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.

Validation of the Iowa Head-Mounted Open-Source Perimeter.

Purpose: To assess the validity of visual field (VF) results from the Iowa Head-Mounted Display (HMD) Open-Source Perimeter and to test the hypothesis that VF defects and test-retest repeatability are similar between the HMD and Octopus 900 perimeters. Methods: We tested 20 healthy and nine glaucoma patients on the HMD and Octopus 900 perimeters using the Open Perimetry Interface platform with size V stimuli, a custom grid spanning the central 26° of the VF, and a ZEST thresholding algorithm. Historical data from the Humphrey Field Analyzer (HFA) were also analyzed. Repeatability was analyzed with the repeatability coefficient (RC), and VF defect detection was determined through side-by-side comparisons. Results: The pointwise RCs were 2.6 dB and 3.4 dB for the HMD and Octopus 900 perimeters in ocular healthy subjects, respectively. Likewise, the RCs were 4.2 dB and 3.5 dB, respectively, in glaucomatous patients. Limits of agreement between the HMD and Octopus 900 perimeters were ±4.6 dB (mean difference, 0.4 dB) for healthy patients and ±8.9 dB (mean difference, 0.1 dB) for glaucomatous patients. Retrospective analysis showed that pointwise RCs on the HFA2 perimeter were between 3.4 and 3.7 dB for healthy patients and between 3.9 and 4.7 dB for glaucoma patients. VF defects were similar between the HMD and Octopus 900 for glaucoma subjects. Conclusions: The Iowa Virtual Reality HMD Open-Source Perimeter is as repeatable as the Octopus 900 perimeter and is a more portable and less expensive alternative than traditional perimeters. Translational Relevance: This study demonstrates the validity of the visual field results from the Iowa HMD Open-Source Perimeter which may help expand perimetry access.

The Open Perimetry Initiative: A framework for cross-platform development for the new generation of portable perimeters.

The Open Perimetry Initiative was formed in 2010 with the aim of reducing barriers to clinical research with visual fields and perimetry. Our two principal tools are the Open Perimetry Interface (OPI) and the visualFields package with analytical tools. Both are fully open source. The OPI package contains a growing number of drivers for commercially available perimeters, head-mounted devices, and virtual reality headsets. The visualFields package contains tools for the analysis and visualization of visual field data, including methods to compute deviation values and probability maps. We introduce a new frontend, the opiApp, that provides tools for customization for visual field testing and can be used as a frontend to run the OPI. The app can be used on the Octopus 900 (Haag-Streit), the Compass (iCare), the AP 7000 (Kowa), and the IMO (CREWT) perimeters, with permission from the device manufacturers. The app can also be used on Android phones with virtual reality headsets via a new driver interface, the PhoneHMD, implemented on the OPI. The use of the tools provided by the OPI library is showcased with a custom static automated perimetry test for the full visual field (up to 50 degrees nasally and 80 degrees temporally) developed with the OPI driver for the Octopus 900 and using visualFields for statistical analysis. With more than 60 citations in clinical and translational science journals, this initiative has contributed significantly to expand research in perimetry. The continued support of researchers, clinicians, and industry are key in transforming perimetry research into an open science.

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.

Deep Learning-Based Retinal Nerve Fiber Layer Thickness Measurement of Murine Eyes.

Purpose: To design a robust and automated estimation method for measuring the retinal nerve fiber layer (RNFL) thickness using spectral domain optical coherence tomography (SD-OCT). Methods: We developed a deep learning-based image segmentation network for automated segmentation of the RNFL in SD-OCT B-scans of mouse eyes. In total, 5500 SD-OCT B-scans (5200 B-scans were used as training data with the remaining 300 B-scans used as testing data) were used to develop this segmentation network. Postprocessing operations were then applied on the segmentation results to fill any discontinuities or remove any speckles in the RNFL. Subsequently, a three-dimensional retina thickness map was generated by z-stacking 100 segmentation processed thickness B-scan images together. Finally, the average absolute difference between algorithm predicted RNFL thickness compared to the ground truth manual human segmentation was calculated. Results: The proposed method achieves an average dice similarity coefficient of 0.929 in the SD-OCT segmentation task and an average absolute difference of 0.0009 mm in thickness estimation task on the basis of the testing dataset. We also evaluated our segmentation algorithm on another biological dataset with SD-OCT volumes for RNFL thickness after the optic nerve crush injury. Results were shown to be comparable between the predicted and manually measured retina thickness values. Conclusions: Experimental results demonstrate that our automated segmentation algorithm reliably predicts the RNFL thickness in SD-OCT volumes of mouse eyes compared to laborious and more subjective manual SD-OCT RNFL segmentation. Translational Relevance: Automated segmentation using a deep learning-based algorithm for murine eye OCT effectively and rapidly produced nerve fiber layer thicknesses comparable to manual segmentation.

