NF VALIDATION – EN ISO 16140 VALIDATION OF THE METHOD VIDAS Listeria monocytogenes II (LMO2 ‐ Ref. 30704) BIO 12/09‐07/02 for the detection of Listeria monocytogenes Protocol for human food products (excluding raw products) and environmental samples SUMMARY REPORT – DECEMBER 2014 – V1 Expert laboratory: Manufacturer: ISHA bioMérieux 25 avenue de la République Chemin de l’Orme 91300 Massy 69280 Marcy L’Étoile France France This test report is only applicable to the items analyzed. Reproduction of this document is authorized only in full photographic facsimile form. It contains 40 pages.
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NF VALIDATION – EN ISO 16140
VALIDATION OF THE METHOD
VIDAS Listeria monocytogenes II (LMO2 ‐ Ref. 30704)
BIO 12/09‐07/02
for the detection of Listeria monocytogenes
Protocol for human food products (excluding raw products) and environmental samples
SUMMARY REPORT – DECEMBER 2014 – V1
Expert laboratory: Manufacturer: ISHA bioMérieux 25 avenue de la République Chemin de l’Orme 91300 Massy 69280 Marcy L’Étoile France France
This test report is only applicable to the items analyzed. Reproduction of this document is authorized only in full photographic facsimile form. It contains 40 pages.
bioMérieux ‐ VIDAS LMO2 30°C Summary report ‐ December 2014
Table of contents 1. Introduction ................................................................................................................................................. 3
1.1. Date(s) and validation history ............................................................................................................. 3
1.2. Principle and protocol for the alternative method ............................................................................. 3
1.2.1. Principle of the method ............................................................................................................... 3
3. Inter‐laboratory study ............................................................................................................................... 11
Appendix 1: Protocol for the alternative method Appendix 2: Protocol for the reference method Appendix 3: List of stressed strains Appendix 4: Results of relative accuracy, relative sensitivity and relative specificity tests Appendix 5: Selectivity results
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1. Introduction
1.1. Date(s) and validation history The VIDAS LMO2 method was validated by AFNOR Certification according to the ISO 16140 standard under the certification number BIO 12/9‐07/02. The validation stages are as follows:
‐ July 2002: initial validation, ‐ September 2002: extension for environmental samples in accordance with EN ISO 16140, ‐ December 2006: first renewal and completion of inter‐laboratory study in accordance with EN ISO 16140 standard, ‐ May 2010: second renewal, ‐ May 2014: third renewal.
The results set out in this report were produced during validation tests carried out by SERMHA, Institut Pasteur in Lille, in accordance with prevailing requirements.
1.2. Principle and protocol for the alternative method
1.2.1. Principle of the method The VIDAS LMO2 method is based on an immuno‐enzymatic assay, enabling the detection of Listeria monocytogenes antigens using the ELFA (Enzyme Linked Fluorescent Assay) method using 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 interior of the SPR is coated with anti‐Listeria monocytogenes antibodies absorbed on its surface.
The strip contains all of the ready‐to‐use reagents required for the test: washing solution, anti‐Listeria monocytogenes antibodies conjugated with alkaline phosphatase and substrate.
An aliquot of the enrichment broth is dispensed in the strip and then all the steps are performed automatically by the VIDAS analytical module. The reaction medium is cycled in and out of the SPR several times, 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 < 0.05 test negative and TV > 0.05 test positive
1.2.2. Protocol The validated protocol is as follows:
‐ pre‐enrichment in half‐Fraser broth, incubated for 24 to 26 hours at 30°C ± 1°C, ‐ subculture in complete Fraser broth (1 ml in 10 ml), incubated for 24 to 26 hours at 30°C ± 1°C
NB: In the comparative study of methods, the minimum incubation time (i.e. 24 hours) was tested.
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The VIDAS LMO2 test is then performed using an aliquot of the unheated complete Fraser broth. Samples found to be positive using the VIDAS LMO2 test are confirmed through isolation on agar: PALCAM, Oxford or a chromogenic agar:
‐ if PALCAM or Oxford agar is used, the characteristic colonies are confirmed using the tests set out in the methods standardized by CEN or ISO, ‐ if a chromogenic agar is used, the presence of typical Listeria monocytogenes colonies is sufficient to confirm the VIDAS LMO2 result.
