This document provides information on antifungal susceptibility testing methods. It discusses the CLSI broth microdilution method (M27-A3) for testing yeasts, using RPMI 1640 medium. Quality control strains, inoculum preparation, and reading results are described. The CLSI method (M38-A2) for filamentous fungi testing is also covered. Finally, the CLSI disk diffusion method (M44-A2) for testing yeasts using Mueller-Hinton agar supplemented with glucose and methylene blue is summarized.

1. Antifungal Susceptibility Testing
Mycology Research Lab
Department of Medical Microbiology. PGIMER, Chandigarh.
Dr Sshrutkirti gupta
Consultant microbiologist

2. Current Antifungal Targets
Cell wall
Echinocandins:
Caspofungin
Micafungin
Anidulafungin
Cell membrane
Azoles:
Fluconazole
Ketoconazole
Itraconazole
Voriconazole
Posaconazole
Cell membrane
Polyenes:
Amphotericin B
Lipid AmB formulations*
Nystatin
DNA and RNA
Antimetabolites:
5-fluorocytosine

3. Antifungal Agents
Polyenes (Alter membrane function)
• Amphotericin B (liposomal), Nystatin, Natamycin
Pyrimidine analogs (Inhibit DNA/RNA)
• 5-Flucytosine
Azoles (Inhibit ergosterol synthesis)
• Immidazoles - Miconazole, Clotrimazole, Ketoconazole
• Triazoles - Fluconazole, Itraconazole,Voriconazole,
Posaconazole, isavuconazole, etc.
Echinocandins (Inhibit glucan synthesis)
• Caspofungin ,Micafungin and Anidulafungin

4.  Fungal infections - Immunocompromised hosts
 Therapeutic monitoring
 Resistance across the board
- Candida - Aspergillus - Cryptococcus
- Histoplasma - Trichosporon
 Newer drugs
 Increased demand
- Drug discovery
- Epidemiology
Need for Susceptibility Testing

5. Standards for antifungal susceptibility
testing
NCCLS is an international, interdisciplinary,
nonprofit, standards-developing, and educational
organization that promotes the development and
use of voluntary consensus standards and guidelines
within the healthcare community.
Providing NCCLS standards and guidelines, ISO/TC 212 standards and ISO/TC 76
standards
National Committee on Clinical Laboratory
Standards(NCCLS)
1 January 2005
USA

6. EUCAST = European Committee for
Antimicrobial Susceptibility Testing
Consensus standards organization
Europe

7. Standard Plates Glucose
Conc.
inoculum Incubation Reading
EUCAST Flat-bottom 2% 0.5×105 -
2.5×105 24 hrs Spectrophot
ometric
CLSI Round
bottom
0.2% 0.5×103 -
2.5×103 24- 48hrs manual
Comparison of CLSI and EUCAST
Similarities
- BMD Methods
- RPMI-1640
- Endpoint criterion same
Differences

8.  CLSI Broth Dilution Method for Yeasts (M27-A/A3/A4)
 CLSI Broth Dilution Method for Non-dermatophyte
molds (M38-A/A2)
 CLSI Disk Diffusion for Yeasts (M44-A/A2)
 CLSI Disk Diffusion for Non-dermatophyte molds(M-
51A)
Methods for Susceptibility Testing

9. Other methods
 E-test
 Flowcytometry based methods
 Other Methods for Yeasts and Molds
 Testing of Dermatophytes

10. CLSI Broth Dilution for Yeasts (M27-A3)
 Method for testing all pathogenic Candida species and
Cryptococcus neoformans.
 Not used for yeast forms of dimorphic fungi.
(NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second
Edition. NCCLS document M27-A2 [ISBN 1-56238-469-4]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania
19087-1898 USA, 2002.)

11. NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
• Antifungal Powders
- Source: Directly from the manufacturer
- Pharmacy stock not recommended
• Properties of stock powder to be kept in consideration
- Generic name
- Assay potency…..amount of active ingredient/gm powder
- expiration date
- Solubility………………………………………….DMSO/DH20
- Storage………………………………………………-20 or below
CLSI Broth Dilution for Yeasts (M27-A3)

12. CLSI Broth Dilution for Yeasts (M27-A3)

13. NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
Therefore, the 182.6 mg of the antifungal powder is to be dissolved in 107.0 mL of diluent.
Make stock solutions of antifungals
CLSI Broth Dilution for Yeasts (M27-A3)

14. Stock solutions
• Stock conc. should be 80/ 100 times the highest
conc. tested
S.No. Solubility Range
Stock
(µg/mL)
1 DMSO 0.0313-16.0 1600
2 water 0.125 - 64.0 5120
3 Echinocandins 0.015 – 8.0 5120/1600

15. NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
Testing Media
• RPMI 1640
- synthetic medium
- with glutamine, without bicarbonate
- Phenol red as a pH indicator
- MOPS [3-(N morpholino propane sulfonic acid] @ 0.165
mol/L)
CLSI Broth Dilution for Yeasts (M27-A3)

17. NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
CLSI Broth Dilution for Yeasts (M27-A3)

18. CLSI Microdilution method Suggested Plating Scheme

19. Inoculation of drug plates
Inoculum (test and control strains)
- very important
- Freshly S/C organisms
- Ensure purity and viability of isolates

20. -
0.5 McFarland standard(1×106
-5×106 cfu/mL)
- Absorbance at 625nm =
0.08-0.13
1000 times dilute in RPMI-
1640 (1:50, 1:20)
2X(standard(1×103 -5×103
cfu/mL)
Dispense 0.1mL in each drug
conc.
1X(standard(0.5×103 -2.5×103
cfu/mL)

21. Quality control strains
16 8 0.03
0.06
0.12
0.25
0.5
1
2
4
Ideal Reference
strain
Acceptable
Ideal reference strain
- AST pattern genetically stable
- Storage -70 in 15% glycerol
- Avoid using after more than 3
passages

23. NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
 Incubation @ 35 °C for 24 to 48 hours. C.neoformans, 70
to 74 hours.
 Reading Results - The amount of growth in the tubes
containing the agent is compared visually with the amount
of growth in the growth-control tubes (no antifungal agent)
used in each set of tests.
 AmpB – well defined, Azole and echinocandins - 50%
inhibition relative to growth control well taken as MIC
CLSI Broth Dilution for Yeasts (M27-A3)

24. Scale for determining MIC
Value Visual Reading % inhibition Agents
0 Optically clear 100 amphotericin B
1 Slightly hazy 80 -
2 Prominent
reduction
50 5- FC and azoles
Echinocandins
3
Slight reduction in
turbidity
20 -
4 No reduction in
turbidity
0 -
Echinocandins: reading taken after 24 Hrs

25. Trailing growth and impact of time of reading
• Trailing growth: partial inhibition of growth over an extended
range of antifungal concentrations
• Some isolates show a dramatic rise in MIC between 24-48hrs
due to significant trailing growth (for example, 5 % isolates in case
of fluconazole)
• Difficult to assign the susceptibility category
• Such isolates should be treated as susceptible

26. QC responsibilities
• Drug manufacturers responsibilities
- antifungal stability
- Potency
- Labelling
- compliance with good manufacturing process,etc.
• Laboratorian
- Storage( drug deterioration)
- operator proficiency
- Adherence to procedure(e g., inoculum, incubation )

27. Trailing Growth
yeasts Molds
CLSI Broth Dilution for Yeasts (M27-A3)

28. CLSI Broth Dilution for Yeasts (M27-A3)
NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.

30. CLSI Broth Dilution for Filamentous fungi (M38-A2)
NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. NCCLS
document M38-A2 (ISBN 1-56238-668-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.

31. Broth Dilution for Filamentous fungi (M38-A2)
 Aspergillus species, Fusarium species, Rhizopus species,
Pseudallescheria boydii, and the mycelial form of Sporothrix
schenckii.
 Rest protocol is similar to M27-A3 except Inoculum preparation.
 0.4 x 104 to 5 x 104 CFU/ml provided the most reproducible MIC
data.
Aspergillus and Sporothrix schenckii - OD @530 - 0.09- 0.11
 Fusarium species, Pseudallescheria boydii- 0.15-0.17.
 P. boydii may require 50% lower dilution factor.
 For inoculum only 1:50 dilution.
 Dermatophytes final 1 x 103 to 3 x 103 CFU/ml
NCCLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard. NCCLS
document M38-A (ISBN 1-56238-470-8). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002.

32. Inoculum preparation of filamentous fungi
• Growth on PDA slants for 7 days at 35 C or until good sporulation
• Cover sporulating colonies with 1mL of sterile NS ,add one drop tween-20
• Prepare a conidial suspension by slighltly probing the colonies with a tip
•
• Transfer the suspension into sterile tubes and keep undisturbed
• After the hyphae and crude material settle, take out upper clear suspension
and adjust its OD with spectrophotometer
• Dilute this inoculum 1:50 in the medium and directly inoculate each well

33. Quality Control strains(M38-A2)

34. CLSI Disk Diffusion for Yeasts (M44-A2)
NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS
document M44-A [ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania
19087-1898 USA, 2004.
 Method for testing of Candida species
 Zone Breakpoints for fluconazole and voriconazole QC parameters
for fluconazole, voriconazole posaconazole and caspofungin
 Qualitative results 24 hours
 Readily available - reduced cost
 Development of testing - other antifungal agents / genera

35. DISC diffusion methods
• Need?
To make antifungal susceptibility testing
- readily availability
- simple, rapid, and cost-effective

