Laboratory exploration of the use of ripasudil in descemetorhexis with a human ex vivo model.

The aim of the study was to investigate the effect of ripasudil on corneal endothelial cell survival and migration after two types of descemetorhexis on a human ex vivo model. Eleven human corneoscleral buttons were incubated in either 50 ml organ culture medium containing 10 µM ripasudil or 50 µl dimethyl sulfoxide (DMSO), the vehicle in ripasudil for 2 days prior to wound creation then for 14 days after. The wound was created with either full trephination scoring or by shallow trephination plus manual peeling. At day 14, immunohistochemistry with vimentin and Na+/K+/ATPase markers was conducted. Tissues were assessed at day 3, 7 and 14 for morphology, cell migration, cell viability and cell density. Full trephination scoring created more damage on tissues compared to shallow trephination with full Descemet membrane peeling. In the full trephination scoring group, no differences in cell viability were noted when ripasudil and DMSO were compared. With the peeling method, Ripasudil could protect the endothelial cell death and maintain the morphology compared to the control. At day 14, no differences in the peripheral cell viability and density were found between ripasudil and DMSO, although the ripasudil group presented significantly increased central cell count and cell viability. Increased cell migration was noted with ripasudil and the initial cell morphology of those migrated cells was similar to that of fibroblasts. In conclusion, ex vivo modelling suggested that peeling resulted in less cell damage than scoring and ripasudil maintained better morphology and promoted migration. These effects might be via transformation of endothelial cells into a more motile spindle-like phenotype.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates.

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Epidemiology of and Genetic Factors Associated with Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK) is a severe, rare protozoal infection of the cornea. Acanthamoeba can survive in diverse habitats and at extreme temperatures. AK is mostly seen in contact lens wearers whose lenses have become contaminated or who have a history of water exposure, and in those without contact lens wear who have experienced recent eye trauma involving contaminated soil or water. Infection usually results in severe eye pain, photophobia, inflammation, and corneal epithelial defects. The pathophysiology of this infection is multifactorial, including the production of cytotoxic proteases by Acanthamoeba that degrades the corneal epithelial basement membrane and induces the death of ocular surface cells, resulting in degradation of the collagen-rich corneal stroma. AK can be prevented by avoiding risk factors, which includes avoiding water contact, such as swimming or showering in contact lenses, and wearing protective goggles when working on the land. AK is mostly treated with an antimicrobial therapy of biguanides alone or in combination with diaminidines, although the commercial availability of these medicines is variable. Other than anti-amoeba therapies, targeting host immune pathways in Acanthamoeba disease may lead to the development of vaccines or antibody therapeutics which could transform the management of AK.

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

BACKGROUND: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. METHODOLOGY/PRINCIPAL FINDINGS: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

Patch endothelial keratoplasty for corneal perforations secondary to ocular surface disease: case series.

BACKGROUND: Corneal perforation is an ophthalmic emergency. The conventional management of corneal perforation can be associated with severe complications especially in patients with ocular surface disease. Endothelial keratoplasty has been suggested as an alternative surgical technique for the management of corneal perforations. We present a case series of nine patients with corneal perforation and ocular surface disease managed with secondary patch endothelial keratoplasty. METHODS: This is a retrospective case series of nine patch endothelial keratoplasties performed between 2016 and 2022 at a quaternary eye hospital in Australia. The surgical technique is similar to conventional endothelial keratoplasty except descemetorhexis was not performed. RESULTS: A total of 9 cases were treated during the review period. Eight of the nine cases had an improvement in visual acuity. One case failed to achieve corneal tectonic objective. CONCLUSION: Patch endothelial keratoplasty is a safe secondary procedure for the management of corneal perforations in patients with ocular surface disease.

Comparison of standard versus accelerated corneal collagen cross-linking for keratoconus: 5-year outcomes from the Save Sight Keratoconus Registry.

OBJECTIVE: To compare long-term effectiveness of Standard (UV intensity: 3 mW/cm2, duration: 30 min) vs Accelerated (UV intensity: 9 mW/cm2, duration: 10 min) corneal cross-linking (CXL) for stabilising keratoconus. METHODS: Data for this observational study were captured through a web-based registry system from the routine clinical practice (15 sites across Australia, New Zealand and Italy). The outcomes were compared using mixed-effects regression models. A total of 100 eyes (75 patients) who had standard CXL and 76 eyes (66 patients) who had accelerated CXL, with a follow-up visit at five-year post-CXL were included. RESULTS: Both CXL protocols were effective and safe in stabilising keratoconus and improving outcomes. The adjusted mean changes (95% CI) in outcomes were better in standard CXL than in accelerated CXL [visual acuity gain, 10.2 (7.9-12.5) vs 4.9 (1.6-8.2) logMAR letters; pinhole visual acuity 5.7 (3.5-7.8) vs 0.2 (-2.2 to 2.5) logMAR letters; Kmax -1.8 (-4.3 to 0.6) vs 1.2 (-1.5 to 3.9)D; K2 -0.9 (-2.2 to 0.3) vs 0.1 (-1.3 to 1.6)D; MCT -3.0 (-13.7 to 7.7) vs -11.8 (-23.9 to 0.4) µm (p values for visual acuity, pinhole visual acuity, Kmax: <0.05; for K2 and MCT: >0.05)]. The frequency of adverse events at the 5-year follow-up visit was low in both groups [standard, 5 (5%; haze 3; scarring 1, epithelial defect 1) and accelerated 3 (3.9%; haze 2, scarring 1)]. CONCLUSIONS: Both standard and accelerated CXL were safe and effective procedures for stabilising keratoconus in the long term. The standard CXL resulted in greater improvements in visual acuity and keratometry.

