Search

Vermamoeba vermiformis (V. vermiformis) is one of the most common free-living amoeba (FLA) and is frequently found in environments such as natural freshwater areas, surface waters, soil, and biofilms. V. vermiformis has been reported as a pathogen with pathogenic potential for humans and animals. The aim is to report a case of non-Acanthamoeba keratitis in which V. vermiformis was the etiological agent, identified by culture and molecular techniques. Our case was a 48-year-old male patient with a history of trauma to his eye 10 days ago. The patient complained of eye redness and purulent discharge. A slit-lamp examination of the eye revealed a central corneal ulcer with peripheral infiltration extending into the deep stroma. The corneal scraping sample taken from the patient was cultured on a non-nutritious agar plate (NNA). Amoebae were evaluated according to morphological evaluation criteria. It was investigated by PCR method and confirmed by DNA sequence analysis. Although no bacterial or fungal growth was detected in the routine microbiological evaluation of the corneal scraping sample that was cultured, amoeba growth was detected positively in the NNA culture. Meanwhile, Acanthamoeba was detected negative by real-time PCR. However, V. vermiformis was detected positive with the specific PCR assay. It was confirmed by DNA sequence analysis to be considered an etiological pathogenic agent. Thus, topical administration of chlorhexidine gluconate %0.02 (8 × 1) was initiated. Clinical regression was observed 72 h after chlorhexidine initiation, and complete resolution of keratitis with residual scarring was noticed in 5 weeks. In conclusion, corneal infections due to free-living amoebae can occur, especially in poor hygiene. Although Acanthamoeba is the most common keratitis due to amoeba, V. vermiformis is also assumed to associate keratitis in humans. Clinicians should also be aware of other amoebic agents, such as V. vermiformis, in keratitis patients., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A 32-year-old contact lens-wearing man with recent travel history to the Caribbean was referred for a corneal infiltrate in the left eye that worsened following 1-week of steroid-antibiotic therapy. Corneal cultures were obtained and sent to our facility's clinical microbiology laboratory for analysis. Same-day in vivo confocal microscopy revealed fungal elements. Nucleic acid sequencing performed on the isolated determined it to be a member of the entomopathogenic genus Metarhizium. Over the course of 3 months, the patient's corneal infiltrate ultimately resolved following topical natamycin 5 % therapy. This is the first reported case to have originated in the Caribbean and to utilize in vivo confocal microscopy to aid diagnosis. Our case also supports previous reports of success with natamycin therapy in treatment of Metarhizium sp. keratitis., Competing Interests: Declaration of competing interest The authors do not have any relevant conflicts of interest to disclose, (Copyright © 2024. Published by Elsevier Inc.)

To evaluate the efficacy of topical vancomycin and povidone iodine (PI) application on methicillin-resistant Staphylococcus aureus (MRSA) keratitis model in rabbits., MRSA keratitis was induced by injecting 0.1 mL MRSA containing 1000 colony-forming units (CFU) into central cornea of right eyes of 24 New Zealand White rabbits. Animals were divided into four groups ( n  = 6): control (treated with balanced salt solution), 50 mg/mL topical vancomycin, 5% topical PI, and combination; examined before and after treatment, and corneal tissues were harvested for analysis at 9th hour of treatment., Bacterial load was determined as: 7.63 ± 0.82 log10 CFU/g in control group, 6.95 ± 1.66 log10 CFU/g in PI group, 4.67 ± 0.77 log10 CFU/g in combination group, and 4.33 ± 0.71 log10 CFU/g in vancomycin group ( p  = 0.001). Median of total clinical score increased significantly from 7 [range: 5-8] to 11.5 [range: 11-15] ( p  = 0.001) in control group, did not change (6 [range: 5-8] to 7 [range: 5-7]; p  = 0.695) in vancomycin group, increased significantly from 7 [range: 5-8] to 12.5 [range: 10-14] ( p  < 0.001) in PI group, increased significantly from 6.5 [range: 5-7] to 8 [range: 7-9] in combination group ( p  = 0.002). Post-treatment clinical scores for chemosis, conjunctival injection, iritis, hypopyon, epithelial erosion, and corneal infiltrate were significantly lower in vancomycin-treated groups compared to others ( p  < 0.05). In PI-treated groups, especially scores for chemosis, conjunctival injection, epithelial erosion and corneal infiltrate were significantly higher than vancomycin ( p  < 0.05)., Topical vancomycin significantly inhibited bacterial growth in MRSA keratitis. However, PI was ineffective in controlling this growth; additionally, exerted toxic effect on ocular surface. When vancomycin was combined with PI, no additional increase in efficacy of treatment was detected compared to only vancomycin.

Background: Keratomycosis is a form of infectious keratitis, an infection of the cornea, which is caused by fungi. This disease is a leading cause of ocular morbidity globally with at least 60 % of the affected individuals becoming monocularly blind., Objective: This bibliometric analysis aimed to comprehensively assess the existing body of literature, providing insights of the evolution of keratomycosis research by identifying key themes and research gaps., Methods: This work used the modeling method Latent Dirichlet Allocation (LDA) to identify and interpret scientific information on topics concerning existing categories in a set of documents. The HJ-Biplot method was also used to determine the relationship between the analyzed topics, taking into consideration the years under study., Results: This bibliometric analysis was performed on a total of 2,599 scientific articles published between 1992 and 2022. The five leading countries with more scientific production and citations on keratomycosis were The United States of America, followed by India, China, United Kingdom and Australia. The top five topics studied were Case Reports and Corneal Infections, which exhibited a decreasing trend; followed by Penetrating Keratoplasty and Corneal Surgery, Ocular Effects of Antifungal Drugs, Gene Expression and Inflammatory Response in the Cornea and Patient Data which have been increasing throughout the years. However Filamentous Fungi and Specific Pathogens, and Antifungal Therapies research has been decreasing in trend., Conclusion: Additional investigation into innovative antifungal drug therapies is crucial for proactively tackling the potential future resistance to antifungal agents in scientific writing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microbial keratitis (MK) is an infection of the cornea, caused by bacteria, fungi, parasites, or viruses. MK leads to significant morbidity, being the fifth leading cause of blindness worldwide. There is an urgent requirement to better understand pathogenesis in order to develop novel diagnostic and therapeutic approaches to improve patient outcomes. Many in vitro, ex vivo and in vivo MK models have been developed and implemented to meet this aim. Here, we present current in vitro and ex vivo MK model systems, examining their varied design, outputs, reporting standards, and strengths and limitations. Major limitations include their relative simplicity and the perceived inability to study the immune response in these MK models, an aspect widely accepted to play a significant role in MK pathogenesis. Consequently, there remains a dependence on in vivo models to study this aspect of MK. However, looking to the future, we draw from the broader field of corneal disease modelling, which utilises, for example, three-dimensional co-culture models and dynamic environments observed in bioreactors and organ-on-a-chip scenarios. These remain unexplored in MK research, but incorporation of these approaches will offer further advances in the field of MK corneal modelling, in particular with the focus of incorporation of immune components which we anticipate will better recapitulate pathogenesis and yield novel findings, therefore contributing to the enhancement of MK outcomes., Competing Interests: Declaration of competing interest The authors jointly do not have any competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The objective of this study was to investigate the culture positivity and distribution of the conjunctival sac bacteria in the perioperative period of corneal refractive surgery. The selected time points of the perioperative period included before the use of antibiotic eye drops, before eye wash (after the use of antibiotic eye drops), after eye wash, and immediately after surgery. Conjunctival specimens obtained at the four time points were cultured to detect the positivity and distribution of bacteria. Before prophylactic antibiotic eye drops were administered, 49 eyes (50%) had positive bacterial culture results, with 45 isolates (91.8%) identified as Staphylococcus epidermidis. The culture positivity rates of the conjunctival sac specimens before eye wash, after eye wash, and immediately after surgery were 19.4%, 3.1%, and 4.1%, respectively. The difference was significant before and after the use of antibiotics and before and after eye wash (both P < 0.001). Staphylococcus epidermidis was the major pathogen in the conjunctival sac before corneal refractive surgery, and the culture positivity rate of the conjunctival bacteria was higher in males. Sixteen of 37 eyes (43.2%) with contact lenses had positive culture results, compared to 33 of 61 eyes (54.1%) without contact lenses (P > 0.05). The judicious preoperative use of antibiotic eye drops combined with the surgical sterile eye wash procedure maximised the removal of conjunctival sac bacteria. Skilled surgical manipulations generally did not increase the risk of infection., (© 2024. The Author(s).)

