Bio-Rad. NF VALIDATION Validation study according to the EN ISO standard. Summary report

Transcription

1 ACCREDITATION N PORTEE DISPONIBLE SUR Bio-Rad 3 boulevard Raymond Poincaré MARNES-LA-COQUETTE NF VALIDATION Validation study according to the EN ISO standard Summary report EN ISO Validation of RAPID Campylobacter for Campylobacter spp. enumeration Quantitative method This report includes 74 pages, with 9 appendixes. Only copies including the totality of this report are authorized. Competences of the laboratory are certified by COFRAC accreditation for the analyses marked with symbol. Version 0 May 30, 2014 ADRIA DEVELOPPEMENT Creac h Gwen - F QUIMPER Cedex - Tél. (33) Fax (33) adria.developpement@adria.tm.fr - Site web : ASSOCIATION LOI DE N SIRET N EXISTENCE N TVA FR

2 1 INTRODUCTION 5 2 METHODS PROTOCOLS Reference method Alternative method 5 3 METHOD COMPARISON STUDY Linearity study Food matrices and protocols Calculations and interpretations Statistical results Conclusion Relative accuracy Number and nature of samples Artificial contaminations Results Statistical interpretation Confirmatory tests Detection limit (LOD) and quantification limit (LOQ) Protocol Results Specificity Selectivity Protocol Results Conclusion Practicability 27 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS Study organisation Verification of experimental parameters Strain stability during transport Logistic conditions Quality Controls and conclusion Aerobic mesophilic flora enumeration Statistical interpretations and calculations Scrutiny of the measurement results for consistency Comparison of the trueness and precision characteristics of the reference method and alternative methods 37 5 CONCLUSION 39 ADRIA Développement 2/74 May 30, 2014

3 Appendix 1 Flow diagram of the reference method test procedure: ISO/TS : horizontal method for detection and enumeration of Campylobacter spp. Part 2: colony count technique 40 Appendix 2 RAPID Campylobacter method: flow diagram of the alternative method 41 Appendix 3 Linearity: raw data 42 Appendix 4 Artificial contaminations 44 Appendix 5 Relative accuracy: raw data and summary table 46 Appendix 6 - Detection limit (LOD) and quantification limit (LOQ) 65 Appendix 7 - Specificity and selectivity 66 Appendix 8 Mandel s graphics 70 Appendix 9 Statistical results 72 ADRIA Développement 3/74 May 30, 2014

4 Before comment Quality assurance documents related to this study can be consulted upon request by Bio-Rad. The technical protocol and the result interpretation were realized according to the EN ISO and the AFNOR technical rules. Company: BIO-RAD 3 boulevard Raymond Poincaré F MARNES-LA-COQUETTE Expert Laboratory: ADRIA Développement ZA Creac h Gwen F QUIMPER Cedex Studied method : RAPID Campylobacter for Campylobacter spp. enumeration Validation standard : ISO (October 2003) : Food microbiology Protocol for the validation of alternative methods Reference method : ISO/TS : horizontal method for detection and enumeration of Campylobacter spp. Part 2: Colony count technique Scope: Meat and meat products Poultry and poultry products Environmental samples Organism certification: AFNOR Certification Analysis performed according to the COFRAC accreditation ADRIA Développement 4/74 May 30, 2014

5 1 INTRODUCTION The RAPID Campylobacter method for Campylobacter spp. enumeration was validated according to the EN ISO protocol and the AFNOR technical rules (Certificate number 07/25-01/14). During the validation study, the following criteria were evaluated: The method comparison study: - the practicability, - the linearity study, - the relative accuracy and sensitivity study, - the determination of the limit of detection & limit of quantification. The inter-laboratory study. 2 METHODS PROTOCOLS 2.1 Reference method The reference method used for this validation was the ISO/TS : horizontal method for detection and enumeration of Campylobacter spp. Part 2: Colony count technique. The protocol of the method is given in Appendix Alternative method The general protocol and the package insert are provided in Appendix 2. The method aims at enumerating thermotolerant Campylobacter strains. A PCR test is proposed to confirm the observed characteristic colonies on RAPID Campylobacter agar after 44 h ± 4 h incubation time in meats, poultry meats and environmental samples. Analysis performed according to the COFRAC accreditation ADRIA Développement 5/74 May 30, 2014

6 A latex test is also proposed to confirm the observed characteristic colonies on the RAPID Campylobacter agar after 44 h ± 4 h incubation time. Both confirmation tests were done on the same characteristic colony. During the validation study, in order to check the performances of the proposed confirmatory protocol, the confirmations were realised with the latex tests and the confirmatory tests described in the ISO/TS reference method on typical colonies according to this scheme: Characteristic colonies on RAPID Campylobacter Confirmatory tests of the ISO/TS reference method after a purification step on Columbia agar (5 colonies) PCR or Campylobacter confirm latex done directly on the observed characteristic colonies (one colony only) Note as well that the characteristic colonies isolated from naturally contaminated samples were identified using the PCR method described by Denis 1 et al. (2001), in order to demonstrate the RAPID Campylobacter recovery performances for various Campylobacter species. At least, in order to improve the method practicability, the storage of the RAPID Campylobacter plates for 72 h at 2-8 C was also tested before reading and confirmation. 1 Denis et al. (2001). J. Appl. Microbiol., 9, ADRIA Développement 6/74 May 30, 2014

7 3 METHOD COMPARISON STUDY 3.1 Linearity study Linearity is the ability of the method when used with a given matrix to give results that are in proportion to the amount of analyte present in the sample, that is an increase in analyte corresponds to a linear or proportional increase in results Food matrices and protocols Three matrix/strain pairs were tested. Five contamination levels were done and five replicates were analyzed per level. The matrices tested, the strains inoculated, the contamination levels and the incubation times tested are listed in the Table 1. Table 1 Food item Pork sausage meat White chicken meat Process water Strain Inoculation levels (CFU/g) Campylobacter coli Ad 1889 isolated from porc carcass Campylobacter jejuni Ad isolated from poultry Campylobacter coli Ad isolated from poultry slaughterhouse The raw data are provided in Appendix Calculations and interpretations The bi-dimensional graphs are given in Figure 1. ADRIA Développement 7/74 May 30, 2014

8 log(alternative method) log(alternative method) log(alternative method) Bio-Rad Figure 1 Linearity: bi-dimensional graphs Pork meat-incubation Pork meat time 48H log(reference method) Chicken meat-incubation Chicken meat time 48H log(reference method) Process water-incubation Process time 48H log(reference method) ADRIA Développement 8/74 May 30, 2014

9 3.1.3 Statistical results The statistical interpretation results obtained per matrix are presented in the Table 2. Table 2 Statistical interpretations Matrix R Selected Rob. F Critical P% Correlation Regression equation regression value coefficient Pork sausage meat White chicken meat 4.33 OLS Log Alt. = 0942 log Réf OLS Log Ref. = log Alt Process water 1.87 GMFR Log Alt = log Ref The regression lines are provided in Figure 2. ADRIA Développement 9/74 May 30, 2014

10 Alternative Reference Alternative Bio-Rad Figure 2 Linearity: regression lines 5,00 4,00 Pork meat 48H y = 0,942x + 0,241 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 Reference Chicken meat meat 48H 5,00 4,00 y = 1,285x - 0,774 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 Alternative 5,00 4,00 Process Process water water 48H y = 1,106x - 0,016 3,00 2,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 Reference ADRIA Développement 10/74 May 30, 2014

11 3.1.4 Conclusion The lack-of-fit tests conclude to the linearity of the method, except for "process water". Anyway, the calculated correlation coefficient, which is equal to 0.972, as well the equation regression, confirms both the linearity of the alternative method for that specific matrix: Log Alt = log Ref In the chicken meat matrix, while the lack-of-fit tests conclude to the linearity of the observed data, the ordinates are both higher than 0.7 Log UFC/g. This is due to the replicate data dispersion of the reference method at the levels 1, 4 and 5. Levels Reference method Alternative method Rep. 1 Rep. 2 Rep. 1 Rep ,04 1,60 2,00 2,04 4 3,20 3,66 3,00 3,30 5 3,87 3,53 3,30 3, Relative accuracy The relative accuracy is the closeness of agreement between a test result and the accepted reference value. Relative specificity is defined as the degree to which a method is affected (or not) by the other components present in a multi-component sample; that is, it is the ability of the method to measure exactly a given analyte, or its amount, within the sample without interference from non-target components such as matrix effect or background noise. Relative sensitivity is defined as the ability of the alternative method to detect two different amounts of analyte measured by the reference method within a given matrix over the whole measurement range; that is, it is the minimal quantity variation (increase of the analyte concentration x) which gives a significant variation of the measured signal (response y) Number and nature of samples Three categories were tested, with 3 different types minimum per category. The samples were analyzed in duplicate by the compared methods. ADRIA Développement 11/74 May 30, 2014

12 Table 3 - Distribution per tested category and type Food category Food type Number of Number of exploited analyzed samples samples Meat and meat products (except poultry) Poultry and poultry products Fresh meat (unprocessed) under different storage conditions 11 4 Carcass samples 3 2 Processed meat products 15 8 Fresh meat (unprocessed) under different storage conditions Total Carcass samples 7 2 Cooked meat products 22 6 Total Wastes 5 2 Environmental testing in food production sites Equipment/production environment 15 6 Water used in the manufacturing process 20 3 Total Total samples were analyzed; 42 were available for statistical interpretation. For 71 samples among the 78 non-exploited results, no typical or few typical colonies were numerated at the lowest dilution tested for the reference and the alternative methods. No typical colony was observed with the reference method, while typical colonies were numerated on RAPID'Campylobacter plates for the following 5 samples: - Meat samples: 4851, 4853; - Poultry meat samples: 3183, 3525; - Environmental samples: Note that the meat sample 4674, artificially contaminated, showed a difference higher than 0.5 Log UFC/g in favor of the alternative method. For 6 samples, inconsistent successive dilution results were observed for one replicate. This is probably due to the contamination heterogeneity. ADRIA Développement 12/74 May 30, 2014

13 3.2.2 Artificial contaminations 55 samples were artificially contaminated with injured cells from 23 different strains. 23 gave exploitable results. The inoculated strains, the injury applied and injury evaluation are provided in the Appendix 4. The repartition of naturally and artificially contaminated samples analyzed and retained for statistical interpretation is presented in the table 4. Table 4 Category Naturally contaminated Artificially contaminated Meat and meat products (except poultry) Poultry and poultry products Environmental testing in food production sites Analyzed samples Exploited samples Analyzed samples Exploited samples Total For naturally contaminated samples, 2 characteristic colonies from RAPID'Campylobacter were identified using a species specific PCR test 2. The following results were observed (See Table 5). 2 Denis M. et al., J. Appl. Microbiol. 9, ADRIA Développement 13/74 May 30, 2014

14 Table 5 - Identification by PCR Naturally contaminated samples Product Campylobacter identification 3173 Water process (turkey industry) coli / coli 3183 Quail jejuni / jejuni 3184 Chicken wings coli / jejuni 3185 Raw duck meat coli / coli 3513 Siphon water (chicken industry) coli / coli 3515 Process water (chicken industry) coli / coli 3516 Siphon water (chicken industry) jejuni / jejuni 3517 Process water (chicken industry) coli / jejuni 3519 Swab (chicken industry) coli / coli 3520 Swab (chicken industry) coli / coli 3521 Swab (chicken industry) jejuni / jejuni 3522 Swab (chicken industry) coli / coli 3523 Swab (chicken industry) coli / jejuni 3524 Chicken meat cut jejuni / jejuni 3525 Chicken gizzard jejuni / jejuni 3526 Poultry neck skin jejuni / jejuni 3527 Chicken carcass jejuni / jejuni 3768 Chicken carcass jejuni / jejuni 4045 Guinea fowl carcass jejuni / jejuni 4046 Guinea fowl gizzard jejuni / jejuni 4048 Guinea fowl wastes jejuni / jejuni 4049 Water process (guinea fowl industry) jejuni / jejuni 4171 Water Guinea fowl carcass jejuni / jejuni 4172 Water Guinea fowl carcass coli / jejuni 4173 Chicken gizzard coli / jejuni 4174 Poultry neck skin jejuni / jejuni 4175 Guinea fowl neck skin jejuni / jejuni 4216 Pork carcass coli / coli 4929 Swab (poultry industry) coli / coli 4931 Process water (poultry industry) jejuni / jejuni 4932 Process water (poultry industry) jejuni / jejuni 4933 Chicken wastes jejuni / coli 4934 Process water (poultry industry) jejuni / jejuni 4935 Process water (poultry industry) jejuni / jejuni ADRIA Développement 14/74 May 30, 2014

15 3.2.3 Results The raw data are provided in Appendix 5. The samples were analyzed in duplicate by the reference and the alternative methods. The contamination range is presented in the table 6. Table 6 Food category Meat and meat products (except poultry) Contamination level (log CFU/g) 1.60 to 4.20 Poultry and poultry products 1.60 to 5.26 Environmental testing in food production sites 1.60 to 6.04 All products 1.60 to 6.04 The bi-dimensional graphs are given figure 3. ADRIA Développement 15/74 May 30, 2014

16 log (Alternative method) log (Alternative method) log (Alternative method) log (Alternative method) Bio-Rad Figure 3 Relative accuracy: bi-dimensional graphs Incubation time: 44 h ± 4 h at 41.5 C Incubation time: 44 h ± 4 h at 41.5 C + 72 h at 4 C 5,00 Meat and meat products except poultry- Incubation 48H 41,5 C 5,00 Meat and meat products except poultry- Incubation 48H 41.5 C + 72H 4 C 4,00 4,00 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 log (Reference method)) 0,00 0,00 1,00 2,00 3,00 4,00 5,00 log (Reference method)) 6,00 Poultry meat-incubation 48h 41,5 C 6,00 Poultry meat-incubation 48h 41.5 C + 72H 4 C 5,00 5,00 4,00 4,00 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log (Reference method)) 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 log (Reference method)) ADRIA Développement 16/74 May 30, 2014

17 log (Alternative method) log (Alternative method) log (Alternative method) log (Alternative method) Bio-Rad Incubation time: 44 h ± 4 h at 41.5 C Incubation time: 44 h ± 4 h at 41.5 C + 72 h at 4 C 8,00 Environmental samples-incubation 48H 41,5 C 8,00 Environmental samples- Incubation 48H 41,5 C + 72H 4 C 7,00 7,00 6,00 6,00 5,00 5,00 4,00 4,00 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 log (Reference method)) 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 log (Reference method)) All product-incubation 48H 41,5 C All products- 48H + 72H 4 C 8,00 9,00 7,00 8,00 6,00 7,00 5,00 4,00 6,00 5,00 4,00 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 log (Reference method) 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 log (Reference method) ADRIA Développement 17/74 May 30, 2014

18 3.2.4 Statistical interpretation The results of the statistical interpretation are given in the Tables 7 and 8. Table 7 Statistical interpretation Category n R Regression a t(a) b t(b) Critical T Slope P % Intercept Meat and meat products (except poultry) Poultry and poultry products Environmental samples GMFR GMFR GMFR All products GMFR Category Meat and meat products (except poultry) Bias D (median) Reference method Repeatability limit Alternative method Poultry and poultry products Environmental samples All products The P % were accepted in all cases. The bias varies from to log CFU/g. The repeatability limit varies from to log CFU/g for the alternative method and from to log CFU/g for the reference method. For all products, the regression curve equation is: log Alt. = log Ref ADRIA Développement 18/74 May 30, 2014

19 Table 8 Statistical interpretation - 72 h at 2-8 C Category n R Regression a t(a) b t(b) Critical T Slope P % Intercept Meat and meat products (except poultry) Poultry and poultry products Environmental samples GMFR GMFR GMFR All products GMFR Category Meat and meat products (except poultry) Bias D (median) Reference method Repeatability limit Alternative method Poultry and poultry products Environmental samples All products Only slight evolution was observed after RAPID'Campylobacter plate storage for 72 h at 2-8 C. The P % were accepted in all the cases. The bias varies from to log CFU/g. The repeatability limit varies from to log CFU/g for the alternative method and from to log CFU/g for the reference method. For all products, the regression curve equation is: log Alt. = log Ref ADRIA Développement 19/74 May 30, 2014

20 Alternative Alternative Alternative Alternative Alternative Alternative Bio-Rad Regression lines (graph and equation representations) for each food category and for all matrices are presented in Figure 4. Figure 4 Relative accuracy: regression lines Incubation time: 44 h ± 4 h at 41.5 C Incubation time: 44 h ± 4 h at 41.5 C + 72 h at 4 C 5,00 Meat and meat products except poultry Incubation 48H 41,5 c 5,00 Meat and meat products except poultry Incubation 48H 41,5 c + 72H 4 C 4,00 y = 0,879x + 0,390 4,00 y = 0,879x + 0,390 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 Reference 0,00 1,00 2,00 3,00 4,00 5,00 Reference Poultry meat Incubation 48H 41,5 C Poultry meat Incubation 48H 41,5 C + 72H 4 C 6,00 6,00 5,00 y = 0,932x + 0,196 5,00 y = 0,929x + 0,211 4,00 4,00 3,00 3,00 2,00 2,00 1,00 1,00 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Reference 0,00 0,00 1,00 2,00 3,00 4,00 5,00 6,00 Reference 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 Environmental samples Incubation 48H 41,5 C y = 0,938x + 0,275 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 Reference 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 Environmental samples Incubation 48H 41,5 C + 72H 4 C y = 0,938x + 0,277 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 Reference ADRIA Développement 20/74 May 30, 2014

21 Alternative Alternative Bio-Rad Incubation time: 44 h ± 4 h at 41.5 C Incubation time: 44 h ± 4 h at 41.5 C + 72 h at 4 C 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 All products-incubation 48H 41,5 c y = 0,934x + 0,224 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 Reference All products-incubation 48H 41,5 c + 72H 4 C 9,00 8,00 7,00 6,00 5,00 4,00 3,00 2,00 1,00 0,00 y = 0,933x + 0,231 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 Reference Confirmatory tests The following confirmatory tests were applied during the validation study. One colony was confirmed per RAPID'Campylobacter plate by latex test and PCR after 44 h ± 4 h incubation time on one typical colony per plate and ISO tests on five colonies per plate.. All the observed characteristic colonies gave positive results by all the tested confirmation procedures, except in the cases described in the table 9. The colonies were identified by 16S rdna sequencing. ADRIA Développement 21/74 May 30, 2014

22 Table 9 - Strains not identified as Campylobacter spp + µ: microcolonies ( ): number of colonies tested N Sample Product Dilution cfu numerated Rep 1 Rep 2 RAPID Campylobacter Confimatory tests by PCR Campylobacter confirm latex Confimatory tests by ISO tests Identification by 16S rdna sequencing 3173 Water process (turkey industry) (2) + (2) + (2) (2) + (2) + (2) Pseudomonas otitidis / Pandoraea sp / Arcobacter butzleri 3177 Swab (turkey industry) µ 71 + µ - (5) - (5) - (5) / / / Pseudomonas otitidis / Pseudomonas spp / Enterococcus gallinarum 3178 Swab (turkey industry) 1000 >150 >150 / / / (5) - (5) - (5) Pseudomonas otitidis / Arcobacter butzleri 3179 Swab (turkey industry) (5) - (5) - (5) (5) - (5) - (5) Pseudomonas otitidis / Pseudomonas aeruginosa / Enterococcus gallinarum 3180 Turkey wastes (5) - (5) - (5) (1) Inconclusive result (1) - (1) Pseudomonas otitidis / Enterococcus faecium ADRIA Développement 22/74 May 30, 2014

23 3.3 Detection limit (LOD) and quantification limit (LOQ) The critical level is the defined as the smallest amount which can be detected (not null), but not quantified as an exact value. Below this value, it cannot be sure that the true value is not null. The detection limit is defined as being higher than the critical level because it involves a power, the probability 1-, which has to be well over 50 %, for example 95 %. The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of organisms) which can be measured and quantified with defined precision and accuracy under the experimental conditions by the method under validation Protocol The detection limit of the alternative method was done with pure cultures. Four different inoculation levels were tested, with six replicates per level, i.e. 24 analyses were done by the alternative method. Quantification limit was calculated for six independent blank samples determinations Results The raw data are given in the Appendix 6. These data are intrinsic to the alternative method and are presented in the following tables: ADRIA Développement 23/74 May 30, 2014

24 1 ml suspension on 3 plates Table 10 - Summary results Calculation Calculated Inoculation Number of positive Standard Bias X0 formula values (cfu/ml) level samples deviation (S0) (Xoi median) LC (1ml) 1.65 So + Xo 1.4 T0 0 / / LOD (1ml) 3.3 So + Xo 2.3 T LOQ (1ml) 10So + Xo 6.0 T T T ml suspension on 1 plate Calculated Calculated Calculation Inoculation Number of positive Standard Bias X0 values values formula level samples deviation (S0) (Xoi median) (cfu/0.1ml) (cfu/1ml) LC (0.1ml) 1.65 So + Xo T0 0 / / LOD (0.1ml) 3.3 So + Xo T LOQ (0.1ml) 10So + Xo T T The calculated values for the detection and quantification limits are satisfying. ADRIA Développement 24/74 May 30, 2014

