VALIDATION ‐ Validation of alternative analytical methods Application to food microbiology Summary report – June 2018 – v0 Validation study according to the standard EN ISO 16140‐2 : 2016 VIDAS UP E. coli O157 including H7 BIO 12/25–05/09 for the detection of Escherichia coli O157 Protocol for raw meat, raw milk and raw milk products, raw vegetables and environmental samples Qualitative method CONFIDENTIAL Expert laboratory : ISHA 25 avenue de la République 91300 MASSY ‐ FRANCE Manufacturer: bioMérieux Chemin de l’Orme 69280 MARCY L’ETOILE – France This report contains 83 pages. The reproduction of this report is authorized only in its complete form.
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VALIDATION ‐ Validation of alternative analytical methods Application to food microbiology
Summary report – June 2018 – v0
Validation study according to the standard EN ISO 16140‐2 : 2016
VIDAS UP E. coli O157 including H7 BIO 12/25–05/09
for the detection of Escherichia coli O157
Protocol for raw meat, raw milk and raw milk products, raw vegetables and environmental samples
Qualitative method
CONFIDENTIAL
Expert laboratory : ISHA 25 avenue de la République 91300 MASSY ‐ FRANCE
Manufacturer: bioMérieux Chemin de l’Orme 69280 MARCY L’ETOILE – France
This report contains 83 pages. The reproduction of this report is authorized only in its complete form.
Preamble
Protocol of validation :
EN ISO 16140‐2 (September 2016) : Microbiology of the food chain — Method validation — Part 2 : Protocol for the validation of alternative (proprietary) methods against a reference method.
Complemented by : Requirements regarding comparison and interlaboratory studies for implementation of the standard EN ISO 16140‐2 (version 6).
Reference method:
EN ISO 16654 /A1: June 2017 : Horizontal method for the detection of Escherichia coli O157.
Application scope:
Raw meat, raw milk and raw milk products, raw vegetables and environmental samples.
Table of contents 1. Introduction ................................................................................................................................................. 5
2. Protocols of the methods ............................................................................................................................ 6
2.1. Alternative method ............................................................................................................................. 6
2.1.1. Principle of the alternative method ............................................................................................ 6
2.1.2. Protocol of the alternative method ............................................................................................. 6
2.3. Study design ........................................................................................................................................ 8
3.2.4. Interpretation of the results and statistical analysis ................................................................. 13
3.3. General conclusion ............................................................................................................................ 16
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Definitions
Method comparison study The method comparison study is the part of the validation process that is performed in the organizing laboratory. It consists of three parts namely the following : ‐ A comparative study of the results of the reference method to the results of the alternative method in (naturally and/or artificially) contaminated samples (so‐called sensitivity study); ‐ A comparative study to determine the relative level of detection (RLOD) in artificially contaminated samples (so‐called RLOD study); ‐ An inclusivity/exclusivity study of the alternative method.
Sensitivity study The sensitivity study aims to determine the difference in sensitivity between the reference and the alternative method. The sensitivity is the ability of the reference method or alternative method to detect the analyte.
Relative level of detection study A comparative study is conducted to evaluate the level of detection (LOD) of the alternative method against the reference method. The evaluation is based on the calculation of the relative level of detection (RLOD). The level of detection at 50% (LOD50) is the measured analyte concentration, obtained by a given measurement procedure, for which the probability of detection is 50%. The relative level of detection level of detection at P = 0,50 (LOD50) of the alternative method divided by the level of detection at P = 0,50 (LOD50) of the reference method.
Inclusivity and exclusivity study The inclusivity study is a study involving pure target strains to be detected or enumerated by the alternative method. The exclusivity study is a study involving pure non‐target strains, which can be potentially cross‐reactive, but are not expected to be detected or enumerated by the alternative method.
Interlaboratory study The interlaboratory study is a study performed by multiple laboratories testing identical samples at the same time, the results of which are used to estimate alternative‐method performance parameters. The aim of the interlaboratory study is to determine the difference in sensitivity between the reference and the alternative method when tested by different collaborators using identical samples (reproducibility conditions).
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1. Introduction
This report introduces the results of the renewal study for the AFNOR Certification validation of the method VIDAS UP Escherichia coli O157 including H7 for the detection Escherichia coli O157 in raw meat, raw milk and raw milk products, raw vegetables and environmental samples according to the standard EN ISO 16140‐2 : 2016.
The method VIDAS UP Escherichia coli O157 including H7 for the detection Escherichia coli O157 is validated under the attestation number BIO 12/25–05/09. The validation history of the method is the following:
‐ May 2009: Initial validation for the category “Beef and veal meats (including seasoned meats)” according to the standard ISO 16140 : 2003
‐ December 2009: Extension for other food products and environmental samples ‐ March 2013: First renewal ‐ May 2014 Extension for the addition of a protocol for raw meats of beef and veal ‐ ‐November 2017 Renewal
Results reported in the present report for the assays performed in 2009 were obtained during the validation assays conducted by SERMHA, Institut Pasteur de Lille within the framework of the NF Validation trademark, according to the current requirements.
Assays of the extension in 2014 were performed by ISHA according to the standard ISO 16140/A1 (2011) and to the specific requirements of the Technical Board linked to this standard.
Other assays were performed by ISHA according to the standard ISO 16140‐2 (2016) and to the specific requirements of the Technical Board linked to this standard (Revision 6).
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2. Protocols of the methods
2.1. Alternative method
2.1.1. Principle of the alternative method The VIDAS UP E.coli O157 including H7 (VIDAS ECPT) test is a phage ligand assay which detects Escherichia coli O157 specific receptors using the ELFA (Enzyme Linked Fluorescent Assay) method on the VIDAS automated system.
Each test is composed of two parts: ‐ the Solid Phase Receptacle (SPR) serves as the solid phase as well as the pipeting device. The interior of the SPR is coated with recombinant phage tail fiber protein for the capture of Escherichia coli O157 including H7, ‐ the strip which contains all the ready‐to‐use reagents for the assay: washing buffer, alkaline phosphatase conjugate and substrate.
All the assay steps are performed automatically by the instrument. The reaction medium is cycled in and out of the SPR several times. Part of the enrichment broth is dispensed into the reagent strip. The E. coli O157 including H7 present are captured by the recombinant phage protein coating the interior of the SPR. Unbound sample components are eliminated during the washing steps. Alkaline phosphatase conjugate is then recycled in and out of the SPR and will bind to any E. coli O157 including H7 which are themselves bound to the phage protein on the SPR wall. A final wash step removes unbound conjugate. During the final detection step, the substrate (4‐Methyl‐umbelliferyl phosphate) is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4‐Methyl‐umbelliferone). The intensity of the fluorescence is measured at 450 nm.
At the end of the assay, the results are analyzed automatically by the instrument. A test value, which is compared to stored standards (thresholds) and an interpretation (positive, negative) are generated for each sample. The RFV (Relative Fluorescence Value) is calculated by subtracting the background reading from the final result. The RFV obtained for each sample is interpreted by the VIDAS® system as follows:
Test value (TV) = RFV sample / RFV standard.
if TV < 0.04, the test is negative and if TV > 0.04, the test is positive
2.1.2. Protocol of the alternative method Six protocols are available depending on the categories and the type. These protocols are presented in the table 1 and detailed in appendix 1. The buffered peptone water (BPW) used in all of the enrichments listed in table 1 should be preheated at 41.5 ° C prior to analysis, except for the category raw milk and raw milk products.
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Table 1: protocols of the VIDAS ECPT method
Category (n°)
Type (n°) Protocol Test
portion Dilution
Broth, incubation: time, temperature
Incubation times tested during assays
of:
2009 2014 2017
Raw meat (1)
Raw beef and veal (t1)
(P1) 25g 1/10
BPW +vancomycine 8mg/L (pre‐warmed at 41,5°C), incubation: 16 to 24h at 41,5°C±1°C
15h & 24h
16h 16h & 24h
(P2) 25g 1/10 BPW (pre‐warmed at 41,5°C), incubation: 7 to 24h at 41,5°C±1°C *
6h & 24h
/ 7h & 24h
(P3) 375g 1/4
BPW +vancomycine 8 mg/L (pre‐warmed at 41,5°C), incubation: 8 to 24h at 41,5°C±1°C
8h & 24h
/ 8h & 24h
Seasonned raw beef and veal (t2)
(P1) 25g 1/10
BPW +vancomycine 8 mg/L (pre‐warmed at 41,5°C), incubation: 16 to 24h at 41,5°C±1°C
15h & 24h
16h 16h & 24h
(P2) 25g 1/10 BPW (pre‐warmed at 41,5°C), incubation: 7 to 24h at 41,5°C±1°C
6h & 24h
/ 7h & 24h
Other raw meats (t3)
(P1) 25g 1/10
BPW +vancomycine 8 mg/L (pre‐warmed at 41,5°C), incubation: 16 to 24h at 41,5°C±1°C
15h & 24h
/ /
Raw vegetal products
(2)
Fruits (t1)
(P4) 25g 1/10
BPW +vancomycine 8 mg/L (pre‐warmed at 41,5°C), incubation: 8 to 24h at 41,5°C±1°C
8h & 24h
/ 8h & 24h
Green plants (t2)
Others plants and mix
vegetables (t3)
Raw milk and raw milk products
(3)
Goat and sheep raw milk cheese
(t1)
(P5) 25g 1/10 BPW +acriflavine 10 mg/L, Incubation: 20 to 26h at 41,5°C±1°C
/ / 20h
Raw milks and others raw milk
products (t2)
Cow raw milk cheese
(t3)
Environ‐mental samples
(4)
Process waters (t1)
(P6) 25 g 1/10
BPW + vancomycin 8 mg/L +cefixime 0.0125 mg/L +cefsulodin 10 mg/L (pre‐warmed at 41,5°C), Incubation: 15 to 24h at 41,5°C±1°C
15h & 24h
/ 15h & 24h
Dust and residues (t2)
Surface samples (t3)
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After enrichment, the VIDAS ECPT test is performed on an aliquot of the enriched broth heated for 5±1 minutes at 95‐100°C.