Purpose: To describe the clinical features, management, and long-term outcome of Infectious crystalline keratopathy (ICK)., Methods: The medical records of clinically diagnosed and microbiologically proven cases of ICK were reviewed from January 2011 to December 2022. Clinical characteristics include the presence of whitish needle-like projections with branching, limited to anterior-mid stroma. Keratoplasty being the most common risk factor, graft-related microbial keratitis during the same period was also studied. The demography, clinical profile, microbiology, treatment, and outcome were analyzed, and compared with secondary graft infiltrate(GI)., Results: Medical records of 24 cases with ICK were reviewed. The mean age was 49.3 ± 20.1 years, with 15(62.5%) males. Prior keratoplasty was done in 18 (75%) cases, with a mean graft size of 10.1 ± 1.5 mm, and mean interval between the last graft and presentation was 9.7 ± 6.2 (3-90) months. In comparison to GI (n = 24), ICK patients (n = 18,75%) were less symptomatic, presented late (7.3 ± 6.5 days vs 16.3 ± 19.4, p = 0.003), using frequent topical steroids (> 3 times/day, p =  0.006), smaller infiltrate size < 4 mm (p =  0.008), central (p =  0.02), less associated with epithelial defect (p =  0.0001), hypopyon (p =  of 0.0002), corneal perforation (p =  0.0006), and surgical management (p =  0.03). On microbiology, 22 (91.6%) ICK cases were culture positive, 14 (63.6%) gram-positive, 3 (13.6%) gram-negative, 2 (9%) mixed bacteria, and 3 (13.6%) fungus, comparable with GI., Conclusion: ICK affects poor ocular surfaces usually following keratoplasty with larger graft size, the use of steroids being the most common association, and it responds to medical management as compared to GI., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: To study the risk factors, clinical features, and treatment outcomes of patients with culture-negative keratitis (CNK)., Methods: A retrospective data review of 933 patients with CNK was performed from January 2018 to December 2020. The variables such as the history of injury, visual acuity, slit-lamp findings with measurements of size and depth of ulcer, microbiological evaluation, duct patency, blood glucose levels, and treatment were considered, and clinical outcome was analyzed., Results: Of the 933 patients with CNK, 763 (81.8%) were medically managed, with a mean treatment duration of 2.08 ± 1.7 weeks. Among them, 622 (66.7%) were both smear and culture-negative, and 311 (33.3%) showed only smear positivity. Smear-positive patients showed a positive correlation with the history of injury. A higher incidence of fungal growth on repeat culture was observed. Surgical interventions were done only in 18.2% of the patients; the rest were treated with topical medications alone., Conclusion: High clinical suspicion, differentiation of causative organisms based on clinical findings, and initiating empirical therapy with broad-spectrum antibiotics and antifungals improve the ultimate prognosis in patients with CNK, even though a standard protocol for empirical medical treatment may differ among institutions and surgeons based on their clinical experience and geographical variations., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Purpose: To assess outcomes of keratoplasty performed in patients diagnosed with keratitis caused by Pythium insidiosum (PI)., Design: Retrospective review., Methods: Preoperative, intra operative and post operative data of patients diagnosed with PI keratitis and who underwent keratoplasty for their condition from January 2020 to December 2021 were collected from the central patient database of a tertiary eye care hospital in India. The data were analyzed for anatomic success, elimination of infection, graft survival, incidence of repeat keratoplasty, final visual acuity and varied complications., Results: In total, 16 eyes underwent penetrating keratoplasty for PI keratitis during the study period. Mean time to keratoplasty from onset of symptoms was 31.3 days and mean graft size was 10.4 mm. Nine out of the 16 cases had recurrence of infection following surgery, seven of which required a repeat keratoplasty for elimination of infection. Mean graft size for repeat keratoplasty performed in recurrent cases was 11.7 mm. Globe was successfully salvaged in 14 out of 16 patients (87.5 %). Three grafts remained clear at 6-month follow up while 11 grafts failed. Mean improvement in uncorrected visual acuity from 2.32 to 2.04 logMAR was observed at last follow up. Endo-exudates, graft infiltration, graft dehiscence, secondary glaucoma and retinal detachment were the various complications noted after keratoplasty., Conclusion: PI keratitis is a tenacious and potentially blinding condition. Keratoplasty remains the choice of treatment in this condition, however recurrence of disease and graft failure are common. Large sized grafts, meticulous per-operative removal of infection, adjuvant cryotherapy, and intraoperative and post operative use of antibiotics can help in improving outcome of keratoplasty in these patients., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Fungal keratitis is a severe corneal infection characterized by suppurative and ulcerative lesions. Aspergillus fumigatus is a common cause of fungal keratitis. Antifungal drugs, such as natamycin, are currently the first-line treatment for fungal keratitis, but their ineffectiveness leads to blindness and perforation. Additionally, the development of fungal resistance makes treating fungal keratitis significantly more challenging. The present study used platelet-derived biomaterial (PDB) to manage A. fumigatus keratitis in the animal model. Freezing and thawing processes were used to prepare PDB, and then A. fumigatus keratitis was induced in the mice. Topical administration of PDB, natamycin, and plasma was performed; quantitative real-time PCR (qPCR) and histopathologic examination (HE) were used to assess the inhibitory effect of the mentioned compounds against fungal keratitis. The qPCR results showed that PDB significantly decreased the count of A. fumigatus compared to the control group (P-value ≤ 5). Natamycin also remarkably reduced the count of fungi in comparison to the untreated animal, but its inhibitory effect was not better than PDB (P-value > 5). The findings of HE also demonstrated that treatment with PDB and natamycin decreased the fungal loads in the corneal tissue. However, plasma did not show a significant inhibitory effect against A. fumigatus. PDB is intrinsically safe and free of any infections or allergic responses; additionally, this compound has a potential role in decreasing the burden of A. fumigatus and treating fungal keratitis. Therefore, scientists should consider PDB an applicable approach to managing fungal keratitis and an alternative to conventional antifungal agents., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Infectious keratitis, a significant contributor to blindness, with fungal keratitis accounting for nearly half of cases, poses a formidable diagnostic and therapeutic challenge due to its delayed clinical presentation, prolonged culture times, and the limited availability of effective antifungal medications. Furthermore, infections caused by rare fungal strains warrant equal attention in the management of this condition., Case Presentation: A case of fungal keratitis was presented, where corneal scraping material culture yielded pink colonies. Lactophenol cotton blue staining revealed distinctive spore formation consistent with the Fusarium species. Further analysis using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) identified the causative agent as Fusarium proliferatum. However, definitive diagnosis of Pseudonectria foliicola infection was confirmed through ITS sequencing. The patient's recovery was achieved with a combination therapy of voriconazole eye drops and itraconazole systemic treatment., Conclusion: Pseudonectria foliicola is a plant pathogenic bacterium that has never been reported in human infections before. Therefore, ophthalmologists should consider Pseudonectria foliicola as a possible cause of fungal keratitis, as early identification and timely treatment can help improve vision in most eyes., (© 2024. The Author(s).)

