1. Evaluation of Bactericidal and Fungicidal Activity of Riboflavin Plus UVA Irradiation for Corneal CXL Authors Ashok Sharma, Cornea Centre, Chandigarh, India Verinder S Nirankari, Eye Consultants of Maryland Author do not have any financial interest in the surgical procedure or the medicines used in this study

2. Collagen Cross-Linking with Riboflavin for Keratoconus & Post LASIK Ectasia CXL using riboflavin & UV-A light is minimally invasive CXL using riboflavin & UV-A light is minimally invasive CXL stabilizes post LASIK & ectasia CXL stabilizes post LASIK & ectasia CXL safe for the corneal endothelium if thickness > 400 micron.* CXL safe for the corneal endothelium if thickness > 400 micron.* Wollensak et al. Eye 2004;18:718-22* Wollensak et al. Eye 2004;18:718-22* Collagen-crosslinking

3. UV-A Irradiation: Antimicrobial Effect Photoactivation of riboflavin by using UV radiation causes microbial inactivation. Photoactivation of riboflavin by using UV radiation causes microbial inactivation. UV radiation in presence of riboflavin has been reported to inactivate a wide spectrum of organisms including viruses, fungi, bacteria, and parasites UV radiation in presence of riboflavin has been reported to inactivate a wide spectrum of organisms including viruses, fungi, bacteria, and parasites Photosenstizer releases triplet reactive oxygen species mainly single O2 Photosenstizer releases triplet reactive oxygen species mainly single O2 Martins et al. Antimcrobial efficacy of Riboflavin/ UV-A … for Bacterial and Fungal Isolates…. Infectious Keratitis. IOVS 2008;49:3402-3408 Martins et al. Antimcrobial efficacy of Riboflavin/ UV-A … for Bacterial and Fungal Isolates…. Infectious Keratitis. IOVS 2008;49:3402-3408

4. Purpose To evaluate antimicrobial effect of UV-A radiation / Riboflavin, Moxifloxacin alone and combined UV-A radiation / Riboflavin on Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. To evaluate antimicrobial effect of UV-A radiation / Riboflavin, Moxifloxacin alone and combined UV-A radiation / Riboflavin on Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus.

5. Methods Prospective comparative study Prospective comparative study Pathogens tested: Pathogens tested:  Pseudomonas aeruginosa (PA)  Streptococcus pyogenes (SP)  Methicillin-sensitive Staphylococcus aureus (SA)  Candida albicance (CA) Three treatment groups: Three treatment groups:  Group 1: UVA / R  Group 2: Antimicrobial (AM) alone  Group 3: Combined AM & UVA / R (AM for CA Voriconazole and Moxifloxacin for the rest)

6. Preparation of Bacteria & Innoculation of Agar Plate PA & SP selected from human clinical isolates PA & SP selected from human clinical isolates SA culture of freeze-dried microorganisms used for quality control SA culture of freeze-dried microorganisms used for quality control SA being freeze-dried subjected to two sub- cultures before testing SA being freeze-dried subjected to two sub- cultures before testing End point: McFarland turbidity =/> 0.5 End point: McFarland turbidity =/> 0.5 Sterile cotton swab dipped into innoculum & excess of innoculum removed Sterile cotton swab dipped into innoculum & excess of innoculum removed Agar plate swabed three times & even distribution ensured. Agar plate swabed three times & even distribution ensured.

7. UV-A Exposure Standard innoculum of microorganisms on culture media. Standard innoculum of microorganisms on culture media. A circle of 25 mm diameter was drawn on the plate and centre was marked. A circle of 25 mm diameter was drawn on the plate and centre was marked. A drop of isotonic riboflavin 0.1% on the inoculated media corresponding to the centre of the circle in treatment groups 1 & 3 for various micro organisms. A drop of isotonic riboflavin 0.1% on the inoculated media corresponding to the centre of the circle in treatment groups 1 & 3 for various micro organisms. Diffusion of drop into agar media for 20 minutes allowed Diffusion of drop into agar media for 20 minutes allowed Irradiance was checked with a calibrated UV meter Irradiance was checked with a calibrated UV meter UV-A (370 nm), 3 mW/cm 2, 30 minutes UV-A (370 nm), 3 mW/cm 2, 30 minutes For group 2 Moxifloxacin alone and group 3 after UVA / R was put on the centre of the circle For group 2 Moxifloxacin alone and group 3 after UVA / R was put on the centre of the circle

8. Results Culture plates were incubated for 48 hrs at 34 o C to 35 o C in an ambient air incubator Culture plates were incubated for 48 hrs at 34 o C to 35 o C in an ambient air incubator Mean growth inhibition zone( GIZ) diameter was measured to the nearest whole mm at 24 & 48 hrs. Mean growth inhibition zone( GIZ) diameter was measured to the nearest whole mm at 24 & 48 hrs. ANOVA test was used for comparison of groups ANOVA test was used for comparison of groups GIZ : Pseudomonas aeruginosa GIZ: Staphylococcus aureus

9. Result: Growth Inhibition Zone( GIZ) For PA, SP & SA Moxifloxacin; For CA Voriconazole was used.

10. Coments The effect of UV-A / Riboflavin with prior or concomitant use of antimicrobial agents evaluated The effect of UV-A / Riboflavin with prior or concomitant use of antimicrobial agents evaluated Study shows in vitro efficacy of UV-A / Riboflavin against PA, SP & SA Study shows in vitro efficacy of UV-A / Riboflavin against PA, SP & SA UVA/Riboflavin enhances the antimicrobial effect of Moxifloxacin UVA/Riboflavin enhances the antimicrobial effect of Moxifloxacin UVA/Riboflavin has potential of treating corneal infections UVA/Riboflavin has potential of treating corneal infections Tissue culture models & animal studies are needed to evaluate efficacy of this treatment for infective keratitis Tissue culture models & animal studies are needed to evaluate efficacy of this treatment for infective keratitis The possibility of toxicity to the cornea or other ocular structures need further evaluation The possibility of toxicity to the cornea or other ocular structures need further evaluation