ACCREDITATION N°1‐6415 Scope available on www.cofrac.fr VALIDATION ‐ Validation of alternative analytical methods Application to food microbiology Summary report – January 2019 – v0 Validation study according to the standard EN ISO 16140‐2 : 2016 VIDAS Listeria (VIDAS LIS ‐ Ref. 30700) BIO 12/2‐06/94 for the detection of Listeria spp Protocol for human food products and environmental samples Qualitative method CONFIDENTIAL Expert laboratory : ISHA 17 rue Doyen Denis Leroy 35000 Rennes ‐ FRANCE Manufacturer: bioMérieux Chemin de l’Orme 69280 MARCY L’ETOILE – France This report contains 58 pages. The reproduction of this report is authorized only in its complete form. The accreditation of the COFRAC (Section Laboratory) gives evidence of the competence of the laboratory for the only assays covered by the accreditation. The assays identified by the symbol (◊) were performed under the accreditation N°1‐6415 at the ISHA: 17 rue Doyen Denis Leroy, 35000 Rennes‐ France. The assays identified by the symbol (□) were performed under accreditaƟon at the ISHA: 25 avenue de la Republique, 93000 Massy – France.
58
Embed
Summary report January 2019 v0 - NF Validation · ACCREDITATION N°1‐6415 Scope available on VALIDATION ‐ Validation of alternative analytical methods Application to food microbiology
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
ACCREDITATION
N°1‐6415
Scope available on
www.cofrac.fr
VALIDATION ‐ Validation of alternative analytical methods
Application to food microbiology
Summary report – January 2019 – v0
Validation study according to the standard EN ISO 16140‐2 : 2016
VIDAS Listeria (VIDAS LIS ‐ Ref. 30700)
BIO 12/2‐06/94
for the detection of Listeria spp
Protocol for human food products and environmental samples
Qualitative method
CONFIDENTIAL
Expert laboratory : ISHA 17 rue Doyen Denis Leroy 35000 Rennes ‐ FRANCE
Manufacturer: bioMérieux Chemin de l’Orme 69280 MARCY L’ETOILE – France
This report contains 58 pages. The reproduction of this report is authorized only in its complete form. The accreditation of the COFRAC (Section Laboratory) gives evidence of the competence of the laboratory for the only assays covered by the accreditation. The assays identified by the symbol (◊) were performed under the accreditation N°1‐6415 at the ISHA: 17 rue Doyen Denis Leroy, 35000 Rennes‐ France. The assays identified by the symbol (□) were performed under accredita on at the ISHA: 25 avenue de la Republique, 93000 Massy –
France.
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 (revision 6, May 2017).
Reference method:
EN ISO 11290‐1 : Horizontal method for the detection and enumeration of Listeria monocytogenes and Listeria spp – Part 1 : detection method (1997) EN ISO 11290‐1 / A1 : Horizontal method for the detection and enumeration of Listeria monocytogenes and Listeria spp – Part 1 : detection method (2005) EN ISO 11290‐1 : Horizontal method for the detection and enumeration of Listeria monocytogenes and Listeria spp – Part 1 : detection method (2017)
Application scope:
All human food products and environmental samples.
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).
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 3/58
Table of contents 1. Introduction ................................................................................................................................................. 6
2. Protocols of the methods ............................................................................................................................ 7
2.1. Alternative method ............................................................................................................................. 7
2.1.1. Principle of the alternative method ............................................................................................ 7
2.1.2. Protocol of the alternative method ............................................................................................. 7
2.5. Study design ........................................................................................................................................ 8
3. Method comparison study .......................................................................................................................... 9
3.1. Sensitivity study ................................................................................................................................... 9
3.1.1. Protocols used for the study ....................................................................................................... 9
3.1.2. Number and nature of the samples ............................................................................................ 9
3.1.3. Artificial contaminations of the samples ................................................................................... 10
4. Interlaboratory study ................................................................................................................................ 19
4.1. Organization of the interlaboratory study ........................................................................................ 19
4.4.4. Analysis of the results and collaborators selected for the statistical analysis .......................... 20
4.5. Interpretation of the results and statistical analysis ......................................................................... 21
4.5.1. Interpretation of the results ...................................................................................................... 21
4.5.2. Specificity of the methods ......................................................................................................... 21
4.5.3. Sensitivity of the two methods, relative trueness and false positive ratio of the alternative method 21
4.5.4. Determination of the acceptability limit and conclusion .......................................................... 22
4.5.5. Determination of the relative level of detection ....................................................................... 22
5. General conclusion .................................................................................................................................... 23
Appendix 1: Protocol of the reference method and the alternative method Appendix 2: Artificial contamination Appendix 3: Sensitivity raw results Appendix 4: RLOD raw results Appendix 5: Inclusivity and exclusivity Appendix 6: Interlaboratory study, raw results
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 5/58
1. Introduction
This report introduces the results of the renewal study for the AFNOR Certification validation of the method VIDAS Listeria spp (LIS) for the detection Listeria monocytogenes for human food products and environmental samples according to the standard EN ISO 16140‐2 : 2016.
The method VIDAS LIS for the detection of Listeria spp is validated under the attestation number BIO 12/02‐06/94. The validation history of the method is the following:
Date Validation stages Reference method Validation standard
June 1994 Initial validation for human food products Experimental standard NF V 08‐055:1993
AFNOR requirements in
progress
June 1998 Renewal for human food products EN ISO 11290‐1:1997 AFNOR
requirements in progress
July 2002 Renewal and extension (modification of enrichment protocol) for human food
products EN ISO 11290‐1:1997
AFNOR requirements in
progress
September 2002
Extension for environmental samples EN ISO 11290‐1:1997 AFNOR
requirements in progress
April 2003 Extension (reduction of incubation times for
enrichment protocol) EN ISO 11290‐1:1997
AFNOR requirements in
progress
May 2006 Renewal/extension EN ISO 11290‐1/A1:2005 ISO 16140:2003
May 2010 Second renewal EN ISO 11290‐1/A1:2005 ISO 16140:2003
May 2014 Third renewal EN ISO 11290‐1/A1:2005 ISO 16140/A1:2011
May 2018 Fourth renewal (with supplementary assays to maintain a validation for a broad range of
foods) EN ISO 11290‐1:2017 ISO 16140:2016
The results of the comparative study and of the interlaboratory study reported in this report were produced during the validation tests conducted by SERMHA – Institut Pasteur de Lille under the mark NF VALIDATION certification, in accordance with the requirements in force.
Assays of the 2018 study 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, May 2017).
The data provided from the previous validation using to the EN ISO 11290‐1/A1: 2005 and the data provided from the renewal study 2018 using to the EN ISO 11290‐1: 2017 could be compiled because the main changes listed in the EN ISO 11290‐1:2017 are considered as major but they were considered to have no significant effect on the method performance characteristics or test results.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 6/58
2. Protocols of the methods
2.1. Alternative method
2.1.1. Principle of the alternative method The VIDAS LIS method is based on an immuno‐enzyme test, enabling the detection of the Listeria antigen using the ELFA (Enzyme Linked Fluorescent Assay) method by means of the VIDAS automated system. Each test is broken down into two components:
The single‐use SPR used both for the solid phase and as a pipetting system for the test. The insideof the SPR is coated with anti‐Listeria antibodies absorbed on its surface.