Educators' consideration of learner motivation in ophthalmology education in medical school: Influences on teaching practice and course design.

BACKGROUND: Ophthalmology education in medical school has historically neglected the impact of autonomous motivation on student learning and wellbeing. This study aimed to understand ophthalmology educators' consideration and application of student motivation in ophthalmology medical education. MATERIAL AND METHODS: Lead ophthalmology educators from Australian and New Zealand medical schools participated in an online semi-structured in-depth interview. Interview transcripts were analysed using thematic analysis. Codes were generated and aligned into overarching themes. FINDINGS: Six educators participated in the study. Five main themes arose from the transcripts: the lack of explicit consideration of student motivation, implicit consideration of motivation in curriculum design and in teaching practices, the impact of innovation on motivation and the relationship between teacher and student motivation. Participants also commented on trends in ophthalmology education including generalists' confidence in managing ophthalmic disease, the role of fundoscopy in medical education and time pressure on ophthalmology in medical schools. CONCLUSION: There has only been an implicit instead of explicit consideration of motivation in ophthalmology education in medical school, which leaves an unfulfilled potential for teaching practices to impact the affective along with cognitive and metacognitive aspects of learning. This study highlights the need for motivation to be explicitly incorporated into the development of teaching practices and curriculum reform.

Case Report: The Use of In Vivo Confocal Microscopy for Diagnosis and Monitoring in a Rare Case of Ancaliia algerae Microsporidial Keratitis in New South Wales, Australia.

We describe the successful management of Ancaliia Algerae microsporidial keratitis in an immunosuppressed 54-year-old woman with refractory linear IgA disease. The case highlights the challenges in diagnosis and management of this infection in immunocompromised individuals and emphasizes the usefulness of in vivo confocal microscopy as a novel, noninvasive tool to aid in the diagnosis and monitoring of microsporidial keratitis. We also discuss the possible mode of acquisition of this rare infection.

Case Series of Rare Fungal Keratitides: Experiences from a Quaternary Eye Hospital in Sydney, Australia.

The present article reports on the management of six different and rare cases of fungal keratitides, two of which have never been documented in previous literature. This is a case series of six patients with rare fungal keratitides managed at a quaternary eye referral unit, Sydney Eye Hospital, Australia over a period of 7 months (May to December, 2022). The order of occurrence of fungi isolated was Scedosporium apiospermum, Lomenstospora prolificans, Cladosporium spp., Paecilomyces, Syncephalastrum racemosum and Quambalaria spp. A combination of medical and surgical interventions was employed, including topical and systemic anti-fungal therapy, with one requiring therapeutic penetrating keratoplasty and another eventuating in evisceration. Two patients were successfully treated with corneal debridement and two others required pars plana vitrectomy with anterior chamber washout. It is important to remain vigilant with monitoring patient symptoms and correlating with clinical signs to guide antifungal therapy even in the context of confirmed culture and sensitivity results.

The multifunctional human ocular melanocortin system.

