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------=_NextPart_01C2D2AB.942AF900 Content-Location: file:///C:/2C644EF9/Suscepty_files/pres.xml Content-Transfer-Encoding: quoted-printable Content-Type: text/xml; charset="utf-8" ------=_NextPart_01C2D2AB.942AF900 Content-Location: file:///C:/2C644EF9/Suscepty_files/slide0001.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Antifungal Susceptibility Testing Methodology
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
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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 = ;
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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.
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Center for Medical Mycology, 2002
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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&= #13;
§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&= #13;
§1997:  Publication of document M-27-A (approved)
–Main chan= ges include:  Development of breakpoints.
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Center for Medical Mycology, 2002
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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>
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Center for Medical Mycology, 2002
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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.
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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.
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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
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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)
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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.
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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.
•
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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.
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        = ;            &n= bsp; 
        = ;      Weight  =3D     Volume = (ml) x Concentration (µg/ml)
         = ;            &n= bsp;            = ;   Assay Potency (µg/mg)
        = ;            OR
 
     Volume =3D    Weight = (mg) x Assay Potency (µg/mg)
        = ;            &n= bsp;            = ;   Concentration (µg/ml)
 
Stock Solutions, cont.
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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:
Weight  =3D
Target Vol. (100ml) x Desired Con. (1280 µg/ml) &= #13;
        = ;            &n= bsp;    Potency (750 µg/mg)
=3D170.7 m= g
Stock Solutions, cont.
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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:
=
Volume&= nbsp; =3D
<= span style=3D'mso-bidi-font-family:"Times New Roman";font-size:108%;display:non= e'>
Weight (182.6 mg) x Potency (750 µg/mg)
        = ;      Desired Con. (1280 µg/ml)
=
=3D 107.0 ml
=   = ;
Thus, in the above example, 182.6 mg of powder is to be dissolved  in= 107.0 ml of diluent.
Stock Solutions, cont.
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