Infectious keratitis is a leading cause of corneal blindness worldwide with little information known about causative etiologies in Malawi, Africa. This area is resource-limited with ophthalmologist and microbiology services. The Department of Ophthalmology at the Kamuzu College of Health Sciences in Blantyre, Malawi, is a participating site of an international corneal ulcer consortium, capriCORN (Comprehensive Analysis of Pathogens, Resistomes, and Inflammatory-markers in the CORNea). In this study, 50 patients with corneal ulcers were swabbed for pathogen identification using RNA-sequencing. Corneal trauma was reported in 41% and 19% of the patients worked in agriculture. A pathogen was identified in 58% of the cases. Fungal pathogens predominated, followed by viruses and bacteria. Aspergillus, Fusarium, HSV-1, and Gardnerella were the most common pathogens detected. 50% of patients reported treatment with an antibiotic before presentation. Pathogens unusual for infectious keratitis, such as Subramaniula asteroids, Aureobasidium pullulans, and Gardnerella vaginalis, were also detected.

Purpose: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis., Methods: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1β, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence., Results: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1β and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody., Conclusions: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

Purpose: This study aimed to evaluate the efficacy and safety of McCarey-Kaufman (MK) medium supplemented with colistin and amphotericin B in inhibiting the growth of multidrug-resistant Pseudomonas (P.) aeruginosa , using an ex vivo experimental model with human donor corneas., Methods: Cadaveric human corneas deemed unsuitable for corneal transplantation were obtained, and MK media were supplemented with colistin and amphotericin B. Multidrug-resistant P. aeruginosa was cultured and used to infect the human donor corneas ex vivo . Infected corneas were placed in the MK media with additional antibiotics (colistin and amphotericin B) and the standard MK media, which served as the control arm for comparison. Corneal opacity due to infiltration and quantitative analysis of colony-forming units (CFUs) were assessed. The viability of the corneal endothelium was assessed using trypan blue staining., Results: Corneas incubated in MK media supplemented with additional antibiotics showed less corneal opacification compared with those in standard MK media at both 48- and 96-hour (hr) time points. Quantitative analysis revealed a lower bacterial load and a significant reduction in CFU in the corneas incubated in MK media with additional antibiotics compared with the control group. At 48 hrs, there was 84% ( P value = 0.024) reduction in bacterial load, and at 96 hr, a 53% ( P value = 0.016) reduction was observed in comparison with those placed in standard MK media. The trypan blue staining tests revealed that the extent of endothelial cell loss in corneas incubated in supplemented MK media was comparable to the ones in standard MK media., Conclusion: The addition of colistin and amphotericin B to MK media demonstrated efficacy in inhibiting the growth of multidrug-resistant P. aeruginosa in an ex vivo cornea infection model. The supplemented media had no detrimental effect on the corneal endothelium. The findings suggest that supplementing the MK media with these broad-spectrum antimicrobial agents may help mitigate the risk of postoperative donor-related infection in the recipients by reducing and containing the load of microbial contamination in donor corneas., (Copyright © 2024 Copyright: © 2024 Indian Journal of Ophthalmology.)

