MicroVal validation of a Campylobacter enumeration medium - SVA

1 

MicroVal validation of 

a Campylobacter 

enumeration medium 

Method Comparison Study and 

Interlaboratory Study results 

Wilma Jacobs, EU-RL Campylobacter Workshop 2010

Why validation of alternative methods? 

● EU Microbiological Criteria Document 

(Commission Regulation (EC) No 2073/2005, on microbiological criteria for 

foodstuffs) 

● Article 5 - Specific rules for testing and sampling 

● Use of alternative methods is acceptable, when the method 

has been validated against the reference method and 

certified by a third party according to EN/ISO 16140 (or 

other similar internationally accepted protocols). 

2 

Wilma Jacobs, EU-RL Campylobacter Workshop October 2010

ISO 16140 validation: what is involved 

● EN ISO 16140:2003 „Microbiology of food and animal feeding 

stuffs - Protocol for the validation of alternative methods‟ 

● EN ISO 16140:2003/Draft Amd 1:2009. Amendment 1: 

Interlaboratory study on quantitative methods 

● At the moment in a process of revision 

3 

– Experiences from the 2003 version 

– New version split up in 5 parts 

– First drafts for comments in May 2009 

– May still take some years… 


Validation process 

● Information exchange (client, MV) 

● Application (client) 

● Contracts (client-MCB, client-EL, MCB-EL) 

● Project proposal (EL) 

– Method Reviewers 

– Technical Committee 

– Approval 

● Project report (by EL, in 2 parts: MCS + ILS) 

– Method Reviewers 

– Technical Committee 

– Approval 

● Certificate (MCB) 

4 


ISO 16140:2003 validation 

● Alternative method description (e.g. the “kit insert”) 

● Reference method (ISO) 

● Scope of the validation 

● E.g.: “All foods and animal feed” 

5 

– A minimum of 5 Categories to be tested (Annex B, ISO 16140) 

› Meat products, poultry products, fish & seafood products, fruits and vegetable 

based products, dairy products, chocolate/bakery products, other products, 

animal feeds 

– Additional Categories: environmental samples, samples from the 

primary production stage. 

● One specific Category, e.g. “Poultry Products”, is also possible 



Qualitative method validation (Detection) 

● A. Methods comparison study: 

– Relative accuracy, relative specificity and relative sensitivity 

› 60 samples per Category 

– Relative detection level 

› 6 samples at 3-5 levels per Category 

– Inclusivity and exclusivity 

› 50 target strains and 30 non-target strains to be tested 

● B. Interlaboratory study: 

– At least 10 collaborative laboratories within 3 (European) 

countries 

› 8 replicates at 3 levels = 24 samples by each laboratory 

6 



Quantitative method validation (Enumeration) 

● A. Methods comparison study: 

– Linearity and relative accuracy 

› Per Category at least 1 sample in duplicate at 5 different levels 

– Relative sensitivity and determination of unknown samples 

› Per Category at least 10 additional samples in duplicate 

– Detection and quantification limits 

› 6 replicates of at least 3 levels 

– Specificity, inclusivity and exclusivity 

› 30 target strains and 20 non-target strains to be tested 

● B. Interlaboratory study: 

– At least 8 collaborative laboratories within 3 (European) countries 

› 2 replicates at 4 levels = 8 samples by each laboratory 

7 


Microval Expert Laboratory (EL) 

● Organisation and elaboration of the lab work 

8 

– Methods Comparison Study 

– Interlaboratory Study 

● To be chosen by the client 

● List of labs available at www.microval.org 

● Labs qualifications 

– Officially approved by MicroVal 

– Accreditation (eg ISO 17025) 

– Confidentiality 

● Collaborative laboratories for participation in the ILS: 

– Contacted by the EL 

– Preferably working under a QA system (eg ISO 17025) 


RIKILT Institute of Food Safety, Wageningen, NL 

9 


RIVM, Bilthoven, NL 

National Institute for Public Health and the Environment 

10 


Example: Enumeration of Campylobacter 

● Confidentiality? (the client agrees, and the certificate is granted!) 

● Example of the quantitative validation process 

– Expert Lab: RIKILT Institute of Food Safety 

– Certification Body: Lloyds Register 

– Method reviewers: Henk Stegeman & Basil Jarvis 

● Alternative method: Oxoid Brilliance CampyCount Agar plate for 

enumeration of Campylobacter species in poultry meat products, 

with confirmation according to the reference method, and also with 

confirmation using the O.B.I.S Campy kit or the Dryspot 

Campylobacter Test. 

