Feb.

6, 2010

Antifungal Susceptibility Test

Department of Laboratory Medicine,

Chung-Ang University

Lee Mi-Kyung

1
Introduction

Antifungal Drugs

Antifungal Susceptibility Test

: CLSI AFST

Summary

2
Fungal infections are a serious
& growing public health problem

Major fungal pathogens are increasing

in incidence (systemic fungal infections)

New pathogens are emerging

Growing number of antifungal agents
Drug-resistant fungal pathogens are
emerging

3
Fungal infections  Resistance to
No. of antifungals  antifungal agents

Need for reproducible, clinically relevant

antifungal susceptibility testing 
▪ Provide a reliable measure of the relative activities of
two or more antifungal agents
▪ Correlate with in vivo activity
▪ Predict the likely outcome of therapy
▪ To monitor the development of resistance
▪ Predict the therapeutic potential of newly discovered
investigational agents
4
Antifungal agents

5
Antifungal susceptibility testing (AFST)
1. Broth-based method (M27-A3, M38-A2)
Macrobroth dilution
Microbroth dilution
Colorimetric
VITEK-2 system
2. Agar-based method
Agar dilution
E-test
Disk diffusion test (M44-A, M51-P)
3. Flow Cytometry (FCM)
4. Sterol Quantitation (SQM)
6
 Current CLSI methods for AFST
M27-A3 Reference Method for Broth Dilution Antifungal Susceptibility 4/28/2008
Testing of Yeasts
M27-S3 Reference Method for Broth Dilution Antifungal Susceptibility 4/28/2008
Testing of Yeasts
M38-A2 Reference Method for Broth Dilution Antifungal Susceptibility 4/28/2008
Testing of Filamentous Fungi

M44-A Method for Antifungal Disk Diffusion Susceptibility Testing of 5/20/2004

Yeasts
M44-S2 Zone Diameter Interpretive Standards, Corresponding Minimal 8/15/2007
Inhibitory Concentration (MIC) Interpretive Breakpoints, and
Quality Control Limits for Antifungal Disk Diffusion Susceptibility
Testing of Yeasts

M51-P Method for Antifungal Disk Diffusion Susceptibility Testing of 6/30/2009

Filamentous Fungi
M51-S1 Method for Antifungal Disk Diffusion Susceptibility Testing of 6/30/2009
Filamentous Fungi Informational Supplement

7
CLSI M27-A3: Foreword

Candida spp. & C. neoformans

In 1985
 20% (>200 beds) performing antifungal testing
 Broth dilution
 C. albicans or other spp. of yeasts
 Tested only a few isolates/yr
 Agreement of MIC among labs: unacceptably low

 it would be useful to work a more reproducible

reference testing procedure

8
CLSI M27-A3: Foreword
Proposed Standard (Dec. 1992): M27-P
 broth macrodilution
Tentative Standard (Oct. 1995): M27-T
 broth microdilution
 reference MIC ranges (2 QC strains)
Approved Standard (Jun. 1997): M27-A
 breakpoints for available antifungal agents
Approved Standard (Aug. 2002): M27-A2
Approved Standard (Apr. 2008): M27-A3 & S3
 24- & 48-hour reference MIC ranges

9
CLSI M27-A3: Advantage

Paralleling the broth macrodilution procedure

Reference MIC ranges were established

Interlaboratory agreement 

Ease to performance

Economy

More rapid results

10
CLSI M27-A3: Antifungal agents
Powder: store at -20℃
Prep. stock solution: 10 times, store at -60℃ ↓
Solvents No. of conc. tested
Amphotericin B DMSO 0.0313  16 g/mL
Fluconazole* Water 0.125  64 g/mL
Itraconazole* DMSO 0.0313  16 g/mL
Voriconazole* DMSO 0.0313  16 g/mL
Anidulafungin* DMSO 0.015  8 g/mL
Micafungin* DMSO 0.015  8 g/mL
Caspofungin* Water 0.015  8 g/mL
Flucytosine* Water 0.125  64 g/mL
* Interpretive breakpoints are available for Candida spp.
11
CLSI M27-A3: Guidelines for selective testing