Immune privilege in the eye involves physical barriers, immune regulation and secreted proteins that together limit the damaging effects of intraocular immune responses and inflammation. The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) normally circulates in the aqueous humour of the anterior chamber and the vitreous fluid, secreted by iris and ciliary epithelium, and retinal pigment epithelium (RPE). α-MSH plays an important role in maintaining ocular immune privilege by helping the development of suppressor immune cells and by activating regulatory T-cells. α-MSH functions by binding to and activating melanocortin receptors (MC1R to MC5R) and receptor accessory proteins (MRAPs) that work in concert with antagonists, otherwise known as the melanocortin system. As well as controlling immune responses and inflammation, a broad range of biological functions is increasingly recognised to be orchestrated by the melanocortin system within ocular tissues. This includes maintaining corneal transparency and immune privilege by limiting corneal (lymph)angiogenesis, sustaining corneal epithelial integrity, protecting corneal endothelium and potentially enhancing corneal graft survival, regulating aqueous tear secretion with implications for dry eye disease, facilitating retinal homeostasis via maintaining blood-retinal barriers, providing neuroprotection in the retina, and controlling abnormal new vessel growth in the choroid and retina. The role of melanocortin signalling in uveal melanocyte melanogenesis however remains unclear compared to its established role in skin melanogenesis. The early application of a melanocortin agonist to downregulate systemic inflammation used adrenocorticotropic hormone (ACTH)-based repository cortisone injection (RCI), but adverse side effects including hypertension, edema, and weight gain, related to increased adrenal gland corticosteroid production, impacted clinical uptake. Compared to ACTH, melanocortin peptides that target MC1R, MC3R, MC4R and/or MC5R, but not adrenal gland MC2R, induce minimal corticosteroid production with fewer adverse systemic effects. Pharmacological advances in synthesising MCR-specific targeted peptides provide further opportunities for treating ocular (and systemic) inflammatory diseases. Following from these observations and a renewed clinical and pharmacological interest in the diverse biological roles of the melanocortin system, this review highlights the physiological and disease-related involvement of this system within human eye tissues. We also review the emerging benefits and versatility of melanocortin receptor targeted peptides as non-steroidal alternatives for inflammatory eye diseases such as non-infectious uveitis and dry eye disease, and translational applications in promoting ocular homeostasis, for example, in corneal transplantation and diabetic retinopathy.

Prevalence and Predictors of Knowledge and Attitudes Toward Eye Donation Among the General Population: A Systematic Review.

PURPOSE: The purpose of this systematic review was to investigate knowledge and attitudes toward eye donation and sources of eye donation information among the general population. METHODS: A search was conducted using MEDLINE through Ovid and Scopus; CINAHL through EBSCOhost and ProQuest; and Embase through Ovid database entries from January 2010 to March 2021. Quantitative studies were selected if they included participants aged 16 years or older from the general population (nonhealthcare) and had a sample size of >200. Studies were included if they measured knowledge and attitudes toward eye donation and sources of eye donation information. Methodological quality was assessed using JBI criteria, and the data were analyzed using SUMARI software. RESULTS: A total of 25 studies were included in this review. Pooled data from 6 studies demonstrated that 30.8% of participants [95% confidence interval (CI) = 11.0-55.4] had some knowledge of eye donation. Seventeen studies reported that 40.6% (95% CI = 39.8-41.3) were willing to donate their eyes, and 5 of these studies found that 7.3% (95% CI = 6.5-8.3) had already pledged their eyes. Eleven studies reported on the source of eye donation information, indicating 50.9% of participants (95% CI = 49.8-52.1) received information from mass media. CONCLUSIONS: The results of this review indicate that understanding eye donation knowledge and attitudes is crucial for developing interventions or tools to increase eye donation rates. Further studies in different populations are required.

Ophthalmology teaching in Australian medical schools: A national survey.

PURPOSE: To survey the current educational trends and methods of ophthalmology teaching in Australian undergraduate and postgraduate medical schools. MATERIALS AND METHODS: Cross-sectional survey; National online survey distributed to Australian university undergraduate and post-graduate medical schools from November 2020 to March 2021. The survey encompassed 35 questions on student demographics, teaching methods, core theoretical topics, clinical skills, and assessment methods in ophthalmology. One survey per institution completed by the relevant individual responsible for curriculum. RESULTS: Total response rate of 90.48% (19 of 21 medical schools) was received with good representation across Australia. Ophthalmology rotations were required in 63.3% (n = 12), while 36.7% (n = 7) did not have mandatory terms. This compares favourably to the USA (16%), Canada (35.7%) and equivalent to UK (65%). 74% (n = 14) state ophthalmology is not a priority in the curriculum. All respondents reported student exposure to at least one clinical day in ophthalmology, with total teaching time ranging from less than six hours (36.9%), up to greater than two weeks (10.5%). Overall, only 31.6% reported utilisation of the International Council of Ophthalmology (ICO) curriculum in curricular development. CONCLUSIONS: Ophthalmology medical school teaching in Australia remains reasonable when compared internationally, but there is significant variation amongst universities. Incorporation of the ICO curriculum and development of shared resources would enhance medical graduates' competence.

Acanthamoeba, an environmental phagocyte enhancing survival and transmission of human pathogens.

The opportunistic protist Acanthamoeba, which interacts with other microbes such as bacteria, fungi, and viruses, shows significant similarity in cellular and functional aspects to human macrophages. Intracellular survival of microbes in this microbivorous amoebal host may be a crucial step for initiation of infection in higher eukaryotic cells. Therefore, Acanthamoeba-microbe adaptations are considered an evolutionary model of macrophage-pathogen interactions. This paper reviews Acanthamoeba as an emerging human pathogen and different ecological interactions between Acanthamoeba and microbes that may serve as environmental training grounds and a genetic melting pot for the evolution, persistence, and transmission of potential human pathogens.

