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Journal of Clinical Microbiology, November 2001, p. 3952-3954, Vol. 39, No. 11
CAST Laboratories and Department of
Pathology, University of Iowa College of Medicine, Iowa
City,1 and The JONES Group, North
Liberty,3 Iowa, and AB BIODISK, Solna,
Sweden2
Received 11 June 2001/Returned for modification 27 July
2001/Accepted 12 August 2001
The performance of the Etest for posaconazole (SCH 56592)
susceptibility testing of 314 isolates of Candida spp.
was assessed against 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. MICs were determined by Etest for all 314 isolates with
RPMI agar containing 2% glucose (RPG agar) and were read after
incubation for 48 h at 35°C. The Candida isolates
included C. albicans (n = 174),
C. glabrata (n = 57), C.
tropicalis (n = 31), C.
parapsilosis (n = 39), C.
krusei (n = 5), C.
guilliermondii (n = 6), and C.
lusitaniae (n = 2). The Etest results
correlated well with reference MICs. Overall agreement was 95%, and
agreements for individual species were as follows: C.
krusei, 100%; C. albicans, 98%; C.
tropicalis, 97%; C. glabrata, 93%; C.
parapsilosis, 85%; C. guilliermondii, 83%; and
C. lusitaniae, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an
established ellipse was ignored. The Etest method using RPG agar
appears to be a useful method for determining posaconazole susceptibilities of Candida species.
Agar-based methods for antimicrobial
susceptibility testing include agar dilution, disk diffusion, and the
Etest and are used widely in clinical laboratories due to flexibility
and ease of performance (9). The Etest stable agar
gradient method has been shown to provide reference quality MIC
determinations for a variety of pathogens and antimicrobial agents,
including Candida spp. and filamentous fungi (7, 9,
12-17; M. A. Pfaller, S. A. Messer, K. Mills, A. Bolmström, and R. N. Jones, submitted for
publication). Studies have shown that when performed
according to the manufacturer's instructions, the Etest provides
excellent performance for testing Candida spp. against a
variety of antifungal agents, including polyenes, flucytosine, and
azoles (4, 6, 12-16, 18). Recent studies have
demonstrated that the Etest method is suitable for testing the
investigational antifungal agents voriconazole and caspofungin against
Candida (15; Pfaller et al., submitted).
Another investigational triazole antifungal agent, posaconazole, has
potent activity against pathogenic yeasts, including most species of
Candida (1, 3, 5, 8, 11, 12). This agent has
been widely tested in broth according to National Committee for
Clinical Laboratory Standards (NCCLS) guidelines but has not yet
been evaluated using an agar-based method. Given the success in testing
other triazoles using the Etest, it is reasonable to assume that
posaconazole may be tested by this method as well. The availability of
a variety of Etest reagents for antifungal testing will provide great
flexibility for laboratories that wish to perform quantitative
antifungal susceptibility testing using selected antifungal agents.
In previous evaluations of the Etest for testing amphotericin B
(14), fluconazole (13), voriconazole
(15), and caspofungin (Pfaller et al., submitted), we have
utilized three different media, RPMI 1640 agar supplemented with 2%
glucose (RPG agar), Casitone agar, and Antibiotic Medium 3 agar. In
every instance we have found performance to be best with RPG agar.
Thus, in the present study we evaluated the Etest for posaconazole
using only RPG agar in comparison to the NCCLS reference
microdilution broth method for testing 314 clinical isolates of
Candida spp.
Test organisms
Three hundred fourteen
clinical isolates of Candida species were selected for
testing. The collection included 174 Candida albicans,
57 Candida glabrata, 39 Candida
parapsilosis, 31 Candida tropicalis, six
Candida guilliermondii, five Candida
krusei, and two Candida 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
(12). The isolates were identified by standard methods
(19) 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 onto potato dextrose agar (Remel, Lenexa,
Kans.) to ensure optimal growth characteristics.
Antifungal agents.
Etest strips containing posaconazole were
supplied by AB BIODISK (Solna, Sweden). Posaconazole was obtained as a
powder from Schering-Plough Research Institute (Kenilworth, N.J.).
