NFI-PTM Proficiency Testing Report
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2 Proficiency Testing Report Microbiological Analysis NFI-PTM : Enumeration of E. coli (cfu/g) in Lyophilized Cultures June 2015
3 CONTENTS 4 SUMMARY A 1. INTRODUCTION 1.1 Proficiency Testing E. coli 1 2. TEST MATERIALS 2.1 Sample Preparation Homogeneity Stability 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, u X 4.2 Standard deviation for proficiency assessment,σˆ Calculation of robust Z-Score Z-Score assessment criteria 5 5. REFERENCES 5 6. RESULTS Table 1 Result summary of participants Z-Score for E. coli (cfu/g) 6 in lyophilized cultures Table 2 Statistics summary 12 Figure 1 Z-Score values for E. coli (cfu/g) in lyophilized cultures TECHNICAL COMMENTS CONTACT DETAILS APPENDIX Table 3 Homogeneity and stability data 15 Table 4 Technical information in relation to the methods 16 COMMITTEE 18
4 SUMMARY This summarizes the microbiological proficiency testing program of NFI-PTM : Enumeration of E. coli (cfu/g) in Lyophilized Cultures. There were 107 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. One hundred and seven 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 90 laboratories (84.1%) had satisfactory performance with Z 2, while 10 laboratories (9.4%) had questionable performance with 2 < Z < 3, and 7 laboratories (6.5%) 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. Page A
5 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 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 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 : 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 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 K 2, and by preventing the establishment of pathogenic bacteria within the intestine. Page 1/18
6 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 an 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 June 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 the 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-random sample in the box for 24 hours, and then keeping at 4-10 C until the final test date. On 16 June 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 the Appendix. Page 2/18
7 2.4 Sample Distribution Upon completion of homogeneity test, foam boxes of test materials with ice packs, and documents were dispatched on 10 June 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 practice. - One vial of test material delivered to each participant. Participants were required to report and submit their enumeration of the test material in cfu/g by the closing date 22 June 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 13528:2005. 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 the Appendix. Page 3/18
8 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 of this proficiency testing program is robust Z-Score that is based on ISO 13528: Calculation of the Assigned value, X and Standard uncertainty of the assigned value, u X 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: Log transformation of participant results from cfu/g to log 10 cfu/g Removal of data that was not considered valid Derivation of the robust mean (x*) of the final data set using a robust statistical procedure that calculates by Algorithm A Assessment of the standard uncertainty of the assigned value, u X. Page 4/18 s * u x = 1.25 where s* is robust standard deviation calculated by Algorithm A and p is number of result. If the standard uncertainty u X of the assigned value is too large in comparison with the standard deviation for proficiency testing σˆ, 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 value shall be established and shall be reported to laboratories participating in proficiency testing schemes. If u X 0.3σˆ then the uncertainty of the assigned value is negligible and need not be included in the interpretation of the results of the proficiency test. p
9 4.2 Standard deviation for proficiency assessment, σˆ 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 log 10 cfu/g. This value for σˆ has been used to calculate Z-Score in this assessment. 4.3 Calculation of robust Z-Score Z-Score = ( log 10 x log 10 X ) σˆ Where and x is participant s reported result, X is assigned value σˆ 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: Conformity assessment general requirements for proficiency testing. ISO 13528: Statistical methods for use in proficiency testing by interlaboratory comparisons. Feng, P., S. D. Weagant (ret.), M. A. Grant (dec.), and W. Burkhardt Chapter 4- Enumeration of Escherichia coli and the Coliform Bacteria, in the Bacteriological Analytical Manual Online (September 2002), Food and Drug Administration. (July 2015). Page 5/18
10 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 E E E E E Note: Z-Score values outside the satisfactory range are shown in bold. Page 6/18
11 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 E , E E Note: Z-Score values outside the satisfactory range are shown in bold. Page 7/18
12 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 E E Note: Z-Score values outside the satisfactory range are shown in bold. Page 8/18
13 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 E E E E Note: Z-Score values outside the satisfactory range are shown in bold. Page 9/18
14 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 E , x x Note: Z-Score values outside the satisfactory range are shown in bold. Page 10/18
15 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 x x x x x x x x x , x Note: Z-Score values outside the satisfactory range are shown in bold. Page 11/18
16 Table 2 Statistics summary Number of result Robust mean, x* (log 10 cfu/g) Assigned Value Robust standard deviation, s* Standard uncertainty, u X Standard Deviation for Proficiency Assessment Derived from Retrospective data σˆ Page 12/18
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18 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 THAILAND. Fax number: ext pt@nfi.or.th Website: Contact-Us Form at http//:pt.nfi.or.th Page 14/18
19 9. APPENDIX Table 3 Homogeneity and stability data Sample No. E. coli (log 10 cfu/g)* Homogeneity (x) Stability (y) Sample No. Replicate 1 Replicate 2 Replicate 1 Replicate mean S S x y σˆ σˆ S S 0.3σˆ PASS - x y 0.3σˆ - PASS Note: * AOAC (2012), , Page 15/18
20 Table 4 Technical information in relation to the methods Accredited Page 16/18 Laboratory number Yes No No detail Reference Laboratory number AOAC AOAC Certificate No AFNOR FDA-BAM ISO Japaness Method, Merck Microbiological Manual 2 66 TEMPO E. coli Method 37 No detail
21 Table 4 Technical information in relation to the methods (continued) Selective plating Laboratory number Compact Dry EC Chromocult Coliform Agar Chromogenic E. coli/coliform Petrifilm RAPID E. coli TBX Agar 94 TEMPO E.coli culture medium 37 VRBA VRBGA 103 VRBA with MUG No detail Page 17/18
22 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 Manager, Division of Proficiency Testing Services (Coordinator & Statistician) 3. Miss Kuntida Duangsee Senior Analyst, Division of Proficiency Testing Services 4. 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 17 July 2015 Page 18/18
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