Spiroplasma infection as a cause of severe congenital keratouveitis, cataract and glaucoma.

BACKGROUND: Only seven cases of ocular Spiroplasma infection have been reported to date, all presenting as congenital cataracts with concomitant intraocular inflammation. We describe the first case of Spiroplasma infection initially presenting as a corneal infiltrate. CASE PRESENTATION: A 1-month-old girl was referred for a corneal infiltrate in the left eye. She presented in our hospital with unilateral keratouveitis. Examination showed a stromal corneal infiltrate and dense white keratic precipitates in the left eye. Herpetic keratouveitis was suspected and intravenous acyclovir therapy was initiated. Two weeks later, the inflammation in the left eye persisted and was also noticed in the right eye. Acute angle-closure glaucoma and a cataract with dilated iris vessels extending onto the anterior lens capsule developed in the left eye. The inflammation resolved after treatment with azithromycin. Iridectomy, synechiolysis and lensectomy were performed. Bacterial metagenomic sequencing (16 S rRNA) and transmission electron microscopy revealed Spiroplasma ixodetis species in lens aspirates and biopsy. Consequently, a diagnosis of bilateral Spiroplasma uveitis was made. CONCLUSIONS: In cases of congenital cataract with concomitant intraocular inflammation, Spiroplasma infection should be considered. The purpose of this case report is to raise awareness of congenital Spiroplasma infection as a cause of severe keratouveitis, cataract and angle-closure glaucoma in newborns. Performing molecular testing on lens aspirates is essential to confirm diagnosis. Systemic macrolides are suggested as the mainstay of treatment.

Formononetin protects against Aspergillus fumigatus Keratitis: Targeting inflammation and fungal load.

PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

BACKGROUND: Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. OBJECTIVES: The aim of this study was to explore a possible pathogenic role of DON in FK. METHODS: We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. RESULTS: The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. CONCLUSIONS: DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

The Therapeutic Role and Mechanism of Glabridin Under Aspergillus fumigatus Infection.

Purpose: To characterize the efficiency of glabridin alone and in combination with clinical antifungals in Aspergillus fumigatus keratitis. Methods: The broth microdilution method was performed to investigate whether glabridin exerted an antifungal role on planktonic cells and immature and mature biofilm. Antifungal mechanism was evaluated by Sorbitol and Ergosterol Assays. The synergistic effect of glabridin and antifungals was assessed through the checkerboard microdilution method and time-killing test. Regarding anti-inflammatory role, inflammatory substances induced by A. fumigatus were assessed by real-time quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Drug toxicity was assessed by Draize test in vivo. Macrophage phenotypes were examined by flow cytometry. Results: Regarding antifungal activity, glabridin destroyed fungal cell wall and membrane on planktonic cells and suppressed immature and mature biofilm formation. After combining with natamycin or amphotericin B, glabridin possessed a potent synergistic effect against A. fumigatus. Regarding anti-inflammatory aspects, Dectin-1, toll­like receptor (TLR)-2 and TLR-4 expression of human corneal epithelial cells were significantly elevated after A. fumigatus challenge and reduced by glabridin. The elevated expression of interleukin-1ß and tumor necrosis factor-alpha induced by A. fumigatus or corresponding agonists were reversed by glabridin, equivalent to the effect of corresponding inhibitors. Glabridin could also contribute to anti-inflammation by downregulating inflammatory mediator expression to suppress macrophage infiltration. Conclusions: Glabridin contributed to fungal clearance by destroying fungal cell wall and membrane, and disrupting biofilm. Combining glabridin with clinical antifungals was superior in reducing A. fumigatus growth. Glabridin exerted an anti-inflammatory effect by downregulating proinflammatory substance expression and inhibiting macrophage infiltration, which provide a potential agent and treatment strategies for fungal keratitis.

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.

The role of corneal biopsy in the management of microbial keratitis.

