NFI-PTM 22-2015

Proficiency Testing Report

Microbiological Analysis

NFI-PTM 22-2015: Detection of Salmonella spp.

(per 25 g) in Lyophilized Cultures

September 2015

NFI-PTM 22-2015

หนา้ ข

CONTENTS

SUMMARY A

1. INTRODUCTION

1.1 Proficiency Testing 1

�. Salmonella spp. 1

2. TEST MATERIALS

2.1 Sample Preparation

Table 1 Target microorganism and background flora

2

2

2.2 Homogeneity 3

., Stability 3

2.4 Sample Distribution 3

3. REPORTING 4

4. LABORATORY PERFORMANCE EVALUATION 4

5. REFERENCES 5

6. RESULTS

Table 2 Result summary of participants’ performance evaluation for Salmonella spp.

(per 25 g) in lyophilized cultures

6

7. TECHNICAL COMMENTS 11

8. CONTACT DETAILS 11

9. APPENDIX

Table 3 Homogeneity data of positive sample 12

Table 4 Homogeneity data of negative sample 12

Table 5 Stability data of positive sample 13

Table 6 Technical information in relation to the methods 14

COMMITTEE 19

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Page A

SUMMARY

This summarizes the microbiological proficiency testing program of NFI-PTM 22-2015:

Detection of Salmonella spp. (per 25 g) in Lyophilized Cultures. There were 94 laboratories

participating in this program. The test materials were quality controlled to ensure that consistent

levels were achieved throughout the batch by conducting homogeneity test prior to dispatch.

Stability testing was conducted at the end of shelf life which was the deadline for all laboratories to

run the test – to ensure the integrity of the test materials.

Ninety four laboratories submitted their results which were assessed by comparison with

assigned values for performance evaluation for each laboratory. The assigned values were assigned

as positive or negative by homogeneity testing’s result. Each laboratory received 2 samples for

testing. For criterion of laboratory performance evaluation, laboratory attained satisfactory

performance if both sample’s results complied with the assigned values.

The overall competency evaluation was that there were 90 laboratories (95.7%) attained

satisfactory performance while 4 laboratories (4.3%) received not satisfactory performance.

This proficiency testing program accomplished with smooth operation as expected, and

every participating laboratory benefits and acquires valuable information for advancement whatever

the performance result is.

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1. INTRODUCTION

1.1 Proficiency Testing

Proficiency testing (PT) is a valuable tool for quality assurance of testing and measurement. It is a requirement of accreditation to ISO/IEC 17025 that laboratories take part in proficiency testing schemes. PT involves the use of interlaboratory comparisons for determination of laboratory performance with the purpose of producing laboratory results of greater reliability. Whatever the performance result is, every participating laboratory benefits and acquires valuable information for further advancement in all degree of their current performance.

For the improvement and satisfaction to the requirement of ISO/IEC 17025 of laboratories, hence the Division of Proficiency Testing Services, Department of Laboratory Services, National Food Institute (NFI) provides PT program to follow the international standard ISO/IEC 17043: 2010, Conformity Assessment – General Requirements for Proficiency Testing. This program was named “Microbiological Analysis NFI-PTM 22-2015: Detection of Salmonella spp. (per 25 g) in Lyophilized Cultures” with the focus on Salmonella spp. since it is one of the important pathogenic bacteria on food safety monitoring program.

To ensure performance confidentiality of all participants, a series of Laboratory Number is used in place of participant’s name. A unique Laboratory Number is assigned to each laboratory.

NFI offers Internet access for proficiency testing at ‘http://pt.nfi.or.th’ for fast, convenient and easy access for participants to register, submit form / data / test result / request, view other information, and communicate with the provider. All participants are encouraged to utilize all on-line features, and more for complete and clear action for their own advantage.

1.2 Salmonella spp.

Salmonella is a genus of rod-shaped, gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella that grade in all directions (i.e., peritrichous). They are chemoorganotrophs, obtaining their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. Most species produce hydrogen sulfide, which can readily be detected by growing them on media containing ferrous sulfate, such as TSI.

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Salmonella spp. are organisms of faecal origin, and the majority of serovars are regarded as potentially pathogenic to humans. They cause illnesses such as typhoid fever, paratyphoid fever, and foodborne illness.

