ISO validation studies

(“the CEN Mandate”)

Wilma Jacobs-Reitsma, RIVM

Ida Jongenburger, NVWA

EURL-Campylobacter Workshop, Aberdeen 19-9-2013 1

History of CEN Mandate M/381

● 2006: EC sent a mandate to CEN/TC275/WG6

– for validation of 15 microbiological methods (in Regulation EC 2073/2005: Microbiological criteria)

– CRLs prefered to become projectleader

● 2007: Project proposals sent to CEN

● 2007/2008: Project leaders assigned

● 2008-2010: amendment of project proposal, several administrative issues to solve

● December 2010: Specific agreement signed between CEN and EC

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Future of CEN Mandate M/381

● Start project: 1 January 2011 for a total of 6 years

● Laboratory studies to be finished before the end of 2013

● 2011/2012: subcontracts made between CEN (at Afnor, France) and 15 project leaders

● Results project in 2017: publication of validation data (performance characteristics) in 15 EN-ISO documents

– standardisation of several new methods

– revision of several existing methods

– 15 validation studies performed

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Methods under CEN mandate (I)

Method Project leader

Viruses in food CEFAS (EURL), UK

Detection and quantification of histamine

ANSES Boulogne, FR

Detection of staphylococcal enterotoxins

ANSES Paris (EURL), FR

Detection of Bacillus cereus enterotoxins

NVWA, Wageningen, NL

Detection of Enterobacter sakazakii Spain

Detection of pathogenic Vibrio spp. CEFAS (EURL), UK

Detection and enumeration of Enterobacteriaceae

NVWA, Wageningen, NL

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Methods under CEN mandate (II)

Method Project leader

Detection of Listeria monocytogenes

ANSES Paris (EURL), FR

Enumeration of Listeria monocytogenes

ANSES Paris (EURL) + Cecalait, FR

Detection of E. coli O157 ISS (EURL), IT

Detection of Campylobacter NVWA, Wageningen, NL

Enumeration of Campylobacter RIVM, NL

Detection of Salmonella spp. in primary production

RIVM (EURL), NL

Detection of Yersinia enterocolitica EVIRA, FI

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Protocol for validation studies

● For the validation studies, interlaboratory studies (ILS) have to be organised

● The organiser needs to test the samples of the ILS for homogeneity and stability

● Basis for statistical analysis of the data from the ILS is ISO 16140 (2003/2013?!)

● General procedures are described, so all studies will be performed in a similar way (but still quite some differences in interpretation possible)

● Performance characteristics (=?!) for each method

● Project leaders meeting in November 2013 in Brussels

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Validation of methods for Campylobacter

● EN-ISO 10272-1:2006, Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method

– Projectleaders: Enne de Boer/Ida Jongenburger (NVWA, Wageningen, NL)

– Standard is currently under revision, with quite some impact on the methods to be used in the validation study (1A, 1B, 1C)

● ISO-TS 10272-2:2006, Microbiology of food and animal feeding stuffs – Horizontal method for detection and enumeration of Campylobacter spp. Part 2: Colony-count technique

– Projectleader: Wilma Jacobs (RIVM, Bilthoven, NL)

– Standard is currently under revision, but with less impact than on part 1

● Both projects will be elaborated in close cooperation, and of course also in contact with the EURL-Campylobacter (Eva Olsson/Ingrid Hansson, SVI, Sweden)

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Poster P254 at CHRO2013

Validation Campylobacter - planning ILS

● Identical samples (5 matrices) in detection and enumeration studies

● Inoculation of samples to be done by the participants

● Vials with strains prepared by CHEK (Groningen, NL: www.vwa.nl/chek)

– Blood-based medium and stable when kept at -80°C

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Matrix Strain Detection method

Frozen minced meat (pork/beef) C. coli ATCC 33559 1-A (Bolton)

Lettuce (Iceberg) C. jejuni ATCC 33291 1-A (Bolton)

Raw milk C. jejuni ATCC 29428 1-B (Preston)

Broiler neck skin C. coli ATCC 43478 1-B (Preston)

Broiler caecal material C. jejuni DSM 24306 1-C (direct plating)

Validation Campylobacter- Participating laboratories

● Samples will be shipped to the laboratories by courier, and testing has to start on the designated date(s)

● Testing has to be carried out according to the ILS instructions, describing the (revised) ISO methods

● Laboratories have to provide for their own media, materials and equipment

● (Small) fee available for each participating laboratory

● Participation to only a part of the studies is also possible

● NRLs-Campylobacter within the EU

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ILS for validation of ISO 10272-1

● At least 15 laboratories (≥ 10 valid data sets needed for statistics)

● 3 levels of contamination on samples of 10 g (0,5 ml for caeca) each:

–Blank,

–Low, 5-10 cfu/sample

–High, 50-100 cfu/sample (but maybe 103 cfu/g for caeca?)

