Disk Diffusion Testing Using Candida sp. Colonies Taken Directly from ...

JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p. 3497–3499 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.7.3497–3499.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 7

Disk Diffusion Testing Using Candida sp. Colonies Taken Directly from CHROMagar Candida Medium May Decrease Time Required To Obtain Results Michael Klevay,1 Alex Ebinger,2 Daniel Diekema,1,2,4* Shawn Messer,2 Richard Hollis,2 and Michael Pfaller2,3 Departments of Internal Medicine1 and Pathology,2 University of Iowa Carver College of Medicine, Department of Epidemiology, University of Iowa College of Public Health,3 and Iowa City Veterans Affairs Medical Center,4 Iowa City, Iowa Received 11 November 2004/Returned for modification 25 December 2004/Accepted 14 March 2005

We compared results of disk diffusion antifungal susceptibility testing from Candida sp. strains passaged on CHROMagar and on potato dextrose agar. The overall categorical agreements for fluconazole and voriconazole disk testing were 95% and 98% with 0% and 0.5% very major errors, respectively. Disk diffusion testing by the CLSI (formerly NCCLS) M44-A method can be performed accurately by taking inocula directly from CHROMagar.

CHROMagar Candida (Hardy Diagnostics, Santa Maria, CA) is a differential culture medium effective for primary isolation and identification of common Candida species from clinical samples (5). Laboratories using CHROMagar for rapid identification of selected Candida spp. must still passage the organisms on nondifferential media such as potato dextrose agar (PDA) prior to performing disk diffusion antifungal susceptibility testing (2). Performing disk diffusion antifungal susceptibility testing on colonies directly from CHROMagar could significantly decrease the time required to obtain results. The organisms tested represented unique patient isolates of Candida spp. acquired from several surveillance programs, including SENTRY (3) and ARTEMIS (4). In total, the isolates were collected from 11 centers in the United States; seven in Europe; six in Latin America; and 15 centers in Africa, Canada, Asia, and Australia. One hundred ninety-two isolates of Candida were used, over 50% of which were from the bloodstream, the others being from respiratory, gastrointestinal, cerebrospinal fluid, abscess, and joint cultures. These included isolates of C. albicans (n ⫽ 59), C. glabrata (n ⫽ 57), C. parapsilosis (n ⫽ 43), and C. tropicalis (n ⫽ 33). These four species represent approximately 95% of Candida strains recovered in most clinical laboratories (3). All fungal isolates were identified at the participating institution by the routine methodology in use at each laboratory. Upon receipt at the University of Iowa, species identification was performed with Vitek and API products (bioMerieux, St. Louis, Mo.), as recommended by the manufacturer, or by conventional methods, as required (1). Isolates were stored as suspensions in water at ambient temperature until used in the study. The isolates were subcultured onto PDA (Remel, Lenexa,

KS) and incubated for 24 h at 30°C. Isolates were then subcultured in parallel onto PDA and CHROMagar and incubated for 24 h at 35°C. Disk diffusion testing of fluconazole and voriconazole on the identical parallel isolates was performed as described in CLSI (formerly NCCLS) document M44-A (2). Mueller-Hinton agar plates supplemented with 2% glucose and methylene blue were used. The agar surface was inoculated by using a swab dipped in a cell suspension adjusted to the turbidity of a 0.5 McFarland standard. The plates were incubated in air at 35°C and read at 24 h. Zone diameter endpoints were read manually with calipers at 80% growth inhibition. The interpretive criteria for fluconazole disk diffusion testing were those published by the CLSI and were as follows: susceptible, zone diameter of ⱖ19 mm; susceptible dose dependent, zone diameter of 15 to 18 mm; resistant, zone diameter of ⱕ14 mm. Provisional interpretive criteria were used for voriconazole, with a zone of inhibition of ⱕ13 mm considered resistant and of ⱖ14 mm considered susceptible. Quality control was performed using C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 for each batch tested. The zone diameters (in millimeters) for fluconazole and voriconazole disks at 24 h, after inoculation from CHROMagar, were plotted against their respective zones of inhibition after inoculation from PDA. The method of least squares was used to calculate a regression line for each comparison. Major errors were classified as resistant by disk diffusion of inocula taken from CHROMagar and susceptible when cultured from PDA. Very major errors were classified as susceptible by disk diffusion of Candida spp. taken from CHROMagar and resistant when cultured from PDA. Minor errors occurred when the result of one isolate was susceptible or resistant and that of the parallel isolate was susceptible dose dependent. There was a strong correlation between disk test results for all Candida isolates taken from CHROMagar and results for isolates taken from PDA for both fluconazole and voriconazole (R2 of ⬎0.8, P ⬍ 0.001; Fig. 1 and 2). In addition, overall and species-specific categorical agreements between disk test re-