● Reference method: 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. 

● Food Category: Poultry products. 

11 


Brilliance CampyCount Agar (BCCA) 

● a newly developed defined 

medium for the selective 

enumeration of thermotolerant 

Campylobacter species, in 

particular C. jejuni and C. coli, 

in poultry products. After 

micro-aerobic incubation for 

48 hours at 41.5 C, 

characteristic campylobacters 

appear as red colonies on the 

clear agar background. 

12 


13 


14 

Per dilution: 

2 x 0,1 ml on 2 mCCDA plates 

10 gram test portion + 90 ml peptone-salt solution 

10-fold dilution series in 9 ml peptone-salt solution tubes 

Microaerobic* incubation 

40-48 hours at 41,5 °C 

Counting of characteristic colonies 

Confirmation of 5 characteristic colonies per plate** 

Per dilution: 

2 x 0,1 ml on 2 BCCA plates 


*Microaerobic incubation in gas jars, using the appropriate Oxoid Campygen atmosphere sachets. 

**The duplicate plates of the best countable dilution per sample. 

15 

Counting of characteristic colonies 

Confirmation of 5 characteristic colonies per plate** 

Purification on Columbia blood agar 

-Microscopy for morphology and motility 

-Oxidase test 

-Growth at 41,5 °C, aerobic 

-Growth at 25 °C, microaerobic* 

-O.B.I.S Campy kit (Oxoid ID 0800M) 

-Dryspot Campylobacter test (DR0150M) 


Methods Comparison Study 

● Linearity and relative accuracy 

16 

– Per Category at least 1 sample in duplicate at 5 different 

levels 

– The concentration range in the samples tested should 

cover a minimum (zero or otherwise), a central, a 

maximum, and two intermediary levels. 

● In this study 5 different concentration levels (in cfu/g) were 

chosen, intended to be circa 150, 500, 1500, 5000, and 

50.000. 

● Chicken breast skin: C. jejuni strain C145 

● Poultry minced meat: C. coli strain C161 



● Relative sensitivity and determination of unknown samples 

17 

– Per Category at least 10 additional samples in duplicate 

● Category “Poultry products” 

● Food types tested: 

– chicken and turkey fillet samples (pieces), 

– chicken skin samples from breast/thigh/leg, 

– chicken liver samples, 

– chicken minced meat samples. 

● A total of 103 raw poultry products were tested 


Example of skin sample result on mCCDA and BCCA 

18 



● 103 raw poultry meat samples 

– 57 samples did not contain Campylobacter in detectable numbers (< 20 

cfu/g) 

– 15 samples only contained Campylobacter in very low levels (less than 10 

cfu per plate) 

● Statistical evaluation: 

– 31 naturally contaminated samples in duplicate 

– 11 artificially contaminated samples in duplicate 

● The naturally and artificially contaminated samples did not behave 

differently in the comparison. 

● For the products minced meat, chicken skin, chicken liver, chicken breast 

skin, there was a good agreement between the methods: 

– no significant bias and no significant deviation from linearity 

● For the product chicken thigh skin, however, there was a significant 

systematic bias between the methods (BCCA finds lower results than 

mCCDA). 

19 


20 

BCCA 

6 

5 

4 

3 

2 

1 

1 2 3 4 5 6 

mCCDA 

Figure 1. Calculated regression line y = -0,14 + 1,05 x with 95% confidence 

limits for all poultry products, except for chicken thigh skin samples. 

naturally contaminated samples artificially contaminated samples. 



● Detection and quantification limits 

21 

– 6 replicates of at least 3 levels 

● Fresh overnight culture of C. jejuni strain C145 

● Three test levels: an unspiked blank, and a lower and higher level 

● Plating 0,1 ml portions on to BCCA plates 

● 6 replicates per level 

● The detection and quantification limits are calculated according to 

clause 6.2.2 of ISO 16140 on the basis of threshold spread S 0. 

● Calculate: S 0 = 0,8 colonies/plate. 

● Detection limit LOD = 3 colonies/plate. 

● Quantification limit LOQ = 10 colonies/plate. 



● Specificity, inclusivity and exclusivity 

– 30 target strains and 20 non-target strains to be tested 

● A total of 37 Campylobacter strains were tested for inclusivity. 

