Mast Discs: Combi Specific Tests Susan Thomson

International Business Manager

Company Confidential

Development of the EUCAST disk diffusion antimicrobial

susceptibility testing method and its implementation in routine

microbiology laboratories

EUCAST

E. Matuschek¹, D. F. J. Brown² and G. Kahlmeter¹

1) EUCAST Laboratory for Antimicrobial Susceptibility Testing, Vaxjo, Sweden and 2)

EUCAST Scientific Secretary, Peterborough, UK

http://www.eucast.org

EUCAST

ANTIMICROBIAL RESISTANCE

Aminoglycoside resistance

Macrolide resistance

Antibiotic

Resistance

Quinolone resistance

Glycopeptide resistance

B-lactam resistance

ESBL family

MRSA

‘Classical phenotypic methods are slow, but are the most

extensively evaluated, and for this reason remain the

recommended methods for laboratories without special

expertise in β-lactamase detection’

EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or

epidemiological importance . EUCAST, December 2012

ESBL detection

The modern lab is faced with many challenges in

the pursuit of identifying these mechanisms:

1. Classification terminology

2. Emerging resistances

3. Methodologies available – which one

4. Detection of mixed resistances

5. Amp C expression

6. Organism exceptions – intrinsic resistance

7. The Modified Hodge Test interpretation

8. Lack of a reliable and affordable genotyping platform

BETA-LACTAMASE CLASSES

Molecular Class β-lactamase

A ESBL (TEM, SHV, CTX-M),

KPC (KPC 1, KPC2

B MBL (VIM, IMP, NDM,)

C AmpC (CMY, FOX, MOX)

D OXA type

CLASSIFICATION

ESBLS

ESBL Extended Spectrum Beta

Lactamases Class A

(inhibited by Clavulanic acid)

AmpC Class C

(inhibited by Cloxacillin or Boronic acid)

Carbapenemases

MBL Metallo Beta Lactamases

Class B (inhibited by Mercaptoacetic Acid or EDTA or Dipicolonic

acid DPA)

KPC Klebsiella pneumoniae

carbapenamase Class A

(limited inhibition by Boronic Acid)

Oxacillinases OXA

Class D

DIFFERENCE BETWEEN ESBL AND AMPC

ESBL’s (TEM, SHV, CTX-M):

• Hydrolyse 3rd and 4th generation cephalosporins

• Most are susceptible to cephamycins

• Susceptible to carbapenems (can be resistant due to porin

loss)

• Inhibited by clavulanate

AmpC’s (MOX, FOX, DHA, ACC, CIT, EBC, CMY)

• Hydrolyse 3rd generation cephalosporins and cephamycins

• Susceptible to carbapenems (can be resistant due to porin

loss)

• Not inhibited by clavulanate

• Inhibited by class C inhibitors such as cloxacillin and

boronic acid

WHY DIFFERENTIATE THE TWO?

• Minimises reporting of false cephalosporin susceptibility

• Confirmation with cefoxitin unreliable

• Some combinations used to treat ESBL infections can induce

AmpC production

• AmpC’s are more likely to develop carbapenem resistance due

to impermeability

•Potentially resistance in AmpC producers is broader in

spectrum than ESBL

WHAT ARE CARBAPENEMASES?

• Carbapenemases are β-lactamase enzymes which

hydrolyse almost all β-lactam antibiotics

• Current and extensive worldwide spread in

Enterobacteriaceae

• High rate of transmissibility

• Causes outbreaks

TERMINOLOGY

• CRO – carbapenem resistant organisms

• CPO - carbapenemase producing organisms

• CRE - carbapenem resistant enterobacteriacae

• CPE - carbapenemase producing

enterobacteriacae

Carbapenemase

producers

Carbapenem

resistant

Not all!

Not all!

The Problem

Do You Know Your CRO from your CPO from your CRE from your CPE – Jon Otter

MicroBlog 2013

CARBAPENEM RESISTANCE

COMPARISON OF CARBAPENEM RESISTANCE

KPC enzymes:

• Hydrolyse all β-lactams

• Activity is inhibited partially in vitro by clavulanic acid, tazobactam

and boronic acid.

Metallo-β-lactamases (MBLs; IMP, VIM and NDM)

• Hydrolyse all β-lactams except aztreonam

• Activity is inhibited in vitro with compounds such as zinc chelators

(EDTA, DPA, MCA)

OXA-48-type enzymes

• Hydrolyse aminopenicillins, ureidopenicillins and carbapenems at

low levels, but do not significantly hydrolyse broad-spectrum

cephalosporins.

• Activity is inhibited in vitro by NaCl

Modified Hodge Test

Anderson KF et al JCM 2007; 45(8): 2723-5

10 µg discs. Method not

recommended: results difficult to

interpret; sensitivity and specificity

are poor.

