Variations in irradiation energy and rose bengal concentration for photodynamic antimicrobial therapy of fungal keratitis isolates.

The purpose is to assess the efficacy of rose bengal photodynamic antimicrobial therapy (PDAT) using different irradiation energy levels and photosensitizer concentrations for the inhibition of fungal keratitis isolates. Seven different fungi (Aspergillus fumigatus, Candida albicans, Curvularia lunata, Fusarium keratoplasticum, Fusarium solani, Paecilomyces variotii, and Pseudallescheria boydii) were isolated from patients with confirmed infectious keratitis. Experiments were performed in triplicate with suspensions of each fungus exposed to different PDAT parameters including a control, green light exposure of 5.4 J/cm2, 2.7 J/cm2 (continuous and pulsed), and 1.8 J/cm2 and rose bengal concentrations of 0.1%, 0.05%, and 0.01%. Plates were photographed 72 h after experimentation, and analysis was performed to assess fungal growth inhibition. PDAT using 5.4 J/cm2 of irradiation and 0.1% rose bengal completely inhibited growth of five of the seven fungal species. Candida albicans and Fusarium keratoplasticum were the most susceptible organisms, with growth inhibited with the lowest fluence and minimum rose bengal concentration. Fusarium solani, Pseudallescheria boydii, and Paecilomyces variotii were inhibited by lower light exposures and photosensitizer concentrations. Aspergillus fumigatus and Curvularia lunata were not inhibited by any PDAT parameters tested. Continuous and pulsed irradiation using 2.7 J/cm2 produced similar results. Rose bengal PDAT successfully inhibits the in vitro growth of five fungi known to cause infectious keratitis. Differences in growth inhibition of the various fungi to multiple PDAT parameters suggest that susceptibilities to PDAT are unique among fungal species. These findings support modifying PDAT parameters based on the infectious etiology.

Rose Bengal Photodynamic Antimicrobial Therapy: A Review of the Intermediate-Term Clinical and Surgical Outcomes.

PURPOSE: To evaluate the intermediate-term clinical outcomes of Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) for infectious keratitis; secondarily, to evaluate the surgical outcomes of individuals who underwent optical keratoplasty after RB-PDAT. DESIGN: Retrospective cohort study. METHOD: A retrospective chart review was performed of 31 eyes from 30 consecutive individuals with infectious keratitis refractory to standard medical therapy who underwent RB-PDAT at the Bascom Palmer Eye Institute between January 2016 and July 2020. Data collected included demographics, risk factors for infectious keratitis, microbiological diagnosis, best spectacle-corrected visual acuity (BCVA), clinical outcomes after RB-PDAT, and complication rates post-keratoplasty. RB-PDAT was performed as described in previous studies. Graft survival was evaluated using Kaplan-Meier curves with log-ranks in individuals who underwent keratoplasty after RB-PDAT. RESULTS: The mean age of the study population was 53 ± 18.0 years. In all, 70% were female; 53.3% self-identified as non-Hispanic White and 43.3% as Hispanic. Mean follow-up time was 28.0 ± 14.4 months. Risk factors included contact lens use (80.6%), history of infectious keratitis (19.3%), and ocular surface disease (16.1%). Cultures were positive for Acanthamoeba (51.6%), Fusarium (12.9%), and Pseudomonas (6.5%). Of the individuals with Acanthamoeba infection, 22.5% were treated with concomitant Miltefosine. Clinical resolution was achieved in 77.4% of patients on average 2.72 ± 1.85 months after RB-PDAT, with 22.5% requiring therapeutic penetrating keratoplasties and 54.8% subsequently requiring optical penetrating keratoplasties. At 2 years, the overall probability of graft survival was 78.7%, and the graft failure rate was 21.3%. CONCLUSION: RB-PDAT is a potential adjunct therapy for infectious keratitis that may reduce the need for a therapeutic penetrating keratoplasty. Patients who undergo keratoplasty after RB-PDAT may have a higher probability of graft survival at 1 year postoperatively.

Rose Bengal and Riboflavin Mediated Photodynamic Antimicrobial Therapy Against Selected South Florida Nocardia Keratitis Isolates.

