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Antimicrobial Agents and Chemotherapy, September 2003, p. 3018-3020, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.3018-3020.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Disk Diffusion-Based Methods for Determining Candida parapsilosis Susceptibility to Anidulafungin

Zekaver Odabasi,^1* Victor Paetznick,¹ Beth P. Goldstein,² John H. Rex,^1, and Luis Ostrosky-Zeichner¹

Laboratory of Mycology Research, Division of Infectious Diseases, University of Texas—Houston Medical School, Houston, Texas 77030,¹ Vicuron Pharmaceuticals, Inc., King of Prussia, Pennsylvania 19406²

Received 25 April 2003/ Returned for modification 23 May 2003/ Accepted 11 June 2003

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Zone diameters for anidulafungin by disk diffusion for 139 isolates of C. parapsilosis were compared with MICs by NCCLS M27-A2 broth microdilution. The comparison was poor unless the disks were prepared by dissolving anidulafungin in 1% dimethyl sulfoxide plus 0.1% Tween 80 and testing on Mueller-Hinton agar flooded with glucose and methylene blue.

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The echinocandin antifungal agents are promising for the treatment of systemic fungal infections. The majority of in vitro work with them to date has been with broth-based methods, and there are few reports of testing by a disk diffusion-based procedure (1, 3, 4). Disk diffusion susceptibility methods are simple and cost-effective and highlight different features of the patterns of in vitro response in ways that can be instructive (7).

Evaluation of antifungal susceptibility testing for the echinocandin antifungal agents is at an early state. Broth-based testing has generally noted higher MICs for Candida parapsilosis than for the other common species (5), but the relevance of this is unknown. For this organism, we have also observed a disparity between the results obtained by disk diffusion and those obtained by the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 broth microdilution procedure (6). In particular, disk diffusion-based testing produced very small zones for some isolates for which broth microdilution MICs were similar to those for isolates that had larger inhibition zones. In this report, we describe a systematic survey of alternative approaches to testing that obtained more congruent zones.

Disk-based testing. A collection of 139 bloodstream isolates of C. parapsilosis was used. The isolates were identified and stored according to standard methods. For disk diffusion testing, we used 150-mm-diameter plates with an agar depth of 4 to 5 mm. Testing was initially performed on 0.75% Bacto Agar (Difco Laboratories, Detroit, Mich.) prepared with RPMI 1640 (Sigma Chemicals Company, St. Louis, Mo.) buffered with 0.165 or 0.0165 M morpholinopropanesulfonic acid (MOPS) to pH 7 or with 0.1 M sodium phosphate to pH 4, 5.5, and 7; antibiotic medium 3 (AM3; BBL, Cockeysville, Md.) buffered with 0.1 M sodium phosphate to pH 7; and yeast nitrogen base (YNB; Difco) supplemented with 0.5% glucose and buffered to pH 7 with 0.165 M MOPS. AM3 was also solidified with Noble agar (Sigma). Mueller-Hinton agar plates were obtained from Difco. Some plates were flooded prior to use with a solution of 2% glucose and 0.5 µg of methylene blue per ml (GMB) (2) as a means of enhancing zone definition.

Drug preparation. Anidulafungin (Versicor, Inc.) was dissolved in 1% dimethyl sulfoxide (DMSO; Fisher Chemicals, Fair Lawn, N.J.), 1% DMSO plus 1.5% bovine serum albumin (BSA; Sigma), or 0.1% Tween 80 (Fisher Chemicals). Disks containing 2.5 or 5 µg of drug were prepared by placing 25 µl of a suitable drug concentration on sterile 6.3-mm-diameter paper disks (Difco Laboratories) and allowing them to dry at room temperature with storage at -70°C.

Inoculum preparation. Inocula were prepared by picking colonies from 24-h-old cultures. Colonies were suspended in 5 ml of a solution containing 0.145 mol of saline per liter. The density was adjusted by spectrophotometer to that of a 0.5 McFarland standard at a wavelength of 530 nm to yield a stock suspension of 1 x 10⁶ to 5 x 10⁶ cells per ml (2). Two different inoculum sizes were evaluated: 1 x 10⁶ to 5 x 10⁶ cells per ml and a 1:100 dilution containing 1 x 10⁴ to 5 x 10⁴ cells per ml. Plates were swabbed in three directions.

Testing and reading procedure. After 24 and 48 h at 30 or 35°C, inhibitory zones were measured at the point at which growth abruptly decreased.

Broth-based testing. Broth microdilution testing was performed according to the NCCLS M27-A2 microdilution method and was performed in RPMI 1640 buffered to pH 7.0 with 0.165 M MOPS. Anidulafungin was prepared in 100% DMSO, and the final concentration range was 0.03 to 16 µg/ml. Quality control isolates C. parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included in all runs and were within quality control ranges (6). MICs were read after 24 and 48 h as the smallest drug concentration that produced a prominent decrease in growth (MIC-2) or an optically clear well (MIC-0). The MIC-2 and MIC-0 readings were similar, so this report focuses on MIC-2 results.