The VIDAS ECPT positive results are confirmed using the following protocols: a) Using the immuno concentration assay, VIDAS ICE, performed from the non‐heated enrichmentbroth, followed by plating onto CT‐SMAC agar and ChromID O157:H7 agar. b) Direct plating of the non‐heated enrichment broth onto CT‐SMAC agar and on CT‐ ChromID O157:H7agar plates,
Note : ChromID O157: H7 agar has been replaced by “ChromID EHEC”. The composition of the two agars is the same.
Typical colonies are confirmed: ‐ by the tests described in the reference method (biochemical confirmation and serology) ‐ from the ChromID O157 :H7 plate, using an O157 latex test performed directly from an isolated colony), ‐ from the CT‐SMAC plate, using an O157 latex test and an API strip, performed directly from an isolated colony
For the short enrichment protocols (7‐8 hours), the kit insert recommends to prolong incubation of the broth to 24 hours before proceeding to confirmation.
2.2. Reference method The reference method used for the validation study is the one described in the standard EN ISO 16654 : 2001: Horizontal method for the detection of Escherichia coli O157.
The analytical workflow of the method is shown in appendix 1.
2.3. Study design It is a unpaired study as the reference and the alternative methods used two different enrichment broths for the first step in the enrichment procedure.
2.4. Application scope The scope of the method concerns the categories: ‐ ① Raw meat products, ‐ ② Raw vegetal products, ‐ ③ Raw milk and raw milk products, ‐ ④ Environmental samples.
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3. Method comparison study
The study was carry out on a diversity of samples and of strains representative of the agri‐food products. This does not constitue an exhaustive list of the different matrices included in the scope. For any comment on the alternative method, you can contact AFNOR Certification by logging on the web page http://nf‐validation.afnor.org/contact‐2/ .
3.1.1. Sensitivity study
3.1.1.1. Protocols used for the study The samples were analyzed by the reference and the alternative method. For the alternative method, the minimum incubation time of the broth was applied for raw milk based products and for 2014 assays on the protocol P1 (cf. table 1). For the other categories, both the minimum and the maximum of the enrichment time were tested.
All positive and discordant samples were confirmed using the following protocol: a) Using the immuno concentration assay, VIDAS ICE, performed from the non‐heated enrichment broth,followed by plating onto CT‐SMAC agar and ChromID O157:H7 agar (“VIDAS ICE + 2 agar media), b) Direct plating of the non‐heated enrichment broth onto CT‐SMAC agar and on CT‐ ChromID O157:H7 agarplates (“direct plating on 2 agar media”).
Typical colonies were confirmed: ‐ by the tests described in the reference method (biochemical confirmation and serology) ‐ from the ChromID O157 :H7 agar, using an O157 latex test performed directly from an isolated colony ‐ from the CT‐SMAC agar, using an O157 latex test and an API strip, performed directly from an isolated colony.
A storage of the enrichment broths for up to 48 hours at 5±3°C (72 hours for raw milk products) before performing the VIDAS ECPT test and the confirmation steps was also tested.
3.1.1.2. Number and nature of the samples
A total of 463 samples were analyzed: 228 from the initial validation study and the extension of 2009, 61 from the extension of 2014 and 174 from the renewal study of 2017. The distribution of the samples is presented in table 2.
Some of the samples of the initial validation study are not taken into account because of a contamination level higher than 5‐10 CFU/test portion. These samples are nevertheless shown in raw results detailed in appendix 3.
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Table 2 : number and nature of the samples analyzed (*: positive by any method at the minimum incubation times with the protocol of confirmationgiving the more positive results)
Total Raw milk and raw milk products 97 60 37 0 37 0 0
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Category Type Protocol Year Number of sample
Level of contamination CFU/sample
Total type
Nega‐tive
Posi‐tive*
NC ≤ 5,0 5‐10 > 10
Environmental samples (4)
1 Process waters
6
2009 19 13 6 0 6 0 0
2017 2 0 2 0 2 0 0
total 21 13 8 0 8 0 0
2 Residues
2009 19 10 9 0 7 2 0
2017 1 0 1 0 1 0 0
total 20 10 10 0 8 2 0
3 Surfaces
2009 15 8 6 0 1 3 2
2017 7 2 6 0 6 0 0
total 22 10 12 0 7 3 2
Total Environment samples 63 33 30 0 23 5 2
Total 463 247 216 1 176 33 6
3.1.1.3. Artificial contaminations of the samples One naturally contaminated sample was analyzed. Spikings or seedings were realized on the 215 other positive samples.
For spikings, several strains were stressed using different treatments and the stress intensity was evaluated (logarithmic difference between enumeration on non selective agar and selective agar, cf. appendix 2). For seedings, bacterial suspensions were calibrated and inoculated in the matrices. The samples so contaminated were stored at 2 – 8° for 48 to 72 hours (cf. appendix 2).
The proportion of naturally and artificially contaminated samples giving positive results is presented in table 3.
Table 3 : proportion of naturally and artificially contaminated samples giving positive results with the protocol of confirmation giving the more positive results
Category
Number and percentage of samples analyzed per contamination levels (CFU/test portion)
≤ 5 (spiking) ≤ 3 (seeding) and naturally contaminated
6 – 10 (spiking) 4 – 10 (seeding)
> 10 Total
Raw Meat (1) 92 22 2 117
Raw Vegetable (2) 24 6 2 32
Raw milk and raw milk products (3) 37 0 0 37
Environmental samples (4)
23 5 2 30
Total 176 (81.5 %) 33 (15.3 %) 6 (2.7 %) 216
3.1.1.4. Results Raw results are presented in appendix 3 by step of validation. For the minimum time of incubation, results from the 2009 study were obtained with an incubation time of 6 hours for the protocol P2 and with an incubation time of 15h for the protocol P1.
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To have the same number of samples and to compare the two time of incubation of the method, the results obtained with the samples tested only with the minimum incubation time were duplicated and noticed on the maximum incubation time. That’s concern the samples tested with protocole P5 and the samples of the category 1‐type 1 and 2 tested with protocol 1 (assay 2014). The following tables present the summary of the results by incubation times and protocols of confirmation.
