Corneal Cultures Research Articles

Effects of the COVID-19 Pandemic on Microbial Keratitis: A 5-Year Comparative Study.

To report the clinical and microbiological profiles of microbial keratitis and its antimicrobial resistance before, during, and after COVID-19. This was a retrospective case-note review of all corneal scrape specimens collected from patients with microbial keratitis from January 2018 to December 2023. Case records were analyzed for demographic characteristics, microbiological diagnosis, and antibiograms. All outcome variables were collected, stratified, and compared between 3 periods: the pre-COVID-19 group (January-December 2019), the COVID-19 group (January 2020-December 2022), and the post-COVID-19 group (January-December 2023). A total of 947 corneal cultures from 947 patients were reviewed. Gram-positive bacteria predominated in all periods, with no significant differences in their distribution. Staphylococcus epidermidis was the most frequently identified organism. Pseudomonas aeruginosa was the most common Gram-negative bacterium, with its incidence significantly lower in the post-COVID period. Fungal infections showed a significant increase in the post-COVID group, with Fusarium sp. being the most common fungus and showing a significant increase in incidence in the post-COVID group. Despite a stable incidence of microbial keratitis, this study highlights a concerning trend in antibiotic resistance. Although some pathogens became less common, those that persisted have become increasingly difficult to treat. Understanding the clinical and microbiological profiles of microbial keratitis and antimicrobial resistance patterns before and after the COVID-19 pandemic is crucial for informed treatment decisions.

Non-Contact-Lens-Related Acanthamoeba Keratitis Caused by Acanthamoeba sp. Group T4D/T4e

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the ‘Nagington’ strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK.

A Case of Fulminant Serratia marcescens Panophthalmitis After Penetrating Keratoplasty

Purpose: To report the first case of fulminant Serratia marcescens panophthalmitis after penetrating keratoplasty (PK). Methods: This is a report of a patient who developed fulminant panophthalmitis shortly after undergoing PK with anterior chamber intraocular lens placement. Slit-lamp examination, B-scan ultrasound (B scan), and orbital computed tomography of the left eye (OS) were performed to further evaluate the patient. Tissue culture and histopathologic examination of the corneal specimen were completed to confirm the diagnosis. Results: A 78-year-old pseudophakic woman presented with 2 days of increasing pain, swelling, and purulent discharge after uneventful PK and secondary anterior chamber intraocular lens placement OS. Examination was notable for light perception without projection, elevated intraocular pressure of 48 mmHg, and a perforated corneal ulcer. B scan demonstrated diffuse vitreous opacities and membranes. Orbital computed tomography demonstrated proptosis and high-attenuation material within the left globe. Canthotomy, vitreous sampling, and antibiotic injections were performed. Corneal tissue cultures grew S. marcescens. Therapeutic PK was performed, but after rapid decompensation, the eye was eviscerated. Conclusions: This is the only reported case of fulminant S. marcescens panophthalmitis after PK. S. marcescens panophthalmitis is an aggressive and rapidly progressive infection with poor visual outcomes despite appropriate intravitreal and systemic antibiotic therapy.

Comparative analysis of the effect of drugs lowering intraocular pressure on a primary culture of human corneal epithelium and A549 immortalized cell line

