This document discusses antifungal susceptibility testing. It provides background on the history of antifungal susceptibility testing and why it is needed. It describes different methods for testing including broth dilution and disk diffusion. It discusses various antifungal agents and their mechanisms of action. The document outlines the procedures for broth microdilution and macrodilution testing according to CLSI guidelines, including preparation of inoculum, drug solutions, reading results, and testing of filamentous fungi.

1. ANTIFUNGAL SUSCEPTIBILITY
Presenter : Dr Abhijit Kumar Prasad
PGT, 2nd Year
Moderator : Dr W. V. Lyngdoh
Associate Professor
Department of Microbiology

2. HISTORY
• Antifungal susceptibility testing was not relevant until the
introduction in the 1950s of amphotericin B
• Nearly 30 years after the discovery of the first antibacterial
agents, antifungal susceptibility testing lay fallow for many
years
• While not all fungal infections responded to amphotericin B,
there were no alternatives
• With the development of Flucytosine and Azole antifungal
agents the differences within and between species started to
become apparent

3. WHY DO WE NEED?
• Increase in incidence of immuno-supressive states
• Increasing incidence of invasive mycosis and life threatening
infections as a significant public health issue
• Emerging resistance
• Correlate with in vivo activity and predict the likely outcome
of therapy
• Provide a reliable measure of the relative activities of two or
more antifungal agents

4. Continued. . .
• Growing concern about a shortage of effective antifungal
agents and an increase in the resistance of fungal pathogens
to the existing agents
• Among the invasive mycoses, none is more important or
common than candidiasis
• Candidiasis, specifically candidemia, has been shown in
numerous studies to be the most frequent mycotic infection
in hospitalized patients and is associated with a significant
attributable mortality and excess length of hospital stay

5. SURVIELLANCE
PROGRAMME
YEAR No. OF
ISOLATE
IN BSI
% of Total Isolates
% OF FLUCONAZOLE RESISTANCE
C.
albicans
C.
glabrata
C.
parapsilopsis
C.
tropicalis
CDC 1998-2K 944 45
1
24
10
13
0
12
6
SWEDEN 1994-98 233 0 40 15 0
QUEBEC 1996-98 442 1 9 0 0
SCOPE 1997-2k 934 53
1
20
7
10
0
12
1
EIEIO 1999-01 254 58
0
20
10
7
0
11
0
JOURNAL OF CLINICAL MICROBIOLOGY,
Oct. 2002, p. 3551–3557 Vol. 40, No. 10
0095-1137/02/$04.000 DOI: 10.1128/JCM.40.10.3551–3557.2002
American Society for Microbiology. All Rights Reserved.

6. Indian Studies
% sensitivity of Candida isolates to Antifungal agents
ORGANISM AMB FLUCYT-
OSINE
FLUCONAZOLE ITRACONAZOLE VORICONAZOLE
C. albicans 100 100 100 95 100
NAC Spp 89.6 90.9 68.8 54.3 88.6
Prevalence of NAC in BSI in a tertiary care hospital at New
Delhi, India. Oberoi et al.
Indian J Med Res 136, December 2012, pp 997-1003

7. ANTIFUNGAL AGENTS
Class Generic
Name & Available
formulation
Mechanism of
action
Azoles
Imidazoles
Triazoles
Imidazoles
Ketoconazole
(oral tab/ shampoo)
Miconazole (topical/oral)
Econazole,Bifonazole,Ticonazole
Triazoles
Fluconazole
(IV,oral susp/tab)
Itraconazole (oral cap/Sol)
Voriconazole (IV,oral tab)
Posaconazole
(Oral suspension)
Interferes with
ergosterol synthesis
via inhibition of CYP
dependent 14α
demythylase,
required for
conversion of
lanosterol to
ergosterol

