NLA Conference 2009: Ingrid Flemming IFM Quality Services Pty Ltd, Australia

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Lee Floyd
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1 NLA Conference 2009: Ingrid Flemming IFM Quality Services Pty Ltd, Australia

2 There are very few proficiency testing programs (PTP) presented in a manner that EXACTLY mimics the situation of a laboratory s day-to-day work. At minimum, the reporting requirements are different At most, in addition to the normal job of testing samples, PTP samples often also require re-constitution, or additional method steps, which can add confusion for participants

3 Participation in PTPs is compulsory for accredited laboratories. Despite the facts of life, in our experience microbiology proficiency testing is well done by participants.

4 True technical issues (training and competence) Pseudo technical issues (the effects of method choice) Non-technical issues (common to all science disciplines) Sample issues (sample shipping and stability) We ll start at the bottom of the list

5 Laboratories rely on the proficiency test samples being shipped in a manner that ensures their good condition on arrival. Sadly, from time to time, delays in delivery occur As an example, we recently had 3 attempts at gaining delivery of samples to South America in a reasonable time frame. The samples were rejected because they took more than 10 days to arrive

6 This means that the storage environment was not in control for the period of the delay. When the delay exceeds what was considered probable by the shipper, sample compromise is possible particularly with respect to getting living microbes to stay alive

7 In excess of 70% of wrong results are due either to: Calculation errors (NB: there is often an extra step in Calculation errors (NB: there is often an extra step in preparation of PT samples, therefore can result in confusion when applying factors to the final result.) Transcription errors Sample Mix-up Interpretation errors People simply trying too hard

8 Not specific to microbiology Common across all fields of test The adherence to data integrity clauses in ISO IEC can mitigate these problems Checking calculations and transfers of data is an essential part of all testing work.

9 17025 deals with unique identification of samples, so should also apply to proficiency testing samples Again not exclusive/specific to microbiology

10 Is that gas in the tube? Is that colony blue or red????

11 Come on! At least ONE of these has to be positive!!!!!

12 hospital pass (figuratively, idiomatic, rugby, Australian football) A poorly executed passto a team-mate causing the receiver to present an easy target for a defender, and thus be tackled hard.

13 Method problems rather than technician problems. (Method is carried out correctly, and organisms are identified by a definition in a method, but others are using differing methods)

14 Occur when the method used by one lab has different limitations to methods used by other labs Examples: MPN vs serial dilutions for counts Sensitivity (MPN) vsprecision (serial dilutions)

20 Be aware that the method, media and the mix of organisms in the sample may affect the result obtained.

21 Does it matter that the MPN method you choose is less precise than other methods? Maybe: For example, if you want to show a process improvement, the only way you can do this reliably is with more definitive counts. Maybe not: if the purpose of testing is to detect very low levels of E.coli, you may have better sensitivity with MPN

22 How do the PTP results compare with your own laboratory s acceptance criteria and measurement uncertainty? Only after you have answered this question, can you answer the one above

23 Verification and validation. Is your method fit for purpose? Can you detect spiked levels of analyte in normal samples you test? Do you know how different strains of target organisms behave with your method? Do you know what interferes with your test methods? Does your media QC show you recover known quantities of organisms?

24 When introducing the method, each technician should check as many examples of atypical colonies as typical colonies until satisfied and confident about the meaning of typical appearance. This has 2 benefits: Satisfies demonstrable training requirements in ISO IEC Gives possibility of determining the statistical level of atypical organisms in the sample types tested

25 Continue to check an agreed percentage of atypical organisms.

26 Most methods rely on two factors for identification of E.coli: Fermentation of lactose at 44.5 C with production of gas Production of indole from Tryptone at 44.5 C

27 In our laboratory, as many as 10% of naturally occurring E.coli do not produce gas at 44.5 C 3% do not ferment lactose 5% do not produce indole Indolenegative E.coliare relatively common in wild birds.

28 Typical Salmonella do not ferment lactose, and do produce H2S Many salmonella species from dairy processing plants are able to ferment lactose (because is it the main sugar available) H2S production is variable according to the strain of Salmonella

29 One needs to know, not only the limitations of a test method, but its applicability to the types of testing samples expected by the lab.

30 If you understand your test method, what the method is trying to do, and how this fits into your normal samples, then you will have a better basis for application of logic to problem solving. PTP reports should disclose sample content if you know limitations of your testing methods, then you can easily provide logical reasoning and show good resolution processes.

31 Bacillus species affect Listeria detection Staphylococcus aureus is surprisingly difficult to enrich. They are very sensitive to the quality of peptone/tryptone. Spreading colonies affect counts (obvious, but ) Pseudomonas species and Bacillus species can form zones of inhibition around Legionellacolonies (not just antibiotic producing moulds)

32 The number one real technical problem is making assumptions about the nature of the organisms observed: These are gram negative These are the same as the organisms growing in anaerobic conditions These are red colonies (because they have to be ) That little bubble in the durhamtube is not really gas

33 The number one problem in bacterial identification is the oxidase test Oxidaseresults can mean the difference between (garden variety) Alcaligenessp and Yersiniapestis(the organism causing the plague) Oxidasetests require fresh reagents to be used, (or fresh kits) and positive and negative control organisms EVERY TIME

34 Dilutions, dilutions, dilutions Too few dilutions = crowding plates Crowding plates means one cannot determine the colony colour, whether (in petrifilm) that colony has gas, which colonies cause the reaction, allow distinction between different colony types Starting too high = low sensitivity

35 Using both positive and negative controls for EVERY test. When control organisms do not behave as expected, there is no confidence in the test result

36 Choice of substrate in fermentation / assimilation tests This is critical in identification of Pseudomonas species Again choose controls that are relevant to the test being performed

37 Many common yeasts grow on general plate count media. Many common bacteria grow on general fungal isolation media Microscopy is the only way to determine with certainty whether the organisms are yeasts It is amazing how many microbiologists have forgotten how to use microscopes

38 Lactic acid bacteria prefer an anaerobic environment, mineral substrate with lower ph But so do many other organisms Lactic acid bacteria are CATALASE negative and are also Gram positive (there s that microscope again.)

39 How often do you measure incubator temperature? Would you know if the incubator was out of action between 11pm and 7am? How reliable is the incubator? How is the temperature on the top shelf vs the bottom shelf?

40 After you open the door, how long does it take for the incubator to recover its temperature? Why does this matter???

41 If your testing temperature is /-0.2 C, for E.coli confirmations and coliform doubling time is 20 minutes how long should it take for the incubator to recover its temperature?

42 How many cell divisions does it take to turn a result positive, or give non-target organisms a leg up to tolerate a higher growth temperature? How many times a day is this incubator opened? How many chances are the non-target organisms getting to mimic an E.coli?

43 Last but most definitely not least..

44 One PTP program = A snapshot of the laboratory s performance for that analyst, at that time, on that day for that sample. Is one bad result in PTP a freak event, or a sign of inherent difficulties that need to be solved? Trending of PTP results per analyst over time gives better confidence about the answer to the above.

SCOPE OF ACCREDITATION TO ISO/IEC 17043:2010 PROFICIENCY TESTING PROVIDER. Valid To: November 30, 2015 Certificate Number:

SCOPE OF ACCREDITATION TO ISO/IEC 17043:2010 IFM QUALITY SERVICES PTY LTD 4/58 Stennett Road Ingleburn, NSW 2565 AUSTRALIA Ingrid Flemming 61296183311 PROFICIENCY TESTING PROVIDER Valid To: November 30,