International Standards and Antimicrobial Susceptibility Testing Antimicrobials SIG Workshop ASM Canberra, 12 July, 2015 Peter Taylor, SEALS Microbiology, St George Hospital, Kogarah, NSW

International Standards and Antimicrobial Susceptibility Testing Where did this start – Fleming, Florey and penicillin The International Collaborative Study 1971 – Minimum Inhibitory Concentration; was it all too hard? What is sensitive, resistant? – Application of MIC’s to outcomes; no-one spoke to the germs Back to MIC’s – Back to basics, MIC, ISO – Part 1 – How to test, AST devices, ISO – Part 2 How will this impact – Patient outcomes – National surveillance of AMR – External QA Programs – Changes in current testing Conclusions

Fleming, Florey and penicillin Agar diffusion, broth dilution, bactericidal (Fleming, 1929) – Susceptible, non-susceptible, – Selective agent for isolation for H influenzae Clinical outcomes (Florey, 1941) – Success and failure Resistance to penicillin in Staph aureus – Qualitative and quantitative measures; still a problem

Minimum Inhibitory Concentration, from the ICS, 1971 Agar dilution – Inoculum size Broth dilution – Inoculum size – Broth and Micro-broth methods Endpoints and other limitations – Medium – Growth conditions and supplements – Inoculum effects

Where did ICS finish in 1971? p 86, General recommendations Basic point of reference is MIC determined under reproducible conditions Agar dilution is generally preferred A Reference broth dilution method should also be available, when agar unsuitable Techniques of both should be adjusted to give comparable results Reference diffusion method quantitatively related to dilution methods Reference strains characterized for all commonly used antibiotics, and readily available in lyophilized form from stock culture collections Technical recommendations Precise descriptions of agar and broth dilutions, disc diffusion methods US FDA control disc potency and performance Recommended disc contents for antibiotic classes Mueller-Hinton medium, as interim reference medium, needs improving Duplicate laboratory testing of same test organisms – true validation studies

Application of MIC’s to outcomes, no-one spoke to the germs When is a wild type strain not really a wild type? – Quinolones and Sal typhi – Penicillin and Pneumococcus Reference strains and MIC reproducibility – Neisseria gonorrhoeae Breakpoints, wild-type and ECOFF, or “Clinical breakpoints”……..(a committee decision) Confirm or change the empirically chosen antimicrobial agent, Resistance surveillance, Epidemiology of susceptibility (S and R), Comparison of new with existing agents. Are MICs comparable?

Back to basics, ISO – Part 1 (2006) Clinical laboratory testing and in vitro diagnostic test systems – Susceptibility testing of infectious agents and evaluation of antimicrobial test devices – Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases. Confirm the selected empirically chosen antimicrobial agent, Resistance surveillance, Epidemiology of susceptibility (S and R), Comparison of new with existing agents.

Back to basics, ISO – Part 1 (2006) Application of dilution procedures Determine MIC, as a reference method for AST When routine/breakpoint tests give equivocal results When routine tests are unreliable When quantitation is needed for clinical management Dilution Method Detection of visible growth on a series of agar plates or broth cultures containing doubling dilutions of Abx Lowest concentration (mg/l) that prevents growth under defined in vitro conditions within a defined period of time, MIC.

Back to basics, ISO – Part 1 (2006) Reproducibility Intra- and inter-laboratory reproducibility Broth MIC tests are reproducible to within one doubling dilution of the real end point, ie., ± one well or tube in a doubling dilution series. Pure cultures, aerobic bacteria, overnight growth, Mueller Hinton broth (± supplements) Derived from essentially similar methods used in France, Germany, Sweden, UK, USA and EUCAST, all based on the ICS Study of Ericsson and Sherris (1971), commenced in No ISO standard for agar dilution yet. Preferred method of ICS