8. ANTIFUNGAL AGENTS
Class Generic
Name& Available formulation
Mechanism of action
Allylamines Terbinafine (oral, topical)
Naftifine (topical)
Butenafine (topical)
Inhibit squalene epoxidase
required for ergosterol
formation
Polyenes Amp B deoxycholate (IV, Oral
sol)
Amp B liposomal(IV)
Amp B lipid complex(IV)
Amp B colloidal disp(IV)
Nystatin (topical)
Pimaricin( Eye oint)
Hamycin(Powder)
Binds to ergosterol m& creates
pores, leading to depolarization
of the membrane & cell leakage
Griseofulvin • Griseofulvin –Insoluble (oral) Acts by inhibiting microtubule
function in dermatophytes

9. ANTIFUNGAL AGENT
Class Generic
Name& Available
formulation
Mechanism of action
Pyrimidine Flucytosine ( 5FC)
Oral
Converted by Cytosine deaminase to 5-
FU, an inhibitor of Thymidylate
synthase, thus blocking nucleic acid
synthesis
Echinocandins Caspofungin (IV)
Micafungin(IV)
Anidulafungin(IV)
Inhibit enzyme Glucan synthase which
in turn inhibits formation of β1,3
Glycan synthesis, a component of
fungal cell wall leading to increase cell
wall permeability and lysis of the cell
Thiocarbamates Tolnafate (topical)
Nikkomycins – Inhibit Chitin synthesis, important component of cell wall

10. ANTIFUNGAL SUSCEPTIBILITY TESTS
1) BROTH DILUTION ANTIFUNGAL SUSCEPTIBILITY TEST
2) DISK DIFFUSION ANTIFUNGAL SUSCEPTIBITY TEST
3) E-TEST
4) VITEK

11. BROTH DILUTION ANTIFUNGAL SUSCEPTIBILITY
TESTING FOR YEASTS (CLSI M27/A3)
• Synthetic Media
RPMI 1640 buffered with MOPS (Morpholine
propanesulfonic acid) for Candida spp
Yeast Nitrogen Broth for Cryptococcus neoformans
• Antifungal agents
Obtained commercially as pure salt
Amphotericin-B, 5-Fluorocytosine, Azoles such as
Fluconazole, Itraconazole, Voriconazole, Ketoconazole,
Posaconazole, Ravuconazole
Pharmaceutical preparations of these agents is not
recommended

12. Preparation of Stock Solution
• Water soluble antifungal agent - Fluconazole,5-
fluorocytosine, Caspofungin, Micafungin are prepared in
sterile distilled water
• Water insoluble antifungal agent - amphotericin B,
itraconazole, ketoconazole, posaconazole, ravuconazole, and
voriconazole are prepared in dimethyl sulfoxide (DMSO)
• Other solvents used are
Ethyl Alcohol,
Polyethylene glycol
Carboxy methyl cellulose

13. Weighing of Antifungal powder
• Weight (mg) = Volume (ml) x Conc (µg/ml) (FORMULA 1)
Assay potency (µg/mg)
Q) What amount of antifungal powder is to be weighed to
prepare 100 ml of the stock solution containing 1280 /ml of
antifungal agent with antifungal that has a potency of 750
µg/mg ?
• Weight (mg) = 100 ml x 1280 µg/ml = 170.7 mg
750 µg/mg

14. Continued. . .
“Because it is advisable to weigh a portion of the powder in
excess of that required, powder was deposited on the balance
until 182.6 mg was reached” – CLSI
• Volume (ml) = Wt (mg) x Assay potency (µg/mg ) (Formula 2)
Conc (µg/ml)
• Volume (ml) = 182.6 mg x 750 µg/mg = 107ml
1280 µg/ml

15. Continued. . .
• Stock solution are to be prepared at 10 times the highest
concentration to be tested
• Drugs dissolved in solvent are to be prepared at 100 times
higher than the desired concentration to be tested
• Each intermediate then should be further diluted to final
strength in the test medium
• Reason - Avoids dilution artifact that result from precipitation
of compound with low solubility in aqueous media
• Storage - at – 60°C or below but never at temperature
greater than – 20°C, 6 months