● 8 blind replicates per level

● 5 different matrices (horizontal application of the method)

● Each lab has to test 24 samples per matrix (120 for total ILS)

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ILS for validation of ISO 10272-2

● At least 12 laboratories (≥ 8 valid data sets needed for statistics)

● 4 levels of contamination on samples of 1 g (caeca) or 10 g each:

–Blank,

–Low, eg 104 cfu/g (but maybe 103 cfu/g for neck skin)

–Medium, eg 105 cfu/g

–High, eg 106 cfu/g (but maybe 107 cfu/g for caeca)

–Levels depending on the matrix

● 2 (blind) replicates per level

– Indications were given on the dilutions to be used per sample

– Samples have to be tested by duplicate plating

● 5 different matrices (horizontal application of the method)

● Each lab has to test 8 samples per matrix (40 for total ILS)

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● ILS enumeration on 12 June 2013, 1 shipment

● ILS detection in November 2013, 1 shipment in week 44

– 4 November: 48 samples part B (Preston)

24 samples part C (direct plating)

– 18 November: 48 samples part A (Bolton)

● Discussion of results ILS enumeration during the EURL-Campylobacter Workshop in Aberdeen (19 September 2013)

● Discussion of results ILS detection during a meeting of participants (eg January 2014, Utrecht, NL)

● 2017 at the latest: Publication of the validation data (performance characteristics) in ISO 10272-1 and 10272-2

Validation Campylobacter – time-frame

Czech Republic France Germany Iceland Macedonia Poland Slovakia Sweden Sweden Switzerland UK UK UK

NVWA RIVM

Participants enumeration study n=15

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● Samples enumeration: 5 x 8 x 15 = 600 portions of 10 gram*

● Samples detection: 5 x 24 x 17 = 2040 portions of 10 gram*

*1 gram respectively 0,5 ml portions for caecal samples

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● Origin of the materials: Local supermarket, Iglo, local dairy farm, Guldfågeln AB Sweden, Albert ter Laak (CVI, Lelystad)

● With many thanks to Ellen and Wendy

60 Swedish, so Campylobacter-free, carcasses…

…ends up in enough broiler skin material for at least 528 portions (and some very nice chicken soup)

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Diagram ILS Campylobacter enumeration method

(DRAFT ISO 10272-2 of 2013-04-15, but for this validation ILS with duplicate plating)

*For caecal material: 1 gram portion + 9 ml peptone-salt solution, **Only for broiler skin samples ***One plate per countable dilution per sample.

10 gram test portion* + 90 ml peptone-salt solution (=initial suspension)

10-fold dilution series

in 9 ml peptone-salt solution tubes

Initial suspension**: 1 ml on 1 large mCCDA plate (14 cm Ø) or on 3 common mCCDA plates (9 cm Ø) Prepare duplicates using 2 large or 6 common plates

All dilutions: 0,1 ml on 1 mCCDA plate (9 cm Ø) in duplicate

Micro-aerobic incubation 40-48 hours at 41,5 °C

Counting of characteristic colonies

Confirmation of 5 characteristic colonies per plate***

Purification on Blood agar plates

-Microscopy for morphology and motility -Oxidase test -Absence of growth at 25 °C, aerobic

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- Inoculation and incubation (Day 0, Wednesday 12 June 2013)

Dry the mCCDA plates for 15-30 minutes before use, or as appropriate, and record the

details. Please dry the plates carefully, as some of the strains in this study have quite some swarming capacity!

Samples C1-C4: Duplicate plate 0,1 ml of the 10-2 and the 10-3 dilutions on mCCDA.

Samples C5-C8: Duplicate plate 0,1 ml of the 10-3 , the 10-4, and the 10-5 dilutions on mCCDA.

Samples F1-F4, M1-M4, R1-R4:

Duplicate plate 0,1 ml of the 10-1 and the 10-2 dilutions on mCCDA. Samples F5-F8, M5-M8, R5-R8: Duplicate plate 0,1 ml of the 10-2 , the 10-3, and the 10-4 dilutions on mCCDA.

