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

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

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

Microsporidial Stromal Keratitis: Epidemiological Features, Slit-Lamp Biomicroscopic Characteristics, and Therapy.

PURPOSE: Microsporidial stromal keratitis is a rare form of infectious keratitis, with only 7 cases reported in the United States to date. This study was performed to evaluate risk factors, clinical features, and response to therapy. METHODS: A retrospective review of the medical records of all patients diagnosed with microsporidial stromal keratitis seen in the practices of the authors between 1999 and 2020 was performed. Diagnosis was determined by cytology or histopathology in corneal specimens. Risk factors, presence or absence of distinctive clinical features, and response to medical and surgical therapies were recorded. RESULTS: Nine patients-7M:2F, aged 7 to 99 years-with microsporidial stromal keratitis were identified. Exposures to recreational water and hymenopteran insect bites, both epidemiologically linked risk factors for systemic microsporidial infection, were identified in our patients. Presence of stromal edema with features of disciform keratitis and a distinctive granular keratitis were observed in 6 of 9 and 5 of 9 patients, respectively. Poor response to medical therapy was noted. Penetrating keratoplasty was effective in curing the infection. Final visual acuity was 20/40 or better in 6 of 9 patients. CONCLUSIONS: In patients with slowly progressive keratitis, history of exposure to recreational water or hymenopteran insects should be sought. In patients with corneal edema consistent with disciform keratitis, with evolution to a granular keratitis, microsporidia should be considered in the differential diagnosis. In cases of established microsporidial stromal keratitis, penetrating keratoplasty should be considered if prompt response to medical therapy is not noted.

Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence.

PURPOSE: S. epidermidis is an ocular pathogen and a leading cause of keratitis. It produces hemolysins and at least 3 proteases. The purpose of the present study is to compare the secretion of hemolysins and proteases between 28 ocular isolates and one non-ocular strain and to determine their relationship to ocular virulence in selected strains using a rabbit model of infection. MATERIALS AND METHODS: Culture supernatants were compared for protease production and hemolysis. Selected strains were injected into rabbit corneas and their virulence and pathology recorded. The major protease activity in a virulent strain was identified and the gene was cloned and expressed as a recombinant protein. The corneal toxicity of this protease was determined. Antibodies to the native protease were generated and tested for neutralizing activity in vivo and in vitro. The corneal pathology of the S. epidermidis protease was compared to the pathology of S. aureus V8 protease. RESULTS: Strains that exhibited the least protease activity in vitro caused significantly less ocular pathology in vivo (p ≤ 0.003). Strains that were hemolytic and secreted a major protease had numerically higher SLE scores. This protease was identified as the serine protease Esp. The recombinant Esp protease caused extensive pathology when injected into the corneal stroma (7.62 ± 0.33). Antibody generated against native Esp did not neutralize the activity of the protease in vivo or in vitro. The antibody reacted with Esp proteases secreted by other S. epidermidis strains. S. epidermidis Esp protease and its homologue in S. aureus caused similar ocular pathology when injected in the rabbit corneal stroma. CONCLUSION: Hemolysins and proteases seem to be important in corneal pathology caused by S. epidermidis infections. The Esp protease mediates significant corneal damage. S. epidermidis Esp and S. aureus V8 protease caused similar and extensive edema in rabbit corneas.

Microsporidial stromal keratitis: characterisation of clinical features, ultrastructural study by electron microscopy and efficacy of different surgical modalities.

AIMS: To report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK). METHODS: Fourteen MSK cases between 2009 and 2018 were recruited. Each patient's clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed. RESULTS: The patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100. CONCLUSION: MSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.

The Acanthamoeba-Fungal Keratitis Study.