16. Concentration of the drug to be tested
• Concentration to be tested should encompass the breakpoint
concentration & the expected results for the quality control
strains
• Based on previous studies, the following drug concentration
ranges is to be used –
Amphotericin B, 0.0313 to 16 ꭒg/ml
Flucytosine, 0.125 to 64 ꭒg/ml
Ketoconazole, 0.0313 to 16 ꭒg/ml
Itraconazole & new triazoles 0.0313 to 16 ꭒg/ml
Fluconazole, 0.125 to 64 ꭒg/ml

19. PROCEDURE
• Inoculum preparation
All organisms should be subcultured onto SDA or PDA
Incubated at 35°C for 24 / 48 Hr
Inoculum should be prepared by picking five well-isolated
colonies of 1 mm in diameter
Suspend the organisms in 5 ml of sterile saline
Suspension equal to a 0.5 McFarland standard
Working suspension is prepared by adding 0.1 ml of the
suspension from above to 9.9 ml of RPMI
Further diluted to 1:20 with RPMI to give a concentration
of 5 x 102 to 2.5 x 103 cells / ml

20. Test inoculation and incubation
(macrodilution method)
• Add 0.1 ml of each antifungal (intermediate concentration for
water-soluble drugs and for water insoluble drug intermediate
concentration diluted to 1:10) to a sterile 12- by 75-mm test
tube
• Growth control tube -
0.1 ml of drug diluent without the antifungal agent
• Second tube of drug diluent -
Uninoculated to serve as a sterility control
• Within 15 min of inoculum preparation, 0.9 ml of the
inoculum is added to each tube in the series (Except sterility
control) bringing antifungal agents to the final concentration
• Incubation – 35°C for 46 to 50 h (Candida spp)

21. Reading Results
• Amount of growth in each tube is compared to that of the
growth control
• For amphotericin B, the MIC is read as the lowest drug
concentration that prevents any visible growth
• For 5-fluorocytosine & Azoles, the MIC is read as the lowest
drug concentration that exhibits 80% inhibition
• “Trailing End Points”

22. Microdilution modifications
• Excellent concordance between results obtained by the broth
macrodilution methodology and broth microdilution
adaptation
• Easy to perform, trays may be sealed in plastic bags and
stored frozen at -70 °C for up to six months without
deterioration of drug potency
• Stock solutions of antifungal agents and their intermediate
concentrations are prepared in the manner described earlier
• Intermediate concentrations are diluted 1:5 with RPMI in case
of water soluble drug & 1:50 times for water insoluble drug to
achieve 2 X (times) concentration needed for the
microdilution method

23. ANTIMICROBIAL SOLUTION (Water Soluble Drug) (Double Strength)
STE
P
CONC
(μg/mL)
SOURCE VOLUME (ml) +MEDIUM(ml) Intermediate
Conc (μg/mL)
Final Conc
1:5 (RPMI)
(μg/mL)
1 5120 STOCK 1 ml 7 ml 640 128
2 640 Step 1 1 ml 1 ml 320 64
3 640 Step 1 1 ml 3 ml 160 32
4 160 Step 3 1 ml 1 ml 80 16
5 160 Step 3 0.5 ml 1.5 ml 40 8
6 160 Step 3 0.5 ml 3.5 ml 20 4
7 20 Step 6 1 ml 1 ml 10 2
8 20 Step 6 0.5 ml 1.5 ml 5 1
9 20 Step 6 0.5 ml 3.5 ml 2.5 0.5
10 2.5 Step 9 1 ml 1 ml 1.25 0.25
11 2.5 Step 9 0.5 ml 1.5 ml 0.625 0.12
12 2.5 Step 9 0.5 ml 3.5 ml 0.3126 0.0625