PURPOSE: Corneal biopsy helps in diagnosing deep-seated or recalcitrant lesions of microbial keratitis (MK). We aim to analyze its role in managing these challenging cases. METHODS: This is a retrospective review of 22 cases of corneal biopsy at our institute from January 2010 to December 2021. Data were retrospectively collected using the electronic medical record (EMR) system. Those cases of indolent, progressive MK or deep-seated lesions where cornea scraping was not possible were considered for corneal biopsy to establish the microbiological diagnosis. The primary aims of our study were to analyze the indications, success rates, and outcomes for biopsy patients in our series. Additional outcomes that were analyzed included the average time from presentation to biopsy, the type of causative organism isolated from the biopsy by either histopathological or microbiological method, and the frequency and outcome of surgical interventions performed. Descriptive statistics using mean (±standard deviation) and median (±range) were used to interpret the demographic data. RESULTS: Overall, 15 of 22 patients (68%) had a positive corneal biopsy after microbiological or histopathological examinations. The most identified organism was microsporidia (n = 4,30.7%), followed by mycobacteria (n = 2,15.4%), gram-negative bacilli (n = 2,15.4%), acid-fast bacilli (n = 1,7.6%), fungus (n = 2,15.4%), gram-positive cocci (n = 1,7.6%), and mixed bacterial infection (n = 1,7.6%). CONCLUSION: Corneal biopsy should be considered a diagnostic modality for patients with deep-seated or unresponsive MK. It can improve the treatment for MK, ensuring targeted therapy.

Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway.

PURPOSE: The purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. METHODS: In order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit-lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6 were determined using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and IF techniques. RESULTS: Schaftoside at a concentration of 160 µM displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1ß, TNF-α, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. CONCLUSIONS: Schaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.

Microbial keratitis in lattice corneal dystrophy: microsporidia as a new cause.

A patient in his sixth decade presented to us with redness, pain and a deterioration of vision in his left eye. He had previously been diagnosed with lattice corneal dystrophy (LCD). He was diagnosed with microbial keratitis, and mixed infection was confirmed on culture (bacteria and fungus) with a protracted healing period before resolution of keratitis. He presented 2 years later with similar issues in the same eye and was noted to have a second episode of microbial keratitis, with microsporidia spores noted on gram, potassium hydroxide and calcofluor white stains. He was diagnosed with microsporidial stromal keratitis and underwent therapeutic penetrating keratoplasty. Unfortunately, he suffered a recurrence of microsporidial keratitis following surgery with eventual transplant failure. Microsporidia as an infection in LCD has, to our knowledge, not been previously reported. We aim to discuss microsporidial infection and recurrent microbial keratitis in the setting of LCD.

Quercetin protects against Aspergillus fumigatus keratitis by reducing fungal load and inhibiting TLR-4 induced inflammatory response.

PURPOSE: To investigate the antifungal and anti-inflammatory effects of quercetin in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Draize eye test was performed in mice to evaluate the toxicity of quercetin, and the antifungal effects on A. fumigatus were assessed via scanning electron microscopy (SEM), propidium iodide uptake, and adherence assay. In fungal keratitis (FK) mouse models, immunostaining was performed for investigating toll-like receptor 4 (TLR-4) expression and macrophage infiltration. Real-time PCR, ELISA, and Western blot were used to evaluate the expression of pro-inflammatory factors IL-1ß, TNF-α, and IL-6 in infected RAW264.7 cells. Cells were also treated with TLR-4 siRNA or agonist CRX-527 to investigate mechanisms underlying the anti-inflammatory activity of quercetin. RESULTS: Quercetin at 32 µM was non-toxic to corneal epithelial and significantly inhibited A. fumigatus growth and adhesion, and also altered the structure and reduced the number of mycelia. Quercetin significantly reduced macrophage infiltration in the mouse cornea, and attenuated the expression of TLR-4 in the corneal epithelium and stroma of mice with keratitis caused by A. fumigatus. In RAW264.7 cells infected by A. fumigatus, quercetin downregulated TLR-4 along with pro-inflammatory factors IL-1ß, TNF-α, and IL-6. RAW cells with TLR-4 knockdown had reduced expression of factors after A. fumigatus infection, which was decreased even further with quercetin treatment. In contrast, cells with CRX-527 had elevated inflammatory factors compared to control, which was significantly attenuated in the presence of quercetin. CONCLUSION: Quercetin plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal load, disrupting hyphae structure, macrophage infiltration, and suppressing inflammation response in macrophages via TLR-4 mediated signaling pathway.

0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response.