PURPOSE: To ascertain the incidence of Acanthamoeba keratitis and the coexistence of Acanthamoeba and fungi in microbial keratitis. DESIGN: Prospective cross-sectional study. METHODS: Patients presenting with stromal keratitis were additionally tested for Acanthamoeba irrespective of the clinical diagnosis. Culture positivity was the gold standard. RESULTS: Of the 401 cases included in the study, 40 were positive for Acanthamoeba (10%); of these 40, 16 were positive for both Acanthamoeba and fungi (4.5% of the study group was Acanthamoeba and fungal keratitis positive); 5 were positive for Acanthamoeba and bacteria; and 2 had triple infection with Acanthamoeba, fungi, and bacteria. Ring infiltrates and stromal edema are frequently associated with Acanthamoeba keratitis, as well as in Acanthamoeba coinfections. Ring infiltrates in particular were more frequently seen in the Acanthamoeba and fungal keratitis group (8/16) and they were often yellowish with hyphate edges (vs ring infiltrates only, which are seen in the patients with Acanthamoeba alone). Only 2 patients were contact lens wearers: however, they presented with history of trauma. CONCLUSIONS: Acanthamoeba coinfections are much more frequent and are not restricted to contact lens users. Anticipating coinfections is necessary for establishing a diagnosis as well as for appropriate and timely therapeutic interventions.

Exophiala phaeomuriformis keratitis in a subarctic climate region: a case report.

PURPOSE: To report a case of Exophiala phaeomuriformis mycotic keratitis in a patient from a subarctic climate region. Dematiaceous fungi (black yeasts) have been gaining importance as corneal keratitis and ulcer causative agents in certain regions, but no cases have been described in Scandinavia. METHODS: Case report of a patient with a persistent corneal erosion that eventually presented a brown-pigmented infiltrate. The patient had a history of several months of topical therapy comprising medication for glaucoma, corticosteroids and antibiotics. A therapeutic contact lens was used, and amniotic membrane transplantation was performed before the development of the pigmented infiltrate. RESULTS: Exophiala phaeomuriformis was identified on the microbiological cultures from the surgically obtained infiltrate scrapes. The patient responded to topical amphotericin and fluconazole, the erosion was cured and a stromal scar subsided. During follow-up, sequential slit-lamp images and anterior segment optical coherence tomography (OCT) scans were obtained. CONCLUSION: This is the first described case of keratitis caused by E. phaeomuriformis in a subarctic region, the first in Europe and, to our knowledge, the second reported case in the literature. It is important to remember that superficial corneal brown-pigmented infiltrates should raise the suspicion of an unusual fungal infection even in this climate. This is particularly important in patients with ocular surface disease treated with steroids and antibiotics for a long time.

Coexistence of herpes simplex virus infection in microsporidial stromal keratitis associated with granulomatous inflammation.

BACKGROUND: Microsporidial stromal keratitis poses several diagnostic challenges. Patients may present with corneal ulceration, marked stromal thinning, or even as a quite corneal scar. The presentation of microsporidial stromal keratitis commonly mimics viral keratitis. Microbiology scrapings are usually helpful; however, scraping and culture-negative cases pose a significant diagnostic dilemma. Histopathological examination is diagnostic but shows varying degree of inflammation, predominantly composed of polymorphonuclear leukocytes. Granulomatous inflammation, in microsporidial stromal keratitis, is never well described, and the authors in this article aim to describe the presence of granulomatous inflammation in microsporidial stromal keratitis, in patients with associated herpes simplex virus (HSV) keratitis. METHODS: This was a retrospective and observational study conducted at a tertiary eye care center. RESULTS: Of 263 patients who underwent therapeutic penetrating keratoplasty for infectious keratitis, during 2011-2013, seven patients were diagnosed as microsporidial stromal keratitis. Microsporidial spores could be demonstrated on microbiological scrapings in 5/7 (71%) of cases, but identified on histopathological examination and also confirmed on polymerase chain reaction (PCR) for microsporidium in 100% of cases. There was evidence of diffuse stromal necrosis with markedly severe degree of polymorphonuclear leukocytic infiltrates, with granulomatous inflammation in 42% of cases. Interestingly, these were positive for HSV-1 DNA on PCR. Review of medical records revealed much severe clinical presentations in patients with granulomatous inflammation, in comparison to cases without granulomatous inflammation. CONCLUSIONS: The authors hereby recommend that severe clinical presentation in patients with microsporidial stromal keratitis, markedly dense polymorphonuclear leukocytic infiltrates or the presence of granulomatous inflammation on the histopathological examination, should be investigated further for the presence of HSV-1 DNA for better patient management and good visual outcome.

Second harmonic generation imaging of corneal stroma after infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a pathogenic gram-negative organism that has the ability to cause blinding corneal infections following trauma and during contact lens wear. In this study, we investigated the directional movement and orientation of an invasive corneal isolate of P. aeruginosa in the corneal stroma during infection of ex vivo and in vivo rabbit corneas using multiphoton fluorescence and second harmonic generation (SHG) imaging. Ex vivo, rabbit corneas were subject to three partial thickness wounds prior to inoculation. In vivo, New Zealand white rabbits were fit with P. aeruginosa laden contact lenses in the absence of a penetrating wound. At all time points tested, infiltration of the corneal stroma by P. aeruginosa revealed a high degree of alignment between the bacteria and collagen lamellae ex vivo (p < 0.001). In vivo, P. aeruginosa traveled throughout the stroma in discrete regions or bands. Within each region, the bacteria showed good alignment with collagen lamellae (P = 0.002). Interestingly, in both the in vitro and in vivo models, P. aeruginosa did not appear to cross the corneal limbus. Taken together, our findings suggest that P. aeruginosa exploits the precise spacing of collagen lamellae in the central cornea to facilitate spread throughout the stroma.

Collagen Cross-linking With Photoactivated Riboflavin (PACK-CXL) for Bacterial Keratitis After Small Incision Lenticule Extraction (SMILE).

PURPOSE: To report a case of infectious keratitis after small incision lenticule extraction (SMILE) treated with collagen cross-linking with photoactivated riboflavin (PACK-CXL). METHODS: Case report and literature review. RESULTS: A patient presented with culture-proven bacterial keratitis 5 days after SMILE was treated with fortified topical antibiotics and PACK-CXL. Irrigation of the corneal cap-stromal bed interface, which can be difficult and affect visual outcomes, was not performed. Clinical improvement was seen shortly after treatment and resolution of keratitis was achieved at 2 weeks. CONCLUSIONS: This is the first reported use of PACK-CXL in the management of infectious keratitis after SMILE. PACK-CXL may help sterilize the infection and limit its spread within the interface. [J Refract Surg. 2017;33(4):278-280.].

Microsporidial Stromal Keratitis: Clinical Features, Unique Diagnostic Criteria, and Treatment Outcomes in a Large Case Series.

PURPOSE: To describe the history, clinical features, and outcomes of a large case series of microsporidial stromal keratitis with emphasis on probable predictors of the etiology in this rare and unspecified form of keratitis. METHODS: Retrospective analysis of cases seen between January, 2002, and December, 2013, diagnosed at LV Prasad Eye Institute as having microsporidial stromal keratitis based on clinical, microbiology, and histopathology examination. Outcomes of medical and surgical management with visual recovery were documented. RESULTS: There were 34 cases of microsporidial stromal keratitis with a mean age of 43.3 years (range 2-77 years) and male preponderance. The duration of symptoms was chronic in most cases (mean duration of 288 days). Nearly half of the cases had a history of trauma, in the age group of 20 to 50 years. The most common clinical misdiagnosis was herpes simplex virus keratitis (26.5%) followed by fungal keratitis (17.6%). This could be attributed to the nonspecific clinical picture of diffuse multifocal stromal lesions in 82.4% of cases. The organisms were detected in microbiological evaluation of corneal scrapings in 47% cases, and histopathological detection of the organisms showed a positivity rate of 92.3%. Surgical management was necessary in the majority of the cases (73.5% of patients). CONCLUSIONS: The typical history of trauma with a smoldering, diffuse form of keratitis refractory to conventional medical therapy, responding to surgical removal for recovery is clearly demonstrated as a recurring feature in the majority of the cases. Patients presenting with this characteristic clinical picture should be suspected to harbor this rare pathogen, and early surgical interventions should be considered.

Femtosecond laser-assisted anterior lamellar keratoplasty in stromal keratitis caused by an Endoreticulatus-like microsporidia.

PURPOSE: To describe a case of stromal keratitis caused by an Endoreticulatus-like microsporidia satisfactorily treated with femtosecond-assisted anterior lamellar keratoplasty (femto-ALK). METHODS: Case report. RESULTS: A 65-year-old healthy woman had a 10-month history of foreign body sensation and blurred vision after a flying insect struck her right eye. The patient presented with focal central to paracentral anterior stromal infiltration and edema with keratic precipitates of the right cornea. Confocal microscopy showed enhanced keratocytes with intracellular hyperreflective round and ovoid bodies confined to the anterior two thirds of corneal stroma. Femto-ALK was performed for excisional biopsy and replacement with anterior stromal donor cornea. A modified trichrome stain of corneal tissue showed numerous microsporidial spores whose small subunit rRNA sequence belonged to that of an Endoreticulatus-like microsporidia. CONCLUSIONS: Besides systemic infection, Endoreticulatus-like microsporidia can cause stromal keratitis. With careful patient selection, femto-ALK may be considered a new surgical alternative with satisfactory treatment outcomes.