The outline of the method is set out in Appendix 1.
1.3. Reference method with which the alternative method was compared The horizontal method for the detection and enumeration of Listeria monocytogenes, as per the EN ISO 11290‐1/A1:2004 standard, was used. The method protocol is set out in Appendix 2.
1.4. Scope The scope is as follows: all human food products (excluding raw products) and environmental samples.
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2. Comparative study of methods
2.1. Relative accuracy, specificity and sensitivity According to the EN ISO 16140 standard, a minimum of 60 products per category must be analyzed, with approximately 50% positive products (at least 30 results) and 50% negative products. In the 2002 studies, samples from these five categories were analyzed. The reference method used Listeria‐selective PALCAM and Oxford agars. This method was amended to facilitate the detection of Listeria monocytogenes by introducing a chromogenic agar (Listeria Agar according to Ottaviani and Agosti). Nevertheless, in the context of this validation, ALOA® agars were isolated following the second enrichment in half‐Fraser when samples appeared to be positive following isolation in half‐Fraser broth. Thus, for the majority of samples tested, the results obtained using the reference methods correspond to those that would have been obtained using the amended reference method. The samples for which no result was available using ALOA® agar are primarily negative samples, in which no other Listeria strain was present; the use of chromogenic agar would have led to the same results. Therefore, the results of the 2002 study could have been interpreted according to the ISO 16140 standard. Nevertheless, it should be noted that the majority of samples processed were raw products (87%) that have been excluded from the scope since 2004. In total, 361 samples contaminated and non‐contaminated with Listeria monocytogenes were tested using:
‐ the EN ISO 11290‐1/A1:2004 reference method, ‐ the VIDAS LMO2 method (with an enrichment step at 30°C).
Artificial contamination was carried out using stressed strains in accordance with the requirements of the EN ISO 16140 standard and the AFNOR Validation Technical Board. These relate to 93 positive results. In total, out of 159 positive Listeria monocytogenes results, 60% were obtained following artificial contamination (see Appendix 3). It must be reiterated that raw products have been excluded from the scope since 2004 and it is difficult to find non‐raw processed products that are positive for Listeria monocytogenes. The various samples analyzed and their results are set out in Appendix 4. The crossed‐out samples are those not taken into account in the calculations in order to balance the number of negative samples with the number of positive samples. The results obtained for the 361 samples analyzed are broken down as follows:
Positive reference method
(R+) Negative reference method
(R‐) Total
Positive alternative method (A+)
Positive agreement (A+/R+) ‐ PA = 148
Positive deviation (R‐/A+) ‐ PD = 3
151
Negative alternative method (A‐)
Negative deviation (A‐/R+) ‐ ND = 5*
Negative agreement (A‐/R‐) ‐ NA = 205*
210
Total 153 208 361
Key: A+ = confirmed positives A‐ = immediate negatives and negatives after confirmation of the presumptive positive * = no unconfirmed VIDAS LMO2 positive results.
All of the results obtained were used to calculate the relative accuracy, relative sensitivity and relative specificity for each of the categories and for all the categories, according to the formulas set out in the EN ISO 16140 standard.
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Category PA NA ND PD Sum N
Relative accuracy AC (%)
[100x(PA+NA])/N
N+ PA + ND
Relative sensitivity SE (%)
[100xPA]/N+
N‐ NA + PD
Relative specificity SP (%)
[100xNA]/N‐
Meat products 27 30 2 1 60 95.0 29 93.1 31 96.8
Dairy products 30 37 0 0 67 100 30 100 37 100
Fish products 26 33 2 2 63 93.7 28 92.9 35 94.3
Vegetable 30 40 0 0 70 100 32 100 40 100
Environment 35 37 1 0 73 98.6 36 97.2 37 100
TOTAL 148 177 5 3 333 97.6 153 96.7 180 98.3
NB: For certain product categories, the number of negative samples was greater than the number of positive samples. So as to avoid introducing any bias in the calculations, certain negative results from 2002, in the "dairy products" and "environmental samples" categories, were eliminated. For the alternative method, the percentage values calculated for the following three criteria according to the EN ISO 16140 standard are:
Relative accuracy: AC 97.6%
Relative sensitivity: SP 96.7%
Relative specificity: SE 98.3%
The AFNOR Technical Board stipulates that the sensitivity of both methods should be recalculated accounting for all confirmed positives (this includes additional positives from the alternative method):
There were eight discrepant results between the reference method and alternative method. The performance of the two methods can be considered to be the same.
