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The Neo-Sensitabs diffusion method was evaluated for determining the antifungal susceptibilities of 30 Cryptococcus gattii isolates to amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC) and voriconazole (VRC). Three different culture media, Müeller-Hinton (MH), RPMI 1640 (RPMI) and Antibiotic medium 3 (AM3), all supplemented with 2% of glucose and 0.5 microg/ml of methylene blue, were tested. The tests were repeated three times on different days at three incubation times (48, 72 and 96 h). Results were compared with those obtained with the CLSI M27-A2 broth microdilution method. The degree of reproducibility of the diffusion test was 100% for VRC and ITC, 98.3-100% for AMB and 43.3-73.3% for FLC. The best reproducibility was observed at 48 h of incubation and no important differences among media were observed at any of the incubation times assayed. Between Neo-Sensitabs and the reference method, VRC showed the best agreement and ITC the worst in all conditions tested (100% and 56.7%, respectively). AMB showed a high agreement between the two methods (93.3% to 96.7%) but Neo-Sensitabs assay failed to detect resistant isolates (discrepancy classified as "very major error") in all times of incubation assayed. Only agreement between both methods for FLC was clearly affected by incubation time and media used, the best results being achieved at 48 h of incubation when MH and RPMI (80.0%, in both media) were used.