Back to basics, ISO – Part 1 (2006) Scope Microdilution reference for MICs Activity of the drug under described test conditions, Clinical management requires application of drug pharmacology and bacterial resistance mechanisms S, I, R, for wild-type and non-wild type bacterial populations Modifications for certain antibiotic-bacteria combinations Reference method when comparing others AST methods ≤200 μl wells in micro dilution trays

Back to basics, ISO – Part 1 (2006) Breakpoints Susceptible – high likelihood of therapeutic success Intermediate – uncertain therapeutic success Resistant – high likelihood of therapeutic failure Wild type – absence of acquired resistance mechanisms Reference strain – stable, defined AST phenotype, or genotype Inoculum – calculated wrt final test volume in cfu/ml μl, or 100+≤5 μl Medium – Mueller-Hinton broth (Appx A, + supplements)

Back to basics, ISO – Part 1 (2006) Antibiotics Potency mg/g, NOT pharmaceutical products Manufacturer or reliable commercial supplier Expiry, lot number, storage conditions Stock solutions ≥1000 mg/l (Table 2, working dilutions) Sterile water unless specified solvent or diluent (Table 1) Membrane filtered, assay before and after Working solutions, trays, storage (≤3 months, ≤ -60 degC)

Back to basics, ISO – Part 1 (2006) Final inoculum – 5x10 5 CFU/ml, (2 – 8x10 5 CFU/ml) Broth culture source, or colony suspension methods Inoculate within 30 minutes, colony count as control Incubate at degC in air, for 18 ± 2 hours Read when obvious turbidity in POS growth control and no growth in NEG growth control, and purity established with correct inoculum control Exceptions in Table 3 Aminoglycosides and E faecalis, E faecium β-lactams and effects of some β-lactamases hVISA; oxacillin/methicillin and mecA containing Staph spp.

Back to basics, ISO – Part 1 (2006) Control strains Staph aureus Enterococcus faecalis Escherichia coli Pseudomonas aeruginosa Strep pneumoniae Sources of control strains ATCC NCTC CIP DSM MIC range is always +/- one doubling dilution from the median Same control strain from different sources Table 4. MIC ranges for control strains

AST devices, ISO – Part 2 (2007) Clinical laboratory testing and in vitro diagnostic test systems – Susceptibility testing of infectious agents and evaluation of antimicrobial test devices – Part 2: Evaluation of performance test devices Applicable to all phenotypic test methods MIC based Break point based SIR based Normative reference – ISO – Part1 (2006)

AST devices, ISO – Part 2 (2007) Susceptible – high likelihood of therapeutic success Intermediate – uncertain therapeutic success - body site, drug levels, dose dependent - technical factors, buffer zone of caution Resistant - high likelihood of therapeutic failure Non-susceptible - >S breakpoint, but no I or R breakpoints yet defined, eg., lack of resistant strains Based on breakpoints from defined phenotypic test system May change with revised dosage, emerging resistance

AST devices, ISO – Part 2 (2007) Agreement of test results Category agreement (CA): SIR results from a breakpoint or MIC test and the reference method (ISO – Part1) Essential agreement (EA): MIC result of test method within one doubling dilution step from the MIC by the reference method. Breakpoint Specific values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical categories of S, I or R Note: can refer to latest publications of organizations using this reference method (eg, CLSI, EUCAST) Breakpoint test provides categorical results, SIR MIC test (mg/l) at least 5 consecutive doubling dilutions for which EA can be determined

AST devices, ISO – Part 2 (2007) Discrepancies comparing AST device with reference method Isolates Freshfrom clinical sample within 7 days, not froze, < 5 subcultures Recentfrom clinical sample with 12 months Stockfrom clinical sample, retained, stored or obtained (collection) DiscrepancyAST deviceReference method ISO – 1 Major (MD)RS Minor (mD)I S or R I Very major (VMD)SR

AST devices, ISO – Part 2 (2007) Results MIC lowest concentration, defined conditions, prevents visible growth, defined period of time Zone diameter (mm) diameter of zone of growth inhibition around an antimicrobial disc in an agar diffusion test On-scale MIC test MIC test result with growth in at least one, but not all concentrations tested

