title: Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: Jawaher M
authors.initials: JM
authors.lastname: Alshehri
authors.affiliation: Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Caballero-Lima
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: M Chantal
authors.initials: MC
authors.lastname: Hillarby
authors.affiliation: School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: Susan G
authors.initials: SG
authors.lastname: Shawcross
authors.affiliation: Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom.
authors.forename: Arun
authors.initials: A
authors.lastname: Brahma
authors.affiliation: Manchester Royal Eye Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom.
authors.forename: Fiona
authors.initials: F
authors.lastname: Carley
authors.affiliation: Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: Nick D
authors.initials: ND
authors.lastname: Read
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom.
authors.forename: Hema
authors.initials: H
authors.lastname: Radhakrishnan
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002102:Cadaver; D003315:Cornea / Q000187:drug effects / Q000473:pathology*; D003432:Cross-Linking Reagents / Q000627:therapeutic use*; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000188:drug therapy* / Q000382:microbiology; D005260:Female; D060585:Fusariosis / Q000175:diagnosis / Q000188:drug therapy* / Q000382:microbiology; D005670:Fusarium / Q000302:isolation & purification; D006801:Humans; D007634:Keratitis / Q000175:diagnosis / Q000188:drug therapy* / Q000382:microbiology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D008875:Middle Aged; D010789:Phototherapy / Q000379:methods; D014019:Tissue Donors; D020858:Tissue and Organ Harvesting / Q000379:methods*; D016896:Treatment Outcome
chemical_list: D003432:Cross-Linking Reagents