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.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

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.

Assessment of rose bengal versus riboflavin photodynamic therapy for inhibition of fungal keratitis isolates.

PURPOSE: To compare the in vitro effect of rose bengal and riboflavin as photosensitizing agents for photodynamic therapy (PDT) on fungal isolates that are common causes of fungal keratitis. DESIGN: Experimental study. METHODS: Three isolates (Fusarium solani, Aspergillus fumigatus, Candida albicans) recovered from patients with confirmed fungal keratitis were used in the experiments. Isolates were grown on Sabouraud-Dextrose agar, swabbed, and prepared in suspension, and 1 mL aliquots were inoculated onto test plates in triplicate. Test plates were separated into 5 groups: Group 1, no treatment; Group 2, 0.1% rose bengal alone; Group 3, 518 nm irradiation alone; Group 4, riboflavin PDT (riboflavin + 375 nm irradiation); and Group 5, rose bengal PDT (rose bengal + 518 nm irradiation). Irradiation was performed over a circular area using either a green light-emitting diode (LED) array (peak wavelength: 518 nm) or an ultraviolet-A LED array (peak wavelength: 375 nm). Test plates were irradiated with an energy density of 5.4 J/cm(2). Later, plates were placed in a 30 C incubator and observed for growth. RESULTS: Rose bengal-mediated PDT successfully inhibited the growth of all 3 fungal isolates in the irradiated area. All other groups exhibited unrestricted growth throughout the plate. CONCLUSIONS: Rose bengal-mediated PDT successfully inhibited the growth of 3 types of fungi. No other experimental groups, including riboflavin-mediated PDT, had any inhibitory effect on the isolates. The results might be useful for the treatment of patients suffering from corneal infection.

Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium.

PURPOSE: To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. METHODS: Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. RESULTS: In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. CONCLUSIONS: The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.

Simultaneous measurement of electrocochleography and cochlear blood flow during cochlear hypoxia in rabbits.

In this study, a new monitoring system is developed to measure cochlear blood flow (CBF) and electrocochleography (ECochG) during transient ischemic episodes of the cochlea. A newly designed otic probe was used for the simultaneous recordings of laser-Doppler CBF and ECochG directly from the round window (RW). The probe enabled the recording of high amplitude compound action potentials (CAP) and cochlear microphonics (CM) with few averages. Experiments were conducted on rabbits to generate episodes of cochlear ischemia by using timed compressions of the internal auditory artery (IAA). The computer monitoring system extracted and measured CAP and CM components from ECochG in real-time. Results indicate that CM and CAP generally followed CBF during compressions and releases of IAA. Both CBF values and CAP amplitudes showed an overshoot following the reperfusion. CAP amplitude measures were found to be very sensitive to ischemia showing very rapid amplitude, latency and morphological changes. CM amplitude decreased more slowly than the CAP and CBF. Simultaneous recordings of CBF and ECochG using the otic probe provide a valuable neuromonitoring tool to investigate the dynamic behavior of the cochlea during ischemia.

Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions.

A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T < 100 degrees C) during LITT than the traditional model using a single exponential function. Analysis of the ellipsoid coagulation volume induced in tissue indicates that the 980 nm wavelength does not penetrate deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 x 10(3) mm3) coagulation volume without unwanted tissue liquefaction and carbonization.

Laser interstitial thermotherapy (LITT) monitoring using high-resolution digital mammography: theory and experimental studies.

Laser interstitial thermotherapy (LITT) is a minimally-invasive laser hyperthermia procedure for the treatment of localized tumours. Real-time monitoring of LITT is essential to control the extent of tumour destruction and ensure safe and effective treatments. The feasibility of using high-resolution digital x-ray mammography to monitor LITT of breast cancer was evaluated. Tissue phantoms including polyacrylamide hydrogel and cadaver porcine tissue were heated using a 980 nm diode laser delivered through optical fibres with diffusing tips. Digital images of the tissue phantoms were recorded with a high-resolution digital stereotactic breast biopsy system during heating. The recorded images were processed and analysed to detect heat-induced changes. No changes were detected during heating of the hydrogel. Pixel-by-pixel subtraction of the initial image from images taken during laser heating shows observable thermally-induced changes around the fibre during laser irradiation that correlate with the thermal denaturation zone observed by gross anatomy. These experiments demonstrate that high-resolution digital x-ray mammography can be used to detect heat-induced tissue changes during experimental LITT in fibro-fatty tissue.