Table 4 : results of the sensitivity study with the confirmation a) at the minimum and maximum incubation times (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive deviation, PP: presumptive positive
before confirmation, A+/R+ : confirmed positive, A‐/R‐ negative immediately and negative after confirmation of the presumptive positive)
Category Response
Minimum incubation times Maximum incubation times
Reference method
positive (R+)
Reference method
negative (R‐)
Reference method
positive (R+)
Reference method
negative (R‐)
Raw Meat (1)
Alternative method positive (A+)
PA= 88 PD= 14 PA= 94 PD= 16
Alternative method negative (A‐)
ND= 15 incl. 3 PPND
NA= 108 incl. 0 PPNA
ND= 9 incl. 1 PPND
NA= 106 incl. 0 PPNA
Raw Vegetable (2)
Alternative method positive (A+)
PA= 16 PD=6 PA= 20 PD= 13
Alternative method negative (A‐)
ND= 8 incl. 3 PPND
NA= 48 incl. 0 PPNA
ND= 4 incl. 0 PPND
NA= 41 incl. 0 PPNA
Raw milk and raw milk
products (3)
Alternative method positive (A+)
PA= 18 PD= 11 PA= 18 PD= 11
Alternative method negative (A‐)
ND= 8 incl. 0 PPND
NA= 60 incl. 2 PPNA
ND= 8 incl. 0 PPND
NA= 60 incl. 2 PPNA
Environmental samples (4)
Alternative method positive (A+)
PA= 26 PD= 1 PA= 26 PD= 1
Alternative method negative (A‐)
ND= 3 incl. 0 PPND
NA= 33 incl. 0 PPNA
ND= 3 incl. 0 PPND
NA= 33 incl. 0 PPNA
All categories
Alternative method positive (A+)
PA= 148 PD= 32 PA= 158 PD= 41
Alternative method negative (A‐)
ND= 34 incl. 6 PPND
NA= 249 incl. 2 PPNA
ND= 24 incl. 1 PPND
NA= 240 incl. 0 PPNA
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Table 5 : results of the sensitivity study with the confirmation b) at the minimum and maximum incubation times (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive deviation, PP: presumptive positive
before confirmation, A+/R+ : confirmed positive, A‐/R‐ negative immediately and negative after confirmation of the presumptive positive)
Category Response
Minimum incubation times Maximum incubation times
Reference method
positive (R+)
Reference method
negative (R‐)
Reference method
positive (R+)
Reference method
negative (R‐)
Raw Meat (1)
Alternative method positive (A+)
PA= 89 PD= 14 PA= 93 PD= 16
Alternative method negative (A‐)
ND= 14 incl. 3 PPND
NA= 108 incl. 0 PPNA
ND= 10 incl. 2 PPND
NA= 106 incl. 0 PPNA
Raw Vegetable
(2)
Alternative method positive (A+)
PA= 18 PD= 8 PA= 20 PD= 13
Alternative method negative (A‐)
ND= 6 incl. 1 PPND
NA= 46 incl. 0 PPNA
ND= 4 incl. 0 PPND
NA= 41 incl. 0 PPNA
Raw milk and raw milk
products (3)
Alternative method positive (A+)
PA= 18 PD= 11 PA= 18 PD= 11
Alternative method negative (A‐)
ND= 8 incl. 0 PPND
NA= 60 incl. 2 PPNA
ND= 8 incl. 0 PPND
NA= 60 incl. 2 PPNA
Environmental samples
(4)
Alternative method positive (A+)
PA= 26 PD= 1 PA= 26 PD= 1
Alternative method negative (A‐)
ND= 3 incl. 0 PPND
NA= 33 incl. 0 PPNA
ND= 3 incl. 0 PPND
NA= 33 incl. 0 PPNA
All categories
Alternative method positive (A+)
PA= 151 PD= 34 PA= 157 PD=41
Alternative method negative (A‐)
ND= 31 incl. 4 PPND
NA= 247 incl. 2 PPNA
ND= 25 incl. 2 PPND
NA= 240 incl. 0 PPNA
3.1.1.5. Statistical interpretation These results were used to calculate the sensitivity for the alternative method and the reference method and the relative sensitivity (cf. tables 6, 7, 8 and 9).
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Table 6 : values for each category of sensitivity, relative trueness and false positive ratio for the alternative method with the confirmation a) (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive
deviation, PP: presumptive positive before confirmation, SEalt: sensitivity for the alternative method, SEref: sensitivity for the reference method, RT: relative trueness, FPR: false positive ratio for the alternative method) Incubation Category/Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Minimum time
Raw Meat (1) / P1+P2+P3
1 59 82 9 8 158 1 0 88,2% 89,5% 89,2% 1,2%
2 15 18 4 * 5 42 2 0 83,3% 79,2% 78,6% 11,1%
3 14 8 2 1 25 0 0 88,2% 94,1% 88,0% 0,0%
Total 88 108 15 14 225 3 0 87,2% 88,0% 87,1% 2,8%
Raw Vegetable (2) / P4
1 9 12 1 3 25 0 0 92,3% 76,9% 84,0% 0,0%
2 2 13 5 * 0 20 3 0 28,6% 100,0% 75,0% 23,1%
3 5 23 2 3 33 0 0 80,0% 70,0% 84,8% 0,0%
Total 16 48 8 6 78 3 0 73,3% 80,0% 82,1% 6,3%
Raw milk and raw milk products (3) /
P5
1 2 13 4 2 21 0 0 50,0% 75,0% 71,4% 0,0%
2 8 11 2 1 22 0 0 81,8% 90,9% 86,4% 0,0%
3 8 36 2 8 54 0 2 88,9% 55,6% 81,5% 5,6%
Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
Environmental samples (4) / P6
1 7 13 0 1 21 0 0 100,0% 87,5% 95,2% 0,0%
2 10 10 0 0 20 0 0 100,0% 100,0% 100,0% 0,0%
3 9 10 3 0 22 0 0 75,0% 100,0% 86,4% 0,0%
Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All categories Total 148 249 34 32 463 6 2 84,1% 85,0% 85,7% 3,2%
Incubation Category/Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Maximum time
Raw Meat (1) / P1+P2+P3
1 63 80 5 10 158 1 0 93,6% 87,2% 90,5% 1,3%
2 17 18 2 5 42 0 0 91,7% 79,2% 83,3% 0,0%
3 14 8 2 1 25 0 0 88,2% 94,1% 88,0% 0,0%
Total 94 106 9 16 225 1 0 92,4% 86,6% 88,9% 0,9%
Raw Vegetable (2) / P4
1 9 9 1 6 25 0 0 93,8% 62,5% 72,0% 0,0%
2 6 12 1 1 20 0 0 87,5% 87,5% 90,0% 0,0%
3 5 20 2 6 33 0 0 84,6% 53,8% 75,8% 0,0%
Total 20 41 4 13 78 0 0 89,2% 64,9% 78,2% 0,0%
Raw milk and raw milk products (3) /
P5
1 2 13 4 2 21 0 0 50,0% 75,0% 71,4% 0,0%
2 8 11 2 1 22 0 0 81,8% 90,9% 86,4% 0,0%
3 8 36 2 8 54 0 2 88,9% 55,6% 81,5% 5,6%
Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
Environmental samples (4) / P6
1 7 13 0 1 21 0 0 100,0% 87,5% 95,2% 0,0%
2 10 10 0 0 20 0 0 100,0% 100,0% 100,0% 0,0%
3 9 10 3 0 22 0 0 75,0% 100,0% 86,4% 0,0%
Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All categories Total 158 240 24 41 463 1 2 89,2% 81,6% 86,0% 1,3%
* 3 meat samples and 3 vegetable samples were confirmed after re‐incubation of the enrichment broth until24 hours
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Table 7 : values for each category of sensitivity, relative trueness and false positive ratio for the alternative method with the confirmation b) (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive
deviation, PP: presumptive positive before confirmation, SEalt: sensitivity for the alternative method, SEref: sensitivity for the reference method, RT: relative trueness, FPR: false positive ratio for the alternative method)
Incubation Category/Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Minimum time
Raw Meat (1) / P1+P2+P3
1 60 82 8 8 158 1 0 89,5% 89,5% 89,9% 1,2%
2 17 18 2 5 42 0 0 91,7% 79,2% 83,3% 0,0%
3 12 8 4* 1 25 2 0 76,5% 94,1% 80,0% 25,0%
Total 89 108 14 14 225 3 0 88,0% 88,0% 87,6% 2,8%
Raw Vegetable (2) / P4
1 9 10 1 5 25 0 0 93,3% 66,7% 76,0% 0,0%
2 4 13 3 * 0 20 1 0 57,1% 100,0% 85,0% 7,7%
3 5 23 2 3 33 0 0 80,0% 70,0% 84,8% 0,0%
Total 18 46 6 8 78 1 0 81,3% 75,0% 82,1% 2,2%
Raw milk and raw milk products (3) /
P5
1 2 13 4 2 21 0 0 50,0% 75,0% 71,4% 0,0%
2 8 11 2 1 22 0 0 81,8% 90,9% 86,4% 0,0%
3 8 36 2 8 54 0 2 88,9% 55,6% 81,5% 5,6%
Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
Environmental samples (4) / P6
1 7 13 0 1 21 0 0 100,0% 87,5% 95,2% 0,0%
2 10 10 0 0 20 0 0 100,0% 100,0% 100,0% 0,0%
3 9 10 3 0 22 0 0 75,0% 100,0% 86,4% 0,0%
Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All categories Total 151 247 31 34 463 4 2 85,6% 84,3% 86,0% 2,4%
Incubation Category/Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Maximum time
Raw Meat (1) / P1+P2+P3
1 63 80 5 10 158 1 0 93,6% 87,2% 90,5% 1,3%
2 17 18 2 5 42 0 0 91,7% 79,2% 83,3% 0,0%
3 13 8 3 1 25 1 0 82,4% 94,1% 84,0% 12,5%
Total 93 106 10 16 225 2 0 91,6% 86,6% 88,4% 1,9%
Raw Vegetable (2) / P4
1 9 9 1 6 25 0 0 93,8% 62,5% 72,0% 0,0%
2 6 12 1 1 20 0 0 87,5% 87,5% 90,0% 0,0%
3 5 20 2 6 33 0 0 84,6% 53,8% 75,8% 0,0%
Total 20 41 4 13 78 0 0 89,2% 64,9% 78,2% 0,0%
Raw milk and raw milk products (3) /
P5
1 2 13 4 2 21 0 0 50,0% 75,0% 71,4% 0,0%
2 8 11 2 1 22 0 0 81,8% 90,9% 86,4% 0,0%
3 8 36 2 8 54 0 2 88,9% 55,6% 81,5% 5,6%
Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
Environmental samples (4) / P6
1 7 13 0 1 21 0 0 100,0% 87,5% 95,2% 0,0%
2 10 10 0 0 20 0 0 100,0% 100,0% 100,0% 0,0%
3 9 10 3 0 22 0 0 75,0% 100,0% 86,4% 0,0%
Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All categories Total 157 240 25 41 463 2 2 88,8% 81,6% 85,7% 1,7%
* 1 meat sample and 1 vegetable were confirmed after re‐incubation of the enrichment broth until 24 hours
