1. Warschau Nov. 20081 Dr. K. Luden Lab Quality Assurance by Interlaboratory Comparisons Proficiency Testing Dr. Katrin Luden

2. Dr. K. Luden Drinking water directive 98/83/EC Annex III Specifications for the analysis of Parameters Each Member State must ensure that any laboratory at which samples are analysed has a system of analytical quality control that is subject from time to time to checking by a person who is not under the control of the laboratory and who is approved by the competent authority for that purpose. Warschau Nov. 20082

3. Dr. K. Luden German drinking water directive § 15 Methods of Analysis and Laboratories (4) The nessessary analysis including sampling are to be conducted by laboratories that work by “generally accepted rules of the technology” (allgemein anerkannte Regeln der Technik), have a system of internal quality control, participate at least once a year in external quality assurance schemes, have sufficiently qualified personell and are accredited by a generally recognized accreditation body. The responsible highest federal authority will publish a list of laboratories in its jurisdiction. (5) The authorities (of the federal state) independent of the labs will have to check regularly wether the conditions of (4) are still fullfilled. Warschau Nov. 20083

4. Dr. K. Luden Proficiency testing Participation in proficiency testing programs allows laboratories to:  identify areas where improvement in their testing and measurement methods is needed  identify further training of their staff  foster confidence in the performance of their testing and measurements  assure laboratory performance in their accredited test and calibration results Warschau Nov. 20084

5. Dr. K. Luden Jungle of standards for PT / EQA Warschau Nov. 20085 There are several standards and guidelines for providing PT / EQA schemes (in chronological order) ISO/IEC Guide 43-1:1996 Proficiency testing by Interlaboratory Comparisons – Part 1: Development and Operation of Proficiency testing Schemes ISO 13528:2005 Statistical Methods for use in Proficiency Testing by Interlaboratory comparisons IUPAC Technical Report 1/2006 The International Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories ILAC-G13:2007 ILAC Guidelines for the Competence of Providers of Proficiency Testing Schemes ISO/TS 20612 Water Quality – Interlaboratory comparisons for proficiency testing of analytical chemistry laboratories ISO/IEC CD 17043:2008 Conformity Assessment – General Requirements for Proficiency Testing

6. Dr. K. Luden External Quality Assurance Recommendation of Federal Environmental Agency 2002 - Microbiology Warschau Nov. 20086

7. Dr. K. Luden External Quality Assurance Warschau Nov. 20087 Parameter successfull participation in proficiency testing schemes E. coli/Coliform bacteria Enterococci Colony Count 2x / year Clostridium perfringens Legionella Pseudomonas aeruginosa 1x / year Enteropathogenic Viruses Cryptosporidium parvum International PTS Recommendation of Federal Environmental Agency 2002 - Microbiology

8. Dr. K. Luden External Quality Assurance Warschau Nov. 20088 Parameter successfull participation in proficiency testing schemes All parameters offered by a laboratory 1x within 2-3 years Recommendation of Federal Environmental Agency 2003 - Chemistry  Proficiency testing (PT) according to DIN 38402-45

9. Dr. K. Luden German drinking water PT providers Warschau Nov. 20089  Microbiology and “hygenic-chemical PT  Chemistry PT Regular meetings, harmonized schedules, identical statistical evaluation → comparability for labs and authorities

10. Dr. K. Luden DIN 38402-45 Warschau Nov. 200810  Standard may be applied if:  Parameter analysed can be considered continuously measureable true for most chemical and phisico-chemical parameters not allways true for biological and/or microbiological parameters  Application in Chemistry: Yes  Application in microbiology: by way of trial / mostly

11. Dr. K. Luden Microbiology PT Warschau Nov. 200811 The governmental Institute of Public Health of Lower Saxony (NLGA) is Proficency Test (PT) provider for microbiological measurements in drinking water in Germany Every year:  4 PTS for E. coli/coliform bacteria, Enterococci, and Colony Counts  2 PTS for Pseudomonas aeruginosa, Clostridium perfringens and Legionella  1 PTS for EU bathing water (E. coli, coliforme B.)  1 PTS for selected “hygenic-chemical” parameters