Purpose: To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis. Methods: The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid-Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting. Result: In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1 minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid-Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1ß. Conclusions: According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1ß expression; and provided a potential treatment for fungal keratitis. Translational Relevance: This study provides a potential treatment for fungal keratitis in the clinic.

A Case Series of Infectious Keratitis After Corneal Cross-linking.

PURPOSE: To present the 7-year experience of a tertiary eye hospital while exploring possible risk factors and incidence of infectious keratitis in patients undergoing standard corneal cross-linking (CXL). METHODS: This retrospective cohort study included patients with progressive keratoconus undergoing standard CXL in the Farabi Eye Hospital and all other patients who had undergone CXL in other facilities and were diagnosed as having infectious keratitis in the 7-year period of the study. RESULTS: Among the total of 4,863 eyes that underwent CXL, 6 eyes developed infectious keratitis, yielding an incidence rate of 0.12%. Additionally, 13 eyes from 10 patients with a CXL history in other facilities who developed infectious keratitis were included. The mean age was 23.75 years, and 75% of patients were men and 25% were women. Gram-positive bacteria and Staphylococcus aureus were the most prevalent pathogens. Meibomian gland dysfunction, dry eye disease, or blepharitis were present in 12 patients. Medical treatment did not arrest the disease progress in 5 patients, which eventually required cases to undergo keratoplasty. CONCLUSIONS: This study supports the need for proper patient selection by using a comprehensive medical history. It also highlights the imperative role of rigorous patient education and follow-up, particularly in the first postoperative week. Finally, the study emphasizes aggressive early therapy for patients with suspicious findings. [J Refract Surg. 2023;39(8):564-572.].

[Clinical analysis of corneal interface infection].

Objective: To analyze the clinical features of corneal interface infection. Methods: A retrospective case series study was conducted to explore the clinical features of interstitial corneal infection. The data of eight patients (eight eyes) who were diagnosed with interstitial corneal infection after undergoing corneal transplant or corneal refractive surgery and visited Beijing Tongren Eye Center from January to December 2018 were collected, including two male and six female patients aged between 18 and 55 years (median age, 27 years). The patients' general information, surgical type, onset time, and clinical manifestations were recorded. The lesions were examined by in vivo corneal laser confocal microscopy (IVCM), and microbial cultures and drug sensitivity tests were performed. Results: Among the 8 patients, 4 had undergone small-incision lenticule extraction (SMILE), 2 had undergone lamellar keratoplasty, and 2 had undergone endothelial keratoplasty. The onset of infection occurred between 2 and 30 days after surgery, with a mean of 9.8 days. Among the 3 patients who had undergone SMILE, the treatment outcome was corneal haze or opacity, while the remaining 5 cases required corneal transplantation for interstitial infections. The pathogens of the 4 cases of interstitial infection after corneal transplantation were all Candida species. Under the IVCM, patients with corneal interstitial bacterial infections showed a large amount of necrotic tissue with no normal tissue structure in the corneal stroma, with infiltration of inflammatory cells and local aggregation of inflammatory cells, but no typical pathogen was observed. Patients with fungal infections showed fungal hyphae under the corneal cap (filamentous fungal infection) or dense, punctate, high-reflection structures in the corneal interstitial space (yeast-like fungal infection). Conclusions: Corneal interlayer infection is difficult to diagnose early and has a poor prognosis. IVCM can assist in early diagnosis. The pathogen spectrum of corneal interlayer infection may differ from that of corneal infection caused by trauma.

Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis: Antibacterial and Corneal Wound Healing.

The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range.

Repeated sessions of PACK-CXL WA for the treatment of resistant bacterial keratitis: a retrospective study.