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Table 8 : values for each protocol of sensitivity, relative trueness and false positive ratio for the alternative method with the confirmation a) (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive
deviation, PP: presumptive positive before confirmation, SEalt: sensitivity for the alternative method, SEref: sensitivity for the reference method, RT: relative trueness, FPR: false positive ratio for the alternative method)
Incubation Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Minimum time
P1 Total 43 44 6 8 101 0 0 89,5% 86,0% 86,1% 0,0%
P2 Total 25 31 4 1 61 3 0 86,7% 96,7% 91,8% 9,7%
P3 Total 20 33 5 5 63 0 0 83,3% 83,3% 84,1% 0,0%
P4 Total 16 48 8 6 78 3 0 73,3% 80,0% 82,1% 6,3%
P5 Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
P6 Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All protocol Total 148 249 34 32 463 6 2 84,1% 85,0% 85,7% 3,2%
Incubation Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Maximum time
P1 Total 44 43 5 9 101 0 0 91,4% 84,5% 86,1% 0,0%
P2 Total 28 31 1 1 61 1 0 96,7% 96,7% 96,7% 3,2%
P3 Total 22 32 3 6 63 0 0 90,3% 80,6% 85,7% 0,0%
P4 Total 20 41 4 13 78 0 0 89,2% 64,9% 78,2% 0,0%
P5 Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
P6 Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All protocol Total 158 240 24 41 463 1 2 89,2% 81,6% 86,0% 1,3%
Table 9 : values for each protocol of sensitivity, relative trueness and false positive ratio for the alternative method with the confirmation b) (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive
deviation, PP: presumptive positive before confirmation, SEalt: sensitivity for the alternative method, SEref: sensitivity for the reference method, RT: relative trueness, FPR: false positive ratio for the alternative method)
Incubation Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Minimum time
P1 Total 41 44 8 8 101 2 0 86,0% 86,0% 84,2% 4,5%
P2 Total 28 31 1 1 61 1 0 96,7% 96,7% 96,7% 3,2%
P3 Total 20 33 5 5 63 0 0 83,3% 83,3% 84,1% 0,0%
P4 Total 18 46 6 8 78 1 0 81,3% 75,0% 82,1% 2,2%
P5 Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
P6 Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All protocol Total 151 247 31 34 463 4 2 85,6% 84,3% 86,0% 2,4%
Incubation Protocol Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Maximum time
P1 Total 43 43 6 9 101 1 0 89,7% 84,5% 85,1% 2,3%
P2 Total 28 31 1 1 61 1 0 96,7% 96,7% 96,7% 3,2%
P3 Total 22 32 3 6 63 0 0 90,3% 80,6% 85,7% 0,0%
P4 Total 20 41 4 13 78 0 0 89,2% 64,9% 78,2% 0,0%
P5 Total 18 60 8 11 97 0 2 78,4% 70,3% 80,4% 3,3%
P6 Total 26 33 3 1 63 0 0 90,0% 96,7% 93,7% 0,0%
All protocol Total 157 240 25 41 463 2 2 88,8% 81,6% 85,7% 1,7%
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Table 10 presents the summary of the results for all categories and all protocols:
Table 10 : summary of the sensitivity study results for all the categories of the application scope
Parameter ISO 16140‐2 formulas
Results for all the categories of the application scope
Protocol of confirmation a) Protocol of confirmation b)
Minimum incubation
time
Maximum incubation
time
Minimum incubation
time
Maximum incubation
time
Sensitivity of the alternative
method 84.1 % 89.2 % 85.6 % 88.8 %
Sensitivity of the reference
method 85.0 % 81.6 % 84.3 % 81.6 %
Relative trueness
85.7 % 86.0 % 86.0 % 85.7 %
False positive ratio
3.2 % 1.3 % 2.4 % 1.7 %
3.1.1.6. Analysis of discordant results Discordant results are examined according to the standard ISO 16140‐2: 2016. The number of discordances between the reference method and the alternative method is variable according to the implemented protocols and incubation times.
During the validations studies of 2009, confirmations performed after 6 or 8 hours of incubation were not always successful and it is recommended to extend incubation of the broth to 20‐24 h. Tables 11 and 12 presents the summary of the discordant results for all categories and all protocols, respectively, with the confirmation a and confirmation b.
For the confirmation a (confirmation: Vidas ICE)Samples F6 (ground beef), Y4 (seasonned ground meat) and Y5 ( meat balls), O2 and O7(lettuce) and O6
(Lamb's lettuce), were considered as negative deviations because the VIDAS positive results were not
confirmed when the VIDAS ICE assay was performed after 6 hours of enrichment for F6, Y4 and Y5 and after
8 hours of enrichment for O2 and O7. However, as the confirmation was positive when the VIDAS ICE assay
was performed after 24 hours of enrichment, as recommended in the kit insert, the final result should be a
positive agreement.
Sample B4 (ground beef) was confirmed only after storage of the broth for 48 hours at 2‐8°C.
Sample VEC94 (sweet green pepper) and VEC96 (sweet red pepper) were given a negative accord because
the VIDAS results were not confirmed when the VIDAS ICE assay was performed at 8 hours of enrichment.
However, as the confirmation was positive when the VIDAS ICE assay was performed after 24 hours of
enrichment, as recommended in the kit insert, the final results should be a positive deviation.
For the confirmation b (confirmation: direct plating)Samples AD8 (smoked bacon) and O6 (Lamb's lettuce), were considered as negative deviations because the
VIDAS positive results were not confirmed when the confirmation was performed after ,respectively,15 or 8
hours of enrichment. However, as the confirmation was positive when the confirmation was performed after
24 hours of enrichment, as recommended in the kit insert, the final result should be a positive agreement.
Sample B4 (ground beef) was confirmed only after storage of the broth for 48 hours at 2‐8°C.
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Table 11 : summary of discordant results for all the categories for the alternative method with the confirmation a
Category Study protocol Sample type Products Discordance Category Study protocol Sample type Products Discordance
sample2017 6 VEC 48 1 Process water: vegetables cleaning water PD
Environmental
sample2017 6 VEC 48 1 Process water: vegetables cleaning water PD
Raw vegetal
products
Raw vegetal
products
Raw milk and
raw dairy
products
Raw milk and
raw dairy
products
Raw meat
products
Raw meat
products
Minimum incubation times Maximum incubation times
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Tables 13 and 14 show the difference between negative deviations and positive deviations and the acceptability limits for each categories.
Table 13: acceptability limits for the confirmation a) at minimum and maximum incubation times for each categories
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Minimum time
Raw Meat (1) / P1+P2+P3
1 9 8
/ /
(ND‐PD) ≤ AL
2 4 5
3 2 1
Total 15 (12*) 14 1 (‐2*) 3
Raw Vegetable (2) / P4
1 1 3
/ / 2 5 * 0
3 2 3
Total 8 (5*) 6 2 (‐1*) 3
Raw milk and raw milk products (3) / P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 1
/ / 2 0 0
3 3 0
Total 3 1 2 3
All categories Total 34 (28*) 32 2 (‐4*) 5
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Maximum time
Raw Meat (1) / P1+P2+P3
1 5 10
/ /
(ND‐PD) ≤ AL
2 2 5
3 2 1
Total 9 16 ‐7 3
Raw Vegetable (2) / P4
1 1 6
/ / 2 1 1
3 2 6
Total 4 13 ‐9 3
Raw milk and raw milk products (3) / P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 1
/ / 2 0 0
3 3 0
Total 3 1 2 3
All categories Total 24 41 ‐17 5
* 6 samples were confirmed after re‐incubation of the enrichment broth until 24 hours
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Table 14: acceptability limits for the confirmation b) at minimum and maximum incubation times for each categories
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Minimum time
Raw Meat (1) / P1+P2+P3
1 8 8
/ /
(ND‐PD) ≤ AL
2 2 (1*) 5
3 4 1
Total 14 (13 *) 14 0 (‐1*) 3
Raw Vegetable (2) / P4
1 1 5
/ / 2 3 (2*) 0
3 2 3
Total 6 (5*) 8 ‐2 (‐3*) 3
Raw milk and raw milk products (3) /
P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 1
/ / 2 0 0
3 3 0
Total 3 1 2 3
All categories Total 31 34 ‐3 5
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Maximum time
Raw Meat (1) / P1+P2+P3
1 5 10
/ /
(ND‐PD) ≤ AL
2 2 5
3 3 1
Total 10 16 ‐6 3
Raw Vegetable (2) / P4
1 1 6
/ / 2 1 1
3 2 6
Total 4 13 ‐9 3
Raw milk and raw milk products (3) /
P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 1
/ / 2 0 0
3 3 0
Total 3 1 2 3
All categories Total 25 41 ‐16 5
* 2 samples was confirmed after re‐incubation of the enrichment broth until 24 hours
Tables 15 and 16 show the difference between negative deviations and positive deviations and the acceptability limits for each protocols.
