000 12 Detected 2 744 NR NR - NR - 983 DNR DNR - DNR - 1498 110000 160000 12 Detected 2 1527 77200 70000 12 Not Examined - 1578 DNP DNP - DNP - 1798 240000 350000 10 Detected 2

PT 48 Page 8 of 20

Distribution SF044: Sample SF0095 Contents – Escherichia coli (102 – 103), Aeromonas spp. (103) and Klebsiella aerogenes (104). Reference results E. coli MPN – 1.4 x 102 – 3.4 x 103 per 100g. Salmonella spp. – Not detected in 25g. Table 10 Participants and reference results median, median ±2.68 and ±4 SD - SF0095

Median MPN/100g







DNP – NRL registered for EQA scheme but did not participate in this distribution DNR – Did not register NR – No results returned.

Discussion – SF0095 Participation - Nineteen NRLs and 1 EFTA country received material for examination. All laboratories except Laboratory 744 returned results and were included in the assessment. Laboratory 1527 did not examine the sample for Salmonella spp.. Laboratory 413 and 1578 did not take part in this distribution. E. coli MPN – Seventeen laboratories reported replicate results within the expected range and received a maximum score of 12. Laboratory 413 reported one replicate results between ±2.68 and ±4SD of the participants’ median and received a scored of 9. Laboratory 649 reported one replicate MPN result inconsistent with the reported tube combination and received a score of 10. Salmonella spp. – All laboratories that reported results for Salmonella spp. correctly reported Salmonella spp. as not detected and received a maximum score of 2.

Lab E. coli (per 100g) Salmonella spp. (per 25g) Number Replicate 1 Replicate 2 Score Salmonella spp. Score 121 790 790 12 Not Detected 2 391 790 490 12 Not Detected 2 403 DNP DNP - DNP - 413 1300 3500 9 Not Detected 2 493 330 700 12 Not Detected 2 583 1300 490 12 Not Detected 2 596 500 500 12 Not Detected 2 597 490 330 12 Not Detected 2 601 490 270 12 Not Detected 2 649 1100 1300 10 Not Detected 2 651 700 330 12 Not Detected 2 653 330 490 12 Not Detected 2 658 DNR DNR - DNR - 660 DNR DNR - DNR - 662 DNR DNR - DNR - 701 790 490 12 Not Detected 2 703 490 330 12 Not Detected 2 715 490 330 12 Not Detected 2 718 700 790 12 Not Detected 2 720 1100 330 12 Not Detected 2 744 NR NR - NR - 983 DNR DNR - DNR - 1498 490 790 12 Not Detected 2 1527 280 300 12 Not Examined - 1578 DNP DNP - DNP - 1798 490 330 12 Not Detected 2

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Participation At the 11th workshop of microbiological NRLs for bivalve shellfish it was noted (Resolution 8) that for NRLs achieving a satisfactory score (>70%), the minimum required participation in PT distributions is participation in the EURL matrix distribution (PT 45) (Appendix III) and at least one other non matrix distribution. Table 12 shows the participation of NRLs for 2012 with 73% (19 NRLs) participating in the EURL matrix distribution and 1 or more EQA distributions. Six NRLs have not registered to the EQA scheme. The EURL recommends that NRLs not registered to the EQA scheme should join this scheme Table 12 : Participation by NRLs in 2012 for E. coli and Salmonella spp. determinands.

Country EURL matrix - PT 45 Number of EQA distributions Yes No None 1 2 3

Austria Belgium/ Luxembourg Bulgaria Croatia Czech Republic Denmark Finland France Germany Greece Hungary Iceland Ireland Italy Italy Latvia Lithuania Netherlands Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden UK

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 45) (Appendix III) and 3 EQA distributions (March to November 2012). The allocated scores are summarised in Tables 13 and 14 respectively. Good performances is identified where a cumulative score of >70% is achieved. Participant’s 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 I.

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E. coli MPN assessment Twenty laboratories participated in the EURL matrix scheme (Appendix III) and 1 or more EQA distributions in 2012 and were therefore subject to a full performance assessment. All laboratories achieved a cumulative total of >70% for the two or distributions analysed. Table 13 Summary of participants’ performance in the EURL matrix scheme and the EQA scheme - E. coli

Lab no.

EURL PT 45 Distribution SF041 Distribution SF042 Distribution SF043 All distributions

Sample 1 Sample 2 SF0090 SF0091 SF0092 SF0093 SF0094 SF0095 Cumulative score

Max score %

121 12 12 12 12 12 12 12 12 96 96 100 391 12 12 12 12 - - 12 12 72 72 100 403 12 12 12 12 12 12 - - 72 72 100 413 12 12 12 12 12 12 4 9 85 96 89 493 12 12 - - - - 12 12 48 48 100 583 12 12 12 12 - - 12 12 72 72 100 596 a - - - - - - 12 12 - - - 597 10 10 12 12 12 12 12 12 92 96 96 601 a - - 12 12 - - 12 12 - - - 649 12 12 12 12 12 12 12 10 94 96 98 651 12 12 12 12 12 12 12 12 96 96 100 653 12 12 12 12 12 12 12 12 96 96 100 658 a 6 12 - - - - - - - - - 660 a 12 12 - - - - - - - - - 662 a - - - - - - - - - - - 701 12 12 12 9 12 12 12 12 93 96 97 703 12 12 12 6 12 12 12 12 90 96 94 715 10 10 - - - - 12 12 44 48 92 718 12 12 12 12 9 12 12 12 93 96 97 720 12 12 12 12 12 12 12 12 96 96 100 744 12 12 12 12 12 12 - - 72 72 100 983 a 12 12 - - - - - - - - - 1498 12 12 12 12 12 12 12 12 96 96 100 1527 12 12 12 12 12 12 12 12 96 96 100 1578 12 10 12 12 12 12 - - 70 72 97 1798 12 12 - - 12 12 10 12 70 72 97

a full performance assessment was not carried as laboratories did not participate in PT 45 and 1 EQA distribution during 2012.

