Purpose: To determine the efficacy and safety of different Amphotericin-B (AmphoB) concentrations during short exposure times against Candida albicans in a cold storage media.

Method: AmphoB was added to a final concentration of 0.255 µg/mL, 1.25 µg/ml, 2.50 µg/mL and 5.0 µg/mL in a DMEM-based hypothermic storage media. AmphoB containing media and control samples (tryptic soy broth with no AmphoB) were inoculated with 10⁵ CFU/ml of C. albicans (ATCC10231) and stored at 4°C for 72 hours (triplicate cultures). The number of living microorganisms in each sample was determined initially and after 6, 24, 48, and 72 hours of storage at 4°C. AmphoB was neutralized before plating on agar plates by dilution and spread plate technique. Cell viability of cornea endothelium were also examined after 72 hours of exposure using Calcein-AM staining and FIJI segmentation.

Results: AmphoB concentrations of 1.25 µg/ml, 2.5 µg/ml and 5.0 µg/ml resulted in 0.77, 1.45 and 2.15 log10 reduction after only 6 hours of storage at 4°C, and continued to decrease to 3.63, 3.98 and 4.35 log10 reductions after 72 h (>99.9%), respectively. In contrast, AmphoB at 0.255 µg/ml showed only a 0.73 log10 decrease after 48h of incubation at 4°C. CFU counts of C. albicans were significantly higher in control samples. Endothelial cell viability was not different in donor corneas exposed to AmphoB (≤ 2.5 mg/mL) for 72h compared to non-exposed controls (P=0.52).

Conclusion: Current use of AmphoB at 0.255 mg/mL is not sufficient for C. albicans suppression. Optimal efficacy of AmphoB against C. albicans is achieved in cold storage conditions at concentrations above 1.25 µg/ml and exposure time of 24-48 hours.

Purpose: Cold corneal storage media available on the market do not contain antimycotics. A new corneal storage medium with an antimycotic tablet (Kerasave, AL.CHI.MI.A. S.r.l.) was developed to prevent fungal contamination of donor corneas intended for transplantation. The aim of this study was to assess the antimycotic activity of the new cold storage medium, Kerasave, and to evaluate the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Method(s): Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10 cfu/ml of C. Albicans (ATCC10231 and clinical isolate). The killing rate of the microorganisms was monitored at 4°C after 5 and 10 days of incubation in Kerasave.
Kerasave performance was assessed on 16 pairs of human corneas not suitable for transplantation, procured and evaluated according to standard procedures of Monza Eye Bank, Italy. One cornea was transferred in Kerasave and the contralateral in Optisol GS. Endothelial cell density (ECD), measured by specular microscopy (Keratoanalyzer, Konan), was evaluated pre-processing, and after 7 and 14 days of storage at 4°C. Endothelial cell morphology and mortality were determined according to Stocker method, and epithelial integrity, and corneal transparency were evaluated using a Slit lamp.

Results: In vitro time-kill studies showed 3.6 log reduction for both Candida strains after 5 days of incubation at 4°C and 4 log reduction was observed for both strains after 10 days of incubation.
Kerasave -and Optisol-GS- treated tissues showed similar ECD, mortality and endothelial morphology after 7 and 14 days of cold storage. Slit lamp analysis showed comparable corneal transparency and epithelial integrity in both groups.

Conclusion: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent antimycotic activity and biocompatibility with donor corneas after corneal storage at 4°C for up to 14 days.