Wedelolactone ameliorates Pseudomonas aeruginosa-induced inflammation and corneal injury by suppressing caspase-4/5/11/GSDMD-mediated non-canonical pyroptosis.

Pseudomonas aeruginosa (P. aeruginosa) keratitis, a worldwide leading cause of corneal perforation and blindness, which is associated with contact lens usage. Increasing evidence has indicated that pyroptosis, a novel proinflammatory programmed cell death, is linked with ocular diseases, little is known about the role of noncanonical pyroptosis in microbial keratitis. Here, we first indicated the involvement of noncanonical pyroptosis in P. aeruginosa keratitis and investigated whether wedelolactone (WDL), a major active component of Eclipta prostrate known to target caspase-11, could alleviate P. aeruginosa keratitis development. We found the expression of caspase-4/5/11 and cleaved GSDMD in corneas of P. aeruginosa keratitis patients, animal models and lipopolysaccharide (LPS)-induced primary cultured human corneal keratocytes (piHCKs) were increased. Combining ciprofloxacin with WDL significantly ameliorated the severity of P. aeruginosa keratitis, as manifested by decreased inflammatory responses and reduced corneal epithelial defects. Consistent with these findings, WDL also dose-dependently alleviated LPS-induced noncanonical pyroptosis by reversing the increased expression of caspase-4/5 and GSDMD in piHCKs. In summary, our results demonstrated that by targeting the activation of caspase-4/5/11, wedelolactone inhibited the development of P. aeruginosa keratitis and suppressed the release of proinflammatory cytokines. Wedelolactone may be a promising anti-inflammatory candidate to combat P. aeruginosa keratitis.

Traumatic laser in situ keratomileusis flap dislocation with epithelial ingrowth, Propionibacterium acnes infection, and diffuse lamellar keratitis: A case report.

RATIONALE: Traumatic flap dislocation might occur anytime after laser in situ keratomileusis (LASIK), but it is rarely concomitantly complicated with epithelial ingrowth, infectious keratitis, and diffuse lamellar keratitis altogether. Here we report a case of traumatic LASIK flap inversion with epithelial ingrowth, Propionibacterium acnes infection, and diffuse lamellar keratitis. PATIENT CONCERNS: A 42-year-old man receiving bilateral LASIK surgery 10 years ago complained of right eye pain for 6 days after twig injury. Temporal flap inversion with epithelial ingrowth and dense infiltration at the interface were noted. DIAGNOSES: Traumatic LASIK flap inversion with epithelial ingrowth, Propionibacterium acnes infection and diffuse lamellar keratitis. INTERVENTIONS: Removal of corneal epithelium around the flap inversion site, flap lifting, scraping of epithelial ingrowth, removal of the dense infiltrate, alcohol soaking, interface irrigation with antibiotics, and flap reposition were performed. Diffuse lamellar keratitis was noted postoperatively. Culture of the infiltrate revealed P acnes. The infiltrate subsided and the cornea cleared up under topical antibiotics and steroid. OUTCOMES: The visual acuity returned to 20/20. No recurrent epithelial ingrowth or infiltrate was noted during the follow-up. LESSONS: This is the first report of Propionibacterium acnes keratitis after traumatic flap inversion. Although epithelial ingrowth, infectious keratitis, and diffuse lamellar keratitis all developed after the flap inversion, early recognition and proper intervention lead to a good result without sequels.

Correlation between pathogenic species and clinical findings, disease severity, and visual outcome in patients with fungal keratitis / Correlação entre espécies patogênicas e achados clínicos, gravidade da doença e desfecho visual em pacientes com ceratite fúngica

ABSTRACT Purpose: To determine whether a correlation exists between pathogenic species and clinical findings, disease severity, and visual outcome in patients with keratitis and fungal growth in microbiological culture. Methods: A retrospective study of patients with fungal growth in the microbiological culture of corneal scrapings. Patients were treated at an ophthalmologic reference center in Southeastern Brazil from January 1992 to October 2015. Results: Medical records of 181 patients (131 males and 50 females) with a mean age of 47 ± 18 years were analyzed. The three most common etiologies were Fusarium sp. (38.7%), Aspergillus sp. (15%), and Candida sp. (13.2%). Among these, Fusarium sp. was the most frequent in patients aged £50 years (p=0.002) and in those with a recent history of a foreign body and/or ocular trauma (p=0.01). Candida sp. was the most frequent etiology in patients aged >50 years (p=0.002), in those with postoperative ocular surgery (p=0.002); in those with a previous ocular pathology (p=0.0007); and in immunodepressed patients (p=0.0004). Conclusion: Fusarium sp. was predominant in patients aged £50 years and those with a recent history of foreign body and/or ocular trauma, whereas Candida sp. was predominant in older adults, in those with a postoperative ocular surgery, in those with a previous ocular pathology, and in immunodepressed patients.

