BSAC Susceptibility Testing Residential Workshop
Transcript of BSAC Susceptibility Testing Residential Workshop
Insert name of presentation on Master Slide
BSAC Susceptibility Testing Residential Workshop
Mandy Wootton
EUCAST – Further developments & UK specific advice
National Breakpoint Committees F, G, N, NL, S, UK
Individual experts, ESCMID Study Groups, ECDC Networks, Industry
National Antimicrobial Susceptibility Testing Committees (NACs)
EUCAST Steering Committee Chairman, Scientific Secretary, Clinical Data Co-ordinator, 6 Breakpoint Committee reps
2 General Committee reps, 2 visiting GC reps
EUCAST General Committee 35 National reps, FEMS and FESCI reps
EUCAST Subcommittees Antifungals, Veterinary agents,
Whole genome sequencing, Expert Rules, Anaerobes, Resistance mechanisms
Consultation process
BSAC Susceptibility Testing Workshop
EUCAST decision making process
Steering committee evaluates available data and proposes breakpoints (and other decisions)
Consultation with general committee and national committees (NACs) on proposed decision – revision of the proposal
Public consultation (EUCAST webpage) after which criticism and responses are published – revision of proposed decision
Final decision (and for breakpoints rationale document) published
BSAC Susceptibility Testing Workshop
National AST Committees (NACs), August 2014
Denmark
Finland
Poland
France
Germany
Spain Portugal
Greec
e
Italy Turkey
Switzer- land
Austria
Czech Republic
Estonia
Latvia
Lithuania
Belarus
Ukraine
Romania Hungary
Slovakia Moldova
Bulgaria
Russia
Ireland Great Britain
Monte- negro
Serbia
Slovenia Croatia Bosnia
- Herze- govina Mace-
donia Albania
Norway
Nether-
lands
Malta
Belgium
Luxembourg
Yes
No In the process of forming a NAC
No information
Australia Iceland Israel South Africa USA Brazil Morocco Countries not on map
BSAC Susceptibility Testing Workshop
Implementation of EUCAST breakpoints
BSAC Susceptibility Testing Workshop
EUCAST remit....
To determine clinical breakpoints for bacteria and fungi together with EMA as part of the process for the ”approval of new agents”
Review established clinical breakpoints
To determine epidemiological cut-off values (ECOFFs) for bacteria and fungi
To develop and standardise AST in Europe (methods, QC, education)
To act as an expert committee for EMA, ECDC, EFSA and ESCMID
BSAC Susceptibility Testing Workshop
EUCAST breakpoint tables
BSAC Susceptibility Testing Workshop
Published every January
Yellow denotes change
Guideline documents – published throughout the
year
Rationale documents
Daptomycin & enterococci
BSAC Susceptibility Testing Workshop
No breakpoint exisits at present
Daptomycin is used in enterococcal
endocarditis
Daptomycin marketed for SSTI in Europe
PKPD data insufficient at this time to produce a
breakpoint
Use ECOFFs for guidance
March 2016
Colistin method of testing
BSAC Susceptibility Testing Workshop
Increasingly used so susceptibility testing increased
Current guidelines (BSAC/EUCAST): disc diffusion not recommended
Disc criteria removed from BSAC v10 2011
Unable to detect low level resistance
Disc criteria removed in CLSI in 1981
Unable to detect low level resistance
MIC testing also problematic
– Large molecule: diffuses slowly in any diffusion test
– Cationic: adheres to any negatively charged plastic
– Colistin methanesulfonate sodium (CMS): BPs rely on PKPD data
Comparison of methods
– 50 MDR isolates (COL R)
Adding a surfactant (Tween-80) is problematic
Colistin - Problematic susceptibility testing
Hindler & Humphries JCM 2013; 51
BSAC Susceptibility Testing Workshop
Colistin - Problematic susceptibility testing
Review by a joint CLSI-EUCAST colistin working group
Gradient strips give lower MICs
Perform BMD for most reliable result
─ In non treated polystyrene
trays
─ No Tween 80
─ Colistin & NOT CMS used
Other methods are being
tested 2016
April 2016
BSAC Susceptibility Testing Workshop
Fosfomycin: Problematic testing
Fosfomycin is difficult to test with all
methods
─ Glucose-6-phosphate required in media
Agar dilution is reference method
Disk & gradient strip difficult to read
─ Kasse definition: >5 colonies per cm2
Disc labelling & concentrations vary
Antimicrobial Discs Manufacturer Code Confirmed
Fos content
Confirmed G6P
content
Fosfomycin MAST FOS50C 50ug 50ug
Fosfomycin/trometamol MAST FOT200 200ug 50ug
Fosfomycin MAST FOT200C 200ug 50ug
