Antimicrobial susceptibility testing

Some (unresolved) issues

Aims of this talkAims of this talk

comparing different susceptibility test

methods

do different test results matter?

specific testing issues

Testing methodsTesting methods

Disc

Broth dilution

Automated methods

Etest

Testing methodsTesting methods

Disc

Broth dilution

Automated methods

Etest

Inoculum

Disc strength

Media type

pH

Anionic content

Reproducibility

Testing methodsTesting methods

Disc

=

Broth dilution

=

Automated methods

=

Etest

Essential agreement

+/- 1 dilution of the

accepted “gold standard”

Testing methodsTesting methods

Essential

agreement

4

2

1

0.5

0.25

MIC (mg/L)

1

Essential agreementEssential agreement

Are these two methods in essential agreement?

MIC testing by Method 1 & Method 2

0

1

2

3

4

5

6

7

8

0.25 0.5 1 2 4 8 16 32

MIC values

no of isolates w

ith MIC

MIC testing by Method 1

0

1

2

3

4

5

6

7

8

0.25 0.5 1 2 4 8 16 32

MIC values

no of isolates w

ith MIC

Categorical agreementCategorical agreement

Method 1 has a lot more “S” than Method 2

– Even though both methods have excellent

essential agreement

Susceptible ≤ 2; Resistant > 2

MIC distributionsMIC distributions

breakpoint

no. of resistant strains

no. of susceptible strains

No

of

stra

ins

MIC

MIC distributionsMIC distributions

breakpoint

no. of resistant strains

(with the second method)

no. of susceptible strains

(with the 2nd method)

+ 1 log dilution

MIC distributionsMIC distributions

breakpoint

no. of resistant

strains

no. of susceptible strains

MIC distributionsMIC distributions

breakpoint

no. of resistant

Strains

(with 2nd method)

no. of susceptible strains

(with 2nd method)

+ 1 log dilution

Categorical errorsCategorical errors

Intermediate

error

Intermediate

error

False

resistance

False

susceptibility

Interpretation

Minor errorIS or R

Minor errorS or RI

Major errorSR

Very major

error

RS

Error

category

“Gold

standard”

Test

system

Susceptibility testing methodsSusceptibility testing methods

Multiple factors may affect susceptibility testing

results

Different testing methods need to agree:

– Essential agreement

– Categorical agreement

Automated systemsAutomated systems

Guidance for Industry and FDA

Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test (AST) Systems

Document issued on: August 28, 2009

EN ISO 20776-1:2006

Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -Part 1: Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria involved in infectious diseases (ISO 20776-1:2006)

AST systems AST systems -- FDAFDA

% categorical and essential agreement

> 89.9%.

major error rate

< 3%

very major error rate (based on the number of resistant organisms tested)

1.5% - 7.5%

AST systems AST systems -- CLSICLSI

% categorical and essential agreement

major error rate

very major error rate (based on the number of resistant organisms tested)

GramGram--positivepositive

agentsagents

DaptomycinDaptomycin

Cyclic lipopeptide antibiotic

Bactericidal

Bacteraemia and skin/soft tissue infections

Acts on cell wall

membrance

Calcium dependant

action

DAPTOMYCIN

DaptomycinDaptomycin

MIC’s decrease with increasing Ca2+

concentration

Etest:

calcium supplemented strips

Microbroth dilution:

calcium supplemented broth

DAPTOMYCIN

DaptomycinDaptomycin

Etest v.s. microbroth dilution

Etest MIC higher by 0.5 – 1 dilution

Lot –related elevation in QC?

DAPTOMYCIN

Sader HS, et al. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009 Jul;53(7):3162-5.Friedrich L, et al. Evidence for daptomycin Etest lot-related MIC elevations for Staphylococcus aureus. Diagn Microbiol Infect Dis. 2009 Nov;65(3):306-11.

VANCOMYCIN

VancomycinVancomycin

Glycopeptide

Bactericidal (bacteriostatic?)

Diffuses slowly into agar

MIC testing (broth, agar), Etest and Vitek

methods

(no disc)

VancomycinVancomycin

Etest v.s. microbroth dilution

Etest MIC higher by 1 dilutionVANCOMYCIN

Sader HS, et al. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009 Jul;53(7):3162-5.

VancomycinVancomycin

Etest v.s. microbroth dilution v.s. Vitek

Vitek < broth microdilution < Etest

VANCOMYCIN LOW HIGH

Phoenix,Microscan

Agar dilution,Sensititre

Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.