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Table 15: acceptability limits for the confirmation a) at minimum and maximum incubation times for each protocols
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Minimum time
P1 Total 6 8 ‐2 3
(ND‐PD) ≤ AL
P2 Total 4 (1*) 1 3 (0*) 3
P3 Total 5 (2*) 5 0 (‐3*) 3
P4 Total 8 6 2 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 34
(28*) 32 2 (‐4*) 5
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Maximum time
P1 Total 5 9 ‐4 3
(ND‐PD) ≤ AL
P2 Total 1 1 0 3
P3 Total 3 6 ‐3 3
P4 Total 4 13 ‐9 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 24 41 ‐17 5
* 6 samples were confirmed after re‐incubation of the enrichment broth until 24 hours
Table 16: acceptability limits for the confirmation b) at minimum and maximum incubation times for each protocols
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Minimum time
P1 Total 8
(7*) 8 0 (‐1*) 3
(ND‐PD) ≤ AL
P2 Total 1
(0*) 1 0 (‐1*) 3
P3 Total 5 5 0 3
P4 Total 6 8 ‐2 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 31 34 ‐3 5
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Maximum time
P1 Total 6 9 ‐3 3
(ND‐PD) ≤ AL
P2 Total 1 1 0 3
P3 Total 3 6 ‐3 3
P4 Total 4 13 ‐9 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 25 41 ‐16 5
* 2 samples was confirmed after re‐incubation of the enrichment broth until 24 hours
The observed values are below or equal to the acceptability limits for each category. For all categories, the observed values are below the acceptability limits.
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The alternative method produces results comparable to the reference method.
3.1.1.7. Study of the storage of the broths A stability study of the enriched broths stored at 5±3°C for 72 hours was performed on all positive and discordant samples. The broths were re‐analyzed and confirmed after storage (results in appendix 3). In 2009, the study of the storage of the broths was applied only after the maximum time of incubation. There was no test with the broths incubated at the minium time and stored at 5±3°C. No modifications appeared for the studies realized in 2009 and 2014.
For the additional tests realized in 2017, several changes appeared. Tables 17, 18, 19 and 20 show the difference between negative deviations and positive deviations and the acceptability limits depending on the confirmation protocols and the incubation times.
Table 17 and 18 present the results of the broths enriched with the maximum time of incubation and stored 72h at 5±3°C for each categories Table 19 and 20 present the results of the broths enriched with the maximum time of incubation and stored 72h at 5±3°C for each protocols.
Table 17: acceptability limits for the confirmation a) at minimum and maximum incubation times after conservation of the enriched broths for each categories
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Maximum time
Raw Meat (1) / P1+P2+P3
1 4 10
/ /
(ND‐PD) ≤ AL
2 2 5
3 2 1
Total 8 16 ‐8 3
Raw Vegetable (2) / P4
1 1 6
/ / 2 1 1
3 2 6
Total 4 13 ‐9 3
Raw milk and raw milk products (3) /
P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 0
/ / 2 0 0
3 3 1
Total 3 1 2 3
All categories Total 23 41 ‐18 5
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Table 18: acceptability limits for the confirmation b) at minimum and maximum incubation times after conservation of the enriched broths for each categories
Incubation Category/Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Maximum time
Raw Meat (1) / P1+P2+P3
1 4 9
/ /
(ND‐PD) ≤ AL
2 2 5
3 4 1
Total 10 15 ‐5 3
Raw Vegetable (2) / P4
1 1 6
/ / 2 1 1
3 2 6
Total 4 13 ‐9 3
Raw milk and raw milk products (3) /
P5
1 4 2
/ / 2 2 1
3 2 8
Total 8 11 ‐3 3
Environmental samples (4) / P6
1 0 0
/ / 2 0 0
3 3 1
Total 3 1 2 3
All categories Total 25 40 ‐15 5
Table 19: acceptability limits for the confirmation a) at minimum and maximum incubation times after conservation of the enriched broths for each protocols
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Minimum time
P1 Total 5 9 ‐4 3
(ND‐PD) ≤ AL
P2 Total 0 1 ‐1 3
P3 Total 4 5 ‐1 3
P4 Total 4 12 ‐8 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 24 39 ‐15 5
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit (AL) Observation
Maximum time
P1 Total 5 9 ‐4 3
(ND‐PD) ≤ AL
P2 Total 0 1 ‐1 3
P3 Total 3 6 ‐3 3
P4 Total 4 13 ‐9 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 23 41 ‐18 5
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Table 20: acceptability limits for the confirmation b) at minimum and maximum incubation times after conservation of the enriched broths for each protocols
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Minimum time
P1 Total 7 8 ‐1 3
(ND‐PD) ≤ AL
P2 Total 0 1 ‐1 3
P3 Total 4 5 ‐1 3
P4 Total 4 12 ‐8 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 26 38 ‐12 5
Incubation Protocol Type ND PD (ND‐PD) Acceptability limit
(AL) Observation
Maximum time
P1 Total 7 8 ‐1 3
(ND‐PD) ≤ AL
P2 Total 0 1 ‐1 3
P3 Total 3 6 ‐3 3
P4 Total 4 13 ‐9 3
P5 Total 8 11 ‐3 3
P6 Total 3 1 2 3
All categories Total 25 40 ‐15 5
The observed values are below the acceptability limit for each category and for all categories.
The alternative method produces results comparable to the reference method.
3.1.2. Relative level of detection study
3.1.2.1. Matrices Different “strain‐matrix” couples were studied in parallel with the reference method and the VIDAS ECPT method, for the studied categories.
The total viable count of each matrix was enumerated. Characteristics of the strain and the matrix are shown in table 21.
Table 21 : « matrix – strain » couples of the relative level of detection
P2 Ground beef 25 g E. coli O157:H7 ATCC 43895 hamburger
P3 Ground beef 375 g
(2) Raw vegetable P4 Spinach E. coli O157:H7 EC44 /
(3) Raw milk P5 Raw milk E. coli O157:H7 ESC.1.110 Beef
(4) Process water P6 Process water E. coli O157:H7 EC55 Environment
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3.1.2.2. Protocol of contamination Data from the studies of 2009 (initial validation and first extension) are re‐interpreted with the calculations of the new standard ISO 16140‐2 : 2016. It concerns the protocols P2, P3, P4 and P6.
For the protocol P1 and P5, analyses and calculations were performed in 2014 and 2017 according to the protocol of the standard ISO 16140‐2 : 2016. This protocol is described below.
Protocol for protocols P2, P3, P4 and P6Six levels of contamination were tested including the negative control. Six replicates for each level of contamination were inoculated and analysed by the reference method and the alternative method. As the two methods have no common step, 12 test portions of 25 or 375 g were prepared for each level of contamination and individually inoculated with a calibrated bacterial suspension. Several dilutions of a calibrated and low‐concentrated suspension of E. coli O157:H7 were used to spike the samples before analysis.
Protocol for protocols P1 and P5Three levels of contamination per type were prepared consisting of a negative control level, a low level, and a higher level. Only one strain of the target analyte is used to contaminate the low and the high level. The negative control level shall not produce positive results. Five replicates are tested for this level. The low level shall be the theoretical detection level, it has been contaminated at 0.7 ‐ 1 CFU per test portion to obtain fractional recovery results. Twenty replicates are tested for this level. The higher level shall be just above the theoretical detection level, it has been contaminated at 2 ‐ 3 CFU per test portion. Five replicates are tested for this level.
Food products were contaminated using the seeding protocol. Bulk contaminations were performed on the matrices for the different levels of contamination, then the matrices were stored at 5±3°C for two days before analysis. Samples were then analyzed by the reference and the alternative method. For the alternative method, only the minimal incubation time of the broth of the alternative method was tested,. Simultaneously, a total viable count was performed on a portion of non‐contaminated matrix to estimate the concentration of mesophilic aerobic flora. A detection of E. coli O157:H7 using the reference method was also performed to check the absence of the target analyte in the matrix.
3.1.2.3. Results and calculation of the RLoDs Raw results are shown in appendix 4.
The RLODs calculations were performed according to the standard ISO 16140‐2 : 2016 using the Excel spreadsheet available for download at http://standards.iso.org/iso/16140. Values of the RLODs are presented in table 22.