Background. Glaucoma as one of the most common eye diseases can be a comorbid condition of an epithelial corneal defect of various etiologies. Maintaining an optimal level of ophthalmotonus includes the prescription of antiglaucoma drugs, including benzalkonium chloride-preserved drugs.The aim of the study. To compare the effect of antiglaucoma drugs, as well as benzalkonium chloride (BC), on a primary culture of human corneal epithelium and A549 immortalized cell line.Methods. The effect of brimonidine, dorzolamide and timolol (1/100, 1/50, 1/20 and 1/10 dilutions; exposure 24 hours) on a monolayer of a human corneal epithelial primary culture and A549 immortalized cell line was assessed by structural changes in cells (phase-contrast microscopy) and MTT assay data. The cytotoxic effect of BC was studied in concentrations corresponding to its content in these dilutions of the antiglaucoma drug. Using a model of a linear defect in the monolayer of a corneal epithelial primary culture and A549 immortalized cell line, the effects of brimonidine, dorzolamide and timolol (1/100 and 1/20 dilutions; exposure 48 hours) were assessed by cell migration activity.Results. Among drugs (BC-free), dorzolamide (1/50, 1/20 and 1/10 dilutions) causes minor structural changes in human corneal epithelium and A549 immortalized cell line; timolol (1/100, 1/50, 1/20 and 1/10 dilutions) causes minor structural changes in A549 immortalized cell line. Structural changes in both types of cells, a decrease in their metabolic and migration activity occur under the influence of dorzolamide, brimonidine and timolol (BC-preserved) in 1/100, 1/50, 1/20 and 1/10 dilutions. BC at the studied concentrations exhibits a similar effect.Conclusion. The cytotoxic effect of antiglaucoma drugs is caused by the presence of BC in their composition. Despite similar morphofunctional changes in cells, A549 immortalized cell line is more resistant to the effects of drugs than the human corneal epithelial primary culture. When using it as a cellular model, it is advisable to change the experimental conditions (duration of exposure and concentration of the studied drug).

Human Conjunctival Transcriptome in Acanthamoeba Keratitis: An Exploratory Study.

The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen. The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen. Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1 , SLC35B4 , STEAP2 , ATP2B2 , NMNAT3 , and AKAP12 . This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets.

Rose Bengal Photodynamic Therapy (RB-PDT) Modulates the Inflammatory Response in LPS-Stimulated Human Corneal Fibroblasts By Influencing NF-κB and p38 MAPK Signaling Pathways

Purpose To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process. Methods Human corneal fibroblast cultures underwent 0–2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon β1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot. Results Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA (p < .0001) and interleukin-6 protein increase (p < .0001), and downregulated intercellular adhesion molecule-1 expression (p < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon β1 expressions remained unchanged (p ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription (p ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation (p ≤ .04) in lipopolysaccharide treated human corneal fibroblasts. Conclusion Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and β expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

Lack of Elevated Expression of TGFβ3 Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas.

To investigate the mechanisms underlying the differential roles of TGFβ1 and TGFβ3 in accelerating corneal epithelial wound healing (CEWH) in diabetic (DM) corneas, with normoglycemia (NL) corneas as the control. Two types of diabetic mice, human corneal organ cultures, mouse corneal epithelial progenitor cell lines, and bone marrow-derived macrophages (BMDMs) were employed to assess the effects of TGFβ1 and TGFβ3 on CEWH, utilizing quantitative PCR, western blotting, ELISA, and whole-mount confocal microscopy. Epithelial debridement led to an increased expression of TGFβ1 and TGFβ3 in cultured human NL corneas, but only TGFβ1 in DM corneas. TGFβ1 and TGFβ3 inhibition was significantly impeded, but exogenous TGFβ1 and, more potently, TGFβ3 promoted CEWH in cultured TKE2 cells and in NL and DM C57BL6 mouse corneas. Wounding induced similar levels of p-SMAD2/SMAD3 in NL and DM corneas but weaker ERK1/2, Akt, and EGFR phosphorylation in DM corneas compared to NL corneas. Whereas TGFβ1 augmented SMAD2/SMAD3 phosphorylation, TGFβ3 preferentially activated ERK, PI3K, and EGFR in healing DM corneas. Furthermore, TGFβ1 and TGFβ3 differentially regulated the expression of S100a9, PAI-1, uPA/tPA, and CCL3 in healing NL and DM corneas. Finally, TGFβ1 induced the expression of M1 macrophage markers iNOS, CD86, and CTGF, whereas TGFβ3 promoted the expression of M2 markers CD206 and NGF in BMDMs from db/db or db/+ mice. Hyperglycemia disrupts the balanced expression of TGFβ3/TGFβ1, resulting in delayed CEWH, including impaired sensory nerve regeneration in the cornea. Supplementing TGFβ3 in DM wounds may hold therapeutic potential for accelerating delayed wound healing in diabetic patients.