Fosfomycin/trometamol Oxoid CT0758 200ug 50ug
Fosfomycin Oxoid CT0183 50ug 50ug
Fosfomycin Liofilchem FOS 50ug 50ug
Fosfomycin Liofilchem FOS 100ug 50ug
Fosfomycin Liofilchem FOS 200ug 50ug
Fosfomycin BD FOS200 200ug 50ug
Fosfomycin BD 50ug 0
Fosfomycin BD 50ug 50ug
BSAC Susceptibility Testing Workshop
Difficult tests: Fosfomycin
BSAC Susceptibility Testing Workshop
Fosfomycin is difficult to test with all
methods
Glucose-6-phosphate required
Agar dilution is reference method
200ug disk + G-6-P
Difficult to read
Kasse definition: >5 colonies per cm2
Antimicrobial Discs Manufacturer Code Confirmed
Fos content
Confirmed G6P
content
Fosfomycin MAST FOS50C 50ug 50ug
Fosfomycin/trometamol MAST FOT200 200ug 50ug
Fosfomycin MAST FOT200C 200ug 50ug
Fosfomycin/trometamol Oxoid CT0758 200ug 50ug
Fosfomycin Oxoid CT0183 50ug 50ug
Fosfomycin Liofilchem FOS 50ug 50ug
Fosfomycin Liofilchem FOS 100ug 50ug
Fosfomycin Liofilchem FOS 200ug 50ug
Fosfomycin BD FOS200 200ug 50ug
Fosfomycin BD 50ug 0
Fosfomycin BD 50ug 50ug
AST results vs. WGS
BSAC Susceptibility Testing Workshop
Resistance genes NOT likely,
but results are uncertain
Resistance genes likely, but
results are uncertain
Read at 1mg/L or 128mg/L ?
Can we give guidance for reading Fosfomycin disk & gradient strips
BSAC Susceptibility Testing Workshop
Defined set of isolates (WGS)
Agar dilution, broth microdilution, disk & gradient
strip.
Using same 0.5 McFarland inoculum
At 105, 107 & 108 CFU/mL
Fosfomycin is difficult to test with all methods
Compare results Expected Late Summer 2016
Agents without EUCAST breakpoints & under consideration
BSAC Susceptibility Testing Workshop
Temocillin
Nitroxoline
Spiramycin
Cefoperazone-sulbactam
Currently EUCAST is preparing a list of “all” agents
available worldwide with explanation for why they do
not have breakpoints
EUCAST disk diffusion method development
BSAC Susceptibility Testing Workshop
Neiserria gonorrhoeae
Anaerobes
Kingella kingae..... Coming soon Aerococcus spp.......Coming soon
N. gonorrhoeae
BSAC Susceptibility Testing Workshop
EUCAST disk method not available
Recommend MIC methods using manufacturers
instructions
Media not available in UK
Continue with BSAC disc method in interim
GC agar base + 1% isovitalex 1% haemoglobin OR
Chocolate Mueller Hinton agar
Anaerobes
BSAC Susceptibility Testing Workshop
EUCAST disk method not available
Recommend MIC methods using manufacturers
instructions
Media not available in UK
Continue with BSAC disc method in interim
Brucella agar + vitaminK + haemin
Organisms with no Breakpoints and EUCAST test method
BSAC Susceptibility Testing Workshop
Aerococcus spp.
Kingella kingae
Nocardia spp. Aeromonas spp. Vibrio spp. Streptomyces spp. Leuconostoc spp. Lactobacillus spp. Pediococcus spp. Other HACEK organisms
On-going
From 2016
Aerococcus spp. & Kingella kingae disk diffusion
BSAC Susceptibility Testing Workshop
EUCAST disk diffusion method for fastidious organisms
MH-F agar
McFarland 0.5 inoculum
5% CO2
16-20h incubation
Calibration with broth microdilution
MH-F broth
A. urinae & A. sanguinicola
BSAC Susceptibility Testing Workshop
0
1
2
3
4
5
6
7
8
9
10
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No
of
iso
late
s
Inhibition zone diameter (mm)
0.125
0.06
≤0.03
0
1
2
3
4
5
6
7
8
9
10
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No
of
iso
late
s
Inhibition zone diameter (mm)
A. urinae A. sanguinicola
0
2
4
6
8
10
12
14
16
18
20
22
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No
of
iso
late
s
Inhibition zone diameter (mm)
≥16
8
4
2
1
0.5
0.25
≤0.125
0
2
4
6
8
10
12
14
16
18
20
22
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
No
of
iso
late
s
Inhibition zone diameter (mm)
A. urinae A. sanguinicola
Benzylpenicillin 1 unit Ciprofloxacin 5 µg
Kingella kingae
BSAC Susceptibility Testing Workshop
0
2
4
6
8
10
12
14
16 6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
No
of
iso
late
s
Inhibition zone diameter (mm)
4
2
1
0.5
0.25
0.125
0.06
0.03
0.016
≤0.008
Benzylpenicillin 1 unit
0
2
4
6
8
10
12
14
16
18
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
No
of
iso
late
s
Inhibition zone diameter (mm)
0.06
0.03
≤0.016
Cefotaxime 5 µg
0
5
10
15
20
25
30
35
40
45
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
No
of
iso
late
s
Inhibition zone diameter (mm)
≥16
8
4
2
1
0.5
≤0.25 0
2
4
6
8
10
12
14
16
18
20