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Salmonella spp. assessment Nineteen laboratories participated in the EURL matrix scheme (Appendix III) and 1 or more EQA distributions in 2012 and were therefore subject to a full performance assessment. All laboratories achieved a cumulative total of >70% for the two or distributions analysed. Table 14 Summary of performance of particiants over three distributions, SF038 to SF040 Salmonella spp.

a full performance assessment was not carried as laboratories did not participate in PT 45 and 1 EQA distribution during 2012. Summary In 2012 twenty laboratories completed one or more EQA distributions between March and November 2012 and participated in the EURL matrix distributions. These laboratories therefore were subject to a full performance assessment. For E. coli examination and Salmonella spp. detection all laboratories achieved a score of >70%.

References Anon 2005. ISO TS 16649-3:2005. 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. 2002. ISO 6579:2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Geneva, Switzerland. European Communities 2004. 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, 30.4.04 : 1-141.

Lab no.

EURL PT 45 Distribution SF041 Distribution SF042 Distribution SF043 All distributions

Sample 1 Sample 2 SF0090 SF0091 SF0092 SF0093 SF0094 SF0095 Cumulative score

Max score %

121 2 2 2 2 2 2 2 2 16 16 100 391 2 2 2 2 - - 2 2 12 12 100 403 2 2 2 2 2 0 - - 10 12 83 413 2 2 2 2 2 2 0 2 14 16 88 493 2 2 - - - - 2 2 8 8 100 583 2 2 2 2 - - 2 2 12 12 100 596 a - - - - - - 2 2 - - - 597 2 2 2 2 2 2 2 2 16 16 100 601 a - - 2 2 - - 2 2 - - - 649 2 2 2 2 2 2 2 2 16 16 100 651 2 2 2 2 2 2 2 2 16 16 100 653 2 2 2 2 2 2 2 2 16 16 100 658 a 2 2 - - - - - - - - - 660 a 2 2 - - - - - - - - - 662 a - - - - - - - - - - - 701 2 2 2 2 2 2 2 2 16 16 100 703 2 2 2 2 2 2 2 2 16 16 100 715 2 2 - - - - 2 2 8 8 100 718 2 2 2 2 2 2 2 2 16 16 100 720 2 2 2 2 2 2 2 2 16 16 100 744 2 2 2 2 2 2 - - 12 12 100 983 a 2 2 - - - - - - - - - 1498 2 2 2 2 2 2 2 2 16 16 100 1527 a 2 2 - - - - - - - - - 1578 2 0 2 2 2 2 - - 10 12 83 1798 2 2 - - 2 2 2 2 12 12 100

PT 48 Page 12 of 20

European Communities 2004. 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, 25.6.04 : 83-127. European Communities 2005. Commission Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Off. J. Eur. Communities L338, 22.12.05 : 1-26.

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Figure 1. Distribution SF041: Sample SF0091

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Appendix I: 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.

Where more than three dilutions have been tested for a sample record, select the tube combination as stated in the following rules: 1. Select the combination of three consecutive dilutions having a category 1 profile to obtain the MPN index.

If more than one combination having a category 1 profile is obtained, use the one with the highest number of positive tubes. An example is given below:

If a tube combination of 5, 5, 4, 2 is recorded, the result for the sample would be reported as 16,000

MPN/100g. 2. If no combination having a category 1 profile is available, use the one having a category 2 profile. If more

than one combination having a category 2 profile is obtained, use the one with the highest number of positive tubes.

2. Culture Medium - 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.

1g 0.1g 0.01g 0.001g MPN/100g Category 5 5 4 2 5 5 4 16,000 1 5 4 2 22,000 1

RT 44 Page 19 of 20

Appendix II: E. coli MPN scores allocated to participants returning 2 replicate results

Result Returning of results

Score allocated Total score Replicate 1 Replicate 2

One replicate MPN result reported is outside the expected range and falls between the median ±2.68SD and median ±4SD value

2 5 2 9

Both replicates MPN results are outside the expected range and fall between the median ±2.68SD and median ±4SD value

2 2 2 6

One replicate MPN result reported is outside the median ±4SD value 2 5 0 7

Both replicates MPN results are outside the expected range. The first falls between the median ±2.68SD and median ±4SD value and the second falls outside the median ±4SD value.

