NFI-PTM 17-2021: Detection of Clostridium perfringens (per 0.1 g)

in Lyophilized Cultures

Issue Date 6 September 2021

DEPARTMENT OF LABORATORY SERVICES

Final Report

NFI-PTM 17-2021

Proficiency Testing Report

Microbiological Analysis

NFI-PTM 17-2021: Detection of Clostridium perfringens (per 0.1 g)

in Lyophilized Cultures

Issue Date 6 September 2021

NFI-PTM 17-2021

หนา้ ข

CONTENTS SUMMARY A 1. INTRODUCTION 1.1 Proficiency Testing 1 1.2 Clostridium perfringens 1 2. TEST MATERIALS 2.1 Sample Preparation

Table 1 Target microorganism and background flora 2 2

2.2 Homogeneity 3 2.3 Stability 3 2.4 Sample Distribution 3 3. REPORTING 4 4. ASSIGNED VALUE AND LABORATORY PERFORMANCE EVALUATION 4.1 Assigned Value 4.2 The Laboratory Performance Evaluation

4 4

5. RESULTS Table 2 Result summary of participants’ performance evaluation for C. perfringens

(per 0.1 g) in lyophilized cultures 5

6. METROLOGICAL TRACEABILITY 7. TECHNICAL COMMENTS

9 9

8. CONTACT DETAILS 8.1 Any Question and Complaint 10 8.2 Appeal 10 9. APPENDIX Table 3 Homogeneity data of positive sample 11 Table 4 Homogeneity data of negative sample 11 Table 5 Stability data of positive sample 12 Table 6 Technical information in relation to the methods 13 10. REFERENCES 15 COMMITTEE 16

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Page A

SUMMARY

This summarizes the microbiological proficiency testing program of NFI-PTM 17-2021: Detection of Clostridium perfringens (per 0.1 g) in Lyophilized Cultures. There were 69 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.

The participant’s results were 69 laboratories 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 67 laboratories (97.1%) attained satisfactory performance while 2 laboratories (2.9%) 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 Laboratory, 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 17-2021: Detection of Clostridium perfringens (per 0.1 g) in Lyophilized Cultures” with the focus on C. perfringens since it is one of the most common causes of food poisoning and 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 online features and more for complete and clear action for their own advantage.

1.2 Clostridium perfringens

C. perfringens is a rod-shaped, gram-positive bacterium, which is a mesophile that has an optimal growing temperature of 37 °C. It is a non-motile pathogen that produces endospores. This bacterium produces energy via anaerobic respiration using compounds other than oxygen, such as nitrate. Although C. perfringens is an inhabitant of human normal intestinal flora, it is a pathogen responsible for many gastrointestinal illnesses with severity ranging from mild enterotoxaemia to fatal gas gangrene.

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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). The sample was prepared on 6 July 2021 for the negative sample and 7 July 2021 for the positive sample. After freeze drying, the glass vials were then sealed with rubber cap topped with aluminium-crimp seal. Each was labeled with ID numbers and stored at 2-8°C for homogeneity test and further steps. The test material per glass vial was equivalent to 100 grams 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 001 up to A 090” and

“B 091 up to B 120”. 2. Negative sample set was labeled with a serial sample code of “A 211 up to A 240” and

“B 121 up to B 210”.

Table 1 Target microorganism and background flora Test Material Target Microorganism Background Flora

Positive Sample

- C. perfringens (ATCC 13124) (contamination level about 86 CFU/0.1 g)

- C. sporogenes (ATCC 11437) (contamination level about 14 CFU/0.1 g) - E. coli (ATCC 25922)

(contamination level about 2.5 x 102 CFU/0.1 g)

Negative Sample None

- C. sporogenes (ATCC 11437) (contamination level about 30 CFU/0.1 g) - E. coli (ATCC 25922) (contamination level about 2.6 x 102 CFU/0.1 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 7 July 2021 for the negative sample and 8 July 2021 for the positive 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 C. perfringens per 0.1 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 2-8°C until the final test or after this date. On 2 August 2021, the test materials were examined for detection of C. perfringens per 0.1 g. The stability data were shown in Table 5 in the Appendix.

The work on homogeneity and stability tests was conducted by the subcontractor – Division of Microbiological Laboratory, National Food Institute (ISO/IEC 17025: 2017: DMSc Acc. No. 1005/42) which using the FDA-BAM standard method according to Chapter 16, 2001.

2.4 Sample Distribution

Upon completion of homogeneity test, foam boxes of test materials with ice packs, and documents were dispatched on 20 July 2021 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) Online Instruction Sheet

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

routine practice. - Two glass vials of test material delivered for each participant.

Participants were required to report and submit their result of the test material as either ‘Detected’ or ‘Not Detected’ per 0.1 g by the closing date – “9 August 2021”.

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.

4. ASSIGNED VALUE AND LABORATORY PERFORMANCE EVALUATION

4.1 Assigned Value

The assigned value can be obtained from the test of homogeneity of the sample as follows. 4.1.1 If the homogeneity test results are reported as detected, the assigned value is Detected. 4.1.2 If the homogeneity test results are reported as not detected, the assigned value is Not Detected.

