Q:Lab00PTPTPT 59ข้อเสนอโครงการNFI-PTMNFI-PTM 24 …pt.nfi.or.th/DocFile/20161130081147.pdf · containing microorganisms of target microorganism as Enterococcus

NFI-PTM 24-2016

Proficiency Testing Report

Microbiological Analysis

NFI-PTM 24-2016: Enterococci (Fecal Streptococci) (cfu/g)

in Lyophilized Cultures

October 2016

NFI-PTM 24-2016

หนา้ ข

CONTENTS

SUMMARY A

1. INTRODUCTION

1.1 Proficiency Testing 1

1.2 Enterococci (Fecal Streptococci) 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 5

4.3 Calculation of robust Z-Score 5

4.4 Z-Score assessment criteria 5

5. REFERENCES 6

6. RESULTS

Table 1 Result summary of participants’ Z-Score for Enterococci (Fecal Streptococci)

(cfu/g) in lyophilized cultures

7

Table 2 Statistics summary 10

Figure 1 Z-Score values for Enterococci (Fecal Streptococci) (cfu/g) in lyophilized

cultures

11

7. TECHNICAL COMMENTS 12

8. CONTACT DETAILS 12

9. APPENDIX

Table 3 Homogeneity and stability data 13

Table 4 Technical information in relation to the methods 14

COMMITTEE 15

NFI-PTM 24-2016

Page A

SUMMARY

This summarizes the microbiological proficiency testing program of NFI-PTM 24-2016:

Enterococci (Fecal Streptococci) (cfu/g) in Lyophilized Cultures. There were 48 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 eight 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 47 laboratories (97.9%) had satisfactory performance with Z≤ 2,

and 1 laboratory (2.1%) had questionable performance with 2 < 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 degrees 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 24-2016: Enterococci (Fecal Streptococci) (cfu/g) in

Lyophilized Cultures ” with the focus on enterococci (fecal streptococci) since it is one of the best

indicator of health risk in salt water used for recreation and as a useful indicator in fresh water as well.

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 Enterococci (Fecal Streptococci)

The enterococci are Gram-positive cocci that often occur in pairs or short chains. The

enterococci and fecal streptococci are the species of Enterococcus and Streptococcus associated with

the gastrointestinal tracts of man and animals and traditionally used as indicator of fecal

contamination.

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Fecal streptococci have the Lancefield’s Group D antigen. Within the fecal streptococci

group there was a subgroup of species known as enterococci, which differed from other fecal

streptococci by their resistance to 0.4% sodium azide, ability to grow in 6.5% of sodium chloride, at

pH 9.6, and at 10oC and 45

oC.

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 Enterococcus faecalis (DMST 4736) and

background flora as Enterobacter aerogenes (DMST 8841). After freeze drying, the vials were then

sealed with rubber cap topped with an 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.

2.2 Homogeneity

To insure that consistent levels of organisms were achieved throughout the batch of test

materials, homogeneity test was conducted on 12 October 2016. 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 enterococci (fecal streptococci) (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-random sample

in the box for 24 hours, and then keeping at 4-10°C until the final test or after this date.

On 28 October 2016, the test materials were examined in duplicate for enumeration of enterococci

(fecal streptococci) (cfu/g).

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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). The homogeneity and stability data were shown in Table 3 in the Appendix.

2.4 Sample Distribution

Upon completion of homogeneity test, foam boxes of test materials with ice packs, and

documents were dispatched on 19 October 2016 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 practice.

- 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 – “4 November 2016”.

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 in 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:

2015.

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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 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. STATISTICAL EVALUATION OF RESULTS

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

13528: 2015.

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.

4.1.2 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.

If the standard uncertainty uX of the assigned value is too large in comparison with the

standard deviation for proficiency testing σ̂ , then there is a risk that some laboratories will receive

action and warning signals because of inaccuracy in the determination of the assigned value, not

because of any cause within the laboratories. For this reason, the standard uncertainty of the assigned

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value shall be established and shall be reported to laboratories participating in proficiency testing

schemes.

If uX ≤ 0.3σ̂

Then the uncertainty of the assigned value is negligible and need not be included in the

interpretation of the results of the proficiency test.

4.2 Standard deviation for proficiency assessment, σ̂

The 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.

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

Z-Score = ( log

10 x – log

10 X )

σ̂

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5. REFERENCES

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

ISO 13528: 2015. Statistical methods for use in proficiency testing by interlaboratory comparison.

Paul, A., H., R., H., Deibel, and L., M., Sieverding. 4th ed., 2001. Chapter 9-Compendium of

Method for the Microbiological Examination of Foods, APHA. (November 2016).