2 2 0 4

Both replicates MPN results reported is outside the median ±4SD value 2 0 0 2

E. coli MPN scores allocated to participants returning 1 single replicate results


Score allocated

Total score

Single replicate MPN result reported is within the expected range 2 5 7

Single replicate MPN result reported only and falls between the median ±2.68SD and median ±4SD value 2 2 4

Single replicate MPN result reported is outside the median ±4SD value 2 0 2

E. coli score deductions

Result Score deducted Replicate 1 Replicate 2

Tube combination inconsistent with MPN reported, ISO 7218 or 5 x 3 MPN tables provided by the EURL. 2 2

High censored result (e.g. MPN = >18000 per 100g) 2 2

Sample not examined or results returned late - no explanation received 12

Salmonella spp scoring

Result Score allocated Fully correct results 2 Misleading result, e.g. failure to isolate Salmonella 0

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Appendix III:

NRL results and allocated scores for the EURL matrix distribution (PT 45)

Lab ID Sample 1 - Oysters Sample 2 - Mussels E. coli MPN/100g Salmonella spp. in 25g E. coli MPN/100g Salmonella spp. in 25g Replicate 1 Replicate 2 Score Result Score Replicate 1 Replicate 2 Score Result Score

3 * a 20 <20 12 Not detected 2 20 110 12 Not detected 2 7 * 20 70 12 Not detected 2 70 50 12 Not detected 2 9 * 80 50 12 Not detected 2 50 90 12 Not detected 2 10 * 50 20 12 Not detected 2 130 80 12 Not detected 2 13 * 20 20 12 Not detected 2 45 20 10 Present 0 19 * 70 40 12 Not detected 2 70 40 12 Not detected 2 21 * a 70 <20 10 Not detected 2 20 70 10 Not detected 2 22 * 50 50 12 Not detected 2 50 50 12 Not detected 2 23 * 490 330 6 Not detected 2 80 130 12 Not detected 2 27 * 130 20 12 Not detected 2 70 20 12 Not detected 2 32 * 20 70 12 Not detected 2 270 50 12 Not detected 2 33 * 20 20 12 Not detected 2 20 110 12 Not detected 2 35 * 170 50 12 Not detected 2 20 20 12 Not detected 2 39 * 20 80 12 Not detected 2 80 170 12 Not detected 2 41 * 110 45 10 Not detected 2 110 78 10 Not detected 2 42 * a b <20 80 12 Not detected 2 50 <20 12 Not detected 2 43 * a 50 <20 12 Not detected 2 20 70 12 Not detected 2 44 * 80 20 12 Not detected 2 40 20 12 Not detected 2 47 * 20 50 12 Not detected 2 80 130 12 Not detected 2 68 * 70 50 12 Not detected 2 20 70 12 Not detected 2 83 * a b 20 50 12 Not detected 2 <20 130 12 Not detected 2 86 * 170 170 12 Not detected 2 50 80 12 Not detected 2 90 * 50 70 12 Not detected 2 80 50 12 Not detected 2 147 * 50 20 12 Not detected 2 220 50 12 Not detected 2 170 * a <200 <200 12 Not detected 2 <200 <200 12 Not detected 2 212 * a <67 230 12 NE - <67 230 12 NE -

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European Union Reference Laboratory (EU-RL) Proficiency Testing Scheme Enumeration of Escherichia coli and the detection of Salmonella spp. in Pacific oysters (Crassostrea gigas) and Common mussels (Mytilus edulis) EURL proficiency testing reference number: PT 45 Sample number: PT 45

Proficiency testing 45 Final 2 Page 1

Contents Page number Sample preparation 2 Results 2 General comments 6 References 8 Result charts 9 Appendices 11

Distribution date: 10th September 2012

Report date: 20th November 2012

Report compiled by: Louise Stockley

Authorisation by: Rachel Hartnell

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/HPA Shellfish Scheme (www.hpa.org.uk) 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 17025 accreditation purposes. The EU reference method for enumeration of E. coli in live bivalve molluscs in ISO TS 16649-3, Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 3: Most probable number technique using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (Anon 2005). The EU reference method for detection of Salmonella spp. in live bivalve molluscs is ISO 6579, Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp. (Anon 2002). A scoring system is used to help assess participants’ performance. Details of this system are included as Appendix I of this report. The purpose of scoring is to help the EURL, NRLs and other participating laboratories identify incorrect or outlying results. Further information on the use of scoring in proficiency testing and on recommended procedures for following up poor performance can be accessed via the EURL website (www.eurlcefas.co.uk) or obtained by contacting the EURL. The European Union has produced a protocol for management of underperformance in comparative testing and/or lack of collaboration of NRLs with EURLs activities. If you are experiencing problems with any aspects of these distributions please contact the EURL (contact details below), or alternately refer to the troubleshooting guide included as Appendix II of this report. Further advice on microbiological testing of bivalve mollluscan shellfish can be obtained via the EURL website (www.eurlcefas.co.uk) Due to the nature of this scheme repeat samples are not available.