-1 +10

doubling dilution

VancomycinVancomycin

Etest v.s. microbroth dilution v.s. Vitek

Vitek

VANCOMYCIN LOW HIGH

Agar dilution,Sensititre

Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.

-1 +10

doubling dilution

VancomycinVancomycin

Etest v.s. microbroth dilution v.s. Vitek

Etest

VANCOMYCIN LOW HIGH

Phoenix,Microscan

Jana M. Swenson, et al. Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus. J Clin Microbiol. 2009 July; 47(7): 2013–2017.

-1 +10

doubling dilution

VancomycinVancomycin

Agar screen plate?

BHI agar with 3 mg/L vancomycin

100% sensitivity, 65% specificity

VANCOMYCIN

Carey-Ann D. et al. Novel Screening Agar for Detection of Vancomycin-NonsusceptibleStaphylococcus aureus. J Clin Microbiol. 2010 March; 48(3): 949–951.

Ability to detect true positives

Ability to detect true negatives

TigecyclineTigecycline

Glycycline

(related structure to tetracyclines)

Liable to oxidation

Available for disc (FDA), broth dilution, Etest and Vitek

TIGECYCLINE

Tigecycline & EtestTigecycline & Etest

broth microdilution < EtestTIGECYCLINE

0 1-1doubling dilution

FOR:

Acinetobacter spp., S. pneumoniae, Serratia marcescens

Pillar CM, et al. In vitro activity of tigecycline against gram-positive and gram-negative pathogens as evaluated by broth microdilution and Etest.J Clin Microbiol. 2008 Sep;46(9):2862-7. Epub 2008 Jul 2.

Tigecycline & broth dilutionTigecycline & broth dilution

Needs fresh Mueller-Hinton broth

– MIC’s in aged broth increased by up to 1 dilution

– caused by oxidative effect of dissolved oxygen

May also be a problem for commercial

microbroth kits (e.g. Sensititre)

For agar dilution, use freshly steamed agar

TIGECYCLINE

Patricia A. Bradford, et al. Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test Method. Antimicrob Agents Chemother. 2005 September; 49(9): 3903–3909.

Hope R, et al. Effect of medium type, age and aeration on the MICs of tigecycline and classical tetracyclines. J Antimicrob Chemother. 2005 Dec;56(6):1042-6.

Tigecycline & agar methodsTigecycline & agar methods

Zone diameters may change depending on

brand of agar used

– affects Acinetobacter spp., Enterobacteriaceae

Applies to disc and Etest

TIGECYCLINE

Fernández-Mazarrasa C, et al. High concentrations of manganese in Mueller-Hinton agar increase MICs of tigecycline determined by Etest. J ClinMicrobiol. 2009 Mar;47(3):827-9.

Canigia LF, Bantar C. Susceptibility testing of tigecycline against Acinetobacter spp. by disc diffusion method: withdrawing a therapeutic option by varying the Mueller-Hinton agar? J Antimicrob Chemother. 2008 Dec;62(6):1463-4.

GramGram--negativenegative

agentagent

Polymyxin B & agar methodsPolymyxin B & agar methods

Difficult antibiotic to test:

narrow zone diameters

affected by cations (especially P. aeruginosa)

Etest values affected by brand of media & different lots of media

Disc testing is not reliable

POLYMYXIN

B

Lo-Ten-Foe JR, et al.Comparative evaluation of the VITEK 2, disk diffusion, etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii strains. Antimicrob Agents Chemother. 2007 Oct;51(10):3726-30.

Polymyxin B & agar methodsPolymyxin B & agar methods

Heterogenous resistance: Enterobacter spp.,

Stenotrophomonas maltophiliaPOLYMYXIN

B

Polymyxin B & other methodsPolymyxin B & other methods

Vitek testing:

caution required: may not detect heterogenous

resistance

MIC’s tend to be lower

Broth dilution:

“Gold standard” method

POLYMYXIN

B

Tan TY, Ng SY. Comparison of Etest, Vitek and agar dilution for susceptibility testing of colistin. Clin Microbiol Infect. 2007 May;13(5):541-4. Epub 2007 Mar 19.

SummarySummary

How to evaluate results from different testing

methods

Problematic antibiotics – importance of quality

assurance

Know your bacterial population and the

meaning of different testing methods