Study Of Experimental Fusarium And Aspergillus Keratitis In Rabbit Model

Fungal keratitis is one of severe suppurative corneal infection. This entity accounts for 30~65% of all cases of culture-proven microbial keratitis, and is being increasingly recognized as an important cause of blindness because fungal keratitis can lead to corneal opacity, stromal melting or corneal perforation.In the previous study, we developed a new slide culture system for identifying the fungal species which were isolated from patients with fungal keratitis. Among 61 fungal isolates, fusarium(n=33, 54.1%) and aspergillus(n=9, 14.8%) are the two most common genera causing corneal infection. The result is close to other studies from India and Southern United States.Although it is well known that the typical corneal infiltrate of fungal keratitis is gray-white opacity with dry rough texture and irregular feathery margin, in clinical practice, the diversity of eye presentation can be observed in each case, especially between fusarium and aspergillus keratitis. In most cases of early stage of fusarium keratitis, the typical corneal infiltrate features are usually observed. But in aspergillus keratitis, the corneal infiltrate commonly presented as uniform density without feathery border. However, in later stage of fungal keratitis, the eye symptoms may progress to similar, such as large amount of necrotic slough, corneal abscess or deep ulcer, even corneal perforation could be found in bothfusarium and aspergillus keratitis.In this study, we focused on producing an experimental fusarium and aspergillus keratitis in rabbit model to clarify the natural course of fungal keratitis and its clinical and pathological features, and to elucidate the differences of clinical and pathological features of this two most common filamentous fungal keratitis.MethodsNewzeland white rabbits of either sex weighing 2 to 2.5 kg were used for animal model. Two wild-type strains of Fusarium.solani and Aspergillus.fumigatus in this study were isolated from two clinical cases of fungal keratitis through corneal scraping. The species identification was made by combining the macroscopic with microscopic criteria. Both strains that were cultured previously on Sabouraud's dextrose agar plate for 4~7 days were harvested in sterile saline with removal of hyphaes. The homogenate was quantified under microscopy using cell counter and adjusted the density to 0.5~1.0×10~7 CFU/ml. An inoculums of 100- μl, containing approximately 0.5~ 1.0×10~6 conidia were injected into the anterior and middle of corneal stroma using 27 gauge needle through three points around the pupil. Animals that served as negative control were mock inoculated with same volume sterile saline. After inoculation, all eyes were treated with 0.1% dexamethasone and 0.3% tobramycin ointment one time a day. The duration of topical steroids using is 10 days in all animals which were inoculated with Fusarium.solani, Aspergillus.fumigatus and negative controls.All rabbits were scored daily and photographed every two days for corneal involvements including corneal infiltrate, corneal opacity and edema, corneal ulceration, and inflammation reaction of the eye after infection such as conjunctival injections and hypopyon. The severity of keratomycosis in rabbits was visually scored with slit lamps and photographs. Groups of rabbits were killed and eyeballs were enucleated 3, 5, 10, 15, 20, 25, 30, 35 days after inoculation. The infected corneas were removed at the limbus and bisected longitudinallythrough the focus of corneal infiltrate. One half was processed for histopathological examination; the other was cut into pieces for fungal culture. Hematoxylin-erosin and periodic acid-Schiff staining were applied in histological testing to evaluate inflammations cell infiltration, corneal structural destruction and hyphal invasion. Lastly, we compared the clinical and histopathological findings of Fusarium.solani and Aspergillus.fumigatus keratitis.ResultsAll rabbits that were infected with Fusarium.solani and Aspergillus.fumigatus progressed to severe keratomycosis and persisted for more than 1 mo...