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Sample preparation Sample 1 Approximately 1900 Pacific oysters (Crassostrea gigas) were collected from a UK commercial harvesting area. Sample 1 comprised of 25 randomly selected oysters. Sample 2 Approximately 2700 common mussels (Mytilus edulis) were collected from a UK commercial harvesting area. Sample 2 comprised of 35 randomly selected cockles. Sample distribution and examination Samples were packed in accordance with Cefas protocol for packaging shellfish for transportation and distributed at 1:30pm on 10th September 2011 to 50 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 and the detection of Salmonella spp.. Sample temperature Temperature recorders (Thermotrack, Progress Plus) were included in each consignment. Participants were requested to record the internal sample temperature on arrival and to return the temperature recorder. Temperatures recorded by participants are shown in Appendix III. Results Reference results - E. coli Ten randomly selected sub-samples were analysed in duplicate on 2 consecutive days (11.09.12 and 12.09.12) for E. coli using EURL SOP No. 1175 http://www.eurlcefas.org/InformationCentre/docs/CRL_SOP_E_coli_04_04_08.pdf (Table 1 and 2). Table 1: E. coli MPN/100g reference results for sample 1 - Oysters

Table 2: E. coli MPN/100g reference results for sample 2 - Mussels

GM - geometric mean, SDT - theoretical standard deviation (0.26) Reference results – Salmonella spp. Ten randomly selected sub-samples were analysed on 2 consecutive days (11.09.12 and 12.09.12) for Salmonella spp. based on the mini-MSRV MPN technique (Fravalo et al, 2003) (Table 3 and 4). Note: Regulation (EC) No. 2073/2005 requires presence/absence testing for Salmonella spp. in live bivalve molluscs. Table 3: Salmonella spp. reference results for sample 1 - Oysters

Table 4: Salmonella spp. reference results for sample 2 – Mussels

Analyses dates Range Median GM Median ±3*SDT 11.09.11 <2.0 x 101 – 3.3 x 102 6.5 x 101 6.8 x 101 1.1 x 101 - 3.9 x 102 12.09.11 <2.0 x 101 – 3.3 x 102 3.5 x 101 4.2 x 101 6.0 x 100 - 2.1 x 102

Analyses dates Range Median GM Median ±3*SDT 11.09.11 2.0 x 101 – 1.3 x 102 5.0 x 101 5.2 x 101 8.0 x 100 - 3.0 x 102 12.09.11 2.0 x 101 – 4.9 x 102 8.0 x 101 7.5 x 101 1.3 x 101 - 4.8 x 102

Analyses dates Salmonella spp. No. of replicates giving negative results 11.09.11 Not detected in 25g 10 12.09.11 Not detected in 25g 10

Analyses dates Salmonella spp. No. of replicates giving negative results 11.09.11 Not detected in 25g 10 12.09.11 Not detected in 25g 10

http://www.crlcefas.org/InformationCentre/docs/CRL_SOP_E_coli_04_04_08.pdf�


Participants’ results Performance assessment was according to the procedures described in the EURL/HPA EQA shellfish scheme for a single distribution, with minor modifications (Appendix III). Participants’ results and scores allocated for PT 45 are shown in Tables 7, 8, Figure 1 and 2. Note: The median and upper and lower limits (±3 SD and ±5 SD) were calculated from participants’ results. SDT calculations were based on the inherent variability of the 5 x 3 MPN method (0.26 log10). Reference values were excluded from the calculation of participants’ median. Table 5: Summary statistics for sample 1 – Oysters

1 expected range = participants’ median ± theoretical 3SD, 2 points deducted from participants returning results inconsistent with ISO 7218 who reported using the reference method.

Table 6: Summary statistics for sample 2 – Mussels

1 expected range = participants’ median ± theoretical 3SD, 2 points deducted from participants returning results inconsistent with ISO 7218 who reported using the reference method.

E. coli summary statistics Participants reporting duplicate results for E. coli MPN 44 Participants reporting MPN results within the expected range for both replicates1 41 Participants reporting MPN results outside the expected range for one replicate 1 Participants reporting MPN results outside the expected range for both replicates 2 Participants reporting a single MPN result 3 Participants reporting MPN results within the expected range for a single replicate1 1 Participants reporting MPN results outside the expected range for a single replicate 2 Participants reporting MPN results inconsistent with ISO 7218 (Anon 2007)2 3 Salmonella spp. summary statistics Participants reporting results for Salmonella spp. 47 Participants reporting the absence of Salmonella spp. 45

Participants not returning results 2 Participants not receiving material due to problems at customs 1

E. coli summary statistics Participants reporting duplicate results for E. coli MPN 44 Participants reporting MPN results within the expected range for both replicates1 37 Participants reporting MPN results outside the expected range for one replicate 4 Participants reporting MPN results outside the expected range for both replicates 3 Participants reporting a single MPN result 3 Participants reporting MPN results within the expected range for a single replicate1 1 Participants reporting MPN results outside the expected range for a single replicate 2 Participants reporting MPN results inconsistent with ISO 7218 (Anon 2007)2 5 Salmonella spp. summary statistics Participants reporting results for Salmonella spp. 47 Participants reporting the absence of Salmonella spp. 46

Participants not returning results 2 Participants not receiving material due to problems at customs 1


Table 7: Participants’ results and allocated scores

Lab ID Sample 1 - Oysters Sample 2 - Mussels E. coli MPN/100g Salmonella spp. in 25g E. coli MPN/100g Salmonella spp. in 25g Replicate 1 Replicate 2 Score Result Score Replicate 1 Replicate 2 Score Result Score

