Efficacy of varying Amphotericin-B concentrations against Candida albicans after short exposure times in cold storage

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.