Sensitivity Specificity

Etest MBL 55 100

Modified Hodge 58 93

Mast D70C 78 93

Neo-Sensitabs 80 93

PCR 100 100

Doyle et al J.Clin.Micro. (2012) 50 3877

IMPORTANCE OF DETECTION

• Major public healthcare concern

• Severely limits treatment options-Carbapenems are ‘last resort’

• Associated with high morbidity/mortality rates

• Increases hospital cost

• Dwindling supply of new effective antibiotics

Early detection is critical !

HOW CAN MAST HELP YOU?

‘Classical phenotypic methods are slow, but are the most

extensively evaluated, and for this reason remain the

recommended methods for laboratories without special

expertise in β-lactamase detection’

EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or

epidemiological importance . EUCAST, December 2012

Cheap, but optimal disc separation essential

Double Disc Test

ESBL production is inferred when clavulanate expands

the zone of either cephalosporin.

Augmentin disc

(Clavulanate) Expanded zone/

“synergy”

Cepahalosporin disc

(ie Ceftazidime) Cepahalosporin disc

(ie Cefotaxime)

AmpC and ESBL Detection Set D68C

AmpC & ESBL Positive

ZD – ZC 5mm

and ZB – ZA <5mm

Negative

zones differ by

2mm

Positive Predictive Value

Screening PPV

Vitek 2 69%

Phoenix 68%

Disc diffusion 92%

Confirmation PPV

Etest 61%

Combination disc 91%

Platteel, T.N. et al Clinical Microbiology and Infection (2011) 17 1435

CTX, CAZ and

CPD mastdiscs

D62C & D64C

mastdiscs

D63C

mastdiscs

AmpC EXPRESSION

AmpC

• Clavulanate induces AmpC

• Need to use Cefepime for ESBLs

• Doesn’t differentiate type of AmpC

• E coli hyper-produce AmpC, doesn’t possess inducible AmpC

• Mutations occur causing AmpC gene to be permanently

expressed at high levels – derepressed

• Chromosomal AmpC – Enterobacter, providencia, aeromonas

Hafnia alvei, Morganella morganii, Citrobacter freundii, Serratia

marscesens and Ps aeruginosa

• Plasmid mediated AmpC – E coli, Klebsiella spp, Salmonella

spp, Proteus spp

• Test D69C

AmpC Detection Set D69

AmpC Positive ZC – ZA and ZC – ZB

5mm

Negative

zones differ by

3mm

Derbyshire et al JAC 2009 63:497-501

ESBL Derepressed AmpC M. morganii AmpC

Inducible AmpC ESBL + AmpC ESBL + inducible AmpC

MAST D63C + D66C

Cefoxitin

Ceftazidime

Inducible AmpC

Use CPD & CPD/CLAV – negate ESBLs

Kit Sensitivity Specificity

Mast AmpC detection

disc set 96 – 100% 98 – 100%

Rosco tablets 96% 92%

AmpC gradient test 84 – 93% 70 – 100%

J.Antimicrob.Chem. (2012) 67 2303; J.Med.Microbiol. (2011) 60 715; APMIS (2012) 120 724;

Eur.J.Clin.Microbiol.Infect.Dis (2013) 32 1205; J.Med.Microbiol. (2011) 60 715;

J.Clin.Microbiol. (2011) 49 2924.

Commercially Available Kits

CTX, CAZ and

FOX mastdiscs

D68C or D69C

mastdiscs

“Although data evaluating these methods is

sparse, reasonably accurate detection with in-

‐house methods has been described (8--‐10) as

well as with commercially available tests such as

the Mast “AmpC Detection Disc Set” (sensitivity

96--‐100%, Specificity 98%--‐100%)”

CARBAPENEM RESISTANCE

Carbapenemase Detection Set D70C

MBL Positive

ZB – ZA 5mm

KPC Positive

ZC – ZA 5mm

ZA

ZC

ZB

ZD

ZA ZB

ZD ZC

D70C – Why use Meropenem?

Ertapenem has high sensitivity with poorer

specificity than either meropenem or imipenem

(especially difficult if AmpC/ESBL mediated

carbapenemase observed over true

carabepenemases)

Meropenem has lower sensitivity BUT higher

specificity

Generally we peer review publications

Carbapenemase Detection

Boronic

acid Cloxacillin

DPA or

EDTA

Clavulanate

or

tazobactam

Temocillin R

Piperacillin-

tazobactam R

Aztreonam

KPC + - - Weak - / + R

AmpC + + - - - / + R

MBL - - + - - / + S

OXA-

48 - - - - + S

Combination discs (Mast, Rosco) - increase of > 5 mm with 10 µg discs;

double-ended Etests; agar dilution with EDTA (320 mg/L) - ≥ 8-fold reduction in MIC.

Temocillin > 32 mg/L. Mostly validated on Mueller Hinton agar. 18 hours.

MEM10C

mastdiscs

D70C

mastdiscs

TEMOCILLIN 30C

mastdiscs “The combination disk test has the advantage of being well‐validated

in studies and is also commercially available (MAST),”

MBLs/KPCs

AmpCs

ESBLs

TEŞEKKŰRLER