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2025 OMIG Abstract

Standard, Repeat, and High Fluence Rose Bengal Photodynamic Antimicrobial Therapy in Clinical Acanthamoeba Keratitis Isolates: In Vitro Cysticidal Effect

Juan Carlos Navia¹, Heather Durkee¹, Mariela Aguilar¹, Emily Delgado¹, Salomon Merikansky³,
Alex Gonzalez¹, Cornelis Rowaan¹, Jaime D. Martinez², Harry W. Flynn^1,3, Jean-Marie Parel¹,
Guillermo Amescua^1-3, Darlene Miller<sup>3

1</sup>Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Health Care System, Miami, Florida; ²Anne Bates Leach Eye Hospital, Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Health Care System, Miami, Florida; ³Microbiology Laboratory, Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Health Care System, Miami, Florida

Purpose: To evaluate whether standard, repeated-session, and high-fluence RB-PDAT combined with antiamoebic agents enhance in vitro cysticidal activity against Acanthamoeba.

Methods: Four Acanthamoeba isolates (one ATCC and three clinical strains) were cultured at the Ocular Microbiology Laboratory of the Bascom Palmer Eye Institute. Cysts were induced on NNA plates, harvested with NaCl, washed, counted via hemocytometer, and standardized to 1×10⁵ CFU/mL. Isolates were exposed to 0.02% polyhexamethylene biguanide (PHMB), 0.02% chlorhexidine (CHX), or NaCl (control) for 24 hours. After drug exposure, cysts were washed 3x, further divided into drug-only and RB-PDAT combination groups. In RB-PDAT groups, 1% RB solution was added and rested for 30 minutes. Solutions are aliquoted (250uL) into a 12 well plate and exposed to four light conditions: (1) no-irradiation (dark), (2) RB-PDAT standard therapy (6mW, 5.4J), (3) repeat RB-PDAT therapy (2x session 6mW, 5.4J), and (4) high fluence RB-PDAT (18mW, 16.2J). For the repeat RB-PDAT group, solutions were washed in between the two light exposures. Dark and light groups were performed on separate microplates to prevent unwanted light exposure. After light exposure, solutions were rinsed 3x again and aliquoted (150uL) on NNA with E. Coli. All groups were performed in triplicate. Cysticidal effect, defined as the absence of tracks or trophozoites, was assessed at 24 hours and 7 days.

Results: In the Standard RB-PDAT and no-irradiation groups, trophozoites were present in all conditions at 24 hours. In contrast, both Repeat RB-PDAT and High-Fluence RB-PDAT combined with PHMB or CHX achieved 100% inhibition across all isolates at 24 hours. PHMB and CHX alone produced partial inhibition that was not sustained at 7 days. RB-PDAT combination groups maintained high cysticidal activity at 7 days, while drug-only and control groups showed declining efficacy or persistent trophozoites.

Conclusions: Standard RB-PDAT alone was insufficient for complete cyst inhibition. However, both repeated-session and high-fluence RB-PDAT, when combined with PHMB or CHX, significantly enhanced and sustained in vitro cysticidal activity against Acanthamoeba isolates compared to drug monotherapy. Extended observation up to 14 days would be ideal to fully assess cysticidal activity.