● A total of 21 non-Campylobacter strains were tested for exclusivity. 

● Overnight cultures of strains 

– in Brain Heart Infusion (BHI) for Campylobacter 

– in Buffered Peptone Water (BPW) for non-Campylobacter 

– Dilutions in peptone saline to contain > 100 times the limit of 

detection (so: > 100 CFU/ml). 

– No matrix added. 

● The cell cultures were tested in duplicate with both the reference 

and alternative method by plating out 0,1 ml portions of the 

appropriate dilutions. 

22 


MCS, strains for inclusivity 

Strain Ref nr. Origin Source Strain Ref nr. Origin Source 

C. coli C 18 NCTC 11366 (ATCC 33559) C. jejuni C 09 Finland turkey (cecal) 

C. coli C 19 The Netherlands chicken legs C. jejuni C 10 Belgium poultry minced meat 

C. coli C 20 The Netherlands chicken neck skin C. jejuni C 11 Belgium chicken fillet 

C. coli C 21 The Netherlands chicken caecum C. jejuni C 12 Belgium chicken breast skin 

C. coli C 22 Belgium chicken breast skin C. jejuni C 13 Belgium chicken neck skin 

C. coli C 23 Belgium chicken neck skin C. jejuni C 14 UK poultry 

C. coli C 24 Belgium poultry minced meat C. jejuni C 15 The Netherlands poultry 

C. coli C 25 Belgium chicken fillet C. jejuni C 16 Denmark poultry 

C. coli C 34 ATCC 43478 (LMG 21266) C. jejuni C 17 Finland marinated broiler meat 

C. coli C 35 The Netherlands Chicken neck skin C. jejuni C 31 Finland marinated broiler meat 

C. hyointestinalis C 30 Ireland poultry C. jejuni C 32 Belgium chicken 

C. jejuni C 01 ATCC 33291 (LMG 18455) C. jejuni C 33 The Netherlands chicken breast skin 

C. jejuni C 03 NCTC 11351 (ATCC 33560) C. jejuni subsp. doylei C 02 NCTC 11951 (LMG 8843) 

C. jejuni C 04 Finland turkey (fecal) C. lari C 26 NCTC 11352 

C. jejuni C 05 The Netherlands chicken legs C. lari C 27 The Netherlands raw oysters 

C. jejuni C 06 The Netherlands poultry C. lari C 28 The Netherlands surface water 

C. jejuni C 07 The Netherlands chicken neck skin C. upsaliensis C 29 NCTC 11541 (LMG 8850) 

C. jejuni C 08 The Netherlands chicken caecum C. upsaliensis C 36 LMG 9124 

C. upsaliensis C 37 CCUG 14913 

23 


MCS, inclusivity results 

● All 19 C. jejuni subsp. jejuni strains showed typical growth on both 

mCCDA and BCCA plates. 

● All 10 C. coli strains showed typical growth on both mCCDA and 

BCCA plates. 

● The C. jejuni subsp. doylei strain did not grow on either of the 

plates, as could be expected because of the incubation temperature 

of 41,5 C. 

● 3 C. upsaliensis strains and 1 C. hyointestinalis strain tested, did 

not grow on mCCDA or BCCA. 

● One of the C. lari strains failed to grow on BCCA but did grow on 

mCCDA. The remaining two C. lari strains did grow on both mCCDA 

and BCCA. 

● Note that BCCA plates are intended for counting in particular C. 

jejuni and C. coli. 

24 


MCS, strains for exclusivity 

25 

Strain Ref nr. Origin Source 

Acinetobacter baumannii NS 1 DSM 30007 Urine, type strain 

Acinetobacter calcoaceticus NS 2 The Netherlands Milk 

Aeromonas hydrophila NS 3 ATCC 7966 

Arcobacter butzleri NS 4 The Netherlands Poultry 

Candida albicans NS 5 The Netherlands Poultry meat 

Citrobacter freundii NS 6 Food 

Enterobacter cloacae NS 7 The Netherlands Environment 

Enterococcus faecalis NS 8 NCTC 775 (ATCC 19433) 

Enterococcus hirae NS 9 LMG 6399 Food 

Escherichia coli NS 10 NCTC 9001 (ATCC 11775) 

