Purpose: Cold storage is the standard for corneal preservation method in the U.S., but none of the three U.S. Food and Drug Administration-approved media contains antimycotics. The aim of this study to evaluate antimycotic activity of the first cold storage medium with dissolvable tablet contained Amphotericin B (Kerasave, ALCHIMIA, S.r.l., Italy)(pending FDA approval), and compare the effect of Kerasave versus Optisol-GS on endothelial cell density and corneal transparency.

Materials: Six not-suitable-for-transplant donor corneoscleral rims prepared at Lions Eye Institute for Transplant and Research, Tampa,USA were contaminated by aqueous suspension of Candida Albicans (clinical isolate) prepared to a value of 0.52 on the McFarland Scale of optical turbidimetry and incubated in Kerasave at 4°C. Two additional donor corneoscleral rims remained unaltered in Optisol-GS as control. Kerasave antifungal efficiency was performed by creation and following assessment of Sabouraud agar plates on days two (2), six (6), ten (10), fourteen (14), and sixteen(16). Endothelial cell density and corneal transparency were measured by specular microscope, slit lamp, back light microscopy and OCT after 3, 7 and 14 days of storage at 2-8°C.

Results: Six experimental specimens grew out an average colonies as follows: showed growth too numerous to count on Day 2 (from the Day 0 plating), eleven (11) colonies on Day 6 (Day 4 plating), three (3) colonies on Day 10 (Day 8 plating), and no growth from plating on Day 12 and Day 16. No significant difference in Kerasave vs Optisol-GS corneal cell density and corneal transparency after 3,7 and 14 days of storage.

Conclusion: Addition of Amphotericin B dissolvable tablet into cornea storage media demonstrates effective consistent decrease in fungal contamination compared to control and biocompatibility with donor corneas.

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.

Background: This study aimed at assessing the stability of new cold storage medium with antimycotic tablet, Kerasave (AL.CHI.MI.A. S.r.l.), in terms of shelf-life and stability under the conditions of use.

Methods: The Kerasave medium shelf-life was determined by monitoring the antimicrobials concentration by HPLC, the tablet integrity and dissolution time, and the medium transparency, pH, and osmolality  after storage at room temperature and at 4°C for 2 years.

Additionally, corneal cold storage was mimicked by using porcine corneas and monitoring the medium pH, osmolality and concentration of antimicrobial and antifungal agents after 14 days of corneal storage. The same media parameters were measured without adding the tissue.

Results:  The Kerasave parameters, including tablet integrity and dissolution time, medium transparency, pH and osmolality remained unchanged after 2 years of storage at room temperature and at 4°C. The antimicrobial concentrations remained within the acceptability range of values for 18 months and 24 months, when media were stored at room temperature and at 4°C, respectively.

Chemical-physical analysis of the media, after mimicking the corneal cold storage, indicated that the pH and osmolality remained unchanged while the antimycotic concentration decreased of approximately 10% after 14 days at 4°C.

In the absence of the cornea, all the measured parameters remained unchanged at 4°C for 14 days.

Conclusions: The stability studies allowed to assign a shelf-life of 2 years to the new cold storage medium with antimycotic tablet, Kerasave, when stored at 4°C. The medium exhibited excellent stability under the conditions of use.

Background: This study aimed at assessing the Candida spp. killing efficacy of the new cornea cold storage medium with antimycotic tablet, Kerasave (AL.CHI.MI.A. S.r.l.).

Material and methods: Kerasave antimycotic activity was determined by in vitro time-kill studies. In order to simulate tissue contamination, sterile porcine corneas were immersed in inoculum solutions respectively containing 10⁵ cfu/ml of 5 Candida spp. clinical isolates (C. albicans ATCC 10231, C. albicans ATCC 90028, C. albicans ATCC MYA-2876, C. tropicalis ATCC 750 and C. parapsilosis ATCC 90018) for 5 h at room temperature. The killing rate at 4°C was monitored by determination of residual contamination in tissue homogenates and storage media after 5 and 10 days of incubation. The samples were treated with RESEP for elimination of antimicrobials before plating by spread-plate technique. Killing rate of the media was evaluated at additional 1, 2 and 3 days time points for C. albicans ATCC 10231, C. albicans ATCC 90028 and C. tropicalis ATCC 750.

Results:  In vitro time-kill studies on cornea homogenates showed at least 3 log10  decrease after 5 days incubation at 4°C and 4 to 6 log10 reduction for all Candida strains was achieved within 10 days of incubation at 4°C.

The media showed almost maximal elimination of the contamination corresponding to 3.1 to 5.6 Log 10 already after 5 days of incubation at 4°C for all tested strains.

Conclusions: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent killing rate of all tested Candida clinical isolates in corneal tissue already after 5 days of tissue cold storage.

Purpose: This study aimed at assessing the antimycotic activity of the new cold storage medium, Kerasave, and at evaluating the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Material and Methods: Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10⁴ 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 a 3 to 4 log₁₀ reduction for both Candida strains within 10 days of incubation at 4°C.
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.

Conclusions: 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.

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.