Management of infections at ocular injury often requires prolonged and high dose of antibiotic, which is associated with challenges of antibiotic resistance and bacterial biofilm formation. Tissue glues are commonly used for repairing ocular tissue defects and tissue regeneration, but they are ineffective in curing infection. There is a critical need for antibacterial ocular bio-adhesives capable of both curing infection and aiding wound closure. Herein, we present the development of an imine crosslinked N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC)‑silver chloride nanocomposites (QAm 1 -Ag x ) and poly-dextran aldehyde (PDA) based bactericidal sealant (BacSeal). BacSeal exhibited potent bactericidal activity against a broad spectrum of bacteria including their planktonic and stationary phase within a short duration of 4 h. BacSeal effectively reduced biofilm-embedded MRSA and Pseudomonas aeruginosa by ∼99.99 %. In ex-vivo human cornea infection model, BacSeal displayed ∼99 % reduction of ocular infection. Furthermore, the hydrogel exhibited excellent sealing properties by maintaining ocular pressure up to 75 mm-Hg when applied to human corneal trauma. Cytotoxicity assessment and hydrogel-treated human cornea with a retained tissue structure, indicate its non-toxic nature. Collectively, BacSeal represents a promising candidate for the development of an ocular sealant that can effectively mitigate infections and may assist in tissue regeneration by sealing ocular wounds., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: The purpose of this study was to analyse the contamination rate of corneal samples stored in OCM at Lions Eye Bank of Western Australia over a 12-year period., Methods: All OCM samples used to preserve corneas from 2011 to 2022 (inclusive) underwent microbiological testing. Samples were collected into aerobic and anaerobic culture bottles on day 3-5 of corneal preservation and 24 h after transfer to thinning medium. Samples were tested for 7 days using the BACTEC FX system. Corneas remained in quarantine until clearance was obtained., Results: From 2011 to 2022, 3009 corneas were retrieved and 2756 corneas were stored in OCM. Thirty one (1.1%) positive samples were reported, with 20 growths of bacterial origin and 11 fungal. Microbial contamination was mostly identified on day 1 of culture (77.5%). Donors of contaminated samples had a mean age of 55 years, with 17 male and 14 female donors. The highest incidence of contamination came from donors whose cause of death was cancer. Death to enucleation times of contaminated samples ranged from 3.5 to 25.5 h (mean = 13.5 ± 7.3) and death to preservation time ranged from 4.1 to 27.5 h (mean = 14.8 ± 7.2). These did not significantly differ from the average time from death to enucleation (mean = 13.9 ± 3) and death to preservation (mean = 16.3 ± 4.2) of non-contaminated samples., Conclusion: Microbiological screening of corneas stored in OCM at LEBWA showed a very low rate of positive cultures with no predictive donor characteristics., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: The objective of this study was to assess the clinical diagnostic value of metagenomic next-generation sequencing (mNGS) in cases of challenging corneal infections using corneal tissue samples., Methods: This retrospective study involved 42 patients with corneal infections, where conventional diagnostic techniques failed to identify the causative pathogen. Corneal tissue specimens underwent mNGS, followed by microbial culture for validation. Sensitivity-guided antimicrobial therapy was administered upon identification of the pathogen. The diagnostic and therapeutic efficacy of mNGS was analyzed to evaluate its clinical utility., Results: A total of 42 patients were included in this study, with mNGS detection results obtained for 38 cases (90.48%). Among them, 30 cases (71.43%) were clinically significant, eight cases (19.05%) had low clinical relevance, and four cases (9.52%) showed no detection. Following corresponding antimicrobial treatment, 30 patients exhibited significant improvement, resulting in a treatment effectiveness of 71.43%. The prognosis of mNGS-positive patients was superior to that of mNGS-negative patients, with statistically significant differences observed (P < 0.001)., Conclusions: Corneal tissue mNGS facilitated the rapid identification of causative agents in challenging corneal infections with unclear clinical diagnoses. It could be seamlessly integrated with traditional diagnostic methods to guide the diagnosis and treatment of corneal diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: Antimicrobial resistance in microbial keratitis has not been previously explored in Alexandria. We aim to recommend effective therapies through identification of etiological agents, determination of antimicrobial susceptibilities, and comparing outcomes of empiric topical antimicrobials., Methods: In this 2022 prospective cohort conducted in Alexandria Main University Hospital cornea clinic, antimicrobial susceptibilities of isolated microorganisms from corneal scrapings were detected and antibiograms were developed. Bacterial (BK), fungal (FK), or mixed fungal/bacterial keratitis (MFBK) patients on empiric regimens were compared for ulcer healing, time-to-epithelialization, best-corrected visual acuity, interventions, and complications., Results: The prevalent microorganisms in 93 positive-cultures were coagulase-negative staphylococci (CoNS, 30.1%), Pseudomonas aeruginosa (14%), and Aspergillus spp. (12.9%). CoNS were susceptible to vancomycin (VAN, 100%) and moxifloxacin (MOX, 90.9%). Gram-negative bacteria showed more susceptibility to gatifloxacin (90.9%) than MOX (57.1%), and to gentamicin (GEN, 44.4%) than ceftazidime (CAZ, 11.8%). Methicillin-resistance reached 23.9% among Gram-positive bacteria. Fungi exhibited 10% resistance to voriconazole (VRC). Percentages of healed ulcers in 49 BK patients using GEN + VAN, CAZ + VAN and MOX were 85.7%, 44.4%, and 64.5%, respectively (p = 0.259). Their median time-to-epithelialization reached 21, 30, and 30 days, respectively (log-rank p = 0.020). In 51 FK patients, more ulcers (88.9%) healed with natamycin (NT) + VRC combination compared to VRC (39.1%) or NT (52.6%) (p = 0.036). Their median time-to-epithelialization was 65, 60, and 22 days, respectively (log-rank p < 0.001). The VRC group required more interventions (60.9%) than NT + VRC-treated group (11.1%) (p = 0.018). In 23 MFBK patients, none healed using NT + CAZ + VAN, while 50% healed using VRC + CAZ + VAN (p = 0.052). Regimens had comparable visual outcomes and complications., Conclusion: Based on the higher detected susceptibility, we recommend empiric MOX in suspected Gram-positive BK, gatifloxacin in Gram-negative BK, and GEN + VAN in severe BK. Due to better outcomes, we recommend NT + VRC in severe FK., Trial Registration: ClinicalTrials.gov identifier, NCT05655689. Registered December 19, 2022- Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05655689?cond=NCT05655689.&draw=2&rank=1., (© 2024. The Author(s).)

The effects of exposure to airborne particulate matter with a size of 10 μm or less (PM 10 ) on C57BL/6 mouse corneas, their response to Pseudomonas aeruginosa (PA) infection, and the protective effects of SKQ1 were determined. C57BL/6 mouse corneas receiving PBS or SKQ1 were exposed to control (air) or PM 10 for 2 weeks, infected, and the disease was documented by clinical score, PMN quantitation, bacterial plate count, RT-PCR and Western blot. PBS-treated, PM 10 -exposed corneas did not differ at 1 day postinfection (dpi), but exhibited earlier (3 dpi) corneal thinning compared to controls. By 3 dpi, PM 10 significantly increased corneal mRNA levels of several pro-inflammatory cytokines, but decreased IL-10, NQO1, GR1, GPX4, and Nrf2 over control. SKQ1 reversed these effects and Western blot selectively confirmed the RT-PCR results. PM 10 resulted in higher viable bacterial plate counts at 1 and 3 dpi, but SKQ1 reduced them at 3 dpi. PM 10 significantly increased MPO in the cornea at 3 dpi and was reduced by SKQ1. SKQ1, used as an adjunctive treatment to moxifloxacin, was not significantly different from moxifloxacin alone. Exposure to PM 10 increased the susceptibility of C57BL/6 to PA infection; SKQ1 significantly reversed these effects, but was not effective as an adjunctive treatment.

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-1β. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1β, 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fungal infections of cornea are important causes of blindness especially in developing nations with tropical climate. However, the challenges associated with current treatments are responsible for poor outcome. Natamycin is the only FDA-approved antifungal drug to treat fungal keratitis, but unfortunately due to its poor water solubility, it is available as suspension. The marketed suspension (5% Natamycin) has rapid precorneal clearance, poor corneal permeability, a higher frequency of administration, and corneal irritation due to undissolved suspended drug particles. In our study, we developed clear and stable natamycin-loaded nanomicelles (1% Natcel) to overcome the above challenges. We demonstrated that 1% Natcel could permeate the cornea better than 5% suspension. The developed 1% Natcel was able to provide sustained release for up to 24 h. Further, it was found to be biocompatible and also improved the mean residence time (MRT) than 5% suspension in tears. Therefore, the developed 1% Natcel could be a potential alternative treatment for fungal keratitis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To report the clinical features, phylogenetic characteristics, microbiological characteristics, and the management of the rare emerging fungal species Cylindrocarpon lichenicola., Methods: A 55-year-old male farmer presented with a history of pain, redness, and defective vision. The corneal scrapings revealed septate hyphae macroconidia and multi-celled chlamydospores with lactophenol cotton blue mount. In addition, the culture revealed velvety to floccose, white growth with a pinkish-brown rim on the Sabouraud's dextrose agar. The growth was suggestive of the rare fungus Cylindrocarpon lichenicola., Results: The course of the infection was rapidly progressive, involving the entire cornea with descemetocele and impending perforation. Reinfection with the rapid spread of disease to the sclera was noted; finally, evisceration with scleral frill excision was done., Conclusion: To our knowledge, this is the first case report of Fulminant Sclero Keratomycosis caused by Cylindrocarpon lichenicola., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi., Methods and Analysis: Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for Acanthamoeba , herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise Staphylococcus aureus isolates., Results: 71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative Staphylococcus 31.8% and Streptococcus species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and Acanthamoeba were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with S. aureus keratitis., Conclusions: In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment., Competing Interests: Competing interests: SBK is an editor of BMJ Open Ophthalmology. There are no other conflicts of interest, (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Fungal keratitis (FK) is an infectious eye disease that poses a significant risk of blindness. However, the effectiveness of conventional antifungal drugs is limited due to the intrinsic ocular barrier that impedes drug absorption. There is an urgent need to develop new therapeutic strategies to effectively combat FK. Herein, we synthesized an ultrasmall positively charged carbon dot using a simple stage-melting method. The carbon dot can penetrate the corneal barrier by opening the tight junctions, allowing them to reach the lesion site and effectively kill the fungi. The results both in vitro and in vivo demonstrated that it exhibited good biocompatibility and antifungal activity, significantly improving the therapeutic effect in a mouse model of FK. Therefore, this biophilic ultrasmall size and positive carbon dot, characterized by its ability to penetrate the corneal barrier and its antifungal properties, may offer valuable insights into the design of effective ocular nanomedicines.