RESUMO Objetivo: Determinar se existe uma correlação entre espécies patogênicas e achados clínicos, gravidade da doença e resultado visual em pacientes com ceratite e crescimento de fungos em cultura microbiológica. Métodos: Estudo retrospectivo de pacientes com crescimento de fungos na cultura microbiológica de raspado de córnea. Os pacientes foram tratados em um centro de referência oftalmológica no Sudeste do Brasil de janeiro de 1992 a outubro de 2015. Resultados: Foram analisados registros médicos de 181 pacientes (131 homens e 50 mulheres) com idade média de 47 ± 18 anos. As três etiologias mais comuns foram Fusarium sp. (38,7%), Aspergillus sp. (15%) e Candida sp. (13,2%). Entre estas, Fusarium sp. foi a mais frequente em pacientes com idade £50 anos (p=0,002) e naqueles com história recente de corpo estranho e/ou trauma ocular (p=0,01). Candida sp. foi a etiologia mais frequente em pacientes com idade >50 anos (p=0,002), naqueles com cirurgia ocular pós-operatória (p=0,002); naqueles com patologia ocular prévia (p=0,0007); e em pacientes imunodeprimidos (p=0,0004). Conclusão: Fusarium sp. foi predominante em pacientes com idade £50 anos e naqueles com história recente de corpo estranho e/ou trauma ocular; enquanto Candida sp. foi predominante em adultos mais velhos, naqueles com cirurgia ocular pós-operatória, naqueles com patologia ocular prévia e em pacientes imunodeprimidos.

Impact of topical corticosteroid pretreatment on susceptibility of the injured murine cornea to Pseudomonas aeruginosa colonization and infection.

Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12 h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12 h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48 h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12 h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48 h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12 h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12 h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.

Correlation between pathogenic species and clinical findings, disease severity, and visual outcome in patients with fungal keratitis.

PURPOSE: To determine whether a correlation exists between pathogenic species and clinical findings, disease severity, and visual outcome in patients with keratitis and fungal growth in microbiological culture. METHODS: A retrospective study of patients with fungal growth in the microbiological culture of corneal scrapings. Patients were treated at an ophthalmologic reference center in Southeastern Brazil from January 1992 to October 2015. RESULTS: Medical records of 181 patients (131 males and 50 females) with a mean age of 47 ± 18 years were analyzed. The three most common etiologies were Fusarium sp. (38.7%), Aspergillus sp. (15%), and Candida sp. (13.2%). Among these, Fusarium sp. was the most frequent in patients aged £50 years (p=0.002) and in those with a recent history of a foreign body and/or ocular trauma (p=0.01). Candida sp. was the most frequent etiology in patients aged >50 years (p=0.002), in those with postoperative ocular surgery (p=0.002); in those with a previous ocular pathology (p=0.0007); and in immunodepressed patients (p=0.0004). CONCLUSION: Fusarium sp. was predominant in patients aged £50 years and those with a recent history of foreign body and/or ocular trauma, whereas Candida sp. was predominant in older adults, in those with a postoperative ocular surgery, in those with a previous ocular pathology, and in immunodepressed patients.

Endophthalmitis caused by Fusarium: An emerging problem in patients with corneal trauma. A case series.

BACKGROUND: Although fortunately very rare in countries with a temperate climate, certain factors, such as clinical or pharmacological immunosuppression, may cause Fusarium-related fungal infections to become an emerging problem. Moreover, Fusarium is one of the most important etiological agents in exogenous endophthalmitis, which is often favored by the disruption of the epithelial barriers. AIMS: The aim of this series of clinical cases is to identify characteristic clinical findings that may allow an early diagnosis and more efficient management of this ophthalmologic emergency. METHODS: Three cases of endophthalmitis due to Fusarium solani and Fusarium oxysporum, diagnosed in 2009, 2010, and 2014 in patients from two different health regions belonging to the same health system and separated by around 43 miles, are presented. The Fusarium isolates were initially identified microscopically and the species subsequently confirmed by sequencing the elongation factor alpha (EFα) and internal transcribed spacers (ITS). Susceptibility to antifungal agents was determined using the EUCAST broth dilution method. RESULTS: Evolution was poor as two of the three patients progressed to phthisis bulbi despite surgical measures and broad-spectrum antifungal antibiotic therapy. CONCLUSIONS: It is essential to rapidly instigate multidisciplinary measures to combat suspected endophthalmitis due to Fusarium given the poor prognosis of this type of infection.

[Fungal keratitis: A 5-year monocentric retrospective study on Reunion Island]. / Kératites fongiques : étude rétrospective monocentrique sur 5ans à l'île de la Réunion.

