ACCREDITATION N 1-0144 PORTEE DISPONIBLE SUR WWW.COFRAC.FR Bio-Rad 3 boulevard Raymond Poincaré 92430 MARNES-LA-COQUETTE NF VALIDATION Validation study according to the EN ISO 16140 standard Summary report EN ISO 16140 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. 29196 QUIMPER Cedex - Tél. (33) 02.98.10.18.18 - Fax (33) 02.98.10.18.08 E-mail : adria.developpement@adria.tm.fr - Site web : http://www.adria.tm.fr ASSOCIATION LOI DE 1901 - N SIRET 306 964 271 00036 - N EXISTENCE 532900006329 - N TVA FR4530696427100036
1 INTRODUCTION 5 2 METHODS PROTOCOLS 5 2.1 Reference method 5 2.2 Alternative method 5 3 METHOD COMPARISON STUDY 7 3.1 Linearity study 7 3.1.1 Food matrices and protocols 7 3.1.2 Calculations and interpretations 7 3.1.3 Statistical results 9 3.1.4 Conclusion 11 3.2 Relative accuracy 11 3.2.1 Number and nature of samples 11 3.2.2 Artificial contaminations 13 3.2.3 Results 15 3.2.4 Statistical interpretation 18 3.2.5 Confirmatory tests 21 3.3 Detection limit (LOD) and quantification limit (LOQ) 23 2.3.1. Protocol 23 2.3.2. Results 23 3.4 Specificity Selectivity 25 3.4.1 Protocol 25 3.4.2 Results 25 3.4.3 Conclusion 25 3.5 Practicability 27 4 INTERLABORATORY STUDY ORGANISATION AND RESULTS 29 4.1 Study organisation 29 4.2 Verification of experimental parameters 30 4.2.1 Strain stability during transport 30 4.2.2 Logistic conditions 31 4.2.3 Quality Controls and conclusion 32 4.3 Aerobic mesophilic flora enumeration 33 4.4 Statistical interpretations and calculations 33 4.4.1 Scrutiny of the measurement results for consistency 36 4.4.2 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
Appendix 1 Flow diagram of the reference method test procedure: ISO/TS 10272-2: 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
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 16140 and the AFNOR technical rules. Company: BIO-RAD 3 boulevard Raymond Poincaré F-92430 MARNES-LA-COQUETTE Expert Laboratory: ADRIA Développement ZA Creac h Gwen F- 29196 QUIMPER Cedex Studied method : RAPID Campylobacter for Campylobacter spp. enumeration Validation standard : ISO 16140 (October 2003) : Food microbiology Protocol for the validation of alternative methods Reference method : ISO/TS 10272-2: 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
1 INTRODUCTION The RAPID Campylobacter method for Campylobacter spp. enumeration was validated according to the EN ISO 16140 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 10272-2: horizontal method for detection and enumeration of Campylobacter spp. Part 2: Colony count technique. The protocol of the method is given in Appendix 1. 2.2 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
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 10272-2 reference method on typical colonies according to this scheme: Characteristic colonies on RAPID Campylobacter Confirmatory tests of the ISO/TS 10272-2 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, 255-67 ADRIA Développement 6/74 May 30, 2014
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. 3.1.1 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 50-100 Campylobacter jejuni Ad 1021 10 2-5 10 2 isolated from poultry 5 10 2-10 3 Campylobacter coli Ad 1087 10 3-5 10 3 isolated from poultry 5 10 3-10 4 slaughterhouse The raw data are provided in Appendix 3. 3.1.2 Calculations and interpretations The bi-dimensional graphs are given in Figure 1. ADRIA Développement 7/74 May 30, 2014