OBJECTIVE: The aim of this work is to evaluate the safety and efficacy of repeated sessions of photo-activated chromophore for keratitis-cross linking (PACK-CXL) window absorption (WA) for the treatment of resistant bacterial keratitis (BK). PATIENTS AND METHODS: This is a retrospective clinical cohort study. Thirty eyes with clinically suspected and lab-confirmed bacterial keratitis, resistant to appropriate antibiotic therapy- which was modified by sensitivity reports- for 2 weeks with failure of epithelialization for 4 weeks after the standard anti-microbial therapy (SAT) together with one setting of PACK-CXL WA were included. If after the first session of PACK-CXL, there is a start of improvement in the form of reduction of the size of corneal ulcer and stromal infiltrates together with the start of epithelialization on clinical examination and AS-OCT, another session of PACK-CXL WA was performed after one week, and so on, till the complete healing and resolution of bacterial keratitis and confirmation by negative bacterial culture. Identification of the micro-organisms was done by lab study before and after treatment. Corneal healing was evaluated by corneal examination and anterior segment OCT (AS-OCT). RESULTS: Thirty eyes of 30 patients were recruited in this study. They were 16 males and 14 females, their mean age was 44.3 ± 5.38 years. The mean ulcer size was 3.96 ± 1.87 (mm3), while the mean size of stromal infiltrates was 4.52 ± 2.24 (mm3). PACK-CXL WA treatment was performed an average of 2.87 times for the 30 eyes. Complete healing and resolution (Successful treatment) was observed in 27 eyes (90%) of cases and failure of epithelialization was observed only in 3 eyes (10%). Complete corneal healing was reported in the second month postoperatively in 90% of eyes. CONCLUSION AND RECOMMENDATION: PACK-CXL WA may be a promising, non-invasive treatment option for resistant bacterial keratitis. It may have a synergistic effect with standard antimicrobial treatment (SAT). Also, it can overcome the antibiotics resistance that has become rapidly spreading worldwide. Repeated sessions of PACK-CXL WA may be more effective for the treatment of resistant bacterial keratitis till complete epithelialization and resolution of BK than a single session with few complications. However, further prospective and comparative studies to support the results are needed.

A positive feedback cycle between the alarmin S100A8/A9 and NLRP3 inflammasome-GSDMD signalling reinforces the innate immune response in Candida albicans keratitis.

OBJECTIVE: Fungal keratitis is a severe sight-threatening ocular infection, without effective treatment strategies available now. Calprotectin S100A8/A9 has recently attracted great attention as a critical alarmin modulating the innate immune response against microbial challenges. However, the unique role of S100A8/A9 in fungal keratitis is poorly understood. METHODS: Experimental fungal keratitis was established in wild-type and gene knockout (TLR4-/- and GSDMD-/-) mice by infecting mouse corneas with Candida albicans. The degree of mouse cornea injuries was evaluated by clinical scoring. To interrogate the molecular mechanism in vitro, macrophage RAW264.7 cell line was challenged with Candida albicans or recombinant S100A8/A9 protein. Label-free quantitative proteomics, quantitative real-time PCR, Western blotting, and immunohistochemistry were conducted in this research. RESULTS: Herein, we characterized the proteome of mouse corneas infected with Candida albicans and found that S100A8/A9 was robustly expressed at the early stage of the disease. S100A8/A9 significantly enhanced disease progression by promoting NLRP3 inflammasome activation and Caspase-1 maturation, accompanied by increased accumulation of macrophages in infected corneas. In response to Candida albicans infection, toll-like receptor 4 (TLR4) sensed extracellular S100A8/A9 and acted as a bridge between S100A8/A9 and NLRP3 inflammasome activation in mouse corneas. Furthermore, the deletion of TLR4 resulted in noticeable improvement in fungal keratitis. Remarkably, NLRP3/GSDMD-mediated macrophage pyroptosis in turn facilitates S100A8/A9 secretion during Candida albicans keratitis, thus forming a positive feedback cycle that amplifies the proinflammatory response in corneas. CONCLUSIONS: The present study is the first to reveal the critical roles of the alarmin S100A8/A9 in the immunopathology of Candida albicans keratitis, highlighting a promising approach for therapeutic intervention in the future.

An X-ray inactivated vaccine against Pseudomonas aeruginosa Keratitis in mice.

Pseudomonas aeruginosa (P. aeruginosa) is one of the most prevalent pathogens of bacterial keratitis. Bacterial keratitis is a major cause of blindness worldwide. The rising incidence of multidrug resistance of P. aeruginosa precludes treatment with conventional antibiotics. Herein, we evaluated the protective efficiency and explored the possible underlying mechanism of an X-ray inactivated vaccine (XPa) using a murine P. aeruginosa keratitis model. Mice immunized with XPa exhibit reduced corneal bacterial loads and pathology scores. XPa vaccination induced corneal macrophage polarization toward M2, averting an excessive inflammatory reaction. Furthermore, histological observations indicated that XPa vaccination suppressed corneal fibroblast activation and prevented irreversible visual impairment. The potency of XPa against keratitis highlights its potential utility as an effective and promising vaccine candidate for P. aeruginosa.