2. TEST MATERIALS

2.1 Sample Preparation

Test materials were produced in glass vials by adding 1-milliliter solution of skim milk and microorganisms of either (1) target microorganism and background flora, or (2) only background flora (as shown in Table 1). After freeze drying, the vials were then sealed with rubber cap topped with aluminium-crimp seal. Each was labeled with ID numbers and stored at 4-10°C for homogeneity test and further steps. The test material per vial was equivalent to 100 g of initial sample after adding 100 milliliters of 0.85% NaCl and mixing well to rehydrate the sample before laboratory testing.

The test materials were labeled in two sets: 1. Positive sample set was labeled with a serial sample code of “A 261 up to A 280” and

“B 141 up to B 260”. 2. Negative sample set was labeled with a serial sample code of “A 001 up to A 120” and

“B 121 up to B 140”.

Table 1 Target microorganism and background flora

Test material Target microorganism Background flora

Positive sample - Salmonella Stanley (DMST 17363) (contamination level about 4.3-9.3 MPN/g)

- Enterobacter aerogenes (DMST 8841) (contamination level about 20 cfu/g)

Negative sample None

- Enterobacter aerogenes (DMST 8841) - Citrobacter freundii (DMST 16368) (total contamination level about 4.0x102

cfu/g)

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2.2 Homogeneity

To insure that consistent levels of organisms were achieved throughout the batch of test materials, homogeneity test was conducted on 20 August 2015 for the positive sample and the negative sample. The random number generator from the website ‘random.org’ was used to randomize the order of the samples. Ten out from each set of the positive and negative samples were randomly picked for homogeneity test by carrying out detection of Salmonella spp. per 25 g prior to dispatch. The homogeneity data were shown in Table 3 and 4 in the Appendix.

2.3 Stability

To ensure the constancy of the test, materials not being diminished over time and the transport effect, stability testing was conducted at the end of shelf life. Therefore, five sets of the test materials of positive sample at NFI were packaged using the same types of materials (boxes, ice packs, etc.) and conditions (temperature, length of time, etc.) as those delivered; leaving the selected-randomly sample in the box for 24 hours, and then keeping at 4-10°C until the final test date. On 10 September 2015, the test materials were examined for detection of Salmonella spp. per 25 g. The stability data were shown in Table 5 in the Appendix.

The work on homogeneity and stability tests was conducted by the supporting laboratory – Division of Microbiological Laboratory, National Food Institute (ISO/IEC 17025: 2005: DMSC Acc. No. 1005/42).

2.4 Sample Distribution

Upon completion of homogeneity test, foam boxes of test materials with ice packs, and documents were dispatched on 1 September 2015 to each participant by an express logistics company with 24-hour guaranteed delivery. Documents enclosed in the box were as follows:

1) Letter of Introduction 2) Test Instruction Sheet 3) Receipt Form 4) Results Form 5) Technical Form 6) On-line Instruction Sheet

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Note: - Filling on-line preferred for 3), 4) and 5) - Using test methods (materials, procedures, conditions, etc.) consistent with each

laboratory routine practice. - Two vials of test material delivered to each participant.

Participants were required to report and submit their result of the test material as either ‘Detected’ or ‘Not Detected’ per 25 g by the closing date – “25 September 2015”. 3. REPORTING

Having finished the testing, each participating laboratory, then submitted its test result by the closing date for the summary report to be made. All these data were compared with assigned values. The assigned values were assigned as positive or negative by homogeneity testing’s result.

The summary results are given in Table 2. Technical data of methods used by each laboratory in relation to accreditation, reference, and others were summarized in Table 6 in the Appendix.

Interim report and final report could be uploaded to the website pt.nfi.or.th within two weeks and one month, respectively. After the closing date, while hard copies of interim and final report could be sent to participants with no Internet access by registered mail.

4. LABORATORY PERFORMANCE EVALUATION

This proficiency testing program is qualitative testing. The testing results of each laboratory were compared with assigned value for performance evaluation.

As for criterion of laboratory performance evaluation, laboratories attained satisfactory performance if only both sample’s results complied with the assigned values.

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5. REFERENCES

ISO/IEC 17043: 2010. Conformity assessment – general requirements for proficiency testing. ISO/IEC 6579: 2002. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp.

Andrews, W. H., A. Jacobson and T. Hammack . 1998. Chapter 5 – Salmonella, in the Bacteriological Analytical Manual – Online (May 2014 Version), Food and Drug Administration. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm

(October 2015).

