Introduction: The prevalence and diversity of methicillin-resistant Staphylococcus aureus (MRSA) virulent factors as well as the host inflammatory response makes managing MRSA keratitis challenging. Alternative treatments are being investigated that can both neutralize the MRSA toxins while also reducing the host immune response. One such alternative is Photodynamic Antimicrobial Therapy (PDAT); however, limited research has been performed to understand the impact of PDAT on the host immune response during MRSA-related infections.
Purpose: To measure the immune response (Interleukin(IL)-1, 6 and 8) of human corneal epithelial cells in an ex-vivo tissue model following MRSA infection and rose bengal PDAT.
Methods: EpiCorneal tissue models (Maktek) were prepared according to manufacturer’s protocol. All groups were tested in duplicate: Control, Infection, Infection-PDAT. Infections were created in the tissue with three separate MRSA inocula (10E5 CFU/mL). For the Infection-PDAT group, 0.1% rose bengal solution was applied and wells were irradiated with a custom-made green LED light source. After 30 minutes, fluid from all of the tissue model wells was collected and IL-1, 6 and 8 were quantified with ELISA kits (Thermo Fisher Scientific).
Results: The EpiCorneal Tissue model simulated the immune response of human corneal epithelial cells during MRSA infection and treatment with PDAT. Compared to the control, the immune response increased in all three MRSA infection groups. Of the cytokines tested, IL-8 showed the greatest response in the tissue models, followed by IL-6 and IL-1. In the Infection-PDAT groups, immune response was mixed depending on the MRSA strain. The largest downregulation of immune factors was observed in the community-associated MRSA strain.
Introduction: In the past 25 years, methicillin-resistant Staphylococcus aureus (MRSA) strains have grown in both magnitude and diversity, making it more difficult for healthcare providers to treat these types of infections. Virulence factors of MRSA allow it to adapt to its environment and develop antibiotic resistance rapidly. One alternative treatment to combat these resistant pathogens is PDAT. Previous studies demonstrated the inhibitory effect of PDAT against MRSA isolates; however, there is limited knowledge regarding the effect of PDAT on virulence factors (toxins, defense mechanisms, stress response).
Purpose: To evaluate the impact of rose bengal (RB) and riboflavin (RI) PDAT on the virulence factors of six different ocular species of MRSA.
Methods: Suspensions were made with six separate MRSA species inocula (108CFU/mL) with either water (control), 0.1% RB, or 0.1% RI solutions. Each suspension was aliquoted onto an agar plate and exposed to either dark or a 5.4J/cm2 irradiation dose with custom-made LED irradiation sources [λ= 525nm (RB) or 375nm (RI)]. Plates were incubated for 48 hours and then photographed for percent growth measurement. Microbial samples were collected from each plate from which DNA was extracted and sent for full genome sequencing at CosmosID.
Results: Rose bengal PDAT completely inhibited the growth of all six MRSA species within the irradiation zone, while riboflavin had minimal inhibition. The dark conditions for both photosensitizers showed minimal inhibition. Full genome sequencing revealed that the virulence factors had varying responses to PDAT, depending on the MRSA species, photosensitizer used, and light exposure.
Glaucoma is the leading cause of irreversible blindness worldwide. Due to its wide prevalence, effective screening tools are necessary. The purpose of this project is to design and evaluate a system that enables portable, cost effective, smartphone based visual field screening based on frequency doubling technology. The system is comprised of an Android smartphone to display frequency doubling stimuli and handle processing, a Bluetooth remote for user input, and a virtual reality headset to simulate the exam. The LG Nexus 5 smartphone and BoboVR Z3 virtual reality headset were used for their screen size and lens configuration, respectively. The system is capable of running the C-20, N-30, 24-2, and 30-2 testing patterns. Unlike the existing system, the smartphone FDT tests both eyes concurrently by showing the same background to both eyes but only displaying the stimulus to one eye at a time. Both the Humphrey Zeiss FDT and the smartphone FDT were tested on five subjects without a history of ocular disease with the C-20 testing pattern. The smartphone FDT successfully produced frequency doubling stimuli at the correct spatial and temporal frequency. Subjects could not tell which eye was being tested. All five subjects preferred the smartphone FDT to the Humphrey Zeiss FDT due to comfort and ease of use. The smartphone FDT is a low-cost, portable visual field screening device that can be used as a screening tool for glaucoma.
Fungal keratitis can lead to pain and impaired vision. Current treatment options include antifungal agents and therapeutic penetrating keratoplasty. An emerging option for the management of keratitis is photodynamic antimicrobial therapy (PDAT) which uses a photosensitizer rose bengal activated with green light. Utilizing a pulsed irradiation, rather than the standard continuous irradiation may have a similar antimicrobial effect with less total energy. This study is to compare pulsed and continuous rose bengal mediated PDAT for inhibition of six fungal isolates on agar plates: Fusarium solani, Fusarium keratoplasticum, Aspergillus fumigatus, Candida albicans, Paecilomyces variotti, and Pseudoallescheria boydii. Isolates were mixed with 0.1% rose bengal and exposed to three irradiation conditions: (1) 30-minute continuous (10.8J/cm2), (2) 15-minute continuous (5.4J/cm2), (3) 30-minute pulsed (5.4J/cm2). Plates were photographed at 72 hours and analyzed with custom software. At 72 hours, 30-minute continuous rose bengal mediated PDAT inhibited all six fungal species. Fungal inhibition was analogous between 30-minute continuous and 30-minute pulsed test groups, with the exception of A. fumigatus. The 15-minute continuous irradiation was less effective when compared to both 30-minute continuous and 30-minute pulsed groups. These in vitro results demonstrate the potential strength of pulsed rose bengal mediated PDAT as an adjunct treatment modality for fungal keratitis.
