cen/tc 275/wg 6 mandate m/381

CEN/TC 275/WG 6 Mandate M/381

ANSES-Food Safety Laboratory, SBCL unit, Maisons-Al fort, France

November 17, 2015 - Turin

CEN Mandate – WI 00275169

2012 2013

Method proposals March 2009 / June 2011

20142009 2015

Interlaboratory studiesPart 1 (2013)Part 2 (2014)

Interlaboratory studiesPart 1 (2013)Part 2 (2014)

BMD study

Draft 19020

Intralaboratory study

Intralaboratory study

• Five food categories tested (two different food products per category)

• The five toxins have been tested in each food category at two levels of contamination (0.1 and 0.25 ng/g for SEA and 0.25 and 0.50 ng/g for the others)

• 8 replicates (4 by EURL and 4 by a participating lab)• Five kits evaluated:

- Ridascreen SET ABCDE- Tecra Staph Enterotoxin VIA- Tecra Staph Enterotoxin ID- Transia Plate SE- Oxoid SET-RPLA

Intralaboratory study: January – May 2013

Intralaboratory study: outcome (1/2)

• Intralab study highlighted the need of DC before performing detection in milk samples. During the future ILS, extraction with and without DC will be tested on other food matrices

• Interferences have been detected on 3 food types (chocolate cake« éclair », mixed salad and rice salad)

• LoD50 has been assessed on the basis of the results obtained• Lack of sensitivity (Transia plate SE) or specificity (Oxoid SET

RPLA) has been demonstrated• Moreover, since the aim of the future Standard method is to perform

a combined detection of SEA to SEE, Ridacreen SET ABCDE and Tecra ID won’t be integrated for the final ILS step

Intralaboratory study: outcome (2/2)

• 3 detection kits have been selected :– Ridascreen SET Total– Vidas SET2– Tecra Staph Enterotoxin VIA

• Number of samples, type of food matrcies and extraction procedure(s) have been decided during the second meeting (28-29 May 2013).

Comments received on the ISO NWIP 19020

• A first draft of a Standard has been sent to the CEN/ISO secretary on March 2013 for comments

• The vote has been closed on May 23th and has been approved

• Technical comments have been made by FR, NL and editorial comments by FR, NL, TH and US

�All these comments have been discussed during the CEN WG (28-29 May 2013)

�Proposals sent to the CEN/ISO secretary by the end of 2013

Design of the interlaboratory studies (1/2)

• Data obtained from previous validation studies were used

• ILVT divided into 2 parts to cover 5 food categories:– From July to November 2013 (part 1): meat products and milk products – From March to July 2014 (part 2): fish products, RTE and pastries

• Based on document ISO TC 34/SC9/WG3 N024 + use of available data

• Requirement : at least 13 participants

• Requirement : 3 levels of contamination: blank, low (slightly above LoD), high (3 to 10 LoD)�� 3 levels have been used:

a blankclose to LoD5X LoD

�� Detection with and without dialysis concentration (e xcept for cheese)

Design of the interlaboratory studies (2/2)

• Homogeneity studies have been performed for the 5 types of food matrices for each levels

• 20 samples have been analysed for each couple food/level by the 3 selected detection kits after performing DC� all the prepared samples proved to be homogeneous � all the samples prepared proved to be stable

Results of the ILVS

Guidelines and decisions

Interpretation of the results

• The accuracy of the detection method was defined by the parameters below:

- Sensitivity (SE): Number of samples found positive divided by the number of samples tested at a given level of contamination.

- Specificity (SP): Number of samples found negative divided by the number of blank samples tested.

- LOD50: Concentration (ng/g of SEs) for which the probability of detection is 50%.This parameter was calculated using the program developed especially for this purpose by Dr Cordula Wilrich and Prof PeterWilrich available at:http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich

�� SE, SP, LOD50 must be defined

SE and SP

• On December 17-18, 2014, the WG defined a criteria for SE and SP higher than 90% to be reached by the tested detection kits.

• Note : toxins other than SEA to SEE have not been tested during SP.

LOD50

• Use of the dedicated Excel sheet

• Main question : Are these LOD50 fulfilled the DG Santé needs to protect consumers

� Use of dose-response model to establish a benchmark dose (BMD)…

• Construction of a dose-response relationship for S. aureus enterotoxin: – Scientific litterature– Outbreaks

• Outbreaks (mainly French)– Period: 2010 to 2014– Information on the effects: Symptoms and appearance time– Information available and / or necessary for the dose response:

nature and quantity of toxins, number of sick people, number of people exposed (who ingested the food containing the toxins)

3 ills out of 200 exposed

Effect characterization

• Effects of enterotoxins identified on 61 TIACs- Diarrhoea (D) - Abdominal pain (DA)- Vomiting (V) - Nausea (N) - Fever (F)• Does one always observe the same symptoms?

• By grouping effects


• Effect: – importance toxin types?

– Same symptoms for large outbreaks?

SEA

toxinOther SE toxins

<10 ills >10 ills


• Appearance time of symptoms:

– Difference between outbreaks (e.g. SFPO SEA) ?

– Difference between individuals within an outbreak?

