NFI-PTM 36-2015

Department of Livestock Development (DLD)

Proficiency Testing Report Proficiency Testing Program for DLD Designated Laboratories

January-March 2015

Proficiency Testing Provider: Division of Proficiency Testing Services, National Food Institute

2008 Soi Arun Ammarin 36, Arun Ammarin Rd., Bangyeekhan, Bangplad, Bangkok 10700 Thailand.

Microbiological Analysis

NFI-PTM 36-2015: Enumeration of E. coli (cfu/g) in Lyophilized Cultures

NFI-PTM 36-2015

หนา ข

CONTENTS SUMMARY A 1. INTRODUCTION 1.1 Proficiency Testing 1 1.2 Escherichia coli 1 2. TEST MATERIALS 2.1 Sample Preparation 2 2.2 Homogeneity 2 2.3 Stability 2 2.4 Sample Distribution 3 3. REPORTING 3

4. STATISTICAL EVALUATION OF RESULTS 4.1 Calculation of the Assigned value, X and Standard uncertainty of

the assigned value, uX

4

4.2 Standard deviation for proficiency assessment,σ̂ 4

4.3 Calculation of robust Z-Score 5 4.4 Z-Score assessment criteria 5 5. REFERENCES 5

6. RESULTS Table 1 Result summary of participants’ Z-Score for E. coli (cfu/g)

in lyophilized cultures 6

Table 2 Statistics summary 9 Figure 1 Z-Score values for E. coli (cfu/g) in lyophilized cultures 10

7. TECHNICAL COMMENTS 11

8. CONTACT DETAILS 11 9. APPENDIX

Table 3 Homogeneity and stability data 12 Table 4 Technical information in relation to the methods 13 COMMITTEE 14

NFI-PTM 36-2015

Page A

SUMMARY

This summarizes the microbiological proficiency testing program of NFI-PTM 36-2015: Enumeration of E. coli (cfu/g) in Lyophilized Cultures, under the project “Proficiency Testing Program for DLD Designated Laboratories”.

There were 44 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.

Forty four laboratories submitted their results which were assessed statistically in the form of a robust Z-Score for performance evaluation for each laboratory. The overall competency evaluation was that there were 42 laboratories (95.5%) had satisfactory performance with ⏐Z⏐≤ 2, and 2 laboratories (4.5%) had unsatisfactory performance with ⏐Z⏐≥ 3.

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 36-2015: Enumeration of E. coli (cfu/g) in Lyophilized Cultures” with the focus on E. coli since it is one of the important pathogenic bacteria on food safety monitoring program and it is used as an indicator of sanitary quality of foods and water.

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 Escherichia coli

E. coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls due to food contamination. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine.

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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 containing microorganisms of target microorganism as Escherichia coli (DMST 4212) and background flora as Enterobacter aerogenes (DMST 8841). After freeze drying, the vials were then sealed with rubber cap topped with aluminium-crimp seal, and all the vials with numbers labeled were stored at 4-10 °C for homogeneity test and other 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.

2.2 Homogeneity

To insure that consistent levels of organisms were achieved throughout the batch of test materials, homogeneity test was conducted on 5 February 2015. The random number generator from the website ‘random.org’ was used to randomize the order of the samples. Ten out of all the vials were randomly picked for homogeneity test by carrying out enumeration of E. coli (cfu/g) in duplicate prior to dispatch.

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 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 18 February 2015, the test materials were examined in duplicate for enumeration of E. coli (cfu/g).

The work on homogeneity and stability tests was conducted by the supporting laboratory – Division of Microbiological Laboratory, National Food Institute (ISO/IEC 17025: DMSC Acc. No. 1005/42). The homogeneity and stability data were shown in Table 3 in Appendix.