For categories for which two incubation times were tested, the results were the same at the minimum and the maximum incubation times.
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Table 22 : RLODs values for the categories of the application scope (RLOD: the estimated relative level of detection value, RLODU: the upper limit of the 95% confidence interval for RLOD, RLODL: the lower limit of the 95% confidence interval for RLOD, b=ln(RLOD): logarithm of the RLOD value, sd(b): standard deviation of b, z‐Test statistic: absolute value of the test statistic of the z‐Test with the null hypothesis H0: b=0, p‐value: p‐value of the z‐Test)
Name Protocol RLOD RLODL RLODU b=ln(RLOD) sd(b)z‐Test statistic
* results from the 2009 study were obtained with an incubation time of 6 hours
3.1.2.4. Interpretation and conclusion The RLODs values are below the acceptability limits, meaning that, as stated in ISO 16140‐2: 2016, the maximum increase in LOD of the alternative versus the reference method is not considered as relevant in consideration of the fitness for purpose of the method.
In conclusion, alternative and reference methods show similar LODs values for the detection of E. coli O157:H7 in the categories tested.
3.1.3. Inclusivity and exclusivity study
3.1.3.1. Test protocols
Protocol for inclusivity : two protocols were tested
‐ Protocol specific for raw beef and raw veal For each strain of E. coli O157:H7, a culture in nutritive broth was performed. Then a buffered peptone water was inoculated with about 10 E. coli O157:H7 per 225 ml and incubated at 41.5°C for 6 hours before VIDAS ECPT testing.
‐ Protocol specific for environmental samples For each strain of E. coli O157:H7, a culture in nutritive broth was realized. Then a buffered peptone water (BPW) supplemented with vancomycin (8 mg/l), cefixime (0,0125 mg/l) and cefsulodine (10 mg/l) was inoculated with about 10 E. coli O157:H7 per 225 ml and incubated at 41.5°C for 15 hours before VIDAS ECPT testing.
Protocol for exclusivityThe different negative strains were cultured and diluted in a nutrient broth to obtain a level of about 105 cells per 225 ml. After incubation for 20‐26 hours at 41.5°C, an aliquot of the BPW was heated for 5 ± 1 minutes at 95‐100°C before VIDAS ECPT testing.
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3.1.3.2. Results Raw results in appendix 5.
InclusivityThe 56 E. coli O157 strains (including 50 E. coli O157:H7 strains, 1 E. coli O157:H4 strain and 5 E. coli O157:H7‐ strains were all detected with the VIDAS ECPT assay, whatever the enrichment protocol used.
ExclusivityThe study of 50 strains not belonging to the serogroup E. coli O157 showed cross‐reactions with 3 Salmonella strains from the N group (Salmonella Urbana, Salmonella Soeranga and Salmonella Hilversum). These strains were not characteristic on the selective confirmation media (CT‐SMAC agar and ChromID O157:H7).
3.1.3.3. Conclusion The selectivity of the method is satisfactory.
3.1.4. Practicability The practicability of the alternative method was informed according to the criteria defined by the Technical Committee.
1. Storage conditions, shelf‐life and and modalities of utilisation after first use
The storage temperature of the kit is 2°C to 8°C. The kit expiration date is shown on the box label and on the different components.
The kit components must be stored at 2°C ‐ 8°C. If stored according to the recommended conditions (pouch correctly resealed with dessiccant after use…), all components are stable until the expiration date indicated on the label.
2. Time‐to‐resultNegative results are obtained in one to two days. Positive results are obtained in three to four days.
3. Common step with the reference methodThe alternative method has no common step with the reference method.
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3.2. Interlaboratory study
3.2.1. Organization of the interlaboratory study
3.2.1.1. Participating laboratories
The interlaboratory study was realized by the expert laboratory and seventeen participating laboratories.
3.2.1.2. Absence of E. coli O157 in the matrix
Before spiking, the absence of E. coli O157 was verified in the batch of ground beef used according to the reference method.
3.2.1.3. Samples labeling
The labelling of the vials was realized as follows: a code to identify the laboratory and a code to identify each sample, only known by the expert laboratory.
Table 23 : sample code by contamination level
Contamination level Sample code
L0 1‐2‐3‐8‐9‐10‐20‐21‐25‐26‐27‐32‐33‐34‐44‐45
L1 4‐5‐11‐12‐13‐17‐18‐19‐28‐29‐35‐36‐37‐41‐42‐43
L2 6‐7‐14‐15‐16‐22‐23‐24‐30‐31‐38‐39‐40‐46‐47‐48
3.2.2. Control of the experimental parameters
3.2.2.1. Samples preparation and spiking
The matrix was inoculated with the target strain suspension to obtain 3 contamination levels: ‐L0: 0 cell in 25 g ‐L1: 3 cells in 25 g ‐L2: 30 cells in 25 g
The levels of contamination of the spiked matrix were determined by a MPN method, 48 hours after contamination of samples. The following table shows the contamination levels obtained:
Table 24 : target level, real level and TVC of the matrix
The temperature readings upon reception and the state of the samples are shown in table 17.
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Table 25 : temperature and state of the samples upon reception
Laboratory T°C laboratory T°C probe Comments
A 4.8°C 4.1°C
B 2.0°C 5.1°C
C 4.2°C 4.1°C
D 9.3°C 7.3°C
E 3.9°C 4.2°C
F 7.7°C 6.2°C
G 3.5°C 3.1°C
H 4.9°C 3.7°C
I 18.9°C 17.3°C Reception at D5
J / 4.8°C Reception at D2
K / 7,1°C
L 6.6°C 4.7°C
M 5.0°C 5.5°C
N 7.6°C 3.0°C
0 9.1°C 3,9°C
P 5.1°C 9.1°C
Q / / Analyses not realized
Among the17 laboratories, 14 laboratories received samples the day after the sending. Laboratory P received samples at D1, but registered shipment temperatures were over 8°C. Its results were not considered. Laboratory J received samples at D2, but the delivery temperatures were acceptable, so, its results were exploitable. And 2 laboratories (I and Q) did not realized analysis because of reception conditions.
Finally, 14 laboratories performed the analysis.
3.2.3. Results The interlaboratory studie was performed with the protocol P2 of the altenative method.
3.2.3.1. Total viable counts
For the whole laboratories, the total viable counts at 30°C vary between 3.4x104 CFU/g and 3,1 x 108 CFU/g. Enumerations by laboratory are set out in appendices 6 and 7.
3.2.3.2. Expert laboratory results
The results obtained by the expert laboratory are summarized in table 26 (raw results in appendix 6).
Table 26 : positive results obtained by expert laboratory by both methods
Contamination level Alternative method Reference method (*)
L0 0/8 0/8
L1 8/8 8/8
L2 8/8 8/8
The results are consistent with those expected.
3.2.3.3. Collaborators results
The results are summarized in tables 27 and 28. Raw results in appendix 7.
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The laboratories applied the minimum incubation times for the enrichment broths, namely 7 hours.
Reference method results
Table 27 : reference method positive results for all laboratories
Reference method
Laboratory Contamination level
L0 L1 L2
A 0/8 7/8 8/8
B 0/8 8/8 8/8
C 0/8 8/8 8/8
D 8/8 8/8 8/8
E 0/8 8/8 8/8
F 0/8 8/8 8/8
G 0/8 8/8 8/8
H 0/8 8/8 8/8
J 0/8 8/8 8/8
K 0/8 8/8 8/8
L 1/8 8/8 8/8
M 0/8 8/8 8/8
N 0/8 8/8 8/8
O 0/8 8/8 8/8
Alternative method results
Table 28 : alternative method positive results for all laboratories
Alternative method
Laboratory Contamination level
L0 L1 L2
A 0/8 8/8 8/8
B 0/8 8/8 8/8
C 0/8 8/8 8/8
D 0/8 6/8 8/8
E 0/8 2/8 6/8
F 0/8 8/8 8/8
G 0/8 8/8 8/8
H 0/8 8/8 8/8
J 0/8 8/8 8/8
K 0/8 7/8 8/8
L 0/8 8/8 8/8
M 0/8 8/8 8/8
N 0/8 0/8 3/8
O 0/8 7/8 8/8
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3.2.3.4. Analysis of the results and collaborators selected for the statistical analysis
The results of the reference method and the alternative method were in agreement for 7 laboratories. For 7 other laboratories, the obtained results are the following:
‐ One laboratory (A) showed a negative result with the reference method for 1 sample spiked at the lowest level. ‐ Two laboratories (K and O) showed a negative VIDAS assay for 1 sample spiked at the lowest level. Colonies were found from the isolated broth and the VIDAS assay tested positive after 24 hours of incubation. The threshold of the method was not reached. ‐ One laboratory (L) found 1 positive with the reference method, among the 8 replicates of the uncontaminated samples, probably due to a cross‐contamination. ‐ One laboratory (D) found all the uncontaminated and contaminated samples positive with the reference method. As, furthermore this lab did not implement correctly the reference method, it was excluded from the study. ‐ Two laboratories (E and N) showed a negative VIDAS assay for respectively 9 and 11 samples from the
16 spiked samples. As the protocol of the alternative method was not correctly implemented (enrichment
temperature not respected), their results were not taken into account. As a consequence, it was asked to
include into the package insert the following sentence :
« Incubation conditions may have repercussions on short detection procedures. The temperatures
indicated must be scrupulously respected. In particular, it is advisable to ensure that the conditions for
pre‐heating the enrichment broth enable the indicated temperature to be reached. The sample
preparation time (time between the end of the enrichment broth pre‐heating phase and the start of the
food sample incubation phase), must not exceed 45 minutes. It is recommended to use a ventilated
incubator for the incubation phase. »
After exclusion of laboratories D, E and N, the results from 11 laboratories were considered for calculations.