The strip contains all of the ready‐to‐use reagents required for the test: washing solution, anti‐Listeria antibodies conjugated with alkaline phosphatase and substrate.
All steps are performed automatically by the VIDAS analytical module. An aliquot of enrichment broth is dispensed in the sample well of the strip and is cycled in and out of the SPR several time, whose duration is specifically calculated to activate the reaction. The fluorescence intensity is measured by the VIDAS optical system at 450 nm and expressed as a relative fluorescence value (RFV), interpreted by the VIDAS system as follows:
Test value (TV) = sample RFV standard RFV
TV < 0.1 test negative and TV > 0.1 test positive
2.1.2. Protocol of the alternative method The validated protocol is as follows:
‐ enrichment in half‐Fraser broth, incubated for 20 to 26 hours at 30°C ± 1°C, ‐ subculture in complete Fraser broth (1 ml in 10 ml), incubated for 20 to 26 hours at 30°C ± 1°C
The VIDAS LIS test is then performed using an aliquot of complete Fraser broth heated for 15 minutes ± 1 minute at 95‐100°C. NB: In the comparative study of methods, the minimum incubation time (i.e. 20 hours) was met.
Samples found to be positive using the VIDAS LIS test are confirmed through isolation on agar according to Ottaviani and Agosti, PALCAM or Oxford agar, followed by conventional tests set out in the methods standardized by CEN, ISO or AFNOR (including the purification stage).
The outline of the method is set out in Appendix 1.
2.2. Reference method Assays of 1994 were performed according to the experimental standard NF V 08‐055:1993 "Routine method for the detection o Listeria monocytogenes”
Assays of 1998, 2002 and 2003 were performed according to the standard EN ISO 11290‐1 (1997) "Horizontal method for the detection and enumeration of Listeria monocytogenes ‐ Part 1: detection method.”
Assays of 2006 were performed according to the standard EN ISO 11290‐1 / A1 (2005) "Horizontal method for the detection and enumeration of Listeria monocytogenes ‐ Part 1: detection method.”
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 7/58
Assays of the 2018 study were performed according to the standard EN ISO 11290‐1 (2017) "Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp‐ Part 1: detection method.”
The analytical scheme is presented in appendix 1.
2.3. Application scope The scope of the method concerns a broad range of foods and environmental samples. In this document, categories are classified as below:
2.5. Study design It is a paired study as the reference and the alternative methods used the same pre‐enrichment broth (Half‐Fraser).
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 8/58
3. Method comparison study
3.1. Sensitivity study
3.1.1. 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.
All positive and discordant samples were confirmed through isolation on agar according to Ottaviani and Agosti, PALCAM or Oxford agar, followed by conventional tests set out in the methods standardized by CEN, ISO or AFNOR (including the purification stage).
3.1.2. Number and nature of the samples Three hundred and twenty seven (327) samples from the previous validation studies were kept for the statistical analysis. One hundred and sixty two (162) samples of the renewal study 2018 were added for the statistical analysis.
A total of 489 samples were thus analyzed. The distribution of the samples is presented in table 1.
Some of the samples of the initial validation study are not taken into account because of a contamination level higher than 10 CFU/test portion. These samples are nevertheless shown in raw results detailed in appendix 3.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 9/58
Table 1 : number and nature of the samples analyzed (PV: Previous validation, I 18: ISHA 2018)
Category Type
Year Negative results
Positive results
Total
(1) Meat products
m1 Prepared meals with meat PV 12 12
24 I 18 0 0
m2 Sausages PV 7 12
21 I 18 2 0
m3 Delicatessen PV 11 12
23 I 18 0 0
Total 32 36 68
(2) Dairy products
d1 Raw milk PV 5 4
20 I 18 5 6
d2 Cheese with raw milk PV 22 12
34 I 18 0 0
d3 Thermised dairy products PV 11 7
20 I 18 0 2
Total 43 31 74
(3) Seafood products
s1 Raw fish, shellfish PV 9 9
22 I 18 1 3
s2 Smoked PV 8 10
24 I 18 2 4
s3 Dishes made from fish PV 12 14
26 I 18 0 0
Total 32 40 72
(4) Vegetable products
v1 Frozen PV 10 12
22 I 18 0 0
v2 Fresh PV 10 8
21 I 18 0 3
v3 Seasoned PV 12 10
22 I 18 0 0
Total 32 33 65
(5) Composite
food
c1 Ready‐to‐eat products PV 0 0
46 I 18 27 19
c2 Read‐to‐reheat products PV 1 0
49 I 18 27 21
c3 Pastries and derivated, egg productsPV 3 5
40 I 18 16 16
Total 74 61 135
(6) Environment samples
e1 Process water PV 14 10
29 I 18 2 3
e2 Surface samples PV 12 11
26 I 18 0 3
e3 Residues PV 11 9
20 I 18 0 0
Total 39 36 75
Total (all categories) 252 237 489
3.1.3. Artificial contaminations of the samples Two hundred and thirty seven (237) positives samples were analyzed including 143 naturally contaminated samples.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 10/58
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 2.
Table 2 : 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/25g)
Naturally contaminated
≤ 5 (spiking) ≤ 3 (seeding)
5‐10 (spiking) 3‐10 (seeding)
>10 Total
(1) Meat products 36 0 0 0 36
(2) Dairy products 14 14 2 1 (type 2) 31
(3) Sea food products 39 1 0 0 40
(4) Vegetal products 17 5 8 3 (2 type 2, 1
type 3) 33
(5) Composite food 13 36 12 0 61
(6) Environmental sample 24 11 0 1 (type 1) 36
Total 143 67 22 5 237
% 60.3% 28.3% 9.3% 2.1% 100.0%
Table 3: proportion of L. monocytogenes, Listeria non monocytogene and mixture for samples giving positive results with the protocol of confirmation giving the more positive results.
Category Listeria spp only (A)
Listeria spp + L. monocytogenes (B)
Total A+B L. monocytogenes
only Total
positive samplesNumber % Number % Number % Number %
(1) 13 36.1 8 22.2 21 58.3 15 41.7 36
(2) 18 58.1 1 3.2 19 61.3 12 38.7 31
(3) 11 32.4 4 11.8 15 44.1 25 73.5 34
(4) 14 43.8 1 3.1 15 46.9 18 56.3 32
(5) 38 62.3 1 1.6 39 63.9 22 36.1 61
(6) 19 52.8 5 13.9 24 66.7 12 33.3 36
All 113 47.7 20 8.4 133 56.1 104 43.9 237
According to the” Requirements regarding comparison and interlaboratory studies for implementation of the standard EN ISO 16140‐2”, revision 6 (May 2017), for Listeria genus studies, compliance per category with a proportion of at least 15 to 25 Listeria spp contaminated samples (alone or combined with Listeria monocytogenes) is requested”. This requirement is fulfilled for each category.