Stock solutions were prepared in polyethylene glycol. Serial twofold
dilutions were prepared exactly as outlined in NCCLS document M27-A
(10). Final dilutions were made in RPMI 1640 medium
buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS)
buffer (Sigma). The final concentration of solvent did not exceed 1%
in any well. Aliquots (0.1 ml) of each antifungal agent at a 2× final
concentration were dispensed into the wells of plastic microdilution
trays using a Quick Spense II System (Dynatech Laboratories, Chantilly,
Va.). The trays were sealed and frozen at Media.
The agar formulation used for the Etest was RPMI 1640 (American Biorganic, Buffalo, N.Y.) supplemented with 1.5% agar and 2% glucose (RPG agar) and buffered with MOPS. The RPMI 1640 broth medium used for the microdilution testing was buffered with MOPS in
accordance with the NCCLS M27-A method (10).
Antifungal susceptibility testing methods.
Broth
microdilution tests were performed as described in NCCLS document
M27-A (10). An inoculum concentration of 0.5 × 103 to 2.5 × 103
cells per ml was standardized spectrophotometrically and validated by
quantitative plate counts. Microdilution trays were incubated at 35°C
and read after 48 h of incubation. For posaconazole, the MIC end
point was defined as the lowest concentration that produced a prominent
decrease in turbidity (approximately a 50% reduction in growth)
compared with that of the drug-free control (10).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3952-3954.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of Etest Method for Determining
Posaconazole MICs for 314 Clinical Isolates of
Candida Species
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 they were
used. The final concentrations of posaconazole were 0.007 to 8 µg/ml.
QC. Quality control (QC) was performed in accordance with NCCLS document M27-A using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (10). QC determinations made on each day of testing were within the control limits for posaconazole as established by Barry et al. (2): C. krusei ATCC 6258, 0.12 to 1 µg/ml; and C. parapsilosis ATCC 22019, 0.06 to 0.25 µg/ml.
Analysis of results. Etest MICs read at 48 h were compared to reference microdilution MICs read at 48 h. Since the Etest scale has a continuous gradient of concentrations, the MICs between twofold dilutions were raised to the next twofold level of the reference method for comparison (13-15). 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 314 Candida isolates to posaconazole as
determined by the reference broth microdilution method. The
posaconazole MICs obtained were consistent with values reported
previously for the individual Candida spp. tested in RPMI
1640 medium (11, 12). Posaconazole MICs of >1 µg/ml
were observed for only three isolates of C. albicans (MICs
of >8 µg/ml) and five isolates of C. glabrata (MICs of 2, 4, and >8 [three isolates] µg/ml).
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Table 2 summarizes the percentages of
48-h posaconazole MICs obtained by the Etest in RPG agar that were
within two dilutions of the reference method result. Overall, the
agreement was 95%. The agreement between Etest and microdilution MICs
was >90% for C. albicans (98%), C. glabrata
(93%), C. tropicalis (97%), and C. krusei
(100%). With the exception of C. glabrata, when a
discrepancy was observed between the results obtained by the Etest and
the reference method, the Etest provided a lower MIC. In the case of
C. glabrata, discrepant MICs determined by the Etest were
always higher than those determined by the reference method.
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The results of this study provide the first documentation of the applicability of the Etest method for determining the in vitro susceptibilities of Candida species to the investigational triazole posaconazole. As in previous studies, 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). Similar to the case with the other triazoles, fluconazole (13) and voriconazole (15), the problem of trailing end points due to partial inhibition of growth by azoles was minimized by use of RPG agar and strict 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 discernible growth within an established ellipse was ignored.
In summary, we have provided the first evidence of the ability of the Etest to generate posaconazole MIC data that are comparable to those obtained by the NCCLS microdilution method. RPMI agar with 2% glucose may be used to determine reference quality MICs with the new investigational triazole (voriconazole and posaconazole) and echinocandin (caspofungin) Etest reagents in tests with Candida spp. (15; Pfaller et al., submitted). The availability of Etest reagents for these new antifungal agents will be useful to clinical laboratories because it will provide the flexibility to test one or more of these agents selectively as they are introduced into clinical practice and as the clinical situation dictates.
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ACKNOWLEDGMENTS |
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The excellent secretarial support of Kay Meyer and Linda Elliott is greatly appreciated.
This study was supported in part by Schering-Plough Research Institute 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.
Present address: 345 Beaver Kreek Centre, North Liberty, IA 52317.
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REFERENCES |
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Top
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References
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Journal of Clinical Microbiology, November 2001, p. 3952-3954, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3952-3954.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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