Abstract
Purpose:
Keratomycosis (KM) refers to an infective process associated to outdoor occupations and contact lenses wear. The main agents triggering KM (Fusarium, Aspergillus and Candida) can be found in the air, soil, dust and plants.Without treatment, the inflammation may lead to corneal scarring, anterior chamber infection, perforation, and vision loss. The goal of this work was to asses if Fusarium solani, Aspergillus fumigatus and Candida albicans isolated from patients with KM are able per-se to induce corneal epithelial cell necrosis, cytotoxicity or to trigger programmed corneal cell death (apoptosis).
Methods:
Fungal suspensions were introduced in microwells containing phenotypically-characterized human corneal epithelial cells.Non-viable Fungi served as negative controls.Viability was measured using the AFC substrate, which penetrates in cells with intact membranes and active metabolism. Fungal cytotoxicity was assessed following the incorporation by dead cells of the ApoTix-Glo TM luminescent reactant. Necrosis was confirmed with bis-AAF-R110,which enters through non-viable cell-pores and releases a fluorophore.Apoptosis was studied by activation of the cascade Caspase Glo 3/7 and cleavage of the luciferine labelled Z-DEVD substrate.
Results:
Fusarium solani directly reduced corneal cell viability after 1 hour of contact with human cells. After 2 hours, viability reduction induced by Fusarium solani was 52%; Candida albicans 55% and Aspergillus fumigatus 43%. Significant fungal induced corneal cell-necrosis rates were confirmed after 2h (Candida albicans: 20%; Aspergillus fumigatus: 40% and Fusarium solani: 60%). Aspergillus fumigatus, Candida albicans and Fusarium solani induced detectable levels of corneal-cell apoptosis after 1 h. Apoptotic rates were significantly higher for all the species while comparing 1 h with 2 h of fungal-cell contact.
Conclusions:
The results of this study show for the first time that viable Fungi isolated from patients with KM induce human corneal epithelial-cell necrosis and apoptosis. In addition to the classic anti-fungal and inflammatory agents, it appears necessary that the cytotoxic effects directly induced by Fungi should be integrated in the future development of therapeutic strategies to improve visual prognosis of people with KM.
Keywords: 530 fungal disease •
573 keratitis •
765 wound healing