log(alternative method) log(alternative method) log(alternative method) Bio-Rad Figure 1 Linearity: bi-dimensional graphs Pork meat-incubation Pork meat time 48H 5 4 3 2 1 0 0 1 2 3 4 5 log(reference method) Chicken meat-incubation Chicken meat time 48H 5 4 3 2 1 0 0 1 2 3 4 5 log(reference method) Process water-incubation Process time 48H 5 4 3 2 1 0 0 1 2 3 4 5 log(reference method) ADRIA Développement 8/74 May 30, 2014
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 OLS1 1.339 2.447 36 0.985 Log Alt. = 0942 log Réf. + 0.241 0.24 OLS2 0.000 2.447 100 0.953 Log Ref. = 1.285 log Alt. - 0.774 Process water 1.87 GMFR 17.857 2.447 0 0.972 Log Alt = 1.106 log Ref. - 0.016 The regression lines are provided in Figure 2. ADRIA Développement 9/74 May 30, 2014
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
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 = 1.106 log Ref. - 0.016 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. 2 1 2,04 1,60 2,00 2,04 4 3,20 3,66 3,00 3,30 5 3,87 3,53 3,30 3,28 3.2 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). 3.2.1 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
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 29 14 22 9 Carcass samples 7 2 Cooked meat products 22 6 Total 51 17 Wastes 5 2 Environmental testing in food production sites Equipment/production environment 15 6 Water used in the manufacturing process 20 3 Total 40 11 Total 120 42 120 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: 3524. 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
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 6 1 23 13 28 7 23 10 31 11 9 0 Total 65 19 55 23 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., 2011. J. Appl. Microbiol. 9, 255-267 ADRIA Développement 13/74 May 30, 2014
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
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
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
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
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 14 1.18 GMFR 0.391 0.962 0.879 0.979 2.179 35 36 17 0.68 GMFR 0.197 0.951 0.933 1.025 2.131 32 36 11 0.54 GMFR 0.276 1.167 0.938 0.935 0.262 37 27 All products 42 0.96 GMFR 0.224 1.551 0.935 1.503 2.021 14 13 Category Meat and meat products (except poultry) Bias D (median) Reference method Repeatability limit Alternative method 0.042 0.254 0.306 Poultry and poultry products 0.017 0.450 0.314 Environmental samples 0.040 0.367 0.201 All products 0.035 0.302 0.295 The P % were accepted in all cases. The bias varies from 0.017 to 0.042 log CFU/g. The repeatability limit varies from 0.201 to 0.314 log CFU/g for the alternative method and from 0.254 to 0.450 log CFU/g for the reference method. For all products, the regression curve equation is: log Alt. = 0.935 log Ref. + 0.224 ADRIA Développement 18/74 May 30, 2014
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 14 1.18 GMFR 0.391 0.962 0.879 0.979 2.179 35 36 17 0.63 GMFR 0.211 1.013 0.930 1.063 2.131 30 33 11 0.54 GMFR 0.277 1.178 0.939 0.936 0.262 37 27 All products 42 0.94 GMFR 0.232 1.599 0.933 1.535 2.021 13 12 Category Meat and meat products (except poultry) Bias D (median) Reference method Repeatability limit Alternative method 0.042 0.254 0.306 Poultry and poultry products 0.017 0.450 0.291 Environmental samples 0.042 0.367 0.201 All products 0.037 0.302 0.291 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 0.017 to 0.042 log CFU/g. The repeatability limit varies from 0.201 to 0.306 log CFU/g for the alternative method and from 0.254 to 0.450 log CFU/g for the reference method. For all products, the regression curve equation is: log Alt. = 0.933 log Ref. + 0.232 ADRIA Développement 19/74 May 30, 2014
