Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model

PURPOSE. Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas. METHODS. Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated. RESULTS. The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group. CONCLUSIONS. A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin-induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.