2.2. Relative detection level The objective was to determine the level of contamination allowing to obtain 50% of positive results. Various " food product matrix ‐ strain" pairs were studied using in parallel the reference method and the VIDAS LMO2 method, for five categories, with six replicates per contamination level tested. Artificial contamination was carried out in accordance with the requirements of the EN ISO 16140 standard and of the AFNOR Technical Board. The detection levels, calculated using the Spearman‐Kärber method (LOD50), obtained for each "matrix‐strain" combination are as follows:
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Matrix Strain Relative detection level of the reference method (CFU / 25 g or 25 ml)
Process water L.monocytogenes 1/2c 0.6 [0.3 – 1.2] 0.6 [0.3 – 1.2]
* "Hitchins A. Proposed Use of a 50% Limit of Detection Value in Defining Uncertainty Limits in the Validation of Presence‐Absence Microbial Detection Methods, Draft of December 10, 2003". The same detection level was obtained for the alternative method and for the reference method: it is between 0.3 and 1.3 cells per 25 grams.
2.3. Selectivity The inclusivity and exclusivity of the method was defined by analyzing, respectively, 50 positive strains and 30 negative strains. Reminder (results obtained during the 2002 validation study): The results are set out in Appendix 5. Fifty Listeria monocytogenes strains and 43 non‐Listeria monocytogenes strains, of which 28 did not belong to the Listeria genus, were tested using the VIDAS LMO2 test. All the Listeria monocytogenes strains provided a positive result and no cross‐reactions were observed. This study remains valid with regard to the EN ISO 16140 standard.
2.4. Practicability Practicability is studied based on the 13 criteria defined by the Technical Board by comparing the reference method to the VIDAS LMO2 method. The criteria defined by AFNOR are entered below:
1. Method component packaging mode (see package insert) 2. Reagent volume (see package insert and bottle packaging)
The kits are packaged in 60‐test kits containing: ‐ the LMO2 strips, made of polypropylene, consisting of 10 wells covered with aluminum foil, ‐ the LMO2 SPRs, in aluminum pouches containing 30 units, with a desiccant, ‐ the vial of LMO2 standard, ‐ the vials of positive and negative LMO2 controls, ‐ an MLE card required for calibration of the test.
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3. Component storage conditions (see package insert) – Shelf‐life of unopened products (see package insert)
The test storage temperature is 2‐8°C. The shelf‐life of tests is indicated on the kits.
4. Conditions for use after first use (see package insert)
Each reagent should be stored between +2°C and +8°C.
5. Specific equipment or premises required (see package insert)
The equipment required includes: - an incubator at 30°C + 1°C - a water bath of boiling water - a VIDAS automated testing system
6. Reagents ready for use or requiring reconstitution (see package insert)
All reagents are ready for use.
7. Training time for operators with no previous experience of the method
for an operator trained in conventional microbiology techniques, training in the technique takes less than one day.
8. Actual handling time – Flexibility of method with respect to number of samples under analysis
Steps
Mean time for one sample (minutes)
Mean time for 30 samples (minutes)
Standard Alternative Standard Alternative
Preparation, weighing, dilution and grinding
7 7 90 90
Subculture in selective broth 1 1 25 25 Isolation of half‐Fraser and Fraser broth on selective media: Listeria agar and other media
2 / 30 /
Readings 2 / 20 /
Performance of VIDAS test / 4 / 10
TOTAL 12 minutes 0 h 12 m
12 minutes 0 h 12 m
165 minutes 2 h 45 m
125 minutes 2 h 5 m
For positive samples, the time required for confirmations must be added. For the alternative method, the time required for isolation on selective agar, i.e. approximately 1 minute per sample, must be added. The mean time for biochemical confirmation of a suspect colony using a selective agar has been estimated at approximately 5 minutes. The benefit of the alternative method lies notably in the possibility of sorting the negative samples from the suspect samples and thus simplifying the confirmations, as well as in the time saved by technicians when analyzing series of samples.
