asst antimicrobial susceptibility · pdf filethe purpose to present the methods used for ast a...

SASTAntimicrobial

Susceptibility TestingMarc V. Assous, MD, PhDShaare Zedek Med. Ctr.

Universite Paris Descartes

Dr Marc Assous, Microbiology Lab, Shaare Zedek Med. Center

The purposeThe purpose To present the methods used for AST

A - Micro-dilution (ISO 20776-2006)

B - Agar dilution C - semi-automated methods D - Agar diffusion E - Gradient diffusion

To define for each one Parameters Parameters Performances Advantages Advantages Disadvantages

Only rapid growing bacteriaDr Marc Assous, Microbiology Lab, Shaare Zedek Med. Center

Only rapid growing bacteria

Choice of routine methodsChoice of routine methods For non fastidious bacteria all methods are satisfactory Depends on : Depends on :

cost Time of processing/work Reagents availability Professional knowledge

A tomation access Automation access More sophisticated methods are:

Micro-dilution Micro dilution Agar dilution Semi-automated

Disk diffusion remains the more accessible and economic method

Most laboratories have 2 methods


Most laboratories have 2 methods

MIC = Minimum Inhibitory MIC = Minimum Inhibitory ConcentrationConcentration

StandardStandardinoculuminoculum

DoublingDoublingdilutions ofdilutions of

64 512481632

dilutions ofdilutions ofantibioticantibiotic(mg/L)(mg/L)

64 .512481632

44 μμg/mlg/mlDr Marc Assous, Microbiology Lab, Shaare Zedek Med. CenterAdapted From Pr C. Block

4 4 μμg/mlg/ml

Broth MicroBroth Micro dilutiondilution 11B th M ll Hi t i dil ti th d i th

Broth MicroBroth Micro--dilution dilution -- 11 Broth Muller-Hinton micro-dilution method is the

international standard reference method (ISO 20776-2006)20776 2006)

This standard was chosen by the ISO ISO EUCAST CLSI CLSI CA-SFM

The method was very carefully evaluated and The method was very carefully evaluated and Was correlated to modern parameters like

pharmokinetics/pharmacodynamics (PK/PD)


p p y ( )(breakpoints)

Broth MicrodilutionBroth Microdilution--22 Microplates 96 wells U In house or reagents In house only Reference Lab (titrated antibiotic

d QC)powder, QC) ISO 20776-1(2006) 96 euros

100 l MH b th ti dj t d (CAMHB) 100 µl MH broth cation-adjusted (CAMHB), 5x104 bacteria/ml, 35°C 18-24h.

QC +++ ATCC and QC target /range ± 1 dilution QC +++ ATCC and QC target /range ± 1 dilution OK for Staphylococcus spp., Enterococcus spp,

Bacillus spp., Corynebacterium spp.,Bacillus spp., Corynebacterium spp., Enterobacteriaceae, Aeromonas spp., Vibriospp., Pseudomonas spp., Acinetobacter spp., Burkholderia spp Stenotrophomonas spp


Burkholderia spp., Stenotrophomonas spp.

AnAn exampleexample of broth microof broth micro--dilutiondilution

0.06 - 0.12- 0.25 - 0.5 - 1- 2- 48 16 32

PC

8 - 16 - 32


Broth MicroBroth Micro--dilutiondilution--33 Other media

several genus needs enriched broth HTM for Haemophilus spp Brucella for Brucella and anaerobies Lysed horse blood Special cases : Daptomycine, Abiotrophia, Granulicatella (B6)...

Quite simple to adapt the method for each purpose ! Quite simple to adapt the method for each purpose ! Problems

Not “clear cut” (“En traine”) Partial growth (80% control) Well(s) with no growth

MBC (minimal bactericidal concentration) ? MBC (minimal bactericidal concentration) ? Important to characterize new antibiotics Bacteriostatic or bactericidal


Not useful to determine in clinical practice

Agar dilution methodAgar dilution method--11 Standard method published : CLSI, EUCAST, CA-SFM

Antibiotic included in the agar plateEach plate represents another dilution

42 tips in each

Each plate represents another dilutionControl

12864643216

88421

Steers apparatus0.5

0.250.125


pp0.06250.0312

Agar dilution methodAgar dilution method--22Agar dilution methodAgar dilution method 22- Very frequently used method- Mueller Hinton plates, p ,Isosensitest etc...- NoNo international standardization

Problems :Not standardized for : H. influenzae and parainfluenzae, B. cepaciap pand other fastidious bacteria-For some bacteria/antibiotic diff. between MIC -Technical problems


Technical problems-Ionic concentration not controlled, PB with Dapto. and Tigecycline.