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
No
of
iso
late
s
Inhibition zone diameter (mm)
8
4
2
1
0.5
0.25
0.125
Tetracycline 30 µg Trimethoprim-sulfamethoxazole 25 µg
Topical antimicrobials
BSAC Susceptibility Testing Workshop
Direct susceptibility testing
BSAC Susceptibility Testing Workshop
Advantages:
Early results for pure organisms
More appropriate antimicrobial therapy
Narrow spectrum antimicrobial earlier (Start Smart & Focus)
Disadvantages: Inoculum not controlled Mixed cultures pH variation or substances in samples which affect
results
Direct susceptibility testing
BSAC Susceptibility Testing Workshop
Unreliable results
No validated methods for direct testing
Repeat tests on pure cultures
Validate in house (compare with standardised test)
30 urines
3 staff
Evaluation of disks from nine manufacturers
Abtek
BD
Bio-Rad
Bioanalyse
HiMedia
Liofilchem
Mast
Oxoid
SirScan
BSAC Susceptibility Testing Workshop
Project layout
150-mm plates
16 antimicrobial disks
Each disk is tested with
– Same inoculum
– Same incubation
– Same reader
3 repetitions per disk and QC strain
Evaluation of disks from nine manufacturers
BSAC Susceptibility Testing Workshop
Tobramycin 10µg E. coli ATCC 25922
Benzylpenicillin 1 unit S. pneumoniae ATCC 49619
Evaluation of disks from nine manufacturers
BSAC Susceptibility Testing Workshop
Mean values from triplicate testing of each disk against QC strain
Results categorised:
Disks with occasional reading out of range marked with asterisk
After performed once Manufacturers asked to submit further disks
Results of first evaluation
BSAC Susceptibility Testing Workshop
Results of Second evaluation
BSAC Susceptibility Testing Workshop
Conclusions
BSAC Susceptibility Testing Workshop
There are significant differences in antimicrobial
activity in disks from different manufacturers
It is crucial for laboratories to perform internal QC,
repeating any out of range. Values should be close
to target (±1mm)
Disk manufacturers must continuosly review
manufacturing process. QC should be close to
target
Early reading of disk tests
E. coli with Cefotaxime 5ug
BSAC Susceptibility Testing Workshop
0
5
10
15
20
25
30
35
40
45
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
6 h
0
5
10
15
20
25
30
35
40
45
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
18 h
0
10
20
30
40
50
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
No
of
read
ing
s
Inhibition zone diameter (mm)
6 h incubation
0
10
20
30
40
50
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
No
of
read
ing
s
Inhibition zone diameter (mm)
8 h incubation
0
10
20
30
40
50
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
No
of
read
ing
s
Inhibition zone diameter (mm)
16-20 h incubation
Early reading of disk tests
Poorer separation between wild type and non-wild type with short incubation time
Testing of additional isolates with known resistance mechanisms is on-going
BSAC Susceptibility Testing Workshop
Subcommittees - VetCAST
to determine antimicrobial breakpoints specific to the veterinary
field
to harmonize veterinary antimicrobial susceptibility testing in the
EU
to provide education on antimicrobial susceptibility testing and
antimicrobial therapy in the veterinary field in Europe
to initiate and coordinate EU research aimed at filling the current
gaps in veterinary antimicrobial susceptibility testing
to ensure that antimicrobial susceptibility testing protocols and
interpretive criteria are freely accessible online through the
EUCAST website
BSAC Susceptibility Testing Workshop
Subcommittees – Role of Whole Genome Sequencing in AST of Bacteria Literature review on the role of WGS in antimicrobial susceptibility and
resistance testing of bacteria
Determine the possible sensitivity and specificity compared to routine standard phenotypic AST
determine how WGS may be applied in routine clinical laboratory practice and the likely implications for phenotypic and other genotypic methods of AST now in use
determine the epidemiological implications of use of WGS to detect antimicrobial resistance
determine the clinical implications of WGS in antimicrobial susceptibility testing for the selection of antimicrobial therapy
determine the principles of how the result of WGS for AST would be best presented to clinical users
define the drivers and barriers to routine use
BSAC Susceptibility Testing Workshop