Descemet Membrane Endothelial Keratoplasty With and Without Graft Deswelling: A Prospective Clinical Study.

The purpose of this study was to assess whether omission of dextran from corneal organ culture medium alters the outcome of Descemet membrane endothelial keratoplasty. Participation in this single-center, multisurgeon, prospective, randomized, comparative clinical trial was offered to patients scheduled for Descemet membrane endothelial keratoplasty between April 2020 and May 2022. Patients received grafts from corneas deswollen in organ culture medium-containing 6% dextran T-500 or from corneas that were not deswollen. Corrected distance visual acuity (CDVA), graft detachment, central corneal thickness (CCT), and corneal endothelial cell counts were measured at different time points up to 12 months postoperatively. Grafts stored with dextran were transplanted in 92 patients, and grafts stored without dextran were transplanted in 102 patients. Mean donor age and endothelial cell counts did not differ significantly between both groups. Mean (±SD) postmortem time in hours was 23.9 ± 11.8 in grafts that were deswollen and 28.2 ± 13.8 in grafts that were not deswollen (P = 0.02). The groups did not show any significant difference at baseline regarding sex, CDVA, or CCT. In the group with dextran, patients had a mean age of 72.5 ± 9.9 years versus 69.5 ± 8.7 in the group without dextran (P = 0.03). CDVA improved and CCT decreased significantly in both groups. No differences were detected between the groups regarding CDVA, CCT, endothelial cell counts, or rebubbling rates. This study did not detect any evidence that the omission of dextran from organ culture medium negatively affects the outcomes of Descemet membrane endothelial keratoplasty.

A Mystery Unsolved: A Spontaneous Lens and Uveal Prolapse in a New-born

Introduction : Corneal rupture in new-born, especially during the first week of life is rare. Some of these cases occur because of ocular trauma during deliveries, systemic infection, and congenital anomalies. We aim to deliver a rare case of spontaneous lens and uveal prolapse in new-born and management in treating the case.&#x0D; Case Illustration : A-2-day-old full-term-baby was referred due to bleeding of the right eye 6 hours post-partum by spontaneous vaginal delivery with no trauma. The mother, a 23-year-old, having the second born with no history of medical illness or vaginal discharged during pregnancy. Measurements of birth weight and length were normal. Eye examination of the right eye (RE) was uveal and lens prolapse and left eye (LE) cloudy cornea with leukoma and prominent neovascularization. Orbital CT-Scan revealed bilateral vitreous bleeding of both eyes. Laboratory examination showed reactive IgM for herpes simplex virus, reactive IgG for both toxoplasma and rubella. Corneal swab culture was sterile. Systemic and topical antibiotics were administered then switched to systemic antivirus. The 12-day-old-patient showed partial epithelization of cornea, less uveal volume with it partially shrank. Close observation was conducted and evisceration was postponed.&#x0D; Discussion : Although etiologic work-up has been addressed, exact etiology remains unknown with the possibility of congenital cause. It has been postulated that structurally malformed eyes are more prone to corneal perforations. Management should be personalized based on the patient’s need.&#x0D; Conclusion : Thorough examination is vital a rare case with undisclosed aetiology especially new-born. Patient monitoring is sufficient if no infections and bleedings found.

