Antifungal Susceptibility Test

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

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

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

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

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.

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

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

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

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

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

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

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

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

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

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

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

Antifungal Susceptibility Test

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

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)

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”

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Antifungal Susceptibility Test

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