Flipped ophthalmology classroom augmented with case-based learning.

BACKGROUND: Although the flipped classroom model provides an effective way to teach ophthalmology to medical students, there are concerns that it overburdens the learner. The purpose of this study was to assess medical students' perceptions of a case-based flipped classroom style compared with a traditional didactic lecture series and to evaluate the effects of case-based learning on students' confidence in managing common ophthalmic complaints. METHODS: We created an interactive, case-based flipped classroom ophthalmology curriculum. Paired pre- and post-clerkship surveys were distributed to students on the first and last day of the 2-week clerkship. Questions were formatted as statements using a 6-point Likert scale to assess students' prior exposure to a flipped classroom, perceptions of the flipped classroom curriculum, and confidence in evaluating ophthalmic complaints. RESULTS: A total of 75 students were included during the period July 2019 to March 2020. Pre-clerkship questionnaires revealed no preference for either teaching modality. Wilcoxon signed-rank testing comparing pre- and post-clerkship data revealed a significant increase in students' favoring the case-based flipped-classroom model. Participants reported significant reductions in pressure to perform, course burden, and overall anxiety as well as increased confidence in triaging common eye complaints. CONCLUSIONS: The case-based flipped classroom modality prioritizes key learning objectives while increasing student participation and confidence. The reproducibility and accessibility of standardized prepared video lectures and cases may help institutions to better incorporate ophthalmology into preexisting rotations.

Alleviating an Increasingly Burdened Healthcare System with Telemedicine: Anterior Segment.

Tele-ophthalmology is a rapidly evolving and exciting field that has become increasingly relevant amidst the current global pandemic. With advancements in technology, many components of an evaluation previously possible only in an office setting are becoming possible remotely, paving the way for at-home visits. Examples of these technologies include measurement of visual acuity using smartphones, remote monitoring of intraocular pressure using self-administered tonometers or implantable sensors, and use of digitally acquired slit-lamp or smartphone images for diagnosis of anterior segment pathology. While many examples showcasing the utility of telemedicine have been shown for diseases such as diabetic retinopathy, concerted efforts are needed to develop similar technology for anterior segment diseases. As technology continues to advance, it may be possible to diagnose and manage more anterior segment disease with telemedicine.

Low-cost, smartphone-based frequency doubling technology visual field testing using a head-mounted display.

BACKGROUND: Current visual field screening machines are bulky and expensive, limiting their accessibility, affordability and use. We report the design and evaluation of a novel, portable, cost-effective system for glaucoma screening using smartphone-based visual field screening using frequency doubling technology (FDT) and a head-mounted display. METHODS: Nineteen eyes of 10 subjects with new-onset or chronic primary open angle glaucoma were tested and compared with the Humphrey Zeiss FDT and the newly developed Mobile Virtual Perimetry (MVP) FDT with the C-20 testing pattern. Mann-Whitney, Bland-Altman and linear regression analyses were performed to assess statistical difference, agreement and correlation, respectively, between the two devices. RESULTS: The average age of the participants was 58±15 years. No statistically significant difference was found between the MVP FDT and the Humphrey Zeiss FDT (p>0.05). Bland-Altman and linear regression analyses demonstrated good agreement and correlation between the two devices. CONCLUSION: The MVP FDT is a low-cost, portable visual field screening device that produces comparable results to the Humphrey Zeiss FDT and may be used as an easily accessible screening tool for glaucoma.