Escherichia coli NS 11 The Netherlands Poultry meat 

Escherichia coli NS 12 Belgium Poultry meat 

Escherichia coli NS 21 CHEK 3E-0808 

Klebsiella oxytoca NS 13 Cheese 

Proteus mirabilis NS 14 Shampoo 

Pseudomonas aeruginosa NS 15 ATCC 27853 

Saccaromyces cerevisiae NS 16 ATCC 9080 

Salmonella Typhimurium NS 17 The Netherlands Poultry meat 

Shigella flexneri NS 18 ATCC 12022 

Staphylococcus aureus NS 19 ATCC 25923 

Vibrio alginolyticus NS 20 The Netherlands Oysters 


MCS, exclusivity results 

● 19 out of the 21 non-Campylobacter strains gave a negative 

result (no growth at all) using mCCDA and BCCA. 

● The Acinetobacter baumanii strain and one of the four E. coli 

strains (an ESBL-producing isolate) did show growth on both 

mCCDA and BCCA plates. 

● On mCCDA this growth was atypical (white versus grey 

colonies). 

● On BCCA plates the growth was typical (red colonies). 

However, confirmation tests (microscopy, Dryspot, O.B.I.S) 

readily indicated these strains not to be Campylobacter. 

26 


The Method Comparison Study conclusions are: 

● There is good agreement between the reference method and the 

alternative method: there is no significant bias between the 

methods and the relation between the methods does not deviate 

significantly from linearity. 

● For the alternative method the detection limit LOD = 3 

colonies/plate. The quantification limit LOQ = 10 colonies/plate. 

● The alternative method, particularly intended for enumeration of C. 

jejuni and C. coli, is selective and specific, as concluded from the 

inclusivity and exclusivity results. 

● These conclusions are valid for the alternative method, using either 

the ISO 10272-2:2006 tests or the O.B.I.S Campy test or the 

Dryspot Campylobacter test for the required confirmation. 

● Note: It may be that in chicken thighs BCCA plates give a lower 

yield than mCCDA plates. 

27 


Interlaboratory study 

● At least 8 collaborative laboratories within 3 (European) countries 

Country Laboratory Contact person 

Belgium Labo voor Levensmiddelenmicrobiologie en conservering, Uni Gent Mrs. A. De Loy-Hendrickx 

Belgium Institute of public health Mrs. N. Botteldoorn 

Belgium Dept. Veterinary Public Health and Food Safety, Uni Gent Mrs. M. Boonaert 

Finland University of Helsinki, Ruralia Institute/EVIRA Mrs. U. Lyhs 

France ADRIA Développement Mrs. M. Rannou 

Germany WESSLING Laboratorien GmbH Mrs. B. Konermann 

Italy Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G.Caporale" Mrs. Prencipe 

Italy Instituto Zooprofilattico Sperimentale Regioni Lazio e Toscane Mrs. Flores Rodas 

Italy Agricola Tre Valli – Laboratorio Dr. P. Spezie 

Sweden National Veterinary Institute; SVA Mrs. I. Hansson 

The Netherlands RIVM-LZO Mrs. W. van Overbeek 

The Netherlands Central Veterinary Institute of Wageningen UR Mrs. R. van der Hulst-van Arkel 

The Netherlands Silliker Netherlands BV Mr. M. van Eck 

The Netherlands VWA Mr. E. de Boer 

The Netherlands Plukon Poultry Mr. F. Nijboer 

The Netherlands CCL-Nutricontrol Mr. P. Enthoven 

The Netherlands Laboratorium Heijs de Vries Mr. A. van den Bosch 

United Kingdom Bernard Matthewsfarms Ltd, Northsite Laboratory Mrs. K. Holmes 

28 


Interlaboratory Study 

● Detailed instructions for the collaborative laboratories were emailed 

by the EL to the participants on the 18th of May. 

● The laboratories were asked to examine 8 blindly coded chicken 

minced meat samples of 10 gram each on the number of 

Campylobacter spp. by both the reference and the alternative 

method, including confirmations. 

● Also, the laboratories were asked to test 1 additional blank 10 gram 

chicken minced meat sample on presence of natural background 

flora by using ISO 4388 (pour plating in PCA, 3 days at 30 C). 

● All plating media, diluents, and reagents for confirmations were 

provided to the labs by Oxoid. 

29 


ILS, sample preparation 

● Chicken minced meat 

purchased at local 

supermarket. 

