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


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


Implementation of EUCAST breakpoints


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


EUCAST breakpoint tables


Published every January

Yellow denotes change

Guideline documents – published throughout the

year

Rationale documents

Daptomycin & enterococci


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


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


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


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 BD FOS200 200ug 50ug

Fosfomycin BD 50ug 0

Fosfomycin BD 50ug 50ug


Difficult tests: Fosfomycin


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 BD FOS200 200ug 50ug

Fosfomycin BD 50ug 0

Fosfomycin BD 50ug 50ug

AST results vs. WGS


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


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


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


Neiserria gonorrhoeae

Anaerobes

Kingella kingae..... Coming soon Aerococcus spp.......Coming soon

N. gonorrhoeae


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


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


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


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


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


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


≥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


A. urinae A. sanguinicola

Benzylpenicillin 1 unit Ciprofloxacin 5 µg

Kingella kingae


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


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


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


≥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


8

4

2

1

0.5

0.25

0.125

Tetracycline 30 µg Trimethoprim-sulfamethoxazole 25 µg

Topical antimicrobials


Direct susceptibility testing


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


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


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



Tobramycin 10µg E. coli ATCC 25922

Benzylpenicillin 1 unit S. pneumoniae ATCC 49619



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


Results of Second evaluation


Conclusions


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


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


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


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


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


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


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 residential workshop

Documents