4.2 The Laboratory Performance Evaluation

This proficiency testing program is qualitative testing. The testing results of each laboratory were compared with the assigned value. The evaluation of the participants, If only both sample’s results complied with the assigned values, the laboratory attained satisfactory performance. The criterion of laboratory performance evaluation as follows. 4.2.1 S means the test results passed the evaluation criteria (Satisfactory) 4.2.2 NS means the test results do not pass the evaluation criteria (Not Satisfactory)

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5. RESULTS Table 2 Result summary of participants’ performance evaluation for C. perfringens (per 0.1 g) in lyophilized cultures

Test Sample C. perfringens (per 0.1 g)

Satisfactory/ Not Satisfactory

A B

Assigned Value Detected Not Detected

Laboratory Number Result Result

2 Detected Not Detected S 3 Detected Not Detected S 4 Detected Not Detected S 5 Detected Not Detected S 6 Detected Not Detected S 7 Detected Not Detected S 8 Detected Not Detected S 9 Detected Not Detected S 10 Detected Not Detected S 11 Detected Not Detected S 12 Detected Not Detected S 14 Detected Not Detected S 16 Detected Not Detected S 18 Detected Not Detected S 19 Detected Not Detected S 20 Detected Not Detected S 21 Detected Not Detected S 22 Detected Not Detected S 23 Detected Not Detected S 24 Detected Not Detected S 25 Detected Not Detected S 26 Detected Not Detected S 27 Detected Not Detected S

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’.

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Table 2 Result summary of participants’ performance evaluation for C. perfringens (per 0.1 g) in lyophilized cultures (continued)

Test Sample C. perfringens (per 0.1 g)

Satisfactory/ Not Satisfactory

A B

Assigned Value Detected Not Detected

Laboratory Number Result Result

28 Detected Not Detected S 29 Detected Not Detected S 30 Detected Not Detected S 31 Detected Not Detected S 32 Detected Not Detected S 34 Detected Not Detected S 35 Detected Not Detected S 37 Detected Not Detected S 39 Detected Not Detected S 40 Not Detected Detected NS 42 Detected Not Detected S 44 Detected Not Detected S 45 Detected Not Detected S 47 Detected Not Detected S 48 Detected Not Detected S 49 Detected Not Detected S 50 Detected Not Detected S 51 Detected Not Detected S 52 Detected Not Detected S 53 Detected Not Detected S 55 Detected Not Detected S 56 Detected Not Detected S 57 Detected Not Detected S 58 Detected Not Detected S

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’.

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Table 2 Result summary of participants’ performance evaluation for C. perfringens (per 0.1 g) in lyophilized cultures (continued)

Test Sample C. perfringens (per 0.1 g)

Satisfactory/ Not Satisfactory

A B

Assigned Value Detected Not Detected

Laboratory Number Result Result

59 Detected Not Detected S 60 Detected Not Detected S 62 Detected Not Detected S 63 Detected Not Detected S 65 Detected Not Detected S 67 Detected Not Detected S 69 Not Detected Detected NS

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’.

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Table 2 Result summary of participants’ performance evaluation for C. perfringens (per 0.1 g) in lyophilized cultures (continued)

Test Sample C. perfringens (per 0.1 g)

Satisfactory/ Not Satisfactory

A B

Assigned Value Not Detected Detected

Laboratory Number Result Result

1 Not Detected Detected S 13 Not Detected Detected S 15 Not Detected Detected S 17 Not Detected Detected S 33 Not Detected Detected S 36 Not Detected Detected S 38 Not Detected Detected S 41 Not Detected Detected S 43 Not Detected Detected S 46 Not Detected Detected S 54 Not Detected Detected S 61 Not Detected Detected S 64 Not Detected Detected S 66 Not Detected Detected S 68 Not Detected Detected S

Note: S indicates ‘Satisfactory’, and NS indicates ‘Not Satisfactory’.

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6. METROLOGICAL TRACEABILITY

The PT provider requires participants to report their test results in Detected/ Not Detected. The traceability of the assigned value in this scheme based on the homogeneity results of an appropriate sample, in which the test method uses the FDA-BAM Online (Chapter 16) standard methods version 2001. This scheme applies sample prepared on the skim milk matrix by adding recognized reference bacterium; C. perfringens (ATCC 13124) as the target microorganism, and C. sporogenes (ATCC 11437) and E. coli (ATCC 25922) are the background flora from an international culture collection, respectively.

7. TECHNICAL COMMENTS

Participating Laboratories No. 40 and 69 having not satisfactory performance must be careful not to mixing up the two test materials during the testing process, and data transfer; ensure complete traceability and sample-tracking system.

NFI is pleased to provide further detail, and welcome any discussion regarding the proficiency testing.

