Principles and Practice of Antimicrobial Susceptibility Testing

Microbiology Technical Workshop 25th September 2013

Scope
History Why Perform Antimicrobial Susceptibility Testing? How to Perform an Antimicrobial Susceptibility Test
Isolate Method (phenotypic, special tests, genotypic) Drug Standards (how breakpoints are derived; CLSI, EUCAST, BSAC, CDS)

Understanding An Antimicrobial Susceptibility Test

Categorical interpretation 90-60 rule

Summary

History

Source: http://www.s1darvel.com/

Source: http://www.biography.com/

Sir Alexander Fleming (1881-1955)

When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionize all medicine by discovering the world's first antibiotic, or bacteria killer. But I suppose that was exactly what I did.

Why Perform Antimicrobial Susceptibility Testing?

Empiric Therapy

Investigations

Definitive Therapy

Patient LAM
89 year old Chinese female Previously hospitalized 2 months ago for cystitis
Urine culture ESBL positive E. coli Blood cultures negative

Now admitted from the A&E with fever, dysuria and increased urinary frequency for 3 days
Urine microscopy RBC 3, WBC > 2000, EC 0 Urine culture no bacterial growth

Started empirically on IV Piperacillin/tazobactam

 Predict treatment outcome Guide selection of most appropriate agent Provide alternatives drug allergy, oral option

How to Perform an Antimicrobial Susceptibility Test

Isolate

Method

Drug

Standards

Results

Isolate
Identity of bacterial isolate Normal flora Clinically significant number of colonies Predictable antibiotic susceptibility profile

Method
Phenotypic
Zone diameter Minimum inhibitory concentration (MIC)

Special tests Genotypic

Source: CLSI M100-S23E Performance Standards for Antimicrobial Susceptibility Testing - 23rd Informational Supplement

Source: http://www.cdc.gov/

Source: http://www.cdc.gov/

Disk Diffusion

Source: http://bacterioweb.univ-fcomte.fr/

Etest (Epsilometer test)

Source: http://www.cdc.gov/

Source: http://web.carteret.edu/

Source: http://web.carteret.edu/

Broth Macrodilution

Source: www.biomerieux.com

Source: www.biomerieux.com

Automated (Vitek2)

Source: www.biomerieux.com

Source: http://microblog.me.uk/

D-Test for inducible clindamycin resistance

Cephalosporin

Clavulanic acid

Cephalosporin

ESBL Detection (double-disk approximation)

Source: http://www.cepheid.com/

Source: http://www.ahsoman.com/

Source: http://www.pih.org/

Source: http://www.pih.org/

Drug
Species to be tested Institution formulary Commonly used antimicrobials Availability of antimicrobial agent for testing Tailored to specific needs of institution
Infectious disease physicians Clinical microbiologists Pharmacists Committees concerned with institutional formulary

Source: http://www.eucast.org/

Standards
MIC distribution
Wild-type Epidemiological cut-off (ECOFF)

Pharmacokinetic Pharmacodynamic Clinical data

susceptible
80 70 60 50 40 30 20 10 0 0.125 0.25 0.5 1 2 4 8 16

resistant

32

64

128

256

increasing concentration of antibiotic

susceptible
80 70 60 50 40 30 20 10 0 0.125 0.25 0.5 1 2 4 8 16

resistant

32

64

128

256

increasing concentration of antibiotic

Source: http://mic.eucast.org/

Source: http://mic.eucast.org/

???
Source: http://mic.eucast.org/

Standard CLSI

Location America, several areas of Europe, Asia, Australia Europe

Media Mueller-Hinton agar Mueller-Hinton agar with 5% sheep blood Mueller-Hinton agar Mueller-Hinton agar with 5% defibrinated horse blood + 20 mg/L -NAD Iso-Sensitest agar Iso-Sensitest agar with 5% defibrinated horse blood + 20 mg/L NAD Sensitest agar Sensitest agar with 5% horse blood

Inoculum 0.5 McFarland

EUCAST

0.5 McFarland

BSAC

United Kingdom

0.5 McFarland then dilute (refer to Table) Refer to Figure

CDS

Australia

Source: http://bsac.org.uk/ (Version 12 May 2013)

Source: http://bsac.org.uk/ (Version 12 May 2013)

Source: http://bsac.org.uk/ (Version 12 May 2013)

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

CLSI

Source: CLSI M100-S23E Performance Standards for Antimicrobial Susceptibility Testing - 23rd Informational Supplement

Source: CLSI M100-S23E Performance Standards for Antimicrobial Susceptibility Testing - 23rd Informational Supplement

EUCAST

Source: http://www.eucast.org/

Source: http://www.eucast.org/

BSAC

Source: http://bsac.org.uk/ (Version 12 May 2013)

CDS

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

Source: http://web.med.unsw.edu.au/ (Sixth Edition)

Understanding An Antimicrobial Susceptibility Test

Identify Test Report

Categorical Interpretation
Category Susceptible Resistant Intermediate Interpretation High likelihood of therapeutic success High likelihood of therapeutic failure Uncertain therapeutic effect Drug concentration at body sites Buffer zone Antifungal susceptibility testing Susceptibility dependent on achieving maximal possible blood level Often seen with new antimicrobial agents

Susceptible-dose dependent (S-DD)

Nonsusceptible

Source: Rex and Pfaller CID 2002 - Has Antifungal Susceptibility Testing Come of Age?

Summary
Antimicrobial susceptibility tests allows us to
Predict treatment outcome Guide selection of most appropriate agent Provide alternatives

Factors to consider
Isolate Method Drug Standards

Understanding the results

Categorical interpretation 90-60 rule

Source: http://www.channelnewsasia.com/

Thank you for your attention!

Dr Jamie Tan (jamie.tan.b.x@sgh.com.sg) Registrar Department of Pathology Singapore General Hospital