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
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 3.2.5 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
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) 100 107 165 + (2) + (2) + (2) 1000 66 5 + (2) + (2) + (2) Pseudomonas otitidis / Pandoraea sp / Arcobacter butzleri 3177 Swab (turkey industry) 100 95 + µ 71 + µ - (5) - (5) - (5) 1000 0 0 / / / Pseudomonas otitidis / Pseudomonas spp / Enterococcus gallinarum 3178 Swab (turkey industry) 1000 >150 >150 / / / 10000 29 28 - (5) - (5) - (5) Pseudomonas otitidis / Arcobacter butzleri 3179 Swab (turkey industry) 10 274 96 - (5) - (5) - (5) 100 21 0 - (5) - (5) - (5) Pseudomonas otitidis / Pseudomonas aeruginosa / Enterococcus gallinarum 3180 Turkey wastes 100 73 32 - (5) - (5) - (5) 1000 1 0 - (1) Inconclusive result (1) - (1) Pseudomonas otitidis / Enterococcus faecium ADRIA Développement 22/74 May 30, 2014
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 2.3.1. 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. 2.3.2. 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
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 T1 3 0.548 0.5 LOQ (1ml) 10So + Xo 6.0 T2 5 1.049 1.5 T3 6 1.472 4.5 T4 6 5.574 14.0 0.1 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 5.8 58 T0 0 / / LOD (0.1ml) 3.3 So + Xo 10.1 101 T4 4 2.608 1.5 LOQ (0.1ml) 10So + Xo 27.6 276 T5 6 0.816 1.5 T6 6 5.992 13 The calculated values for the detection and quantification limits are satisfying. ADRIA Développement 24/74 May 30, 2014
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. 3.4.1 Protocol After growth according to appropriate conditions, decimal dilutions were realized and enumerated in duplicate by RAPID'Campylobacter method and ISO/TS 10272-2 standard. Targeted strains and non-targeted strains dilutions were numerated in parallel with a non selective agar (Columbia blood agar or PCA). 3.4.2 Results The raw data are given in the Appendix 7. 3.4.3 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 17512. 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 272-2. ADRIA Développement 25/74 May 30, 2014
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
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 356-4295) and dried supplement (code 356-4296) 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 30 30 Dilution and stomaching 20 20 Dilution and spreading 60 60 Reading 5 5 Total for negative samples analyses 115 115 Total/negative sample 7.7 7.7 Presumptive sample or positive sample Reading 45 45 Latex test / PCR test / 30 Confirmatory test 406 / Total for positive samples 614 185 Total/positive sample 41 12.3 ADRIA Développement 27/74 May 30, 2014
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
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: 100 1 000 CFU/ml, - Level 2: 1 000 10 000 CFU/ml, - Level 3: 10 000 100 000 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
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, 2013. 4.2 Verification of experimental parameters 4.2.1 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 10272-2 (in CFU/g) Day of analysis D0 D1 D2 Inoculation level Campylobacter enumeration (cfu/g) Reference method Alternative method Replicate1 Replicate2 Replicate1 Replicate2 1 1 400 1 400 1 300 1 400 2 13 000 16 000 13 000 9 200 3 95 000 130 0000 75 000 52 000 1 1 000 1 300 1 100 890 2 5 200 3 800 6 200 8 200 3 71 000 40 000 56 000 160 000 1 9 00 1 200 730 790 2 7 400 6 300 8 500 2 500 3 7 800 40 000 70 000 110 000 Aerobic mesophilic numeration(cfu/g) ISO 4833 1.4 10 5 2.4 10 6 7.4 10 6 No evolution of the inoculated strain was observed between Day 0 and Day 2. ADRIA Développement 30/74 May 30, 2014