Keratitis associated with Pseudomonas aeruginosa is difficult to manage. Treatment includes antibiotic eye drops, however, some strains of Pseudomonas aeruginosa are resistant. Current research efforts are focused on finding alternative and adjunct therapies to treat multi-drug resistant bacteria. One promising alternate technique is photodynamic therapy (PDT). The purpose of this study was to evaluate the effect of riboflavin- and rose bengal-mediated PDT on Pseudomonas aeruginosa keratitis isolates in vitro. Two isolates (S+U- and S-U+) of Pseudomonas aeruginosa were derived from keratitis patients and exposed to five experimental groups: (1) Control (dark, UV-A irradiation, 525nm irradiation); (2) 0.1% riboflavin (dark, UV-A irradiation); and (3) 0.1% rose bengal, (4) 0.05% rose bengal and (5) 0.01% rose bengal (dark, 525nm irradiation). Three days after treatment, in dark conditions of all concentration of riboflavin and rose bengal showed no inhibition in both S+U- and S-U+ strains of Pseudomonas aeruginosa. In 0.1% and 0.05% rose bengal irradiated groups, for both S+U- and S-U+ strains, there was complete inhibition of bacterial growth in the central 50mm zone corresponding to the diameter of the green light source. These in vitro results suggest that rose bengal photodynamic therapy may be an effective adjunct treatment for Pseudomonas aeruginosa keratitis.
As there is no clinically available instrument to systematically and reliably determine the photosensitivity thresholds of patients with dry eyes, blepharospasms, migraines, traumatic brain injuries, and genetic disorders such as Achromatopsia, retinitis pigmentosa and other retinal dysfunctions, a computer-controlled optoelectronics system was designed. The BPEI Photosensitivity System provides a light stimuli emitted from a bi-cupola concave, 210 white LED array with varying intensity ranging from 1 to 32,000 lux. The system can either utilize a normal or an enhanced testing mode for subjects with low light tolerance. The automated instrument adjusts the intensity of each light stimulus. The subject is instructed to indicate discomfort by pressing a hand-held button. Reliability of the responses is tracked during the test. The photosensitivity threshold is then calculated after 10 response reversals. In a preliminary study, we demonstrated that subjects suffering from Achromatopsia experienced lower photosensitivity thresholds than normal subjects. Hence, the system can safely and reliably determine the photosensitivity thresholds of healthy and light sensitive subjects by detecting and quantifying the individual differences. Future studies will be performed with this system to determine the photosensitivity threshold differences between normal subjects and subjects suffering from other conditions that affect light sensitivity.
Mariela Aguilar, Hideo Yamamoto, Edgar Espana, Ana Carolina Acosta, Marcia Orozco, Mohamed Aly, Esdras Arrieta, Eleut Hernandez, John Martin, Sander Dubovy, William Smiddy, Leonard Pinchuk, Jean-Marie Parel
Purpose: To demonstrate the biocompatibility of SIBS implants as compared to PDMS implants in the treatment of retinal detachment in New Zealand White (NZW) rabbit model.1,2Introduction: Scleral encircling bands, fixation rings and buckles are utilized for closure of retinal breaks and retina reattachment. The FDA approved PDMS-implant is associated with several post-operative complications, involving thick-fibrotic encapsulations. SIBS, an elastomeric triblock copolymer, was recently FDA approved for use in a cardiovascular drug eluting stent (TAXUSTM, Boston Scientific Corp., MA) and showed excellent biocompatibility and slow drug release capability. Materials and Methods: SIBS (9-mol%-styrene) implants were fabricated (InnFocus LLC, USA) to match PDMS implants (Labtician, Inc, Canada) dimensions. 5 NZW rabbits received SIBS and 4, PDMS-implants. Post-operative exam sequence: day 1 and 2, week 1, 2, 3, 4 and 6, and monthly thereafter for up to 1 year. Anatomohistopathology exams sequence: one SIBS animal at 6 weeks and one animal of each treatment group at 3 and 6-months, and two at 12-months. Results: SIBS compared to PDMS animals exhibited less inflammation and a better buckling effect during the first 6 weeks. At POD 9 months, the conjunctival injection in the SIBS rabbit was none as opposed to the PDMS value and the buckling effect for both groups were equal. There were no visible signs of encapsulation with SIBS. There were no infections in the 9 animals and none of the implants extruded thus far (<10 months). Conclusion: SIBS encircling bands, sleeves, and buckle implants are well tolerated in the rabbit model.
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