25 3.4 Specificity Selectivity The specificity is defined as the degree to which a method is affected (or not) by the other components present in a multi-component sample. That is the ability of a method to measure exactly a given analyte, or its amount, within the sample without interference from non-target components such as a matrix effect, or background noise. The selectivity is defined as a measure of the degree of non-interference in the presence of non-target analytes. A method is selective if it can be used to detect the analyte under examination, and that a guarantee can be provided that the detected signal can only be a product by that specific analyte Protocol After growth according to appropriate conditions, decimal dilutions were realized and enumerated in duplicate by RAPID'Campylobacter method and ISO/TS standard. Targeted strains and non-targeted strains dilutions were numerated in parallel with a non selective agar (Columbia blood agar or PCA) Results The raw data are given in the Appendix Conclusion Inclusivity 35 Campylobacter strains were tested. All gave typical colonies on RAPID'Campylobacter plates. All the strains gave a positive latex test, except 2 Campylobacter lari (Ad 1067 and Ad 1130). Exclusivity Among the 21 non-targeted strains, typical colonies were observed on RAPID'Campylobacter plates for 2 strains: Ralstonia mannitolilytica Ad 1059 and DSM One of them (Ad 1059) gave also typical colonies onto mccda plates. These 2 strains gave an inconclusive result with a clear atypical reaction as show on the following photographies (See figure 5). Six additional Ralstonia mannitolilytica strains were tested and gave the same reaction. Of course, they are easily excluded by using the PCR based confirmation procedure and the ISO/TS ADRIA Développement 25/74 May 30, 2014

26 Figure 5 Agglutination will be atypical i.e. slow to develop (up to 2 minutes), weak (1+ after 2 minutes, ) and atypical in appearance (mucoid/ curd like appearance rather than the normal particulate agglutination with poor background clearing) Campylobacter spp.: Positive agglutination at 30 seconds Campylobacter spp.: Positive agglutination at 2 minutes Ralstonia mannitolilytica. inconclusive result or clear atypical reaction agglutination at 30 seconds. Ralstonia mannitolilytica. inconclusive result or clear atypical reaction agglutination at 2 minutes The alternative method shows satisfying specificity and selectivity. ADRIA Développement 26/74 May 30, 2014

27 3.5 Practicability The alternative method practicability was evaluated according to the AFNOR criteria relative to method comparison study. Packaging and reagents Storage conditions and shelflife Specific equipment Reagents Training Workflow (in minutes) for 15 samples RAPID'Campylobacter media : Dehydrated: x 500 g (code ) and dried supplement (code ) Campylobacter confirm latex: 50 tests RAPID'Campylobacter media powder is stored at room temperature. The expiration date is mentioned on the flask. The supplement is stored at 2-8 C. The expiration date is mentioned on the package. The Campylobacter confirm latex is stored at 2-8 C. The expiration date is mentioned on the box. No specific equipment is required. No specific reagent is needed. No specific training is needed. Steps Reference method Alternative method Negative samples Sampling Dilution and stomaching Dilution and spreading Reading 5 5 Total for negative samples analyses Total/negative sample Presumptive sample or positive sample Reading Latex test / PCR test / 30 Confirmatory test 406 / Total for positive samples Total/positive sample ADRIA Développement 27/74 May 30, 2014

28 Time to result Steps Reference method Alternative method Negative samples Sampling, analyis Day 0 Day 0 Numeration Day 2 Day 2 Final result Day 2 Day 2 Presumptive positive or positive results Sampling, analysis Day 0 Day 0 Numeration Day 2 Day 2 Latex test / Day 2 (Latex and PCR) Confirmatory tests Day 5 - Day 6 / Final result Day 5 - Day 6 Day 2 (Latex and PCR) Technician background The same that needed for the reference method Common step with the Sampling reference method Traceability of the results / Maintenance / ADRIA Développement 28/74 May 30, 2014

29 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS 4.1 Study organisation Collaborators number Samples were sent to 21 laboratories. Matrix and strain used Minced poultry meat samples were inoculated with Campylobacter jejuni Ad 1000, isolated from poultry. The samples were packaged in vacuum conditions, in order to ensure the targeted strain viability and recovery by the tested methods. Inoculation Inoculation levels targeted were: - Level 0: <10 CFU/ml, - Level 1: CFU/ml, - Level 2: CFU/ml, - Level 3: CFU/ml. Each laboratory received eight samples of 10 g, i.e. two samples per inoculation level. Furthermore, one non-inoculated sample was added to the package for total viable count microflora enumeration by the ISO 4833 method. Labelling and shipping Blind coded samples (code is only known by the expert laboratory) were placed in isothermal boxes, which contained cooling blocks, and expressshipped to the different laboratories. A temperature control flask containing temperature register was added to the package in order to register temperature profile during transport and package delivery. Samples were shipped in 48 h to laboratories of the collaborative study. Sample temperature was lower or equal to 8.4 C during transport, and between 0 C 8.4 C at arrival. ADRIA Développement 29/74 May 30, 2014

30 Analyses Collaborative study laboratories and the expert laboratory carried out the analyses with the alternative and reference methods. Strain stability during transport In order to evaluate the used Campylobacter jejuni strain variability during transport, Campylobacter enumerations were performed at different time, i.e. inoculation time, after 24 h and 48 h of storage at 2 C. Expedition conditions The collaborative study instructions were sent on November 25, Verification of experimental parameters Strain stability during transport In order to evaluate the Campylobacter jejuni Ad 1000 strain viability during transport, bacterial count of samples were checked at different times, i.e. inoculation time, after 24 h and 48 h of storage at 4 C. Six samples (3 contamination levels x 2 samples) were enumerated. The results are reported in table 11. Table 11 Campylobacter count with ISO/TS (in CFU/g) Day of analysis D0 D1 D2 Inoculation level Campylobacter enumeration (cfu/g) Reference method Alternative method Replicate1 Replicate2 Replicate1 Replicate Aerobic mesophilic numeration(cfu/g) ISO No evolution of the inoculated strain was observed between Day 0 and Day 2. ADRIA Développement 30/74 May 30, 2014