Samples S1-S4: Duplicate plate 1 ml of the 10-1 dilution on mCCDA, by using 2 mCCDA plates Ø 14 cm, or 6 mCCDA plates Ø 9 cm. Duplicate plate 0,1 ml of the 10-1 dilution on mCCDA. Samples S5-S8:

Duplicate plate 0,1 ml of the 10-1 , the 10-2, and the 10-3 dilutions on mCCDA.

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ANNEX B. General questions

Laboratory Code:

Contact person: e-mail:

Arrival date of samples: Arrival time of samples: Condition of the package and the samples at receipt: Was the water in the “Temp. Control” vial still frozen?: Temperature of water in “Temp. Control” vial at receipt, in case not frozen:

Did you store the samples at -20°C upon receipt?:

Did you store the inoculation suspensions at -80°C upon receipt?: Did you prepare/inoculate the samples according to the instructions given?: Did you Stomach the samples for 1 minute before testing:? Incubation temperature of the mCCDA plates:

Incubation temperature of the blood agar plates (purification):

Incubation temperature of the blood agar plates (confirmation):

11 parcels on 11 June, 4 parcels on 12 June

All still frozen and ok

Retrospectively, no for 1 lab

2 labs reported 42°C instead of 41,5°C

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Diluent: peptone saline solution or BPW: Ready to use, or from dehydrated powder: Manufacturer and product code: mCCDA plates Ready to use, or from dehydrated powder: Manufacturer and product code: How and for how long did you dry the plates before use: Blood agar plates Ready to use, or from dehydrated powder: Manufacturer and product code: How and for how long did you dry the plates before use: Microaerobic atmosphere Anoxomat: Gas-generating sachets in jars: Manufacturer: Microaerobic incubator: Other: Oxidase test Manufacturer and product code:

8x jars, 3x Anoxomat, 4x gas incubator

7x peptone saline, 8x BPW

5x ready to use, 10 own prep.

7x Merck, + various others

Large variety: from 10 min at 50°C, 30-60 min laminar flow, overnight at room temp.

Oxoid, LabM, Merck 5x ready to use, 10 own prep.

8x ready to use, 7 own prep.

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Lab 2 cfu/g log10 cfu/g

C1 0 1 0 0 <100 <2,0

C2 142 117 6 10 1,3E+05 5,1

C3 125 118 9 12 1,2E+05 5,1

C4 0 6 3 4 <100 <2,0

C5 43 19 4 3 0 0 3,1E+05 5,5

C6 >150 >150 48 80 4 3 6,1E+06 6,8

C7 15 19 1 1 0 0 1,6E+05 5,2

C8 >150 >150 55 65 4 12 6,2E+06 6,8

0,1 ml from 10-2 0,1 ml from 10-3 0,1 ml from 10-4 0,1 ml from 10-5

Lab 1 cfu/g log10 cfu/g

C1 0 0 0 0 <100 <2,0

C2 108 104 12 10 1,1E+05 5,0

C3 110 87 24 15 1,1E+05 5,0

C4 0 0 0 0 <100 <2,0

C5 19 18 4 0 0 0 2,0E+05 5,3

C6 224 220 21 55 7 9 4,2E+06 6,6

C7 9 9 1 1 0 1 9,1E+04 5,0

C8 >300 >300 34 56 4 4 4,5E+06 6,6

0,1 ml from 10-2 0,1 ml from 10-3 0,1 ml from 10-4 0,1 ml from 10-5

numbers used in the calculations

numbers <10, uncertainty too high, not to be used in calculations (?)

colonies counted but all confirmed negative

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Confirmations ● 5 suspect colonies per countable plate (< 150 colonies)

– Enumeration in single would be: maximum of 10 confirmations per sample

● Purification on non-selective medium, e.g. blood agar

● Microscopy: characteristic morphology and motility

– Also possible to test directly from mCCDA

● Oxidase test: positive for Campylobacter

● Aerobic growth at 25°C: no growth by Campylobacter

● In this study 5 x 6* x 10 = 300 confirmations per lab (maximum) !