12. Dr. K. Luden Microbiology PT Warschau Nov. 200812 ParameterQualified methods 1986 - 1999 20002001200220032004200620072008 E. coli-coliform bacteria presence/absence test DIN EN ISO 9308-1 Colilert ® -18/Quanti-Tray ® + (+) + (+) + (+) + (+) + (+) + (+) + (+) + (+) + Colony Count German DWD 1990 DIN EN ISO 6222 ++++++ ++++ ++++ ++++ ++++ ++++ ++++ Enterococci EN ISO 7899-2 Chromocult ® -Enterococci-Agar (+) +++++++ ++++ ++++ Clostridium perfringens DWD (mCP-Agar) TSC (+) ++++ ++++ ++++ ++++ ++++ ++++ Pseudomonas aeruginosa EN 12780/ISO 16266++++++ Legionella spec.GVPC (ISO 11731-2)+++++ EC bathing water directive Feacal coliform bacteria ++++++(+) E. coli-coliform bacteria DIN EN ISO 9803-3(+)++ Intestinal Enterococci DIN EN ISO 7899-2 DIN EN ISO 7899-1 (+) ++++ ++++

13. Dr. K. Luden Microbiology PT Warschau Nov. 200813 ParameterQualified methods 1986 - 1999 20002001200220032004200620072008 E. coli-coliform bacteria presence/absence test DIN EN ISO 9308-1 Colilert ® -18/Quanti-Tray ® + (+) + (+) + (+) + (+) + (+) + (+) + (+) + (+) + Colony Count German DWD 1990 DIN EN ISO 6222 ++++++ ++++ ++++ ++++ ++++ ++++ ++++ Enterococci EN ISO 7899-2 Chromocult ® -Enterococci-Agar (+) +++++++ ++++ ++++ Clostridium perfringens DWD (mCP-Agar) TSC (+) ++++ ++++ ++++ ++++ ++++ ++++ Pseudomonas aeruginosa EN 12780/ISO 16266++++++ Legionella spec.GVPC (ISO 11731-2)+++++ EC bathing water directive Feacal coliform bacteria ++++++(+) E. coli-coliform bacteria DIN EN ISO 9803-3(+)++ Intestinal Enterococci DIN EN ISO 7899-2 DIN EN ISO 7899-1 (+) ++++ ++++

14. Dr. K. Luden PT samples - two options Warschau Nov. 200814 Liquid samplesFreeze dried material Bacterial solution in defined mineral medium Bacteria adsorbed to milk powder Very realistic samples Handling exactly like a real sample A solution has to be prepared and first step of handling procedure is different from real samples Stability 4-6 days after shipment at <10°C Stability at -20°C weeks or months GermanyUnited Kingdom, France, Sweden, Australia...

15. Dr. K. Luden Preparation at NLGA Warschau Nov. 200815

16. Dr. K. Luden Shipping Warschau Nov. 200816 300 - 500 participants/PT Registered: > 700 Labs from Germany Austria (26) Switzerland (8) Lithuania (2) Hungary (2) Great Britain (1) Luxembourg (1)

17. Dr. K. Luden Schedule of a proficiency testing scheme Warschau Nov. 200817  Registration of participants  Confirmation of the registration (4 weeks before PT)  Preparation of the samples and shipment  Internal quality control (two weeks before and 6 days after shipment)  Reporting of the results (within 16 days of shipment)  Evaluation of the results usually within 4 weeks after the deadline for reporting

18. Dr. K. Luden What do the labs get? Warschau Nov. 200818  Report on the PT design, special problems, interesting results etc.  Certificate and evaluation report (target value as consensus value of participants results)  Quality control charts about the influence of temperature and the duration of shipping  Z(u)-Scores of all measurements in tables  Z(u)-Scores in graphic form

19. Dr. K. Luden Warschau Nov. 200819 Statistics of DIN 38402-45  Robust statistics (no outlier tests); consensus mean (used as assigned value) calculated by Hampel Estimator  Z U -scores “normalize” the results for comparing different levels of bacterial concentration Z-score:lZ U l  2 - sufficient lZ U l  3 - questionable lZ U l > 3 - insufficient m-4,5s m-3s m-1,5s m m+1,5s m+3s m+4,5s