3.1.4. Results Raw results are presented in appendix 3 by step of validation.
The table 4 present the summary of the results.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 11/58
Table 4 : results of the sensitivity study (PA: positive agreement, NA: negative agreement, ND: negative deviation, PD: positive
deviation, PP: presumptive positive before confirmation)
Category Type PA NA ND PD N PPND PPNA
Meat products (1)
m1 12 12 0 0 24 0 0
m2 12 9 0 0 21 0 0
m3 12 11 0 0 23 0 0
Total 36 32 0 0 68 0 0
Dairy products (2)
d1 10 10 0 0 20 0 0
d2 11 22 1 0 34 0 0
d3 8 11 0 1 20 0 0
Total 29 43 1 1 74 0 0
Sea food products (3)
s1 12 10 0 0 22 0 0
s2 14 10 0 0 24 0 0
s3 14 12 0 0 26 0 0
Total 40 32 0 0 72 0 0
Vegetal products (4)
v1 12 10 0 0 22 0 0
v2 11 10 0 0 21 0 0
v3 10 12 0 0 22 0 0
Total 33 32 0 0 65 0 0
Composite food (5)
c1 16 27 3 0 46 0 0
c2 21 28 0 0 49 0 0
c3 21 19 0 0 40 0 0
Total 58 74 3 0 135 0 0
Environmental sample (6)
e1 12 16 1 0 29 0 0
e2 14 12 0 0 26 0 0
e3 9 11 0 0 20 0 0
Total 35 39 1 0 75 0 0
Total all category 231 252 5 1 489 0 0
3.1.5. Statistical interpretation These results were used to calculate the sensitivity for the alternative method and the reference method and the relative sensitivity (cf. tables 5).
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 12/58
Table 5 : values in % of sensitivity for the two methods, relative trueness and false positive ratio for the alternative method (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)
Category Type PA NA ND PD N PPND PPNA SEalt SEref RT FPR
Meat products (1)
m1 12 12 0 0 24 0 0 100.0% 100.0% 100.0% 0.0%
m2 12 9 0 0 21 0 0 100.0% 100.0% 100.0% 0.0%
m3 12 11 0 0 23 0 0 100.0% 100.0% 100.0% 0.0%
Total 36 32 0 0 68 0 0 100.0% 100.0% 100.0% 0.0%
Dairy products (2)
d1 10 10 0 0 20 0 0 100.0% 100.0% 100.0% 0.0%
d2 11 22 1 0 34 0 0 91.7% 100.0% 97.1% 0.0%
d3 8 11 0 1 20 0 0 100.0% 88.9% 95.0% 0.0%
Total 29 43 1 1 74 0 0 96.8% 96.8% 97.3% 0.0%
Sea food products (3)
s1 12 10 0 0 22 0 0 100.0% 100.0% 100.0% 0.0%
s2 14 10 0 0 24 0 0 100.0% 100.0% 100.0% 0.0%
s3 14 12 0 0 26 0 0 100.0% 100.0% 100.0% 0.0%
Total 40 32 0 0 72 0 0 100.0% 100.0% 100.0% 0.0%
Vegetal products (4)
v1 12 10 0 0 22 0 0 100.0% 100.0% 100.0% 0.0%
v2 11 10 0 0 21 0 0 100.0% 100.0% 100.0% 0.0%
v3 10 12 0 0 22 0 0 100.0% 100.0% 100.0% 0.0%
Total 33 32 0 0 65 0 0 100.0% 100.0% 100.0% 0.0%
Composite food (5)
c1 16 26 3 0 45 0 0 84.2% 100.0% 93.5% 0.0%
c2 21 29 0 0 50 0 0 100.0% 100.0% 100.0% 0.0%
c3 21 19 0 0 40 0 0 100.0% 100.0% 100.0% 0.0%
Total 58 74 3 0 135 0 0 95.1% 100.0% 97.8% 0.0%
Environmental sample (6)
e1 12 16 1 0 29 0 0 92.3% 100.0% 96.6% 0.0%
e2 14 12 0 0 26 0 0 100.0% 100.0% 100.0% 0.0%
e3 9 11 0 0 20 0 0 100.0% 100.0% 100.0% 0.0%
Total 35 39 1 0 75 0 0 97.2% 100.0% 98.7% 0.0%
All categories Total 231 252 5 1 489 0 0 98.2% 99.5% 99.0% 0.0%
Table 6 presents the summary of the results for all categories and all protocols:
Table 6 : summary of the sensitivity study results for all the categories of the application scope Parameter ISO 16140‐2 formulas Results for all the categories
Sensitivity of the alternative method
98.2%
Sensitivity of the reference method
99.5%
Relative trueness 99.0%
False positive ratio 0.0%
3.1.6. Analysis of discordant results Discordant results are examined according to the standard ISO 16140‐2: 2016.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 13/58
Table 7 presents the summary of the discordant results for all categories.
Table 7 : summary of discordant results for all the categories (PV: Previous Validation, I18: ISHA 2018, TC: type of contamination (sp: spinking, se: seeding, LC: level of contamination, L. in: L. innocua, L. se: L. seeligeri, L. we: L.welshimeri, L. gra: L. grayi, L. mono: L. monocytogenes)
Categ‐ory
Study TypeN°
Sample Sample Code Strain TC
LC (CFU/25g)
Reference method: final result
Alternative method
Discor‐ dance
Vidas test
Confirma‐tion
Final results
(2) PV d1 2003
Raw cow milk
cheese / / / / P ‐
L. se L.we
A ND
PV d2 C22 Picodon L64 L. in sp 3.4 A + L. in P PD
(5)
I18 c1 26
Wrap with chiken,
letuce and parmesan
/ / / / P ‐ / A ND
I18 c1 27
Chiken kebab
sandwich with
crudeness
/ / / / P ‐ L. gra A ND
I18 c1 15
Cooked potatoes
and strasbourg sausages salad
LIS. 6.24
L. we se 3.8 P ‐ L.we A ND
(6) PV e1 E4 Water
outlet filter L28
L. mono 1/2a
sp 2.2 P ‐ L. mono A ND
Negative deviation:A positive result is obtained by the reference method whereas a negative result is obtained by the alternative method.
Five (5) negative deviations were observed (including 3 for samples naturally contamined):
‐ For 1 negative deviation (c1+:26), the protocol of the alternative method was applied and non typicals colonies was observed. The first enrichment in half‐Fraser is identical for the two methods, however the transfer volume as well as time and temperature applied for the second enrichment in Fraser, are different between the two methods (appendix 1). It is probable that the enrichment of the alternative method did not allow to reach the threshold of the Vidas LIS method
‐ Four (4) negatives deviations (d1+: 2003; c1+: 15 and 27; e1+: E4) are false negative highlighted by the confirmation protocol of the alternative method. It is probable that the enrichment did not allow to reach the threshold of the Vidas LIS test.
NB : 3 negatives deviations were obtained for the category “composite foods”, but the total number of
positive samples is equal to 61 instead of the minimum requierement of 30.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 14/58
Positives deviations:A positive result is obtained by the alternative method whereas a negative result is obtained by the reference
method.
‐ One (1) positive deviations (d2+: C22) was observed. The first enrichment in half‐Fraser is identical for the two methods, however the transfer volume as well as time and temperature applied for the second enrichment in Fraser, are different between the two methods (appendix 1). It is probable that the enrichment of the reference method did not allow to reach the threshold.
Table 8 shows the difference between negative deviations and positive deviations and the acceptability limits
for each categories.
