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 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 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 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 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)
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
50+50 μl, or 100+≤5 μl
Medium – Mueller-Hinton broth (Appx A, + supplements)
Back to basics, ISO 20776 – 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 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; oxacillin/methicillin and mecA containing Staph spp.
Back to basics, ISO 20776 – Part 1 (2006)
Table 4. MIC ranges for control strains
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
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)
AST devices, ISO 20776 – 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 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 determined
AST devices, ISO 20776 – Part 2 (2007)
Discrepancies
comparing AST device with reference method
Discrepancy
AST device
Reference method
ISO 20776 – 1
Major (MD)
R
S
Minor (mD)
I
S or R
S or R
I
S
R
Very major (VMD)
Isolates
Fresh
from clinical sample within 7 days, not froze, < 5 subcultures
Recent
Stock
from clinical sample with 12 months
from clinical sample, retained, stored or obtained (collection)
AST devices, ISO 20776 – 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 20776 – 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 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 error
AST devices, ISO 20776 – Part 2 (2007)
Acceptance criteria
Accuracy 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% results
Has ISO 20776 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)*
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)
VMD/MD/mD
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
*(#) 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 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