Purpose: Corneal infections are a leading cause of visual impairment and blindness worldwide. Here we applied high-resolution transcriptomic profiling to assess the general and pathogen-specific molecular and cellular mechanisms during human corneal infection., Methods: Clinical diagnoses of herpes simplex virus (HSV) (n=5) and bacterial/fungal (n=5) keratitis were confirmed by histology. Healthy corneas (n=7) and keratoconus (n=4) samples served as controls. Formalin-fixed, paraffin-embedded (FFPE) human corneal specimens were analyzed using the 3' RNA sequencing method Massive Analysis of cDNA Ends (MACE RNA-seq). The cellular host response was investigated using comprehensive bioinformatic deconvolution (xCell and CYBERSORTx) analyses and by integration with published single cell RNA-seq data of the human cornea., Results: Our analysis identified 216 and 561 genes, that were specifically overexpressed in viral or bacterial/fungal keratitis, respectively, and allowed to distinguish the two etiologies. The virus-specific host response was driven by adaptive immunity and associated molecular signaling pathways, whereas the bacterial/fungal-specific host response mainly involved innate immunity signaling pathways and cell types. We identified several genes and pathways involved in the host response to infectious keratitis, including CXCL9, CXCR3, and MMP9 for viral, and S100A8/A9, MMP9, and the IL17 pathway for bacterial/fungal keratitis., Conclusions: High-resolution molecular profiling provides new insights into the human corneal host response to viral and bacterial/fungal infection. Pathogen-specific molecular profiles may provide the foundation for novel diagnostic biomarker and therapeutic approaches that target inflammation-induced damage to corneal host cells with the goal to improve the outcome of infectious keratitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lapp, Kammrath Betancor, Schlunck, Auw-Hädrich, Maier, Lange, Reinhard and Wolf.)

Standard bacteriological examinations, which involve culturing microorganisms at 37 °C, are commonly used in clinical practice for diagnosing infectious diseases. However, the growth temperature of microorganisms on the ocular surface (OS) during infectious keratitis (IK) may not coincide with the laboratory standard, which is due to the characteristic features of heat exchange in the eye., Purpose: This exploratory study examines the distribution and properties of OS microorganisms isolated under different temperature cultivation conditions in patients with IK and healthy volunteers without ophthalmic pathology., Material and Methods: Fifteen participants were divided into two groups. Group 1 ( n =10) consisted of patients with signs of unilateral infectious keratitis, while group 2 ( n =5) served as the control group. A novel microbiological method was employed to isolate pure cultures of microorganisms. This method involved cultivating microorganisms at two temperature regimes (37 °C and 24 °C) and subsequently identifying them using biochemical, immunological, and physicochemical techniques, including mass spectrometry. Scanning electron microscopy (SEM) with lanthanide staining used as the reference method. The temperature status of the ocular surface was assessed using non-contact infrared thermography., Results: The study demonstrated the presence of psychrotolerant microorganisms on the ocular surface, which exhibited growth at a relatively low temperature of 24 °C. These psychrotolerant microorganisms were found to be isolated from the ocular surface displaying signs of temperature dysregulation. Among such microorganisms are Acinetobacter lwoffii, Achromobacter xylosoxidans, Bacillus licheniformis, Enterococcus faecalis, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas luteola, Streptococcus spp., Conclusion: When identifying the causative agent of infectious keratitis, it is crucial to consider the divergence of growth temperature of ocular surface microorganisms. The presence of psychrotolerant microorganisms on the ocular surface, which can effectively grow at room temperature, should be taken into account, especially in cases of temperature dysregulation.

Atypical mycobacteria or non-tuberculous mycobacteria (NTM) are a group of acid-fast bacteria that are pathogenic to different parts of the eye. The organisms can cause a spectrum of ocular infections including keratitis, scleritis, uveitis, endophthalmitis and orbital cellulitis. Trauma, whether surgical or nonsurgical, has the highest correlation with development of this infection. Common surgeries after which these infections have been reported include laser in situ keratomileusis (LASIK) and scleral buckle surgery. The organism is noted to form biofilms with sequestration of the microbe at different inaccessible locations leading to high virulence. Collection of infective ocular material (corneal scraping/necrotic scleral tissue/abscess material/vitreous aspirate, etc.) and laboratory identification of the organism through microbiologic testing are vital for confirming presence of the infection and initiating treatment. In cluster infections, tracing the source of infection in the hospital setting via testing of different in-house samples is equally important to prevent further occurrences. Although the incidence of these infections is low, their presence can cause prolonged disease that may often be resistant to medical therapy alone. In this review, we describe the various types of NTM-ocular infections, their clinical presentation, laboratory diagnosis, management, and outcomes., (Copyright © 2023 Copyright: © 2023 Indian Journal of Ophthalmology.)

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., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: To compare results from a commercial next-generation sequencing (NGS) service to corneal cytology and culture for identification of causative organisms in veterinary patients presenting for infectious ulcerative keratitis (IUK)., Procedure: Swabs for corneal aerobic and fungal cultures and DNA swabs for NGS were submitted for canine and equine normal controls (n = 11 and n = 4, respectively) and IUK patients (n = 22 and n = 8, respectively) for which microbrush cytology specimens confirmed the presence of infectious organisms. The sensitivity of the NGS results was compared with bacterial and fungal culture results. Concordance between the NGS and culture results was determined., Results: The NGS results were positive for bacterial and fungal organisms in 5 and 1 normal and 18 and 1 IUK cases, respectively. Bacterial and fungal cultures were positive for 7 and 2 normal and 20 and 5 IUK cases, respectively. Sensitivity of NGS was 82.14% (95% confidence interval (CI), 63.11% to 93.94%) and specificity was 76.47% (95% CI, 50.10% to 93.19%). Concordance (complete and partial) between identified bacterial and fungal organisms was found in 79% and 100% of cases, respectively. NGS identified organisms in 3 culture-negative IUK samples., Conclusion: A commercial NGS service may be useful in the identification of causative agents in IUK cases with a sensitivity greater than the sensitivity previously reported for aerobic culture. Further testing is needed to determine the clinical significance of additional organisms isolated by NGS from infected cases, as well as organisms isolated from normal corneas., (© 2023 American College of Veterinary Ophthalmologists.)

