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Journal of Clinical Microbiology, September 2000, p. 3359-3361, Vol. 38, No. 9
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 13 June
2000/Accepted 12 July 2000
The performance of the Etest for itraconazole susceptibility
testing of 50 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)
proposed standard 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 and with Casitone agar and were read after incubation for
24 h (Aspergillus spp. and Rhizopus spp.) and 48 h (all species except Rhizopus spp.) at 35°C.
The isolates included Aspergillus flavus, Aspergillus
fumigatus, Aspergillus niger, Aspergillus
terreus, Fusarium spp., Pseudallescheria
boydii, Rhizopus spp., Paecilomyces
variotii, and an Acremonium sp. Overall agreement
between Etest and microdilution MICs was 96% with RPMI agar and 80%
with Casitone agar. The agreement was 100% for all species except
Rhizopus spp. (83%) and Paecilomyces varioti
(0%) with RPMI agar. When Casitone agar was used, the agreement ranged from 50% with Rhizopus spp. to 100% with
Fusarium spp., P. boydii, P. varioti, and an Acremonium sp. Notably, for
Aspergillus spp., the agreement between itraconazole Etest
MICs read at 24 h and reference microdilution MICs read at 48 h was 100% with both RPMI and Casitone agar. Both media supported the
growth of all filamentous fungi tested. Where a discrepancy was
observed between Etest and the reference method, the Etest MIC was
generally higher. The Etest method using RPMI agar appears to be a
useful method for determining itraconazole susceptibilities of
Aspergillus spp. and other filamentous fungi.
Testing of susceptibility to
antifungal agents has now been standardized for yeasts, and additional
efforts to develop more user-friendly testing methods for use in the
clinical laboratory have been successful (2, 10, 13).
Efforts to adapt the National Committee for Clinical Laboratory
Standards (NCCLS) M27-A broth microdilution methodology to testing of
molds have also been successful (3, 5, 6, 11). A recent
multicenter study documented excellent reproducibility of the
microdilution method for testing amphotericin B, ketoconazole, and
itraconazole against molds in 11 laboratories (5). A
reference method for broth dilution antifungal agent susceptibility
testing of filamentous fungi has been proposed by the NCCLS (M38-P)
(11). As with yeasts, there is a need to validate
alternative approaches for testing of molds.
The Etest stable gradient MIC method (AB BIODISK, Solna, Sweden) has
proven to be a remarkably flexible means of performing MIC testing for
a wide variety of microbial pathogens and antimicrobial agents (1,
8, 9). In addition to the testing of routine and fastidious
bacteria (9), the Etest has proven to be useful in testing
yeasts (4, 12, 14, 15, 19). To our knowledge there have been
only two published reports describing the adaptation of the Etest for
antifungal agent susceptibility testing of molds. Szekely et al.
(18) showed that the Etest was a suitable alternative to
broth microdilution for testing the susceptibility of molds to
amphotericin B and itraconazole. Johnson et al. (7)
demonstrated differences in amphotericin B MICs among isolates of
Aspergillus spp. using the Etest but failed to find a close
clinical correlation with these results in a murine model of
aspergillosis. The present report describes the use of the Etest to
determine the in vitro susceptibility of 50 clinical mold isolates to a
clinically useful antifungal agent, itraconazole. The itraconazole MIC
results determined by the Etest using two different agar media are
compared to MICs determined by the NCCLS proposed reference
microdilution method, NCCLS M38-P (11).
Test isolates.
A total of 50 isolates were tested. These
included clinical isolates of Aspergillus flavus (10 isolates), Aspergillus fumigatus (12 isolates),
Aspergillus niger (1 isolate), Aspergillus
terreus (1 isolate), Fusarium oxysporum (5 isolates),
Fusarium solani (5 isolates), other Fusarium spp.
(3 isolates), Pseudallescheria boydii (5 isolates),
Rhizopus spp. (6 isolates), Paecilomyces variotii
(1 isolate) and an Acremonium sp. (1 isolate). The isolates came from the culture collection of the University of Iowa and were
stored on slants or in water suspension at ambient temperature until
used in the study. Prior to use in the study isolates were passaged at
least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure
viability and adequate sporulation.