3.2.4. Interpretation of the results and statistical analysis
3.2.4.1. Interpretation of the results
The interpretation of the results is shown in the table below.
Table 29 : tests results for both methods (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive deviation)
Level Alternative method (AM) Reference method (RM)
RM+ RM‐ Total
L0
AM+ PA=0 PD=0 0
AM‐ ND=1
including 0 PPND NA=87
including 0 PPNA 88
Total 1 87 88
L1
AM+ PA=85 PD=1 86
AM‐ ND=2
including 0 PPND NA=0
including 0 PPNA 2
Total 87 1 88
L2
AM+ PA=88 PD=0 88
AM‐ ND=0
including 0 PPND NA=0
including 0 PPNA 0
Total 88 0 88
L1+L2
AM+ PA=173 PD=1 174
AM‐ ND=2
including 0 PPND NA=0
including 0 PPNA 2
Total 175 1 176
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3.2.4.2. Specificity of the methods
The percentage specificity of the reference method and the alternative method is calculated using the data after confirmation, based on th results of level L0.
‐ Specificity of the reference method: 1 100% = 98.9%,
‐ Specificity of the alternative method: 1 100% = 100%,
where: N_ is the number of all L0 tests; P0 is the total number of false‐positive results obtained with the blank samples before confirmation; CP0 is the total number of false‐positive results obtained with blank samples.
3.2.4.3. Sensitivity of the two methods, relative trueness and false positive ratio of
the alternative method
The sensitivity of the two methods, the relative trueness of the alternative method and the false positive ratio of the two methods are calculated. Results are presented in the table 30.
Table 30 : summary of the sensitivity study results for all the categoriesof the application scope
Parameter ISO 16140‐2 formulas Results
Sensitivity of the alternative method
98.9%
Sensitivity of the reference method
99.4%
Relative trueness 98.3%
False positive ratio 0%
3.2.4.4. Determination of the acceptability limit and conclusion
The difference between (ND – PD) for the level where fractional recovery was obtained (L1) is calculated. The observed value found for (ND – PD) shall not be higher than the acceptability limit (AL). The AL is defined as [(ND – PD)max] and calculated per level where fractional recovery was obtained as described below using the following three parameters:
‐ ,
where Px = number of samples with a positive result obtained with the reference method at level x, (L1 or L2) for all laboratories; Nx = number of samples tested at level x (L1 or L2) with the reference method by all laboratories.
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‐ ,
where CPx = number of samples with a confirmed positive result obtained with the alternative method at level x (L1 or L2) for all laboratories; Nx = number of samples tested at level x (L1 or L2) with the alternative method by all laboratories.
‐ 3 2 ,
where Nx = the total number of samples tested for level x (L1 or L2) by all laboratories. The AL is not met when the observed value is higher than the AL. When the AL is not met, investigations should be made (e.g. root cause analysis) in order to provide an explanation of the observed results.
Based on the AL and the additional information, it is decided whether the alternative method is regarded as not fit for purpose. The reasons for acceptance of the alternative method in case the AL is not met shall be stated in the study report.
In this study, fractional positive results are observed at level L1 only. The different parameters obtained by the calculation are detailed in the table below:
Table 31 : values obtained for the determination of the acceptability limit
Parameter Value
NL1 : number of samples tested at level 1 88
(p+)ref 0.9886
(p+)alt 0.9773
(ND‐PD)max 2.98
(ND‐PD) 1
The value (ND‐PD) is inferior to the AL, so the requirements of the standard ISO 16140‐2 : 2016 are fulfilled.
The performance of the alternative method and the reference method can be considered as equivalent.
3.2.4.5. Determination of the relative level of detection
This evaluation is performed according to Annex F of ISO/FDIS 16140‐2:2015 and using the excel spreadsheet as described in this standard.
As there is limited experience with the interpretation of this approach, the results are used only for information. Results are shown in the table below :
Table 32 : values obtained for the determination of the relative level of detection
Number of sets of data RLOD RLODL RLODU b=ln(RLOD) sd(b) z‐Test statistic p‐value
11 1,229 0,689 2,192 0,206 0,289 0,712 0,476
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3.3. General conclusion
Method comparison studyThe performances of the VIDAS ECPT test are comparable to those of the standard ISO 16654 : 2001.
This study concerned 463 samples of four categories of products:
‐Raw meat
‐ Raw vegetal products for test portions of 25 g
‐ Raw milk and raw milk products for test portions of 25 g
‐ Environmental samples
Values obtained for the criteria of the sensitivity study are the following, depending on the incubation times and the protocol of confirmation:
‐ sensitivity of the alternative method : from 84.1% to 89.2% ‐ sensitivity of the reference method : from 81.6% to 85.0% ‐ relative trueness: from 85.7% to 86.0% ‐ false positive ratio: from 1.3% to 3.2%
Some discordant results were observed. These discordances may be mostly linked to the sampling which is different between the two methods.
The relative level of detection of the alternative method and the reference method was evaluated for all categories. The results are comparable between the two methods. It varies between 0.868 and 1.591 CFU in 25 g for the alternative method for all categories.
The specificity of the method is satisfactory.
The study of the practicability of the alternative method shows a simple and easy‐to‐use method and significant time savings compared to the reference method.
Interlaboratory studyConcerning the interlaboratory study, the results obtained for the selected laboratories showed that the performance of the alternative method and the reference method can be considered as equivalent. For protocol P2 (enrichment for 6‐24 hours), as the minimum of the incubation time was not fully respected in few Labs, the AFNOR Technical Board asked that this minimum was increased to 7 hours.