24. ANTIMICROBIAL SOLUTION (Water Insoluble Drug) (Double Strength)
STE
P
CONC
(μg/mL)
SOURCE VOLUME (ml) + SOLVENT(ml) Intermediate
Conc (μg/mL)
Final Conc
1:50 (RPMI)
(μg/mL)
1 1600 STOCK 1600 32
2 1600 STOCK 0.5 ml 0.5 ml 800 16
3 1600 STOCK 0.5 ml 1.5 ml 400 8
4 1600 STOCK 0.5 ml 3.5 ml 200 4
5 200 Step 4 0.5 ml 0.5 ml 100 2
6 200 Step 4 0.5 ml 1.5 ml 50 1
7 200 Step 4 0.5 ml 3.5 ml 25 0.5
8 25 Step 7 0.5 ml 0.5 ml 12.5 0.25
9 25 Step 7 0.5 ml 1.5 ml 6.25 0.125
10 25 Step 7 0.5 ml 3.5 ml 3.13 0.0625

25. Continued. . .
• A stock yeast suspension of 0.5 Mc Farland is prepared
• 0.5 McF corresponds to 1 x 106 to 5 x 106
• Suspension is then diluted 1:50 (2 x 104 to 5 x 106)
• Further diluted 1:20 with RPMI to achieve the 2 x test
inoculum (5 x 102 to 2.5 x 103) = (1 x 103 to 5 x 103) CFU/ml)
• When combined with drug, the final concentration will be 5 x
102 to 2.5 x 103 CFU/ml

26. Continued . . .
• Using a multichannel pipette, dispense 100 ꭒl of the 2x
antifungal concentrations into columns 1 to 10 of sterile-
disposal 96-well (U-shaped) microdilution plates
• Column 1 will contain the highest concentration, and column
10 will contain the lowest concentration of drug
• Columns 11 and 12 should each receive 100 ꭒl of diluent
• Using a multichannel pipette, dispense 100 ꭒl of working
yeast suspension into each well of columns 1 to 11
• Column 11 will serve as Growth Control
• Column 12 should remain uninoculated & is used as a
sterility control

27. AmB
5FC
FLU
ITRA
VORI
CASPO
Increasing Concentrations
candida
Decreasing concentration

28. Continued. . .
• Tubes should be incubated without agitation at 35°C for 46 to
50 hr (Candida spp.) or 70 to 74 (C. neoformans) hr
• Plates are placed on a microdilution plate reader with a
magnifying mirror
• MICs are read in the same manner as described for
macrodilution tubes

29. Testing of Filamentous Fungi
• As per CLSI M38/A2
• Test medium of choice for filamentous moulds is RPMI 1640
• Preparation of both stock and working solutions of antifungal
agents remains the same as described
• Allylamine terbinafine can be added to the list of antifungal
agents, in particular when testing dermatophytes
• Terbinafine is dissolved in DMSO and tested at final conc. of –
16 to 0.03 ꭒg/ml for filamentous moulds
0.5 to 0.001 ꭒg/ml for dermatophytes

30. Continued. . .
• Aspergillus spp., Pseudallescheria boydii, Rhizopus, and
Sporothrix -
Grown on PDA slants at 35°C for 7 days
• Fusarium spp
Grown on PDA slants at 35°C for 2 to 3 days and then at
room temperature until day 7
• Trichophyton rubrum isolates can be induced to form conidia
on cereal agar plates incubated at 30°C for 5 to 7 days

31. Continued. . .
• Slants are covered with sterile saline and conidia are
harvested by agitation with a Pasteur pipette
• One drop of Tween 20 is added to facilitate inoculum
formation
• Resulting mixture of conidia & hyphal particles is removed to
a sterile conical tube and allowed to settle for 3 to 5 min
• Upper layer is removed to a second vial and vortexed
• Optical density (OD) of this suspension is measured using
Densitometer and adjusted with sterile saline -
Aspergillus spp. and Sporothrix, 0.09 to 0.11 OD units
Fusarium, P. boydii, and Rhizopus, 0.15 to 0.17 OD units

32. Continued. . .
• Stock suspensions of conidia is to be diluted
1:50 in RPMI 1640 for microdilution testing
• Final test inocula suspension should be in the range of 0.4 x
104 to 5 x 104
• Procedures for setting up the macro- and microdilution tests
are the same as described above for yeast isolates