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 3.0 3.6 Day 1 15H30 B 0.5 2.7 Day 1 11H30 C 1.5 Not communicated Day 1 14H00 D 2.0 3.5 Day 1 11H50 E 2.5 4.0 Day 1 11H10 F 2.5 9.0 Day 1 10H45 G 1.5 3.0 Day 1 16H00 H 2.0 4.3 Day 1 11H40 J 0.5 3.0 Day 1 11H30 K 2.5 8.1 Day 1 09H56 L 1.5 5.9 Day 1 13H00 M 1.0 5.5 Day 1 13H00 N 1.5 4.3 Day 1 12H40 O 2.0? Day 1 12H05 P 1.5 5.2 Day 1 10H15 Q 1.5 3.0 Day 1 11H30 R 1.5 3.8 Day 1 11H00 S 2.3 4.3 Day 1 12H30 T 2.5 Not communicated Day 1 11H00 U 1.5 Not communicated Day 1 15H33 V 8.0 9.4 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
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
4.3 Aerobic mesophilic flora enumeration Depending on the Lab results, the enumeration levels varied from 5.3 10 5 to 7.3 10 8 CFU/g. 4.4 Statistical interpretations and calculations The calculations were realised according to the amendment number 1 of the ISO 16140 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
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
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 <10 1200 1500 1300 1600 7300 12000 8500 11000 100000 100000 54000 85000 D <10 <10 <10 <10 870 1000 460 690 9700 8700 8400 6600 81000 82000 76000 76000 E <10 <10 <10 <10 980 610 1100 860 5900 5600 1700 7200 23000 26000 15000 16000 F <10 <10 <10 <10 640 760 170 250 2500 6500 1400 4800 19000 31000 11000 12000 G <10 <10 <10 <10 680 730 160 260 7700 6000 2500 3400 34000 25000 11000 9100 H <10 <10 <10 <10 1100 790 620 750 4300 9400 6900 7400 75000 100000 41000 41000 J <10 <10 <10 <10 910 740 660 570 4900 4100 1400 1700 14000 51000 13000 34000 K <10 <10 <10 <10 1300 1400 400 490 15000 15000 8200 6800 130000 110000 69000 47000 L <10 <10 <10 <10 2500 3100 850 1200 7100 7200 1600 1700 99000 81000 15000 20000 M <10 <10 <10 <10 130 160 230 270 780 1500 1500 4200 7800 3100 6200 4600 N <10 <10 <10 <10 250 860 350 790 2700 5600 4800 1800 4200 41000 9600 20000 Q <10 <10 <10 <10 1200 1100 660 640 7600 11000 5100 4300 93000 88000 47000 36000 T <10 <10 <10 <10 1800 1500 1700 2700 8400 7700 7500 6600 60000 100000 70000 95000 U <10 <10 <10 <10 1000 1000 810 550 11000 15000 5700 5500 180000 130000 70000 65000 V <10 <10 <10 <10 1200 960 660 750 5000 8100 4300 1800 99000 50000 25000 78000 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 <1.00 3.079 3.176 3.114 3.204 3.863 4.079 3.929 4.041 5.000 5.000 4.732 4.929 D <1.00 <1.00 <1.00 <1.00 2.940 2.785 2.663 2.839 3.987 3.940 3.924 3.820 4.908 4.914 4.881 4.881 E <1.00 <1.00 <1.00 <1.00 2.991 2.881 3.041 2.934 3.771 3.748 3.230 3.857 4.362 4.415 4.176 4.204 F <1.00 <1.00 <1.00 <1.00 2.833 2.863 2.230 2.398 3.398 3.813 3.146 3.681 4.279 4.491 4.041 4.079 G <1.00 <1.00 <1.00 <1.00 3.041 2.898 2.204 2.415 3.886 3.778 3.398 3.531 4.531 4.398 4.041 3.959 H <1.00 <1.00 <1.00 <1.00 3.041 2.898 2.792 2.875 3.633 3.973 3.839 3.869 4.875 5.000 4.613 4.613 J <1.00 <1.00 <1.00 <1.00 2.959 2.869 2.820 2.756 3.690 3.613 3.146 3.230 4.146 4.708 4.114 4.531 K <1.00 <1.00 <1.00 <1.00 3.114 3.146 2.602 2.690 4.176 4.176 3.914 3.833 5.114 5.041 4.839 4.672 L <1.00 <1.00 <1.00 <1.00 3.398 3.491 2.929 3.079 3.851 3.857 3.204 3.230 