Live 4D monitoring of fungal infection in an ex vivo model of fungal keratitis reveals enhanced efficacy of combined PACK-CXL and natamycin treatment

Fungal keratitis (FK) is a major cause of visual impairment worldwide, yet the development and evaluation of new therapeutic approaches are limited by experimental models that inadequately reproduce deep stromal infection or permit longitudinal quantification of fungal burden. Here, we developed an ex vivo porcine corneal model of Aspergillus fumigatus keratitis that supports reproducible fungal invasion into the deeper corneal stroma and enables non-destructive monitoring of infection over time. Optimisation of the culture conditions demonstrated that reducing nutrient availability markedly promoted stromal penetration compared with nutrient rich conditions. Using a YFP expressing A. fumigatus strain combined with confocal microscopy and three-dimensional image analysis, fungal burden could be quantified repeatedly within individual corneas, allowing changes in established infection to be monitored longitudinally. We applied the model to evaluate natamycin and photoactivated chromophore for keratitis corneal cross-linking (PACK-CXL), alone and in combination. Both monotherapies significantly restricted the increase in fungal burden observed in untreated corneas, but neither consistently reduced pre-existing fungal burden. In contrast, combined natamycin and PACK-CXL treatment reduced fungal burden in all treated corneas and was significantly more effective than either treatment alone. Together, these findings establish a quantitative ex vivo platform for investigating invasive A. fumigatus corneal infection and demonstrate its ability to distinguish between treatments that restrict fungal expansion and those that reduce established fungal burden. The model provides a useful approach for the longitudinal evaluation and optimisation of therapeutic interventions for fungal keratitis.