Etest method.
The Etest was performed in accordance with the
manufacturer's instructions. Isolates were grown on potato dextrose
agar slants (Remel) at 35°C for seven days to ensure adequate
sporulation. Spore suspensions were prepared in sterile saline and
adjusted to a concentration of 106 spores/ml, corresponding
to 68 to 82% transmittance at 530 nm (14). Agar
formulations used for the Etest were RPMI 1640 (American Biorganics,
Buffalo, N.Y.) supplemented with 1.5% agar and 2% glucose and
buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS)
buffer (Sigma, St. Louis, Mo.) and Casitone agar (Difco). The
90-mm-diameter plates contained RPMI or Casitone agar at a depth of 4.0 mm. The plates were inoculated by dipping a sterile swab into the cell
suspension and streaking it across the surface of the agar in three
directions. The plates were dried at ambient temperature for 15 min
before applying the itraconazole Etest strips. The plates were
incubated at 35°C and read at 24 (Aspergillus spp. and
Rhizopus spp.) and 48 h (all species except
Rhizopus spp.). The Etest MIC was read as the drug
concentration at the point where dense colonial growth intersected the
strip, ignoring sparse subsurface hyphae at the margins (Fig.
1). Microcolonies within the ellipse were
ignored.
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In Vitro Susceptibility Testing of Filamentous Fungi:
Comparison of Etest and Reference Microdilution Methods for
Determining Itraconazole MICs
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
View larger version (99K):
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FIG. 1.
Itraconazole (IT) Etest reading pattern for A. flavus. A clear ellipse on RPMI agar is shown; the MIC is 1.0 µg/ml. The numbers on the scale correspond to the itraconazole
concentrations on the strip (in micrograms per milliliter).
Reference broth microdilution method.
The broth
microdilution method was performed according to NCCLS proposed
guidelines (11) and as described by Espinel-Ingroff et al.
(5, 6). Itraconazole was obtained from Janssen Research Foundation (Beerse, Belgium). Stock solutions were prepared in 100%
dimethyl sulfoxide (Sigma), diluted to 100 times the final concentrations in dimethyl sulfide, further diluted in RPMI 1640 medium
buffered to pH 7.0 with MOPS buffer, and dispensed into 96-well
microdilution trays. Trays containing a 0.1-ml aliquot of itraconazole
solution (two times the final concentration) in each well were
subjected to quality control (QC) and then sealed and stored at
70°C until use in the study. The final concentration of
itraconazole in the wells ranged from 0.007 to 8.0 µg/ml. The stock
conidial suspension (106 spores/ml) was diluted to a final
inoculum concentration of 0.4 × 104 to 5 × 104 CFU/ml and dispensed into the microdilution wells. The
inoculated microdilution trays were incubated at 35°C and read at 24 (Rhizopus spp.) and 48 h (all other species). The MIC
endpoint for itraconazole was defined as the lowest concentration that
produced prominent inhibition of growth (approximately 50% inhibition)
relative to the drug-free growth control (11).
QC. QC was performed in accordance with NCCLS guidelines (11) and as recommended by Espinel-Ingroff et al. (6) using Candida krusei ATCC 6258 (MIC range, 0.12 to 0.5 µg/ml), Candida parapsilosis (MIC range, 0.06 to 0.25 µg/ml), and P. variotii ATCC 22319 (MIC range, 0.03 to 1.0 µg/ml).
