principles and practice of antimicrobial susceptibility testing...

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.biography.com/

Source: http://www.s1darvel.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

InvestigationsDefinitive 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

Results

Standards

Method Drug

Isolate

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/

Broth Macrodilution

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

Automated (Vitek2)

Source: www.biomerieux.com

Source: www.biomerieux.com

Source: www.biomerieux.com

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

D-Test for inducible clindamycin resistance

Clavulanic acid Cephalosporin

ESBL Detection (double-disk approximation)

Cephalosporin

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

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

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

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

0

10

20

30

40

50

60

70

80

0.125 0.25 0.5 1 2 4 8 16 32 64 128 256

increasing concentration of antibiotic

susceptible resistant

0

10

20

30

40

50

60

70

80

0.125 0.25 0.5 1 2 4 8 16 32 64 128 256

increasing concentration of antibiotic

susceptible resistant

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

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

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

???

Standard Location Media Inoculum

CLSI America, several

areas of Europe,

Asia, Australia

Mueller-Hinton agar

Mueller-Hinton agar with 5% sheep blood

0.5 McFarland

EUCAST Europe Mueller-Hinton agar

Mueller-Hinton agar with 5% defibrinated horse

blood + 20 mg/L β-NAD

0.5 McFarland

BSAC United Kingdom Iso-Sensitest agar

Iso-Sensitest agar with 5% defibrinated horse

blood + 20 mg/L NAD

0.5 McFarland

then dilute

(refer to Table)

CDS Australia Sensitest agar

Sensitest agar with 5% horse blood

Refer to Figure

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)

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

CLSI

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

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

EUCAST

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

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

BSAC

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 Interpretation

Susceptible • High likelihood of therapeutic success

Resistant • High likelihood of therapeutic failure

Intermediate • Uncertain therapeutic effect

• Drug concentration at body sites

• Buffer zone

Susceptible-dose dependent (S-DD) • Antifungal susceptibility testing

• Susceptibility dependent on achieving

maximal possible blood level

Nonsusceptible • Often seen with new antimicrobial

agents

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

Thank you for your attention!

Dr Jamie Tan (jamie.tan.b.x@sgh.com.sg)

Registrar

Department of Pathology

Singapore General Hospital

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