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Antifungal Susceptibility Testing Methodology
Mahmoud A. Ghannoum, Ph.D., Professor, Director=
Center for Medical Mycology, UHC and CWRU
<=
/b>
11100 Euclid Ave., LKS 5028
Cleveland, OH&=
nbsp;
44106
Center for Medical Mycology, 2002
2
§The need for a standardized, clinically relevan=
t, =
antifungal susceptibility testing method became=
obvious as a result of recent developments in t=
he =
field of mycology.
§<=
b>The
developments include:
–An alarm=
ing
increased in the rate of fungal infections
–An increase in the number=
of
reports of resistance to all of the available
antifungals
–Introduct=
ion
of new antifungals such as fluconazole, itraconazo=
le,
terbinafine
Center for Medical Mycology, 2002
3
A=
ntifungal
Susceptibility Testing Methodology
•Historical perspective:&=
#13;
§A large number of investigators from around the globe contributed to the development of antifungal=
<=
span
style=3D'font-size:94%'>susceptibility testing techniques.
&sec=
t;This workshop will focus on the contribution of =
the National Committee for Clinical Laboratory =
Standards (NCCLS) Subcommittee of Antifungal =
Susceptibility testing.
§Established in1982, the committee has dedicated =
15 years of collaborative efforts to standardizing=
=
the method.
Center for Medical Mycology, 2002
4
Anti=
fungal
Susceptibility Testing Methodology
§Method development passed through several phases:
<=
/div>
&se=
ct;
§1992: Publication of do=
cument
M27-P (proposed)
=
;
–Set the i=
nitial
conditions for the standardized method (medium, inoculum, incubation time and temperatur=
e, endpoint criteria)
–Macrodilu=
tion
format
–Candid=
a spp. and <=
i>Cryptococcus
neoformans
s
§1995:
Publication of document M-27T (tentative)
–Main chan=
ges
include: Microdilution forma=
t and modifications for determining the susceptibility =
of Cr. neoformans
and amphotericin B.
s
§1997:
Publication of document M-27-A (approved)
–Main chan=
ges
include: Development of
breakpoints.
Center for Medical Mycology, 2002
5
An=
tifungal
Susceptibility Testing Methodology
§1998:
Publication of document M38-P(proposed) - a reference method for determining the antifungal
susceptibility of
conidium-forming filamentous fungi.
§National Committee for Clinical Laboratory Standar=
ds.
1997. Reference method for broth dilution antifungal
susceptibility testing=
of
yeasts. Approved standard. NCCLS document M27-A-National Committee for Clinical Laboratory <=
/span>=
Standards, Wayne, PA.
§National Committee for Clinical Laboratory Standar=
ds.
1998. Reference method =
for
broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed =
standard. NCCLS document M27-A-National Committee =
for Clinical Laboratory Standards, Wayne, PA. <=
/span>
Center for Medical Mycology, 2002
6
Prin=
ciple
§Adopts a broth dilution methodology which quantitatively measures the in vitro
activity of an antifun=
gal
agent against a fungal isolate.
§Various antifungal concentrations are inoculated with a standardized suspension of test organisms.&=
#13;&se=
ct;The minimum inhibitory concentration (MIC) is determined following 48 to 96 hour incubation at <=
/span>35 ° C.
Center for Medical Mycology, 2002
7
Test
Medium
§S=
ynthetic
medium RPMI-1640
–With MOPS
(morpholinepropanesulfonic acid), without sodium
bicarbonate
–Supplemented with L-glutamine
and a pH indicator
–Should be
filter sterilized with a final pH of 7.0 at 25° C
–Suitable for testing the
susceptibility of Candida spp. to amphotericin B, 5-FC, =
and
the azoles.
–Yeast nitrogen broth is prop=
osed
for use in testing Cr. neoformans.
Center for Medical Mycology, 2002
8
Stock
Solutions of Antifungal Agents
<=
/div>
§=
The
susceptibility of yeast is tested against
the following antifungal agents:
̵=
1;Amphotericin B
̵=
1;5-fluorocytosine (5-FC)
̵=
1;Azoles including fluconazole, ketoconazole, and itraconazole
Center for Medical Mycology, 2002
9
Stock
Solutions, cont.
§Drugs are obtained as pure reference powders directly from the manufacturer.
&se=
ct;If pure powder is not available, as is often the case with amphotericin B, pharmaceutical preparations may be =
substituted (e.g. FungizoneÒ<=
/span> for injection,
Bristol Myers Squibb)
Center for Medical Mycology, 2002
10
Stock
Solutions, cont.
§Stock solutions of antifungal agents do not commonly support the growth of =
contaminating organisms and can be =
considered sterile.
&se=
ct;Fluconazole and 5-FC are prepared in sterile distilled water and normal saline, respectively.
Center for Medical Mycology, 2002
11
Stock
Solutions, cont.
§Amphotericin B, itraconazole, and ketoconazole
are prepared in dimethyl sulfoxide (DMSO).
§Pharmaceutical preparation=
s of
amphotericin B are dissolved in sterile distilled
water.
Center for Medical Mycology, 2002
12
Stock
Solutions, cont.
§Assay units of a particular antifungal=
powder
can differ widely from the actual weight of the powder.
=
§Antifungal solutions must =
be
standardized according to the assays of each batch lot.
§The following formulae are used to determine
the amount of powder or diluent needed.
Center for Medical Mycology, 2002
13
Stock
Solutions, cont.
Center for Medical Mycology, 2002
14
If possible, more than 100 mg of powder
should be weighed initially. F=
or
example, to prepare 100 ml of a stock solution at a
concentration of 1280 µg/ml using a powder that has a =
potency of
750µg/mg, use the following formula to establish the weight=
of
powder needed:
Target Vol. (100ml) x Desired Con. (1280 µg/ml) &=
#13;
=
; &n=
bsp;
Potency (750 µg/mg)
Weight =3D
=3D170.7 m=
g
Stock
Solutions, cont.
Center for Medical Mycology, 2002
15
Using the second formula, and a weight in
excess of that required (e.g.
182.6 mg), the volume of diluent is determined, as in this example:
Thus, in the above example, 182.6 mg of
powder is to be dissolved in=
107.0
ml of diluent.
Stock
Solutions, cont.
Center for Medical Mycology, 2002
16
Weig=
hing
the Antifungal Powder