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9. Lead time to obtain results Negative samples
Step Lead time obtained VIDAS LMO2 method
Lead time obtained reference method
ISO 11290‐1
Performance of primary enrichment D0 D0
Inoculations of various secondary enrichment broths (half‐Fraser)
D1 D1
Performance of VIDAS LMO2 test D2 /
Isolation of selective broths on selective agar / D1 & D3
Negative results obtained - if there are no characteristic colonies - if the VIDAS LMO2 test is negative - if the VIDAS LMO2 test is positive and the confirmation
is negative
D2
D3 to D5
D5
Positive samples:
Step Lead time obtained VIDAS LMO2 method
Lead time obtained reference method
ISO 11290‐1
Performance of primary enrichment D0 D0
Inoculations of various secondary enrichment broths D1 D1
Performance of VIDAS LMO2 test and isolation on selective agars
D2 /
Isolation of selective broths on selective agar / D1 & D3
Confirmation tests: Genus - Isolation on TSAYE - Gram stain, catalase Species - CAMP test, hemolysis, TSBYE broth - Use of carbohydrates - Isolation on chromogenic agar
D3 D4
D4 D5 D2
D2 to D5 D3 to D6
D3 to D6 D4 to D7
Positive results obtained - after confirmation with reference method tests - if API strips used - if isolated on chromogenic agar
D10
D5 D3 to D4
D9 to D12
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10. Type of operator qualification
Identical level to that required for reference method
11. Steps common to reference method
None
12. Traceability of analysis results
A results sheet is printed stating the reagent references, the date and time, the test result and the sample identification.
13. Laboratory maintenance
The VIDAS user manual provides explanations for certain problems. bioMérieux offers technical support by telephone. Various preventive maintenance contracts are available.
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3. Inter‐laboratory study A validation extension was obtained in December 2006 following the completion of the inter‐laboratory study in accordance with the EN ISO 16140 standard. Sixteen laboratories received samples of pasteurized milk. The strain used for contamination was a strain of Listeria monocytogenes (L32), originating from a raw milk cheese. The contamination rates obtained and the estimated precisions are set out in the table below:
Level Samples
Targeted theoretical rate (b/25
ml)
Actual level (b/25 ml of sample)
Estimation of lower contamination limit per 25 ml of sample
Estimation of upper contamination limit per 25 ml of sample
Level 0 6‐7‐8‐14‐15‐19‐20‐21 0 0 / /
Low level 1‐2‐9‐1011‐16‐22‐23 3 4.5 1.2 11.5
High level 3‐4‐5‐12‐13‐17‐18‐24 30 46.6 34 62
As a result of transport conditions, only 14 laboratories carried out the tests, as two laboratories did not receive the samples within the lead time.
