Current Status of Antifungal Susceptibility Testing: Methods and Clinical Application

Jong Hee Shin

doi:10.5145/KJCM.2009.12.4.154

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Shin: Current Status of Antifungal Susceptibility Testing: Methods and Clinical Application

Original Article

Korean J Clin Microbiol 2009;12(4):154-158.

Published online: 22 December 2009

DOI: https://doi.org/10.5145/KJCM.2009.12.4.154

Current Status of Antifungal Susceptibility Testing: Methods and Clinical Application

Jong Hee Shin

Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea

Correspondence: Jong Hee Shin, Department of Laboratory Medicine, Chonnam National University Medical School, 671, Jebong-ro, Dong-gu, Gwangju 501-757, Korea. (Tel) 82-62-220-5342, (Fax) 82-62-224-2518, (E-mail) shinjh@chonnam.ac.kr

Received 18 August 20099 September 2009 Accepted 15 October 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

During the past two decades, Clinical and Laboratory Standards Institute (CLSI) antifungal susceptibility testing methods for both yeasts and molds have been developed and established in response to increasing invasive fungal infections and the release of multiple new antifungal agents. In addition, other methods including Etest, the disk diffusion test, and some CLSI modification methods have been intensively studied. Antifungal susceptibility tests are now routinely used for local epidemiological surveys to determine the susceptibility patterns of clinical isolates of fungi, the degree of antifungal activity of newly developed antifungal agents, and to predict the clinical outcomes of antifungal therapy for patients with Candida infections. It is anticipated that in the near future, antifungal susceptibility tests that can detect amphotericin B resistance, that can be used to establish the minimum inhibitory concentration (MIC) breakpoints of molds, and that can provide increased clinical guidance for antifungal therapy, will be developed. This review focuses on the various methods used for antifungal susceptibility testing and the clinical utility of antifungal susceptibility testing.

Keywords: Antifungal susceptibility, Candida, Molds, Amphotericin B, CLSI

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Table 1.

Interpretive guidelines for in vitro susceptibility testing of Candida spp. using CLSI M27 reference methods∗

Antifungal agent	Minimum inhibitory concentration (μg/mL)
Antifungal agent	Susceptible (S)	Susceptible-dose dependent (SDD)	Intermediate (I)	Resistant (R)	Nonsusceptible (NS)
Anidulafungin	≤2				＞2
Caspofungin	≤2				＞2
Fluconazole	≤8	16∼32		≥64
Flucytosine	≤4		8∼16	≥32
Itraconazole	≤0.125	0.25∼0.5		≥1
Micafungin Voriconazole	≤2 ≤1	2		≥4	＞2
Voriconazole	≤1	2		≥4

^∗ Table is adopted from CLSI M27-S3 (2008).

Table 2.

Current methods commonly used for antifungal susceptibility testing

Method	Comments
CLSI M27-A3	Reference method for broth dilution antifungal susceptibility testing of yeasts (Candida spp. and Cryptococcus neoformans); testing amphotericin B, flucytosine, azoles (fluconazole, ketoconazole, itraconazole, posaconazole, ravuconazole, and voriconazole), and echinocandins (anidulafungin, caspofungin, and micafungin; test medium, RPMI 1640 medium with 0.2% glucose; inoculums density, 0.5-2.5×10³ cfu/mL; microdilution plates, 96 flat-bottom wells; MIC reading time point (general) and method, 48 h and visual, respectively.
CLSI M38-A2	Reference method for broth dilution antifungal susceptibility testing of filamentous fungi (Aspergillus spp., Fusarium spp., Rhizopus spp., Pseudallescheria boydii, and the mycelial form of Sporothrix schenckii); testing amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, posaconazole, ravuconazole, voriconazole, anidulafungin, caspofungin, micafungin, ciclopirox, and griseofulvin; test medium, same as M-27; inoculums density, 0.4-5×10⁴ cfu/mL; MIC reading time point, 24 h (Rhizopus), 48 h most others (Aspergillus), 72 h (Psuedallescheria boydii); MIC reading method, visual.
CLSI M44-A	Methods for antifungal disk diffusion testing of yeasts; for testing fluconazole and voriconazole; use Mueller-Hinton agar with methylene blue and 2% glucose provides sharper zones with fluconazole disk.
Etest	Commercially available agar-based diffusion method, (AB BioDisk, Solna, Sweden) which enables the determination of MIC values; useful for yeasts and molds; testing amphotericin B, fluconazole, itraconazole, flucytosine, voriconazole and caspofungin; most sensitive method for detecting amphotericin B resistance.
EUCAST method	Standard methods of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing; test medium, RPMI 1640 medium with 2% glucose; inoculums density, 1-5×10⁵ cfu/mL; microdilution plates, 96 flat-bottom wells; MIC reading time point and method for Candida spp., 24 h and spectrophometric (530 nm), respectively; MIC reading time and endpoint for Aspergillus and molds, 48 h and no growth, respectively.
YeastOne	Commercially available Colorimetric microdilution method (Trek, Westlake, Ohio, USA) based on the CLSI methodology; use of RPMI with 2% glucose and Alamar blue; testing amphotericin B, fluconazole, itraconazole, ketoconazole, flucytosine, voriconazole and caspofungin.
Vitek 2	Fully automated commercial antifungal susceptibility test system (bioMérieux, Hazelwood, MO, USA) that determines the MIC endpoint spectrophotometrically; testing amphotericin B, fluconazole, flucytosine, and voriconazole.

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