Photodynamic Therapy of Onychomycosis with Rose Bengal

Photodynamic therapy (PDT) is emerging as a treatment for onychomycosis; a fungal infection of the nail often caused by Trichophyton rubrum. The antimicrobial action of PDT occurs when a photosensitive molecule absorbs light irradiation and produces reactive oxygen species (ROS). Rose bengal (RB) is a photosensitive dye that has demonstrated antifungal capacity in vitro, warranting further investigation and development of RB-PDT into a clinically feasible treatment for onychomycosis. This thesis aims to establish a clinical treatment for onychomycosis using PDT with RB and green light, and to understand the mechanisms that affect the tolerance to this therapy. Low dosage RB-PDT to eradicate T. rubrum spores was carried out using 140 µM RB and green light (532 nm) at fluences of 25 – 228 J/cm2. The production of ROS by xanthomegnin, an endogenous pigment of T. rubrum, was also investigated. A screen of the entire non-essential gene deletion library of Saccharomyces cerevisiae was completed to ascertain the mechanisms influencing altered tolerance to RB-PDT. Finally, a pilot patient study was carried out to treat onychomycosis in six patients (seven toenails) using 140 µM RB and ~763 J/cm2 green light irradiation. A low PDT dosage of 25 J/cm2 was found to eradicate T. rubrum spores in vitro, with xanthomegnin also shown to produce singlet oxygen photochemically. Sensitivity to RB-PDT was associated with disrupted ergosterol biosynthesis and vacuolar acidification leading to compromised membrane integrity and Ca2+ homeostasis. Resistance was related to purine/S-adenosyl-L-methionine biosynthesis, leading to potential accumulation of ROS scavengers and reduced RB uptake. Complete cure (mycological and clinical) of onychomycosis was achieved in all six patients within three to five PDT sessions in the pilot study. No pain or recurrence of infection was reported. RB-PDT has been demonstrated, for the first time, to be a safe and effective treatment for onychomycosis caused by T. rubrum. Cellular processes affecting the tolerance to RB-PDT have also been revealed in the genetic screen of S. cerevisiae. Future work focusing on the improved delivery of RB and light through the nail plate will enhance the clinical efficacy of this treatment.