Ocular Microbiology and Immunology Group
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2025 OMIG Abstract

Photochemical Interactions Between Rose Bengal Photodynamic Therapy and Topical Antifungals Against Filamentous Fungal Keratitis Isolates

Brandon Chou¹, Katherine Krishna¹, Leonardo Gonzalez¹, Heather Durkee¹, Suraj Paudyal^1,2,
Anam Ahmed¹, Felipe Echeverri Tribin¹, Braulio C. L. B. Ferreira^1,2, James Lai¹, Mariela C. Aguilar¹, Alejandro Arboleda³, Roger Leblanc², Harry W. Flynn, Jr.^1,4,5, Guillermo Amescua^1,4,5, Jean-Marie Parel^1,5, Darlene Miller<sup>1,5

1</sup>Ophthalmic Biophysics Center, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; ²Department of Chemistry, University of Miami, Coral Gables, Florida; ³Cullen Eye Institute, Baylor College of Medicine, Houston, Texas; ⁴Ocular Microbiology Laboratory, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida; ⁵Anne Bates Leach Eye Center, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida

Purpose: To evaluate the in vitro activity of rose bengal photodynamic antimicrobial therapy (RB-PDAT) combined with conventional topical antifungals (amphotericin B, natamycin, and voriconazole) against clinical fungal keratitis isolates.

Methods: UV-visible spectroscopy was used to assess the structural stability of antifungals following exposure to RB and light irradiation (518 nm, 5.4 J/cm² for 15 minutes). Clinical isolates of Fusarium and Aspergillus spp. were tested on agar plates containing three concentrations of amphotericin B (1.0, 2.0, 4.0 mg/L), natamycin (8.0, 16.0, 32.0 mg/L), or voriconazole (0.5, 1.0, 2.0 mg/L) in addition to control agar plates without any antifungal. The treatment groups included antifungal monotherapy, RB-PDAT monotherapy, and antifungal with RB-PDAT combination therapy in both dark and light conditions. After seven days, fungal growth inhibition was quantified with ImageJ. Statistical significance (p<0.05) was determined using Kruskal-Wallis tests. A combination score was calculated by subtracting the sum of the percent inhibition of the two monotherapy conditions from the observed experimental percent inhibition of the combination therapy condition.

Results: Spectroscopy revealed 70% degradation of amphotericin B and complete degradation of natamycin following RB-PDAT, while voriconazole remained stable. Across all conditions, there was a significant difference in percent inhibition between antifungal monotherapy, RB-PDAT monotherapy, and combination therapy (p < 0.001). Fusarium isolates were more susceptible than Aspergillus to both antifungal and RB-PDAT monotherapies, with A. fumigatus showing the greatest response among Aspergillus strains. RB-PDAT monotherapy resulted in average inhibition of 20.8% for Fusarium and only 3.5% for Aspergillus. At the highest tested concentration (4.0 mg/L), amphotericin B monotherapy inhibited Fusarium and Aspergillus by 46.2% and 3.7%, respectively; combination therapy yielded 47.5% and 5.6% inhibition, with mostly neutral to mildly positive combination scores. Natamycin monotherapy (32 mg/L) produced 91.7% inhibition in Fusarium and 37.2% in Aspergillus, but addition of RB-PDAT reduced inhibition to 74.2% and 10.2%, respectively, corresponding to neutral to negative combination scores, particularly in Fusarium. Voriconazole monotherapy (2.0 mg/L) inhibited Fusarium by 20.3% and Aspergillus by 69.9%; when combined with RB-PDAT, Fusarium inhibition increased to 68.2%, while inhibition in Aspergillus was maintained. In both Fusarium and Aspergillus, combination therapy conditions resulted in increased fungal inhibition compared to RB-PDAT monotherapy conditions.

Conclusions: RB-PDAT leads to photodegradation of polyene antifungals (amphotericin B and natamycin) and resulted in decreased fungal inhibition. Voriconazole remained stable and enhanced inhibition in Fusarium, supporting its potential compatibility alongside RB-PDAT. These findings suggest that topical antifungal dosing may need adjustment following RB-PDAT, although the clinical impact remains uncertain.