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2.4 Sample Distribution

Upon completion of homogeneity test, foam boxes of test materials with ice packs, and documents were dispatched on 11 February 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 Note: - Filling on-line preferred for 3), 4) and 5). - Using test methods (materials, procedures, conditions, etc.) consistent with each

laboratory routine practices. - One vial of test material delivered for each participant.

Participants were required to report and submit their enumeration of the test material in cfu/g by the closing date – “23 February 2015”.

3. REPORTING

Having finished the calculation of the test result, each participating laboratory then submitted its test result by the closing date for analysis, and for the summary report to be made. With all these data, together with those received through PT provider’s quality control, statistical analysis for homogeneity test, stability test, and others were carried out according to the standard ISO 13528:2005.

The summary results are given in Table 1. Table 2 summarizes the statistics calculated. Technical data of methods used by each laboratory in relation to accreditation, reference, and others are summarized in Table 4 in Appendix.

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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. STATISTICAL EVALUATION OF RESULTS

Statistical use for this proficiency testing program is robust Z-Score that is based on ISO 13528:2005.

4.1 Calculation of the Assigned value, X and Standard uncertainty of the assigned value, uX

4.1.1 Assigned value, X (assigned value for proficiency assessment) was set as the consensus of the results submitted by participants. The procedure used to derive this consensus involved some or all of the following:

4.1.1.1 Log transformation of participant results from cfu/g to log10cfu/g. 4.1.1.2 Removal of data that was not considered valid. 4.1.1.3 Derivation of the robust mean (x*) of the final data set using a robust

statistical procedure that calculate by Algorithm A. 4.1.1.4 Assessment of the standard uncertainty of the assigned value, uX .

p

sux

*

25.1 ×=

where s* is robust standard deviation calculated by Algorithm A and p is number of result.

4.2 Standard deviation for proficiency assessment, σ̂

Standard deviation for proficiency assessment used here has been estimated from the previous standard deviation of NFI-PT microbiological enumeration program and the advisory group has agreed that an appropriate value is 0.300 log10cfu/g. This value for σ̂ has been used to calculate Z-Score in this assessment.

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4.3 Calculation of robust Z-Score

Where x is participant’s reported result,

X is assigned value and σ̂ is standard deviation for proficiency assessment.

4.4 Z- Score assessment criteria ⏐Z⏐ ≤ 2 is Satisfactory

2 <⏐Z⏐ < 3 is Questionable ⏐Z⏐ ≥ 3 is Unsatisfactory

5. REFERENCES

ISO/IEC 17043:2010. Conformity assessment – general requirements for proficiency testing.

ISO 13528:2005. Statistical methods for use in proficiency testing by interlaboratory comparisons.

Feng, P., S. D. Weagant (ret.), M. A. Grant (dec.), and W. Burkhardt. 1998. Chapter 4- Enumeration of Escherichia coli and the Coliform Bacteria, in the Bacteriological Analytical Manual – Online (September 2002), Food and Drug Administration. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm064948.htm (March 2015).

Z-Score = ( log10 x – log10 X )

σ̂

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6. RESULTS

Table 1 Result summary of participants’ Z-Score for E. coli (cfu/g) in lyophilized cultures

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

1 1.2E+04 4.079 0.9

2 8.3E+03 3.919 0.4

3 1.0E+04 4.000 0.7

4 1.1E+04 4.041 0.8

5 15000 4.176 1.3

6 8600 3.934 0.4

7 1.3E+04 4.114 1.0

8 7.1E+03 3.851 0.2

9 6600 3.820 0.1

10 6.1E+03 3.785 -0.1

11 8500 3.929 0.4

12 13000 4.114 1.0

13 6250 3.796 0.0

14 75500 4.878 3.6

15 2600 3.415 -1.3

16 3700 3.568 -0.8

17 4700 3.672 -0.4

18 7500 3.875 0.2

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 1 Result summary of participants’ Z-Score for E. coli (cfu/g) in lyophilized cultures (continued)

