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JOURNAL OF CLINICAL MICROBIOLOGY, May 2003, p. 2141–2143
0095-1137/03/$08.00ϩ0 DOI: 10.1128/JCM.41.5.2141–2143.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.
Detection of Fluconazole-Resistant Isolates of Candida glabrata
Susan M. Nelson1 and Charles P. Cartwright1,2*
Department of Laboratory Medicine and Pathology, Hennepin County Medical Center, Minneapolis, Minnesota
1 and Department of Laboratory Medicine and Pathology, University of Minnesota Medical School,
Received 10 October 2002/Returned for modification 14 January 2003/Accepted 25 January 2003
The ability of a fluconazole-containing agar screen assay to accurately detect isolates of Candida glabrata
resistant to the azole antifungal agent fluconazole was evaluated on a collection of 100 clinical isolates of this
organism. Results were correlated with the MIC of fluconazole for these isolates and compared with the results
of a previously published disk diffusion-based fluconazole resistance screening test. Agar screen assay results
were in categorical agreement with MIC-based determinations for 97% (97/100) of the isolates tested. This
correlation was higher than that obtained with the disk diffusion technique, which categorized only 87%
(87/100) of isolates correctly, and suggests that the agar screening approach can effectively expedite fluconazole
susceptibility testing of C. glabrata isolates.
The relatively recent publication of approved procedures
excellent 97% correlation between agar screen testing and
and interpretive categories for determining the susceptibility of
MIC determination for a small group of 30 isolates of C.
clinical isolates of Candida
spp. to antifungal agents (12, 18)
. These results, and the relative lack of data evaluating
provides a basis for susceptibility testing to become a valuable
fluconazole disk diffusion testing on C. glabrata
tool for the optimization of antifungal therapy for patients with
gested to us that it would be worthwhile to perform a compar-
invasive yeast infections (5, 7). Unfortunately, both the ap-
ative evaluation of a screening agar-based method and disk
proved broth macrodilution method (12) and microdilution
diffusion for the rapid detection of fluconazole resistance in a
adaptations of this technique (6, 12) are relatively expensive
large collection of clinical isolates of C. glabrata
and laborious and consequently are used in only a minority of
All organisms used in the study were originally isolated from
clinical microbiology laboratories that routinely perform anti-
clinical specimens submitted for culture to the Microbiology
bacterial susceptibility testing (3). The absence of institution-
Laboratory, Hennepin County Medical Center, between April
specific susceptibility information is unfortunate given con-
1998 and December 2000. The clinical sources of the isolates
cerns about the emergence of antifungal resistance in Candida
were as follows: urine (42 isolates), blood (31), surgically col-
spp. (17). Candida glabrata
in particular has become a more
lected tissue or aspirate (11), abscess or drainage fluid (9), and
frequent isolate in many laboratories (2, 16), and the flucon-
wound (7). Using standard methods (4), isolates were identi-
azole MIC at which 50% of tested isolates of this organism are
fied as C. glabrata
. The MICs of fluconazole for isolates were
inhibited is typically 8- to 16-fold higher than that seen for C.
determined using the NCCLS reference broth microdilution
(15, 16, 19). In addition, the typical fluconazole MICs
method (12). Final fluconazole concentrations tested ranged
for 10 to 15% of invasive isolates of C. glabrata
place them in
from 0.125 to 64 g/ml. Microtiter plates were incubated for
the resistant category, compared with less than 1% of C. albi-
48 h at 35°C, and the MIC was read as the lowest concentration
isolates (15, 20). Antifungal susceptibility testing of C.
of fluconazole that effected a visually distinct decrease (Ͼ50%)
is, therefore, of greater clinical import than for many
in turbidity relative to the growth control. The fluconazole
species, and techniques that facilitate the per-
screen agar was formulated as described by Patterson et al.
formance of fluconazole susceptibility testing of C. glabrata
(13). To prepare the inoculum for the agar-screening assay,
routine clinical microbiology laboratories need to be devel-
several isolated colonies of each isolate were picked and sus-
oped. Previous studies have described the use of simple disk
pended in sterile saline (0.9%). The optical density of each
diffusion-based techniques for detecting resistance to flucon-
suspension was then adjusted until it corresponded to a 0.5
azole in Candida
spp. (1, 8, 9, 10, 11, 13, 19), but with the
McFarland standard, and a sterile calibrated loop (1 l) was
notable exception of the study of Sandven (19), these studies
used to streak a set of three plates containing 0, 8, and 16 g
have concentrated on testing C. albicans
isolates. Use of flu-
of fluconazole/ml. Inoculated plates were incubated at 30°C for
conazole-containing agar plates has also been reported as a
a total of 48 h. Growth characteristics of individual isolates
possible means of identifying fluconazole-resistant isolates of
were recorded after 24 and 48 h of incubation by visually
spp. (15, 20), and Patterson et al. (13) reported an
comparing the diameters of 15 to 20 colonies on the flucon-azole-containing plates with those on the fluconazole-freeplate. To provide for a more objective analysis of the data,
* Corresponding author. Mailing address: Clinical Laboratories,
results observed in the fluconazole-agar screen assay were di-
MC #812, Hennepin County Medical Center, 701 Park Ave., Minne-
apolis, MN 55415. Phone: (612) 347-3026. Fax: (612) 904-4229. E-mail:
vided into four categories: category I (colonies indistinguish-
able in size on media with and without fluconazole); category
TABLE 1. Distribution of 48-h broth microdilution fluconazole
TABLE 3. Correlation between results obtained by disk diffusion
MIC for C. glabrata
isolates evaluated in the present study
and MIC broth microdilution testing of fluconazole susceptibility
No. of isolates with fluconazole MIC (g/ml) of:
No. of isolates with disk diffusion zone sizes (mm)
II (colonies visually smaller on fluconazole-containing media
but with a typical colony diameter Ͼ50% of the diameter seen
on fluconazole-free media); category III (colonies significantly
Categories: susceptible (Յ8 g/ml), susceptible-dose dependent (16 to 32
smaller on fluconazole-containing media and typical colony
diameter Ͻ50% of the diameter seen on fluconazole-free me-
dia); and category IV (no growth or only pinpoint colonies on
fluconazole-containing media). Fluconazole disk diffusion test-
study (14). Indeed, a majority of isolates with fluconazole
ing was performed as described by Barry and Brown (1). Zone
MICs of 16 or 32 g/ml (13 of 18 [72%] at 24 h and 15 of 18
diameters were measured after 48 h of incubation at 35°C in
[83%] at 48 h) were designated susceptible (category III or IV)
ambient air, and breakpoints of Ն19 mm (susceptible), 15 to 18
when 16 g of fluconazole/ml was added to the medium. All
mm (intermediate), and Յ14 mm (resistant) were used, corre-
isolates determined to be in the R category by the reference
sponding (according to previously published data) to flucon-
MIC method were designated category I (little or no decrease
azole MICs of Յ8, 16 to 32, and Ն64 ug/ml, respectively (1).