MANAGEMENT OF CORNEAL ULCER PRESENTED WITH RARE PATHOGEN : STREPTOCOCCUS MITIS

Introduction : Corneal ulcers can arise from various corneal diseases, including infections, inflammation, and autoimmune disease. Staphylococcus aureus is the most common bacterial pathogen, but lesser- known bacteria like Streptococcus mitis can also cause corneal ulcers. Accurate diagnosis and treatment are essential to prevent vision loss and complications.&#x0D; Case Illustration : A 19-year-old female patient presented with a chief complaint of white spots on her right eye for three months. Previously, her right eye was hit by a foreign object and subsequently, the patient experienced redness and pain in the affected eye. One week later, white spot appeared in the patient's eye and reported to enlarge in the last month. The patient also complained of pain, blurry vision with visual acuity of 1/300, redness, glare, and a foreign body sensation. Corneal swab culture discovered Streptococcus mitis to be the causative agent. The patients was given hourly drop of topical levofloxacin. One month after initial treatment, patient’s symptoms was improving with visual acuity of 6/60 on her affected eye and the ulcer was healed.&#x0D; Discussion : Streptococcus mitis is a commensal pathogen in oral cavity and not normally cause infection of ocular tissue. It may cause ulcer due to prolonged use of topical steroid and previous ocular trauma, since Streptococcus mitis had low virulence rate.&#x0D; Conclusion : Corneal scraping culture should be carried out before treatment of infectious corneal ulcer. Streptococcus mitis is considered rare for corneal ulcer pathogen. Topical quinolones are the first- line drug for bacterial corneal ulcer and worked exceptional for wide range of bacteria.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Ocular irritation reversibility assessment of a laundry detergent using the Porcine Corneal Opacity Reversibility Assay (PorCORA): a focused study

Consumer product manufacturers utilise a spectrum of alternative ocular irritation assays, as these tests do not require the use of live animals. Despite their usefulness, no regulatory-accepted assay assesses the reversibility of ocular damage, a key criterion of GHS ocular classification, like the rabbit eye test (i.e., Draize Rabbit Eye Test [DRET]) . The Porcine Corneal Opacity Reversibility Assay (PorCORA), an ex vivo intact corneal tissue culture model, predicts the reversibility of damage by ocular irritants. Inclusion of the damage reversibility endpoint in the PorCORA supplements other alternative test methods for ocular irritation, by assessing induced eye damage and the ability of this damage to reverse (heal) without the use of live animals to distinguish between Globally Harmonised System of Classification and Labelling of Chemicals (GHS) ocular classifications. In this focused study, results of a Bovine Corneal Opacity and Permeability (BCOP) test of a laundry detergent, neat and 10% dilution, (product mixture from S.C. Johnson & Son, Inc. [SCJ]) classified the product into GHS Category 1; however, the BCOP test cannot assess the reversibility of ocular damage. The laundry detergent was evaluated using the PorCORA, where ocular damage induced by the detergent was fully reversed within seven days. Evaluation of the reversibility of ocular damage using the PorCORA in this focused study can add strength to the weight-of-evidence (WoE) analysis approach in ocular hazard assessment. This WoE approach strengthens the argument that the PorCORA can be used to supplement BCOP data, and that this laundry detergent is not an irreversible eye irritant.

In the eye of the ophthalmologist: the corneal microbiome in microbial keratitis.

To describe the bacterial findings by a targeted sequencing approach from corneal samples of patients with microbial keratitis and factors influencing culture outcome of indirectly inoculated corneal specimen. Prospective inclusion of patients fulfilling predefined criteria of microbial keratitis. Samples from the corneal lesion were collected and dispensed in liquid transport medium, from which both culture and targeted amplification and sequencing of the V3-V4 region of the 16S rRNA gene were carried out. Additional standard corneal culture from the corneal lesions was also performed. Factors influencing culture outcome of indirectly inoculated corneal samples were identified by a multivariate regression model incorporating quantitative data from sequencing. Among the 94 included patients with microbial keratitis, contact lens wear (n = 69; 73%) was the most common risk factor. Contact lens wearers displayed significant differences in the bacterial community composition of the corneal lesion compared to no lens wearers, with higher abundance of Staphylococcus spp., Corynebacterium spp., and Stenotrophomonas maltophilia. Targeted sequencing detected a potential corneal pathogen in the highest proportional abundance among 9 of the 24 (38%) culture-negative patients with microbial keratitis. Age, bacterial density in the sample, and prior antibiotic treatment significantly influenced culture outcome of indirectly inoculated corneal samples. Targeted sequencing may provide insights on pathogens in both culture negative episodes of microbial keratitis and among subgroups of patients with microbial keratitis as well as factors influencing culture outcome of indirectly inoculated corneal samples.

A Novel Riboflavin Formulation for Corneal Delivery Without Damaging Epithelial Cells.