Table 8: acceptability limits
Category Type ND PD (ND‐PD) (ND+PD) Acceptability limit (AL) Observation
(1) Meat products
m1 0 0
/ / /
(ND‐PD) ≤ AL and (ND+PD) ≤ AL paired
data
m2 0 0
m3 0 0
Total 0 0 0 0 (ND – PD)AL=3 ; (ND+
PD)AL=6
(2) Dairy products
d1 0 0
/ / / d2 1 0
d3 0 1
Total 1 1 0 2 (ND – PD)AL=3 ; (ND+
PD)AL=6
(3) Sea food products s
s1 0 0
/ / / s2 0 0
s3 0 0
Total 0 0 0 0 (ND – PD)AL=3 ; (ND+
PD)AL=6
(4) Vegetal products
v1 0 0
/ / / v2 0 0
v3 0 0
Total 0 0 0 0 (ND – PD)AL=3 ; (ND+
PD)AL=6
(5) Composite food
c1 3 0
/ / / c2 0 0
c3 0 0
Total 3 0 3 3 (ND – PD)AL=3 ; (ND+
PD)AL=6
(6) Environmental
sample
e1 1 0
/ / / e2 0 0
e3 0 0
Total 1 0 1 1 (ND – PD)AL=3 ; (ND+
PD)AL=6
All categories Total 5 1 4 6 (ND – PD)AL=6 ; (ND+
PD)AL=16
The observed values are below or equal to the acceptability limits for each category and for the combined categories. The alternative method produces results comparable to the reference method.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 15/58
3.2. Relative level of detection study
3.2.1. Matrices Different “strain‐matrix” couples were studied in parallel with the reference method and the VIDAS LIS 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 9.
Table 9 : « matrix – strain » couples of the relative level of detection
Category Matrix Strain Code strain Strain origin Study
Protocol for categories 1, 2, 3, 4 and 6 (previous validation)One negative control and 3 to 4 level of contaminations were tested. Six replicates for each level of contamination were inoculated and analysed by the reference method and the alternative method. Artificial contamination was carried out in accordance with the requirements of the EN ISO 16140 standard and of the AFNOR Technical Board in force.
As the two methods have the same enrichment step in half‐Fraser, the same test portions of 25 g was tested by the two method. Test portion 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 Listeria spp were used to spike the samples before analysis.
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 Listeria spp using the reference method was also performed to check the absence of the target analyte in the matrix.
Protocol for the category 5 (assays 2018)Three levels of contamination 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.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 16/58
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 Listeria spp using the reference method was also performed to check the absence of the target analyte in the matrix.
3.2.3. 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 10.
Table 10 : 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)
Category Name AL RLOD RLODL RLODU b=ln (RLOD) sd(b) z‐Test statistic p‐value
3.2.4. 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 Listeria spp in the categories tested.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 17/58
3.3. Inclusivity and exclusivity study The inclusivity and exclusivity of the method are defined by analyzing, respectively, 50 positive strains and 30 negative strains. The specificity study was performed again in its entirety in 2006.
The strains were first grown in a non selective broth for 24 hours at 30°C. For inclusivity, a half Fraser broth was then inoculated with 10 to 100 Listeria and the full VIDAS Listeria enrichment protocol was then applied before VIDAS testing. For exclusivity, a non selective broth was then inoculated with about 105 cells/mL and then incubated for 24 hours at 30°C before WIDAS testing. The results are set out in Appendix 5.
3.3.1. Results The 50 Listeria strains (25 Listeria monocytogenes strains and 25 other Listeria strains) were all tested using the VIDAS LIS test. All the Listeria spp strain provided a positive result and no cross‐reactions were observed with non‐target strains.
3.3.2. Conclusion The selectivity of the method is satisfactory.
3.4. Practicability
1. Storage conditions of thecomponents (see package insert) – Expiration date of unopened products (see package insert)
The storage temperature of the VIDAS LIS kit is 2‐8°C. The kit expiration date is shown on the box label and on the various vials.
2. Conditions of use after first use(see package insert)
The kit components should be stored at 2‐8°C. If stored as recommended (pouch correctly resealed with desiccant after use, etc.), all the components will remain stable until the expiration date indicated on the label.
3. Time‐to‐result:
Step Time required (Day) VIDAS LIS method
Time required (Day) EN ISO 11290‐1 standard
Pre‐enrichment D0 D0
Inoculation of Fraser D1 D1
Streaking on selective media / D1 & D3
Perform VIDAS LIS test D2 /
Plate reading / D4
Obtention of negative results (if no characteristic colonies)
D2 D4
Confirmation testing / D2 to D5
Obtention of negative results (after negative confirmation testing if necessary)
D4 to D6 D4 to D6
Obtention of positive results (confirmation of typical colonies) D5 to D6 D5 to D6
4. Steps common to the referencemethod
The two enrichment steps, confirmation testing (reference method tests including purification)
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 18/58
4. Interlaboratory study
4.1. Organization of the interlaboratory study The interlaboratory study was realized by the expert laboratory and 15 participating laboratories.
Number of samples per laboratory: 24 samples per method were prepared to represent 3 levels ofcontamination, with 8 samples per level for each method (48 samples per parcel).
4.3. Control of the experimental parameters
4.3.1. Samples preparation and spiking The following table shows the contamination rates obtained and the estimated accuracies:
Table 11: contamination levels
Level Samples Targeted theoretical level
(CFU/25 g) Real level (CFU/25 g)
Level 0 (L0)
3‐4‐9‐10‐17‐18‐21‐22 0 0
Low level (L1)
1‐2‐7‐8‐13‐14‐19‐20 3 2.5
High level (L2)
5‐6‐11‐12‐15‐16‐23‐24 30 22.8
4.3.2. Temperature of the samples The measured temperatures at reception are listed in the table below.
Table 12: temperature measurements
Laboratory Temperatures (°C)
Comments communicated by the laboratory
measured by the temperature probe
A 4,7°C 2,7°C
B 5,0°C 4,7°C
C 2,8°C 3,2°C
D 4,3°C 2,7°C
E 4,7°C 2,7°C
F 2,1°C 2,7°C
G 1,8°C 2,2°C
H / / Receipt at D2
I 3,6°C 3,5°C
J 5,0°C 2,7°C
K 5,2°C 2,7°C
L 3,5°C 3,7°C
M 4,5°C 4,2°C
N 3,0°C 4,7°C
O 4,0°C 3,2°C
Fourteen of the 15 laboratories received their samples the day after they were sent.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 19/58
4.4. Results Fourteen laboratories out of the 15 laboratories were finally retained for the study (exclusion of laboratory H).
4.4.1. Total viable counts For the whole laboratories, the total viable counts at 30°C vary between 800 CFU/g and 9900 CFU/g.
4.4.2. Expert laboratory results The results obtained by the expert laboratory are summarized in table 13 (raw results in appendix 6).
Table 13 : 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.
4.4.3. Collaborators results The results are summarized in tables 14 Raw results in appendix 6.
Table 14 : results for all laboratories
Alternative method Reference method
Labora‐tory
L0 L1 L2 Labora‐tory
L0 L1 L2 Before Conf.
After. Conf.
Final result
BeforeConf.
After. Conf.
Final result
BeforeConf.
After. Conf.