Early-Onset Candida glabrata Interface Keratitis after Deep Anterior Lamellar Keratoplasty.

PURPOSE: Fungal interface keratitis by Candida species can occur several weeks to several months after deep anterior lamellar keratoplasty (DALK). Here, we report a case of early-onset fungal interface keratitis by Candida glabrata after DALK. CASE REPORT: A 31-year-old Chinese man complained of decreased vision 4 days after an uneventful DALK for keratoconus. White to cream-colored interface deposits were identified under slit-lamp examination. The confocal scan disclosed clusters of hyperreflective granular deposits of 2 to 4 µm at the interface, without evidence of inflammation or hyphae-like structures. The graft was then removed, along with interface irrigation, and another graft was sutured. Finally, a penetrating keratoplasty was performed because the interface opacities recurred and deteriorated after graft replacement. Histopathological examination disclosed yeast-like structures at the retrocorneal side. The microbiologic results of both corneal scrapings taken from the recipient stromal bed and the removed half cornea button showed C. glabrata. CONCLUSIONS: Candida glabrata interface keratitis can occur early after DALK, which can only be effectively treated with penetrating keratoplasty. Confocal microscopy is a promising tool to diagnose this rare complication. The importance of donor corneoscleral rim cultures should be emphasized during DALK.

Intrastromal voriconazole for deep recalcitrant fungal keratitis: a case series.

AIM: To evaluate the outcomes of treating deep recalcitrant fungal keratitis with intrastromal voriconazole injection. METHODS: Twenty-five patients with culture proven fungal keratitis, not responding to a combination of topical 5% natamycin and 1% voriconazole were treated with intrastromal voriconazole (50 µg/0.1 mL) injected in five divided doses around the infiltrate to form a depot of the drug around the circumference of the lesion. RESULTS: The mean age of the patients was 52.52±12.21 years and mean time to presentation was 17.12±13.75 days from the onset of symptoms. The mean area of the infiltrate was 30.41±17.2 mm(2), hypopyon was present in 88% and all cases had infiltrates that extended beyond the mid-stromal level. Intrastromal voriconazole helped to resolve the infection in 18 (72%) patients and about 15% of these needed more than one injection. Smaller ulcers responded better to treatment. Fusarium spp were responsible for six of the seven cases that failed treatment. CONCLUSIONS: Targeted delivery of voriconazole by intrastromal injection (50 µg/0.1 mL) is a safe and effective way to treat deep recalcitrant fungal keratitis, though some may need repeated injections. Fusarium keratitis may show suboptimal response but this needs further study.

TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis.

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.

Microsporidial stromal keratitis masquerading as acute graft rejection.

PURPOSE: To describe the clinical features, disease course, management, and visual outcome of stromal microsporidial keratitis involving the corneal graft in an immunocompetent patient. METHODS: Case report. RESULTS: We report the case of a 21-year-old immunocompetent woman, who underwent penetrating keratoplasty for advanced keratoconus in her right eye and presented with features of acute graft rejection after 15 months. Standard treatment using topical and systemic steroids failed to reverse the graft rejection. A regraft was performed in that eye. Histopathology of the donor corneal button revealed microsporidiosis involving the deep stroma. CONCLUSIONS: Microsporidiosis is rarely encountered in a corneal graft and masquerades as acute graft rejection. Histopathology could help in making a final diagnosis.

Staphylococcus aureus Blepharitis Associated with Multiple Corneal Stromal Microabscess, Stromal Edema, and Uveitis.

We report a case of an immunocompetent woman with atypical marginal keratitis. She presented with recurrent episodes of multiples microabscess distributed in a triangular pattern associated with stromal oedema and anterior chamber uveitis, affecting both eyes, but not simultaneously. The episodes responded to steroid drops, corneal inflammation was coincidental with a worsening of her blepharitis in the affected eye and S. aureus was isolated from the lids.