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Journal of Clinical Microbiology, November 1998, p. 3429-3432, Vol. 36, No. 11
Departments of
Medicine,1
Pathology,2 and
General
Dentistry,
Received 24 April 1998/Returned for modification 2 June
1998/Accepted 4 August 1998
We describe a simple procedure for detecting fluconazole-resistant
yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or
50-µg fluconazole disks. With 25-µg disks, zones of inhibition of
Advances in supportive therapy for
immunocompromised patients have led to increased rates of fungal
infections (9, 20). With the widespread use of fluconazole
for treatment and prevention of oropharyngeal candidiasis, the
most common fungal infection in patients infected with human
immunodeficiency virus (18, 22), clinical resistance is
becoming a serious problem (5, 11, 18, 22). Consequently, a
rapid, reproducible method of fluconazole susceptibility testing would
be useful in determining the epidemiology of and optimal treatment for
infection with resistant isolates (4, 8, 13, 21).
Candida albicans is usually acutely susceptible to
fluconazole; fluconazole MICs for approximately 90% of C. albicans isolates are Screening clinical yeast isolates for decreased susceptibility to
fluconazole has been difficult. Methodologies such as the standard
National Committee for Clinical Laboratory Standards (NCCLS) broth
macrodilution test (12) and alternative methods such as the
E test or microdilution adaptations of the NCCLS method generally
compare favorably for determining MICs for isolates (3, 10, 15,
23); however, these are not easily adapted to the screening of
yeasts for fluconazole susceptibility. A disk diffusion method
analogous to that used for testing antibacterial agents could be easily
incorporated into a clinical laboratory and serve as an effective means
for fluconazole susceptibility screening. Previously described
disk diffusion methods utilize disks or employ media not available
for routine use (2, 24). We have developed a disk diffusion
method for susceptibility screening which uses disks that are
simple to prepare and standard media, such that it could be easily
implemented in routine clinical mycology laboratories.
(This study was presented in part at the 97th General Meeting of the
American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997 [25]).
Medium.
RPMI 1640 with L-glutamine and
morpholinepropanesulfonic acid (MOPS) organic buffer (ABI, Niagara
Falls, N.Y.) was prepared from a powdered medium at double the desired
concentration with 500 ml of deionized H2O, and the
solution was sterile filtered. Bacto Agar (Difco Laboratories,
Detroit, Mich.) was also prepared at a 2× concentration by
adding 20 g of agar to 500 ml of deionized H2O
(14). The solution was mixed over heat to dissolve the
agar, autoclaved, and then cooled to 48°C in a water bath. The agar and RPMI 1640 solutions were combined at 48°C and stirred, and approximately 20 ml was dispensed into sterile 100-mm-diameter petri
plates. The medium was allowed to cool and harden at room temperature
for 3 to 5 days, and the plates were then stored at 4°C and used
within 1 to 2 weeks.
Organisms and reference methods.
Forty clinical isolates of
Candida spp., representing forty consecutive clinical
samples, which were submitted to the Fungus Testing Lab (The University
of Texas Health Science Center, San Antonio, Tex.) for MIC
determination by both the NCCLS broth macrodilution procedure
(12) and a broth microdilution adaptation (1, 6) were selected for evaluation. Both 24- and 48-h MIC readings were evaluated, coordinating, respectively, with 24- and 48-h disk results.
The breakdown of Candida species was as follows: 35 isolates were C. albicans, 2 were C. krusei, 1 was
C. tropicalis, 1 was C. parapsilosis, and 1 was
C. glabrata.
Fluconazole disks.
Twenty-five-microgram disks were prepared
by pipetting 12.5-µl volumes of stock fluconazole (2 mg/ml;
Pfizer-Roerig, New York, N.Y.) onto sterile blank disks (Difco
Laboratories), while 50-µg disks were similarly prepared by using 25 µl of fluconazole solution. The disks were dried and then stored at
4°C until use within 1 to 2 weeks.
Inoculum.
The yeast isolates were stored at room
temperature in sterile deionized H2O, subcultured
onto Sabouraud dextrose agar (BBL, Cockeysville, Md.) to
ensure purity and viability (2), and then subcultured again
to select for isolated colonies. Three to five colonies were
then suspended in 5.0 ml of sterile deionized H2O and mixed
thoroughly on a vortex mixer. The suspension was adjusted to a 0.5 McFarland turbidity standard (106 CFU/ml) by using a
spectrophotometer (17). Serial log10 dilutions of the adjusted suspension were prepared and quantitatively cultured to
determine counts of CFU. A 1-ml inoculum from a suspension of
104 CFU/ml yielded optimal confluent growth for lawn
formation.
Zone determination and comparative MIC interpretation.
Inhibitory zone diameters were measured at the transitional point
where growth abruptly decreased, as determined by a marked reduction in colony sizes (Fig. 1). The
zone sizes measured at 24 and 48 h were then plotted against
respective 24- and 48-h MICs determined by the reference methods. To
interpret zone diameters, the NCCLS method was followed, in which MIC
breakpoints of
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Fluconazole Disk Diffusion Susceptibility Testing of
Candida Species
ABSTRACT
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20 mm at 24 h accurately identified 29 of 29 isolates for which
MICs were 
8 µg/ml, and with 50-µg disks, zones of 27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were
>8 µg/ml were identified by using either disk. Disk diffusion may be
a useful screening method for clinical microbiology laboratories.
TEXT
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1 µg/ml (16, 19). Some
non-C. albicans yeasts have been noted to have decreased
susceptibility or resistance to fluconazole (19, 22);
these species include C. glabrata and C. krusei,
which are being isolated more frequently (7, 24).