● nearly 4500* confirmations, all but 5 were Campylobacter

*blank samples excluded 28

Blank samples and background flora

● All matrices were tested for presence (absence) of Campylobacter

– 10 random samples per matrix tested

– Enrichment in Preston (24 h) and isolation on mCCDA

– Direct plating on mCCDA

– No Campylobacter detected in the 50 samples

● All matrices tested on total viable count (PCA, 3 days at 30°C)

– Means for 3 samples per matrix:

● Generally, not much background flora reported on the mCCDA

C ceacal samples 8,3 log10 kve/g

F frozen spinach 3,2 log10 kve/g

M minced meat 6,8 log10 kve/g

R raw milk 3,5 log10 kve/g

S chicken skin 5,3 log10 kve/g

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Campylobacter on brand A and brand B mCCDA (identical sample)

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Lab 15 cfu/g log10 cfu/g

M1 0 0 0 0 <100 <2,0

M2 0 0 0 0 <100 <2,0

M3 52 49 8 16 5,7E+03 3,8

M4 76 96 14 18 9,3E+03 4,0

M5 >150 >150 49 24 4 1 3,5E+05 5,5

M6 >150 >150 80 55 13 3 6,9E+05 5,8

M7 >150 >150 101 69 20 5 8,9E+05 5,9

M8 >150 80 20 12 5 1 9,3E+04 5,0

>150 80 20 12 5 1 1,7E+05 5,2

>150 80 20 12 5 1 1,6E+05 5,2

0,1 ml from 10-1 0,1 ml from 10-2 0,1 ml from 10-3 0,1 ml from 10-4

What are “valid data” ?

● Duplicate plating used in this ILS

● Single plating described in ISO 10272-2

● Calculations according to ISO 10272-2/ISO 7218

● Interpretations and common practice ?

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First general overview of the data (in log10 cfu/g or ml), obtained in the interlaboratory study on validation of ISO 10272-2 enumeration of Campylobacter (12-6-2013)