● Ten samples of 10 gram each 

of the batch of chicken 

minced meat to be used for 

the ILS were tested for 

presence and numbers of 

Campylobacter by both ISO 

10272-1 and -2. 

30 

– Campylobacter was not 

detected in those samples. 



● ILS samples: 

31 

– 10 gram portions 

– Individually inoculated with 1 ml of inoculation suspension 

– C. jejuni strain C145 



32 

● Intended levels of contamination were: 

In cfu/gram In log 10 cfu/gram 

Level 0 (Blank): < 100 < 2,0 

Level 1 (Low): 2500 3,4 

Level 2 (Medium): 50.000 4,7 

Level 3 (High): 1.000.000 6,0 


ILS, shipment of samples 

● Transport of samples was 

under refrigerating 

conditions (isolating boxes 

with ice packs), monitored 

by means of a temperature 

probe. 

● Each laboratory also 

received a “Temp Control” 

vial containing 10 ml of 

water, for a temperature 

measurement at actual 

time of receipt. 

33 



● Samples were transported to 

each of the collaborative 

laboratories on Monday 31 May 

2010. 

● Starting of the analysis was 

intended to be on Tuesday 1 

June 2010. 

● Italy has a national holiday on 

the 2nd of June! 

34 



● Sample receipt form 

● To be returned as soon as the samples arrived 

35 

– Laboratory Name: 

– Contact person: 

– e-mail: 

– Arrival date of samples: 

– Arrival time of samples: 

– Condition of the package and the samples at receipt: 

– Temperature of vial “Temp. Control” at receipt: 

● N.B. Please also send back the temperature probe (without the 

tube) in the provided return-envelope as soon as possible. 


36 

Laboratory Date of 

receipt 

Time of 

receipt 

Temperature of 

water vial upon 

arrival (°C) 

Maximum 

temperature 

iButton during 

transport (°C) 

Day of 

starting 

analysis 

TVC result 

(log10 cfu/g) 

A 1-6-2010 14:00 2,1 2,0 1 5,5 

B 1-6-2010 15:05 5,0 2,0 1 5,8 

C 2-6-2010 12:50 3,8 1,5 2 5,8 

D 3-6-2010 12:00 15,2 14,0 3 > 6,5 

E 2-6-2010 9:55 3,3 1,0 2 6,0 

F 1-6-2010 7:35 4,9 8,0 1 5,9 

G 1-6-2010 13:10 3,6 3,5 1 6,1 

H 1-6-2010 10:55 7,7 7,0 1 5,8 

I 1-6-2010 7:30 5 7,0 1 6,2 

J 1-6-2010 16:00 10 1,5 1 6,1 

K 1-6-2010 8.15 8,0 7,0 1 6,2 

L 1-6-2010 10:00 2,2 1,5 1 5,9 

M 1-6-2010 13:20 19,1 7,0 1 6,1 

N 3-6-2010 10:30 12,1 9,5 3 6,2 

O 1-6-2010 12:45 8,4 7,0 1 6,3 

P 1-6-2010 14:25 4,4 2,5 1 5,9 

Q 3-6-2010 nt nt 

EL 1-6-2010 7:30 5,0 2,0 1 5,7 

Average: 6,0 


ILS, Homogeneity and stability of samples 

● The homogeneity, stability and the level of contamination of the 

prepared samples was tested in 10-fold per level of contamination 

on 

37 

– Monday 30 May (day of preparation = day 0), 

– Tuesday 1 June (day 1), 

– Wednesday 2 June (day 2). 


● No significant trends were seen in the homogeneity and stability 

results. Stability data on day 0 and day 1 show very much the same 

results. For day 2 there is a possibility of some deterioration which 

may cause some additional dispersion. 

38 


Counting colonies on mCCDA and BCCA plates 

39 


ILS, couting results returned by the labs 

40 


ILS, confirmation results returned by the labs 

41 


ILS, confirmation results 

● 12 out of the 17 labs 

reported (slight) 

growth of isolates at 

41,5C aerobically. 

● “Officially”, this 

should not happen. 

● Data brought in for 

consideration in the 

revision of ISO-10272 

confirmations. 

42 


Summary of results, expressed in log 10 cfu/gram. 