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

19 7.3E+03 3.863 0.2

20 5600 3.748 -0.2

21 6.0E+03 3.778 -0.1

22 490 2.690 -3.7

23 8200 3.914 0.4

24 3750 3.574 -0.8

25 6.00E+3 3.778 -0.1

26 5600 3.748 -0.2

27 3200 3.505 -1.0

28 5000 3.699 -0.3

29 10200 4.009 0.7

30 2900 3.462 -1.1

31 7.9E+03 3.898 0.3

32 6.7E+3 3.826 0.1

33 4.0E+03 3.602 -0.7

34 4.2E+03 3.623 -0.6

35 11000 4.041 0.8

36 2200 3.342 -1.5

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 1 Result summary of participants’ Z-Score for E. coli (cfu/g) in lyophilized cultures (continued)

Laboratory Number

Result cfu/g

Result Log 10 cfu/g

Z-Score

37 3800 3.580 -0.7

38 6.8E+03 3.833 0.1

39 3400 3.531 -0.9

40 7.5E+03 3.875 0.2

41 6100 3.785 -0.1

42 4600 3.663 -0.5

43 7.0E+03 3.845 0.1

44 6.2x103 3.792 0.0

Note: Z-Score values outside the satisfactory range are shown in bold.

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Table 2 Statistics summary

Assigned Value Standard Deviation

for Proficiency Assessment

Number of result

Robust mean, x* (log10 cfu/g)

Robust standard deviation, s*

Standard uncertainty, uX

Derived from σ̂

44 3.801 0.210 0.040 Retrospective

data 0.300

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7. TECHNICAL COMMENTS

For this proficiency testing program there are two main viewpoints to be considered by those in need of improvement:

7.1 Calculation of the test result and the data transfer The laboratory must carefully calculate the test result, e.g. multiplication of the dilution

and checking of the data transfer before reporting. 7.2 Analytical technique

The laboratories must be careful to all details in the testing process, such as incubation temperature, and incubation time.

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 and stability data

E. coli (log10 cfu/g)*

Homogeneity (x) Stability (y) Sample No.

Replicate 1 Replicate 2 Sample No.

Replicate 1 Replicate 2

1 3.519 3.716 1 3.748 3.580 2 3.813 3.771 2 3.681 3.716 3 3.833 3.820 3 3.519 3.591 4 3.672 3.653 4 3.568 3.580 5 3.708 3.833 5 3.580 3.643 6 3.792 3.763 - - - 7 3.602 3.690 - - - 8 3.724 3.732 - - - 9 3.568 3.613 - - - 10 3.580 3.740 - - -

mean 3.707 3.621 SS 0.066 -

yx − - 0.086

σ̂ 0.300 0.300 0.3σ̂ 0.090 0.090

SS ≤0.3σ̂ PASS - yx − ≤0.3σ̂ - PASS

Note: * AOAC (2012), 991.14, 998.08

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Table 4 Technical information in relation to the methods

Accredited Laboratory number

Yes 1 2 4 6 7 8 9 10 11 12 14 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 35 36 37 38 39 40 41 42 43 44

No 3 5 13 25 34

Reference Laboratory number

AOAC 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 38 39 40 41

APHA 4 43 FDA-BAM 37 ISO 16649-2 42 44

Selective plating Laboratory number

Chromogenic E. coli/Coliforms 42 Petrifilm 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 38 39 40 41 43

TBX Agar 44 VRBA with MUG 37

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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. Mrs. Nitaya Pirapatrungsuriya Vice President, Department of Laboratory Services 2. Miss Tanaporn Borisut Senior Analyst, Division of Proficiency Testing Services (Coordinator & Statistician) 3. Miss Thatsanee Kansan Scientist, Division of Proficiency Testing Services

Proficiency testing provider accreditation ISO/IEC 17043: BLA-DSS Acc. No. PTP-0002 Approved by (Mrs. Nitaya Pirapatrungsuriya) Technical Manager 9 March 2015