in mean colony diameter) in the fluconazole agar screen assay
The fluconazole MICs for the 100 isolates of C. glabrata
irrespective of the concentration of fluconazole used or length
evaluated in this study are shown in Table 1. Significant clus-
of incubation. Unfortunately, under none of the tested condi-
tering of isolates around the susceptible breakpoint of 8 g/ml
tions were isolates in the S-DD category able to be accurately
was observed, with fluconazole MICs either at or within one
differentiated from those isolates demonstrating outright resis-
doubling dilution of this value for 71% (71/100) of the isolates.
tance to fluconazole (Table 2). The results of disk diffusion
The fluconazole MICs for a total of 31 isolates were Ͼ8 g/ml,
testing are shown in Table 3. All 31 isolates placed in the R or
with 18 (58%) of these showing fluconazole MICs in the
S-DD category by MIC testing were identified as either inter-
NCCLS susceptible–dose-dependent (S-DD) range and 13
mediate or resistant by disk diffusion testing. A total of 13 of
(42%) testing in the resistant (R) range (Ն64 g/ml). The
the 69 (18.9%) isolates in the S category were also classified as
results of the agar screen assay are shown in Table 2. The use
resistant (7 isolates) or intermediate (6 isolates) by disk diffu-
of 8 g of fluconazole/ml and an incubation time of 24 h
sion, resulting in an overall accuracy of 87% (87/100).
enabled the most accurate differentiation of fluconazole-sus-
The results of our investigation of simple techniques for
ceptible C. glabrata
isolates (MIC Յ 8 g/ml) from those in the
screening isolates of C. glabrata
for susceptibility to fluconazole
S-DD and R categories. Under these conditions, all 31 isolates
are in general agreement with those reported by previous in-
with fluconazole MICs of Ͼ8 g/ml were placed in category I
vestigators (1, 11, 13, 19). Both the agar screen method that we
or II (less than 50% decrease in mean colony diameter), with
developed and the disk diffusion technique correctly desig-
only 3 of the 69 isolates (4.3%) with fluconazole MICs of Յ8
nated resistant all 31 isolates with fluconazole MICs of Ͼ8
g/ml being similarly designated. Using 8 g of fluconazole/ml
g/ml. Neither methodology effectively discriminated between
but prolonging the incubation period to 48 h resulted in a
isolates that were determined to be in the S-DD category by
marginal improvement in sensitivity but greatly decreased the
MIC testing and those determined to be resistant outright to
specificity of the assay (Table 2). The use of 16 g of flucon-
fluconazole. In the agar screen assay, 56% (10/18) of S-DD
azole/ml did not improve differentiation of isolates with flu-
isolates appeared fully resistant to fluconazole, while only 33%
conazole MICs of 8 g/ml or less from those with fluconazole
(6/18) of S-DD isolates would have been classified as interme-
MICs of 16 g/ml or above, as had been reported in a previous
diate by the disk diffusion assay. These results strongly suggest
TABLE 2. Correlation between decrease in colony diameter observed on fluconazole-screening agar and NCCLS interpretive categorization
of C. glabrata
isolates based on broth microdilution fluconazole MICs
No. of isolates of indicated colony size categoryb
in test conditions (g) of fluconazole/incubation time (h) of:
Categories: susceptible (Յ8 g/ml), susceptible-dose dependent (16 to 32 g/ml), resistant (Ն64 g/ml).
Definitions of colony size categories are as follows: category I (no discernible difference in colony size on fluconazole-containing media); category II (colonies
smaller on fluconazole-containing media but typically Ͼ50% of the size of control colonies); category III (colonies significantly smaller on fluconazole-containing media
and typically Ͻ50% of the size of control colonies); and category IV (no growth or only pinpoint colonies on fluconazole-containing media).
that qualitative test methodologies cannot be used to defini-
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