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Farhad Hafezi, Zisis Gatzioufas, Arthur Hammer, David Tabibian, Olivier Richoz; Fluorescein Competes Riboflavin in CXL (Corneal Collagen Cross-Linking) for the Treatment of Infectious Keratitis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5268.
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Infectious keratitis is a potentially sight-threatening condition which may cause severe visual loss. Antimicrobial treatment does not always guarantee rapid and complete healing, since the development of resistance to antimicrobial agents and misdiagnosis of fungal keratitis contribute to an increased rate of refractory keratitis. Recently, the antimicrobial properties of CXL against common pathogens in vitro, and CXL has been successfully used in the treatment of refractory infectious keratitis with corneal melting. In the present report we provide evidence that fluorescein competes riboflavin in UV-A absorption during CXL.
We analyzed the UVA-absorption of riboflavin and fluorescein by spectrophotometry (Lightwave S2000, Biochrom WPA, Cambridge, UK). The following concentrations were tested: 0.1%, 0.01%, 0.001% for riboflavin and 10%, 1%, 0.1%, 0.01% for fluorescein. NRS1 strains ‘Network on Antimicrobial Resistance in Staphylococcus aureus’ were used in all experiments. The number of colony-forming units was evaluated after a 24 h incubation period at 37°C. For each experiment, the difference between the S. aureus killing rate with UV-A (UVA+) and without UV-A-irradiation (UVA-) were evaluated. The effective killing-rate was determined using the formula [TKL= (killing-rate UVA+) - (killing-rate UVA-) = Δ killing rate].
Absorption analysis revealed that 0.1% riboflavin displayed a 100% absorption capacity for UV-A, while absorption decreased to approximately 45% for 0.01% riboflavin. Fluorescein demonstrated a UV-A absorption rate of 10% for 0.01% fluorescein and 100% for 1% fluorescein. When analyzing the killing-rate at 1 minute, the highest value (82%) was measured in the suspension containing riboflavin only (no fluorescein), wheares the lowest value (19%) was obtained in the suspension containing riboflavin and 1% fluorescein.
Fluorescein absorbs UV-A in a significant and concentration-depending manner. The addition of fluorescein led to a significant reduction in the killing-rate of S. aureus in vitro. Our results indicate that fluorescein competes riboflavin for the absorption of UV-A during CXL, limiting the therapeutic effect. In consequence, preoperative staining of the ulcus or infiltrate at the slit-lamp using fluorescein must be avoided prior to CXL, since it may significantly reduce the therapeutic effect of CXL.
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