Purpose: There is a need to understand physicians' diagnostic uncertainty in the initial management of microbial keratitis (MK). This study aimed to understand corneal specialists' diagnostic uncertainty by establishing risk thresholds for treatment of MK that could be used to inform a decision curve analysis for prediction modeling., Methods: A cross-sectional survey of corneal specialists with at least 2 years clinical experience was conducted. Clinicians provided the percentage risk at which they would always or never treat MK types (bacterial, fungal, herpetic, and amoebic) based on initial ulcer sizes and locations (<2 mm 2 central, <2 mm 2 peripheral, and >8 mm 2 central)., Results: Seventy-two of 99 ophthalmologists participated who were 50% female with an average of 14.7 (SD = 10.1) years of experience, 60% in academic practices, and 38% outside the United States. Clinicians reported they would "never" and "always" treat a <2 mm 2 central MK infection if the median risk was 0% and 20% for bacterial (interquartile range, IQR = 0-5 and 5-50), 4.5% and 27.5% for herpetic (IQR = 0-10 and 10-50), 5% and 50% for fungal (IQR = 0-10 and 20-75), and 5% and 50.5% for amoebic (IQR = 0-20 and 32-80), respectively. Mixed-effects models showed lower thresholds to treat larger and central infections ( P < 0.001, respectively), and thresholds to always treat differed between MK types for the United States ( P < 0.001) but not international clinicians., Conclusions: Risk thresholds to treat differed by practice locations and MK types, location, and size. Researchers can use these thresholds to understand when a clinician is uncertain and to create decision support tools to guide clinicians' treatment decisions., Competing Interests: Funding for this research was provided by the National Eye Institute (R01EY031033, M.A.W.) (P30 EY005722, S.F.) and a Research to Prevent Blindness Career Advancement Award (M.A.W.). The remaining authors have no funding or conflicts of interest to disclose. The funding support played no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To ascertain the frequency of coinfections in Acanthamoeba keratitis, the nature of copathogens involved, and to analyze the implications in the context of current research on amoebic interactions., Methods: A retrospective case review from a Tertiary Care Eye Hospital in South India. Smear and culture data for coinfections in Acanthamoeba corneal ulcers were collected from records over a 5-year period. The significance and relevance of our findings in the light of current research on Acanthamoeba interactions were analyzed., Results: Eighty-five cases of culture-positive Acanthamoeba keratitis were identified over a 5-year period (43 of them being coinfections). Fusarium was most commonly identified species, followed by Aspergillus and the dematiaceous fungi. Pseudomonas spp was the commonest bacterial isolate., Conclusion: Coinfections with Acanthamoeba are common at our centre, and account for 50% of Acanthamoeba keratitis. The diverse nature of the organisms involved in coinfections suggest that such amoebic interactions with other organisms are probably more widespread than recognized. To the best of our knowledge, this is the first documentation from a long-term study of pathogen diversity in Acanthamoeba coinfections. It is possible that Acanthamoeba itself may be virulence enhanced and secondary to the co-organism, breaching the ocular surface defenses in an already compromised cornea. However, observations from the existing literature on Acanthamoeba interactions with bacteria and certain fungi are based mainly on nonocular or nonclinical isolates. It would be illuminating if such studies are performed on Acanthamoeba and coinfectors from corneal ulcers-to ascertain whether interactions are endosymbiotic or virulence enhanced through amoebic passage., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2023 Contact Lens Association of Ophthalmologists.)

Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Purpose: Rapid and accurate diagnosis of microbial keratitis (MK) could greatly improve patient outcomes. Here, we present the development of a rapid, accessible multicolour fluorescence imaging device (FluoroPi) and evaluate its performance in combination with fluorescent optical reporters (SmartProbes) to distinguish bacterial Gram status. Furthermore, we show feasibility by imaging samples obtained by corneal scrape and minimally invasive corneal impression membrane (CIM) from ex vivo porcine corneal MK models., Methods: FluoroPi was built using a Raspberry Pi single-board computer and camera, light-emitting-diodes (LEDs), and filters for white-light and fluorescent imaging, with excitation and detection of bacterial optical SmartProbes: Gram-negative, NBD-PMX (exmax 488 nm); Gram positive, Merocy-Van (exmax 590 nm). We evaluated FluoroPi with bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) isolated from ex vivo porcine corneal models of MK by scrape (needle) and CIM with the SmartProbes., Results: FluoroPi provides <1 µm resolution and was able to readily distinguish bacteria isolated from ex vivo models of MK from tissue debris when combined with SmartProbes, retrieved by both scrape and CIM. Single bacteria could be resolved within the field of view, with limits of detection demonstrated as 103 to 104 CFU/mL. Sample preparation prior to imaging was minimal (wash-free), and imaging and postprocessing with FluoroPi were straightforward, confirming ease of use., Conclusions: FluoroPi coupled with SmartProbes provides effective, low-cost bacterial imaging, delineating Gram-negative and Gram-positive bacteria directly sampled from a preclinical model of MK., Translational Relevance: This study provides a crucial stepping stone toward clinical translation of a rapid, minimally invasive diagnostic approach for MK.

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production., (Copyright © 2023. Published by Elsevier Ltd.)

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-1β, 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.

Purpose: To study predisposing factors, clinical presentation and management strategies for Klebsiella keratitis., Methods: A retrospective case review was performed on clinical records of culture-proven Klebsiella keratitis cases in a tertiary referral center over an 8-year period (from 2012 to 2020)., Results: Thirty eight episodes of culture-proven Klebsiella keratitis were identified in 37 patients. The mean age of the patients was 62.9 years (range, 24-101). Multiple predisposing factors were identified in 33 eyes including history of previous keratoplasty (n = 11) history of ocular trauma (n = 7), preexisting ocular surface disease (n = 7) and diabetes (n = 6). Corrected distance visual acuity (CDVA) at presentation was light perception (LP) in 16 patients, hand motion (HM) in 12, counting fingers (CF) at 50 cm in 5, CF at 1 m in 1, CF at 2 m in 2. One patient had a CDVA of 3/10. On initial examination Hypopyon was detected in 21 eyes. Descemet's membrane folds were present in 1 eye. Corneal thinning was identified in 20 eyes and perforation occurred in 4 patients. Corneal ulcer progressed to endophthalmitis in one patient. Microbiologic sensitivity testing showed that 89.5% isolates were sensitive to amikacin (34/38),88.9%sensitive to ceftazidime (32/36),94.4% were sensitive to gentamicin (34/36),97.2% sensitive to ciprofloxacin (35/36), and 100% to levofloxacin (26/26).Ultimately, one or more surgical procedures was needed in 21 patients., Conclusion: Previous keratoplasty, history of ocular trauma, ocular surface disease and systemic disease such as diabetes are major risk factors for Klebsiella keratitis. In most of the patients, surgical and tectonic procedures were necessary to control the infection., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: The true disease status of a population with suspected microbial keratitis (MK) cannot be verified. There is not an accurate (gold) reference standard to confirm infection and inter-test comparisons of sensitivity and specificity therefore lead to bias with questionable estimates of test utility. We present an alternative method to report results., Methods: We used a decision to treat as the definition for MK. We retrospectively compared the results of corneal culture and polymerase chain reaction (PCR) as these are objective tests available for the three principal groups of pathogens. We then estimated the potential contribution of positive results, either alone or in combination, to support the working diagnosis., Results: We included 2021 (77.4%) eyes with suspected bacterial keratitis, 365 (14.0%) with suspected acanthamoeba keratitis, and 226 (8.6%) with suspected fungal keratitis, all treated between July 2013 and December 2019. In these groups, there were 51.6% positive culture and 6.5% positive PCR results for bacteria, 19.0% and 40.5% for acanthamoeba, and 28.3% and 15.0% for fungi. Between groups the differences in the proportions of positive results from culture and PCR was statistically significant (P < 0.001). The added benefit of PCR to the result of culture in identifying a potential pathogen was 1.4% for bacteria (P = 0.6292), 24.4% for acanthamoeba (P = 0.0001), and 5.8% for fungi (P = 0.3853)., Conclusions: For suspected MK a comparison of the test positivity rate is an easily comprehensible outcome measure of test utility., (© 2022. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015-2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015-2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1-3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections., (© 2021. The Author(s).)