Blank Low concentration Medium concentration High concentration 

Reference Alternative Reference Alternative Reference Alternative Reference Alternative 

Lab C3 C8 C3 C8 C4 C7 C4 C7 C1 C6 C1 C6 C2 C5 C2 C5 

A < 2,0 < 2,0 < 2,0 < 2,0 3,65 3,74 3,64 3,86 4,75 4,92 4,88 4,85 6,13 6,04 6,14 6,13 

B < 2,0 < 2,0 < 2,0 < 2,0 3,18 3,19 3,21 3,71 4,00 4,31 4,49 4,85 5,06 5,54 5,15 5,79 

C < 2,0 < 2,0 < 2,0 < 2,0 3,78 3,48 3,75 3,62 4,62 4,84 4,66 4,80 5,75 6,03 6,08 6,07 

D* < 2,0 < 2,0 < 2,0 < 2,0 2,50 < 2,0 2,86 2,61 4,46 4,54 4,62 4,51 5,88 5,92 5,96 5,93 

E < 2,0 < 2,0 < 2,0 < 2,0 3,53 3,72 3,73 3,59 4,89 4,84 4,92 4,83 6,16 6,14 6,15 6,08 

F < 2,0 < 2,0 < 2,0 < 2,0 3,73 3,76 3,68 3,88 4,81 4,85 4,94 4,96 6,13 6,22 6,19 6,23 

G < 2,0 < 2,0 < 2,0 < 2,0 3,46 3,65 3,78 3,48 4,76 4,87 4,82 4,83 6,07 6,16 6,28 6,27 

H < 2,0 < 2,0 < 2,0 < 2,0 3,56 3,42 3,78 3,64 4,57 4,66 4,79 4,75 5,82 5,86 6,15 6,11 

I < 2,0 < 2,0 < 2,0 < 2,0 2,74 2,66 3,30 2,86 4,48 4,63 4,97 4,94 6,26 5,79 6,11 5,71 

J < 2,0 < 2,0 < 2,0 < 2,0 < 2,0 2,50 2,36 2,89 2,96 3,80 2,66 3,13 5,23 4,30 4,98 4,81 

K < 2,0 < 2,0 < 2,0 < 2,0 2,48 3,06 2,88 3,00 4,81 3,10 5,04 3,56 3,73 4,73 3,36 5,30 

L < 2,0 < 2,0 < 2,0 < 2,0 3,88 3,81 3,88 3,89 4,92 4,91 4,91 5,00 5,98 5,83 6,05 6,26 

M < 2,0 < 2,0 < 2,0 < 2,0 3,60 3,72 3,73 3,90 4,68 4,24 4,87 4,83 6,07 5,71 6,25 6,07 

N* < 2,0 < 2,0 < 2,0 < 2,0 2,56 3,21 3,30 3,63 4,70 4,65 4,74 4,72 6,04 6,04 6,22 6,04 

O < 2,0 < 2,0 < 2,0 < 2,0 3,64 3,70 3,84 3,77 4,79 4,61 4,89 4,60 5,92 5,94 6,09 6,00 

P* < 2,0 < 2,0 < 2,0 < 2,0 3,62 2,66 3,70 3,42 4,66 4,15 4,35 4,65 4,88 5,67 5,79 5,02 

EL < 2,0 < 2,0 < 2,0 < 2,0 3,16 3,50 3,34 3,82 4,29 4,20 4,37 4,74 5,77 5,23 6,02 5,92 

43 


ILS, Scrutiny of the results for consistency 

The Mandel‟s h-statistics represent the between-laboratory 

consistency 

CCDA: 

BCCA: 

44 

4 

2 

0 

0 

-2 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 

-4 

-6 

-8 

3 

1 

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 

-3 

-5 

-7 



The Mandel‟s k-statistics represents the within-laboratory consistency. 

CCDA: 

BCCA: 

45 

8 

7 

6 

5 

4 

3 

2 

1 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 

10 

8 

6 

4 

2 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 



● Examination of the Mandel‟s h values and the Mandel‟s k values 

clearly shows that laboratories J and K are markedly different from 

the other participating laboratories. This means that these 

laboratories show both systematic deviations as well as large 

differences between the replicates. 

● The other participators show stable results. 

● Because of the robust statistics applied, laboratories J and K have 

not been removed from the data set, notwithstanding their 

deviating results. After contacting both laboratories, there was still 

no sound microbiological information to do so. 

46 


ILS, statistical analysis on the results 

● Calculations were according to section 6.3.4 of Amendment 1 of ISO 

16140:2003/2009. 