3 * a 20 <20 12 Not detected 2 20 110 12 Not detected 2 7 * 20 70 12 Not detected 2 70 50 12 Not detected 2 9 * 80 50 12 Not detected 2 50 90 12 Not detected 2 10 * 50 20 12 Not detected 2 130 80 12 Not detected 2 13 * 20 20 12 Not detected 2 45 20 10 Present 0 19 * 70 40 12 Not detected 2 70 40 12 Not detected 2 20 70 20 12 Not detected 2 110 330 12 Not detected 2 21 * a 70 <20 10 Not detected 2 20 70 10 Not detected 2 22 * 50 50 12 Not detected 2 50 50 12 Not detected 2 23 * 490 330 6 Not detected 2 80 130 12 Not detected 2 27 * 130 20 12 Not detected 2 70 20 12 Not detected 2 28 a <10 NE 4 Not detected 2 <10 NE 4 Not detected 2 30 230 80 12 Not detected 2 80 80 12 Not detected 2 32 * 20 70 12 Not detected 2 270 50 12 Not detected 2 33 * 20 20 12 Not detected 2 20 110 12 Not detected 2 35 * 170 50 12 Not detected 2 20 20 12 Not detected 2 39 * 20 80 12 Not detected 2 80 170 12 Not detected 2 41 * 110 45 10 Not detected 2 110 78 10 Not detected 2 42 * a b <20 80 12 Not detected 2 50 <20 12 Not detected 2 43 * a 50 <20 12 Not detected 2 20 70 12 Not detected 2 44 * 80 20 12 Not detected 2 40 20 12 Not detected 2 47 * 20 50 12 Not detected 2 80 130 12 Not detected 2 48 40 40 12 Not detected 2 70 70 8 Not detected 2 68 * 70 50 12 Not detected 2 20 70 12 Not detected 2 69 170 50 12 Not detected 2 130 80 12 Not detected 2 76 c 12 15 8 Not detected 2 66 51 8 Not detected 2 83 * a b 20 50 12 Not detected 2 <20 130 12 Not detected 2 86 * 170 170 12 Not detected 2 50 80 12 Not detected 2


Lab ID Sample 1 – Oysters Sample 2 – Mussels E. coli MPN/100g Salmonella spp. in 25g E. coli MPN/100g Salmonella spp. in 25g Replicate 1 Replicate 2 Score Result Score Replicate 1 Replicate 2 Score Result Score

87 a b 20 20 12 Not detected 2 <20 <20 12 Not detected 2 90 * 50 70 12 Not detected 2 80 50 12 Not detected 2 92 230 330 9 Not detected 2 170 130 12 Not detected 2 95 3.6 NE 4 Not detected 2 3.6 NE 4 Not detected 2 106 a b c <20 20 12 Not detected 2 140 <20 12 Not detected 2 111 a b <20 20 12 Not detected 2 <20 50 12 Not detected 2 119 20 <20 12 Present 0 130 130 12 Not detected 2 130 IP IP - IP - IP IP - IP - 141 20 20 12 Not detected 2 230 230 12 Not detected 2 147 * 50 20 12 Not detected 2 220 50 12 Not detected 2 155 a <20 <20 12 Not detected 2 230 170 12 Not detected 2 160 a <20 50 12 Not detected 2 20 80 12 Not detected 2 166 a c 15 20 12 Not detected 2 <3 <3 2 Not detected 2 170 * a <200 <200 12 Not detected 2 <200 <200 12 Not detected 2 177 80 50 12 Not detected 2 80 80 12 Not detected 2 185 a b <20 <20 12 Not detected 2 20 <20 12 Not detected 2 196 50 20 12 Not detected 2 20 80 12 Not detected 2 208 NE NE 2 Not detected 2 NE NE 2 Not detected 2 212 * a <67 230 12 NE - <67 230 12 NE - 219 NR NR 0 NR 0 NR NR 0 NR 0 226 c 45 78 12 Present 0 78 20 12 Not detected 2 241 c 230 NE 7 Not detected 2 490 NE 7 Not detected 2

a Low censored values were halved for data assessment. b No score deduction for reported MPN values of <20 (sample 2 only) as participants’ medium range was12 – 420 MPN/100g. c Laboratory did not use an approved method. No marks were deducted as not an NRL or OCL, * Designated NRL’s, IP – Laboratory experienced import problems and was unable to receive material, NE – Not examined, NR – Not returned.