Purpose: The purpose of this study was to assess the effect of a second povidone-iodine (PVP-I) application at the time of donor tissue recovery on overall tissue quality and to analyze the rate of positive fungal and bacterial rim cultures before and after implementing increased PVP-I exposure., Methods: The left cornea was recovered after a single application of PVP-I, while the right cornea was recovered after double PVP-I application in research-consented donors. The epithelial cell death rate was estimated using viability assay in corneal whole mounts under 10× objective (n = 5). Clinical characteristics of epithelium, stroma, and endothelium; positive rim culture rate; and incidences of infectious postoperative adverse reactions were compared for a period of 14 months before and after implementation of increased PVP-I protocol., Results: The average epithelial cell death rate was unaltered between single and double PVP-I exposure groups. We observed a modest 10% increase in the number of tissues with mild edema after implementation of increased PVP-I exposure. Nonetheless, the percentage of tissues with moderate or severe edema was unaltered. The average positive rim culture rate decreased from 1.17% to 0.88% (P = 0.075) after implementation of the double PVP-I soak procedure. There has been only one report of infectious postoperative adverse reactions since this procedure change. By contrast, there were 5 reports for a period of 14 months before implementation of this protocol., Conclusions: These results indicate that new donor preparation methods with an additional 5 minutes of PVP-I exposure do not affect tissue quality, reduce positive rim cultures, and lead to lower incidence of postoperative infection., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Background: Corneas procured post mortem are at risk of microbiology contamination, therefore decontamination procedures before storage, aseptic techniques during processing and antimicrobials used in the storage medium are routinely used. Despite that, corneas are discarded due to microbiology contamination. According to professional guidelines, corneas can be procured preferably within 24 hours after cardiac arrest but up to 48 hours. Our goal was to evaluate the risk of contamination depending on the post mortem time and the spectrum of microbes isolated., Methods: Corneas were decontaminated before procurement using 0,5% povidone iodine and tobramycin, stored in the organ culture medium and microbiologically tested after four to seven days of storage. Ten millilitres of cornea preservation medium were inoculated in two blood bottles (aerobic, anaerobic/fungi, Biomeriex) and incubated for seven days.Microbiology testing results in the period of four years (2016-2020) were retrospectively analysed. Corneas were divided in four groups depending on the duration of post mortem interval: group A post mortem interval < 8 h, group B post mortem ranging from 8 to 16 h, group C post mortem ranging from 16 to 24 h and group D post mortem > 24 h. Contamination rate and spectrum of isolated microorganisms in all four groups were analysed., Results: 1426 of 2019 procured corneas were stored in organ culture and microbiologically tested. 65/1426 of tested corneas were contaminated (4,6%). In total, 28 strains of bacteria and fungi were isolated.Contamination rate of post mortem groups are as following: group A 3,1% (14/455), group B 4,1% (23/561), group C 6,7% (27/402) and group D 12,5% (1/8).In the group A bacteria family Staphylococcaceae, Moraxellaceae, Morganellaceae were predominately isolated (64,3%). In the group B fungi Saccharomycetaceae, bacteria Moraxellacea, Staphylococcaceae, Morganellaceae and Enterococcaceae were predominately isolated (78,1%). In the group C, bacteria family Enterococcaceae, Moraxellaceae and fungi Saccharomycetaceae are most often isolated (70,3%). In the group D bacteria family Enterobacteriaceae was isolated (100%)., Conclusion: Organ culture allows detection and discard of microbiology contaminated corneas. Our results show higher microbiology contamination rate for corneas with longer post mortem intervals, suggesting these contaminations can be rather related to donor post mortem changes and contamination than previous infection. In order to keep the best quality and safety of the donor cornea, all efforts should be directed in disinfection of the cornea and keeping post mortem interval shorter., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The purpose of this study was to report a case of fungal keratitis resistant to standard-of-care antimicrobial treatment and successful resolution, thanks to the repeated high-fluence accelerated photoactivated chromophore for keratitis-corneal cross-linking (PACK-CXL)., Methods: This was a case report., Results: A 79-year-old male patient with previous Descemet membrane endothelial keratoplasty presented with a corneal ulcer that was resistant to topical antimicrobial therapy and amniotic membrane placement. Fungal keratitis was diagnosed, and the cornea was on the verge of perforation. After over a month of topical and systemic therapy without marked improvement, the patient underwent 2 repeated high-fluence accelerated CXL procedures (7.2 J/cm2 using a UV irradiation of 30 mW/cm2 for 4 minutes) over an interval of 8 days (accumulated fluence of 14.4 J/cm2), which resulted in significant clinical improvement, with consolidation into a quiescent scar., Conclusions: PACK-CXL protocols delivering a total UV fluence of 5.4 J/cm2 (as per the original Dresden protocol for corneal ectasia cross-linking) can be an effective primary therapy for initial or superficial corneal infections because approximately half of the energy is absorbed in the first 100 μm of a riboflavin-soaked cornea. However, fungal keratitis may require higher fluences than 5.4 J/cm2 because, unlike ectatic corneas, corneal ulcers are not transparent, and the infection may involve deep stroma. This case illustrates how repeated high-fluence accelerated PACK-CXL can be used to successfully treat fungal keratitis resistant to conventional topical and systemic medications., Competing Interests: F. Hafezi is the chief scientific officer of EMAGine AG (Zug, Switzerland) and coinventor of the PCT applications CH2012/0000090 and PCT2014/CH000075 regarding CXL technology. The remaining authors have no conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Fungal keratitis is a devastating and difficult-to-treat ocular infection with high morbidity. Understanding geographic microbiological and clinical trends helps to guide rapid and effective treatment. We therefore report the characteristics and outcomes of fungal keratitis in Toronto,ON Canada, over a 20-year period. An electronic search of microbiology records at University Health Network, Toronto, ON, Canada identified all patients with positive corneal fungal culture over a 20-year period seen at our tertiary referral cornea practice. Review of corresponding patient charts identified demographic and microbiological details, clinical course, treatment regimen, and final outcomes associated with each episode of culture-positive fungal keratitis. A total of 46 patients with 51 discrete fungal keratitis episodes were included. Five patients experienced recurrent fungal keratitis. Candida species accounted for 60.8% of positive fungal cultures, followed by Filamentous species at 35.3%. Preferred initial anti-fungal treatment was topical amphotericin at 36.7% followed by topical voriconazole at 32.6%. Surgical intervention was required in 48.9% with therapeutic penetrating keratoplasty being the most common procedure (22.4%). Final visual acuity (VA) of <20/200 was attributed to 58% of patients in this study. Risk factors for poor outcomes included poor VA, topical steroid use at presentation, Candida involvement, history of ocular surface disease, organic ocular trauma, or prior corneal transplantation. Candida is the most frequent keratomycotic pathogen in Toronto. Risk factors for poor visual outcome include prior corneal transplantation, ocular surface disease/trauma, or pre-existing topical steroid use. Early suspicion, diagnosis and treatment are paramount for best clinical outcomes., Lay Summary: Fungal keratitis can cause severe vision loss without effective treatment. In Toronto, Candida had been the most common species over a 20-year period. Poor clinical outcome was associated with low presenting visual acuity, previous corneal transplant, topical steroid use, trauma, and Candida involvement., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Bacterial keratitis is a common corneal infection that is treated with topical antimicrobials. By the time of presentation there may already be severe visual loss from corneal ulceration and opacity, which may persist despite treatment. There are significant differences in the associated risk factors and the bacterial isolates between high income and low- or middle-income countries, so that general management guidelines may not be appropriate. Although the diagnosis of bacterial keratitis may seem intuitive there are multiple uncertainties about the criteria that are used, which impacts the interpretation of investigations and recruitment to clinical studies. Importantly, the concept that bacterial keratitis can only be confirmed by culture ignores the approximately 50% of cases clinically consistent with bacterial keratitis in which investigations are negative. The aetiology of these culture-negative cases is unknown. Currently, the estimation of bacterial susceptibility to antimicrobials is based on data from systemic administration and achievable serum or tissue concentrations, rather than relevant corneal concentrations and biological activity in the cornea. The provision to the clinician of minimum inhibitory concentrations of the antimicrobials for the isolated bacteria would be an important step forward. An increase in the prevalence of antimicrobial resistance is a concern, but the effect this has on disease outcomes is yet unclear. Virulence factors are not routinely assessed although they may affect the pathogenicity of bacteria within species and affect outcomes. New technologies have been developed to detect and kill bacteria, and their application to bacterial keratitis is discussed. In this review we present the multiple areas of clinical uncertainty that hamper research and the clinical management of bacterial keratitis, and we address some of the assumptions and dogma that have become established in the literature., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Empirical antimicrobial therapy is linked to a surge in antimicrobial resistant infections. However, an insight on the bacteria etiology of ocular infections is essential in the appropriation of choice of antimicrobial among clinicians, yet there remains a dearth of data from Ghana. We investigated the bacteria etiology of external ocular and periocular infections and antimicrobial treatment patterns among a Ghanaian ophthalmic population. A multicenter study design with purposive sampling approach was employed. Patients demographics and clinical data were collated using a pretested structure questionnaire. Cornea specimens and conjunctival swabs were obtained for bacterial isolation following standard protocols. About 95% (98/103) of ocular samples were positive for bacteria culture. The proportion of Gram-negative bacteria was 58.2%, and the predominant bacteria species were Pseudomonas aeruginosa 38.8% and Staphylococcus aureus 27.6%. Conjunctivitis 40.0% and keratitis 75.0% were mostly caused by Pseudomonas aeruginosa. The routinely administered antimicrobial therapy were polymyxin B 41.2%, neomycin 35.1% and ciprofloxacin 31.6%. Participants demographic and clinical characteristics were unrelated with positive bacteria culture (p > 0.05). Our results showed a markedly high burden of ocular bacterial infections and variations in etiology. Bacterial infection-control and antimicrobial agent management programs should be urgently institutionalized to prevent the emergence of resistant infections., (© 2022. The Author(s).)