Lab C1 C4 C2 C3 C5 C7 C6 C8 Lab R2 R4 R1 R3 R7 R8 R5 R6

7 7

1 <2,0 <2,0 5,0 5,0 5,3 5,0 6,6 6,6 1 <2,0 <2,0 3,7 3,7 4,5 4,6 5,9 3,5

2 <2,0 <2,0 5,1 5,1 5,5 5,2 6,8 6,8 2 <2,0 <2,0 3,1 3,4 4,6 4,3 6,0 5,8

3 <2,0 <2,0 5,4 5,3 5,6 5,4 7,1 7,0 3 <2,0 <2,0 3,9 4,1 4,5 4,7 5,6 5,6

4 <2,0 <2,0 5,0 5,0 5,4 5,3 6,6 6,8 4 <2,0 <2,0 4,0 4,0 4,5 5,0 6,1 5,9

5 <2,0 <2,0 4,5 4,9 4,8 4,8 6,1 6,1 5 <2,0 <2,0 2,7 2,6 4,7 4,3 5,6 5,4

6 <2,0 <2,0 pos 4,7 5,1 pos pos 6,3 6 <2,0 <2,0 3,4 3,5 3,7 4,1 5,8 5,9

8 <2,0 <2,0 5,2 5,3 4,9 4,9 6,8 6,7 8 <2,0 <2,0 3,8 3,5 4,9 4,6 5,8 6,3

9 <2,0 <2,0 5,1 5,1 5,9 6,1 6,7 6,4 9 <2,0 <2,0 3,7 4,8 5,7 5,9 6,4 6,2

10 <2,0 <2,0 5,2 5,0 5,2 5,2 6,8 7,1 10 <2,0 <2,0 4,1 4,2 5,1 <3,0 6,9 6,8

11 <2,0 <2,0 5,3 5,2 5,5 5,5 7,0 6,8 11 <2,0 <2,0 3,6 3,3 5,6 5,4 6,1 5,9

12 <2,0 <2,0 4,8 4,9 4,9 4,7 6,4 6,7 12 <2,0 <2,0 3,2 2,5 4,2 4,2 5,8 5,7

13 <2,0 <2,0 4,5 4,6 4,5 4,8 6,4 6,2 13 <2,0 <2,0 3,6 3,3 3,5 3,8 5,7 5,6

14 <2,0 <2,0 5,4 5,5 5,6 5,4 7,0 7,0 14 <2,0 <2,0 3,8 3,5 4,7 4,6 6,0 6,0

15 <2,0 <2,0 6,0 6,1 6,4 7,1 6,4 7,7 15 <2,0 <2,0 4,5 4,1 5,4 5,1 6,6 6,6

AVERAGE 5,1 5,1 5,4 5,4 6,7 6,7 AVERAGE 3,7 3,7 4,8 4,7 6,0 6,0

STDEV 0,4 0,4 0,3 0,3 0,3 0,3 STDEV 0,4 0,3 0,4 0,5 0,4 0,4

Lab F2 F3 F1 F4 F5 F6 F7 F8 Lab S3 S4 S1 S2 S6 S7 S5 S8

7 7 <2,0 <2,0 2,1 2,3 3,1 2,9 3,8 4,2

1 <2,0 <2,0 4,2 3,7 4,6 4,6 5,6 5,6 1 <2,0 <2,0 3,3 3,2 4,2 4,0 4,8 5,4

2 <2,0 <2,0 3,4 3,5 4,2 4,4 5,1 5,4 2 <2,0 <2,0 2,1 1,7 3,0 3,6 5,1 3,5

3 <2,0 <2,0 3,4 3,0 4,0 3,9 5,2 5,1 3 <2,0 <2,0 3,2 3,3 3,5 3,2 4,5 4,8

4 <2,0 <2,0 4,1 4,0 4,9 4,9 5,6 5,7 4 <2,0 <2,0 2,8 2,5 3,4 3,8 5,0 4,9

5 <2,0 <2,0 3,1 2,7 pos pos 4,7 5,1 5 <2,0 <2,0 2,2 2,1 2,7 3,4 3,9 4,1

6 <2,0 <2,0 3,1 3,1 3,9 4,0 5,0 4,8 6 <2,0 <2,0 1,9 2,7 3,4 3,2 4,1 4,7

8 <2,0 <2,0 3,6 3,7 4,5 4,4 5,2 5,1 8 <2,0 <2,0 pos 1,7 3,2 2,6 4,6 4,7

9 <2,0 <2,0 4,7 4,7 5,6 5,6 6,0 6,1 9 <2,0 <2,0 4,3 4,2 5,0 5,1 >6,2 >6,2

10 <2,0 <2,0 4,0 4,0 4,4 4,7 5,8 5,8 10 <2,0 <2,0 2,4 2,6 4,2 3,1 5,0 5,1

11 <2,0 <2,0 3,9 3,9 4,5 4,4 5,4 5,6 11 <2,0 <2,0 3,1 3,1 3,9 3,7 5,1 5,3

12 <2,0 <2,0 2,5 pos 4,0 4,0 4,4 4,8 12 <2,0 <2,0 1,9 2,7 4,0 3,7 4,7 5,2

13 <2,0 <2,0 2,7 pos 3,9 3,5 4,2 4,4 13 <2,0 <2,0 2,2 2,8 3,2 4,0 5,0 5,1

14 <2,0 <2,0 4,1 3,9 4,5 4,5 5,5 5,5 14 <2,0 <2,0 3,2 3,2 3,8 4,0 5,5 5,4

15 <2,0 <2,0 3,6 3,5 4,4 4,5 5,5 5,2 15 <2,0 <2,0 2,3 2,3 4,3 4,0 5,4 5,0

AVERAGE 3,8 3,7 4,5 4,6 5,2 5,3 AVERAGE 2,7 2,8 3,7 3,6 4,8 4,9

STDEV 0,5 0,5 0,5 0,4 0,5 0,5 STDEV 0,5 0,4 0,4 0,4 0,5 0,5

Lab M1 M2 M3 M4 M5 M8 M6 M7 Data on Reference Material valid data, to be used in the calculations

7 in cfu per CCDA plate all plates cfu <10, not used in calculations

8 Lab RM 1st RM 2nd all plates cfu >150 but <200, not used in calculations

1 <2,0 <2,0 3,9 neg 5,0 5,1 4,5 5,8 7 0 0 non-valid data, eg due to probable mistake in inoculation

2 <2,0 <2,0 3,5 3,1 4,3 3,9 5,2 4,6 1 pos pos or due to problems in storage of samples (lab 7)

3 <2,0 <2,0 3,4 2,9 4,3 4,3 4,9 5,1 2 32 48

4 <2,0 <2,0 3,3 3,3 4,7 4,6 5,0 5,0 3 61 38 Matrices studied: C Caecal material

5 <2,0 <2,0 2,9 2,9 3,7 3,4 4,8 4,4 4 52 58 F Frozen spinach

6 <2,0 <2,0 3,6 3,7 4,6 4,3 4,8 5,0 5 45 24 M Minced meat

9 <2,0 <2,0 3,6 3,5 4,8 4,9 5,6 5,4 6 3 4 R raw milk

10 <2,0 <2,0 3,8 3,6 4,7 4,7 5,6 5,5 8 34 45 S Chicken Skin

11 <2,0 <2,0 3,4 3,1 4,3 4,4 4,6 5,0 9 27 29 Per matrix: Samples were inoculated at 4 levels (including a blank), in duplicate

12 <2,0 <2,0 2,9 2,8 3,6 4,4 4,5 5,0 10 56 44

13 <2,0 <2,0 2,8 2,6 3,8 4,0 4,2 4,8 11 21 30 AVERAGE and STDEV are just given as a general indication on results.