Final result
A 0/8 0/8 0/8 8/8 8/8 8/8 8/8 8/8 8/8 A 0/8 8/8 8/8
B 0/8 0/8 0/8 6/8 6/8 6/8 8/8 8/8 8/8 B 0/8 6/8 8/8
C 1/8 0/8 0/8 8/8 8/8 8/8 8/8 8/8 8/8 C 0/8 8/8 8/8
D 0/8 0/8 0/8 7/8 7/8 7/8 8/8 8/8 8/8 D 0/8 7/8 8/8
E 0/8 0/8 0/8 7/8 7/8 7/8 8/8 8/8 8/8 E 0/8 7/8 8/8
F 0/8 0/8 0/8 8/8 8/8 8/8 8/8 8/8 8/8 F 0/8 8/8 8/8
G 0/8 0/8 0/8 7/8 7/8 7/8 8/8 8/8 8/8 G 0/8 7/8 8/8
I 0/8 0/8 0/8 7/8 7/8 7/8 8/8 8/8 8/8 I 0/8 7/8 8/8
K 0/8 0/8 0/8 8/8 8/8 8/8 8/8 8/8 8/8 K 0/8 8/8 8/8
L 0/8 0/8 0/8 6/8 6/8 6/8 8/8 8/8 8/8 L 0/8 6/8 8/8
M 0/8 0/8 0/8 7/8 7/8 7/8 8/8 8/8 8/8 M 0/8 7/8 8/8
N 0/8 0/8 0/8 8/8 8/8 8/8 8/8 8/8 8/8 N 0/8 8/8 8/8
O 0/8 0/8 0/8 5/8 5/8 5/8 8/8 8/8 8/8 O 0/8 5/8 8/8
Total 0
/112 0
/112 0
/112 99 /112
99 /112
99 /112
112 /112
112 /112
112 /112
Total 0
/112 99 /112
112 /112
4.4.4. Analysis of the results and collaborators selected for the statistical analysis The results of 14 laboratories were considered.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 20/58
4.5. Interpretation of the results and statistical analysis
4.5.1. Interpretation of the results The interpretation of the results is shown in the table below.
Table 15 : 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= 0 NA= 112
112 including PPND= 0 including PPNA= 1
Total 0 112 112
L1
AM+ PA= 99 PD= 0 99
AM‐ ND= 0 NA= 13
13 including PPND= 0 including PPNA= 0
Total 99 13 112
L2
AM+ PA= 112 PD= 0 112
AM‐ ND= 0 NA= 0
0 including PPND= 0 including PPNA= 0
Total 112 0 112
4.5.2. Specificity of the methods The percentage specificity of the reference method and the alternative method is calculated using the data after confirmation, based on the results of level L0.
‐ Specificity of the reference method: 1 100% = 100.0%,
‐ 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.
4.5.3. 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 16.
In this study, fractional positive results are observed at level L1 only.
Table 16 : summary of the sensitivity study results for all the categoriesof the application scope Parameter ISO 16140‐2 formulas Results
Sensitivity of the alternative method
∗ 100% 100.0%
Sensitivity of the reference method SE refPA ND
PA ND PD∗ 100% 100.0%
Relative trueness RTPA NA
N∗ 100% 100.0%
False positive ratio FPRFP
∗ 100% 0%
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 21/58
4.5.4. Determination of the acceptability limit and conclusion The difference between (ND – PD) and the addition (ND+PD) for the level where fractional recovery was obtained (L1) are calculated. The observed value found for (ND – PD) and (ND+PD) shall not be higher than the acceptability limit (AL). Results are shown in the table 17.
Table 17: acceptability limits
Level N lab (ND‐PD) (ND+PD) Acceptability limit (AL)
L1 14 0 0 (ND – PD)AL=4 ; (ND + PD)AL=6
The values (ND‐PD) and (ND+PD) for the level L1 are 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.
4.5.5. Determination of the relative level of detection This evaluation is performed according to Annex F of ISO/FDIS 16140‐2:2016 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 19 : values obtained for the determination of the relative level of detection
Name RLOD RLODL RLODU b=ln(RLOD) sd(b) z‐Test statistic
The following calculation is performed according to the new excel spread sheet “ RLOD_inter‐lab‐study_16140‐2_AnnexF_ver1_28‐06‐2017”.
Reference method Alternative method
Detection limit
Lower conf. limit
Upper conf. limit
Detection limit
Lower conf. limit
Upper conf. limit
LOD50% (50% limit of detection in CFU/sample size)
0.80 0.63 1.03 0.80 0.63 1.03
LOD95% (95% limit of detection in CFU/sample size)
3.48 2.73 4.43 3.48 2.73 4.43
rLOD 1.00 (0.75 – 1.33)
Conclusion : The methods are not significantly different at the 5% significance level (change in deviance of the model with method effects to the null model Dmethod = 0 with 1 degree of freedom, p‐value 1). The relative limit of detection (RLOD) of the alternative method, as compared to the reference method, is 1 with a 90% confidence interval of 0,75 ‐ 1,33.
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 22/58
5. General conclusion
Method comparison studyThe performances of the VIDAS LIS test are comparable to those of the standard ISO 11290‐1 : 2017.
This study concerned 489 samples of six categories of products:
‐ Meat products
‐ Dairy products
‐ Seafood products
‐ Vegetal products
‐ Composite food
‐ 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 : 98.2% ‐ sensitivity of the reference method : 99.5% ‐ relative trueness: 99.0% ‐ false positive ratio: from 0.0%
Some discordant results were observed. The first enrichment in half‐Fraser is identical for the two methods, however time and temperature applied for the second enrichment in Fraser, are different between the two methods (appendix 1). That could be explain the discordant results observed.
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 1.000 and 1.315 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.
Massy, the 24th of January 2019, Olivier Mathia
Innovation Biologie Unit Manager
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 23/58
Appendix 1 :
Protocols of the reference method
and
the alternative method
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 24/58
EN ISO 11290-1/A1 : 2005
X g (mL) sample + 9X mL half Fraser broth
Incubation for 24±2 h at 30±1 °C
Isolation on selective agars Transfer of 0.1 mL enriched broth O.A.(1) and Palcam (2) in 10 mL Fraser broth
(1) Incubation 24±3 h and 24±3 h at 37±1 °C Incubation 48±2 h (2) Incubation 48±3h at 37±1 °C
at 37±1 °C
Isolation on selective agars O.A. (1) and Palcam (2)
(1) Incubation 24±3 h and 24±3 h at 37±1 °C (2) Incubation 48±3 h
at 37±1 °C
Presence of typical colonies of Listeria spp (5 colonies per medium for testing)
Confirmation of the genus Listeria spp
Species identification: - Catalase - Gram
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 25/58
EN ISO 11290-1 / 2017
Test portion (25g or 25 mL) + 225g or 225 mL half Fraser broth
Incubation: 25 h ± 1 h at 30 °C ± 1 °C
0,1 mL culture + 10 mL Fraser broth
Incubation: 24 h ± 2 h at 37 °C ± 1 °C
Plating out on Agar Listeria according to O&A + second selective medium
O&A Listeria agar incubation : 24h ± 2 h and an additional 24 h ± 2 h at 37 °C ± 1 °C Second selective medium incubation: according to manufacturer
Confirmation:
Inoculation of non selective agar medium, incubation: 37±1°C
Confirmation mandatory of L.monocytogenes:Microscopic aspect, β haemolysis,
L-Rhamnose, D-xylose
Confirmation mandatory of L.spp:Microscopic aspect, catalase
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 26/58
PROTOCOL FOR THE ALTERNATIVE METHOD
Dilute to 1/10 in half-Fraser broth
Incubate for 20-26 hrs at 30°C
± 1°C
Transfer 1 ml of primary enrichment broth into 10
ml of complete Fraser medium.
Presence of
characteristic
colonies
Reincubate for 24 hrs at 37°C
Confirmations
Prepare the test sample by weighing 25 g of product.
Heat for 15 minutes 1 minute at 95-100°C.
