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Journal of Clinical Microbiology, October 2000, p. 3715-3717, Vol. 38, No. 10
Department of Pathology, University of Iowa College of
Medicine, Iowa City, Iowa,1 and AB BIODISK,
Solna, Sweden2
Received 26 April 2000/Returned for modification 4 July
2000/Accepted 4 August 2000
Performance of the Etest for voriconazole susceptibility testing of
312 isolates of Candida spp. was assessed against that of
the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth
medium, and MICs were read after incubation for 48 h at 35°C.
Etest MICs were determined with RPMI agar containing 2% glucose (RPG),
Casitone agar (CAS), and antibiotic medium 3 (AM3) agar and were read
after incubation for 48 h at 35°C. The Candida spp.
isolates included C. albicans (n = 174),
C. glabrata (n = 55), C. tropicalis (n = 31), C. parapsilosis
(n = 39), C. krusei (n = 5), C. lusitaniae (n = 2), and C. guilliermondii (n = 6). The Etest results
obtained using RPG correlated well with the reference MICs. Overall
agreement ranged from 91% for C. glabrata to 100% for
C. tropicalis, C. parapsilosis, C. guilliermondii, C. krusei, and C. lusitaniae. When CAS was used, agreement ranged from 80% for
C. krusei to 100% for C. parapsilosis,
C. guilliermondii, and C. lusitaniae. With AM3,
agreement ranged from 58% for C. glabrata to 100% for
C. lusitaniae and C. guilliermondii. The Etest
method using RPG appears to be a useful method for determining voriconazole susceptibilities of Candida species.
The Etest stable agar gradient
susceptibility testing method has been shown to be extremely flexible
in testing a variety of fastidious and nonfastidious organisms,
including bacteria, yeasts, and moulds (2-4, 6-9, 12-14,
18, 19; our unpublished data). The major perceived advantage
of Etest for susceptibility testing of fungi is that laboratories
wishing to test only one or two agents against an occasional yeast
isolate may do so and generate quantitative MICs (7).
Numerous studies have now been published documenting that the
performance of Etest is comparable with that of reference broth
dilution testing of amphotericin B (6, 9, 14, 19),
fluconazole (2-4, 6, 13), itraconazole (2-4, 6,
18), and ketoconazole (2-4, 6). Notably, Etest may be
the preferred method for detecting amphotericin B-resistant strains of
Candida spp. and Cryptococcus neoformans (7,
9, 12, 14, 19). Presently, however, there are few or no data available describing the performance of Etest with the newer
investigational triazole and echinocandin antifungals.
Voriconazole is a new investigational monotriazole antifungal agent
with systemic activity against a broad spectrum of pathogenic fungi
including Candida spp., Cryptococcus neoformans,
and Aspergillus spp. (5, 7, 10, 15, 16, 17). This
agent has been tested extensively in vitro using broth dilution methods
but has not been evaluated using agar-based methods such as Etest. In the present study, we evaluate the Etest for voriconazole using three
agar media, RPMI, Casitone, and antibiotic medium 3 agar, by comparing
the results with those obtained using the National Committee for
Clinical Laboratory Standards (NCCLS) reference broth microdilution
method for testing 312 clinical isolates of Candida spp.
Test organisms.
Three hundred twelve clinical isolates of
Candida species were selected for testing. The collection
included 174 C. albicans isolates, 55 C. glabrata
isolates, 31 C. tropicalis isolates, 39 C. parapsilosis isolates, 5 C. krusei isolates, 6 C. guilliermondii isolates, and 2 C. lusitaniae isolates.
The members of this collection were all recent clinical isolates from
geographically diverse medical centers in North and Latin America. The
majority were isolated from blood or normally sterile body fluids
(15). The isolates were identified by standard methods
(20) and were stored as suspensions in water at ambient
temperature until used in the study. Prior to testing, each isolate was
subcultured at least twice to Sabouraud dextrose agar (Remel, Lenexa,
Kans.) to ensure optimal growth characteristics.
Antifungal agents.
Etest strips containing voriconazole were
supplied by AB BIODISK (Solna, Sweden). Voriconazole was obtained as a
reagent-grade powder from Pfizer Inc., Central Research Division
(Groton, Conn.). Stock solutions were prepared in dimethyl sulfoxide
and further diluted in RPMI 1640 medium buffered to pH 7.0 with 0.165 M
morpholinepropanesulfonic acid (MOPS) buffer (Sigma, St. Louis, Mo.)
and were dispensed into 96-well microdilution trays. Trays containing a
0.1-ml aliquot of appropriate drug solution (two times the final
concentration) in each well were subjected to quality control (QC) and
then sealed and stored at Media.
Agar formulations used for the Etest were RPMI 1640 (American Biorganics, Buffalo, N.Y.) supplemented with 1.5% agar and
2% glucose (RPG) and buffered with MOPS, Casitone agar (CAS) (Difco), and antibiotic medium 3 (AM3) agar (BBL). The RPMI 1640 broth medium
used for the microdilution testing was buffered with MOPS in accordance
with the NCCLS M27-A method (11).
Antifungal susceptibility test methods.
Broth microdilution
tests were performed according to NCCLS document M27-A (11).