As part of periodic batch surveys

To aid in the management of refractory
oropharyngeal infections due to Candida
spp. in pts. who appear to be experiencing
therapeutic failure
To aid in the management of invasive
infections due to Candida spp. when the
utility of azole antifungal agents is
uncertain (non-C. albicans)

12
CLSI M27-A3: Test procedures

Broth medium
: RPMI 1640, 0.165 mol/L MOPS, pH 6.9-7.1
Preparing diluted antifungal agents (2x serial)
: microdilution plate (96 U-shaped wells)
plastic test tubes 64 32 16 8 4 2 1 ……. 0.125 GC SC

GC (growth control)
SC (sterility control)

0.1 mL

FCZ 0.125  64 g/mL
stored at -70℃ for up to 6 months
13
CLSI M27-A3: Test procedures
Inoculum prepararion
1. subcultured onto SDA at 35℃ (purity & viability)
2. Candida spp.(24h), C. neoformans (48h)
3. 0.5 McF (15X106 cells/mL)
1:1000 dilution (0.52.5X103 cells/mL)


Incubation at 35℃
Candida spp.(2448h), C. neoformans (7074h)
14
CLSI M27-A3: Reading results
Amount of growth-compared visually with GC
0: optically clear
1: slightly hazy
2: prominent decrease (50%) in turbidity
3: slight reduction in turbidity
4: no reduction in turbidity
GC

 Score of 0: AMB
 Score of 2: 5-FC, Azole, Echinocandin

15
 Interpretive guidelines for in vitro susceptibility
testing of Candida spp.
Minimum inhibitory concentration (㎍/mL)
Antifungal Susceptible-dos
Agent Susceptible Intermediate Resistant Nonsusceptible
e dependent
(S) (I) (R) (NS)
(SDD)

Anidulafungin ≤2 >2

Caspofungin ≤2 >2

Fluconazole ≤8 16-32 ≥ 64

Flucytosine ≤4 8-16 ≥ 32

Itraconazole ≤ 0.125 0.25-0.5 ≥1

Micafungin ≤2 >2

Voriconazole ≤1 2 ≥4

16
CLSI M27-A3: Time of reading

24 h 48h
Amphotericin B Yes Yes
Fluconazole Yes Yes
Itraconazole No Yes
Posaconazole No Yes
Ravuconazole No Yes
Voriconazole No Yes
Flucytosine No Yes
Echinocandins Yes No

AMB: No approved interpretive breakpoints are available

17
CLSI M27-A3: Problems

Trailing phenomenon with some strains of Candida

spp. / azoles (5%)

Unreliable detection of resistance to AMB

Poor growth of some organisms

Labour-intensive

Hard to determine objectively through visual

reading

18
CLSI M38-A2: Foreword & Scope

Filamentous fungi (Aspergillus spp., Fusarium spp.,

Rhizopus spp., Pseudallescheria boydii, Sporothrix
schenckii, & other pathogenic molds)
Dermatophytes (Trichophyton, Microsporum, &
Epidermophyton spp.)

 Proposed Standard (Nov. 1998) ): M38-P

 Approved Standard (Aug. 2002): M38-A
 Approved Standard (Apr. 2008): M38-A2

19
CLSI M38-A2: Guidelines for selective testing

As part of periodic batch surveys

To aid in the management of invasive & cutaneous
infections due to filamentous fungi when the utility
of the azole is uncertain
Interpretive breakpoints are not available
Definition
 Minimal effective concentration (MBC)
: the lowest conc. of an antimicrobial agent that
leads to the growth of small, rounded, compact
hyphal forms as compared to the hyphal growth
seen in the GC - echinocandin

20
CLSI M38-A2: Test procedures

Inoculum prepararion
 Final conc.
Nondermatophytes: 0.45X104 CFU/mL
Dermatophytes: 13X103 CFU/mL

Incubation
 at 35℃ or 30℃ (some of Alternaria spp.)
 2126 hrs, 4650 hrs, 4672hrs

21
CLSI M38-A2: Reading results
Amount of growth-compared visually with GC
 AMB: 100% inhibition
 5-FC, FCZ, Ketokonazole
50% or more inhibition (nondermatophytes)
80% or more inhibition (dermatophytes)
 other azole: 100% inhibition
Interpretation
: The clinical relevance of testing ramains uncertain,
and breakpoints with proven relevance have yet to
identified or approved by CLSI or any regulatory
agency.