Table 8: Participants results

GM - geometric mean, SDT – theoretical standard deviation General comments Fifty laboratories (26 NRL and 24 other laboratories) were sent material with only laboratory 130 not receiving the material due to incorrect or absence of import permissions required at customs. Information provided by laboratories on the temperature and arrival time of the material, 59% (29) of laboratories received the material the day after dispatch (11.09.12) with 52% (15) of these laboratories analysing the material on arrival. Sixteen percent (8) of laboratories received material within 48 hours after dispatch with the remaining 22% of laboratories receiving their material between 72 and 240 hours after dispatch. In general the delays in laboratories receiving material were associated with incorrect or absence of import permissions. One laboratory did not provide any information of the material temperature or arrival time. Temperature recorders stored in each consignment showed an in transit temperature range of 0.5 – 10.5°C. All temperature data, arrival and analysis dates and times recorded by participants are shown in Appendix I. Methodology Forty-five laboratories provided information on the methodology used for E. coli and Salmonella spp. analyses and are shown in Tables 9 and 10. E. coli Seventy-six percent (21 NRLs and 16 other laboratories) of laboratories cited ISO TS 16649-3 (Anon 2005) as their laboratory method for the enumeration of E. coli. Three laboratories cited use of FDA/BAM method. It is noted that this is not an approved method for the official control testing of live bivalve molluscs in the EU. The 5 x 3 MPN tables in Donovan et al (1998) and those contained in ISO 7251 (2005) differ slightly from those contained in ISO 7218. Laboratories are reminded that for enumeration of E. coli in live bivalve molluscs for official control testing using ISO TS 16649-3 should use 5 x 3 MPN tables in ISO 7218 or those provided by the EURL should be used. Table 9: E. coli methodology E. coli methods Number of laboratories ISO TS 16649-3 (Anon 2005) 37 FDA/BAM Chapter 4 (2002) 3 Donovan et al (1998) 2 ISO 7251 (Anon 2005) 1 ISO 16649-2 (Anon 2001) 1 NMKL 96 1

Salmonella spp. Fifty-nine percent (19 NRLs and 10 other laboratories) of laboratories cited ISO 6579 (Anon 2005) as their laboratory method for the detection of Salmonella spp.. with a further 8 laboratories citing ISO 6579 with supplementary confirmation tests as their laboratory method. Laboratories are reminded that for official control testing of live bivalve molluscs for Salmonella spp the EU reference method is ISO 6579 (Anon 2002).

E. coli MPN/100g Range Median GM Median±3*SDT

Sample 1 - Oysters <2.0 x 101 - 4.9 x 102 4.5 x 101 3.8 x 101 7.0 x 100 - 2.7 x 102

Sample 2 - Mussels <2.0 x 101 - 4.9 x 102 7.0 x 101 5.2 x 101 1.2 x 101 - 4.2 x 102


Table 10: Salmonella spp. methodology Salmonella spp. methods Number of laboratories EN ISO 6579 (Anon 2002) 37 Biomerieux vidas 7 NMKL 71 4 FDA/BAM Chapter 5 (2003) 2 BAX system (PCR) 2 Bio-Rad 1 iQ-Check Salmonella II 1 Real time PCR 1 Oxidase Sal rapid test 1

Sample analyses Forty-seven laboratories returned the report form for this PT distribution. Laboratory 130 did not examine this distribution Laboratory 208 did not examine this distribution for E. coli and Laboratory 219 did not return their results. E. coli Sample 1 – Oysters Forty-two laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants’ median with 39 laboratories obtaining a score of 12. Laboratory 241 reported a single result which was within ±3 SD of the participants’ median and scored 7. Laboratories 23, 28, 92 and 95 reported one or both replicate results between ±3 SD and ±5 SD of the participants’ median. Laboratories reporting MPN results not consistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables or those previously supplied to NRLs by the EURL and perform official control testing of live bivalve molluscs in the EU were deducted points (Laboratories 21, 41 and 76). Note: Points were only deducted from NRL s or official control laboratories who were not using an approved reference method or MPN table. No points were deducted for use of alternative methods by non-EU countries. Sample 2 – Mussels Thirty-seven laboratories returned duplicate E. coli MPN/100g results falling between ±3 SD of the participants’ median with 38 obtained a score of 12. Laboratory 241 reported a single result which was within ±3 SD of the participants’ median and scored 7. Laboratories 28, 42, 83, 87, 95, 106, 111 and 185 reported one or both replicate results between -3 SD and -5 SD of the participants’ median. Laboratories 166 reported both replicate results outside -5 SD of the participants’ median. Laboratories reporting MPN results not consistent with the guidance given in ISO 7218 for interpretation of 5 x 3 MPN tables or those previously supplied to NRLs by the EURL and perform official control testing of live bivalve molluscs in the EU were deducted points (Laboratories 13, 21, 41, 48 and 76) Note: Points were only deducted from NRL s or official control laboratories who were not using an approved reference method or MPN table. No points were deducted for use of alternative methods by non-EU countries. Salmonella spp. Sample 1 – Oysters Forty-five laboratories returned expected results for Salmonella spp. as not detected in sample 1. Laboratories 119 and 226 reported the presence of Salmonella spp. in the sample. Sample 2 – Mussels Forty-six laboratories returned expected results for Salmonella spp. as not detected in sample 2. Laboratories 13 reported the presence of Salmonella spp. in the sample.


References Anon 2005. ISO TS 16649-3. 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 2002. ISO 6579. Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp. Geneva, Switzerland. Fravalo P, Hascoet Y, Le Fellic M, Queguiner S, Petton J and Salvat G. (2003). ‘Convenient method for rapid and quantitative assessment of Salmonella enterica contamination: The mini-MSRV MPN technique. Anon 2007. ISO 7218. Microbiology of food and animal feeding stuffs - General recommendations and guidance for microbiological examinations. Geneva, Switzerland. Donovan TJ, Gallacher S, Andrews NJ, Greenwood MH, Graham J, Russel JE, Roberts D, Lee R. (1998). ‘Modification of the standard method used in the united kingdom for counting Escherichia coli in live bivalve molluscs’. Communicable disease and public health 1: 188-96. Anon 2001. ISO TS 16649-2. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli Part 2: Colony-count technique at 44̊ C using 5 -bromo-4-chloro-3-indolyl-β-D-glucuronide. Geneva, Switzerland. Anon 2005. ISO 7251. Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of presumptive Escherichia coli – Most probable number technique. Anon 2002, BAM: Enumeration of Escherichia coli and the Coliform Bacteria. Peter Feng, Stephen D. Weagant, Michael A. Grant, William Burkhardt. Bacteriological Analytical Manual, Chapter 4.