This study aimed to evaluate the trans-epithelial permeability enhancement and cell damage caused by a novel riboflavin composition for corneal delivery. We developed a trans-epithelial formulation of riboflavin for corneal delivery using 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and isostearic acid (ISA). The permeation enhancement was evaluated using an in vitro corneal epithelial cell culture system by measuring the amount of transferred riboflavin with high-performance liquid chromatography. Riboflavin permeation of MedioCROSS TE, a commercially available riboflavin formulation containing benzalkonium chloride, was also evaluated and compared to that of the DODAP/ISA formulation by changing the riboflavin concentration. The trans-epithelial electrical resistance (TEER) was measured after exposure to the samples in an in vitro corneal epithelial cell culture system to assess cytotoxicity. The DODAP/ISA formulation demonstrated greater permeation when used together than when each component was used individually. The permeation enhancement effect of the DODAP/ISA formulation was almost the same as that of MedioCROSS TE. However, when a 10-fold higher riboflavin concentration was used in the DODAP/ISA formulation, the permeation enhancement effect surpassed that of MedioCROSS TE. After 24hours of exposure, the TEER of the DODAP/ISA formulation was higher than that of MedioCROSS TE, indicating that the DODAP/ISA formulation was less cytotoxic than MedioCROSS TE. This study indicated that the DODAP/ISA formulation could serve as a less cytotoxic alternative to MedioCROSS TE. Further studies are required to determine the clinical efficacy and safety of the DODAP/ISA formulation in vivo. This study may provide alternative procedures for corneal collagen crosslinking with less of a cytotoxic effect on corneal epithelial cells.

Comparison of cytotoxic, apoptotic and oxidative properties of Akacid plus and chlorhexidine in corneal epithelial cell culture.

This study aims to compare the cytotoxic, apoptotic, and oxidative effects of a new cationic disinfectant, Akacid Plus, with chlorhexidine, on the human corneal epithelial cell line. Time-dependent cytotoxicity studies were performed with the Alamar Blue method. Apoptotic activity was investigated by flow cytometric methods. Reactive oxygen species levels were measured with the ROS cellular test kit. BAX, BCL2 and caspase 3, 9, 12 mRNA expressions were evaluated by PCR, as well as BAX and BCL2 protein expressions by Western-Blot. At the fifth minute of the treatment, the viability was 68.15% with Akacid Plus and 43.95% with chlorhexidine. At the 15th minute, no significant difference was observed with both solutions. In the apoptotic evaluation, Akacid Plus significantly increased the early and late apoptotic activity in the cell line (p < 0.0001), while a significant increase was observed in late apoptosis and necrosis levels with chlorhexidine (p < 0.001). Chlorhexidine also induced gene expression of BAX, BCL2, caspase 3, 9 and BAX proteins (p < 0.05), while reducing protein expression of BCL2 (p < 0.001). Akacid Plus induced the gene expressions of BCL2, CASP3 and caspase 9, reduced gene expressions of BAX and caspase 12 and protein expression of BCL2 (p < 0.05). No significant difference was observed in the ROS level with both solutions (p > 0.05). Due to the widespread use of cationic polymers in ophthalmology, this new molecule with high antimicrobial activity and relatively low cytotoxicity may be of interest for clinical use. Further investigations are necessary to fully understand the ophthalmologic potential of this solution.

Phenylarsine oxide induced corneal injury involves oxidative stress mediated unfolded protein response and ferroptotic cell death: Amelioration by NAC