14 <2,0 <2,0 3,5 4,0 4,7 4,9 5,4 5,5 12 11 7 Statistical analysis to elaborate the "performance characteristics"

15 <2,0 <2,0 3,8 4,0 5,5 5,2 5,8 5,9 13 7 9 still have to be done.

AVERAGE 3,6 3,6 4,7 4,6 5,1 5,2 14 28 21

STDEV 0,2 0,3 0,4 0,4 0,5 0,4 15 35 38 Wilma Jacobs, RIVM, Bilthoven NL, 29-7-2013 page 1 of 132

“Performance characteristics”

● Which ones and how to calculate ?!

● Example from ISO 6888-2:1999/Amd.2003 (enumeration staphylococci)

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Example from ISO 6888-2:1999/Amd.2003

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Example from ISO 6888-2:1999/Amd.2003

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Poster P255 at CHRO2013

Matrix

Strain used for inoculation

Level of inoculation Low Medium High

Number of participating laboratories 15 15 15

Number of samples per laboratory 2 2 2

Number of samples accepted for statistical analysis 26 16 22

Median value (in log10 cfu/g) 5,1 5,4 6,7

Repeatability standard deviation S r (in log10 cfu/g) 0,08 0,13 0,13

Repeatability relative standard deviation (in %) 1,59 2,39 1,87

Repeatability limit r:

- as an absolute difference between log10-transformed

test results (in log10 cfu/g) 0,23 0,36 0,35

- as a ratio of the higher to the lower of the two test results

on a normal scale 1,7 2,3 2,3

Reproducibility standard deviation S R (in log10 cfu/g) 0,29 0,24 0,24

Reproducibility relative standard deviation (in %) 5,68 4,46 3,52

Reproducibility limit R:

- as an absolute difference between log10-transformed

test results (in log10 cfu/g) 0,81 0,68 0,66

- as a ratio of the higher to the lower of the two test results

on a normal scale 6,5 4,8 4,6

C. jejuni DSM 24306

Caecal material

NB Preliminary data !

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Low Medium High Low Medium High Low Medium High Low Medium High Low Medium High

15 15 15 15 15 15 15 15 15 15 15 15 15 15 15

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

26 16 22 22 20 28 16 18 24 22 20 24 16 22 26

5,1 5,4 6,7 3,9 4,5 5,3 3,6 4,7 5,0 3,7 4,6 5,9 2,9 3,6 5,0

0,08 0,13 0,13 0,10 0,09 0,17 0,21 0,14 0,28 0,18 0,23 0,12 0,02 0,34 0,31

1,59 2,39 1,87 2,63 2,06 3,21 5,71 2,98 5,64 4,88 5,06 1,97 0,81 9,35 6,22

0,23 0,36 0,35 0,29 0,26 0,47 0,58 0,39 0,79 0,50 0,65 0,33 0,07 0,95 0,86

1,7 2,3 2,3 1,9 1,8 3,0 3,8 2,5 6,2 3,2 4,5 2,1 1,2 9,0 7,3

0,29 0,24 0,24 0,58 0,27 0,54 0,28 0,39 0,44 0,40 0,32 0,39 0,49 0,49 0,51

5,68 4,46 3,52 14,64 6,03 10,27 7,83 8,31 8,87 10,80 7,04 6,57 17,14 13,39 10,34

0,81 0,68 0,66 1,61 0,76 1,52 0,79 1,09 1,24 1,11 0,91 1,09 1,39 1,37 1,43

6,5 4,8 4,6 40,9 5,7 33,0 6,2 12,4 17,4 13,0 8,1 12,4 24,3 23,2 27,2

C. jejuni DSM 24306 C. jejuni WDCM 00005 C. coli ATCC 33559 C. jejuni WDCM 00156 C. coli WDCM 00004

Caecal material Frozen spinace Minced meat (pork/beef) Raw milk Broiler skin

NB Preliminary data !

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Just some more statistical analysis to do…

Thank you for your attention (and participation) !

39 wilma.jacobs@rivm.nl & ida.jongenburger@vwa.nl