Analysis of results. Etest MICs read at 48 h on the two media were compared to reference microdilution MICs read at 48 h. Due to rapid growth on agar, Etest results for Rhizopus spp. were read at 18 to 24 h. Etest results for Aspergillus spp. were read at both 24 and 48 h. The reference microdilution MICs and Etest MICs were determined in two physically separate laboratories and were read independently; i.e., the testing was blinded. Since the Etest scale has a continuous gradient of concentrations, the MICs in between twofold dilutions were rounded to the next twofold level of the reference method for comparison (14, 15, 18). Off-scale MICs at the upper limit were converted to the next-higher concentration, and off-scale results at the lower end were left unchanged. Discrepancies between MICs of no more than 2 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 50 clinical mold isolates to itraconazole as
determined by the reference microdilution method. The data are
presented as MIC ranges and, where appropriate, as the drug
concentrations necessary to inhibit 50 and 90% of the isolates of each
species. A broad range of MICs was observed. In general, itraconazole
MICs obtained were similar to those reported previously for the
individual fungal species (16, 18). In each batch of
microdilution tests, the MICs of itraconazole for the three QC strains
were within the accepted limits (11).
|
Table 2 summarizes the percentage of 48-h
itraconazole MICs obtained by the Etest on the two agar media that were
within 2 dilutions of the reference method result. Overall, the percent agreement was 96% with RPMI and 80% with Casitone agar. The agreement between Etest and microdilution MICs was 100% for all species except
Rhizopus spp. (83%) and P. variotii (0%) with
RPMI agar. Lower levels of agreement were observed when Casitone agar
was used; however, 100% agreement was observed with
Fusarium spp., P. boydii, P. variotii,
and the Acremonium sp. In virtually every instance when a
discrepancy was observed between Etest and reference method results,
the Etest provided a higher MIC.
|
It is notable that for Aspergillus spp. the agreement
between itraconazole Etest MICs read at 24 h and reference
microdilution MICs read at 48 h was 100% with both RPMI and
Casitone media (Table 3). Thus for the
most common species of filamentous fungi encountered clinically, the
Etest may be read earlier than the broth method and the choice of test
medium may be flexible.
|
The results of this study confirm and extend those of Szekely et al. (18) regarding the applicability of the Etest stable agar gradient method for determining the in vitro susceptibilities of filamentous fungi to itraconazole. Szekely et al. (18) used RPMI agar and 48 h of incubation and found that the Etest procedure was reproducible and served as a suitable method for antifungal susceptibility testing of molds. The overall agreement between Etest and broth microdilution was 88% for itraconazole. The agreement was 97.5% when testing Aspergillus spp. (18). Furthermore, they demonstrated that the Etest was able to detect resistance to itraconazole among A. fumigatus and other molds (18).
We found the Etest to be a very simple means of determining the in vitro susceptibilities of molds to itraconazole. RPMI agar with glucose (final concentration, 2%) supported optimal growth of all species tested and provided excellent (96%) agreement with the MICs obtained with the broth microdilution method (Table 2). As noted by Szekely et al. (18), the itraconazole inhibition ellipses were clear for most isolates tested on RPMI medium (Fig. 1).
Although Casitone agar did not perform as well as RPMI agar, it supported growth of the test isolates and achieved 80% agreement with results for the reference method (Tables 2 and 3). Importantly, when read at 24 h, Casitone agar and RPMI agar produced results that were in complete agreement with the 48-h reference MICs in testing of Aspergillus spp. (Table 3). Since Aspergillus spp. constitute the most frequently encountered pathogen among the filamentous fungi (17), an method for determining MICs whose results can be read as early as 24 h may be useful in the clinical laboratory.
In summary, we have provided additional documentation of the ability of Etest to generate itraconazole MIC data for filamentous fungi 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 of itraconazole as well as amphotericin B (18) when tested against Aspergillus spp. and other less common molds, as well as Candida spp. and Cryptococcus neoformans (14, 15, 18). This will be attractive to laboratories, since it will provide the flexibility to test one or more commonly used antifungal agents selectively against a wide variety of yeasts and molds that may be encountered clinically. It is prudent to keep in mind that these results, as noted by Johnson et al. (7) for amphotericin B and aspergillosis, may not be entirely predictive of clinical outcome.
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ACKNOWLEDGMENTS |
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The excellent secretarial support of Kay L. Meyer is greatly appreciated.
This study was supported in part 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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References
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Journal of Clinical Microbiology, September 2000, p. 3359-3361, Vol. 38, No. 9
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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