4.996 4.908 4.176 4.301 M <1.00 <1.00 <1.00 <1.00 2.114 2.204 2.362 2.431 2.892 3.176 3.176 3.623 3.892 3.491 3.792 3.663 N <1.00 <1.00 <1.00 <1.00 2.398 2.934 2.544 2.898 3.431 3.748 3.681 3.255 3.623 4.613 3.982 4.301 Q <1.00 <1.00 <1.00 <1.00 3.079 3.041 2.820 2.806 3.881 4.041 3.708 3.633 4.968 4.944 4.672 4.556 T <1.00 <1.00 <1.00 <1.00 3.255 3.176 3.230 3.431 3.924 3.886 3.875 3.820 4.778 5.000 4.845 4.978 U <1.00 <1.00 <1.00 <1.00 3.000 3.000 2.908 2.740 4.041 4.176 3.756 3.740 5.255 5.114 4.845 4.813 V <1.00 <1.00 <1.00 <1.00 3.079 2.982 2.820 2.875 3.699 3.908 3.633 3.255 4.996 4.699 4.398 4.892 ADRIA Développement 35/74 May 30, 2014
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 4.2.3. 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 16140 standard (2013 DIS Version). ADRIA Développement 36/74 May 30, 2014
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 1 2.970 0.087 0.194 2.813 0.120 0.298 1.382 1.537 2 3.832 0.154 0.261 3.544 0.145 0.277 0.947 1.060 3 4.889 0.188 0.262 4.613 0.139 0.387 0.742 1.478 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 1-0.176 1.82 Non significant bias 2-0.216 2.42 Significant bias 3-0.202 2.51 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 - 0.18 to - 0.22 log CFU/g, which correspond to values usually observed and acceptable. ADRIA Développement 37/74 May 30, 2014
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. 1 0.087 0.244 0.120 0.337 1.382 2 0.154 0.430 0.145 0.407 0.847 3 0.188 0.525 0.139 0.390 0.742 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
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. 1 0.194 0.543 0.298 0.835 1.537 2 0.261 0.732 0.277 0.776 1.060 3 0.262 0.732 0.387 1.083 1.478 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 - 0.18 to - 0.22 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
Appendix 1 Flow diagram of the reference method test procedure: ISO/TS 10272-2: 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 40-48 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
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 10272-2 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 10272-2) after a purification step on Columbia agar. ADRIA Développement 41/74 May 30, 2014
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 10272-2 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 0 0 <10 <10 <1.00 <1.00 10 0 0 <10 <10 <1.00 <1.00 100 0 0 100 0 0 4559 10 14 15 140 150 2.15 2.18 10 13 12 150 130 2.18 2.11 4560 100 1 1 100 3 2 4561 10 41 55 400 530 2.60 2.72 10 51 73 520 700 2.72 2.85 4562 100 3 3 100 6 4 4896 10 137 136 1300 1300 3.11 3.11 100 19 23 1800 2100 3.26 3.32 4897 100 8 5 1000 1 0 4567 100 30 30 2800 2800 3.45 3.45 100 59 34 5800 3300 3.76 3.52 4568 1000 1 1 1000 5 2 4569 1000 30 32 30000 31000 4.48 4.49 1000 28 17 29000 18000 4.46 4.26 4570 10000 3 2 10000 4 3 Control 10 0 0 <10 <10 <1.00 <1.00 10 0 0 <10 <10 <1.00 <1.00 100 0 0 100 0 0 3646 10 12 4 110 40 2.04 1.60 10 11 11 100 110 2.00 2.04 3648 100 0 0 Ne 100 0 1 3652 10 49 47 470 460 2.67 2.66 10 40 51 380 460 2.58 2.66 3653 100 3 3 100 2 0 3657 10 77 65 720 650 2.86 2.81 10 93 105 920 1000 2.96 3.00 3658 100 2 6 100 8 5 3661 100 15 47 1600 4600 3.20 3.66 100 10 22 1000 2000 3.00 3.30 3662 1000 3 4 1000 1 0 3669 100 47 5 4500 successive dilution 3.65 successive dilution 100 20 18 2000 1900 3.30 3.28 3670 1000 3 4 inconsistent result inconsistent result 1000 2 3 4509 100 79 33 7400 3400 3.87 3.53 100 35 22 3700 2500 3.57 3.40 4510 1000 2 4 1000 6 5 Analyses performed according to the COFRAC accreditation ADRIA Développement 42/74 May 30, 2014