‐ Results obtained by participating laboratories Positive results after confirmation obtained with the reference method
Contamination levels
Laboratories L0 L1 L2
Obtained No. of samples Obtained No. of samples Obtained No. of samples
Laboratory A 0 8 8 8 8 8
Laboratory D 0 8 8 8 8 8
Laboratory E 0 8 8 8 8 8
Laboratory F 0 8 4 4 8 8
Laboratory G 0 8 8 8 8 8
Laboratory H 0 8 8 8 8 8
Laboratory I 0 8 8 8 8 8
Laboratory J 0 8 8 8 8 8
Laboratory K 0 8 8 8 8 8
Laboratory L 0 8 8 8 8 8
Laboratory M 0 8 8 8 8 8
Laboratory N 0 8 8 8 8 8
Laboratory O 0 8 8 8 8 8
Laboratory P 0 8 8 8 8 8
Total 0 112 108 108 112 112
(a) (b) (c)
Key: (a): false positive, (b): true positive obtained at level 1, (c): true positive obtained at level 2
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Positive results after confirmation obtained with the alternative method
Contamination levels
Laboratories L0 L1 L2
Obtained No. of samples Obtained No. of samples Obtained No. of samples
Laboratory A 0 8 8 8 8 8
Laboratory D 0 8 8 8 8 8
Laboratory E 0 8 8 8 8 8
Laboratory F 0 8 4 4 8 8
Laboratory G 0 8 8 8 8 8
Laboratory H 0 8 8 8 8 8
Laboratory I 0 8 8 8 8 8
Laboratory J 0 8 8 8 8 8
Laboratory K 0 8 8 8 8 8
Laboratory L 0 8 8 8 8 8
Laboratory M 0 8 8 8 8 8
Laboratory N 0 8 8 8 8 8
Laboratory O 0 8 8 8 8 8
Laboratory P 0 8 8 8 8 8
Total 0 112 108 108 112 112
(a) (b) (c)
Key: (a): false positive, (b): true positive obtained at level 1, (c): true positive obtained at level 2 Laboratory F was excluded from the final statistical analysis of the results, as four samples had not been analyzed owing to leaks. The results for the reference method and alternative method were concordant for all 13 laboratories included. The specificity (SP) and sensitivity (SE) rates were as follows:
Level Reference method Alternative method
SP/SE LCL* % SP/SE LCL* %
L0 SP% = 100 98 SP% = 100 98
L1 SE% = 100 98 SE% = 100 98
L2 SE% = 100 98 SE% = 100 98
L1+L2 SE% = 100 98 SE% = 100 98
* LCL: low critical value, defined by the EN ISO 16140 standard. Relative accuracy was 100%. No discrepancies were observed between the two methods. Comparison of relative accuracy (AC), specificity (SP) and sensitivity (SE) values
Inter‐laboratory study Comparative study
Relative accuracy (AC) 100% 97.6%
Sensitivity (SE) 100% 96.7%
Specificity (SP) 100% 98.3%
The relative accuracy and specificity values obtained from the inter‐laboratory study are comparable to those of the collaborative study.
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The AFNOR Technical Bureau stipulates that the sensitivity of both methods should be recalculated accounting for all confirmed positives (actual positive samples) (this includes additional positives from the alternative method):
Degrees of agreement for each method, at all levels, were identical:
Level Reference method Alternative method
L0 DA % = 100% DA % = 100%
L1 DA % = 100% DA % = 100%
L2 DA % = 100% DA % = 100%
Concordance rates for each method, at all levels, were identical:
Level Reference method Alternative method
L0 Concordance % = 100% Concordance % = 100%
L1 Concordance % = 100% Concordance % = 100%
L2 Concordance % = 100% Concordance % = 100%
The odds ratios for each method, at all levels, were identical:
Level Alternative method Reference method
L0 COR % = 1.00 COR % = 1.00
L1 COR % = 1.00 COR % = 1.00
L2 COR % = 1.00 COR % = 1.00
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4. Conclusions The validation study was performed according to the EN ISO 16140 standard. The comparative study of methods enabled the following results to be obtained:
‐ relative accuracy, relative specificity and relative sensitivity, ‐ relative detection level ‐ inclusivity and exclusivity.
The performance of the VIDAS LMO2 method is equivalent to that of the EN ISO 11290‐1/A1:2004 reference method. This performance was determined by analyzing 333 samples from five product categories. The relative accuracy obtained is 97.6%, the relative sensitivity is 96.7% and the relative specificity is 98.3%, based on the calculations stipulated by the EN ISO 16140 standard. There were eight discrepant results: three positive deviations and five negative deviations. As the samples found to be positive using the alternative method were confirmed positive samples, the sensibility and specificity values were recalculated relative to all of the positive results. The following values were obtained:
‐ 96.8% sensitivity for the alternative method ‐ 98.1% sensitivity for the reference method
The 50% relative detection level of the VIDAS LMO2 method and the reference method was evaluated by artificially contaminating five different products representative of the five categories tested. It is between 0.3 and 1.3 Listeria monocytogenes cells per 25 g or ml of sample for both methods. The method offers good specificity, as all the strains of Listeria monocytogenes were detected (inclusivity) and no cross‐reactions were observed among the Listeria non‐monocytogenes or the non‐Listeria strains tested (exclusivity). The inter‐laboratory study results obtained for all 13 laboratories selected demonstrate that the alternative method and the reference method have equivalent relative accuracy, specificity and sensitivity values in the same level as those obtained in the preliminary study. The variability of the alternative method (degree of agreement, concordance, odds ratio) is identical to that of the reference method, as all of the contaminated samples were found to be negative using both methods.