Phenylarsine oxide (PAO), an analog of lewisite, is a highly toxic trivalent arsenical and a potential chemical warfare agent. PAO-induced toxicity has been studied in lung, liver, and skin tissues. Nevertheless, very few studies have been published to comprehend the impact of PAO-induced toxicity on ocular tissues, even though eyes are uniquely vulnerable to injury by vesicants. Notably, arsenical vesicants such as lewisite have been shown to cause edema of eyelids, inflammation, massive corneal necrosis, and blindness. Accordingly, human corneal epithelial cells were used to study the effects of PAO exposure. PAO (100 and 200 nM) induced significant oxidative stress in corneal epithelial cells. Simultaneous treatment with N-acetyl-l-cysteine (NAC), an FDA-approved antioxidant, reversed the PAO-induced toxicity in human corneal epithelial cells. Furthermore, oxidative stress induction by PAO was accompanied by unfolded protein response (UPR) signaling activation and ferroptotic cell death. Further, to validate the findings of our in vitro studies, we optimized injury biomarkers and developed an ex vivo rabbit corneal culture model of PAO exposure. Investigations using PAO in ex vivo rabbit corneas revealed similar results. PAO (5 or 10 μg) for 3, 5, and 10 min caused moderate to extensive corneal epithelial layer degradation and reduced the epithelial layer thickness in a concentration- and time-dependent manner. Similar to human corneal cells, injuries by PAO in ex vivo cultured rabbit corneas were also associated with elevated oxidative stress, UPR signaling, and ferroptosis induction. NAC mitigated PAO-induced corneal injuries in rabbit ex vivo cornea culture as well. The reversal of PAO toxicity upon NAC treatment observed in our studies could be attributed to its antioxidant properties. These findings suggest that PAO exposure can cause significant corneal injury and highlight the need for further mechanistic studies to better understand the pathobiology of different arsenical vesicants, including PAO and lewisite.

A Comparison of Culture Results and Visual Acuity in Contact Lens Related Microbial Keratitis

Purpose Evaluate the effect of corneal and contact lens-related (CLR) culture results on visual acuity (VA) in patients with microbial keratitis (MK). Methods MK patients with corneal and CLR cultures were identified in the University of Michigan electronic health record from August 2012 to April 2022. Test results were classified as laboratory-positive or laboratory-negative. Linear regression was used to examine trends of VA and associations between changes in VA (differences of VA at 90-day and baseline VA) and corneal and CLR culture results, after adjustment for baseline VA. One-sample t-tests were used to test if the slope estimates were different from zero. Results MK patients (n = 50) were on average 49 years old (standard deviation = 20.9), 56% female, and 90% White. Positive corneal and CLR cultures were reported in 60% and 64% of patients, respectively, and 38% reported both. The agreement rate between corneal and CLR culture results was 30% (n = 15/50). LogMAR VA improved over time in patients with positive corneal and CLR cultures (Estimate=-0.19 per 10-day increase, p = 0.002), and in those with negative corneal and positive CLR cultures (Estimate=-0.17 per 10-day increase, p = 0.004). Compared to patients with negative corneal and CLR cultures, there was a trend toward improvement in VA for patients with positive corneal and CLR cultures (Estimate=-0.68, p = 0.068), and those with negative corneal and positive CLR cultures (Estimate=-0.74, p = 0.059), after adjusting for baseline VA. Conclusions Positive CLR cultures are associated with significant improvement in VA over time. These additional cultures can provide guidance on appropriate antimicrobial selection, especially when corneal cultures are negative.

Deficiency in the Midst of Abundance: Xerophthalmia Case Series and Vitamin A Levels in a New England Hospital

Purpose: To describe 3 adult cases of keratitis secondary to vitamin A deficiency (VAD) in relation to vitamin A levels Dartmouth-Hitchcock Medical Center (DHMC) population and published literature. Methods: Records of 3 patients with xerophthalmia were reviewed. All serum vitamin A levels obtained at DHMC during the same time period of our 3 cases (2019–2020) were analyzed. Outcomes were examined by age and range of deficiency. Results: Three patients, with short gut syndrome, chronic esophagitis, and alcohol use disorder, presented with xerophthalmia over 1 year. Serum vitamin A levels were 6.4 μg/dL, 16.1 μg/dL, and undetectable (&lt;5.0 μg/dL), respectively (normal: 32.5–78.0 μg/dL). Findings ranged from conjunctival keratinization to corneal perforation. Corneal cultures in patient 3 grew methicillin-sensitive Staphylococcus aureus. The ocular surface improved significantly in 2 patients following vitamin A supplementation. Two patients died during treatment. Analysis of 1596 vitamin A levels at DHMC revealed 431 patients with VAD, including 158 levels at or below those of our presented symptomatic cases. Conclusions: Vitamin A deficiency can occur in adults in high resource settings and lead to severe ocular morbidities, and is commonly associated with comorbidities such as alcohol use disorder and gastrointestinal disease. Vitamin A supplementation improved ocular findings in 2 of our patients. VAD was identified in 431 patients at DHMC over one year, indicating a surprisingly large population of patients at risk for xerophthalmia.