Process water Campylobacter coli Ad1087 Bio-Rad ISO 10272-2 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 0 0 <1 <1 <0.00 <0.00 1 0 0 <1 <1 <0.00 <0.00 10 0 0 10 0 0 4178 1 84 79 78 81 1.89 1.91 1 80 164 75 170 1.88 2.23 4179 10 2 10 10 2 22 4791 10 13 10 120 100 2.08 2.00 10 13 10 130 91 2.11 1.96 4792 100 0 1 100 1 0 4180 10 17 13 180 160 2.26 2.20 10 54 53 510 520 2.71 2.72 4181 100 3 5 100 2 4 4182 10 62 52 620 470 2.79 2.67 10 80 69 870 760 2.94 2.88 4183 100 6 0 100 16 15 4184 100 11 18 1100 1700 3.04 3.23 100 35 37 4000 3900 3.60 3.59 4185 1000 1 1 1000 9 6 4186 100 65 66 6500 6500 3.81 3.81 100 90 138 9800 14000 3.99 4.15 4187 1000 7 6 1000 18 11 Analyses performed according to the COFRAC accreditation ADRIA Développement 43/74 May 30, 2014
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 0.51 4051 Escalope de dinde milanaise Cooked meat turkey C. coli Ad1125 Chicken 12d / 4 C 0.51 4052 Filet poulet cuit Cooked meat chicken C. jejuni Ad1000 Turkey neck skin 12d / 4 C 0.51 4053 Aiguillettes poulet au paprika Cooked meat chicken C. jejuni Ad1000 Turkey neck skin 12d / 4 C 0.51 4217 Chiffonnette carcasse porc (avant éviscération) Pork carcass C. coli Ad1481 Pork carcass 6d / 4 C 0.71 4218 Chiffonnette Tablier (atelier porc) Swab (pork industry) C. coli Ad1481 Pork carcass 6d / 4 C 0.71 4219 Chiffonnette pomme jet (atelier porc) Swab (pork industry) C. coli Ad1478 Pork carcass 6d / 4 C 0.49 4220 Eau bac d'échaudage (atelier porc Water process (pork industry) C. coli Ad1478 Pork carcass 6d / 4 C 0.49 4221 Déchets parage (atelier porc) Pork wastes C. coli Ad1480 Pork carcass 6d / 4 C 0.51 4222 Terrine de volaille Pocessed poultry meat product C. coli Ad1125 Chicken 18d / 4 C 1.10 4223 Mousse de canard Pocessed duck meat product C. coli Ad1124 Chicken meat 18d / 4 C 1.97 4224 Blanc de poulet cuit Pocessed chicken meat product C. jejuni Ad1000 Turkey neck skin 18d / 4 C 1.26 4225 Roti dinde aux herbes Pocessed turkey meat product C. jejuni Ad1013 Chicken neck skin 18d / 4 C 1.67 4324 Blanc de poulet cuit Pocessed chicken meat product C. coli Ad1006 Chicken skin 24d / 4 C 1.81 4325 Dés de poulet Pocessed chicken meat product C. coli Ad1017 Turkey meat 24d / 4 C 1.47 4326 Mousse de canard Pocessed duck meat product C. coli Ad1018 Chicken leg 24d / 4 C 1.55 4327 Jambon de dinde Pocessed turkey meat product C. coli Ad1018 Chicken leg 24d / 4 C 1.55 4328 Pâté de volaille Pocessed poultry meat product C. coli Ad1018 Chicken leg 24d / 4 C 1.55 4329 Eau bac échaudage (porc) Water process (pork industry) C. coli Ad1479 Pork carcass 14d / 4 C 0.56 4330 Eau de process (porc) Water process (pork industry) C. coli Ad1481 Pork carcass 14d / 4 C 0.86 4331 Lingette pomme jet surpressé (porc) Swab (pork industry) C. coli Ad1478 Pork carcass 14d / 4 C 0.70 4332 Lingette carcasse porc Swab (pork industry) C. coli Ad1123 Pork meat 14d / 4 C 0.68 4503 Haché de volaille Pocessed poultry meat product C. coli Ad1087 Chicken skin 2d / 4 C 0.35 4504 Terrine de poulet Pocessed chicken meat product C. coli Ad1087 Turkey skin 4d / 4 C 0.48 4505 Haché de poulet grillé Pocessed chicken meat product C. coli Ad1087 Turkey skin 4d / 4 C 0.48 4506 Roti dinde cuit Pocessed turkey meat product C. jejuni Ad1089 Turkey neck skin 4d / 4 C 0.37 4507 Blanc de poulet cuit Pocessed chicken meat product C. jejuni Ad1088 Turkey neck skin 4d / 4 C 0.41 4508 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