* Corresponding author. Mailing address: C 606 General Hospital, University of Iowa Hospital, 200 Hawkins Drive, Iowa City, Iowa 52246. Phone: (319) 356-8615. Fax: (319) 356-4916. E-mail: daniel-diekema @uiowa.edu. 3497

FIG. 1. Zones of inhibition (mm) around voriconazole disks for Candida spp. inoculated from CHROMagar compared to those inoculated from PDA. The numbers plotted in the graph represent the number of Candida isolates at each pair of inhibition zones.

FIG. 2. Zones of inhibition (mm) around fluconazole disks for Candida spp. inoculated from CHROMagar compared to those inoculated from PDA. The numbers plotted in the graph represent the number of Candida isolates at each pair of inhibition zones. 3498

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TABLE 1. Categorical interpretive agreement for disk diffusion from PDA and that from CHROMagar No. of errors (% by row) Organism (n)

C. albicans (59) C. glabrata (57) C. parapsilosis (43) C. tropicalis (33) All Candida strains (192)

Agent

% Agreement

Fluconazole Voriconazole Fluconazole Voriconazole Fluconazole Voriconazole Fluconazole Voriconazole Fluconazole Voriconazole

sults after inoculation from CHROMagar and results after inoculation from PDA for all Candida strains were excellent (90 to 100%; Table 1). Given current patterns of resistance among Candida species (3), clinicians are usually most interested in quickly knowing the species and the fluconazole susceptibility of sterile-site Candida isolates. For laboratories that do not perform broth microdilution susceptibility testing of yeast species on site, sending isolates to a reference laboratory can result in delays in reporting of results that limit the usefulness of the information. For these laboratories, disk diffusion susceptibility testing, limited to just one or two drugs, including fluconazole, is often enough to guide therapy. We have demonstrated that disk diffusion testing using the CLSI M44-A method can be performed directly from either CHROMagar or PDA, with categorical agreement between the two of 95% for fluconazole and 98% for voriconazole. This allows for more timely determination of antifungal susceptibility test results in laboratories using

93.2 96.6 96.5 98.3 90.7 100 100 100 94.8 98.4

Minor

Major

Very major

1 (1.7)

3 (5.1) 1 (1.7) 1 (1.8) 1 (1.8) 0 (0) 0 (0) 0 (0) 0 (0) 4 (2.1) 2 (1.0)

0 (0) 1 (1.7) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.5)

1 (1.8) 4 (9.3) 0 (0) 6 (3.1)

CHROMagar for rapid identification of common Candida species. REFERENCES 1. Hazen, K. C., and S. A. Howell. 2003. Candida, Cryptococcus, and other yeasts of medical importance, p. 1693–1711. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, D.C. 2. National Committee for Clinical Laboratory Standards. 2004. Method for antifungal disk diffusion susceptibility testing of yeasts: approved guideline M44-A. National Committee for Clinical Laboratory Standards, Wayne, Pa. 3. Pfaller, M. A., D. J. Diekema, R. N. Jones, et al. 2002. Trends in antifungal susceptibility of Candida spp. isolated from pediatric and adult patients with bloodstream infections: SENTRY Antimicrobial Surveillance Program, 1997– 2000. J. Clin. Microbiol. 40:852–856. 4. Pfaller, M. A., K. C. Hazen, S. A. Messer, et al. 2004. Comparison of results of fluconazole disk diffusion testing for Candida species with results from a central reference laboratory in the ARTEMIS global antifungal surveillance program. J. Clin. Microbiol. 42:3607–3612. 5. Pfaller, M. A., A. Houston, and S. C. Coffmann. 1996. Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata. J. Clin. Microbiol. 34:58–61.