Incubate for 20-26 hrs at 30°C ± 1°C
Collect 1 ml of secondary
enrichment broth in a test
tube.
Store the secondary enrichment broth
at 2-8°C for subsequent confirmation
of tests + After cooling, place 0.5 ml in a strip and pass it
through the VIDAS system.
NO Response:
Absence of Listeria
in 25 g
Kit results:
positive
Isolate on Listeria-selective agar
YES
Incubate for 24 hrs at 37°C
NO
YES
YES
NO Response:
Absence of Listeria in 25 g Presence of
characteristic
colonies
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 27/58
Appendix 2 :
Artificial contamination
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 28/58
Category TypeSample
numberSample
Code
strainStrain Origin Inoculation by: Protocol of seeding Stress intensity
Inoculation
level
(CFU/25g)
Global result
d1 18‐1 Raw milk 1 LIS.3.5 L. ivanovii Ewe raw milk Seeding 48h at 5 ± 3 °C / 2.2 +
d1 18‐3 Raw milk 2 LIS.3.5 L. ivanovii Ewe raw milk Seeding 48h at 5 ± 3 °C / 2.2 +
d1 18‐5 Raw milk 3 LIS.3.5 L. ivanovii Ewe raw milk Seeding 48h at 5 ± 3 °C / 2.2 +
d1 18‐7 Raw milk 4 LIS.3.1 L. ivanovii Raw milk Seeding 48h at 5 ± 3 °C / 2,0 +
d1 18‐9 Raw milk 5 LIS.3.1 L. ivanovii Raw milk Seeding 48h at 5 ± 3 °C / 2,0 +
d1 18‐11 Raw milk 6 LIS.3.1 L. ivanovii Raw milk Seeding 48h at 5 ± 3 °C / 2,0 +
d2 C21 Goat's cheese with raw milk L64 L. innocua Époisses cheese Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.6 5.1 +
d2 C24 Goat's cheese made from raw milk L64 L. innocua Époisses cheese Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.6 5.1 +
d2 C25 Goat's cheese made from raw milk L37 L. monocytogenes ½ b Maroilles cheese made with raw milk Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.2 14.6 +
d2 D1 Goat's cheese log made from raw milk, salt‐free L111 L. innocua Munster cheese made with raw milk Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 1.4 +
d2 D2 Le Chevrot cheese L111 L. innocua Munster cheese made with raw milk Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 2.8 +
d2 F21 Goat's cheese made from raw milk L72 L. innocua Boulette d’Avesnes cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C >1 ND ‐
d2 G8 Goat's cheese mini‐log made from raw milk L72 L. innocua Boulette d’Avesnes cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.4 0.6 ‐
d2 G9 Goat's cheese made from raw milk L72 L. innocua Boulette d’Avesnes cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.4 0.4 ‐
d2 H4 Goat's cheese made from raw milk L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 5,0 ‐
d2 H5 Crottin de Chavignol goat's cheese L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 8,0 ‐
d2 H7 Goat's cheese made from raw milk L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 0.2 ‐
d2 H8 Crottin de Chavignol goat's cheese L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 0.3 ‐
d2 J3 Etorki cheese L62 L. monocytogenes Reblochon cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.5 2.6 +
d3 C20 Powdered milk L64 L. innocua Époisses cheese Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.6 3.4 +
d3 C22 Picodon goat's cheese L64 L. innocua Époisses cheese Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.6 3.4 +
d3 C23 Picodon goat's cheese L37 L. monocytogenes ½ b Maroilles cheese made with raw milk Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.2 14.6 ‐
d3 D3 Goat's cheese L111 L. innocua Munster cheese made with raw milk Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 4.2 +
d3 F20 Rocamadour goat's cheese L72 L. innocua Boulette d’Avesnes cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C >1 ND ‐
d3 H3 Goat's cheese log L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 2.5 ‐
d3 H6 Goat's cheese log L72 L. innocua Boulette d’Avesnes cheese Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.2 0.1 ‐
d3 18‐13 Emmental LIS.2.12 L. innocua Raw milk Seeding 48h at 5 ± 3 °C 2.6 +
d3 18‐15 Gouda LIS.2.12 L. innocua Raw milk Seeding 48h at 5 ± 3 °C 2.6 +
Seafood
productss1 18‐17 Cod fillet LIS.5.6 L. seeligeri Smoked halibut Seeding 48h at 5 ± 3 °C 1.8 +
v2 D15 Heart of lettuce L66 L. innocua Spinach Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.3 12.8 +
v2 D16Mixed raw vegetables with carrots and white
cabbageL66 L. innocua Spinach Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.3 12.8 +
v2 D22 Mixed salad L112 L. innocua Potato fries Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 3.8 +
v2 E16 Soya L125 L. monocytogenes Pan‐fried vegetables Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 8.1 +
v2 F19 Crottin de Chavignol goat's cheese L72 L. innocua Boulette d’Avesnes cheese Spiking 50 mins at 55°C, 35 mins at ‐80°C >1 ND ‐
v2 G4 Mushrooms L47 L. monocytogenes Fried potatoes Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 7.1 +
v2 G5 Red cabbage L112 L. innocua Potato fries Spiking 50 mins at 55°C, 35 mins at ‐80°C 1.5 9.6 +
v3 D17 Catalan salad mix L112 L. innocua Potato fries Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 6,0 +
v3 D18 Red cabbage L112 L. innocua Potato fries Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 4,0 +
v3 D19 Cucumbers with vinaigrette L66 L. innocua Spinach Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.3 9.6 +
v3 D21 Diced mixed vegetables L112 L. innocua Potato fries Spiking 45 mins at 55°C, 30 mins at ‐80°C, 5 mins at 46°C 0.4 3.8 +
v3 E17 Celery remoulade L125 L. monocytogenes Pan‐fried vegetables Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 8.1 +
v3 E18 Grated carrots with vinaigrette L125 L. monocytogenes Pan‐fried vegetables Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 16.2 +
v3 G1 Seasoned pasta salad L47 L. monocytogenes Fried potatoes Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 7.1 +
v3 G3 Celery remoulade L112 L. innocua Potato fries Spiking 50 mins at 55°C, 35 mins at ‐80°C 1.5 9.6 +
v2 18‐19 Letuce LIS.6.7 L. welshimeri Spinach Seeding 48h at 5 ± 3 °C / 2.8 +
v2 18‐21 Tomato LIS.6.7 L. welshimeri Spinach Seeding 48h at 5 ± 3 °C / 2.8 +
c1 11 Sandwich with tomato, letuce and chicken LIS.5.12 L. seeligeri Chicken burger Seeding 48h at 5 ± 3 °C / 1,0 ‐