Purpose: To report a new entity called "toxic non-inflammatory fungal keratitis.", Methods: Eyes manifesting infective keratitis with a history of prior administration of topical steroids were included in the study. The details pertaining to the type of injury, duration of injury, and primary treatment for corneal trauma were meticulously documented. The corneal tissues were scraped from the patients and were analyzed for fungal filaments by using a 10% KOH mount under a compound microscope. Moreover, these scraped materials were plated on blood agar and Sabouraud dextrose agar plates., Results: The corneal ulcers displayed a disproportionately reduced intensity of pain and improved visual acuity. Further, 10% KOH revealed profuse fungal filaments with few inflammatory cells in all the patients. The anterior chamber cells and flare were either reduced or entirely absent. There was no evidence of lid edema and surrounding corneal edema in any of the patients. The mean healing period was 28.8 days (standard deviation (SD): 10.05). The KOH mount revealed the presence of confluent fungal hyphae with a few inflammatory cell infiltrates. The Aspergillus species and Fusarium species were found in 47% and 40% of the cases, respectively., Conclusion: Toxic non-inflammatory fungal keratitis following steroid therapy needs to be considered in fungal ulcers with disproportionately less pain and good visual acuity. The fungal ulcers with altered clinical signs of classical inflammation need to be assessed for topical steroid misuse., Competing Interests: None

Purpose: To determine the degree of microbiological agreement between corneal scrapings and contact lens cultures in cases of contact lens-associated keratitis., Methods: Electronic medical records of all cases of contact lens-associated keratitis at a single institution from April 2006 to May 2019 were reviewed. Cases where both corneal scrapings and contact lens cultures were obtained were included in the study. Group 1 demonstrated agreement between corneal scrapings and contact lens cultures. Group 2 demonstrated growth on both cultures, but disagreement in isolated organism. Group 3 demonstrated negative corneal cultures but growth on contact lens cultures and the diagnostic yield of contact lens microbiological cultures and agreement between corneal and contact lens cultures., Results: A total of 80 eyes of 72 patients were included in the study. 135 total incidences of microbiological results were included for data analysis. Group 1 contained nine incidences (6.7%), group 2 contained 60 incidences (44.4%) and group 3 contained 66 incidences (49%). In group 3, 50% of the cases were treated based on contact lens culture data. There was no statistically significant difference between all three groups in terms of baseline characteristics, presenting vision, vision at last follow-up, number of antibiotics used or complications from keratitis. Pseudomonas was the most common microorganism isolated, and in 89% of these cases, there was disagreement between corneal and contact lens cultures., Conclusions: Although there was a disagreement in the microbiological yield between contact lens and corneal cultures, contact lens cultures were useful in management of patients while achieving similar outcomes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Fungal keratitis (FK) is a serious and sight-threatening corneal infection with global reach. The need for prompt diagnosis is paramount, as a delay in initiation of treatment could lead to irreversible vision loss. Current "gold standard" diagnostic methods, namely corneal smear and culture, have limitations due to diagnostic insensitivity and their time-consuming nature. PCR is a newer, complementary method used in the diagnosis of fungal keratitis, whose results are also sample-dependent. In vivo confocal microscopy (IVCM) is a promising complementary diagnostic method of increasing importance as it allows non-invasive real-time direct visualization of potential fungal pathogens and manifesting infection directly in the patient's cornea. In numerous articles and case reports, FK diagnosis by IVCM has been evaluated, and different features, approaches, sensitivity/specificity, and limitations have been noted. Here, we provide an up-to-date, comprehensive review of the current literature and present the authors' combined recommendations for fungal identification in IVCM images, while also looking to the future of FK assessment by IVCM using artificial intelligence methods., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)