Signed at Massy, December 19, 2014
François Le Nestour Biology Innovation Unit Manager
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APPENDICES
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APPENDIX 1
Protocol for the reference method
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ISO 11290-1/A1:2004 STANDARD
YES
YES
YES
NO
NO
Incubation for 24 h at 37°C ± 1°C
Incubate for 24 h
Prepare test sample (x g ou x mL)
Dilute to 1:10 in Fraser ½ broth
Isolate onto Listeria agar
OAA and Palcam
Transfer 0,1 mL of Fraser ½
enrichment in 10 mL Fraser 1
Plating on selective Listeria agar : OAA and
Palcam
Re‐incubation 24 h à 37°C ±1°C
Confirmation Response :
Presence of Listeria monocytogenes
Incubation for 24 h at 30°C ± 1°C
Incubation for 48 h at 37°C ± 1°C
NO Response :
Absence of Listeria monocytogenes
Presence of typical
colonies
Presence of typical
colonies
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APPENDIX 2
Protocol for the alternative method
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VIDAS LMO2 (with an enrichment step at 30°C)
Dilute in 225 ml of half-Fraser broth
Incubate for 24-26 hrs at 30°C ± 1°C
Transfer 1 ml of primary enrichment broth into 10 ml of complete Fraser
medium.
Reincubate, if necessary, for 24 hrs at 37°C ± 1°C.
Prepare the test sample by weighing 25 g of product.
Incubate for 24-26 hrs at 30°C ± 1°C
NO
Presence of characteristic
colonies
Confirmations, as required
Store the secondary enrichment broth at 2-8°C for subsequent confirmation of tests
Place 0.5 ml in a strip and pass it through the VIDAS system.
NO Absence of Listeria monocytogenes
in 25 g Kit results:
positive
Isolate on Palcam or Oxford agar or chromogenic medium
YES
Incubate for 24 hrs at 37°C ± 1°C
YES
YES
NOResponse: Absence of Listeria monocytogenes in 25 g
Presence of characteristic
colonies
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APPENDIX 3
List of stressed strains
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Code Name Cat Strain
Type of stress Evaluation of
stress
CFU / 25
g Results
N° Name Origin
E1 Water from outlet at sauce station PV2 L144 Listeria innocua 6b Bin surface 50 minutes at 55°C, 35 minutes at ‐80°C 0,6 3,0 +
E2 Steriflow water PV2 L144 Listeria innocua 6b Bin surface 50 minutes at 55°C, 35 minutes at ‐80°C 0,6 3,0 ‐
E4 Water from filter outlet PL1 L28 Listeria monocytogenes ½ Surface sponge 50 minutes at 55°C, 35 minutes at ‐80°C 0,9 2,2 +
E5 Doser rinsing water PL1 L28 Listeria monocytogenes ½ Surface sponge 50 minutes at 55°C, 35 minutes at ‐80°C 0,9 4,4 +
F25 Swab from wall‐floor join PL1 L132 Listeria innocua Cheese counter chopping board 50 minutes at 55°C, 35 minutes at ‐80°C 1,3 1,0 ‐
F26 Cold store floor PL2 L132 Listeria innocua Cheese counter chopping board 50 minutes at 55°C, 35 minutes at ‐80°C 1,3 0,6 ‐
F28 Residue from cheese‐cutting table PL2 L132 Listeria innocua Cheese counter chopping board 50 minutes at 55°C, 35 minutes at ‐80°C ND ND ‐
F29 Water from final rinsing sink PL2 L115 Listeria seeligeri Pool water 50 minutes at 55°C, 35 minutes at ‐80°C ND ND ‐
F30 Process water PL1 L115 Listeria seeligeri Pool water 50 minutes at 55°C, 35 minutes at ‐80°C ND ND ‐
G10 Water from final rinsing sink PL1 L132 Listeria innocua Cheese counter chopping board 50 minutes at 55°C, 35 minutes at ‐80°C 0,8 21,6 +
J1 Pasteurized goat's cheese PL2 L7 Listeria monocytogenes ½ a Munster cheese rind 50 minutes at 55°C, 35 minutes at ‐80°C >0.9 0,2 ‐