8 µg/ml define susceptibility and 64 µg/ml
define resistance, with MICs of between 16 and 32 µg/ml reflecting
dose-dependent susceptibility (12). Zones corresponding to
these MIC breakpoints were determined graphically through regression
analysis for 25-µg (Fig. 2A) and 50-µg (panel B) disks at 24 and 48 h (data not shown).
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FIG. 1.
(A) A 25-µg fluconazole disk on a lawn of
104 CFU of C. albicans after 24 h of
incubation. (B) A 50-µg fluconazole disk on a lawn of 104
CFU of C. albicans after 48 h of incubation. Inhibitory
zone diameters were measured at the transitional point where growth
abruptly decreased (interior edges of bars), as determined by a marked
reduction in colony sizes.
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FIG. 2.
(A) Regression analysis correlating zones of inhibition
(24 h) obtained with 25-µg fluconazole disks with NCCLS 24-h broth
macrodilution MICs. (B) Regression analysis correlating zones of
inhibition (24 h) obtained with 50-µg fluconazole disks with NCCLS
24-h broth macrodilution MICs.
20 mm
for susceptibility (MIC 8 µg/ml) with 25-µg disks and
27 mm with 50-µg disks at both 24 and 48 h. Zones which were smaller than these breakpoints were defined as indicating either dose-dependent susceptibility or resistance.
Three separate trials with 25- and 50-µg disks were performed
on all 40 isolates for verification of reproducibility.
Regression analysis indicated that zones could vary up to 4 mm and
remain within ±1 reference tube dilution (NCCLS broth macrodilution
method). Zones from all three trials met these criteria for 95% of
isolates at 24 h, but at 48 h criteria were met for only 86%
(data not shown).
Zone diameters measured at 24 h for 25- and 50-µg disks showed
excellent correlation with predicted macrodilution 24-h MICs for
susceptible yeasts. Diameters for 25-µg drug concentration disks
accurately identified 29 of 29 (100%) of the isolates for which MICs
were 8 µg/ml. Disks with 50 µg of fluconazole provided similar
results, identifying 28 of 29 (97%) of the isolates for which MICs
were 8 µg/ml. The zone diameters also correlated well with
macrodilution 24-h MICs in predicting either dose-dependent susceptibility or resistance in yeast strains. Zones at 24 h with either 25- or 50-µg disks identified 11 of 11 (100%) isolates for which MICs were 16 µg/ml. Although yeast strains for which MICs were 16 µg/ml were accurately identified by the disk method, results could not adequately differentiate strains with dose-dependent susceptibility (MICs of 16 to 32 µg/ml) from fully resistant strains (MICs of 64 µg/ml). Comparison of disk diameters to microdilution MICs showed correlation similar to the correlation with broth macrodilution reference method MICs (data not shown).
Zone diameters after 48 h correlated less well with the
reference macrodilution 48-h MICs at either drug concentration, with the 25- or 50-µg disks accurately predicting MICs of 8 µg/ml for
23 of 26 (88%) isolates. Sensitivity in identification of yeasts for
which MICs were 16 µg/ml was much lower at 48 h: predictions were correct for only 11 of 14 (79%) isolates with 25-µg disks and
12 of 14 (86%) isolates with 50-µg disks. As with 24-h results, comparison of disk diameters to microdilution MICs showed similar correlation (data not shown).
As a screening tool, the disk diffusion procedure correlated well with
the NCCLS reference broth macrodilution method and the microdilution
method in identifying fluconazole-susceptible yeasts. In this study,
both 25- and 50-µg fluconazole disks allowed correct determination of
all fluconazole-susceptible isolates examined. Quick discernment
of susceptibility would allow clinical laboratories to focus more
effort on characterization of less-susceptible isolates. Yeasts which
have dose-dependent susceptibility or frank resistance could not be
readily differentiated from each other based on disk diffusion results.
Such isolates could be reexamined by other, more discriminating tests
such as the E test or a broth microdilution adaptation of the NCCLS
method.
Reading inoculated plates with 25-µg disks after 24 h
resulted in the most sensitive method, accurately
identifying 100% of susceptible Candida spp. with a
diameter of 20 mm. Although 50-µg disks also yielded good results,
no distinct advantage of using the higher-concentration disk rather
than the 25-µg disk was found. Zone diameter results noted after
48 h were much less sensitive than the corresponding 24-h results
for disks of both drug concentrations. Disk diffusion testing could
probably be performed at 24 h with most Candida
species; however, some species, such as C. krusei, do not
always show resistance at 24 h and results should be read at
48 h (2).
The fluconazole disk diffusion procedure showed very good
reproducibility at 24 h (95%), with some decline after 48 h
(86%). The presence of microcolonies necessitated measurement of zone diameters at the transitional point where growth abruptly decreased from normal-sized colonies (Fig. 1). Thus, a certain amount of subjectivity was introduced into zone determination, as is the case for
endpoint determination decisions made with the E-test (4) or
NCCLS M27-A (12) methods.
The use of the fluconazole disk diffusion procedure appears to be a
convenient and sensitive method for susceptibility screening or
detection of fluconazole-resistant yeasts. Additional studies with a
wider variety of yeast isolates should be conducted to determine the
appropriateness of screening clinical isolates in this manner.
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FOOTNOTES |
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* Corresponding author. Mailing address: The University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Infectious Diseases, 7703 Floyd Curl Dr., San Antonio, TX 78284-7881. Phone: (210) 567-4823. Fax: (210) 567-4670. E-mail: PATTERSON{at}UTHSCSA.EDU.
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Journal of Clinical Microbiology, November 1998, p. 3429-3432, Vol. 36, No. 11
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