Identification of infected corneal epithelial cells using an innovative in vivo fluorescent multilaser confocal microscope: Proofs of concept

Purpose To make the proof of concept of using fluorescent markers specific of certain bacteria or viruses (Herpes simplex virus, HSV) to make the aetiologic diagnosis of infectious keratitis directly at patient bedside, using fluorescent in vivo confocal microscopy (FIVCM) Methods We fully adapted for ophthalmology, the dermatology multilaser FIVCM vivascope 1500ML (Lucid Inc, NY, MAVIG GmbH, Germany) equipped with 488, 658, and 785nm class IB lasers. Human corneas unsuitable for graft were infected with either a specific bacteria species or HSV suspensions after mechanichal superficial central wound to mimick a superficial infectious keratitis. They were incubated with the suspension during 2h, rinsed, and returned to storage medium for 24h at 31°C. For controls, bacteria or viruses were replaced by balanced salt solution. Corneas were incubated with the corresponding fluorescent marker, one for HSV, the other for bacteria during 1h at 37°C. After meticulous rinsing, corneas were mounted on an anterior artificial chamber and observed with the FIVCM Results Control corneas showed little to no fluorescence, whereas numerous epithelial cells were fluorescent using the corresponding couple marker/wavelength Conclusion For the first time we obtained the POCs, on ex vivo human corneas, of specific diagnosis of bacteria or HSV keratitis using FIVCM. Animal experiments are ongoing in our laboratory on infectious keratitis models to confirm the feasibility and safety of this new promising diagnosis method. Grant: Appel a projet jeune chercheur GIRCI Rhone Alpes Auvergne 2012