Effect of Riboflavin/Rose Bengal-Mediated PACK-CXL on Acanthamoeba Trophozoites and Cysts in Vitro.

PURPOSE: To evaluate the antiamoebic properties of photo-activated chromophore for keratitis (PACK)-corneal cross-linking (CXL) (PACK-CXL), in combination with riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%), for treatment of Acanthamoeba trophozoites and cysts. MATERIALS AND METHODS: Cultures of Acanthamoeba castellanii were grown in a fluid medium at a concentration of 2.7 × 105 cell/ml. PACK-CXL was used on A. castellani cells in combination with either riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%). Riboflavin-containing wells were irradiated with ultraviolet-A (UVA) light (365-nm wavelength). Rose bengal-containing wells were irradiated with green light (523-nm wavelength). A power density of 9 mW/cm2 for 10 min and total irradiation dose of 5.4 J/cm2 was used for both riboflavin and rose bengal. After UVA and green light irradiation, cell viabilities were evaluated, and percentage of dead cells calculated. RESULTS: No significant amoebicidal activity was observed following PACK-CXL/riboflavin at either concentration. PACK-CXL/rose bengal, however, was observed to be highly effective in eradicating Acanthamoeba cells at either concentration, with no significant difference observed between the two concentrations. The percentage of dead cells was 63% following treatment at either rose bengal concentration. CONCLUSION: PACK-CXL with rose bengal demonstrated pronounced antiamoebic activity against A.castellanii. Further in vitro and in vivo studies are needed to confirm this finding.

Evaluation of Antifungal Efficacy of 0.1% and 0.25% Riboflavin with UVA: A Comparative In Vitro Study.

PURPOSE: Antifungal efficacy of photochemical cross-linking (PACK-CXL) with 0.1% and 0.25% riboflavin was evaluated with a comparative in vitro study. METHODS: Candida albicans and Aspergillus fumigatus ATCC reference strains, Candida parapsilosis, Aspergillus fumigatus, Fusarium solani, Scedosporium apiospermum, and Alternaria alternata strains isolated from keratitis cases were chosen as targeted microorganisms. Unique "black plate method" was developed in polystyrene microplates. Riboflavin suspensions in 0.1% and 0.25% were separately added into inoculated wells. Non-inoculated wells were filled by black colored dye in order to protect treated wells from reflection of UV treatment. After ultraviolet A (UVA) treatment, each well was evaluated by microbiological culture in order to count viable fungal colonies. Fungal killing rate was calculated by comparing fungal counts (CFU/mL) before and after UVA application of riboflavin-added wells. RESULTS: Four different fungal inoculum concentrations of targeted microorganisms, including 104, 103, 102, and 101 CFU/mL, were assayed. PACK-CXL with 0.25% riboflavin was found to be highly effective on fungal cells even in 104 CFU/mL of concentration. CONCLUSIONS: PACK-CXL appears as a promising treatment option for difficult-to-treat cases of fungal keratitis and 0.25% riboflavin concentration increases fungicidal effect of the procedure dramatically.