31 4.2.2 Logistic conditions Temperature conditions are given below: Laboratories Table 12 - Sample temperatures at receipt Temperature measured by the sensor ( C) Temperature measured at receipt ( C) Receipt date and time A Day 1 15H30 B Day 1 11H30 C 1.5 Not communicated Day 1 14H00 D Day 1 11H50 E Day 1 11H10 F Day 1 10H45 G Day 1 16H00 H Day 1 11H40 J Day 1 11H30 K Day 1 09H56 L Day 1 13H00 M Day 1 13H00 N Day 1 12H40 O 2.0? Day 1 12H05 P Day 1 10H15 Q Day 1 11H30 R Day 1 11H00 S Day 1 12H30 T 2.5 Not communicated Day 1 11H00 U 1.5 Not communicated Day 1 15H33 V Day 1 17H34 (3) Even if some labs measured a temperature at receipt above 8.4 C, all the measured temperatures by the probes were correct. 3 Received at Day 2 at 10H00 in the lab. ADRIA Développement 31/74 May 30, 2014

32 4.2.3 Quality Controls and conclusion Some problems were encountered during the ring trial, they are listed below: - Lab A The protocol for the preparation of the RAPID Campylobacter plates was not respected. This lab was not retained for interpretation. - Lab C The RAPID Campylobacter plates for samples C1, C2, C3 and C4 were incubated in the same jar; no colony was numerated on the plates after 24 h incubation time for these samples ONLY. Bad incubation conditions are suspected. This lab was not retained for interpretation. - Lab O This lab didn t realise RAPID Campylobacter enumeration after 44 ± 4 h incubation and didn t follow the instructions to run correctly the confirmatory tests of both tested methods, i.e. the reference and the alternative methods). This lab was not retained for interpretation. - Lab P This lab mentioned a lack of microaerobic conditions during the incubation step. This lab was not retained for interpretation. - Lab R Lab R didn t provide results; the RAPID Campylobacter media was not prepared in good conditions and no colony was numerated on the plates. - Lab S This lab didn t incubate the RAPID Campylobacter plates in microaerobic conditions for the first 4 h of the required incubation time. This lab was not retained for interpretation. Il manqué le laboratoire C Finally, the following labs were retained: Labs Total B, D, E, F, G, H, J, K, L, M, N, Q, T, U, V 15 ADRIA Développement 32/74 May 30, 2014

33 4.3 Aerobic mesophilic flora enumeration Depending on the Lab results, the enumeration levels varied from to CFU/g. 4.4 Statistical interpretations and calculations The calculations were realised according to the amendment number 1 of the ISO standard (2011): Accuracy: closeness of agreement between a measurement result and the accepted reference value. Note: Accuracy refers to a combination of trueness and precision Trueness: closeness of agreement between the expectation of a measurement result and the accepted reference value. Note: the measure of trueness is usually expressed in terms of bias Precision: closeness of agreement between independent measurement results obtained under stipulated conditions. Note: quantitative measures of precision depend critically on the stipulated conditions. Repeatability conditions and reproducibility conditions are particular sets of extreme stipulated conditions Repeatability: precision under repeatability conditions Repeatability conditions: measurement conditions where independent measurement results are obtained with the same method on identical measurement items in the same laboratory by the same operator using the same equipment within short interval of time. Repeatability standard deviation: standard deviation of measurement results obtained under repeatability conditions Repeatability limit (r): value less than or equal to which the absolute difference between two measurement results obtained under repeatability conditions is expected to be with a probability of 95% Reproducibility: precision under reproducibility conditions ADRIA Développement 33/74 May 30, 2014

34 Reproducibility conditions: measurement conditions where measurement results are obtained with the same method on identical measurement items in different laboratories with different operators using different equipment Reproducibility standard deviation: standard deviation of measurement results obtained under reproducibility conditions Reproducibility limit (R): value less than or equal to which the absolute difference between two measurement results obtained under reproducibility conditions is expected to be with a probability of 95% All the available results are summarised in Tables 13 and 14. ADRIA Développement 34/74 May 30, 2014

35 Table 13 Results synthesis (CFU/g) Bio-Rad Level 0 Level 1 Level 2 Level 3 Labs Alternative method Alternative method Alternative method Alternative method Reference method Reference method Reference method Reference method 44 h ± 4 h 44 h ± 4 h 44 h ± 4 h 44 h ± 4 h B <10 <10 <10 < D <10 <10 <10 < E <10 <10 <10 < F <10 <10 <10 < G <10 <10 <10 < H <10 <10 <10 < J <10 <10 <10 < K <10 <10 <10 < L <10 <10 <10 < M <10 <10 <10 < N <10 <10 <10 < Q <10 <10 <10 < T <10 <10 <10 < U <10 <10 <10 < V <10 <10 <10 < Table 14 Results summary (log CFU/g) Level 0 Level 1 Level 2 Level 3 Labs Alternative method Alternative method Alternative method Alternative method Reference method Reference method Reference method Reference method 44 h ± 4 h 44 h ± 4 h 44 h ± 4 h 44 h ± 4 h B <1.00 <1.00 <1.00 < D <1.00 <1.00 <1.00 < E <1.00 <1.00 <1.00 < F <1.00 <1.00 <1.00 < G <1.00 <1.00 <1.00 < H <1.00 <1.00 <1.00 < J <1.00 <1.00 <1.00 < K <1.00 <1.00 <1.00 < L <1.00 <1.00 <1.00 < M <1.00 <1.00 <1.00 < N <1.00 <1.00 <1.00 < Q <1.00 <1.00 <1.00 < T <1.00 <1.00 <1.00 < U <1.00 <1.00 <1.00 < V <1.00 <1.00 <1.00 < ADRIA Développement 35/74 May 30, 2014