Figure 1: Results chart sample 1 - Oysters Note: The median and upper and lower limits (±3 SD and ±5 SD) were calculated from participants’ results. SD calculations were based on the inherent variability of the 5 x 3 MPN method (0.26 log10). Reference values were excluded from the calculation of participants’ median.


Figure 2: Results chart sample 2 - Mussels Note: The median and upper and lower limits (±3 SD and ±5 SD) were calculated from participants’ results. SD calculations were based on the inherent variability of the 5 x 3 MPN method (0.26 log10). Reference values were excluded from the calculation of participants’ median.


Appendix I E. coli MPN scores allocated to participants returning 2 replicate results


Score allocated Total score Replicate 1 Replicate 2

Both replicate MPN results reported fall within the median ±3SD value 2 5 5 12

One replicate MPN result reported is outside the expected range and falls between the median ±3SD and median ±5SD value

2 5 2 9

Both replicates MPN results are outside the expected range and fall between the median ±3SD and median ±5SD value

2 2 2 6

One replicate MPN result reported is outside the median ±5SD value 2 5 0 7

Both replicates MPN results are outside the expected range. The first falls between the median ±3SD and median ±5SD value and the second falls outside the median ±5SD value.

2 2 0 4

Both replicates MPN results reported is outside the median ±5SD value 2 0 0 2

E. coli MPN scores allocated to participants returning 1 single replicate results


Score allocated

Total score

Single replicate MPN result reported is within the expected range 2 5 7

Single replicate MPN result reported only and falls between the median ±3SD and median ±5SD value 2 2 4

Single replicate MPN result reported is outside the median ±5SD value 2 0 2

E. coli score deductions

Result Score deducted Replicate 1 Replicate 2

Tube combination inconsistent with MPN reported, ISO 7218 or 5 x 3 MPN tables provided by the EURL. 2 2

High censored result (e.g. MPN = >18000 per 100g) 2 2

Sample not examined or results returned late - no explanation received 12 Salmonella spp scoring

Result Score allocated Fully correct results 2 Misleading result, e.g. failure to isolate Salmonella 0


Appendix II: 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. Where more than three dilutions have been tested for a sample record, select the tube combination as stated in the following rules: 1. Select the combination of three consecutive dilutions having a category 1 profile to obtain the MPN index.

If more than one combination having a category 1 profile is obtained, use the one with the highest number of positive tubes. An example is given below:

If a tube combination of 5, 5, 4, 2 is recorded, the result for the sample would be reported as 16,000

MPN/100g.

1g 0.1g 0.01g 0.001g MPN/100g Category 5 5 4 2 5 5 4 16,000 1 5 4 2 22,000 1

2. If no combination having a category 1 profile is available, use the one having a category 2 profile. If more

than one combination having a category 2 profile is obtained, use the one with the highest number of positive tubes.