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6. RESULTS

Table 2 Result summary of participants’ performance evaluation for Salmonella spp. (per 25 g) in lyophilized cultures

Laboratory Number

Salmonella spp. (per 25 g) Satisfactory/

Not Satisfactory

Serological test of detected sample

(Laboratory result) Test sample A (Not detected)

Test sample B (Detected)

1 Not detected Detected S - 2 Not detected Detected S - 3 Not detected Detected S group B 4 Not detected Detected S - 5 Not detected Detected S - 6 Not detected Detected S - 7 Not detected Detected S - 8 Not detected Detected S Group B 9 Not detected Detected S Group B

11 Not detected Detected S - 12 Not detected Detected S - 13 Not detected Detected S - 14 Not detected Detected S - 15 Not detected Detected S - 16 Not detected Detected S - 17 Not detected Detected S - 18 Not detected Detected S - 19 Not detected Detected S - 20 Not detected Detected S - 22 Not detected Detected S - 23 Not detected Detected S -

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’. Serological test information was not considered here.

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Table 2 Result summary of participants’ performance evaluation for Salmonella spp. (per 25 g) in lyophilized cultures (continued)

Laboratory Number

Salmonella spp. (per 25 g) Satisfactory/

Not Satisfactory

Serological test of detected sample

(Laboratory result)

Test sample A (Not detected)

Test sample B (Detected)

24 Not detected Detected S - 25 Not detected Detected S B 26 Not detected Detected S - 27 Detected Detected NS - 28 Not detected Detected S - 29 Not detected Detected S - 30 Not detected Detected S - 31 Detected Not detected NS - 32 Not detected Detected S OMA group B 33 Not detected Detected S - 34 Not detected Detected S - 35 Not detected Detected S - 36 Not detected Detected S Group B 37 Not detected Detected S - 39 Not detected Detected S - 40 Not detected Detected S - 41 Not detected Detected S gr.B 42 Not detected Detected S - 43 Not detected Detected S - 44 Not detected Detected S - 45 Not detected Detected S - 46 Not detected Detected S B

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’. Serological test information was not considered here.

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Table 2 Result summary of participants’ performance evaluation for Salmonella spp. (per 25 g) in lyophilized cultures (continued)

Laboratory Number

Salmonella spp. (per 25 g) Satisfactory/

Not Satisfactory

Serological test of detected sample

(Laboratory result) Test sample A (Not detected)

Test sample B (Detected)

47 Not detected Detected S Gr. B non ST 48 Not detected Detected S - 49 Not detected Detected S - 51 Not detected Detected S - 52 Not detected Detected S gr. B 53 Not detected Detected S - 54 Not detected Detected S - 55 Not detected Detected S - 56 Not detected Detected S - 57 Detected Detected NS - 58 Not detected Detected S - 59 Not detected Detected S - 60 Not detected Detected S - 61 Not detected Detected S - 62 Not detected Detected S OMA group B 63 Not detected Detected S A-I 64 Not detected Detected S - 66 Not detected Detected S OMA Gr. B 68 Not detected Detected S - 69 Not detected Detected S Group B 70 Not detected Detected S OMA 71 Not detected Detected S - 72 Not detected Detected S -

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’. Serological test information was not considered here.

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Table 2 Result summary of participants’ performance evaluation for Salmonella spp. (per 25 g) in lyophilized cultures (continued)

Laboratory Number

Salmonella spp. (per 25 g) Satisfactory/

Not Satisfactory

Serological test of detected sample

(Laboratory result)

Test sample A (Not detected)

Test sample B (Detected)

73 Not detected Detected S OMA gr. B 74 Not detected Detected S - 75 Not detected Detected S - 76 Not detected Detected S - 78 Not detected Detected S Group B 79 Not detected Detected S Salmonella Group. B 80 Not detected Detected S - 81 Not detected Detected S - 82 Not detected Detected S - 83 Not detected Detected S - 84 Not detected Detected S - 85 Not detected Detected S - 86 Not detected Detected S - 87 Not detected Detected S - 88 Not detected Detected S Group B 89 Not detected Detected S - 90 Not detected Detected S - 91 Not detected Detected S - 92 Not detected Detected S gr B 93 Not detected Detected S -

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’. Serological test information was not considered here.

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Table 2 Result summary of participants’ performance evaluation for Salmonella spp. (per 25 g) in lyophilized cultures (continued)

Laboratory number

Salmonella spp. (per 25 g) Satisfactory/

Not Satisfactory

Serological test

of detected sample

(Laboratory result)

Test sample A (Detected)

Test sample B (Not detected)

10 Detected Not detected S Group B 21 Not detected Detected NS - 38 Detected Not detected S Gr. B 50 Detected Not detected S OMA 65 Detected Not detected S Group B 67 Detected Not detected S - 77 Detected Not detected S Salmonella gr. B 94 Detected Not detected S gr. B

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’. Serological test information was not considered here.