33. Reading Result
• For conventional microdilution procedure, the growth in each
MIC well is compared with that of the growth control with the
aid of reading mirror
• Each microdilution well is then given a numerical score as
follows :
4 – No reduction in growth
3 – Approximately 75% reduction in growth
2 – Approximately 50% reduction in growth
1 - Approximately 50% reduction in growth
0 – Optically clear or no growth

34. Continued. . .
• For AMB –
End point well defined
MIC is read as the lowest concentration that prevents any
discernible growth (Score 0)
• Flucytosine, Fluconazole & Ketoconazole
End point typically less defined;
Less stringent end point is allowed
Turbidity that corresponds to approximately 50% reduction
in growth compared to the growth control well (Score 2)

35. Continued. . .
• Itraconazole & newer triazoles - Posaconazole, Ravuconazole
& Voriconazole
End point well defined (Score 0) – For Aspergillus spp
When testing for Dermatophyte isolate, 80% or more
reduction in turbidity relative to the growth control
• Terbinafine
Turbidity allowed is corresponds to 80% or more
reduction in growth relative to growth control
• Recommended MIC / MEC limits for Quality Control &
Reference strains are given in CLSI M27/A3 & M38/A2
document

36. CALORIMETRIC METHOD
• Based on microdilution
format as CLSI
• Reading end points are
enhanced by the use of
Calorimetric indicator
(Modified Resazurin)
• Blue – No growth
• Red – Growth
• Purple – Partial inhibition

37. ANTIFUNGAL DISK DIFFUSION
SUSCEPTIBILITY TESTING (CLSI M44/A2)
• Method described is intended for testing Candida spp
• Does not encompass any other genera & has not been used in
the studies of the yeast form of the Dimorphic fungi
REAGENTS FOR THE DISK DIFFUSION TEST
• Mueller-Hinton Agar + 2% Glucose + 0.5 mcg/ml Methylene
Blue Dye medium (GMB); pH 7.2 to 7.4
• 2% glucose provides for suitable fungal growth
• Methylene blue enhances zone edge definition

38. Continued. . .
Preparation of test inoculum
Inoculation of the test plate (Same as that for bacteria)
Application of Disks to inoculated agar plate

39. Reading plates & interpreting results
• Examine each plate after 24 to 24 Hr of incubation
• Measure the zone diameter to the nearest whole millimeter
at the point at which there is a prominent reduction in growth
• Pinpoint microcolonies at the zone edge or larger colonies
within a zone are encountered frequently & should be ignored

40. Continued. . .
INTERPRETIVE CATEGORIES
• Susceptible (S) : Infection due to the strain may be
appropriately treated with the dose of the antimicrobial agent
recommended
• Susceptible Dose Dependent (S-DD) : includes isolates with
antimicrobial agent MICs that approach usually attainable
blood & tissue levels but the response rate may be lower than
for susceptible isolates (Only applies when multiple approved
dosage options exist)
• Resitant (R) : Isolates are not inhibited by the usually
achievable concs of the agent with normal dosage schedules

41. ANTIFUNGAL AGENTS, ZONE DIAMETER & MIC INTERPRETIVE
CRITERIA FOR C. albicans & C. tropicalis
Antimicr
obial
agent
Dics Diffusion Zone Diameter
(mm)
MIC (mcg/ml)
Suscep
-tible
Interme-
diate
Resistant Suscept-
ible
S-DD Interm-
ediate
Resistant
Anidul-
afungin
- - - ≤ 0.25 - 0.5 ≥ 1
Caspof-
ungin
≥17 15-16 ≤14 ≤ 0.25 - 0.5 ≥ 1
Micafu-
ngin
≥22 20-21 ≤19 ≤ 0.25 - 0.5 ≥ 1
Flucon-
azole
≥17 14-16 ≤13 ≤ 2 4 - ≥ 8
Itracon-
azole
≤ 0.125 0.25-0.5 - ≥ 1
Voricon-
azole
≥17 15-16 ≤14 ≤ 0.125 0.25-0.5 - ≥ 1