SemiSemi automated methodsautomated methods 11SemiSemi--automated methodsautomated methods--113 A t Si Mi S ® 3 Apparatus : Siemens MicroScan®, Biomerieux Vitek2®, BD Phoenix®

Liquid medium growth and turbidimetric readingreading

Antibiotic concentrations are chosen between breakpoints

Validation versus the micro-dilution method Validation versus the micro dilution method correlation with the Ref. method is

S ft i t dDr Marc Assous, Microbiology Lab, Shaare Zedek Med. Center

Software assisted

SemiSemi automated methodsautomated methods 22Mi ® d BD Ph i ®

SemiSemi--automated methodsautomated methods--22 Microscan® and BD Phoenix® true MIC,

but only 1 or 2 dilutions around breakpoints Biomerieux Vitek2® growth algorithm Whatever, limitation in the results Whatever, limitation in the results Limitation of the card panel

D l b t f t Delay between occurrence of new events and updates.

Cost is higher than other phenotypic methods


Agar diffusion methodAgar diffusion method--11Agar diffusion methodAgar diffusion method 11 The most used routine method for routine ASTThe most used routine method for routine AST Available for a lot of bacteria species, fastidious bacteria

included (Streptococcus, Haemophilus, pneumococcus...) A lot of International experience and experts from all over

the world.A lot of variation on the same principle But now A lot of variation on the same principle. But now, EUCAST and CLSI

XXXX

Ad t d F P C Bl k


Zone diameter Adapted From Pr C. Block

Agar diffusion methodAgar diffusion method--22gg

Regression curve

MICMIC

Zone diameter (mm)Zone diameter (mm)


Zone diameter (mm)Zone diameter (mm)Adapted From Pr C. Block

Agar diffusion methodAgar diffusion method--33Agar diffusion methodAgar diffusion method--33

Regression depends on the species but the method uses an average RC.

There is a need to use RC according to the species (EUCAST)S tibi ti hibit diff i bl Some antibiotics exhibit diffusion problems (glycopeptides, polypeptides, daptomycine, linezolide) so Disc Diffusion is not adapted.p

The USA standard is CLSI (375 $ !!!) widespread European standard is EUCAST (Free of charge), rather

new, but progressively replaces national European standards (BASC, CA-SFM, SRGA)


Agar diffusion methodAgar diffusion method--44CLSI (Cli i l d L b t St d d I tit t )

Agar diffusion methodAgar diffusion method--44 CLSI (Clinical and Laboratory Standard Institute)

Mueller Hinton rapid growing bacteria Haemophilus Test Medium. Haemophilus Test Medium. MH + 5% sheep blood Streptococcus (pneumoniae) Other fastidious bacteria

EUCAST (European Union Committee for AST) Mueller Hinton rapid growing bacteria Mueller Hinton rapid growing bacteria MH + 5% horse blood + 20mg β-NAD/L Haemophilus AND

Streptococcus (pneumoniae)Correlation bet een Zone Diameters and MIC Correlation between Zone Diameters and MIC

Breakpoints from EUCAST


Agar diffusion methodAgar diffusion method –– 5

Standardization +++

Agar diffusion method Agar diffusion method –– 5

Standardization +++ Standard Operating Procedures

Z Di t Di t ib ti Zone Diameter Distribution CLSI and EUCAST : same MIC/ZD for rapid

i b t igrowing bacteria CLSI and EUCAST : same MIC for fastidious but

t ZDnot same ZD. For better validation , comparison with histogram

di t ib tidistribution QC for routine is necessary to implement.


Agar diffusion methodAgar diffusion method -- PerformancesAgar diffusion method Agar diffusion method -- Performances


Agar diffusion methodAgar diffusion method

eucast.org


ECOFF


Interpretative reading of the antibiogram


This is a sub-population p p


EE-- Gradient Diffusion Gradient Diffusion 1988, new method

antibiotic gradient strip E t t® (AB Bi di k)E-test® (AB-Biodisk)

Also fungus and fastidious etc..

Apparently, simple to use but needs to be performed accordingperformed according to manufacturer instructions and to implement QC/readingimplement QC/reading

Quite expensive but simply allows to obtain

da very good approximation of MIC for a lot of


microorganisms

ConclusionsMi dil ti d A dil ti t MIC b t

Conclusions Micro-dilution and Agar dilution true MIC but

not possible to use in routine For routine

Semi-automated method good approximation of the MIC but expensive, not flexible. OK for SOP. Problems for detection of new mechanisms of resistanceresistance.

Disk diffusion not MIC but EUCAST approach is of interest low cost but needs carefully implementationinterest, low cost but needs carefully implementation of SOP and QC. Need for another method like E-test for some special cases (glycopeptides, polypeptides,


p (g y p p , p yp p ,etc...).

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