OBJECTIVES: Fungal keratitis is rare in France, but could be a severe sight-threatening condition. Here, we aimed to describe the epidemiology of fungal keratitis in Réunion Island. METHODS: In a retrospective study, we analyzed 13 culture-proven keratitis episodes, occurred between January 2013 and July 2017 in the ophthalmology ward of a University Hospital, Saint-Pierre. Twelve isolates were genotyped and antifungal susceptibility testing was performed. RESULTS: Corneal abrasion caused by vegetable matter was the main predisposing factor. Stromal infiltration was observed in 12 patients. Six patients did not response to medical treatment, requiring surgical care, including two enucleations surgery. Fusarium solani (n = 6) and Fusarium dimerum (n = 4) were the main fungal species involved in fungal keratitis. Clinical failures were more prevalent with F. solani infections. The lowest minimal inhibitory concentrations for Fusarium sp. were observed with voriconazole and amphotericin B. CONCLUSION: In Reunion Island, the epidemiology of fungal keratitis is characterized by the predominance of Fusarium species, potentially involved in visual loss. This pattern is consistent with the epidemiology usually observed in tropical areas.

Corneal abrasion and microbial contamination in horses following general anaesthesia for non-ocular surgery.

OBJECTIVE: To evaluate the incidence of corneal abrasions/ulceration and microbial contamination in horses undergoing general anaesthesia. STUDY DESIGN: Prospective, observational, clinical study. ANIMALS: A total of 40 client-owned healthy horses scheduled for elective non-ophthalmic procedures. METHODS: Conjunctival sac swabs were taken, fluorescein dye applied and digital images recorded from both eyes of the horses after preanaesthetic medication and 24 hours after recovery from general anaesthesia. A paraffin-based bland ophthalmic ointment was applied on the ocular surface intraoperatively following collection of a sample into a sterile container. All samples underwent aerobic, anaerobic and fungal culture. Subject demographics, chronology of ophthalmic ointment use, anaesthesia duration, recumbency after induction, during surgery and recovery, fluorescein uptake and culture results were recorded. Descriptive statistics were performed. RESULTS: Complete data were collected from 34 horses; six (17.6%) developed mild unilateral generalized fluorescein uptake consistent with corneal abrasions. Recumbency on the operating table was the only risk factor significantly associated with corneal abrasions. A total of 11 bacterial species were identified; Staphylococcus spp. (15 eyes) and Micrococcus spp. (eight eyes) were the most frequently isolated bacteria. Two fungal species were isolated postoperatively (Aspergillus spp., Saccharomyces spp.) in two eyes. Ointment contamination was recorded in two cases (5%) but cross-contamination was not recognized. CONCLUSIONSAND CLINICAL RELEVANCE: Incidence of corneal abrasion/ulceration in horses undergoing general anaesthesia and contamination rate of ophthalmic solutions are similar to those previously reported in dogs.

Corneal Response to Injury and Infection in the Horse.

This article describes the natural responses of the immune system and the cornea to injury and infection. The process of reepithelialization and reformation of stromal collagen is discussed, as are the clinical signs and manifestations of the effects of the healing response when it is routine and when it is pathologic. Excessive inflammatory or immune responses by host tissues can cause further damage that may be present from the antecedent injury or the effect of a pathogen. The clinical signs and manifestations of wound healing as well as potential therapeutic interventions are described.

Pediatric posttraumatic endophthalmitis.

BACKGROUND: To identify the etiology, pars plana vitrectomy results, culture results, and visual outcome of endophthalmitis in the pediatric age group. METHODS: Fifteen eyes of 15 consecutive pediatric patients who were treated and followed up for endophthalmitis between July 2011 and December 2012 were included in this study. Combined pars plana vitrectomy and intravitreal norvancomycin plus ceftazidime injection was performed on all 15 eyes. The vitreous samples were obtained by a standard pars plana vitrectomy. The specimens were sent to microbiology facility for gram staining, culturing, and sensitivity testing. RESULTS: The mean age of the patients was 5.3 ± 2.8 years (3-10 years). The mean follow-up time was 9.7 ± 1.5 months (7-12 months). Preoperatively, the visual acuities of the patients were as follows: light perception (n = 7), hand movements (n = 4), and counting fingers from 30 cm (n = 1), and these values were not available in three patients (n = 3). Postoperatively, the final best corrected visual acuity was 20/200 or better in six (40 %), counting of fingers in four (26.7 %), light perception to hand movements in two (13.3 %), and no light perception in one (6.7 %), with results being unavailable for two patients. Positive culture results were obtained from ten eyes (66.7 %). A single species was isolated in eight eyes, and multiple organisms were isolated in two eyes. CONCLUSIONS: Penetrating ocular trauma was the most common cause of pediatric endophthalmitis in this consecutive series of endophthalmitis. Staphylococci and Streptococcus species were the most common isolated organisms. Patients with multiple organisms had poor visual recovery. Visual outcomes were poor in this series in spite of vitrectomy being performed immediately on all patients.

The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88-/- epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88-/- mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain.