Diagnostic utility of in vivo confocal microscopy in Acanthamoeba keratitis following corneal crosslinking.

Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening complication of corneal collagen crosslinking (CXL) for keratoconus. In this report, we describe an early adolescent male who underwent routine CXL for progressive keratoconus in his left eye. Preprocedural left visual acuity (VA) was 6/9. At day 5 postprocedure, multifocal corneal infiltrates were identified. Corneal scrape, bandage contact lens cultures and herpetic and Acanthamoeba PCR were negative. In vivo, confocal microscopy (IVCM) identified Acanthamoeba cysts within the corneal stroma. Intensive amoebicidal therapy was initiated, but recovery was complicated by significant inflammation, resulting in widespread aggressive corneal vascularisation necessitating topical steroids and steroid-sparing agents. At 10 months, his left VA was 6/24. This report emphasises the importance of maintaining a high index of suspicion for AK in cases of post-CXL microbial keratitis and highlights the diagnostic value of IVCM, particularly in culture-negative and PCR-negative cases.

Case Report: Suspected Donor Transmission of Acanthamoeba Keratitis After Deep Anterior Lamellar Keratoplasty.

PURPOSE: In our report, we present a suspected case of donor-derived Acanthamoeba keratitis after deep anterior lamellar keratoplasty. To the authors' knowledge, there have been no confirmed cases of Acanthamoeba keratitis transmission through corneal transplantation. METHODS: Deep anterior lamellar keratoplasty was performed on the right eye of a 33-year-old man with severe bilateral keratoconus and an intolerance to all forms of contact lenses. The postoperative visual acuity deteriorated, while inflammation, rising ocular pressure, increasing corneal thickness, and severe eye pain began to present. Confocal imaging revealed hyperreflective cysts and trophozoite figures representative of amoebic keratitis. Despite an additional penetrating keratoplasty, antiamoeba therapy, and corneal crosslinking, the patient's condition worsened, resulting in stromal melt and corneal perforation. Emergent combined surgery of temporary keratoprosthesis, vitrectomy, lensectomy, and iridectomy was performed, along with Ahmed valve shunt placement and another penetrating keratoplasty. RESULTS: The infection was resistant to aggressive antiamoeba therapy, but after the emergent combined surgery, the graft re-epithelialized quickly and has since remained clear, with no presence of keratitis. CONCLUSIONS: Several signs led us to believe that this case was donor-derived. There was little opportunity for graft exposure to the amoeba, and deep amoebic cysts and trophozoites were present on postoperative week 1-a highly unusual time course and depth of invasion for primary amoebic infection. In addition, pathological analysis revealed cysts only within the confines of the donor tissue and none in the recipient; Acanthamoeba cysts would have been present in the recipient rim tissue if the infection originated from the patient himself.

18S rDNA sequencing aided diagnosis of Acanthamoeba jacobsi keratitis -A case report.

Identification of Acanthamoeba cysts and trophozoites in cases of keratitis is traditionally done with microbiological techniques such as smear examination with 10% potassium hydroxide (KOH) and culture. Double walled cyst with hexagonal inner wall is characteristic of Acanthamoeba. We report a unique case of a 9 year old boy who presented with dense anterior corneal stromal infltration, which on smear examination showed atypical double walled spherical cysts, leading to a diagnostic dilemma. An 18S rRNA gene-based PCR done on the growth on culture, subsequently identifed a rarely reported species of Acanthamoeba. The patient was advised combination therapy with polyhexamethylene biguanide (PHMB 0.02%) and chlorhexidine (0.02%) eye drops. Three weeks post treatment, the keratitis resolved with scarring and vascularisation and visual acuity improved to 20/60. At 8 weeks follow up Best corrected visual acuity further improved to 20/30 with contact lens.

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.

A report of 10 patients with Acanthamoeba keratitis. / Casuística de 10 pacientes con queratitis por Acanthamoeba.

CLINICAL CASES: Cases are presented of 10 patients with Acanthamoeba keratitis treated between 2008 and 2017. All were contact lens wearers. All of them received treatment with a biguanide combined with a diamidine. In 3 cases the infestation did not exceed the superficial stroma, responding to topical treatment. In 7, the infection reached the deep stroma, with 6 of these cases requiring penetrating keratoplasty (PKP), 3 of them therapeutic PKP because of perforation risk or ocular spreading. The visual acuity improved in all the cases. CONCLUSION: The infestation depth at the time of diagnosis appears to be the main risk factor for requiring a PKP.

Keratitis After Post-Kala-Azar Dermal Leishmaniasis.

PURPOSE: To report 2 cases of keratitis occurring after post-kala-azar dermal leishmaniasis (PKDL) in immunocompetent patients. METHODS: Case report and literature review. RESULTS: Two male patients presented with a dense stromal infiltrate in the temporal and superior aspects of the cornea, respectively. Microbiological workup in both patients did not show any offending organism. Both patients were undergoing treatment for PKDL in the form of oral miltefosine. After careful deliberation, topical prednisolone was started under antibiotic cover for both patients with close follow-up. There was a dramatic improvement with resolution of the infiltrate and a good visual outcome. CONCLUSIONS: We present the first reported cases of keratitis after PKDL, which responded to topical corticosteroid therapy. The treating clinician must be made aware of such a presentation of leishmaniasis especially in endemic areas.

Intracorneal rhinosporidiosis managed with deep anterior lamellar keratoplasty.

A healthy 22-year-old male presented to Institutional Cornea Clinic with an intracorneal mass overlying the pupil with lobulated edges having many tiny greyish white dots. The patient had a history of trauma while swimming in a pond with subsequent removal of intracorneal foreign body in the left eye approximately a year prior to presentation. Anterior segment optical coherence tomography (OCT) confirmed that an intracorneal mass sparing deep stroma and Descemet's membrane. A deep anterior lamellar keratoplasty (DALK) was performed in left eye and the mass was sent for histology examination. Histology evaluation was suggestive of rhinosporidiosis. The patient achieved 20/60 BCVA with -1.25 Χ× 120° 1 year postoperatively without any evidence of recurrence at the graft-host interface. This unique presentation (as an 'intracorneal mass') of ocular rhinosporidiosis emphasizes that clinicians from our region of the world must consider rhinosporidiosis in the differential diagnosis especially with a history of penetrating injury while swimming in pond or river water.

Mixed Acanthamoeba and multidrug-resistant Achromobacter xyloxidans in late-onset keratitis after laser in situ keratomileusis.

A 31-year-old woman developed a spontaneous flap interface keratitis in the left eye 6 years after a laser in situ keratomileusis (LASIK) enhancement. Cultures and polymerase chain reaction (PCR) were positive for Achromobacter xyloxidans resistant to first- and second-generation cephalosporin, aminoglycosides, and quinolones and also positive for Acanthamoeba T4. Treatment with topical fortified ceftazidime, topical chlorhexidine, and voriconazole and oral voriconazole did not stop the progression of the disease. Flap amputation revealed persistence of Acanthamoeba but not Achromobacter. Six weeks after flap amputation, the infiltrate had resolved, PCR was negative for Acanthamoeba, and the cornea had fully epithelialized. To our knowledge, this is the first report of post-LASIK infectious keratitis caused by mixed infection of Achromobacter xyloxidans and Acanthamoeba occurring years after the procedure without apparent ocular trauma. It is also the first report of the use of combined systemic and topical voriconazole as a therapy for Acanthamoeba keratitis after LASIK.

Cytopathic effect of Acanthamoeba on human corneal fibroblasts.

PURPOSE: Acanthamoeba keratitis is associated with keratocyte depletion in humans. We investigated how Acanthamoebae isolated from corneas affected by Acanthamoeba keratitis interacted with human corneal stromal cells in vitro. METHODS: Acanthamoebae were isolated from 6 patients with Acanthamoeba keratitis and genotyping was done. Whether the isolated Acanthamoebae could invade the corneal stroma was assessed with denuded corneal stroma ex vivo. The cytopathic effect of Acanthamoeba on cultured corneal fibroblasts from donor corneas was quantitatively evaluated by the MTT assay after culture under various conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Annexin V staining were employed to detect apoptotic cells among the corneal fibroblasts co-cultured with Acanthamoebae. RESULTS: All 6 Acanthamoebae isolated from the patients with Acanthamoeba keratitis were shown to have the T4 genotype by 18S rDNA sequence analysis. Acanthamoebae invaded the denuded corneal stroma in the ex vivo experiments and had a cytopathic effect on human corneal fibroblasts after direct adhesion, but not via chemical mediators. A cytopathic effect was detected with all 6 Acanthamoebae and corneal fibroblasts mainly died by apoptosis, as evidenced by Annexin V staining. CONCLUSIONS: Acanthamoebae isolated from patients with Acanthamoeba keratitis had a cytopathic effect on human corneal fibroblasts, mainly via induction of apoptosis after direct adhesion. Our findings may provide some clues to the pathophysiology of corneal keratocyte depletion in patients with Acanthamoeba keratitis.

Successful treatment of chronic stromal acanthamoeba keratitis with oral voriconazole monotherapy.

PURPOSE: To describe the treatment of chronic stromal Acanthamoeba keratitis (AK) with oral voriconazole monotherapy. METHODS: All cases of chronic stromal AK recalcitrant to traditional therapy subsequently treated with systemic voriconazole seen at the University of Illinois Eye and Ear Infirmary between June 2003 and July 2009 were reviewed for clinical presentation, clinical course, and outcome. RESULTS: Three eyes of 2 patients were identified with culture-confirmed chronic stromal AK unresponsive to traditional antiacanthamoebal therapies, requiring topical corticosteroids to maintain corneal clarity. Oral voriconazole 200 mg twice daily achieved a rapid but transient reduction of inflammation and elimination of corticosteroid dependency but, in both patients, recrudesced approximately 6 weeks after its discontinuation. Subsequent repeated and/or extended use of oral voriconazole alone resulted in complete resolution ranging from 7 to 11 months off all medications with final best-corrected visual acuity ranging from 20/20 to 20/25. CONCLUSIONS: Recalcitrant chronic Acanthamoeba stromal keratitis was successfully treated with extended systemic voriconazole administration with good preservation of vision. The clinical resolution of chronic stromal keratitis in our 2 cases suggests that voriconazole may have a larger role in the treatment of AK.

Penetrating keratoplasty in active Acanthamoeba keratitis.

PURPOSE: To report the results of penetrating keratoplasty (PK) in active Acanthamoeba keratitis (AK). METHODS: Nine patients with deep stromal infiltrates because of AK were treated with intensive antiamoebic medical therapy followed by PK during the acute infectious phase because of poor clinical response or poor compliance. Antiamoebic therapy was tapered after PK. RESULTS: Visual acuity ranged from 20/15 to 20/50 after an average of 17 months after PK with no signs of recurrences. Patients had rapid resolution of symptoms. CONCLUSION: PK is a viable option for active AK not responding to maximum medical treatment.

Gamma interferon and interleukin-1 receptor 1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis.

Toll-like receptor 2 (TLR2) is an essential mediator of corneal inflammation induced by the filarial nematode Onchocerca volvulus, which harbors endosymbiotic Wolbachia bacteria. TLR2 is also required for dendritic cell activation, gamma interferon (IFN-gamma) production, and neutrophil recruitment to the cornea. To examine the role of IFN-gamma in O. volvulus keratitis, C57BL/6 and IFN-gamma(-/-) mice were immunized subcutaneously, and a soluble antigen extract from O. volvulus adult worms (OvAg) was injected into the corneal stroma of each animal. We found that, in the absence of IFN-gamma, neutrophil recruitment to the cornea was significantly impaired, whereas there was no effect on eosinophil infiltration. Since the cornea contains resident macrophages and fibroblasts and our previous studies showed that CXC chemokines mediate neutrophil recruitment, we examined the role of recombinant IFN-gamma (rIFN-gamma) on each cell type. We found no effect of rIFN-gamma on CXC chemokine production by macrophages or corneal fibroblasts, either alone or with filarial extracts; in contrast, rIFN-gamma was found to enhance OvAg-induced tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-1alpha, and IL-1beta in macrophages. Furthermore, we found that rTNF-alpha, rIL-1alpha, or rIL-1beta induced CXC chemokine production by corneal fibroblasts but not by macrophages. To determine the relative contributions of endogenous cytokines, we injected OvAg into the corneal stroma of C57BL/6, IL-1 receptor 1(-/-) (IL-1R1(-/-)), and TNF-alphaR1/2(-/-) mice and examined neutrophil recruitment. We found that neutrophil infiltration was impaired in IL-1R1(-/-) mice but not in TNF-alphaR1/2(-/-) mice. IFN-gamma therefore appears to regulate neutrophil recruitment to the corneal stroma by enhancing TLR2 expression and OvAg-induced IL-1alpha and IL-1beta production by macrophages in the cornea, which then induce IL-1R1-dependent production of CXC chemokine by resident corneal fibroblasts.

Acanthamoeba keratitis: persistent organisms without inflammation after 1 year of topical chlorhexidine.

PURPOSE: To report a non-contact lens wearer with persistent Acanthamoeba organisms in the cornea after being treated with medical therapy that included topical chlorhexidine as 1 agent for 1 year. METHODS: A 53-year-old man with Acanthamoeba keratitis was treated with medical therapy for >1 year, followed by a penetrating keratoplasty. RESULTS: Histopathologic examination of the keratoplasty specimen revealed viable-appearing Acanthamoeba cysts and trophozoites within the deep corneal stroma in a focus of corneal scarring. CONCLUSIONS: The use of chlorhexidine as 1 agent in the medical management of Acanthamoeba keratitis may not eradicate the organisms.

Unusual case of acanthamoeba keratitis in a contact lens wearer.

PURPOSE: To present an atypical case of acanthamoeba keratitis in a soft contact lens wearer. METHODS: A case report of a 28-year-old female soft contact lens wearer with a 6-day history of redness and blurred vision in her right eye. Examination showed stromal keratitis that did not respond to therapy for herpes simplex virus. Four weeks later, the patient had increased stromal infiltration, an endothelial plaque, and a hypopyon. She did not complain of pain apart from mild discomfort. Corneal smears and anterior chamber tap were negative for bacteria, fungi, acanthamoeba, and herpes simplex virus, and the patient was prescribed fortified antibiotics. One week later, a large epithelial defect with a surrounding ring infiltrate was apparent. Corneal biopsy showed acanthamoeba. RESULTS: The acanthamoeba keratitis resolved with successful treatment. One year later, the patient had a residual visual deficit secondary to stromal scarring and her best-corrected visual acuity was 20/29. CONCLUSIONS: This case emphasizes the importance of considering acanthamoeba species in the differential diagnosis of keratitis, even without the classic symptom of severe pain. A high degree of suspicion with rapid and appropriate treatment may result in improved recovery of vision.

A keratitis rat model for evaluation of anti-Acanthamoeba polyphaga agents.

PURPOSE: To develop a rat model of chronic Acanthamoeba polyphaga keratitis suitable for pharmacologic assessment of therapeutic agents. METHODS: An A. polyphaga isolate (ATCC #50495) was grown in peptone-yeast extract-glucose medium. Five-weeks-old, Sprague-Dawley male rats were injected with 10(3) or 10(4) trophozoites in the left cornea stromal layer. A subconjunctival injection of 0.14, 0.28, or 0.57 mg long-acting betamethasone was performed weekly. At the end of experiments, rats were killed; the superficial corneal epithelium gently scraped and cultured; and globes histologically examined. Topical polyhexamethylene biguanide (PHMB), hexamidine diisethionate, and miltefosine (hexadecylphosphocholine) were administered topically as eye drops 3 times a day at concentrations of 0.02%, 0.1%, and 0.01% respectively. In vitro minimal inhibitory concentration (MIC) and fractional inhibitory concentration values were measured in A. polyphaga cultures. RESULTS: In infected eyes, lesions consisted of the sequential appearance within 2 weeks of edema, infiltrates, and/or abscesses. On day 35 postinfection, a combination of 10(4) parasites with a regimen of 0.28 mg/week betamethasone resulted in the highest ratio of rats with abscesses. Presence of A. polyphaga was confirmed histologically and inconsistently in cultures. In rats optimally prepared as said earlier, agents were administered on day 6 postinfection. A combination of PHMB and hexamidine diisethionate exerted a synergistic effect and was more effective than PHMB, hexamidine diisethionate, or miltefosine alone. In vitro, PHMB (MIC = 14.6 microM) and hexamidine diisethionate (MIC = 555 microM) exerted a synergistic effect (fractional inhibitory concentration = 0.06), and miltefosine exhibited antiamoebal activity (MIC = 27.4 microM). CONCLUSIONS: In this study, a rat model of chronic A. polyphaga keratitis was obtained and found suitable for assessment of pharmacologic agents. It provides an in vivo approach of drug resistance, pathogenicity, and physiopathologic mechanisms of chronic amoebic keratitis.

Chronic microsporidial stromal keratitis in an immunocompetent, non-contact lens wearer.