36 4.4.1 Scrutiny of the measurement results for consistency In order to identify other measurement results or laboratories that could be inconsistent, two graphical consistency techniques were realised: the robust Mandel s h-ank-statistics. These graphics are given in Appendix 8. All the statistical results are given in Appendix 9. Mandel indicators h and k at 5 % significance highlight some possible inconsistent data: Table 15 Mandel s values Number of values above the threshold Reference method Alternative method h > 1 % Lab M Levels 1, 2 and 3 / Lab N Level 3 h > 5 % Lab E Level 3 Lab M Level 3 Lab F Level 3 Lab J Level 3 Lab L Level 1 Lab M Levels 1, 2 and 3 Lab N Level 3 k> 1 % Lab N Levels 1 and 3 Lab E Level 2 Lab F Level 2 k > 5 % Lab F Level 2 Lab E Level 3 Lab J Level 3 Lab F Level 3 Lab N Levels 1 and 3 Lab J Level 3 Lab M Level 2 Lab N Levels 1 and 2 Lab V Level 3 Some labs were already excluded, as mentioned chapter The exclusions were based on data inconsistency, with clear evidence that a part of the protocols were not respected. Moreover, the heterogeneity within the data generated by the reference method for Campylobacter spp enumeration has been already shown and discussed 4. This heterogeneity is indeed expected for both methods, i.e. the alternative and the reference methods. 4 Stakeholder Panel Meeting held in April 2012 in Brussels for the determination of the Acceptability Limits in the Revised ISO standard (2013 DIS Version). ADRIA Développement 36/74 May 30, 2014

37 4.4.2 Comparison of the trueness and precision characteristics of the reference method and alternative methods The statistical values are summarised hereafter: Table 16 Level Median Reference method Repeatability Reproducibility s.d. s.d. Median Alternative method Repeatability Reproducibility s.d. s.d. Ratios repeatability Ratios reproductibility Bias of the alternative method In order to estimate the bias of the alternative method with respect to the reference method for each level, Dij and t are calculated as described below: Dij Y ij, Alt Y ij, Ref t median i ( Dij) / (2 p) Diff If t is larger than 2, the alternative method is significantly biased with respect to the reference method. The values obtained for t are given in table 17. Table 17 Values obtained for t(d) Level Bias D t (d) Interpretation Non significant bias Significant bias Significant bias According to the statistical tests, the bias is not significant for level 1, and significant for levels 2 and 3. The bias varies between to log CFU/g, which correspond to values usually observed and acceptable. ADRIA Développement 37/74 May 30, 2014

38 Comparison of the repeatability standard deviations If the ratio Srj, Alt / Srj, Ref. of the repeatability standard deviations of the alternative method and the reference method is larger than 2, the precision under repeatability 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 conditions of the alternative method is considered to be greater than that of the reference method. The ratio values are given in table 18. Table 18 Contamination level Reference method Alternative method Ratio Sr Ref. r Ref. Sr Alt. r Alt. Sr Alt. / Sr Ref The ratios of the repeatability standard deviations are comprised below 2 for all the tested contamination levels; the precision under repeatability conditions of the alternative method is equivalent to that of the reference method. Comparison of the reproducibility standard deviations If the ratio Srj, Alt / Srj, Ref. of the reproducibility standard deviations of the alternative method and the reference method is larger than 2, the precision under 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 reproducibility conditions of the alternative method is considered to be greater than that of the reference method. The ratio values are given in table 19. ADRIA Développement 38/74 May 30, 2014

39 Table 19 Contamination level Reference method Alternative method Ratio S R Ref. R Ref. S R Alt. R Alt. S R Alt/S R Ref The ratios of the reproducibility standard deviations are comprised below 2 for the contamination levels; the precision under reproducibility conditions of the alternative method is equivalent to that of the reference method. 5 CONCLUSION The method comparison study conclusions are: The RAPID'Campylobacter method shows satisfying linearity results. Accuracy results are satisfying for the three tested categories (meat, poultry meat and environmental samples). The RAPID'Campylobacter method is specific and selective. Note that some Ralstonia mannitolilytica can show characteristic conlonies on RAPID'Campylobacter. They are excluded by using the PCR base confirmation procedure, and they show an atypical latex reaction. The inter-laboratory study conclusions are: The bias values between the reference and the alternative methods vary between to log CFU/g. The repeatability and reproducibility of the alternative method are clearly similar to the repeatability and the reproducibility of the reference method. ADRIA Développement 39/74 May 30, 2014

40 Appendix 1 Flow diagram of the reference method test procedure: ISO/TS : horizontal method for detection and enumeration of Campylobacter spp. Part 2: colony count technique Meat and waste samples: 10 g test sample + 90 ml peptone-salt Sawbs samples: swab + 90 ml peptone-salt Decimal dilutions in 9 ml peptone-salt tubes 1 ml on 3 mccda plates (for low contamination level) Or 0.1 ml on mccda plates per dilution Incubation in microaerobic conditions h at 41.5 C 1 C Characteristic colonies count Purification on Columbia agar for: - microscopy (morphology and mobility) - oxidase test - aerobic growth at 41,5 C - microaerobic growth at 25 C ADRIA Développement 40/74 May 30, 2014

