Disk Diffusion-Based Methods for Determining Candida parapsilosis ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2003, p. 3018–3020 0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.9.3018–3020.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 47, No. 9

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 and Vicuron Pharmaceuticals, Inc., King of Prussia, Pennsylvania 194062 Received 25 April 2003/Returned for modification 23 May 2003/Accepted 11 June 2003

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.

dium phosphate to pH 4, 5.5, and 7; antibiotic medium 3

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 so-

(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 ␮g 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 ␮g of drug were prepared by placing 25 ␮l 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 ⫻ 106 to 5 ⫻ 106 cells per ml (2). Two different inoculum sizes were evaluated: 1 ⫻ 106 to 5 ⫻ 106 cells per ml and a 1:100 dilution containing 1 ⫻ 104 to 5 ⫻ 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 ␮g/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 prom-

* 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 @uth.tmc.edu. † Present address: AstraZeneca Pharmaceuticals, Macclesfield, United Kingdom SK10 4TF. 3018

VOL. 47, 2003

NOTES TABLE 2. Initial observations for selected isolatesa

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

A

Zone diam (mm) 6b 7 8 9 10 11 12 13 14 15 16 Total

B

Zone diam (mm) 6b 9 11 12 13 14 15 16 17 18 19 20 21 22 23 25 27 29 31 34 Total

0.03125

0.0625

0.125

1 1

1 1

4

0.5

1

1 4 2

7

2 1 1 1

1

5

1 1

1

1 2 1

1 2 1 1

1 1 4

2

26

57

7 8 5 2 2 2 1

2

53

1

1

1

1

1 1 2 1 1 13 5 8 9 4 3 3

1

4

4

2 1 2 1 65

4

1 1 4 9 6 10 11 10 7 2 1 1

3 1

1 1

1 7

1

5

53

MIC-0

Zone diam (mm)

037-0012 24 h 48 h

0.06 0.03

0.125 0.125

8 7

031-0032 24 h 48 h

0.06 1

1 1

6 6

041-0071 24 h 48 h

1 1

1 2

12 11

025-0047 24 h 48 h

2 2

2 2

6 6

65

4

Total

89 1 9 10 7 8 7 3 2 2 1 139

2 2 1 1 6 11 7 15 26 17 18 11 7 5 4 1 2 1 1 1 139

a

Shown is the comparison between MIC-2 at 48 h (NCCLS M27-A2) and the zone diameter observed with the different methods. Results for method A were obtained after 24 h by using disks containing 2.5 ␮g of anidulafungin in 1% DMSO on RPMI agar swabbed with the higher inoculum. r2 ⫽ 0.16. Results for method B were obtained after 24 h by using 2.5-␮g anidulafungin disks (in 1% DMSO plus 0.1% Tween 80 carrier) applied to Mueller-Hinton agar flooded with GMB. r2 ⫽ 0.06. Note that while there is no significant correlation for either method, method B does produce inhibition zones in more isolates. b Growth filled the diameter of the disk, and thus no zone was seen.

inent 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 diffusionbased 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

Inhibitory concn (␮g/ml) MIC-2

Isolate

No. of isolates at MIC-2 (␮g/ml):

3019

a MICs were determined at least three times by the NCCLS M27-A procedure, and the median result is shown. For disk diffusion-based testing, plates were incubated at 35°C and zone diameters were determined as described in the text. Inhibition zones were determined on RPMI agar by using the higher inoculum and 2.5-␮g anidulafungin disks with no supplements.

less growth than other conditions (data not shown), and subsequent work thus focused on testing at 35°C and with the higher inoculum (1 ⫻ 106 to 5 ⫻ 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-␮g 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. 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-␮g anidulafungin disks prepared with 1% DMSO and 0.1% Tween 80, the higher inoculum (1 ⫻ 106 to 5 ⫻ 106 cells per ml), and MuellerHinton 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.

3020

NOTES

ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Comparison of results obtained under different conditions Zone diam (mm) for isolate with supplementb:

Anidulafungin combination

a

RPMI 1640 ⫹ 0.165 M MOPS (pH 7) RPMI 1640 ⫹ 0.0165 M MOPS (pH 7) RPMI 1640 ⫹ 0.1 M phosphate (pH 7) RPMI 1640 ⫹ 0.1 M phosphate (pH 5.5) RPMI 1640 ⫹ 0.1 M phosphate (pH 4) AM3 (pH 7) AM3 (pH 7) ⫹ Noble agar YNB (pH 7) Mueller-Hinton agar Mueller-Hinton agar flooded with GMB

037-0012

031-0032

041-0071

025-0047

None

Tween 80

BSA

None

Tween 80

BSA

None

Tween 80

BSA

None

Tween 80

BSA

13 13 13 12 12 12 13 15

19

16

15

13

13

11

22 18

14 18

21 20

14 18

7 7 0 9 9 8 7 0

11

17 32

9 9 9 10 10 10 9 10

16

28 32

9 9 9 9 10 9 9 9

20 12

13 12

a

Each disk contained anidulafungin dissolved in 1% DMSO plus the additives shown. Data shown are zone diameters after 24 h of incubation at 35°C using a 2.5-␮g anidulafungin disk and the higher inoculum. Tween 80 was used at 0.1%, and BSA was used at 1.5%. Similar results were obtained with a 5-␮g disk. b

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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. 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. 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.