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6. RESULTS

Table 1 Result summary of participants’ Z-Score for Enterococci (Fecal Streptococci) (cfu/g) in

lyophilized cultures

Laboratory Number Result

cfu/g

Result

Log10cfu/g Z-Score

1 4.3E+03 3.633 0.4

2 12000 4.079 1.9

3 3.55E+03 3.550 0.1

4 3700 3.568 0.1

5 3800 3.580 0.2

6 3.6E+03 3.556 0.1

7 3050 3.484 -0.1

8 3.7E+03 3.568 0.1

9 3,500 3.544 0.1

10 2.9E+03 3.462 -0.2

11 7000 3.845 1.1

12 3200 3.505 -0.1

13 3700 3.568 0.1

14 4800 3.681 0.5

15 3300 3.519 0.0

16 3.1E+03 3.491 -0.1

17 2900 3.462 -0.2

18 3100 3.491 -0.1

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

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lyophilized cultures (continued)


cfu/g

Result

Log10cfu/g Z-Score

19 1.3E+03 3.114 -1.4

20 3600 3.556 0.1

21 2900 3.462 -0.2

22 3100 3.491 -0.1

23 3.1E+03 3.491 -0.1

24 3000 3.477 -0.2

25 3500 3.544 0.1

26 2900 3.462 -0.2

27 3800 3.580 0.2

28 3500 3.544 0.1

29 3300 3.519 0.0

30 2900 3.462 -0.2

31 3000 3.477 -0.2

32 1600 3.204 -1.1

33 2.5E+03 3.398 -0.4

34 4000 3.602 0.3

35 3400 3.531 0.0

36 2900 3.462 -0.2

37 3.0E+03 3.477 -0.2


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lyophilized cultures (continued)


cfu/g

Result

Log10cfu/g Z-Score

38 3600 3.556 0.1

39 3600 3.556 0.1

40 3.2x103 3.505 -0.1

41 3.6x103 3.556 0.1

42 5.2x103 3.716 0.6

43 5.4x102 2.732 -2.6

44 3,000 3.477 -0.2

45 3.3x103 3.519 0.0

46 4,400 3.643 0.4

47 4.9x103 3.690 0.6

48 2.1x103 3.322 -0.7


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Table 2 Statistics summary

Assigned Value Standard Deviation

for Proficiency Assessment

Number of Result

Robust Mean, x* (log10cfu/g)

Robust Standard Deviation, s*

Standard Uncertainty, u

X

Derived from σ̂

48 3.524 0.079 0.014 Retrospective

data 0.300

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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 Reporting, 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, Bangphlat, Bangkok

10700 THAILAND.

Fax number: 0 2883 5022/ 0 2886 8088 ext. 5555

E-mail: [email protected]

Website: Contact-Us Form at http//:pt.nfi.or.th

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9. APPENDIX

Table 3 Homogeneity and stability data

Enterococci (Fecal Streptococci) (log10 cfu/g)*

Sample No. Homogeneity (x)

Sample No. Stability (y)

Replicate 1 Replicate 2 Replicate 1 Replicate 2

1 3.663 3.613 1 3.505 3.556

2 3.663 3.580 2 3.580 3.602

3 3.491 3.580 3 3.623 3.519

4 3.602 3.580 4 3.556 3.623

5 3.556 3.415 5 3.672 3.580

6 3.623 3.643 - - -

7 3.580 3.447 - - -

8 3.613 3.505 - - -

9 3.580 3.580 - - -

10 3.602 3.602 - - -

mean 3.576 3.582

SS 0.032 -

yx − - 0.006

σ̂ 0.300 0.300

0.3σ̂ 0.090 0.090

SS ≤0.3σ̂ PASS -

yx − ≤0.3σ̂ - PASS

Note: *Compendium of Method for the Microbiological Examination of Foods (APHA),

4th ed., 2001, Chapter 9.

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Table 4 Technical information in relation to the methods

Accredited Laboratory Number

Yes 1 3 4 5 7 9 12 13 14 15 16 17 18 19

22 23 24 27 29 32 35 36 37 38 39 41

42 44 48

No 2 6 8 10 11 20 21 25 26 28 30 31 33

34 40 43 45 46 47

Reference Laboratory Number

APHA 8 9 10 12 13 14 16 20 21 22 24 34 37

38 44 47

Compact Dry 2

Compendium of Methods for The Microbiological

Examination of Foods

7 18 25 30 32 40

Nordic Committee on Food Analysis (NMKL) 1 3 4 5 6 11 15 17 19 23 27 28 29 31

33 35 36 39 41 42 43 45 46 48

Nordic Committee on Food Analysis (NMKL),

NordVal: 047

26

Media: Selective Plating Laboratory Number

Compact Dry 2 26

Enterococcus Agar 4 19 43 48

KF Streptococcus Agar 7 8 9 10 12 13 14 16 18 20 21 22 24

25 30 32 37 38 40 44 46 47

Slanetz and Bartley Medium 1 5 6 11 15 17 23 27 28 29 31 33 34

35 36 39 41 45

Slanetz and Bartley Medium, Bile Aesculin 42

TSA, Slanetz 3

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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, Division of Proficiency Testing Services

(Statistician)

2. Miss Kuntida Duangsee Senior Analyst, Division of Proficiency Testing Services

3. Miss Thatsanee Kansan Scientist, Division of Proficiency Testing Services

(Coordinator)

Proficiency testing provider accreditation

ISO/IEC 17043: 2010: BLA-DSS Acc. No. PTP-0002

Approved by

(Mrs. Nitaya Pirapatrungsuriya)

Technical Manager

30 November 2016