J2 Petit Billy goat's cheese PL2 L37 Listeria monocytogenes ½ b Maroilles cheese made with raw milk 50 minutes at 55°C, 35 minutes at ‐80°C 0,7 0,9 ‐
J3 Etorki cheese PC3 L62 Listeria monocytogenes Reblochon cheese 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 2,6 +
K5 Pasteurized milk PP1 L37 Listeria monocytogenes ½ b Maroilles cheese made with raw milk 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 20,6 +
K6 Pasteurized milk PP1 L62 Listeria monocytogenes Reblochon cheese 50 minutes at 55°C, 35 minutes at ‐80°C 0,1 3,2 +
K16 Pasteurized goat's cheese log PV3 L37 Listeria monocytogenes ½ b Maroilles cheese made with raw milk 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 20,6 +
O21 Egg custard pie PV1 L7 Listeria monocytogenes ½ a Munster cheese rind 50 minutes at 55°C, 35 minutes at ‐80°C 0,4 7,0 +
O22 Frozen leek and carrot patties PV1 L47 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 0,9 ‐
O23 Pan‐fried mushrooms and vegetables PV1 L47 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 0,7 ‐
O24 Tuscan purée PV2 L47 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 0,4 ‐
O25 Cooked potatoes PV2 L47 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 0,7 ‐
O26 Frozen fries PV2 L47 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 0,9 ‐
P1 Potted seafood PV2 L20 Listeria monocytogenes ½ Smoked salmon offcuts 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C >1.4 3,8 ‐
P2 Salmon terrine with crème fraîche PV3 L20 Listeria monocytogenes ½ Smoked salmon offcuts 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C >1.4 3,8 +
P3 Salmon terrine with vegetables PL1 L20 Listeria monocytogenes ½ Smoked salmon offcuts 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C >1.4 2,5 +
P4 Minced tuna with peppers PL1 L20 Listeria monocytogenes ½ Smoked salmon offcuts 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C >1.4 5,0 ‐
P5 Pasteurized Tomme cheese PL2 L18 Listeria monocytogenes ½ c Munster cheese rind 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,0 11,0 ‐
P6 Brin de paille cheese PL2 L51 ND ND ‐
P7 Époisses cheese PL2 L51 ND ND ‐
P8 Limousin goat's cheese PP1 L18 Listeria monocytogenes ½ c Munster cheese rind 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,0 8,3 ‐
P9 Pasteurized goat's cheese log PP1 L18 Listeria monocytogenes ½ c Munster cheese rind 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,0 5,5 ‐
P10 Pasteurized goat's cheese log PV1 L51 Listeria monocytogenes ½ b Matured Germain cheese 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C ND ND ‐
P11 Petit Billy goat's cheese PP3 L51 Listeria monocytogenes ½ b Matured Germain cheese 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C ND ND ‐
P12 Pan‐fried mushrooms and vegetables PP3 mixture with naturally contaminated rice +
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P14 Frozen cauliflower and carrot patties PP1 mixture with naturally contaminated rice +
P15 Frozen leek and carrot patties PV2 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 16,0 +
P16 Pan‐fried "Romaine" vegetables PV2 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 8,0 +
P17 Vegetables for casserole PV2 mixture with naturally contaminated rice +
P19 Potatoes and courgettes with sauce PL1 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 12,0 +
P20 Tuscan purée PL1 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 8,0 +
P21 Wheat PL2 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 16,0 +
