Ocular Microbiology and Immunology Group
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2024 OMIG Abstract

Alternative Photosensitizer Evaluation in Fungal Keratitis:
In vitro Inhibition and Biophysical Profile Analysis

James Lai¹, Brandon Chou¹, Katherine Krishna¹, Heather Durkee¹, Felipe Echeverri Tribin¹, Anam Ahmed¹, Mariela C. Aguilar¹, Braulio C.L.B. Ferreira^1,3, Jaime Martinez², Noel Ziebarth¹, Roger M. Leblanc³,
Harry W. Flynn Jr.^1,2,4, Guillermo Amescua^1,2,4, Darlene Miller^2,4, Jean-Marie Parel<sup>1,2

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

Purpose: Corneal infections caused by fungal species such as Aspergillus and Fusarium are often serious and difficult to treat with currently approved antifungals. Rose bengal (RB) photodynamic antimicrobial therapy (PDAT) as an adjunct solution has demonstrated efficacy against fungal keratitis but is likely limited by its poor penetration depth into the cornea. Alternative photosensitizers such as erythrosin B (EB) and eosin Y (EY) may present alternatives to RB but their biophysical profiles and antifungal activity are yet to be fully described. This study will compare both the in vitro antifungal effect of RB against EB and EY PDAT, as well as their respective penetration depths into the cornea.

Methods: In Vitro: Twelve fungal isolates (Fusarium spp., n=6, Aspergillus spp., n=6) were diluted to 1x10E5 CFU/ml. 0.1% solution of RB, EB, EY were prepared in NaCl. Three experimental groups were plated on Sabouraud-Dextrose agar for each of the 12 isolates. 1. Isolate only 2. Isolate mixed with photosensitizer without irradiation 3. Isolate mixed with photosensitizer with irradiation. Fungal growth inhibition was assessed by analysis of growth within the irradiated zone on agar plates.

Ex-Vivo: For ex-vivo penetration comparison, 20 human eyes (n<30 days postmortem) were de-epithelialized as per Dresden protocol. They were then soaked for 30 minutes anterior side down in either 0.0075% RB, EB, EY or NaCl prior to imaging with confocal microscopy. Penetration depth was assessed using full-width half maximum calculations from normalized intensity graphs.

Results: In Vitro: All twelve fungal isolates showed no inhibition to EB, EY and RB without irradiation. Among the Fusarium spp., analysis revealed that while there was a significant difference in percent inhibition between irradiated EB, EY and RB with the control (p<0.05), there was no significant difference in percent inhibition between any of the experimental groups (p=1.0). Among the Aspergillus spp., analysis revealed that while there was a significant difference in percent inhibition between irradiated EB and EY with the control, there was no difference between irradiated RB with the control, indicating slightly better antifungal activity of EB and EY. There was no significant difference between the percent inhibition of irradiated EB and EY.

Ex-Vivo: There was significantly greater penetration between the RB (99 ± 13 µm), EB (163 ± 13 µm) and EY (163 ± 13 µm) groups as compared to control (0 µm). Both EB and EY penetrated significantly deeper than RB into ex vivo human corneas.

Conclusions: Against 12 isolated strains of either Aspergillus or Fusarium, EB and EY had either equivalent or slightly better antifungal profiles as compared to RB. This non-inferiority is encouraging, as the improved penetration profile of EB and EY as compared to RB may indicate that alternative photosensitizers are viable options for targeting deep infections that RB may not currently reach.


Disclosure:

N (JL, BC, KK, FET, AA, BCLBF, JM, NZ, RML, HWF, J-MP); P (HD, MCA, GA, DM)