International Standards and - PowerPoint Presentation

Slide1

International Standards and Antimicrobial Susceptibility Testing

Antimicrobials SIG Workshop ASM Canberra, 12 July, 2015

Peter Taylor, SEALS Microbiology,

St George Hospital,

Kogarah

, NSWSlide2

International Standards and Antimicrobial Susceptibility Testing

Where did this startFleming, Florey and penicillinThe International Collaborative Study 1971Minimum 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

ConclusionsSlide3

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

Slide4

Minimum Inhibitory Concentration,from the ICS, 1971

Agar dilutionInoculum sizeBroth dilution

Inoculum size

Broth and Micro-broth methods

Endpoints and other limitations

Medium

Growth conditions and supplements

Inoculum effectsSlide5

Where did ICS finish in 1971?

p 86, General recommendationsBasic point of reference is MIC determined under reproducible conditionsAgar 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 studiesSlide6

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 S

al

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?Slide7

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,

E

pidemiology of susceptibility (S and R),

Comparison of new with existing agents.Slide8

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

Application of dilution proceduresDetermine MIC, as a reference method for ASTWhen routine/breakpoint tests give equivocal resultsWhen 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.Slide9

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

ReproducibilityIntra- and inter-laboratory reproducibilityBroth 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 ICSSlide10

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

ScopeMicrodilution reference for MICsActivity 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 traysSlide11

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

Susceptible – high likelihood of therapeutic successIntermediate – 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

50+50

μl

, or 100+≤5

μl

Medium – Mueller-Hinton broth (

Appx

A, + supplements)Slide12

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

AntibioticsPotency mg/g, NOT pharmaceutical productsManufacturer 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

)Slide13

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

Final inoculum – 5x105 CFU/ml, (2 – 8x105 CFU/ml)Broth culture source, or colony suspension methods

Inoculate within 30 minutes, colony count as control

Incubate at 34-37

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

;

o

xacillin

/methicillin

and

mec

A

containing Staph spp.Slide14

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

Staph aureusEnterococcus 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 strainsSlide15

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 methods

MIC based

Break point based

SIR based

Normative reference – ISO 20776 – Part1 (2006)Slide16

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

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 resistanceSlide17

AST devices, ISO 20776 – Part 2 (2007)

Agreement of test results

Category 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 test

provides categorical results , SIR

MIC test (mg/l)

at least 5 consecutive doubling dilutions for which EA can be determinedSlide18

AST devices, ISO 20776 – Part 2 (2007)

Discrepancies comparing AST device with reference method

Isolates

Fresh from clinical sample within 7 days, not froze, < 5 subcultures

Recent from clinical sample with 12 months

Stock from clinical sample, retained, stored or obtained (collection)

Discrepancy

AST device

Reference method

ISO

20776 – 1

Major (MD)

R

S

Minor (

mD

)

I

S or R

S or R

I

Very major (VMD)

S

RSlide19

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 test

MIC test result with growth in at least one, but not all concentrations testedSlide20

AST devices, ISO 20776 – Part 2 (2007)

Governance – manufacturer, co-ordinator, investigator, reportTest 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 (

mec

A

, 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 5.2.5

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 errorSlide21

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 strains

BP test CA ≥ 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% resultsSlide22

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 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

✓Slide23

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

150ResistanceESBL Kl pneumMBL Cit freundiiMRO E cloacaeMBL E coli

* Numbers of Australian laboratories using these AST methods for each exerciseSlide24

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 carbapenemsSlide25
Slide26

International Standards and Antimicrobial Susceptibility Testing

Where did this startFleming, Florey and penicillin – life in the pre-AMR eraThe 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