An 82-year-old healthy man with unilateral chronic stromal keratitis, initially diagnosed to have viral keratitis and refractory to medical therapy, showed numerous oval, microsporidial organisms, measuring 4-5 m in length in the corneal biopsy. Penetrating keratoplasty, followed by treatment with systemic albendazole and topical propamidine isethionate resulted in resolution of the infection. Electron microscopy of the keratoplasty specimen demonstrated sporoblasts with diplokaryotic nuclei and multiple coils of the filament. The light and electron microscopic features were consistent with microsporidial keratitis.

Local and systemic humoral immune response during acute and chronic Acanthamoeba keratitis in rabbits.

PURPOSE: To study local and systemic antibody responses during the course of acute and chronic Acanthamoeba keratitis in a rabbit model. The role of secretory IgA (sIgA) in innate and adaptive immunity is investigated. METHODS: Acanthamoeba keratitis was induced by intrastromal injection of live trophozoites in rabbits with and without prior oral immunization with aqueous Acanthamoeba antigen. The severity score of the ensuing keratitis was followed. Anti-Acanthamoeba antibodies in tears and sera were determined before infection and on a weekly basis postinfection for 6 weeks. The role of mucosal IgA as a link between the innate and adaptive immunity to Acanthamoeba was evaluated. RESULTS: Acanthamoeba keratitis was significantly milder in animals infected after oral immunization than in naïve animals. Mucosal sIgA bound Acanthamoeba in a concentration-dependent, mannose-mediated manner. It significantly inhibited Acanthamoeba binding to rabbit corneal epithelium in vitro and in vivo. Anti-Acanthamoeba IgA significantly influenced amoebic lysis by neutrophils. CONCLUSIONS: Mucosal IgA protects the external ocular surface by virtue of anti-adhesin properties displayed by the mannosylated residues on the heavy chains of IgA molecule (innate immunity) as well as specific antigen-binding sites (adaptive immunity). Immune IgA also augmented neutrophil-mediated amoebic lysis (adaptive immunity).

An atypical presentation of Acanthamoeba keratitis in a noncontact lens wearer.

This article presents the case of a 49-year-old man who did not have a history of wearing contact lenses and who developed a rapidly progressive course of Acanthamoeba keratitis. The patient developed stromal keratitis that did not respond to herpes simplex virus therapies. Within 1 week after presentation, the patient progressed from mild anterior stromal haze and edema to a ring infiltrate, epithelial loss, and significant corneal edema. Corneal scrapings demonstrated cysts consistent with Acanthanmoeba keratitis. The patient was admitted to the hospital and placed on intensive medical therapy. He responded to therapy, and at 5 months showed central scarring in a quiet eye. This article presents a case of Acanthamoeba keratitis in a non-contact lens wearer, who was diagnosed clinically and histopathologically within 1 week of onset of symptoms. His case was atypical given his lack of contact lens wear or antecedent trauma and rapid progression to a ring infiltrate, usually seen as late findings.

IL-4 and IL-13 regulation of ICAM-1 expression and eosinophil recruitment in Onchocerca volvulus keratitis.

PURPOSE: The presence of eosinophilic granulocytes in ocular tissue is a hallmark of the host response to environmental and parasite allergens. Using a mouse model of Onchocerca volvulus-mediated keratitis (river blindness), the present study examined the role of the cytokines interleukin (IL)-4 and IL-13 in regulating recruitment of eosinophils to the cornea through expression of intercellular cell adhesion molecule (ICAM)-1. METHODS: C57BL/6 mice received an intrastromal injection of recombinant IL-4 and IL-13 (rIL-4 and IL-13) or were immunized by subcutaneous injection prior to receiving an intrastromal injection of a soluble O. volvulus extract. Expression of ICAM-1 and recruitment of eosinophils to the cornea were monitored by immunohistochemistry. RESULTS: Expression of ICAM-1 was elevated after injection of rIL-4 or IL-13 together with recombinant tumor necrosis factor (rTNF)-alpha. Conversely, expression of ICAM-1 in O. volvulus-mediated keratitis was significantly reduced after subconjunctival injection of a monoclonal antibody (mAb) to IL-4 or IL-13. In addition, combined in vivo neutralization of IL-4 and IL-13 inhibited recruitment of eosinophils, but not of neutrophils, to the corneal stroma. CONCLUSIONS: These findings demonstrate that expression of ICAM-1 and recruitment of eosinophils to the cornea are tightly regulated by IL-4 and IL-13, and indicate that these cytokines are a potential target for immune intervention in ocular allergy and parasitic infections of the eye.