AST devices, ISO – Part 2 (2007) Governance – manufacturer, co-ordinator, investigator, report Test methods Strain selection: 300 clinical isolates relevant to one antibiotic 100 clinical isolates for a single species plus QC strain(s) Isolate testing protocol - manufacturer's instructions v reference method, and additional genetic or gene product tests (mecA, PBP 2a, Carba-NP) Inoculum preparation – same for test and reference method, same day Reproducibility – triplicate testing of ten strains with on-scale MIC, on three days, at each site. Strains NOT within one dilution of breakpoint. Daily QC strain testing using reference method, reject out of range results according to rules, section Results (5.2.6) – MIC tests EA (%), all tests CA (%) Discrepancy resolution (5.2.7) – repeat testing for Major and Very major Discrepancies. Repeat triplicate test if no obvious technical error

AST devices, ISO – Part 2 (2007) Acceptance criteria Accuracy of AST device MIC testEA ≥ 90%, VMD and MD ≤ 3% each, depends on # of R strains BP testCA ≥ 90%, VMD and MD ≤ 3% each, check for species QC of AST device QC strains with expected range for at least 95% of results during study period, MICs and zone diameters, ± 3mm of the mode for ≥ 95% results

Has ISO fulfilled ICS recommendations? p 86, General recommendations Basic point of reference is MIC determined under reproducible conditions ✔ Agar dilution is generally preferred ✗ A Reference broth dilution method should also be available, when agar unsuitable ✔ Techniques of both should be adjusted to give comparable results ✗ Reference diffusion method quantitatively related to dilution methods ✗ Reference strains characterized for all commonly used antibiotics, and readily available in lyophilized form from stock culture collections ✓ Technical recommendations Precise descriptions of agar and broth dilutions, disc diffusion methods ✗ / ✓ US FDA control disc potency and performance ✓ Recommended disc contents for antibiotic classes ✗ Mueller-Hinton medium, as interim reference medium, needs improving ✓ Duplicate laboratory testing of same test organisms ✓ – true validation studies ✓

What’s the score? Australian laboratories, RCPA QAP (MRO’s)* Method2013/32013/42013/72014/1 Agar dilution4343 CDS CLSI disc EUCAST disc Phoenix2121 Replicator1111 Vitek 2 CLSI Vitek 2 EUCAST18 Total ResistanceESBL Kl pneum MBL Cit freundii MRO E cloacae MBL E coli * Numbers of Australian laboratories using these AST methods for each exercise

What’s the score? Australian laboratories, RCPA QAP (MROs) Method2013/32013/4 (#)*2013/72014/1 CDS0/17/16/2/0 (5/40)0/9/01/2/0 (1/37) CLSI disc4/30/3 (0/15)6/4/3 (4/14)1/1/5 (0/19)8/0/0 (1/13) EUCAST disc0/0/11/0/0 (1/1)0/0/10/0/0 Vitek 2 CLSI2/34/3 (0/30)4/0/11 (0/33)2/7/20/0/0 Vitek 2 EUCAST0/0/0 Total ResistanceESBL Kl pneum MBL Cit freun MRO E cloac MBL E coli VMD/MD/mD *(#) major errors for carbapenem tests reported Note low numbers of CLSI users reporting carbapenems

International Standards and Antimicrobial Susceptibility Testing Where did this start – Fleming, Florey and penicillin – life in the pre-AMR era The International Collaborative Study 1971 – Much has been achieved What is sensitive, resistant? – Application of MIC’s to outcomes; no-one spoke to the germs Back to MIC’s – Back to basics, MIC, ISO – Part 1 – How to test, AST devices, ISO – Part 2 How will this impact – Patient outcomes in the era of AMR – National surveillance of AMR – External QA Programs – detection of AMR – Changes in current testing – wait and see