Correlation of Neo-Sensitabs Tablet Diffusion Assay Results on Three Different Agar Media with CLSI Broth Microdilution M27-A2 and Disk Diffusion M44-A Results for Testing Susceptibilities of Candida spp. and Cryptococcus neoformans to Amphotericin B, Caspofungin, Fluconazole, Itraconazole, and Voriconazole

Abstract

The Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) Subcommittee on Antifungal Susceptibility Tests has developed reproducible procedures for antifungal susceptibility testing of yeasts by broth microdilution (document M27-A2) and disk diffusion (document M44-A for fluconazole and voriconazole) methods (4-7). The Neo-Sensitabs assay (A/S Rosco Diagnostica, Taastrup, Denmark) utilizes a 9-mm-diameter (1-mm-thickness) tablet for antimicrobial susceptibility testing including several antifungal agents (3). This assay has been favorably investigated for testing yeasts with 15-g fluconazole tablets on either Shadomy (SHA) or RPMI 1640 agar (2, 21, 22) and more recently with posaconazole (9). Amphotericin B, fluconazole, itraconazole, and voriconazole tablets (A/S Rosco Diagnostica) and 6-mm disks (Abtek Biologicals Ltd., Liverpool, United Kingdom) are available in Europe but are not yet available in the United States.The purpose of this study was to compare the Neo-Sensitabs tablet assay to both CLSI reference broth microdilution (document M27-A2) and disk diffusion (document M44-A) methods for testing susceptibilities of 10 to 20 isolates each of Candida (eight species) and Cryptococcus neoformans to five antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole). The evaluation included the following determinations: (i) determination of reference MICs of the five agents by CLSI broth microdilution method M27-A2, (ii) determination of inhibition zone diameters (in millimeters) by the reference M44-A disk diffusion method and by commercial Neo-Sensitabs tablet diffusion methods, (iii) determination of the correlation coefficient between inhibition zone diameters (in millimeters) and reference MICs of the five antifungal agents, and (iv) determination of the agreement by breakpoint category of disk and tablet inhibition zone diameters (in millimeters) with reference MICs of the five agents. MATERIALS AND METHODSIsolates. A total of 110 isolates of Candida spp. and Cryptococcus neoformans were evaluated by each method. The set of isolates included strains with different patterns of susceptibility to caspofungin and amphotericin B as well as fluconazole-, voriconazole-, and itraconazole-resistant, -susceptible-dose-dependent (S-DD), and -susceptible isolates ( (10,13). The CLSI quality control (QC) isolates Candida krusei ATCC 6258 and C. parapsilosis ATCC 22019 were tested each time a set of isolates was evaluated by the three procedures; MICs of each antifungal agent for both QC isolates were within the established MIC limits (6).CLSI broth microdilution procedure (document M27-A2).

Clinical Evaluation of the Sensititre YeastOne Colorimetric Antifungal Panel for Antifungal Susceptibility Testing of the Echinocandins Anidulafungin, Caspofungin, and Micafungin

“…These high-MIC strains are sufficiently rare that they have not been encountered with any frequency in clinical trials (15,17,22,25,26,33), although several isolates with echinocandin MICs of Ͼ2 g/ml have recently been associated with clinical resistance to echinocandin therapy in published case reports (2,5,6,11,13,14,16,18,20,23,27,28,34). These observations underscore the importance of antifungal susceptibility testing of echinocandins in detecting unusual resistance profiles, as these agents are used more broadly worldwide (32).The Clinical and Laboratory Standards Institute (CLSI) BMD method for the testing of caspofungin has served as the reference standard for the development of both broth-and agar-based procedures designed to provide simple, flexible, and commercially available susceptibility testing methods for use in the clinical laboratory (1,9,10,19). Among the commercially available BMD antifungal testing systems, only the Sensititre YeastOne system (Trek Diagnostic Systems, Cleveland, OH) offers an echinocandin (caspofungin) on a dried 96-well BMD panel (1, 9, 21).…”

“…The Clinical and Laboratory Standards Institute (CLSI) BMD method for the testing of caspofungin has served as the reference standard for the development of both broth-and agar-based procedures designed to provide simple, flexible, and commercially available susceptibility testing methods for use in the clinical laboratory (1,9,10,19). Among the commercially available BMD antifungal testing systems, only the Sensititre YeastOne system (Trek Diagnostic Systems, Cleveland, OH) offers an echinocandin (caspofungin) on a dried 96-well BMD panel (1,9,21).…”

Fungal Infections in Immunosuppressed Patients

“…• Nonculture Candida detection assays [18][19][20] • The Candida mannan assay yields a sensitivity of 31-90% (less for non-albicans Candida species).…”

“…• Antifungal susceptibility testing [18] • In vitro susceptibility testing for Candida species is now standardized using the Clinical Laboratory Standards Institute (CLSI) microbroth dilution (CLSI M27-A2, 2002) or the disk diffusion (CLSI M44-P, 2003) methodology. This was formerly known as the National Committee for Clinical Laboratory Standards (NCCLS) microbroth dilution.…”

Comparison between Disk Diffusion and Microdilution Methods for Determining Susceptibility of Clinical Fungal Isolates to Caspofungin

“…In addition, the DD assay does not generate an MIC of the drug but generates only a zone of inhibition. In the last few years, several investigators compared the results of triazole MICs for yeasts and filamentous fungi obtained by the BD and the DD methods (4,7,8,11,13,17). Few data are still available for echinocandin drugs.…”