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8. CONTACT DETAILS

8.1 Any Question and Complaint Upon receiving the final report, if there is any question and complaint regarding the proficiency testing assessment incur, should be contacted to the project coordinator by the following channels:

Miss Thatsanee Kansan (Project coordinator) Division of Proficiency Testing Laboratory, Department of Laboratory Services, National Food Institute, 2008 Soi Arun Amarin 36, Arun Amarin Rd., Bang Yi Khan, Bang Phlat, Bangkok 10700 THAILAND.

Fax number: 0 2422 8554 E-mail: pt@nfi.or.th Website: Contact-Us Form at http://pt.nfi.or.th

8.2 Appeal Upon receiving the final report, if there is an appeal regarding the proficiency testing

assessment incur, written notification should be submitted to the President of National Food Institute (NFI) within thirty days by the following channel:

President of the National Food Institute, 2008 Soi Arun Amarin 36, Arun Amarin Rd., Bang Yi Khan, Bang Phlat, Bangkok 10700 THAILAND.

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9. APPENDIX

Table 3 Homogeneity data of positive sample

Sample No. C. perfringens Results (per 0.1 g)

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

10 Detected Note: FDA-BAM Online, 2001 (Chapter 16)

Table 4 Homogeneity data of negative sample

Sample No. C. perfringens Results (per 0.1 g)

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: FDA-BAM Online, 2001 (Chapter 16)

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Table 5 Stability data of positive sample

Sample No. C. perfringens Results (per 0.1 g)

1 Detected 2 Detected 3 Detected 4 Detected 5 Detected

Note: FDA-BAM Online, 2001 (Chapter 16)

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Table 6 Technical information in relation to the methods

Test Method Accredited by ISO/IEC 17025 Laboratory Number

Yes 3 6 9 12 13 14 15 17 18 21 22 23 25 26 29 30 31 33 34 35 38 40 41 42 43 44 47 48 49 50 51 53 54 55 56 57 58 59 60 61 62 63 64 65 66 68

No 1 2 4 5 7 8 10 11 16 19 20 24 27 28 32 36 37 39 45 46 52 67 69

Reference Method Laboratory Number

Compendium 40 FDA-BAM Chapter 16 2 4 5 6 7 8 9 11 12 13 14 15 18 19 20

21 22 23 24 25 26 29 31 33 34 35 36 37 39 41 42 44 45 46 47 48 49 50 51 52 53 54 56 57 59 60 61 62 63 64 65 66 67 68

In-House Method 1 In-House Method Based on FDA-BAM Chapter 16 3 10 16 17 27 32 38 43 55 58 69

ISO 7937: 2004 28 30

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

Media: Enrichment Media Laboratory Number

Cooked Meat Medium 1 2 3 6 7 8 9 10 11 12 14 15 16 17 18 20 21 22 23 24 25 26 29 30 31 32 33 34 35 36 37 38 39 41 42 43 44 45 46 47 49 50 51 52 55 56 58 59 60 61 62 63 64 65 66 67 68 69

Cooked Meat Medium (Modified) 53 54 57 Liver Broth 40 48

Unused 4 5 13 19 27 28

Media: Selective Plating Media Laboratory Number

SFP Agar Base 13 16 33 44 61 Tryptose Sulfite Cycloserine (TSC) Agar 1 4 11 12 19 27 28 30 35 40 47 48 50

60 67 Tryptose Sulfite Cycloserine (TSC) Agar with Egg Yolk 2 3 5 6 7 8 9 10 14 15 17 18 20 21 22

23 24 25 26 29 31 32 34 36 37 38 39 41 42 43 45 46 49 51 52 53 54 55 56 57 58 59 62 63 64 65 66 68 69

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10. REFERENCES

ISO/IEC 17043: 2010. Conformity assessment – general requirements for proficiency testing. Rhodehamel, E.J. (ret.) and S.M. Harmon (ret.). 1998. Chapter 16-Clostridium perfringens, in the Bacteriological Analytical Manual - Online (January 2001), Food and Drug Administration. https://www.fda.gov/food/laboratory-methods-food/bam-chapter-16-clostridium-perfringens (September 2021).

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COMMITTEE

Project Coordinator 1. Miss Thatsanee Kansan Analyst, Division of Proficiency Testing Laboratory

Technical Consultants 1. Mrs. Nitaya Pirapatrungsuriya Executive Vice President, Food Industry Laboratory

Service Center 2. Miss Preeyaporn Jaengkarnkit Vice President, Department of Laboratory Services

3. Mrs. Prachern Nakpan Manager, Division of Microbiological Laboratory Statistician Consultant

1. Asst. Prof. Dr. Chutima Waisarayutt Department of Agro-Industrial Technology Faculty of Agro-Industry, Kasetsart University 2. Assoc. Prof. Dr. Kunchit Judprasong Institute of Nutrition, Mahidol University

Operators 1. Miss Tanaporn Borisut Manager, Division of Proficiency Testing Laboratory

2. Miss Kuntida Duangsee Expert, Division of Proficiency Testing Laboratory 3. Miss Thatsanee Kansan Analyst, Division of Proficiency Testing Laboratory

Approved by

(Miss Preeyaporn Jaengkarnkit) Technical Manager 6 September 2021

kuntida

Stamp