42. Antifungal
Agents
Disk Content
(mcg)
Recommended Quality Control Zone
Diameter (mm) Ranges
C. albicans C.
parapsilosis
C. tropicalis
Fluconazole 25 mcg 28 -39 22 – 33 26 - 37
Voriconazole 1 mcg 31 – 42 28 – 37 *
Quality control ranges have not been established for these strains /
antimicrobial agent combination due to extensive inter-laboratory variation

43. E–TEST (EPSILOMETER TEST)
• A predefined stable antimicrobial gradient is present on a thin
inert non-porous plastic carrier strip 5mm wide, 60 mm long
known as E-test strip
• Device consists of a predefined, continuous, and exponential
gradient of antibiotic concentrations immobilized along a
rectangular plastic test strip
• Following incubation, a symmetrical inhibition ellipse is
produced
• Intersection of the inhibitory zone edge and the calibrated
carrier strip indicates the MIC value

44. Continued. . .
• Following incubation, a
symmetrical inhibition
ellipse is produced
• Intersection of the
inhibitory zone edge
and the calibrated
carrier strip indicates
the MIC value

45. Fluconazole E-Test Strip

46. Continued. . .
Microorganism YEAST MOULD
Medium RPMI 1640 + 2% glucose +
MOPS + 1.5% Bacto agar
RPMI 1640 + 2% glucose +
MOPS + 1.5% Bacto agar
Inoculum 0.5 McF (for Candida spp)
(1McF for Cryptococcus
neoformans); Suspension
made in Saline
0.5 McF Aspergillus spp.
(1 for Fusarium, Rhizopus
spp);
Suspension is made in
Saline + Tween 20
Time/ Temp 24 Hrs at 35°C for Candida
spp
16 to 72 Hrs depending on
genus at 35°C
MIC Panel FL, IT, VO, AMB, CS, FC AMB, IT, CS, POS, VO

47. VITEK 2 COMPACT

48. VITEK 2 COMPACT
Suspension Preparation
• Transfer a sufficient number of colonies of a pure culture in
3.0 mL of sterile saline (aqueous 0.45% to 0.50% NaCl, pH 4.5
to 7.0) in a 12 x 75 mm clear plastic (polystyrene) test tube
• Turbidity is adjusted accordingly & measured using DensiChek
• For Yeast & Yeast like organism turbidity should be between
1.8 – 2.2 McF
• In a second tube containing 3.0 mL of saline, transfer 280 μL
of the the suspension prepared earlier & the AST card is put
on the corresponding slot

49. Continued. . .
• Reagent cards have 64 wells that can each contain an
individual test substrate
• Each AST card contains 64 microwells
• Control well contains only the microbial culture media, with
the remaining wells containing premeasured amounts of
specific antimicrobials combined with culture medium
• Card is then filled, sealed, and thereafter placed into the
instrument incubator/reader manually / automatically
• Each card is removed from the carousel incubator once every
15 minutes
• Transported to the optical system for reaction readings, and
then returned to the incubator until the next read time

50. Continued. . .
• Data are collected at 15-minute intervals during the entire
incubation period
• Test data from an unknown organism are compared to the
respective database to determine a quantitative value for
proximity to each of the database taxa
• Each of the composite values is compared to the others to
determine if the data are sufficiently unique or close to one or
more of the other database taxa
• If a unique identification pattern is not recognized, a list of
possible organisms is given, or the strain is determined to be
outside the scope of the database

51. Continued. . .
AST CARD
• Instrument monitors the growth of each well in the card over
a defined period of time upto 36 Hrs for Yeast
• At the completion of the incubation cycle, MIC values are
determined for each antimicrobial contained on the card
automatically by the VITEK machine

53. CONCLUSION
• While promising, antifungal susceptibility testing is still a
research tool
• In view of the AIDS epidemic & increasing incidence of
invasive mycosis and life threatening infections there is a need
to institute antifungal susceptibility in all tertiary care
hospitals
• Helps to evaluate therapeutic outcome
• Surveillance required to assess the in vitro activity of newer
antifungal drugs & know the emerging resistance pattern

54. THANK YOU