1. Correlation of Neo-Sensitabs on Three Media with NCCLS Reference Disk Diffusion and Broth Microdilution Methods for Testing the Susceptibility of Candida spp. and Cryptococcus neoformans to Voriconazole and Fluconazole A. Espinel-Ingroff, E. Cantón, D. Gibbs and A. Wang. VCU Medical Center, USA; Unidad de Microbiologia Experimental, Centro de Investigación, Hospital Universitario La Fe, Valencia, Spain; Giles Scientific Inc., Santa Barbara, CA, USA Introduction Neo-Sensitabs tablets (Rosco Diagnostica, Taastrup, Denmark) of fluconazole, amphotericin B, voriconazole and caspofungin are commercially available for testing yeasts by an agar diffusion assay. The correlation between antifungal susceptibility results obtained using Rosco Neo-Sensitabs and both NCCLS M-44 and M27-A2 methods has not been evaluated with the new 1 μg voriconazole and the 25 μg fluconazole tablets. Purpose To assess the correlation between both NCCLS methods for yeasts and Rosco results of fluconazole and voriconazole for each isolate evaluated. Study Design Each isolate was tested by each method (NCCLS M27-A2 broth microdilution method, NCCLS M44-A disk diffusion agar, and Rosco Neo-Sensitabs diffusion agar) with the two triazoles, fluconazole and voriconazole. Materials and Methods Isolates: 10 to 20 isolates/per specie of Candida albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Cryptococus neoformans and NCCLS QC isolates C. krusei ATCC 6258, C. parapsilosis 22019, C. albicans ATCC 90028 and C. tropicalis ATCC 750. QC isolates were included each time isolates were evaluated by each method with each drug. B. M44-A disk diffusion method 1. The same stock inoculum suspension as for the M27-A2 method. 2. Each Mueller-Hinton agar plate with 2% dextrose and 0.5 μg/ml of methylene blue (Remel) was inoculated as described in the M44-A method. 3. Zone diameters were measured to the nearest whole mm at the point in which there is a prominent reduction of growth after 20 to 24 h (Candida spp.) and 48 to 72 h (C. neoformans) of incubation. Note: Voriconazole and fluconazole disks were supplied by Pfizer. C. Neo-sensitabs tablet method This method was performed according to the manufacturer’s instructions using three media (RPMI-1640 with 2% dextrose [Remel], modified Shadomy [E & O Laboratories] and Mueller-Hinton [with 2% dextrose and 0.5 μg/ml of methylene blue [Remel]) agars and 9 mm tablets (25 μg fluconazole and 1 μg voriconazole). Zone diameters were measured to the nearest whole mm at the point in which there is a prominent reduction of growth after 20 to 24 h (Candida spp.) and 48 to 72 h (C. neoformans) of incubation. Note: Neo-sensitabs tablets were supplied by Rosco, Denmark. A. NCCLS broth microdilution M27-A2 method 1. Each isolate, including QC isolates, was grown on Sabouraud dextrose agar at 35 o C for 24 (Candida spp.) and 48 h (C. neoformans). 2. Each yeast suspension was adjusted spectrophotometrically (530 nm wavelength) to the OD of a 0.5 Mcfarland standard. MIC determination NCCLS MICs were determined after 48 h (Candida spp.) and 72 h (C. neoformans) of incubation as described in M27-A2 document.. Results The performance of Neo-sensitabs method (24h for Candida spp. and 48h for C. neoformans) was similar to that of NCCLS M44-A disk diffusion method as demonstrated by the comparison of zone diameters and M27-A2 MIC values (Above Figures provide regresion statistic R values for fluconazole and voriconazole by each method. The overall categorical agreement (>90%) was also similar to that obtained between M44-A zone diameters and M27-A2 MICs. Both fluconazole and voriconazole tablets were able to identify fluconazole-resistant strains (MICs >64 µg/mL) as well as those with decreased susceptibility to voriconazole (MICs >2 µg/mL). Conclusions Neo-sensitabs method had suitable and similar performance on the three media with both antifungal agents. Therefore, Neo-sensitabs tablet method is a potential alternative to the M44-A method for testing the susceptibility of yeasts to fluconazole and voriconazole using the standard Mueller-Hinton agar with 2% dextrose and 0.5 μg/ml of methylene blue. Corresponding author: A. Espinel-Ingroff E-mail: avingroff@hsc.vcu.edu 15 th ECCMID Fig. 1. Neo-sensitabs tablet zone diameters as determined on three media