c1 12 Torti with surimi LIS.5.12 L. seeligeri Chicken burger Seeding 48h at 5 ± 3 °C / 1,0 ‐
c1 15 Coocked potatoes with strasbourg sausages LIS.6.24 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 3.8 +
c1 32 Falafels, chickpea, and carrots LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 1.2 ‐
c1 34 Alaska salad : surimi and pineapples LIS.4.5 L. monocytogenes 1/2a Ham and crudeness Seeding 48h at 5 ± 3 °C / 1.2 +
c1 68 Chicken crudeness salad LIS.3.13 L. ivanovii Raw lamb meat Seeding 48h at 5 ± 3 °C / 8,0 ‐
c1 69 Potatoes and sausages salad LIS.3.13 L. ivanovii Raw lamb meat Seeding 48h at 5 ± 3 °C / 8,0 +
c1 70 Farfalles with tomatoes and parmesan LIS.3.13 L. ivanovii Raw lamb meat Seeding 48h at 5 ± 3 °C / 8,0 +
c1 71 Salami sandwich LIS.6.24 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 5,0 ‐
c1 72 Sandwich with rosette LIS.6.24 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 5,0 +
c1 73 beets LIS.6.24 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 5,0 +
c1 18‐23 Sandwich with tuna and crudeness LIS.5.8 L. seeligeri Sea food terrine Seeding 48h at 5 ± 3 °C / 2.8 +
c1 18‐25 Sandwich with salmon and cream LIS.5.8 L. seeligeri Sea food terrine Seeding 48h at 5 ± 3 °C / 2.8 +
c1 18‐27 Sandwich with salmon and chives LIS.5.8 L. seeligeri Sea food terrine Seeding 48h at 5 ± 3 °C / 2.8 +
c1 18‐29 Sandwich with surimi LIS.5.8 L. seeligeri Sea food terrine Seeding 48h at 5 ± 3 °C / 2.8 +
c1 18‐31 Tabbouleh with chicken LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 2.4 +
c1 18‐33 Sandwich with chicken LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 2.4 +
c1 18‐35 Chicken rice salad LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 2.4 +
c1 18‐37 Crudeness salad LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 2.4 +
c1 18‐39 Grated carrot with dressing LIS.4.81 L. monocytogenes Mixed vegetables Seeding 48h at 5 ± 3 °C / 2,0 +
c1 18‐41 Cuncumber with dressing LIS.4.81 L. monocytogenes Mixed vegetables Seeding 48h at 5 ± 3 °C / 2,0 +
c2 7 Kebab with chicken LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 0.8 ‐
c2 6 Lorraine quiche LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 0.8 ‐
c2 8 Three cheese pizza LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 0.8 ‐
c2 9 Gratin of endive with ham LIS.5.11 L. seeligeri Chicken sandwich with crudeness Seeding 48h at 5 ± 3 °C / 0.8 ‐
c2 13 Moussaka LIS.5.12 L. seeligeri Chicken burger Seeding 48h at 5 ± 3 °C / 1,0 ‐
c2 16 Chicken caramelized with rice and vegetables LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 8.6 +
c2 17 Porc with caramel, rice and oignon sauce LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 8.6 +
c2 18 Tortilla with oignons, potatoes and eggs LIS.4.6 L. monocytogenes 1/2a Ham and emmental sandwich Seeding 48h at 5 ± 3 °C / 0.8 +
c2 19 Flammkuchen with bacon and white cheese LIS.4.6 L. monocytogenes 1/2a Ham and emmental sandwich Seeding 48h at 5 ± 3 °C / 0.8 +
c2 23 Pasta with poultry and, tomato and basilic sauce LIS.2.1 L. innocua Sandwich with vegetables Seeding 48h at 5 ± 3 °C / 1.8 +
c2 24 Polenta with duck and vegetables LIS.2.1 L. innocua Sandwich with vegetables Seeding 48h at 5 ± 3 °C / 1.8 ‐
c2 29 Carbonara tagliatelle LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 1.2 +
c2 30 Pizza with 4 cheeses LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 1.2 +
c2 31 Pizza with mushroom and ham LIS.2.7 L. innocua Sandwich with chicken and bacon Seeding 48h at 5 ± 3 °C / 1.2 +
c2 33 Croissant with chicken and emmental LIS.4.5 L. monocytogenes 1/2a Ham and crudeness Seeding 48h at 5 ± 3 °C / 1.2 +
c2 35 Bouche à la reine LIS.4.5 L. monocytogenes 1/2a Ham and crudeness Seeding 48h at 5 ± 3 °C / 1.2 +
c2 18‐43 Pizza with goat cheese and bacon LIS.4.4 L. monocytogenes 1/2a skewer of goat cheese and zucchini Seeding 48h at 5 ± 3 °C / 2.4 +
c2 18‐45 Pizza with three cheeses LIS.4.4 L. monocytogenes 1/2a skewer of goat cheese and zucchini Seeding 48h at 5 ± 3 °C / 2.4 +
c2 18‐47 Zucchini gratin with porc meat LIS.4.4 L. monocytogenes 1/2a skewer of goat cheese and zucchini Seeding 48h at 5 ± 3 °C / 2.4 +
c2 18‐49 Hamburger LIS.6.11 L. welshimeri Ground beef with green bean Seeding 48h at 5 ± 3 °C / 1.6 +
c2 18‐51 Beef bourgignon LIS.6.11 L. welshimeri Ground beef with green bean Seeding 48h at 5 ± 3 °C / 1.6 +
c2 18‐53 Blanquette of veal LIS.6.11 L. welshimeri Ground beef with green bean Seeding 48h at 5 ± 3 °C / 1.6 +
c2 18‐55 Porc with caramel LIS.3.8 L. ivanovii Bacon Seeding 48h at 5 ± 3 °C / 2.8 +
c2 18‐57 Spaghetti carbonara LIS.3.8 L. ivanovii Bacon Seeding 48h at 5 ± 3 °C / 2.8 +
c2 18‐59 Flamenkuche LIS.3.8 L. ivanovii Bacon Seeding 48h at 5 ± 3 °C / 2.8 +
c2 18‐61 Quiche lorraine LIS.3.8 L. ivanovii Bacon Seeding 48h at 5 ± 3 °C / 2.8 +
c3 10 Apple pie LIS.5.12 L. seeligeri Chicken burger Seeding 48h at 5 ± 3 °C / 1,0 ‐
c3 14 Fruit tart LIS.6.24 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 3.8 +
c3 20 Bears' paws LIS.4.6 L. monocytogenes 1/2a Ham and emmental sandwich Seeding 48h at 5 ± 3 °C / 0.8 ‐
c3 21 Pudding LIS.2.1 L. innocua Sandwich with vegetables Seeding 48h at 5 ± 3 °C / 1.8 +
c3 22 ParisBrest LIS.2.1 L. innocua Sandwich with vegetables Seeding 48h at 5 ± 3 °C / 1.8 +
c3 74 Baba with whipped cream LIS.6.25 L. welshimeri Tabbouleh Seeding 48h at 5 ± 3 °C / 3.4 +
c3 75 Rolls with butter cream and vanilla LIS.6.25 L. welshimeri Coconut pearl Seeding 48h at 5 ± 3 °C / 3.4 +
c3 76 Rolls with butter cream and coffee LIS.6.25 L. welshimeri Coconut pearl Seeding 48h at 5 ± 3 °C / 3.4 +
c3 77 Cornet with pastry cream and vanilla sugar LIS.6.25 L. welshimeri Coconut pearl Seeding 48h at 5 ± 3 °C / 3.4 +
c3 18‐63 Pudding LIS.6.5 L. welshimeri Cream dessert Seeding 48h at 5 ± 3 °C / 1.4 +
c3 18‐65 Pudding with coconuts LIS.6.5 L. welshimeri Cream dessert Seeding 48h at 5 ± 3 °C / 1.4 +
c3 18‐67 Apple pie LIS.6.5 L. welshimeri Cream dessert Seeding 48h at 5 ± 3 °C / 1.4 +
c3 18‐69 Lemon pie LIS.5.4 L. seeligeri Pastry cream Seeding 48h at 5 ± 3 °C / 2,0 +
