Evaluation of the NCCLS M44-P Disk Diffusion Method for Determining Susceptibilities of 276 Clinical Isolates of Cryptococcus neoformans to Fluconazole

Abstract

We evaluated the NCCLS M44-P fluconazole disk diffusion method in comparison with the NCCLS M27-A2 broth microdilution method for determining the susceptibility of 276 isolates of Cryptococcus neoformans. Disk diffusion testing was performed using Mueller-Hinton agar supplemented with 2% glucose and 0.5 g of methylene blue/ml. Among the 276 isolates, 259 (93.8%) were susceptible, 16 (5.8%) were susceptible-dose dependent, and 1 (0.4%) was resistant to fluconazole as determined by the NCCLS broth microdilution method. The overall categorical agreement between the two methods was 86%, with 0% very major errors, 2% major errors, and 12% minor errors. The disk diffusion method using Mueller-Hinton agar supplemented with glucose and methylene blue appears to be a useful approach for determining the fluconazole susceptibility of C. neoformans.Cryptococcus neoformans remains an important cause of opportunistic mycosis in both immunocompromised and immune-intact individuals (8, 10, 12-14, 18, 19). Treatment of cryptococcal meningitis includes the administration of amphotericin B with or without flucytosine as first-line agents, often followed by fluconazole for maintenance or consolidation therapy (19,24). Given that in certain patient groups fluconazole therapy may be required for extended periods of time (24,25), there is a concern about the development of resistance to this agent (6, 7). Performance of antifungal susceptibility testing on the infecting isolate has recently been suggested as an aid in the management of these difficult patients (23).In vitro susceptibility testing of fluconazole may be performed using either broth-based or agar-based methods (1,11,20,26). Recently the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Testing has proposed an agar disk diffusion method, M44-P, for testing of fluconazole against yeasts (2,3,5,17). This method has been shown to be accurate and precise and correlates well with the NCCLS broth microdilution (BMD) MIC method when testing Candida (5, 21). Zone diameter interpretive criteria have been developed along with reference MIC correlates for the categories of susceptible (S) (19 mm [Յ8 g/ml]), susceptible-dose dependent (SDD) (15 to 18 mm [16 to 32 g/ml]), and resistant (R) (Յ14 mm [Ն64 g/ml]) (17). Although the NCCLS BMD method has been used to determine the susceptibility of C. neoformans to fluconazole (6,20,26), comparable studies of the NCCLS proposed disk diffusion method have not been published. As with Candida, disk diffusion testing may provide a faster, simpler method for determining the in vitro susceptibility of C. neoformans to fluconazole (5).The purpose of this study was to evaluate the M44-P disk test (17) for determining the in vitro susceptibility of C. neoformans to fluconazole. The disk diffusion zone diameters obtained for each isolate were compared to the MICs determined by the M27-A2 BMD method (16).A total of 276 clinical isolates of C. neoformans were obtained from 60 different medical centers worldwide dur...

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

“…The higher performance of MGM was similar to that reported previously for fluconazole disk testing (20). The interpretive agreement for Neo-Sensitabs was comparable to disk results in this study (95.5 to 96.4%) and previous fluconazole and voriconazole studies (86 to 99.4%) (12,(17)(18)(19). Both disk and tablet were able to identify the C. neoformans isolate that was S-DD to fluconazole (18-mm zone diameter) as previously reported (19).…”

“…and 48 and 72 h (C. neoformans). The correlation of the Neo-Sensitabs assay for fluconazole on MGM agar was similar or superior to that of the disk in our study (tablet R, 0.889; disk R, 0.8768) and previous studies (R, 0.6 to 0.7) (17,19). Results were slightly higher when RPMI agar, which has been a medium recommended by Rosco for Neo-Sensitabs (Neo-Sensitabs user's guide; A/S Rosco Diagnostica), was used.…”

“…MIC breakpoints have also been established for itraconazole, but guidelines for disk testing are not available. Although triazole MIC breakpoints have been established only for Candida spp., breakpoint category analysis has been applied to C. neoformans using fluconazole interpretive criteria (19). We have conducted similar analyses for the three triazoles including isolates of this species.…”

Cryptococcus neoformansMitochondrial Superoxide Dismutase: an Essential Link between Antioxidant Function and High-Temperature Growth

“…Disk diffusion assays were performed to assess the sensitivity of sod2⌬, sod2⌬ SOD2, and wild-type H99 strains to oxidative stress in vitro. The disk diffusion method has previously been used to assess antifungal drug susceptibility for C. neoformans strains (32). All strains were grown in YPD medium at 25°C overnight with shaking and washed with sterile phosphatebuffered saline, and the concentration of cells was adjusted to an optical density at 600 nm of 0.2.…”

Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: 10.5-Year Analysis of Susceptibilities of Noncandidal Yeast Species to Fluconazole and Voriconazole Determined by CLSI Standardized Disk Diffusion Testing

“…Although there is no standardized method for testing most of these fungi, the vast majority grow well on the supplemented Mueller-Hinton agar plates used in the study, and the zone diameters are easily determined (47,56). For the purposes of the study, we utilized the interpretive breakpoints for fluconazole and voriconazole that have been established for Candida (52, 53), and we recognize that they may need to be adjusted for noncandidal yeasts in the future.…”