In a survey of 139 C. parapsilosis isolates, a sharp difference was observed between the broth- and disk diffusion-based results (Table 1). Seventy three isolates showed no zone of inhibition despite broth microdilution MICs that suggested relative susceptibility. Four isolates that illustrated different MIC-zone relationships were identified for further testing under all proposed conditions (Table 2). The 1:100-diluted inoculum and incubation at 30°C produced less growth than other conditions (data not shown), and subsequent work thus focused on testing at 35°C and with the higher inoculum (1 x 10⁶ to 5 x 10⁶ cells per ml). Drug diluted in 1% DMSO and delivered via dried paper disks or by adding the same volume of drug to a 2-mm-diameter well punched into the agar gave similar zone diameters (data not shown), suggesting that inhibition zones were not related to the use of disks (i.e., were not due to binding of the drug to the paper). Among the media, the largest zone diameters were obtained on Mueller-Hinton agar with GMB (Table 3). The combination of 0.1% Tween 80 with 1% DMSO produced the largest zone diameters, and both 2.5- and 5-µg disks prepared in this fashion produced similar and acceptable zone diameters. Nevertheless, no method showed a significant correlation with the broth-based microdilution method when r² was calculated with EpiInfo 2002 software.

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TABLE 1. Comparison between MIC-2 at 48 h and zone diameter observed with different methodsa

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TABLE 2. Initial observations for selected isolatesa

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TABLE 3. Comparison of results obtained under different conditions

On the basis of the results obtained with four isolates, the following conditions were chosen for surveying 139 of the original C. parapsilosis isolates: 2.5-µg anidulafungin disks prepared with 1% DMSO and 0.1% Tween 80, the higher inoculum (1 x 10⁶ to 5 x 10⁶ cells per ml), and Mueller-Hinton agar with GMB. By this method, anidulafungin inhibition zone diameters seemed more congruent with broth MICs (Table 1) than by the method used originally, since this method produced inhibition zones for isolates that did not have them when tested by the original method. Only one isolate now showed no zone of inhibition. The causes of the remaining discrepant results are unknown, and the clinical implications of isolates with high MICs and/or small zone will require further investigation. While this study failed to show a mathematical correlation between any disk-based method and microbroth susceptibility testing, we have provided information to be considered when the disk-based methods of antifungal susceptibility for the echinocandins are further standardized.

 
* Corresponding author. Mailing address: Laboratory of Mycology Research, Division of Infectious Diseases, University of Texas—Houston Medical School, 6431 Fannin, JFB 1.728, Houston, TX 77030. Phone: (713) 500-5383. Fax: (713) 500-5495. E-mail: zekaver.odabasi{at}uth.tmc.edu.

Present address: AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom SK10 4TF.

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1. Arikan, S., V. Paetznick, and J. H. Rex. 2002. Comparative evaluation of disk diffusion with microdilution assay in susceptibility testing of caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 46:3084-3087.[Abstract/Free Full Text]
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2. Barry, A. L., M. A. Pfaller, R. P. Rennie, P. C. Fuchs, and S. D. Brown. 2002. Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods. Antimicrob. Agents Chemother. 46:1781-1784.[Abstract/Free Full Text]
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3. Espinel-Ingroff, A., S. Shadomy, and S. White. 1991. Agar disk diffusion susceptibility tests with cilofungin (LY 121019). J. Med. Vet. Mycol. 29:93-98.[Medline]
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4. Lozano-Chiu, M., P. W. Nelson, V. L. Paetznick, and J. H. Rex. 1999. Disk diffusion method for determining susceptibilities of Candida spp. to MK-0991. J. Clin. Microbiol. 37:1625-1627.[Abstract/Free Full Text]
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5. Marco, F., M. A. Pfaller, S. A. Messer, and R. N. Jones. 1998. Activity of MK-0991 (L-743,872), a new echinocandin, compared with those of LY303366 and four other antifungal agents tested against bloodstream isolates of Candida spp. Diagn. Microbiol. Infect. Dis. 32:33-37.[CrossRef][Medline]
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6. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard NCCLS M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
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7. Wanger, A., K. Mills, P. W. Nelson, and J. H. Rex. 1995. Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Antimicrob. Agents Chemother. 39:2520-2522.[Abstract]

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Antimicrobial Agents and Chemotherapy, September 2003, p. 3018-3020, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.3018-3020.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Odabasi, Z. Articles by Ostrosky-Zeichner, L. Search for Related Content PubMed PubMed Citation Articles by Odabasi, Z. Articles by Ostrosky-Zeichner, L.