36. CLSI Disk Diffusion for Yeasts (M44-A2)
NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A
[ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.
Equipment & Reagents
 Incubator set at 350C (± 20C)
 McFarland 0.5 Turbidity Standard
 Sterile cotton swabs (not synthetic polyester fiber)
 Sterile physiologic saline (8.5 g/L NaCl; 0.85%)
 Mueller-Hinton Agar
 Antimicrobial (antifungal) disks

37. Mueller-Hinton Agar (GMB) Medium
(2% Glucose and 0.5 μg/mL Methylene Blue Dye)
• Readily available
• Good batch-to-batch reproducibility
• Supplemented with glucose - suitable fungal growth
• Methylene blue dye enhances zone edge
• Medium supplemented either pre- or postproduction
CLSI Disk Diffusion for Yeasts (M44-A2)

38. • pH of Mueller-Hinton Agar - 7.2 and 7.4 @RT
• Moisture on Agar Surface - if excess, plates dried
• Storage of Antimicrobial Disks – store@ < 8 °C or freeze at
-14 °C or below
• Turbidity Standard for Inoculum Preparation - inoculum
density, turbidity standard (BaSO4, equivalent to a 0.5
McFarland standard or its optical equivalent (e.g., latex
particle suspension)
CLSI Disk Diffusion for Yeasts (M44-A2)

39. Standard strains
• Candida albicans ATCC 90028.
• Candida parapsilosis ATCC 22019.
• Candida tropicalis ATCC 750.
• Candida krusei ATCC 6258.
CLSI Disk Diffusion for Yeasts (M44-A2)

40. Inoculum Preparation: Direct Colony Suspension Method
• Subculture to ensure purity and viability, @35 °C (±2 °C)
• Inoculum preparation - Pick 5 colonies 1 mm, suspend in 5 ml
of sterile 0.145 mol/L saline
• Vortex suspension - 15 seconds, adjust turbidity visually/
spectrophotometer (530 nm wavelength) to 0.5 McFarland
standard
• Yeast stock suspension of 1 x 106 to 5 x 106/ ml - produce semi-
confluent growth with most Candida species
M44-A2 Procedure

41. Inoculation of Test Plates
• Within 15 minutes – dip sterile cotton swab into the
suspension, rotate several times and press firmly against the
inside wall of the tube above the fluid level.
• Inoculate - evenly streak the swab on surface X 2, rotate the
plate 60°, finally swab rim of agar.
• Lid ajar for 3-5min, not >15 min - apply drug-impregnated
disks.
M44-A2 Procedure

42. Application of Disks
• Antimicrobial disks - placed 24 mm apart (center- center).
• 12 disks 150-mm plate, 5 disks 100-mm plate.
• Do not move once in place, instead a new disk in new
location (drug diffuses instantaneously).
• Incubate @ 35 °C (± 2 °C) within 15 minutes
M44-A2 Procedure

43. Reading
• Read 20 to 24 hours of incubation,
Zones - uniformly circular, semi
confluent growth.
• Zone diameter to the nearest mm -
• Pinpoint microcolonies at zone edge
ignored
• Read at 48 hours if insufficient growth
• Highly subjective, experience greater
accuracy.
M44-A2 Procedure
This image shows a Candida albicans tested with
fluconazole and voricionazole disks.

45. Interpretation
Categories
• Susceptible (S) - Treated with the recommended dose of
antifungal.
• Susceptible-Dose Dependent (S-DD) - MICs that approach
attainable blood and tissue levels - response rates may be
lower than for susceptible isolates.
• Resistant (R) - not inhibited by usual achievable
concentrations.
M44-A Procedure

46. Interpretation
Zone Diameter Interpretive Standards and Corresponding Minimal
Inhibitory Concentrations (MIC) Breakpoints for Candida spp.
M44-A2 Procedure

47. Quality Control
Recommended Quality Control Zone Diameter (mm) Ranges
M44-A Procedure

48. Disk Diffusion Daily Quality Control Testing Protocol
NCCLS Disk Diffusion for Yeasts
(M44-A).
Reproduced with permission,
from NCCLS publication M44-
A—Method for Antifungal
Disk Diffusion Susceptibility
Testing of Yeasts; Approved
Guideline (ISBN 1-56238-532-
1). Copies of the current
edition may be obtained from
NCCLS, 940 West Valley
Road,Suite 1400, Wayne,
Pennsylvania 19087-1898,
USA.

50. • Microtitre broth dilution method
• Each test – disposable microtitre
plate
• Dried serial dilutions antifungal
• Wells contain Alamar Blue
• Colorimetric indicator
• Improves the end point
readability
• Colour change from blue to pink.
Sensititre® Yeast One™ Test Panel
Manufactured by TREK