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.

BAP1 Loss Is Associated with DNA Methylomic Repatterning in Highly Aggressive Class 2 Uveal Melanomas.

PURPOSE: The strong association between BAP1 mutations and metastasizing Class 2 uveal melanoma (UM) suggests that epigenetic alterations may play a significant role in tumor progression. Thus, we characterized the impact of BAP1 loss on the DNA methylome in UM.Experimental Design: Global DNA methylation was analyzed in 47 Class 1 and 45 Class 2 primary UMs and in UM cells engineered to inducibly deplete BAP1. RNA-Seq was analyzed in 80 UM samples and engineered UM cells. RESULTS: Hypermethylation on chromosome 3 correlated with downregulated gene expression at several loci, including 3p21, where BAP1 is located. Gene set analysis of hypermethylated and downregulated genes identified axon guidance and melanogenesis as deregulated pathways, with several of these genes located on chromosome 3. A novel hypermethylated site within the BAP1 locus was found in all Class 2 tumors, suggesting that BAP1 itself is epigenetically regulated. Highly differentially methylated probes were orthogonally validated using bisulfite sequencing, and they successfully distinguished Class 1 and Class 2 tumors in 100% of cases. In functional validation experiments, BAP1 knockdown in UM cells induced methylomic repatterning similar to UM tumors, enriched for genes involved in axon guidance, melanogenesis, and development. CONCLUSIONS: This study, coupled with previous work, suggests that the initial event in the divergence of Class 2 UM from Class 1 UM is loss of one copy of chromosome 3, followed by mutation of BAP1 on the remaining copy of chromosome 3, leading to the methylomic repatterning profile characteristic of Class 2 UMs.

Automated instrument designed to determine visual photosensitivity thresholds.

The Ocular Photosensitivity Analyzer (OPA), a new automated instrument to quantify the visual photosensitivity thresholds (VPT) in healthy and light sensitive subjects, is described. The OPA generates light stimuli of varying intensities utilizing unequal ascending and descending steps to yield the VPT. The performance of the OPA was evaluated in healthy subjects, as well as light sensitive subjects with achromatopsia or traumatic brain injury (TBI). VPT in healthy, achromatopsia, and TBI subjects were 3.2 ± 0.6 log lux, 0.5 ± 0.5 log lux, and 0.4 ± 0.6 log lux, respectively. Light sensitive subjects manifested significantly lower VPT compared to healthy subjects. Longitudinal analysis revealed that the OPA reliably measured VPT in healthy subjects.

Quantification of the ciliary muscle and crystalline lens interaction during accommodation with synchronous OCT imaging.

Two SD-OCT systems and a dual channel accommodation target were combined and precisely synchronized to simultaneously image the anterior segment and the ciliary muscle during dynamic accommodation. The imaging system simultaneously generates two synchronized OCT image sequences of the anterior segment and ciliary muscle with an imaging speed of 13 frames per second. The system was used to acquire OCT image sequences of a non-presbyopic and a pre-presbyopic subject accommodating in response to step changes in vergence. The image sequences were processed to extract dynamic morphological data from the crystalline lens and the ciliary muscle. The synchronization between the OCT systems allowed the precise correlation of anatomical changes occurring in the crystalline lens and ciliary muscle at identical time points during accommodation. To describe the dynamic interaction between the crystalline lens and ciliary muscle, we introduce accommodation state diagrams that display the relation between anatomical changes occurring in the accommodating crystalline lens and ciliary muscle.

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.

Therapeutic Strategies to Treat Dry Eye in an Aging Population.

Dry eye (DE) is a prevalent ocular disease that primarily affects the elderly. Affecting up to 30% of adults aged 50 years and older, DE affects both visual function and quality of life. Symptoms of DE, including ocular pain (aching, burning), visual disturbances, and tearing, can be addressed with therapeutic agents that target dysfunction of the meibomian glands, lacrimal glands, goblet cells, ocular surface, and/or neural network. This review provides an overview of the efficacy, use, and limitations of current therapeutic interventions being used to treat DE.

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.