Effect of Corneal Collagen Cross-Linking on Subsequent Corneal Fungal Infection in Rats.

Purpose: The purpose of this study was to determine whether corneal collagen cross-linking (CXL) alters fungal susceptibility and increases the severity of keratitis through macrophage activation in rats. Methods: Four weeks following CXL pretreatment, the corneal epithelium of adult rats was removed and inoculated with Candida albicans (C. albicans; CXL + inoculation group). The non-CXL-pretreated corneas were also inoculated with C. albicans (inoculation group). Clinical scoring and histopathological examination were performed to determine the severity of fungal keratitis. Immunofluorescence and confocal microscopy imaging were applied to determine the effects of CXL treatment on corneal local macrophage content. Real-time polymerase chain reaction (RT-PCR) and Western blots were used to evaluate mRNA and protein expression. Flow cytometry assays were performed to detect M1- and M2-type macrophages. Results: CXL pretreatment (CXL + inoculation) resulted in higher infection success rate and more severe fungal keratitis than inoculation alone (inoculation group). On days 1, 3, and 7 following fungal infection, the increase in macrophage infiltration and IL-1ß, MMP-9, and VEGFA expression was greater in the CXL + inoculation group than in the inoculation group. Number of M1- and M2-type macrophages, M1 to M2 ratio, M1-type macrophage genes, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNFα) expression were higher in the CXL + inoculation group compared with the inoculation group. Conclusions: Our data demonstrate that CXL may increase the colonization of macrophages and activate more M1-type macrophages to increase fungal susceptibility and severity of keratitis. Translational Relevance: This study may aid long-term risk assessment and treatment of the complications of CXL.

Trachipleistophora hominis: molecular characterization of a rare pathogen causing microsporidial stromal keratitis.

The authors report the clinical and microbiological findings of a unique case of stromal keratitis caused by a rare microsporidium, Trachipleistophora hominis. This case of stromal keratitis was in a 49-year-old male with a history of COVID-19 infection and diabetes mellitus. Corneal scraping specimens revealed numerous microsporidia spores upon microscopic examination. PCR of the corneal button revealed the presence of T. hominis infection, which could be controlled by penetrating keratoplasty surgery. The graft was clear with no recurrence of infection until the last follow-up 6 weeks postsurgery. This is the first case of human stromal keratitis caused by this organism in a post-COVID infection, confirmed by molecular diagnosis.

Thymosin beta 4: A potential novel adjunct treatment for bacterial keratitis.

Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification or scarring, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts.Pseudomonas aeruginosaandStaphylococcus aureusare the two bacteria most commonly associated with this type of infection. Risk factors include patients who are immunocompromised, those who have undergone refractive corneal surgery, and those with prior penetrating keratoplasty, as well as extended wear contact lens users. Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics. Bacterial clearance is of utmost importance yet does not guarantee good visual outcome. Clinicians are often left to rely upon the eye's innate ability to heal itself, as there are limited options beyond antibiotics and corticosteroids for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, and anti-inflammatory drops, do not fully accommodate clinical needs and have many potential harmful complications. To this end, treatments are needed that both regulate the inflammatory response and promote corneal wound healing to resolve visual disturbances and improve quality of life. Thymosin beta 4 is a small, naturally occurring 43-amino-acid protein that promotes wound healing and reduces corneal inflammation and is currently in Phase 3 human clinical trials for dry eye disease. Our previous work has shown that topical Tß4 as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages) while enhancing bacterial killing and wound healing pathway activation in an experimental model ofP. aeruginosa-induced keratitis. Adjunctive thymosin beta 4 treatment holds novel therapeutic potential to regulate and, optimally, resolve disease pathogenesis in the cornea and perhaps other infectious and immune-based inflammatory disease. We plan to establish the importance of thymosin beta 4 as a therapeutic agent in conjunction with antibiotics with high impact for immediate clinical development.