P22 Braised chicory PL2 L47 Listeria monocytogenes ½ a Fried potatoes 24 hrs at 4°C, then 50 minutes at 55°C, 30 minutes at ‐80°C 1,8 6,4 +
Q1 Pasteurized goat's cheese log PL2 L32 Listeria monocytogenes 4b Munster cheese rind 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 3,3 <1 ‐
Q2 Pasteurized goat's cheese log PL1 L32 Listeria monocytogenes 4b Munster cheese rind 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 3,3 <1 ‐
Q3 Pasteurized brie cheese PL1 L32 Listeria monocytogenes 4b Munster cheese rind 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 3,3 <1 ‐
Q4 Camembert PL2 L32 Listeria monocytogenes 4b Munster cheese rind 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 3,3 <1 ‐
Q5 Shrimps PL2 L12 Listeria monocytogenes ½ a Smoked salmon 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,4 0,4 ‐
Q6 Shrimps PC3 L12 Listeria monocytogenes ½ a Smoked salmon 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,4 0,3 ‐
Q7 Potted salmon PP1 L12 Listeria monocytogenes ½ a Smoked salmon 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,4 0,2 ‐
Q8 Salmon terrine PP1 L12 Listeria monocytogenes ½ a Smoked salmon 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,4 0,3 +
Q9 Country‐style pâté PP2 L53 Listeria monocytogenes ½ c Minced beef burger 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,1 5,7 +
Q10 Ham PP3 L53 Listeria monocytogenes ½ c Minced beef burger 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,1 7,6 +
Q11 Mixed vegetables PV1 L58 Listeria monocytogenes 4b Salad 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,5 2,4 ‐
Q12 Ratatouille PV1 L58 Listeria monocytogenes 4b Salad 24 hrs at 4°C, 50 minutes at 55°C, 30 minutes at ‐80°C 0,5 1,8 ‐
R1 Carré de l'Est cheese PV1 L62 Listeria monocytogenes Reblochon cheese 50 minutes at 55°C, 35 minutes at ‐80°C 0,6 6,4 ‐
R2 Brie cheese PV2 L18 Listeria monocytogenes ½ c Munster cheese rind 50 minutes at 55°C, 35 minutes at ‐80°C >1.3 0,2 ‐
R3 Époisses cheese PV2 L18 Listeria monocytogenes ½ c Munster cheese rind 50 minutes at 55°C, 35 minutes at ‐80°C >1.3 0,3 ‐
R4 Petit Billy goat's cheese PV2 L18 Listeria monocytogenes ½ c Munster cheese rind 50 minutes at 55°C, 35 minutes at ‐80°C >1.3 0,2 ‐
S10 Frozen fries PL2 L58 Listeria monocytogenes 4b Salad 50 minutes at 55°C, 35 minutes at ‐80°C 0,3 3,6 +
S11 Broccoli and carrot patties PC3 L58 Listeria monocytogenes 4b Salad 50 minutes at 55°C, 35 minutes at ‐80°C 0,3 2,4 ‐
S12 Cooked green beans PP1 L58 Listeria monocytogenes 4b Salad 50 minutes at 55°C, 35 minutes at ‐80°C 0,3 2,4 +
T2 Crab PP1 L20 Listeria monocytogenes ½ Smoked salmon offcuts 50 minutes at 55°C, 35 minutes at ‐80°C >0.8 ND ‐
T3 Shrimps PP2 L20 Listeria monocytogenes ½ Smoked salmon offcuts 50 minutes at 55°C, 35 minutes at ‐80°C >0.8 ND ‐
T4 Zucchini PP3 L129 Listeria monocytogenes ½ a Fried potatoes 50 minutes at 55°C, 35 minutes at ‐80°C 0,5 ND ‐
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APPENDIX 4
Relative accuracy, relative specificity and relative sensitivity per sample category
‐ Detailed results
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Bacterial burden Ø: no culture L = low M = moderate H = high Breakdown of flora A = pure culture of suspect colonies B = mixture with a majority of suspect colonies C = mixture with a minority of suspect colonies D = mixture with rare suspect colonies E = absence of suspect colonies (x): x colonies characteristic of Listeria if x ≤ 5
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