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7. TECHNICAL COMMENTS

It is very important to find errors and correct them. For this proficiency testing program, there are two main viewpoints to be considered by those in need of improvement:

7.1 Sample identification and the data transfer Be careful not to mixing up the two test materials during the testing process, and data transfer; ensure complete traceability and sample-tracking system.

7.2 Analytical technique The laboratory must be careful to all details in the testing process, such as enrichment, plating, screening, confirming, and biochemical test. Furthermore, high attention must be maintained to address cross contamination. NFI is pleased to provide further detail, and welcome any discussion regarding the proficiency testing.

8. CONTACT DETAILS

Upon receiving the final report, if there is any question regarding the proficiency testing assessment incurs, written notification should be submitted to the project coordinator by one of the following channels:

Mail: Project coordinator Division of Proficiency Testing Services, Department of Laboratory Services, National Food Institute, 2008 Soi Arun Ammarin 36, Arun Ammarin Rd., Bangyeekhan, Bangplad, Bangkok 10700 THAILAND.

Fax number: 0 2886 8088 ext. 5555 E-mail: pt@nfi.or.th Website: Contact-Us Form at http//:pt.nfi.or.th

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9. APPENDIX

Table 3 Homogeneity data of positive sample

Sample No. Salmonella spp. Results (per 25 g) Serological Test

1 Detected Group B 2 Detected Group B 3 Detected Group B 4 Detected Group B 5 Detected Group B 6 Detected Group B 7 Detected Group B 8 Detected Group B 9 Detected Group B

10 Detected Group B

Note: ISO 6579: 2002/Cor. 1: 2004 Table 4 Homogeneity data of negative sample

Sample No. Salmonella spp. Results (per 25 g) Serological Test

1 Not detected - 2 Not detected - 3 Not detected - 4 Not detected - 5 Not detected - 6 Not detected - 7 Not detected - 8 Not detected - 9 Not detected -

10 Not detected -

Note: ISO 6579: 2002/Cor. 1: 2004

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Table 5 Stability data of positive sample

Sample No. Salmonella spp. Results (per 25 g) Serological Test

1 Detected Group B 2 Detected Group B 3 Detected Group B 4 Detected Group B 5 Detected Group B

Note: ISO 6579: 2002/Cor. 1: 2004

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Table 6 Technical information in relation to the methods

Accredited Laboratory Number

Yes 6 8 9 10 11 14 26 27 32 36 38 40 42 45 46 47 52 53 54 55 57 58 59 60 62 63 65 66 70 73 88 89 93

No 1 2 3 4 5 7 12 13 15 16 17 18 19 20 21 22 23 24 25 28 29 30 31 33 34 35 37 39 41 43 44 48 49 50 51 56 61 64 67 68 69 71 72 74 75 76 77 78 79 80 81 82 83 84 85 86 87 90 91 92 94

Reference Laboratory Number

AJIS (Ajinomoto Method) 2 AFNOR 3 32 76 92 94 AOAC 4 11 15 20 27 30 31 46 53 54 67 69

74 75 82 87 European Pharmacopoeia 8.0 18 FDA-BAM 8 9 14 16 19 22 23 25 26 28 33 34 35

37 48 49 51 57 58 59 61 71 72 80 83 84 86 89 90 91 93

ISO 6579 5 6 10 12 13 17 21 24 29 36 38 39 41 42 44 45 47 50 52 55 56 60 62 63 64 65 66 68 70 73 77 78 79 81 85 88

USP 37-NF32 40 3M Petrifilm Salmonella Express System 1 3M Tecra VIA Salmonella spp. 43 Merck: Microbiology Manual 12th Edition 7

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Table 6 Technical information in relation to the methods (continued)

Pre-Enrichment Media Laboratory Number

Buffered Peptone Water 3 5 6 10 12 13 14 16 17 21 24 27 29 31 32 34 36 38 39 41 42 44 45 47 50 52 55 56 60 62 63 64 65 66 67 68 69 70 73 74 76 77 78 79 81 85 86 88 91 92 94

Lactose Broth 8 9 11 19 23 25 26 28 33 35 37 43 48 49 51 58 59 61 72 75 80 82 83 84 89 90 93