c3 18‐71 Strawberry pie LIS.5.4 L. seeligeri Pastry cream Seeding 48h at 5 ± 3 °C / 2,0 +
e1 E1 Water from outlet at sauce station L144 L. innocua 6b Bin surface Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 3,0 +
e1 E2 Steriflow water L144 L. innocua 6b Bin surface Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 3,0 ‐
e1 E3 Residual water in gray container L144 L. innocua 6b Bin surface Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.6 3.8 +
e1 E4 Water from filter outlet L28 L. monocytogenes ½ Surface sponge Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.9 2.2 +
e1 E5 Doser rinsing water L28 L. monocytogenes ½ Surface sponge Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.9 4.4 +
e1 F29 Water from final rinsing sink L115 L. seeligeri Pool water Spiking 50 mins at 55°C, 35 mins at ‐80°C ND ND ‐
e1 F30 Process water L115 L. seeligeri Pool water Spiking 50 mins at 55°C, 35 mins at ‐80°C ND ND ‐
e1 G10 Water from final rinsing sink L132 L. innocua Cheese counter chopping board Spiking 50 mins at 55°C, 35 mins at ‐80°C 0.8 21.6 +
e1 82 Rince water 3 LIS.2.11 L. innocua Raw cow milk filter Seeding 48h at 5 ± 3 °C / 1.2 ‐
e1 83 Rince water 1 LIS.2.11 L. innocua Raw cow milk filter Seeding 48h at 5 ± 3 °C / 1.2 +
e1 84 Rince water 2 LIS.2.11 L. innocua Raw cow milk filter Seeding 48h at 5 ± 3 °C / 1.2 +
e1 85 Water from wash station LIS.4.16 L. monocytogenes 1/2a Surface control sewer Seeding 48h at 5 ± 3 °C / 1.2 ‐
e1 86 Water from washing station 2 LIS.4.16 L. monocytogenes 1/2a Surface control sewer Seeding 48h at 5 ± 3 °C / 1.2 +
e2 87 Swab 1 LIS.4.16 L. monocytogenes 1/2a Surface control sewer Seeding 48h at 5 ± 3 °C / 1.2 +
e2 88 Swab 2 LIS.4.16 L. monocytogenes 1/2a Surface control sewer Seeding 48h at 5 ± 3 °C / 1.2 +
e2 89 Sponge 1 LIS.4.16 L. monocytogenes 1/2a Surface control sewer Seeding 48h at 5 ± 3 °C / 1.2 +
e2 F25 Swab from wall‐floor join L132 L. innocua Cheese counter chopping board Spiking 50 mins at 55°C, 35 mins at ‐80°C 1.3 1,0 ‐
e2 F26 Cold store floor L132 L. innocua Cheese counter chopping board Spiking 50 mins at 55°C, 35 mins at ‐80°C 1.3 0.6 ‐
e2 88 Swab 2 LIS.5.3 L. seeligeri Goat milk filter Seeding 48h at 5 ± 3 °C / 1.4 ‐
e2 89 Sponge 1 LIS.5.3 L. seeligeri Goat milk filter Seeding 48h at 5 ± 3 °C / 1.4 ‐
e3 F28 Residue from cheese‐cutting table L132 L. innocua Cheese counter chopping board Spiking 50 mins at 55°C, 35 mins at ‐80°C ND ND ‐
e3 H12 Residue from cutting facility L132 L. innocua Cheese counter chopping board Spiking 24 hours at ‐80°C, 50 mins at 55°C 0.3 2.2 +
Dairy
products
Vegetal
product
Composite
food
Environ‐
mental
sample
Artificial contamination
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 29/58
Appendix 3 :
Sensitivity:
raw results
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 30/58
sp : spikingse : seedingAC : artificial contaminationLevel of conta.: level of contaminationmix: contamination by mixtureOAA : Ottaviani & Agosti agarOX: Oxford agarPAL: Palcam agarChromo: Chromogenic agar+ : positive result- : negative result/ : test not realizedPA : positive agreementNA : negative agreementPD : positive deviationND : negative deviation
Ø : absence of colonies Ø : absence of annex floraØ, L, M, H: level of bacterial load from absence to high 0 / 1 / 2 / 3 / 4 : level of typical flora, from absence to highA: pure culture of suspected colonies Ø / L / M / H : level of annex flora, from absence to highB: mixing with a majority of suspected colonies L. welshimeri: L. wC: mixing with a minority of suspected colonies L. monocytogenes: L. mD: mixing with rare suspected colonies L. innocua: L. inE: absence of suspected colonies L. seeligeri: L. se(x): x colonies characteristic of Listeria if x ≤ 5 L. ivanovii: L. iv
ComparisonYear Code Matrices (french name) cat. CA Final resultCFU / 25g
FRASER 1/2
Raw milkRaw milkRaw milkRaw milkRaw milk
Dairy products
EN ISO 11290-1 methodFRASER CONFIRMATION
Raw cow milk cheeseRaw cow milk cheeseReblochonRaw cow milk cheeseGoat cheese with raw milkGoat cheese with raw milkGoat cheese with raw milkBrie de MeauxRaw milk cheeseCamembert with raw milkGoat cheese with raw milkCrottin de ChavignolCrottin de ChavignolCrottin de ChavignolCrottin de ChavignolGoat cheese with raw milkGoat cheese cap with raw milkGoat cheese with raw milkGoat cheese with raw milkCrottin de ChavignolGoat cheese with raw milkCrottin de ChavignolGoudaPont l'EvequeMaroillespicodonSt MaureGoat cheeseSt MaureGoat cheese
Reblochon
Goat logGoat logMilk powder
Raw milk
Raw milk
Raw milk
Raw cow milk cheese
Goat cheese with raw milk, without salt
Goat cheese with raw milk
Goat cheese with raw milk
Goat cheese with raw milk
Raw milk
Raw cow milk cheese
Raw cow milk cheese
Heart of Neufchâtel with raw milk
Raw cow milk cheese
Goat
Milk powder
Matrices
Goat cheese with raw milk
Maroilles
Epoisses
Maroilles
Sheep's milk cheesepicodon
Raw cow milk cheese
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 33/58
Final resultYear Code Matrices (french name) Cat. CA
EN ISO 11290-1 method VIDAS LIS
Comparison
CONFIRMATION
Environmental samples
FRASER FRASER 1/2
Water outlet washing machineStanding waterRinsing water DosingStagnant water soil storageNetwork waterFrozen waterNetwork waterFrozen waterStagnant water clean trayWater washing machineStériflow waterSteriflow WaterWater final rinse tankProcess water
Siphon swabSiphon swabWorkplanProduction sector work planSurface hoodStainless steel table - cheese stand
Institut Scientifique d'Hygiène et d'Analyse 45/58
Caption
ST : sample typeSN : sample number# : sample identity◊ : level determined by 3 to 5 enumerationssp : spikingse : seedingnc : naturally contaminatedcm: contamination by mixture+ : positive result- : negative result/ : test not realizedØ : absence of coloniesA : absenceP : presence 0 / 1 / 2 / 3 / 4 : level of typical flora, from absence to highØ / L / M / H : level of annex flora, from absence to highListeria welshimeri : L.wListeria seeligeri: L. se
RLOD composite food (2016, 2018)
VIDAS LIS January 24th, 2019 Summary report - v0
Institut Scientifique d'Hygiène et d'Analyse 46/58