Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation

ObjectiveTo study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with postkeratoplasty infections.Methods and AnalysisThe antimicrobial activity of Kerasave was determined after 0, 3 and 14 days of incubation at 2°C–8°C, inoculating Kerasave and the control medium with 105–106 colony forming units (CFU) of Candida albicans (CA), Fusarium solani (FS), Aspergillus brasiliensis (AB), Staphylococcus aureus (SA), Enterococcus faecalis (EF), Bacillus subtilis spizizenii (BS), Pseudomonas aeruginosa (PA), Enterobacter cloacae (EC) and Klebsiella pneumoniae (KP). Log10 reductions at different time intervals were determined by assessing the number of viable CFU using the serial dilution plating technique.ResultsAfter 3 days, Kerasave induced the highest log10 decrease in the concentrations of KP, PA, CA and EC (5.37, 4.15, 2.97 and 2.67, respectively; all p<0.001). The log10 decreases of SA and EF were 2.27 and 2.11, respectively (all p<0.001). The lowest log10 decrease was observed in BS, AB and FS concentrations (0.25, 0.30 and 0.67, respectively; p<0.001 for BS and AB and p=0.004 for FS). After 14 days, the microbial count of CA, FS, SA, EF, PA and EC further decreased (p=0.006 for FS; p<0.001 for the others).ConclusionKerasave effectively reduced or kept unchanged the microbial concentration of almost all tested strains after 3 days. Thus, this novel medium represents a valuable tool to control the microbial contamination of human donor corneas during hypothermic storage for up to 14 days before transplantation.

Possibility of application different cryopreservatives for tissue-engineered corneal constructions storage

Purpose. Conduct a comparative analysis of corneal tissue–engineered constructs that have undergone cryopreservation of three different cryoprotectants. Materials and methods. After refractive surgery Relex SMILE, corneal tissue was obtained which is called a lenticule. Corneal tissue engineering constructs (RTK) have created using decellularization methods that relate to the technologies of tissue and regenerative medicine. For decellularization of the lenticules, a protocol with a 1.5 M solution of Sodium Chloride with DNase 5 U / ml and RNase 5 U / ml was used. After decellularization, RTK was cryopreserved using: 1) 10% DMSO and 90% “corneal storage solution” (Borzenok–Moroz medium), 2) «Cryoderm» cell cryopreservation medium, 3) 100% glycerin. Transparency was assessed using spectrophotometry. The thickness of collagen fibers was assessed by scanning electron microscopy (SEM). Results. When assessing the transparency of the control and experimental groups, a statistically significant decrease in transparency was revealed in the groups using cryoprotectants – Glycerin and Cryoderm (p<0.001), the groups using DMSO did not differ from the control (p=0.99). Also, statistically significant differences were found between the experimental groups «Cryoderm»–DMSO (p=0.02), «Cryoderm»–Glycerin (p<0.001) and Glycerin–DMSO (p<0.001). When evaluating the SEM data, it was found that the thickness of collagen fibers did not differ in the DMSO–Control and Cryoderm–Control groups (p>0.05). The Glycerol–Control group was statistically different (p<0.001). Conclusions. In our work, we have shown the use of DMSO for storing RTK causes thickening of collagen fibers after decellularization of similar to native lenticules. This protocol can be useful for long–term storage and creation of the RTK cryobank. Key words: storage, lenticule, cryoprotectants, cryopreservation, cornea.

Fungal infection after keratoplasty and the role of antifungal supplementation to storage solution: a review

Fungal infection after corneal transplantation is a rare, yet potentially devastating, postoperative complication and has become a growing concern for the transplant surgeon and eye banking community. The Eye Bank Association of America (EBAA) has reported an increasing trend in the rate of postkeratoplasty fungal infections and a reversal in the previously documented predominance of bacterial over fungal infections. Additionally, several studies have confirmed a high correlation between positive corneoscleral donor rim fungal cultures and postoperative infections. Optisol GS (Bausch & Lomb, Irvine, California, USA), the most extensively used corneal storage solution in US eye banks, does not currently contain any antifungal supplementation. Although large randomised control trials evaluating the efficacy and safety of routine antifungal supplementation to corneal storage solution are lacking, several investigative studies have assessed the role of antifungal agents in reducing fungal contamination of donor corneas without causing undue corneal toxicity. This review will present the current epidemiology of postkeratoplasty fungal infections and evidence for obtaining routine fungal rim cultures and antifungal supplementation of storage solution.