2. Culture Medium - 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.


Appendix III: Sample arrival and temperature

Lab ID Date arrived

Time of arrival

Temp. recorder (°C) Sample (°C) Storage (°C) Date

analysed Time of analysis 3 * 11.09.12 14:00 4.0 - 9.5 4 - 11.09.12 14:30

7 * 11.09.12 14:20 5.5 - 9.0 3.8 1 to 5 12.09.12 12:20 9 * 11.09.12 10:30 8 11.09.12 11:00 10 * 11.09.12 14:00 4.0 - 9.5 6.2 - 11.09.12 16:00 13 * 11.09.12 10:00 5 - 9 5.3 3.5 11.09.12 14:00 19 * 12.09.12 07:45 1.5 - 8.5 3.6 4 12.09.12 09:00 20 11.09.12 08:20 3.5 - 7.5 6 4 11.09.12 10:30 21 * 11.09.12 13:30 4.5 - 6.5 8.8 5.8 11.09.12 15:30 22 * 11.09.12 13:00 3.5 - 9.0 6.9 1 to 5 12.09.12 10:30 23 * 11.09.12 13:30 6.0 - 10.5 7.7 3.5 12.09.12 10:15 27 * 11.09.12 11:50 7.0 - 10.0 5 1 to 5 12.09.12 09:30 28 18.09.12 14:30 1.0 - 9.0 10 4 18.09.12 17:30 30 11.09.12 12:00 3.5 - 9.5 2.5 2.2 11.09.12 13:00 32 * 11.09.12 11:45 3.5 - 7.0 6.8 - 11.09.12 13:00 33 * 11.09.12 12:30 4.5 - 9.5 6.4 1 to 5 12.09.12 12:30 35 * 11.09.12 14:00 3.5 - 9.5 4.5 4.5 12.09.12 11:15 39 * 11.09.12 12:00 4.5 - 9.5 5.5 1 to 5 12.09.12 11:00 41 * 11.09.12 12:00 6.4 4 11.09.12 13:55 42 * 11.09.12 11:40 -12 4 11.09.12 13:30 43 * 12.09.12 15:30 5.0 - 9.0 5 7 13.09.12 10:00 44 * 11.09.12 12:40 3.5 - 9.0 4.2 2 12.09.12 10:40 47 * 11.09.12 14:00 6.5 - 10.0 6.4 4 11.09.12 15:45 48 11.09.12 10:00 6.0 - 9.0 5.9, 5.7 5 11.09.12 11:00 68 * 11.09.12 10:30 6.0 - 9.5 6.5 11.09.12 11:00 69 12.09.12 12:30 3.0 - 9.0 4 - 12.09.12 13:00 76 11.09.12 14:10 3.5 - 8.0 5.3, 7.2 1 to 5 12.09.12 83 11.09.12 13:50 3.5 - 7.0 5.8 1 12.09.12 12:00 86 * 11.09.12 10:50 4.5 - 9.0 5.9 5 11.09.12 14:05 87 12.09.12 16:00 2.5 - 9.5 4 4 13.09.12 14:00 90 12.09.12 10:30 5.5 - 9.0 11.2 1 to 3 13.09.12 10:15 92 12.09.12 09:54 4.0 - 9.0 3.5 4 12.09.12 12:10 95 13.09.12 16:30 2.5 - 7.5 12.8, 13.6 13.09.12 17:30 106 11.09.12 15:00 3.5 - 9.5 10, 9.5 4 12.09.12 13:00 111 11.09.12 13:30 4.5 - 8.0 3 2 12.09.12 11:00 119 11.09.12 14:00 4 4 12.09.12 09:00 130 Problems at customs 141 13.09.12 12:30 6 3 13.09.12 147 * 11.09.12 am 5.5 - 10.0 12.09.12 48h


Sample arrival and temperature continued

* Designated NRL’s

Lab ID Date arrived

Time of arrival

Temp. recorder (°C) Sample (°C) Storage (°C) Date

analysed Time of analysis 155 13.09.12 09:45 4.5 - 9.5 4 4 13.09.12 13:00

160 13.09.12 16:00 7 4.3 14.09.12 10:00

166 18.09.12 22:00 0.5 - 7.5 5 1 to 5 18.09.12 22:00

170 * 12.09.12 07:45 1.5 - 7.0 5.8 4 12.09.12 09:00

177 20.09.12 12:30 1.5 - 3.5 10 3 20.09.12 13:00

185 13.09.12 16:37 4.1 4 14.09.12 09:00

196 13.09.12 12:20 4.2 4 14.09.12 21:30

208 12.09.12 21:30 2.5 - 8.5 1.8 1 to 5 15.09.12 17:32

212 * 11.09.12 12:00 11.09.12

219

226 20.09.12 14:00 1.5 - 10.5 6.6 4 22.09.12 08:00

241 17.09.12 17.09.12 14:00













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• international and UK government departments

• the European Commission

• the World Bank

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(FAO)

• oil, water, chemical, pharmaceutical, agro-chemical,


• non-governmental and environmental organisations

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property

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European Community Reference laboratory

for monitoring bacteriological and



Science


Barrack Road,

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Dorset DT4 8UB UK

Tel: +44 (0) 1305 206600, Fax +44 (0) 1305 206601


Dear Participant PT 46 NOROVIRUS (GENOGROUP I AND II) AND HEPATITIS A VIRUS PROFICIENCY TESTING The intended results were: Sample Norovirus HAV GI GII

Shellfish sample 1 a + (1.38 x 10

1 - 3.44 x 10

3) + (5.89 x 10

2 - 8.54 x 10

4) + (1.76 x 10

2 - 4.98 x 10

4)

Shellfish sample 2 a

+ (2.12 x 102 - 6.21 x 10

2) + (1.68 x 10

3 - 5.47 x 10

3) -


- - -


- - + (3.06 x 104 - 9.24 x 10

4)

LENTICULE™ 1 b

+ (9.23 x 103 - 3.55 x 10

4) - -

LENTICULE™ 2 b

- + (3.96 x 103 - 5.75 x 10

3) + (1.15 x 10

5 - 7.84 x 10

5)

a Copies/g;

b Copies/ LENTICULE.

The ranges provided are based on a 95% confidence limit determined as 2 geometric standard deviations above and below the geometric mean. A full report including an analysis of all participants' results will be available shortly. Yours sincerely

Louise Stockley

CEFAS is an Executive Agency of the Department for Environment, Food and Rural Affairs













(www.cefas.co.uk)
















international and UK government departments

the European Commission

the World Bank

Food and Agriculture Organisation of the United Nations

(FAO)

oil, water, chemical, pharmaceutical, agro-chemical,


non-governmental and environmental organisations

regulators and enforcement agencies

local authorities and other public bodies




property

.


Tel +44 (0) 1502 56 2244 Fax +44 (0) 1502 51 3865 Web www.cefas.co.uk


Tel +44 (0) 1305 206600 Fax +44 (0) 1305 206601


european union reference laboratory for monitoring ... · european union reference laboratory for...

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