Purpose: To examine and compare the efficacy of in vitro growth inhibition using rose bengal and riboflavin photodynamic antimicrobial therapy (PDAT) for Nocardia keratitis isolates. Methods: Nocardia asteroides complex, Nocardia amikacinitolerans, and Nocardia farcinica species were isolated from patients with confirmed Nocardia keratitis. Isolates were tested against three experimental groups: (1) no photosensitizer/no irradiation, (2) photosensitizer/no irradiation, and (3) photosensitizer/irradiation. Each isolate was prepared in suspension to a concentration of 1.5 × 108 CFU/mL. Bacterial suspensions were mixed with water or prepared 0.1% photosensitizer solution for a final bacterial concentration of 1.5 × 107 CFU/mL. Aliquots of 1 mL were plated on 5% sheep blood agar. Rose bengal and riboflavin PDAT plates were irradiated for 15 minutes with a 525- or 375-nm custom 6-mW/cm2 powered light source for a total fluence of 5.4 J/cm2. All experimental groups were repeated in triplicate. Plates were incubated in a 35°C non-CO2 incubator for 96 hours and photographed. Percent inhibition was evaluated using LabVIEW-based software. Results: All strains of Nocardia tested with 0.1% rose bengal and irradiated for 15 minutes demonstrated statistically significant inhibition of growth (P < 0.05). No other experimental groups displayed any bacterial inhibition. Conclusions: Rose bengal is superior to riboflavin PDAT against selected Nocardia isolates. In vivo testing is warranted to investigate the utility of rose bengal PDAT for severe Nocardia keratitis. Translational Relevance: In vitro results for three clinical strains of Nocardia support the possible use of rose bengal PDAT as a complementary treatment of Nocardia keratitis.

Rose Bengal Photodynamic Antimicrobial Therapy: A Pilot Safety Study.

PURPOSE: To evaluate the in vivo corneal changes after Rose Bengal photodynamic antimicrobial therapy (RB-PDAT) treatment in New Zealand White rabbits. METHODS: Sixteen rabbits were divided into 5 groups. All groups underwent deepithelialization of an 8 mm diameter area in the central cornea. Group 1: balanced salt solution drops only, group 2: 0.2% RB only, group 3: green light exposure (525 nm, 5.4 J/cm2) only, group 4: 0.1% RB-PDAT, and group 5: 0.1% RB-PDAT. All rabbits were followed clinically. Group 5 rabbits were followed using anterior segment optical coherence tomography (AS-OCT) and clinically. On day 35 after initial treatment, 1 rabbit from group 5 was re-exposed to green light (5.4 J/cm2) to evaluate reactivation of the remaining RB dye, and terminal deoxynucleotyl transferase-mediated UTP-biotin-nick-end labeling assay was performed on corneal cryosections. RESULTS: Complete reepithelization was observed, and corneas remained clear after treatment in all groups. In group 5, AS-OCT revealed a cross-linking demarcation line. AS-OCT showed RB fluorescence and collagen cross-linking in all treated eyes of group 5 animals after 5 weeks of treatment. Photobleached RB retention in the corneal stroma was corroborated by fluorescence confocal microscopy on frozen sections. There was no evidence of a sustained cytotoxic effect through terminal deoxynucleotyl transferase-mediated UTP-biotin-nick-end labeling at 5 weeks. CONCLUSIONS: RB-PDAT with 0.1% RB is a safe procedure. There was no difference clinically and on histopathology compared with control groups. In eyes where RB dye is retained in the corneal stroma after 1 month of treatment, oxidative stress is not evidenced at long term.

Rose bengal photodynamic antimicrobial therapy to inhibit Pseudomonas aeruginosa keratitis isolates.

To evaluate the in vitro efficacy of rose bengal and riboflavin photodynamic antimicrobial therapy for inhibition the growth of four Pseudomonas aeruginosa (P. aeruginosa) isolates. Four different clinical P. aeruginosa isolates were collected from patients with confirmed keratitis. Each strain was mixed with either sterile water, 0.1% riboflavin solution, or 0.1% rose bengal solution to yield a final bacteria concentration of 1.5 × 107 CFU/mL. Aliquots from each suspension were plated onto nutrient agar in triplicate. Plates were separated into two groups: (1) no irradiation and (2) 5.4 J/cm2 of radiant exposure with custom-made LED irradiation sources. Separate irradiation sources were used for each photosensitizer. The riboflavin groups used a UV-A light source (375 nm) and rose bengal groups used a green light source (525 nm). Plates were photographed at 72 h and custom software measured bacterial growth inhibition. Growth inhibition to riboflavin and rose bengal PDAT showed strain-dependent variability. All four strains of P. aeruginosa showed greatest growth inhibition (89-99%) in the green irradiated-rose bengal group. The UV-A-irradiated riboflavin showed inhibition of 24-44%. UV-A irradiation only showed minimal inhibition (7-14%). There was little inhibitory effect in the non-irradiated photosensitizer groups. Rose bengal PDAT had the greatest inhibitory effect on all four P. aeruginosa isolates. In the UV-A-irradiated riboflavin group, there was moderate inhibition within the irradiation zone; however, there was no inhibition in the non-irradiated groups. These results suggest that rose bengal PDAT may be an effective alternative treatment for Pseudomonas aeruginosa infections.

Rose Bengal Photodynamic Antimicrobial Therapy for Patients With Progressive Infectious Keratitis: A Pilot Clinical Study.

