Methods for AST: diffusion or dilution? (pro s en con s)

Transcription

1 7 th EURL-AR workshop 4-5 April 2013 DTU, Kgs. Lyngby, Denmark Methods for AST: diffusion or dilution? (pro s en con s) Kees Veldman

2 Introduction Work as a senior technician at the Central Veterinary Institute in Lelystad (NL) since 1991 Responsible for the daily routine regarding resistance monitoring (NRL for antibiotic resistance in animals) PhD on plasmid mediated quinolone resistance in Salmonella and E. coli (defense of thesis in 2014)

3 Outline Overview of techniques Methods Dilution & Diffusion Pro s en Con s Conclusions

4 Why perform susceptibility tests? Reasons for susceptibility testing of bacteria Monitoring National programs Diagnostics Focused at treatment

5 Techniques MIC testing Agar dilution (CLSI golden standard) Broth (micro)dilution (ISO-reference method) Diffusion Paper disks Tablets Other tests E-test

6 Agar dilution method

7 Broth dilution method

8 Broth microdilution ISO-Reference method for MIC testing

9 Broth microdilution Different companies Sensititre (Trek Diagnostic Systems, UK) VETMIC (SVA, Uppsala, Sweden) Micronaut (Merlin, Germany)

10 Broth microdilution Performance and reading Manual, semi-automatic or fully automated systems

11 Tablet diffusion (Rosco-Neo sensitabs) Old standard method IST agar Semi-confluent growth (according to ICS, Ericsson & Sherris, 1971) Criteria: Rosco manual (Dutch CRG, 2002) Other methods (Kirby/Bauer EUCAST) MH agar Confluent growth Criteria: Rosco criteria derived from to CLSI/EUCAST (validated??)

12 Tablet diffusion (Rosco-Neo sensitabs) Shift to other packages and dispensers

13 Disk diffusion (paper disks) Method Disk diffusion test (according to EUCAST or CLSI) Media: MH agar Antibiotics: paper disks different loads! Inoculum: confluent Criteria: EUCAST or CLSI

14 Zone diameter sizes Reading: Manual (calipers) Semi-automated (with electronic calipers) Fully automated by image analyses with a camera

15 Epsilometer test Strips E-test (Bio-Merieux) M.I.C.E. test (Oxoid)

16 Fully automated susceptibility (and identification) systems Phoenix (Becton-Dickinson) Microscan WalkAway System Sensititre ARIS Vitek (2) System

17 Distribution of MIC s

18 Distribution of inhibtion zones

19 Linear regression

20 Critical steps in susceptibility testing should be standardized at all times Inocula Media Antibiotics End point determination

21 Inocula Standard: 0.5 McFarland Using a turbidometer/ densitometer

22 Media for disk diffusion Easy growers: Mueller Hinton agar (both CLSI and EUCAST) Fastidious bacteria: EUCAST: MH-F agar (= MH agar + 5 % sheep blood + % NAD) for all difficult growers, including campylobacters CLSI: MH agar + 5% defribinated sheep blood for Streptococci and Pasteurellae MH agar % lysed horse blood for Listeria Chocolate Mueller Hinton agar for APP and HSO

23 Media for broth microdilution Easy growing bacteria: ISO, EUCAST and CLSI : Cation-adjusted Mueller Hinton Broth(CAMHB) Fastidious bacteria: ISO, EUCAST and CLSI: CAMHB + Lysed Horse Blood for testing streptococci, campylobacter, H.influenzae, Pasteurella spp and others CLSI (veterinary): Veterinary Fastidious Medium for APP and HSO

24 Antibiotics Diffusion Choose a reliable company for tablets or disks Use the correct load Dilution Choose a reliable company for the microtiter plates Choose a suitable panel Buy a custom made panel

25 End point reading Diffusion Difficult antibiotics Sulfanomides and trimethoprim: 80% growth inhibition Penicillin and staphylococci : sharp edge means resistant Dilution Sulfanomides and trimethoprim: 80% growth inhibition Skips are ignored unless...

26 Guidelines Diffusion EUCAST disk diffusion method (new European standard) CLSI several documents (veterinair + human) Dilution CLSI several documents ISO document :2006

27 CLSI Recommend QC-ranges of reference strains Clinical breakpoints are recommended: Susceptible Intermediate resistant Resistant

28 EUCAST Recommend QC-ranges of reference strains Both clinical breakpoint and epidemiological cut-off values Cut-off values defined by EUCAST by wild-type distribution Strictly for monitoring purposes! All bacteria with MIC above WT are defined as NWT, but are often called resistant

29 Epidemiological cut-off value & clinical breakpoints

30 Pro s en con s dilution Pro s Internationally standardized Hard (quantitative) figures (MIC s) Reproducible and robust Automated systems available Con s Expensive Requires more lab facilities

31 Pro s en con s diffusion Pro s Cheap Easy to perform Con s Results derived from MICs Many methods available (e.g. ISO, CLSI etc) Less robust and reproducible??

32 Summary Parameter Dilution Diffusion Validated method and criteria available available Information hard figures (MIC s) derived MIC s Reproducibility and robustness high medium Potential for automation high only for reading Costs expensive cheap Equipment required variable minor Laborious depending on equipment depending on equipment

33 Dilution or diffusion Choice is dependent of: Purpose of the test Monitoring Diagnostics Budget Lab facilities Frequency of testing

34 Discussion Dilution or diffusion? No confusion, standardize your test carefully!

35 Standardization means: 1 methods for all labs (dilution or diffusion) Identical concentration ranges/disk loads/media/inocula 1 set of interpretive criteria/cut-off values/breakpoints Internal and external QAS Only then results are truly comparable

36 ECDC ringtrials for Salmonella Enternet lab s

37 Acknowledgements Project group for Antibiotic Resistance (CVI) Prof. Dr D. Mevius Drs C. Dierikx Mrs M. Japing Mr J. Testerink Mrs A. van Essen Mr A. Kant

38 Microbroth dilution (MBD) vs Disk diffusion Robert Skov Statens Serum Institut / EUCAST

39 Reason for making a standard To have ONE value to measure against Needed for manufacturers of AST devices! MIC is the natural choice as clinical data is correlated to MIC when establishing the efficacy of the antibiotics MBD was chosen not the least because it is the most widely used! ISO (2006) Non-fastidious organisms only!

40 For fastidious organisms no gold standard has been defined CLSI: CAMHB % LHB (streptococci, campylobacter) HTM (haemophilus) EUCAST:

41 EUCAST AST of fastidious organisms MH-F is Mueller-Hinton agar (or broth) with 5% (mechanically) defibrinated horse blood and 20 mg/l β-nad. Developed for: Haemophilus influenzae Moraxella catarrhalis Streptococci A, B, C, G and viridans group Streptococcus pneumoniae Campylobacter jejuni and C. coli Listeria monocytogenes Corynebacterium spp. Pasteurella multocida

42 Do MBD always give the right value/answer? Are MBD always better than all other systems? Especially DD

43 Well calibrated MBD has a precision of +/- one dilution i.e. when close to a breakpoint it will sometimes give a false result MBD NEEDS to be well calibrated and quality controlled just as well as all other systems! The discrete values of MBD mask some of the variation (makes it appear less variable than DD)

44 Ciprofloxacin (MBD) QC readings from the manufacturer for ciprofloxacin were at the lower limit of the QC range < Resistance Mechanism None qnr QRDR aac6

45 40 Ofloxacin Resistance Mechanism None qnr QRDR aac6 5 0 <

46 Repeated MICs for 17 retested isolates Ceftaroline for S. aureus Isolate Etest BMD Mean BMD "True MIC"

47 MBD is fairly expensive i.e. truncated ranges are often used May need alternative systems for testing other antibiotics than is on your panel

48 Disk diffusion Just like with MIC dependant of a range of factors Needs to be well calibrated! DD is not suitable for all drug bug combinations and can only be used where developed Usually very high correlation between MBD and DD Salmonella and ciprofloxacin is an example where DD still needs refinement this is not clear from the present table will be fixed

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50 Stability of DD 10 years QC data displayed as a yearly median value for all Swedish laboratories

51 Cipro 5 vs 128 salmonellae tested 4 times Breakpoint for susceptibility in CLSI M100 Zone diameter Resistance mechanism none qnr QRDR aac

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53 Tetracycline 30 μg vs. MIC S. aureus, 172 clinical isolates MIC (mg/l) >8 8 No of isolates Inhibition zone diameter (mm) Breakpoints ECOFF MIC S 1, R>2 mg/l WT 1 mg/l Zone diameter S 22, R<19 mm

54 40 Ciprofloxacin 5 µg vs. MIC, Campylobacter jejuni and coli 57 clinical isolates tested in duplicate No of isolates ECOFF: WT 1 mg/l Inhinition zone diameter (mm) NL and FI isolates read in Växjö. All isolates tested in duplicate on in-house MH-F plates from Oxoid and BBL MH.

55 70 S. aureus vs. Ceftaroline 5 µg (Oxoid and MAST disks) 17 selected isolates tested x15 (N=323) No of readings Inhibition zone diameter (mm)

56 DD hold significant advantages Cheaper Always provide the full range DD gives continous values whereas MIC only gives discrete values (1,2,4.) Inexpensive to test alternative antibiotics

57 Provided well calibrated both MBD and DD gives reproducible results Do include some variation! MBD is a natural choice for gold standard DD produces equally good results For those drug/bug combinations where it is developed Both systems have high reproducibility Provided well calibrated DD is cheaper