Massy, the 25th June 2018, Olivier Mathia
Innovation Biologie Unit Manager
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APPENDIX 1
ANALYTICAL PROTOCOLS
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ALTERNATIVE METHOD PROTOCOL ‐ VIDAS ECPT
Category Raw meat Raw vegetal products
Raw milk and raw milk products
Environmental samples
Type
‐Raw beef and veal (t1)
and ‐Seasonned raw beef
and veal (t2) and
‐Other raw meats (t3)
‐Raw beef and veal (t1)
and ‐Seasonned raw beef and
veal (t2)
Raw beef and veal (t1)
Fruits (t1) Green plants
(t2) Others
plants and mix
vegetables (t3)
Goat and sheep raw milk cheese
(t1) Raw milks and others raw
milk products (t2)
Cow raw milk cheese (t3)
Process waters (t1) Dust and
residues (t2) Surface
samples (t3)
Protocol P1 P2 P3 P4 P5 P6
Test portion 25 g 25 g 50 to 375
g 25 g 25 g
25 g or other test portion (swab, wipe, sponge…)
Dilution 1/10 1/10 1/4 1/10 1/10
1/10 Swab in 10
mL Wipe in 100
mL
Broth Pre‐warmed
BPW + vancomycine
Pre‐warmed BPW
Pre‐warmed BPW +
vancomycine
Pre‐warmed BPW +
vancomycine
BPW + acriflavine
Pre‐warmed BPW +
vancomycine + cefixime + cefsulodin
Incubation time and
temperature
41.5±1°C for 16 – 24 h
41.5±1°C for 7 – 24 h
41.5±1°C for
8 – 24 h
41.5±1°C for 8 – 24 h
41.5±1°C for 20 – 26 h
41.5±1°C for 15 – 24 h
Volume to transfer in a tube and
heat at 95 – 100°C for 5±1 min
1 – 2 mL or Heat and Go
(except poultry products)
1 – 2 mL or Heat and Go
2 – 3 mL 1 – 2 mL or Heat and Go
1 – 2 mL or Heat and Go
1 – 2 mL or Heat and Go
↓
VIDAS protocol detection
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VIDAS protocol detection
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EN ISO STANDARD 16654: 2001
Test portion (x g or x ml)
+9 x ml mTSB + novobiocin pre-warmed to 41.5°C, and homogenization
Incubation for 6 h at 41.5±1.0°C
IMS 1 : Concentration of E. coli O157 by capture onto immunomagnetic particles, washing, and resuspension in 0,1 ml of sterile wash buffer
Inoculation of 50 µl of the washed and re-suspended magnetic particles on selective medium to obtain isolated : - 50 µl on CT SMAC medium - 50 µl on second isolation medium
Incubation for 18 h to 24 h at 37±1°C
Incubation for a further 12 h to 18 h (total elapsed time of 18 h to 24 h) at 41.5±1.0°C
IMS 2 and inoculation on selective media (CT SMAC medium and second isolation medium)
Confirmation of pure colonies (maximum of 5 typical colonies by medium) by indole formation or by commercially available biochemical identification kits and by serological identification with antiserum to E. coli O157
Presence of E. coli O157 in x g or x ml Absence of E. coli O157 in x g or x ml
Presence of characteristic colonies
YES
YES
NO
NO
If presence of typical colonies
Enrichment
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APPENDIX 2
BACTERIAL STRAINS FOR CONTAMINATION
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N° Name Origin
AB5 Aglet of duck EC53 E. coli O 157 Environment 24 h at 4°C 0,7 13,2 +
AB6 Turkey cutlet EC53 E. coli O 157 Environment 24 h at 4°C 0,7 17,6 +
AB8 Aglet of duck EC54 E. coli O 157 Environment 24 h at 4°C 0,5 1,4 +
AD1 Green pepercorn pavé EC31 E coli O157 H7 ATCC 43895 origin : hamburger 20 min at ‐80°C 0,7 4,0 +
AD8 Smoked bacon EC31 E coli O157 H7 ATCC 43895 origin : hamburger 24 h at 4°C 0,7 6,0 +
AD9 Sausages EC31 E. coli O 157 ATCC 43895 origin : hamburger 24 h at 4°C 0,7 8,0 +
AF3 Gizzard of poultry EC62 E. coli O 157 Environment 20 min at ‐80°C 0,9 25,2 +
AF4 Heart of beef EC62 E. coli O 157 Environment 20 min at ‐80°C 0,9 33,6 +
AF7 Chicken breast EC82 E. coli O 157 Beef 20 min at ‐80°C >3,4 33,2 +
AF8 Chicken wings EC82 E. coli O 157 Beef 20 min at ‐80°C >3,4 41,5 +
AP1 Chicken legs EC31 E coli O157 H7 ATCC 43895 origin : hamburger 48 h at 4°C 0,5 3,6 +
AP2 Filet mignon of pork EC31 E. coli O 157 ATCC 43895 origin : hamburger 48 h at 4°C 0,5 2,5 +
AP3 Calf sweetbread EC31 E. coli O 157 ATCC 43895 origin : hamburger 48 h at 4°C 0,5 4,1 +
AP4 Sausage meat pure pork EC42 E coli O157 H7 ATCC43890 (human faeces) 48 h at 4°C 0,4 3,6 +
AP5 Prok rib fillet EC31 E coli O157 H7 ATCC 43895 origine : hamburger 48 h at 4°C 0,5 2,4 +
AP6 Pork shoulder chop EC42 E coli O157 H7 ATCC43890 (human faeces) 48 h at 4°C 0,4 3,0 +
AP7 Raw guinea fowl EC42 E. coli O 157 ATCC43890 (human faeces) 48 h at 4°C 0,4 4,2 +
AP8 Kidneys of pork EC42 E. coli O 157 ATCC43890 (human faeces) 48 h at 4°C 0,4 4,8 +
AQ1 Duck filet EC82 E coli O157 H7 Beef 48 h at 4°C 0,8 17,0 +
AQ10 Turkey cutlet EC81 E coli O157 H7 Pork 48 h at 4°C 0,8 8,2 +
AQ11 Magret of duck dried EC56 E.coli O157 H7‐ Environment 48 h at 4°C 0,4 5,4 +
AQ12 Shank of pork EC82 E coli O157 H7 Beef 48 h at 4°C 0,8 12,0 +
AQ2 Chicken filet EC81 E coli O157 H7 Pork 48 h at 4°C 0,8 16,0 +
AQ3 Flank steak maritaned with shallot EC82 E coli O157 H7 Beef 48 h at 4°C 0,8 3,4 +
AQ4 Sliced porck sauce Toscane EC81 E coli O157 H7 Pork 48 h at 4°C 0,8 3,2 +
AQ5 Merguez beef‐sheep EC82 E coli O157 H7 Beef 48 h at 4°C 0,8 2,6 +
AQ6 Sausages with herbals EC56 E.coli O157 H7‐ Environment 48 h at 4°C 0,4 12,0 +
AQ7 Shank of lamb EC81 E coli O157 H7 Pork 48 h at 4°C 0,8 9,5 +
AQ8 Flank steak of horse EC56 E.coli O157 H7‐ Environment 48 h at 4°C 0,4 3,4 +
AQ9 Chicken cutlet EC54 E. coli O 157 Environment 48 h at 4°C 0,4 8,3 +
AR1 Sausage of turkey EC83 E coli O157 H7 Cider 48 h at 4°C 0,3 6,1 +
AR10 Beef meat for fondue EC9 E.coli O157 H7‐ clinical origin 48 h at 4°C 1,7 7,6 +
AR11 Ground horse EC80 E coli O157 H7 Ground beef 48 h at 4°C 0,4 9,0 +
AR12 Ground beef EC80 E coli O157 H7 Ground beef 48 h at 4°C 0,4 7,5 +
AR13 ground beef (5% fat) EC3 E.coli O157 H7‐ Faeces 48 h at 4°C 0,5 10,8 +
AR2 Veal sauté EC48 E coli O157 H7 Collection 48 h at 4°C 0,5 6,9 +
AR3 Ribs of lamb EC48 E coli O157 H7 Collection 48 h at 4°C 0,5 7,9 +
AR4 Leg of sliced lamb EC80 E coli O157 H7 Ground beef 48 h at 4°C 0,4 12,0 +
AR5 Sausages with herbals EC9 E.coli O157 H7‐ clinical origin 48 h at 4°C 1,7 10,2 +
AR6 Magret of duck EC9 E.coli O157 H7‐ clinical origin 48 h at 4°C 1,7 8,9 +
AR7 Quail EC3 E.coli O157 H7‐ Faeces 48 h at 4°C 0,5 12,3 +
AR8 Flank steak maritaned EC80 E coli O157 H7 Ground beef 48 h at 4°C 0,4 10,5 +
AR9 Rump of beef EC48 E coli O157 H7 Collection 48 h at 4°C 0,5 5,9 +
2009 4 AM11 1 Eau de réseau Network water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM12 1 Eau de réseau Network water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM13 1 Eau glacée Frozen water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM14 1 Eau de process Process water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM15 1 Eau glacée Frozen water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM16 1 Eau glacée Frozen water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM17 1 Eau de rinçage légumes Rinsing water vegetables no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM18 1 Eau siphon Siphon water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM19 1 Eau récupérateur Reclaimed water no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM20 1 Eau stagnante Backwater no / Neg 0,00 ‐ ‐ Neg NA 0 ‐ ‐ ‐ NA / / / / / /
2009 4 AM2 1 Eau de réseau Network water sp 3,3 Pos 2,53 + + Pos PA 2,47 + + Pos PA / / / 2,46 Pos PA
2009 4 AM3 1 Eau glacée Frozen water sp 4 Pos 2,57 + + Pos PA 2,49 + + Pos PA / / / 2,43 Pos PA
2009 4 AM4 1 Eau de process Process water sp 3,2 Pos 2,62 + + Pos PA 2,48 + + Pos PA / / / 2,49 Pos PA
2009 4 AM5 1 Eau glacée Frozen water sp 4,1 Pos 2,60 + + Pos PA 2,53 + + Pos PA / / / 2,54 Pos PA
2009 4 AM6 1 Eau glacée Frozen water sp 2,9 Pos 2,63 + + Pos PA 2,56 + + Pos PA / / / 2,49 Pos PA
2009 4 AM7 1 Eau de rinçage légumes Rinsing water vegetable sp 4,8 Pos 2,49 + + Pos PA 2,42 + + Pos PA / / / 2,56 Pos PA
2017 4 VEC48 1 Eau de lavage de végétaux Process water: vegetables cleaning water se 1.6 Neg 1.85 + + Pos PD 1.88 + + Pos PD 1.90 Pos PD 1.87 Pos PD
2017 4 VEC152 1 Eau de process (préparation poisson) Process water: fish preparation se 2.2 Pos 1.86 + + Pos PA 1.83 + + Pos PA 1.97 Pos PA 1.76 Pos PA
Environmental samples
ISO 16654
VIDAS ECPT
15 h incubation time 24 h incubation time 15 h + storage 3 days at 5°C 24h + storage 3 days at 5°CStudy Code Type
Artificial conta.
Protocol Produit Product
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BPW: Buffered Peptone WaterBPW+VCC: Buffered Peptone Water supplemented with Vancomycin, Cefixime and CefsulodineRFV: Relative Fluorescence ValueTV: Test Value
Strains
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