20. Dr. K. Luden Warschau Nov. 200820 Name/Adress of PT-provider Address of participant Identification of PT (e.g. 3-2008) Table of parameters, successfully analyzed param. And method used Signatures of PT provider Certificate

21. Dr. K. Luden Microbiology PT Warschau Nov. 200821 Z U -score Group B III-2008 E. coli (assigned value 21 CFU/100 ml) Labcode

22. Dr. K. Luden Interlaboratory comparisons Warschau Nov. 200822  method validation studies: aptitude/qualification of the lab is prerequisite reference material is fit for purpose  to certify reference material: method is supposed to be fit for purpose/valid labs are expert labs  Quality control/proficiency testing: performance of a lab is tested, reference material and method are supposed to be valid/fit for purpose

23. Dr. K. Luden Warschau Nov. 200823 PT 1-2003 E. coli

24. Dr. K. Luden Warschau Nov. 200824 PT 2-2004 Clostridium perfringens CFU/100 ml Labcode

25. Dr. K. Luden Warschau Nov. 200825 Clostridium perfringens Medium/method used Recovery rate Columbia blood agar / membrane filtration100 % Columbia blood agar / membrane filtration - no shaking of the sample 39 % DEV-Nutrient agar / pour plate method78 % mCP Agar / Filtration (24 hours TNT)43 % mCP Agar / Filtration (24 hours 4°C)43 % mCP Agar / Filtration (PT participants mean)11 %

26. Dr. K. Luden Warschau Nov. 200826 Clostridium perfringens Medium/method used Recovery rate Columbia blood agar / membrane filtration100 % Columbia blood agar / membrane filtration - no shaking of the sample 39 % DEV-Nutrient agar / pour plate method78 % mCP Agar / Filtration (24 hours TNT)43 % mCP Agar / Filtration (24 hours 4°C)43 % mCP Agar / Filtration (PT participants mean)11 %

27. Dr. K. Luden Warschau Nov. 200827 

28. Dr. K. Luden Pseudomonas aeruginosa Warschau Nov. 200828 E. coli P. species P. putida Negative contr. P. aeruginosa In PT 1-2007 30 % of labs failed because they reported a numerical result for P. aeruginosa when the strain provided was not a P. aeruginosa Possible explanation: problems with confirmation reaction - Ammonia formation from acetamide

29. Dr. K. Luden E. coli ISO 9308-1 (n = 246) E. coli Coliform bacteria delta 61687 025 305424 15161 21221 24251 Warschau Nov. 200829 A pure culture of E. coli was used in PT 4-2004 False results: ISO 9308-1: 2,4 % Colilert ® -18:18,8 % Colilert (n = 80) E. coli Coliform bacteria delta 066 704 62708 74784 0 083 74839 304313 48502 48535 50533 596 19212 24251 274518

30. Dr. K. Luden Explanation? Warschau Nov. 200830 ß-Galactosidase ß-Glucuronidase

31. Dr. K. Luden Conclusions Warschau Nov. 200831  Interaction between filter and nutrient agar might be unfavorable for growth of the target organism  Performance of the material combinations used has to be checked (even if supplied with a certificate)  Negative controls are as important as positive controls  One can learn about the methods while testing performance of the lab ....

32. Dr. K. Luden Thank you for your attention! Warschau Nov. 200832

33. Dr. K. Luden Most likely errors - E. coli / coliforms Warschau Nov. 200833  Formal mistakes in the results: E. coli is part of the group of coliforms, therefore the number of E. coli cannot be larger than the number of total coliforms  Commercial identification systems (that are not to be used for the PT) might give conflicting results with the PT method  ISO 9803-1: 44°C Indole formation, wrong or not exactly controlled incubation temperature (use a waterbath)  Colilert ® -18: measurements carried out at the wrong wavelength; cavities not properly filled.

34. Dr. K. Luden Most likely errors colony counts Warschau Nov. 200834  Contamination of the molten agar or the sample  Temperature of the agar too high: a slight increase in temperature might cause considerable decrease in numbers of bacteria that are able to form colonies; too low temperatures might lead to uneven distribution of bacteria as the agar starts clumping and proper mixing is prevented  Volume of agar is too small