Nutrient Broth 57 RapidChek SELECT Salmonella Primary Enrichment 4 RV (R10) 46 Salmosyst Broth Base 7 Tryptic Soy Broth 18 22 40 71 87 3M Salmonella Enrichment Base 20 3M Enrichment Supplement Base 53 3M Salmonella Enrichment Base + 3M Salmonella Enrichment Supplement

1 15 54

Unused 2 30

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Table 6 Technical information in relation to the methods (continued)

Enrichment Media Laboratory Number

BHI 11 69 Mossel Broth 2 MSRV 36 38 41 47 88 RapidChek SELECT Salmonella Secondary Enrichment 4 RV 18 25 31 82 86 R-VR10 Broth 20 RV 10 54 RV, MKTTn 55 63 RV, M Broth 87 RVS, M Broth 16 23 27 40 43 75 RVS, MKTTn 3 5 6 9 12 17 21 24 29 39 42 45 50

52 56 60 62 64 65 68 70 72 73 77 78 79

RVS, MKTTn, SX2 66 RV, MSRV 10 SEB, SESUP 1 SX2 13 32 76 Salmosyst Selective Supplement Tablets 7 TT 14 34 TT, RV 8 19 22 26 28 33 35 37 44 48 49 51

57 58 59 61 71 80 81 83 84 85 89 91 93

3M Salmonella Enrichment Base (SEB) 30 3M Salmonella Enrichment + Supplement 46 3M Petrifilm Salmonella 53 3M Rappaport-Vassiliadis R10 (R-VR 10) Broth 15 Unused 67 74 90 92 94

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Table 6 Technical information in relation to the methods (continued)

Selective Plating Laboratory Number

BS, HE, XLD 8 9 22 26 33 37 48 49 51 57 59 61 72 80 89 91 93

BS, XLD 85 86 DHL Agar 2 23 HE, XLD 6 11 16 19 21 24 28 32 34 35 39 42

55 58 62 64 65 68 70 71 73 81 83 84 Rambach Agar 7 SM2, XLD, BGA 66 XLD 4 12 14 17 18 25 40 43 69 XLD, BGA 3 5 10 13 29 36 38 41 44 45 47 50 52

56 60 63 77 78 79 88 3M Petrifilm Salmonella Confirmation Disk 53 3M Petrifilm Salmonella Express Plate 1 15 20 46 3M Petrifilm Salmonella Express System 54 Unused 27 30 31 67 74 75 76 82 87 90 92 94

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Table 6 Technical information in relation to the methods (continued)

Rapid Method Laboratory Number

API 20E 23 33 35 56 58 61 62 BAX Automated PCR System 11 69 Bio-Rad 48 Bio Rad iQ-Check TM Salmonella II Kit 67 Compact Dry Salmonella spp. 22 90 In House Method 2 Microgen Bio Products (Microgen GN-A ID) 60 RT PCR 47 Salmonella AD Test Kit 39 3M Molecular Detection System 49 94 3M Molecular Detection Assay Salmonella 74 3M Petrifilm Salmonella Express System 1 30 46 54 3M Petrifilm TM Salmonella Express Confirmation Disk 20 3M Tecra VIA Salmonella spp. 12 17 24 31 43 75 82 87 Singlepath® Salmonella 27 RapidCheck SELECT Salmonella Test 4 Vidas Salmonella 3 13 45 66 76 91 92 Vidas Easy Salmonella 32 Unused 5 6 7 8 9 10 14 15 16 18 19 21 25 26

28 29 34 36 37 38 40 41 42 44 50 51 52 53 55 57 59 63 64 65 68 70 71 72 73 77 78 79 80 81 83 84 85 86 88 89 93

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COMMITTEE

Technical consultants 1. Mrs. Nitaya Pirapatrungsuriya Vice President, Department of Laboratory Services 2. Mrs. Prachern Nakpan Manager, Division of Microbiological Laboratory

Statistician consultant

1. Chutima Waisarayutt, Ph.D. Department of Agro-Industrial Technology Faculty of Agro-Industry, Kasetsart University

Operators 1. Miss Tanaporn Borisut Manager, Proficiency Testing Services Division (Coordinator) 2. Miss Kuntida Duangsee Senior Analyst, Division of Proficiency Testing Services 3. Miss Thatsanee Kansan Scientist, Division of Proficiency Testing Services

Proficiency testing provider accreditation ISO/IEC 17043: 2010: BLA-DSS Acc. No. PTP-0002 Approved by (Mrs. Nitaya Pirapatrungsuriya) Technical Manager 22 October 2015