22
CLSI M44-A: Disk diffusion of Yeasts

Mueller-Hinton agar
+ 2% Glucose (fungal growth)
& 0.5 g/mL Methylene Blue dye (zone edge
definition)
0.5 McF standard (15X106 cells/mL)
Reading: after 20-24 hrs of incubation

23
Zone diameter interpretive standards for Candida spp.

Inhibition zone diameter (mm)

Susceptible Dose-dependent Resistant

(S) susceptible (S-DD) (R)

< 14
Fluconazole > 19 15 - 18

< 13
Voriconazole > 17 14 - 16

24
 3227 Candida spp. performed by 47 centers
 The overall categoric agreement between participant disk diffusion
test results and reference MIC results was 87% for fluconazole and
95.2% for voriconazole.
25
CLSI M51-P: Disk diffusion of Filamentous fungi

Nonsupplemented Mueller-Hinton agar

0.45X106 CFU/mL
Reading: after 24-72 hrs of incubation
Zone diameter
: prominent reduction in growth (80%)
Microcolonies or Slight trailing
ignored: azole, caspofungin
not ignored for AMB

26
Zone diameter epidemiological cutoff values (ECV) and corresponding
MIC or minimal effective concentration (MEC) for filamentous fungia

Equivalent MIC
Zone diameter, Nea
or MECb ECV
Antifungal agent Disk Content rest whole (mm)
(μg/mL)
ECV ECV
Amphotericin B 10 μg 15 1
Caspofungin 5 μg 17 1
Itraconazole 10 μg 17 1
Posaconazole 5 μg 17 1
Voriconazole 1 μg 17 1
aECV: The value shown the highest MIC/MEC (lowest zone diameter) of isolates
belonging to the wild-type (WT) distribution. The ECV is expressed as WT<X mcg/mL
(WT>Xmm).
bMEC applies to caspofungin only.

27
 Vitek 2 Yeast AST card
(bioMerieux, France)
The first automatic AFST
More rapid results
Eliminates the difficulties in reading of
other methods (trailing zone)
AMB, 5-FC, FCZ, VOR
(Oct 2006: licence for FCZ)

28
If the Vitek 2
system is used as
a routine test for
fluconazole MIC
measurement, it
is recommended
that Candida spp.
where the MIC is
above 4 μg/mL
be examined
using a different
method.

29
 E-Test (AB Biodisk, Sweden)

Quantitative agar
diffusion method using
antifungal gradient strips:
direct reading of MICs.
Available for all
systemic AFST, excellent
alternative to the CLSI
standard.

30
 Sterol quantitation method (SQM)

Susceptible
Resistant

31
Flowcytometry method: PI (Propidium iodide)
Flu 0 μg /mL
MCF 63

Flu 64 μg /mL
MCF 173

Heating
MCF 250

ATCC22019

32
C. tropicalis

(A) Flu 0 (B) Flu 1 (C) Flu 2

MCF 107 MCF 199 MCF 216

(D) Flu 4 (E) Flu 8 (F) Flu 16

MCF 191 MCF 200 MCF 216

(G) Flu 32 (H) Flu 64 M27-A2 (0.5 μg /mL)

MCF 202 MCF 165 FCM (1 μg /mL)

33
 Future Direction of AFST
Improved clinical correlation
Decreased subjectivity of endpoint
determination
Decreased time to result (rapid)
Personalized tests (simple)
Cost-effective
Automation
34