PURPOSE: To report clinical outcomes of rose bengal photodynamic antimicrobial therapy (RB-PDAT) as an adjunct treatment for severe, progressive infectious keratitis. DESIGN: Consecutive interventional case series. METHODS: Patients with progressive infectious keratitis unresponsive to standard medical therapy underwent RB-PDAT at the Bascom Palmer Eye Institute from January 2016 through March 2018. RB-PDAT was performed by applying a solution of rose bengal (0.1% or 0.2% RB in balanced salt solution) to the de-epithelialized cornea for 30 minutes, followed by irradiation with a 6 mW/cm2 custom-made green LED source for 15 minutes (5.4 J/cm2). RESULTS: The current study included 18 patients (7 male and 11 female) ranging from 17 to 83 years old. Acanthamoeba was the most frequent microbe (10/17; 59%), followed by Fusarium spp. (4/17; 24%), Pseudomonas aeruginosa (2/17; 12%), and Curvularia spp. (1/17; 6%); 1 patient had no confirmed microbiologic diagnosis. Main clinical risk factor for keratitis included contact lens wear (79%). The average area of epithelial defect prior to first RB-PDAT was 32 ± 27 mm2 and average stromal depth hyperreflectivity measured with anterior segment optical coherence tomography was 269 ± 75 µm. Successful RB-PDAT (avoidance of therapeutic keratoplasty) was achieved in 72% of the cases, with an average time to clinical resolution (decreased pain and inflammation with re-epithelialization and infiltrate resolution) of 46.9 ± 26.4 days after RB-PDAT. Time of follow-up after RB-PDAT was 13.3 ± 5.7 months. CONCLUSION: RB-PDAT can be considered as an adjunct therapy for cases of severe, progressive infectious keratitis before performing a therapeutic keratoplasty.

Rose Bengal Photodynamic Antimicrobial Therapy: A Novel Treatment for Resistant Fusarium Keratitis.

PURPOSE: To evaluate the efficacy of rose bengal PDAT for the management of a patient with multidrug-resistant Fusarium keratoplasticum keratitis unresponsive to standard clinical treatment. METHODS: This case report presents a clinical case of F. keratoplasticum keratitis not responsive to standard medical care. In vitro studies from patients culture isolated responded to rose bengal PDAT. Patient received two treatments with rose bengal 0.1% and exposure to green light with a total energy of 2.7 J/cm. RESULTS: In vitro results demonstrated the efficacy of rose bengal PDAT a multidrug-resistant F. keratoplasticum species. There was complete fungal inhibition in our irradiation zone on the agar plates. In the clinical case, the patient was successfully treated with 2 sessions of rose bengal PDAT, and at 8-month follow-up, there was neither recurrence of infection nor adverse effects to report. CONCLUSIONS: Rose bengal PDAT is a novel treatment that may be considered in cases of aggressive infectious keratitis. Further studies are needed to understand the mechanisms of PDAT in vivo.

Rose Bengal- and Riboflavin-Mediated Photodynamic Therapy to Inhibit Methicillin-Resistant Staphylococcus aureus Keratitis Isolates.

PURPOSE: To evaluate the in vitro efficacy of rose bengal- and riboflavin-mediated photodynamic therapy for inhibition of methicillin-resistant Staphylococcus aureus (MRSA) isolates. DESIGN: Experimental study. METHODS: Two different multidrug-resistant, clinical MRSA isolates were grown on nutrient agar, prepared in suspension, and adjusted to concentrations of 1.5 × 10(4) colony-forming units per milliliter. Bacterial suspensions were mixed with rose bengal, riboflavin, or water according to experimental group. Tested in triplicate, groups included: Group I, MRSA control; Group II, MRSA with 0.1% rose bengal; Group III, MRSA with 0.03% rose bengal; and Group IV, MRSA with 0.1% riboflavin. All experimental groups were exposed to 3 lighting conditions: dark, ambient room light for 30 minutes, and 5.4 J/cm(2) with either green light-emitting diode (LED) or ultraviolet-A (UV-A) irradiation. Plates were photographed at 72 hours and custom software measured bacterial growth inhibition. RESULTS: Complete growth inhibition of both MRSA strains was demonstrated (1) for both rose bengal concentrations under ambient and green LED irradiation, and (2) for the 0.1% rose bengal in the dark. The 0.03% rose bengal in dark conditions showed complete inhibition of strain 2 but incomplete inhibition of strain 1. Riboflavin showed almost complete inhibition with UV-A irradiation but demonstrated minimal inhibition for both strains in dark and ambient light conditions. CONCLUSIONS: Rose bengal- and riboflavin-mediated photodynamic therapy demonstrated complete growth inhibition in vitro of 2 multidrug-resistant MRSA strains. Rose bengal was also effective in dark and ambient conditions. These results may have implications for in vivo therapy.