An inoculum concentration of 0.5 × 103 to 2.5 × 103 cells per ml was standardized spectrophotometrically
and by quantitative plate counts. Microdilution trays were incubated in
air at 35°C and read after 48 h of incubation. For voriconazole,
the MIC endpoint was defined as the lowest concentration that produced
a significant decrease in turbidity relative to the control (drug-free)
well (10, 11).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of the Etest Method for Determining
Voriconazole Susceptibilities of 312 Clinical Isolates of
Candida Species by Using Three Different Agar
Media
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
70°C until used in the study. The final
concentration of voriconazole in the wells ranged from 0.007 to 8 µg/ml.
QC. QC was performed in accordance with NCCLS document M27-A using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (11). QC determinations made on each day of testing were within the control limits for voriconazole established by Barry et al. (1): C. krusei ATCC 6258, 0.12 to 1.0 µg/ml, and C. parapsilosis ATCC 22019, 0.03 to 0.25 µg/ml.
Analysis of results. Etest MICs read at 48 h on the three media were compared to reference microdilution MICs read at 48 h. The reference microdilution MIC and Etest MIC determinations were performed in two physically separate laboratories and the results were read independently; i.e., the testing was blind. Since the Etest scale has a continuous gradient of concentrations, the MICs between twofold dilutions were rounded to the next twofold level of the reference method for comparison (13, 14). Off-scale MICs at the upper limit were converted to the next higher concentration, and off-scale results at the lower limit were left unchanged. Discrepancies between MICs of no more than two dilutions were used to calculate the percent agreement.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Table 1 summarizes the in vitro
susceptibilities of 312 Candida isolates to voriconazole, as
determined by the reference broth microdilution method. A broad range
of MICs was observed for most organism groups. In general, the
voriconazole MICs obtained were similar to those reported previously
for the individual Candida species (5, 10, 15,
17). Voriconazole MICs of >1 µg/ml were observed for only
three isolates: one strain of C. albicans (MIC, >8 µg/ml)
and two strains of C. glabrata (MICs of 2 and 8 µg/ml,
respectively).
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Table 2 summarizes the percentage of 48-h
voriconazole MICs obtained by the Etest on the three agar media that
were within two dilutions of the reference method result. Overall, the
percent agreement was 98% with RPG, 89% with CAS, and 89% with AM3
agar. The agreement between Etest and microdilution MICs was 
90% for all species with RPG agar. Slightly lower levels of agreement were
observed when either CAS or AM3 agar was used; however, 80% agreement was observed on both media for all species with the exception
of C. glabrata (58% agreement on AM3). When a discrepancy was observed between the results obtained by the Etest and the reference method, the Etest provided a lower MIC with AM3 (79% of
discrepancies) and a higher MIC with CAS (85%) and RPG (57%). These
discrepancies reflect, in part, slower growth of the isolates on AM3
and improved growth on CAS and RPG agar relative to RPMI broth.
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The results of this study provide the first documentation of the applicability of the Etest stable agar gradient method for determining the in vitro susceptibilities of Candida species to the investigational triazole voriconazole. We found that RPMI agar with glucose (2% final concentration) supported optimal growth of all species tested and provided excellent agreement with the MICs obtained with the broth microdilution method (Table 2). As was seen with fluconazole (13), the problem of trailing end points due to partial inhibition of growth by azoles was minimized with the use of RPG agar and adherence to specific criteria for reading Etest MICs as described in the Etest package insert and technical guide for yeasts (AB BIODISK). Good agreement with broth dilution MICs was observed when discernable growth within the ellipse was ignored.
Although CAS and AM3 agar did not perform as well as RPG, both media supported the growth of most of the test isolates and performed reasonably well compared to the reference method (Table 2). Trailing growth within a discernable ellipse was observed with certain strains with both of these media. In most instances, the MIC for voriconazole was underestimated relative to the broth MIC when determined on AM3 and overestimated relative to the broth MIC when determined on CAS. The influence of reduced growth on AM3 agar was most prominent with C. glabrata, resulting in MICs that were more than two dilutions lower than those of the reference method in 42% of the isolates tested.
In summary, we have provided the first documentation of the ability of Etest to generate voriconazole MIC data that are comparable to those obtained by the NCCLS broth microdilution method. RPMI agar with 2% glucose may be used to determine reference quality MICs with triazole (fluconazole, itraconazole, and voriconazole), polyene (amphotericin B), and echinocandin (caspofungin [MK-0991]) Etest reagents in tests with Candida spp. and Cryptococcus neoformans (2-4, 6, 9, 13, 14; our unpublished data). This will be very attractive to laboratories because it will provide the flexibility to test only one agent or a panel of antifungal agents representing different classes of systemic antifungal agents as the clinical situation dictates.
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ACKNOWLEDGMENTS |
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The excellent secretarial support of K. L. Meyer is greatly appreciated.
This study was supported in part by Pfizer Pharmaceuticals and by AB BIODISK.
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FOOTNOTES |
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* Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail: michael-pfaller{at}uiowa.edu.
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References
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Journal of Clinical Microbiology, October 2000, p. 3715-3717, Vol. 38, No. 10
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