Recently, the photodynamic therapy (PDT) has been researched as an alternative tool to photoeradicate bacteria, protozoa, viruses, and fungal infections. It uses a combination of a photosensitive substance activated by a light source in the presence of oxygen. The resulting reaction induces cell death due to the production of toxic oxygen species, such as singlet oxygen and/or free radicals. Objective: To evaluate the effectiveness of methylene blue and light-emitting diode (LED) against Cryptococcus neoformans and C. gattii resistant to antifungal drugs.Methods: A total of 15 (9 clinical and 6 environmental) C. neoformans and two clinical C. gattii isolates resistant to conventional antifungal drugs were selected. Of 9 clinical strains of C. neoformans, 4 (44.4%), 7 (77.7%) and 2 (22.2%) exhibited resistant to amphtotericin B (AMB), itraconazole (ITZ) and fluconazole (FLZ), respectively, whereas 2 (33.3%) and 3 (50%) environmental isolates exhibited resistant to ITZ and FLZ, respectively. The C. gattii clinical isolates were resistant to FLZ, 50% to ITZ and 50% to AMB. The light source to evaluate the PDT was a LED emitting at λ660nm with output power of 6.2 mW/cm2 for 15, 30 and 45 min of irradiation, resulting fluences at 5.6, 11.2 and 16.9 J/cm2. The methylene blue (MB) was used as photosensitizer at 50 µM, 250 µM e 500 µM. A mixture of MB and fungal suspension (106 CFU ml−1) was stored in a dark room at 25 ºC for 10 min prior to laser irradiation. All PDT were repeated in triplicate. The CFU counts were made up to 96 h after PDT. The susceptibility to PDT and antifungal drugs were analyzed by nonparametric Friedman and Wilcoxon tests, P value equal to 0.01. Results: The LED or MB alone did not reduce cell viability of strains evaluated. LED energy density of 16.9 J cm-2 together with 250 µM of MB significantly reduced the survival rate of clinical C. neoformans isolates in 3.1 log10 CFU ml-1 (p ≤ 0.01). Clinical C. neoformans isolates were more susceptible to PDT than the environmental ones without statistical significance. Light doses of 16.9 J cm-2 killed both clinical and environmental C. neoformans strains at 250 µM of MB, but only the strains susceptible to AMB showed statistical significance (p ≤ 0.05). The C. gattii strains resistant to FLZ exhibited more tolerance to PDT than the C. neoformans (p = 0.05). Conclusion: Herein, it was observed the photodynamic activity of MB in combination with LED irradiation decreasing the cryptococcal viability of both, clinical and environmental isolates resistant to several antifungal drugs. In vivo tests must be performed to verify the viability of MB and LED as potential therapeutic to inhibit cryptococcal cellular growth in infected sites.

Action of methylene blue photodynamic therapy on Cryptococcus spp. resistant to conventional antifungals