● The results of the statistical analysis on 14 datasets are given 

below. 

Contamination 

Level 

47 

Number of 

samples 

analysed 

Number of 

samples 

evaluated Median 

Reference method Alternative method 

s.d. 

Repeatabilty 

S r 

s.d. 

Reproducibility 

S R 

Median 

s.d. 

Repeatabilty 

S r 

s.d. 


S R 

Low 34 28 3,59 0,17 0,30 3,67 0,24 0,27 0,08 

Medium 34 28 4,66 0,19 0,36 4,80 0,13 0,20 0,14 

High 34 28 5,90 0,26 0,38 6,09 0,14 0,21 0,19 

Wilma Jacobs, EU-RL Campylobacter Workshop October 2010 

D (bias)

ILS, statistical analysis of the results 

● The bias of the alternative method with respect to the reference 

method for each level, Dj , is calculated as described in section 

6.3.6.1 of amendment 1 of ISO 16140:2003/2009. 

● At all three levels the alternative method BCCA is not significantly 

biased with respect to the reference method mCCDA. 

48 

Level (j) j 

D j, 

0. 

95 

D Conclusion 

low 0,14 0,35 D 1 D1, 

0. 

95 not biased 

medium 0,13 0,29 D 2 D2, 

0. 

95 not biased 

high 0,17 0,26 D 3 D3, 

0. 

95 not biased 



● Comparison of the repeatability standard deviations 

● Comparison of the reproducibility standard deviations 

● If the ratio of the repeatability or reproducibility standard deviations 

of the alternative method and the reference method is larger than 

2, the precision under repeatability or reproducibility conditions of 

the alternative method is considered to be lower than that of the 

reference method. 

● If this ratio is smaller than 0,5, the precision under repeatability or 

reproducibility conditions of the alternative method is considered to 

be greater than that of the reference method. 

49 



Contamination 

Level 

● None of the repeatability or reproducibility ratios is < 0,5 or >2. 

● The differences between the repeatability standard deviations and 

the reproducibility standard deviations of both methods are not 

considered as significant. 

50 

Reference method Alternative method 

s.d. 

Repeatabilty 

S r 

s.d. 


S R 

s.d. 

Repeatabilty 

S r 

s.d. 


S R 

Ratio 

Repeatability 

Ratio 


Low 0,17 0,30 0,24 0,27 1,39 0,90 

Medium 0,19 0,36 0,13 0,20 0,68 0,56 

High 0,26 0,38 0,14 0,21 0,54 0,56 


The Interlaboratory Study conclusions are: 

● The alternative method is not biased with respect to the reference 

method. 

● The repeatability standard deviations of both methods are not 

considered as significantly different. The average repeatability 

across levels is 0,21 (reference method) and 0,18 (alternative 

method). 

● The reproducibility standard deviations of both methods are not 

considered as significantly different. The average reproducibility 

across levels is 0,35 (reference method) and 0,23 (alternative 

method). 

● These conclusions are valid for the alternative method, using either 

the ISO 10272-2:2006 tests or the O.B.I.S Campy test or the 

Dryspot Campylobacter test for the required confirmation. 

51 


Overall Conclusion: 

● The method comparison study and the interlaboratory study showed 

comparable results for the Brilliance CampyCount Agar count 

method and the ISO/TS 10272-2:2006 mCCDA agar count method 

for the enumeration of Campylobacter spp. in poultry products. 

● This conclusion is valid for the alternative method, using either the 

ISO 10272-2:2006 tests or the O.B.I.S Campy test or the Dryspot 

Campylobacter test for the required confirmations. 

● Certificate will be on www.microval.org by the end of this week. 

52 


Any questions? 

53 

wilma.jacobs@wur.nl 

Nowadays: 

wilma.jacobs@rivm.nl 

Wilma Jacobs, EU-RL Campylobacter Workshop 2010

List of abbreviations 

● MV MicroVal 

● MGC MicroVal General Committee 

● MCB MicroVal Certification Body 

● EL Expert Laboratory 

● MR Method Reviewer 

● MV TC MicroVal Technical Committee 

● MCS Method Comparison Study 

● ILS Interlaboratory Study 

● BCCA Brilliance CampyCount Agar 

● COS COlumbia Sheep blood agar 

● mCCDA modified Charcoal Cefoperazone Deoxycholate Agar 

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MicroVal validation of a Campylobacter enumeration medium - SVA

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