Case Report of Candida Parapsilosis Keratitis After Cataract Surgery Leading to Corneal Perforation

Purpose:The objective of this study was to report and document a rare case ofCandida parapsilosiskeratitis that occurred following a routine clear corneal incision cataract surgery, ultimately resulting in a perforated corneal ulcer and subsequent need for a penetrating keratoplasty (PK). By presenting this case, the study aims to contribute to the existing literature and raise awareness about the potential risk ofC. parapsilosiskeratitis as a complication of cataract surgery.Methods:In this case report, we describe the presentation and management of a rare fungal infection following routine cataract extraction in an 80-year-old male patient. The patient initially received treatment for suspected endophthalmitis and bacterial keratitis but experienced worsening symptoms, including a small corneal perforation. Upon referral to the clinic, the patient exhibited a fluffy stromal infiltrate near the cataract wound site, an epithelial defect, and a hypopyon. Treatment with compounded topical 1% voriconazole was initiated but did not prevent the progression of the corneal perforation. Attempts to seal the perforation with corneal glue were unsuccessful, leading to the decision to perform a PK. During the surgery, corneal tissue was biopsied and cultured to identify the causative agent.Results:Following PK, the corneal cultures revealed the growth ofC. parapsilosis, confirming the fungal nature of the infection. The patient was then reinitiated on topical voriconazole for a prolonged course of 7 months. Throughout this period, the patient was closely monitored and followed up in the clinic. Fortunately, there were no recurrences of the fungal lesion during this time.Conclusions:No prior cases of a perforated corneal ulcer resulting fromC. parapsilosiskeratitis after routine cataract surgery have been reported in the literature to our knowledge. Our patient experienced postoperative complications characterized by recurrent inflammation, which was treated with potent and frequent topical steroids. This treatment masked the underlying mycotic infection and contributed to corneal perforation. Ultimately, the patient underwent a successful PK procedure to remove the infectious nidus, followed by an extended 7-month course of topical voriconazole. No recurrence of the lesion was observed. Therefore, clinicians should be vigilant for mycotic keratitis, specificallyC. parapsilosis, in patients who display late-onset, persistent inflammation and white plaques at the clear corneal incision site after cataract surgery. The combination of PK and an extended postoperative treatment with topical antifungal agents proves to be an effective strategy for managing corneal perforations caused byC. parapsilosiskeratitis.

Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway

Severe dry eye (SDE) can cause grievous damage of the ocular surface and result in vision impairment and even blindness. To investigate the fate of limbal stem cells in SDE and the underlying mechanism, we established an SDE rat model by removing the extraorbital and infraorbital lacrimal glands and maintaining them in a low-humidity environment. One month after the surgery, aqueous tear secretion was reduced dramatically, blood vessels invaded into the central cornea, and inflammatory cells infiltrated into the limbal stroma. The expression of K12 and Pax6 were down-regulated dramatically, while those of K10, Sprr1b, and MUC5AC were up-regulated in the corneal epithelium of the SDE rats. Cell proliferation in the limbal epithelium was up-regulated, while the stem/progenitor marker adenosine 5'-triphosphate-binding cassette member 2 and the limbal epithelial colony-forming efficiency were decreased in the SDE condition. Furthermore, the p38 mitogen-activated protein kinase signaling pathway was activated in the limbal corneal epithelium of SDE rats. The abnormal differentiation and stemness loss in the cornea epithelium could be reversed upon treatment with a p38 inhibitor in a SDE invivo model and invitro hyperosmolar corneal epithelial culture conditions. In conclusion, SDE can lead to limbal stem cell dysfunction, and p38 mitogen-activated protein kinase signaling pathway activation plays an essential role in this process.