Purpose: Infectious keratitis caused by Aspergillus species is difficult to treat with conventional therapies. Novel treatments have been used to treat these severe infections, such as Rose Bengal (RB) photodynamic antimicrobial therapy (PDAT), which uses a photosensitizer (PS) activated with light. This activation leads to the generation of reactive oxygen species, which can kill infectious organisms. Resistant fungal strains, like Aspergillus, are not inhibited by RB. Other green light activated photosensitizers, such as Erythrosin B (EB), may inhibit fungal isolates. This study will investigate singlet oxygen (1O2) generation and fungal growth inhibition in RB and EB.

Methods: To quantify 1O2, one mL aliquots of RB and EB (1.0, 2.0, and 4.9 mM) were plated onto SabDex agar. The plates were then measured with an optical dosimeter to detect the 1O2 luminescence signal. To evaluate in vitro inhibition, two Aspergillus strains (A. fumigatus and A. glaucus) were isolated from patients with keratitis and prepared in solutions of 1x10E4 CFU/mL. Organisms were mixed with RB or EB at the same three concentrations as the 1O2 experiments. Organism/photosensitizer solutions were plated in triplicate and irradiated with a custom green (518 nm) LED light (5.4 J/cm2). Plates were then incubated for 72 hours and photographed to analyze fungal growth.

Results: For RB and EB,1O2 signal increased as the concentration increased. Also, EB had more 1O2 signal than RB at the highest concentration (4.9 mM) (Figure 1). The 1O2 findings were supported by the in vitro inhibition experiments which showed increased growth inhibition with increased PS concentration. Furthermore, EB had more inhibition of the Aspergillus organisms than RB (Figure 2).

Conclusions: This study shows 1O2 generation alongside strain specific inhibition of PDAT in support of high concentrations of EB to kill Aspergillus species. Increasing concentrations of RB and EB boosts the 1O2 signal and Aspergillus species growth inhibition. These findings support the idea that 1O2 dose is an important metric for evaluating the efficacy of PDAT.