Antifungal Susceptibility Testing

Notes on the disk diffusion and ETEST methods.

Introduction

The disk agar gradient method can accurately and reproducibly determine the susceptibility of fungi to antifungal agents eg fluconazole. Improvements include the use of RPMI glucose agar, inoculum density adjustment with a 0.5 McFarland Standard, and incubation at 35C for 24 hours. These changes standardise the test to current CLSI guide-lines and make it relatively comparable to standard bacterial test methods. This method and interpretive criteria apply to rapidly growing yeast species, defined as strains producing growth within a 24-48 hour incubation period. Data on slower growing moulds is largely in-house at present.

Antifungal agents available for testing:

Store both disks and Etest refrigerated with desiccant.

Disk Method

1. Melt and pour RPMI-Glucose Agar plates [media may be stored in 60 ml aliquot's and then melted in a microwave to pour plates when required]. For a standard screen using disks of all the above antifungal agents we typically use 3 plates set up as follows (Additional plates may need to be poured for Etest and other investigational purposes. Note susceptibility tests for Ampohtericin may be unreliable on RPMI media; the use of Antibiotic Medium 3 may enhance the detection of resistance, but this medium is not standardised and substantial lot-to-lot variability is possible).

1. Amphotericin B 20 ug.

2. Voriconazole 1.0 ug, Itraconazole 8 ug and Fluconazole 25 ug

3. 5-Fluorocytosine 1.0 ug/ml for yeasts 10 ug for Aspergillus.

2. Prepare an inoculum by suspending a single isolated colony in about 5 ml of normal saline or sterile water. Agitate to achieve a smooth suspension on the Vortex mixer. Adjust the suspension with saline or water to approximate a density of a #0.5 McFarland Turbidity Standard [we use a bioMerieux Densimat]. For filamentous fungi, which do not sporulate profusely, add one drop of the wetting agent Tween 20 to the sterile distilled water and then break up the mould by shaking it with small glass beads or by grinding it into a suitable suspension.

3. Moisten a sterile cotton (not dacron) swab in the adjusted inoculum suspension. Express excess moisture by rolling the swab on the inside of tube above fluid level. Streak the surface of an RPMI-glucose agar plate in 4 different directions (at 90 degree angles) to cover the entire surface. Let the surface of RPMI-glucose agar plates dry at 35 C with lids ajar until no droplets of moisture are on the agar surface.

4. Using a pair of flamed sterilised forceps apply the disks containing the antifungal agent to be tested onto the surface of the inoculated agar plate and press lightly to insure complete contact with agar.

5. Incubate plates at 35C for 24-48 hours or until sufficient growth has occurred. Plates should be read as early as possible after 24 hours incubation and results recorded in the susceptibility book.

Etest method

1. Melt and pour one plate of RPMI-Glucose Agar for each Etest to be performed.

2. Follow the exact method as described above for disks.

3. Using a pair of flamed sterilised forceps apply one Etest strip and one Neo-sensitab of the antifungal agent to be tested onto the surface of the inoculated agar plate and press lightly to insure complete contact with agar.

Measurement of Zone Diameters

Measure zone diameter to the nearest whole millimetre. For Amphotericin B and 5-Fluorocytosine the zone of inhibition should be determined at the point of complete (100%) or almost complete (95%) inhibition. For the azoles Ketoconazole, Fluconazole and Itraconazole the zone of inhibition should be read at the first point of significant inhibition/marked decrease in growth intensity ie (80%) inhibition. Pinpoint microcolonies at the zone edge or within the zone of inhibition should be ignored.

References

NCCLS Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard NCCLS document M27-A [ISBN 1-56238-328-0], 1997.
Rex et al. 1997. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vitro correlation data for fluconazole, itraconazole, and Candida infections. Clin. Infect. Dis. 24:235-247.
Barry AL and SD Brown. 1996. Fluconazole disk diffusion procedure for the determining susceptibility of Candida species. J. Clin. Micro. 34:2154-2157.
Etest Technical Guide 4B. Antifungal susceptibility testing of yeasts. AB Biodisk, Sweden.

Interpretation of antifungal susceptibility zone diameters using

Neosenstiabs on Mueller Hinton Agar + Methylene Blue Agar and and Etest MIC'S on RPMI-Glucose Agar.

The following zone diameter and MIC standards should be interpreted with caution. They are in house standards currently used by the WCH Mycology Unit and are subject to change at any time. Currently, the most reliable data available is for fluconazole and itraconazole against Candida infections; the least reliable data is for the moulds. SSD (susceptibility - dose dependent) is a new category recently described by the NCCLS subcommittee on antifungal susceptibility testing for isolates where susceptibility is dependent on achieving the maximal possible blood level of the antifungal agent. For fluconazole doses of 400 mg/d or more may be required for adults with normal renal function and habitus. For itraconazole plasma concentrations of >0.5 ug/ml may be required for optimal response [Rex et al. CID 1997;24:235-47].

* Note susceptibility tests for Ampohtericin may be unreliable on RPMI media; the use of Antibiotic Medium 3 may enhance the detection of resistance, but this medium is not standardised and substantial lot-to-lot variability is possible.

INTERPRETATION OF ZONE DIAMETERS AND MIC’S

The following zone diameter and MIC standards are for NeoSensitabs on RPMI media [note these are in house standards and are subject to change at any time]:

RPMI 1640 Media

It is essential to use the exact reagents from Sigma as detailed below.

RPMI-1640 Cell Culture Reagent with Phenol Red pH indicator and 0.2% glucose (Sigma #R-7755, St. Louis, USA).

MOPS buffer as a 0.165M solution (Sigma #M-6270).

Agar ( Bacto agar, Difco, Detroit, USA; or comparable brand).

Method

1. Add 34.53 gm MOPS to one litre of deionised water

2. To 900 ml of MOPS buffer, ADD:

1. 10.4 gm of RPMI-1640 reagent

2. 18 gm of glucose (final concentration 2 gm/100 ml)

3. 15 gm of agar

4. If necessary, adjust pH to 6.9-7.1 by adding 1N Na0H or IN HC1.

5. Additional MOPS buffer to a final volume of 990 ml.

3. Autoclave at 121C for 15 minutes, then cool to 56 C.

4. To the cooled broth medium, add 0.3g L-glutamine in 10 ml deionised water (filter sterilised). NOTE: This solution can be prepared and frozen until needed. Glutamine will redissolve when thawed in a 35C water bath or incubator.

5. Mix and pour 60-70 ml in 150 mm petri-plates, or 25-30 ml in 90-100 mm plates, to a depth of 4 mm. NOTE: These plates may be sealed in a plastic bag and stored in a refrigerator for about 4 weeks until used.

Quality Control Tests Acceptable Performance Range

C. albicans ATCC 90028: = 32-43 mm for fluconazole

optional additional QC strains:

C. parapsilosis ATCC 22019 : = 26-37 mm for fluconazole

C. krusei ATCC 6258: = 6-17 mm for fluconazole