Development of an In Situ Printing System With Human Platelet Lysate-Based Bio-Adhesive to Treat Corneal Perforations.

Purpose: Corneal perforation is a clinical emergency that can result in blindness. Currently corneal perforations are treated either by cyanoacrylate glue which is toxic to corneal cells, or by using commercial fibrin glue for small perforations. Both methods use manual delivery which lead to uncontrolled application of the glues to the corneal surface. Therefore, there is a need to develop a safe and effective alternative to artificial adhesives. Methods: Previously, our group developed a transparent human platelet lysate (hPL)-based biomaterial that accelerated corneal epithelial cells healing in vitro. This biomaterial was further characterized in this study using rheometry and adhesive test, and a two-component delivery system was developed for its application. An animal trial (5 New Zealand white rabbits) to compare impact of the biomaterial and cyanoacrylate glue (control group) on a 2 mm perforation was conducted to evaluate safety and efficacy. Results: The hPL-based biomaterial showed higher adhesiveness compared to commercial fibrin glue. Treatment rabbits had lower pain scores and faster recovery, despite generating similar scar-forming structure compared to controls. No secondary corneal ulcer was generated in rabbits treated with the bio-adhesive. Conclusions: This study reports an in situ printing system capable of delivering a hPL-based, transparent bio-adhesive and successfully treating small corneal perforations. The bio-adhesive-treated rabbits recovered faster and required no additional analgesia. Translational Relevance: The developed in situ hPL bio-adhesives treatment represents a new format of treating corneal perforation that is easy to use, allows for accurate application, and can be a potentially effective and pain relief treatment.

Visual Acuity and Number of Amniotic Membrane Layers as Indicators of Efficacy in Amniotic Membrane Transplantation for Corneal Ulcers: A Multicenter Study.

BACKGROUND: To evaluate new indicators in the efficacy of amniotic membrane transplantation (AMT) for non-healing corneal ulcers (NHCUs). METHODS: Retrospective, multicenter study. In total, 223 AMTs for NHCU in 191 patients were assessed. The main outcomes studied were the success rate of AMT (complete re-epithelization), postoperative visual acuity (VA) gain, and number of AM layers transplanted. RESULTS: The overall AMT success rate was 74.4%. In 92% of our patients VA stability or improvement. Postoperative VA was significantly higher than preoperative VA in the entire cohort (p < 0.001) and in all etiological groups of ulcers (post-bacterial, p ≤ 0.001; post-herpetic, p ≤ 0.0038; neurotrophic ulcers, p ≤ 0.014; non-rheumatic peripheral, p ≤ 0.001; and ulcers secondary to lagophthalmos and eyelid malposition or trauma, p ≤ 0.004). Most participants (56.5%) presented a preoperative VA equal to or less than counting fingers (≤0.01). Of these, 13.5% reached a postoperative VA equal to or better than legal blindness (≥0.05) after AMT. A higher success rate was observed in the monolayer than in the multilayer AMT (79.5% and 64.9%, respectively; p = 0.018). No statistically significant values were found between the number of layers transplanted and VA gain (p = 0.509). CONCLUSION: AMT is not only beneficial in achieving complete re-epithelialization in NHCUs but also in improving postoperative VA; these improvements are independent of etiologies of ulcers. Furthermore, the use of monolayer AMT seems to be a more appropriate option than multilayer AMT for NHCU since the multilayer AMT did not present better outcomes (success rate and VA gain) compared to monolayer AMT in the different types of ulcers studied.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells.

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

20 years since the Herpetic Eye Disease Study: Lessons, developments and applications to clinical practice.

Herpes Simplex Virus (HSV) is the most common virus that causes eye disease. Although around 60% of the world's population are seropositive for HSV antigens, fortunately, it is estimated that only 1% of seropositive individuals develop eye disease. The most common ocular manifestation of HSV is keratitis, while uveitis and retinal necrosis occur in a small number of cases. HSV keratitis is a debilitating disease, for several reasons: pain , photophobia, and vision loss in acute disease, latency of the virus which leads to infection reactivation from various triggers, scarring, and neovascularisation, leading to permanent vision loss with poor visual rehabilitation prospects. The Herpetic Eye Disease Study (HEDS) was a landmark series of randomised controlled trials in the 1990s that set the benchmark for evidence-based treatment guidelines for anterior eye herpetic disease. Since this time, there has been a change in the distribution of seroprevalence of herpes in the community, a simplified diagnostic classification, advances in treatment options, an emergence of new and a better understanding of risk factors, and discoveries in science that show promise for vaccine and novel future treatments. However, many of the principles of the HEDS study remain rightly entrenched in clinical practice. In this article, the HEDS study is revisited 20 years on through the lens of published literature, to determine current best practise and look towards the future.