41 Appendix 2 RAPID Campylobacter method: flow diagram of the alternative method Meat and waste samples: 10 g test sample + 90 ml peptone-salt Sawbs samples: swab + 90 ml peptone-salt Decimal dilutions in 9 ml peptone-salt tubes 1 ml on 3 RAPID Campylobacter plates for low contamination level or 0.1 ml on 1 RAPID Campylobacter plate per dilution Incubation in microaerobic condition at 41.5 C ± 1 C 44 h ± 4 h incubation time Characteristic colonies count Campylobacter confirm latex on one of the observed characteristic colony on the same colony* Direct PCR on one of the observed characteristic colonies* ISO/TS confirmatory tests on 5 colonies per dilution (including the colony tested by PCR and latex tests) Purification on Columbia agar for: - microscopy (morphology and mobility) - oxidase test - aerobic growth at 41,5 C - microaerobic growth at 25 C *During the validation study, the same colony was tested by both confirmation procedures. During the method comparison study, the observed characteristic colonies were also confirmed with the confirmatory tests of the reference method (ISO/TS ) after a purification step on Columbia agar. ADRIA Développement 41/74 May 30, 2014

42 White chicken meat Campylobacter jejuni Ad 1021 Sausage pork meat Campylobacter coli Ad1889 Bio-Rad Appendix 3 Linearity: raw data Sample N Strain inoculated ISO Method RAPID'Campylobacter method Matrix CFU/plate CFU/plate CFU/g log CFU/g Dilution confirmed Dilution confirmed CFU/g log CFU/g Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Rep1 Rep2 Control <10 <10 <1.00 < <10 <10 <1.00 < Control <10 <10 <1.00 < <10 <10 <1.00 < Ne successive dilution 3.65 successive dilution inconsistent result inconsistent result Analyses performed according to the COFRAC accreditation ADRIA Développement 42/74 May 30, 2014

43 Process water Campylobacter coli Ad1087 Bio-Rad ISO Method RAPID'Campylobacter method Sample Strain Matrix N inoculated CFU/plate CFU/plate Dilution CFU/g log CFU/g Dilution CFU/g log CFU/g confirmed confirmed Control <1 <1 <0.00 < <1 <1 <0.00 < Analyses performed according to the COFRAC accreditation ADRIA Développement 43/74 May 30, 2014

44 Appendix 4 Artificial contaminations Artificial contaminations (seeding protocol) Sample N Product (French name) Product Strain Origin Storage Injury measurement (Columbia -RAPID'Campylobacter) 4050 Foie gras Cooked duck liver (foie gras) C. coli Ad1125 Chicken 12d / 4 C Escalope de dinde milanaise Cooked meat turkey C. coli Ad1125 Chicken 12d / 4 C Filet poulet cuit Cooked meat chicken C. jejuni Ad1000 Turkey neck skin 12d / 4 C Aiguillettes poulet au paprika Cooked meat chicken C. jejuni Ad1000 Turkey neck skin 12d / 4 C Chiffonnette carcasse porc (avant éviscération) Pork carcass C. coli Ad1481 Pork carcass 6d / 4 C Chiffonnette Tablier (atelier porc) Swab (pork industry) C. coli Ad1481 Pork carcass 6d / 4 C Chiffonnette pomme jet (atelier porc) Swab (pork industry) C. coli Ad1478 Pork carcass 6d / 4 C Eau bac d'échaudage (atelier porc Water process (pork industry) C. coli Ad1478 Pork carcass 6d / 4 C Déchets parage (atelier porc) Pork wastes C. coli Ad1480 Pork carcass 6d / 4 C Terrine de volaille Pocessed poultry meat product C. coli Ad1125 Chicken 18d / 4 C Mousse de canard Pocessed duck meat product C. coli Ad1124 Chicken meat 18d / 4 C Blanc de poulet cuit Pocessed chicken meat product C. jejuni Ad1000 Turkey neck skin 18d / 4 C Roti dinde aux herbes Pocessed turkey meat product C. jejuni Ad1013 Chicken neck skin 18d / 4 C Blanc de poulet cuit Pocessed chicken meat product C. coli Ad1006 Chicken skin 24d / 4 C Dés de poulet Pocessed chicken meat product C. coli Ad1017 Turkey meat 24d / 4 C Mousse de canard Pocessed duck meat product C. coli Ad1018 Chicken leg 24d / 4 C Jambon de dinde Pocessed turkey meat product C. coli Ad1018 Chicken leg 24d / 4 C Pâté de volaille Pocessed poultry meat product C. coli Ad1018 Chicken leg 24d / 4 C Eau bac échaudage (porc) Water process (pork industry) C. coli Ad1479 Pork carcass 14d / 4 C Eau de process (porc) Water process (pork industry) C. coli Ad1481 Pork carcass 14d / 4 C Lingette pomme jet surpressé (porc) Swab (pork industry) C. coli Ad1478 Pork carcass 14d / 4 C Lingette carcasse porc Swab (pork industry) C. coli Ad1123 Pork meat 14d / 4 C Haché de volaille Pocessed poultry meat product C. coli Ad1087 Chicken skin 2d / 4 C Terrine de poulet Pocessed chicken meat product C. coli Ad1087 Turkey skin 4d / 4 C Haché de poulet grillé Pocessed chicken meat product C. coli Ad1087 Turkey skin 4d / 4 C Roti dinde cuit Pocessed turkey meat product C. jejuni Ad1089 Turkey neck skin 4d / 4 C Blanc de poulet cuit Pocessed chicken meat product C. jejuni Ad1088 Turkey neck skin 4d / 4 C Blanc de dinde cuit Pocessed turkey meat product C. jejuni Ad1084 Turkey neck skin 4d / 4 C 0.39 ADRIA Développement 44/74 May 30, 2014