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 20776 – Part 1– How to test, AST devices, ISO 20776 – 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 recommendationsBasic point of reference is MIC determined under reproducible conditionsAgar dilution is generally preferredA Reference broth dilution method should also be available, when agar

unsuitableTechniques of both should be adjusted to give comparable resultsReference diffusion method quantitatively related to dilution methodsReference strains characterized for all commonly used antibiotics, and readily

available in lyophilized form from stock culture collections

Technical recommendationsPrecise descriptions of agar and broth dilutions, disc diffusion methodsUS FDA control disc potency and performanceRecommended disc contents for antibiotic classesMueller-Hinton medium, as interim reference medium, needs improvingDuplicate 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 20776 – 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 20776 – 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 MethodDetection 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 20776 – 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 1962.•No ISO standard for agar dilution yet. Preferred method of ICS

Back to basics, ISO 20776 – 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 20776 – Part 1 (2006)

BreakpointsSusceptible – high likelihood of therapeutic successIntermediate – uncertain therapeutic successResistant – high likelihood of therapeutic failure

Wild type – absence of acquired resistance mechanismsReference strain – stable, defined AST phenotype, or genotypeInoculum – calculated wrt final test volume in cfu/ml

50+50 μl, or 100+≤5 μlMedium – Mueller-Hinton broth (Appx A, + supplements)

Back to basics, ISO 20776 – Part 1 (2006)

AntibioticsPotency mg/g, NOT pharmaceutical productsManufacturer or reliable commercial supplierExpiry, lot number, storage conditionsStock solutions ≥1000 mg/l (Table 2, working dilutions)Sterile water unless specified solvent or diluent (Table 1)Membrane filtered, assay before and afterWorking solutions, trays, storage (≤3 months, ≤ -60 degC)

Back to basics, ISO 20776 – Part 1 (2006)

Final inoculum – 5x105 CFU/ml, (2 – 8x105 CFU/ml)Broth culture source, or colony suspension methodsInoculate within 30 minutes, colony count as controlIncubate at 34-37 degC in air, for 18 ± 2 hoursRead when obvious turbidity in POS growth control and no growth in NEG growth control, and purity established with correct inoculum controlExceptions in Table 3

Aminoglycosides and E faecalis, E faeciumβ-lactams and effects of some β-lactamaseshVISA; oxacillin/methicillin and mecA containing Staph

spp.

Back to basics, ISO 20776 – Part 1 (2006)

Control strainsStaph aureusEnterococcus faecalisEscherichia coliPseudomonas aeruginosaStrep pneumoniae

Sources of control strainsATCCNCTCCIPDSM

MIC range is always +/- one doubling dilution from the medianSame control strain from different sources

Table 4. MIC ranges for control strains

AST devices, ISO 20776 – 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 methodsMIC basedBreak point basedSIR based

Normative reference – ISO 20776 – Part1 (2006)

AST devices, ISO 20776 – Part 2 (2007)

Susceptible – high likelihood of therapeutic successIntermediate – uncertain therapeutic success

- body site, drug levels, dose dependent- technical factors, buffer zone of caution

Resistant - high likelihood of therapeutic failureNon-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 20776 – Part 2 (2007)

Agreement of test resultsCategory agreement (CA): SIR results from a breakpoint or MIC test and the reference method (ISO 20776 – 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 testprovides categorical results , SIR

MIC test (mg/l)at least 5 consecutive doubling dilutions for which EA can be determined

AST devices, ISO 20776 – Part 2 (2007)

Discrepanciescomparing AST device with reference method

IsolatesFresh from clinical sample within 7 days, not froze, < 5 subculturesRecent from clinical sample with 12 monthsStock from clinical sample, retained, stored or obtained (collection)

Discrepancy AST device Reference methodISO 20776 – 1

Major (MD) R S

Minor (mD) IS or R

S or RI

Very major (VMD) S R

AST devices, ISO 20776 – Part 2 (2007)

ResultsMIC

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 testMIC test result with growth in at least one, but not all concentrations tested

AST devices, ISO 20776 – Part 2 (2007)

Governance – manufacturer, co-ordinator, investigator, reportTest methodsStrain 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 dayReproducibility – 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 5.2.5Results (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 20776 – Part 2 (2007)

Acceptance criteriaAccuracy of AST device

MIC test EA ≥ 90%, VMD and MD ≤ 3% each, depends on # of R strainsBP test CA ≥ 90%, VMD and MD ≤ 3% each, check for species

QC of AST deviceQC 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 20776 fulfilled ICS recommendations?p 86, General recommendationsBasic 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 recommendationsPrecise 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)*

Method 2013/3 2013/4 2013/7 2014/1

Agar dilution 4 3 4 3

CDS 37 44 32 36

CLSI disc 43 49 42 46

EUCAST disc 4 10 4 11

Phoenix 2 1 2 1

Replicator 1 1 1 1

Vitek 2 CLSI 46 49 47 34

Vitek 2 EUCAST

18

Total 137 157 132 150

Resistance ESBL 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)

Method 2013/3 2013/4 (#)* 2013/7 2014/1

CDS 0/17/1 6/2/0 (5/40) 0/9/0 1/2/0 (1/37)

CLSI disc 4/30/3 (0/15) 6/4/3 (4/14) 1/1/5 (0/19) 8/0/0 (1/13)

EUCAST disc 0/0/1 1/0/0 (1/1) 0/0/1 0/0/0

Vitek 2 CLSI 2/34/3 (0/30) 4/0/11 (0/33) 2/7/2 0/0/0

Vitek 2 EUCAST

0/0/0

Total 137 157 132 150

Resistance ESBL Kl pneum MBL Cit freun MRO E cloac MBL E coli

VMD/MD/mD

*(#) major errors for carbapenem tests reportedNote 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 20776 – Part 1– How to test, AST devices, ISO 20776 – 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