Rela

tive f

req

uen

cy

Rela

tive f

req

uen

cy

Appearance time (h) Appearance time (h)

Rela

tive f

req

uen

cy

Rela

tive f

req

uen

cy

Appearance time (h)Appearance time (h)

Toward one (or more) dose-response relationship(s)?

• From 61 French outbreaks + european outbreaks (NRLs) + international outbreaks (publication)

→ To establish a dose-response relationship (based on BMD models)

• Conditions : – To be sure of the implicated food (excluding

outbreaks for which analyzed several foods contain one or toxins) and the involvement of S. aureus

– To have an amount of toxin> LQ – To know the number of ill and exposed consumers

Toward one (or more) dose-response relationship(s)?

• Modelling strategy:– Working on outbreaks that involve single toxin

– To establish a dose response toxin (SEA ok, SED ∼, other probably not feasible due to insufficient data set)

– Then check that the response in case of presence of several toxins is consistent or not with the dose-response set for SEA

• September 2015 communications on results for SEA (Guillier L, Bergis H, Guillier F, Noel V, Auvray F, Hennekinne JA. Dose-response modelling of staphylococcal enterotoxins using outbreakdata. Procedia Food Science 2015)

Toward one (or more) dose-response relationship (s)?

• US EPA benchmark dose-response modelling software

• BMD for SEA ∼ 3-5 ng • Food intake of 100 - 150 g → BMD ∼ 0.02 - 0.03 ng/g

0

0.2

0.4

0.6

0.8

1

0 5000 10000 15000 20000

Fra

ctio

n A

ffect

ed

dose

Gamma Multi-Hit Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

22:37 12/14 2014

BMDL BMD

Gamma Multi-Hit

0

0.2

0.4

0.6

0.8

1

0 1000 2000 3000 4000 5000 6000 7000

Fra

ctio

n A

ffect

ed

dose

Weibull Model, with BMR of 10% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

00:21 12/17 2014

BMDL BMD

Weibull

Performance criteria defined by the working group

• SE > 90%• SP > 90%• For SEA 0.05 ng/g must be detected

Overall results: parts 1 and 2

Specificity

• Satisfactory using DC whatever kit used

Matrices Detection kits With DC Without DC

RTE Food(SEA)

Vidas SET2 100% 100%Ridascreen SET Total 100% 100%Tecra VIA 90% 100%

Fish(SEC)


Pastry(SEE)


Cheese(SED)

Vidas SET2 98%Not

performedRidascreen SET Total 100%Tecra VIA 100%

Chicken(SEA)


Sensitivity RTE food (SEA)0.055, 0.11 , 0.22 ng/g

Fish (SEC)0.15 and 0.25 ng/g

Pastry (SEE) 0.15, 0.25, 0.40 ng/g

With

DC

Without

DC

With

DC

Without

DC

With

DC

Without

DC

Vidas SET 2 Level 1 100 87 100 3 100 1

Level 2 100 100 100 68 100 86

Level 3 100 100 * * 100 100

Ridascreen

SET Total

Level 1 98 10 98 10 94 0

Level 2 100 52 100 44 100 26

Level 3 100 100 * * 100 67

Tecra VIA Level 1 33 10 97 23 20 0

Level 2 57 17 100 30 37 10

Level 3 100 30 * * 50 10

* Not performed

Sensitivity

Cheese (SED)0.2 and 0.9 ng/g

Chicken (SEA)0.22 and 1.10 ng/g

With

DC

Without

DC

With

DC

Without

DC

100 * 100 99

100 * 100 100

* * * *

87 * 100 37

98 * 100 100

* * * *

83 * 95 10

97 * 100 100

* * * *

� DC increases sensitivity whatever the kit and/or the matrix used � The Vidas SET2 seems to be the most sensitive followed by Ridascreen SET

Total� Low sensitivity of Tecra VIA on RTE and pastry cate gories

�� Need of DC !!

LoD50 ng/g using DC (parts 1 and 2)

LOD50 ng/gRTE food

(SEA)Chicken

(SEA)Fish

(SEC)Pastry(SEE)

Cheese(SED)

Vidas SET 2 0.007Not

calculated0.017 0.017

Not calculated

Ridascreen SET Total

0.010Not

calculated0.027 0.036 0.076

Tecra VIA 0.082 0.047 0.031 0.408 0.092

Vidas SET2 : from 0.004 to 0.02 ng/gRidascreen SET total : from 0.005 to 0.04 ng/gTecra VIA : from 0.02 to 0.4 ng/g

�� Mandatory use of DC, 2 kits over the 3 satisfied the defined criteria

Next steps: administrative paperwork

• Revise the EN ISO 19020 draft and send to WGSummer 2015

• Receive comments from WGDeadline: August 14, 2015

• Prepare a consolidated version for the CEN/ISO secretary for submission to public enquiryDeadline: September 15, 2015

• CD vote in progress� Deadline: December 20, 2015

• DIS vote� From January to March 2016

• Final WG meeting� April/May 2016

Acknowledgments

Séverine Gentil, Lucie Neveux, the Staphylococcus team,

Laurent Guillier and of course all the WG members and

the participating labs !

26

Grazie per la vostra attenzione !

Avete domande?

cen/tc 275/wg 6 mandate m/381

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