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

Collagen Cross-Linking with Riboflavin for Keratoconus & Post LASIK Ectasia

• CXL using riboflavin & UV-A light is minimally invasive

• CXL stabilizes post LASIK & ectasia

• CXL safe for the corneal endothelium if thickness > 400 micron.*

Wollensak et al. Eye 2004;18:718-22* 

Collagen-crosslinking

UV-A Irradiation: Antimicrobial Effect

• 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

• 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

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.

Methods• Prospective comparative study• Pathogens tested:

Pseudomonas aeruginosa (PA) Streptococcus pyogenes (SP) Methicillin-sensitive Staphylococcus aureus

(SA) Candida albicance (CA)

• 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)

Preparation of Bacteria & Innoculation of Agar Plate

• PA & SP selected from human clinical isolates• SA culture of freeze-dried microorganisms

used for quality control• SA being freeze-dried subjected to two sub-

cultures before testing• End point: McFarland turbidity =/> 0.5• Sterile cotton swab dipped into innoculum &

excess of innoculum removed• Agar plate swabed three times & even

distribution ensured.

•

UV-A Exposure• Standard innoculum of microorganisms on culture

media.• 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.

• Diffusion of drop into agar media for 20 minutes allowed

• Irradiance was checked with a calibrated UV meter• UV-A (370 nm), 3 mW/cm2, 30 minutes• For group 2 Moxifloxacin alone and group 3 after

UVA / R was put on the centre of the circle

Results• Culture plates were

incubated for 48 hrs at 34oC to 35oC in an ambient air incubator

• 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

GIZ : Pseudomonas aeruginosa

GIZ: Staphylococcus aureus

PA SP SA CA0

5

10

15

20

25

30

35

40

12.159.54

6.5

0

32.92

21.6220

0

34.31

24.92

23.25

0

UVA/R Moxifloxacin

Combined UVA/R &AM

Result: Growth Inhibition Zone( GIZ)

For PA, SP & SA Moxifloxacin; For CA Voriconazole was used.

Coments• 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• UVA/Riboflavin enhances the antimicrobial effect

of Moxifloxacin• UVA/Riboflavin has potential of treating corneal

infections• 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