Endothelial Biopsy for the Diagnosis and Management of Culture-Negative Retrocorneal Fungal Keratitis With the Assistance of Optical Coherence Tomography Imaging.

PURPOSE: To report a case of a culture-negative deep fungal corneal infection that was diagnosed after histopathology of an anterior segment optical coherence tomography-guided endothelial biopsy. METHODS: A 22-year-old woman with history of contact lens wear and concomitant topical steroid use presented with a mid-stromal corneal infiltrate that failed to respond to oral acyclovir and topical fortified antibiotics. Although cornea stains, cultures, and confocal microscopy showed negative results, there was high clinical suspicion for fungal keratitis. After 2 months on topical natamycin, oral voriconazole, and serial intrastromal and intracameral voriconazole injections, the infiltrate enlarged and deepened. Imaging with anterior segment optical coherence tomography revealed that the infection had progressed to an endothelial plaque. RESULTS: Diagnostic endothelial biopsy was performed in the operating room. Cultures showed again negative results, whereas histopathology of the removed specimen revealed fungal elements. The postoperative edema at the site of the biopsy resolved over the course of 4 weeks, and a posterior stromal scar formed. Serial intrastromal and intracameral voriconazole injections were continued for the first postoperative month. At the 1-year and the 3-year follow-up examinations, the patient's vision was 20/20 without recurrence. CONCLUSIONS: Intraoperative scraping of the endothelial plaque and histopathologic evaluation of the specimen proved to be of utmost importance for definitive diagnosis and resolution of the culture-negative deep fungal infection in this case. This young patient's cornea was retained and vision remains excellent.

Efficacy and Safety of Various Amphotericin B Concentrations on Candida albicans in Cold Storage Conditions.

PURPOSE: To determine the concentration of amphotericin B that would be both effective against Candida albicans contamination and safe for corneal endothelial cells (CECs) in cold storage conditions. METHODS: Triplicate media cultures were inoculated with 10 colony-forming units (CFUs)/mL of C. albicans (American Type Culture Collection 10231), supplemented with amphotericin B (0-20 µg/mL), stored in cold conditions (2°C-8°C) for 72 hours, and analyzed quantitatively for CFUs. C. albicans concentration in each sample was determined initially and after 6, 24, 48, and 72 hours of storage. CEC mitochondrial function (oxygen consumption rate), apoptosis, and necrosis were examined in donor corneas after 7 days of amphotericin B exposure and compared with untreated controls. CEC viability was also examined by calcein-AM staining and Fiji segmentation after 72 hours or 2 weeks of amphotericin B exposure to mimic potential eye bank practices. RESULTS: Amphotericin B concentrations of 1.25, 2.5, and 5.0 µg/mL resulted in 0.47, 1.11, and 1.21 log10 CFU reduction after only 6 hours of cold storage and continued to decrease to 3.50, 3.86, and 4.49 log10 reductions after 72 hours, respectively. By contrast, amphotericin B 0.255 µg/mL showed only 1.01 log10 CFU reduction after 72 hours of incubation. CEC mitochondrial function and viability did not differ in donor corneas exposed to amphotericin B ≤2.59 µg/mL compared with the controls. CONCLUSIONS: Optimal efficacy of amphotericin B against C. albicans is achieved in cold storage conditions at concentrations ≥1.25 µg/mL, and 2.5 µg/mL reduces Candida contamination by >90% after 6 hours of cold storage without sacrificing CEC health.

Infectious crystalline keratopathy post-Descemet stripping endothelial keratoplasty.

We report a case of a 46-year-old female who developed infectious crystalline keratopathy (ICK) after Descemet's stripping endothelial keratoplasty (DSEK). She underwent DSEK for pseudophakic corneal edema in her left eye. Ten weeks after the procedure, the patient presented with complaints of blurred vision, redness in eye, and ocular pain. Slit lamp examination revealed white nonsuppurative branching deep stromal infiltrate. Microscopic examination of the Gram-stained smear showed gram-positive cocci. Streptococcus viridans was isolated on cultures. Isolated organism was sensitive to linezolid. Based on antibiotic sensitivity report, fortified linezolid (0.2%) eye drop was started on hourly basis. After 10 weeks of topical fortified linezolid (0.2%) therapy, complete resolution of infiltrate with significant corneal scarring and vascularization was seen. Infectious crystalline keratopathy can occur after DSEK.

Interface Scopulariopsis gracilis fungal keratitis following Descemet's stripping automated endothelial keratoplasty (DSAEK) with a contaminated graft.

PURPOSE: To report for the first time a case of interface Scopulariopsis gracilis fungal keratitis following Descemet's stripping automated endothelial keratoplasty (DSAEK) with a contaminated graft. METHODS: A 57-year-old man with bilateral keratoconus and previous bilateral penetrating keratoplasties (PK) developed graft failure in association with marked corneal ectasia. He underwent a successful DSAEK. Unfortunately, a contaminated graft was transplanted and the following morning we were contacted by the eye bank to inform us a slow-growing fungus had been detected in the culture plates inoculated with dextran solution used to store the issued corneoscleral button. Immediate patient review revealed four infiltrates in the interface between the donor and the recipient tissue. The patient returned to theatre for the removal of the infected graft and was successfully treated with topical amphotericin 0.15%, voriconazole 1% and oral voriconazole and later oral itraconazole. Two intracameral injections of 5 µg in 0.1 ml of amphotericin B were also performed. RESULTS: A reference laboratory cultured and identified the fungus as Scopulariopsis gracilis species. The patient responded to treatment and eventually achieved a spectacle-corrected logMAR visual acuity of 0.3 following a delayed PK. CONCLUSION: Scopulariopsis gracilis fungal keratitis is a rare infection, and the species can be difficult to eradicate. This is the first case report of an infection secondary to a contaminated graft with the species, and we report its successful treatment with an excellent visual outcome.

Association Between Fungal Contamination and Eye Bank-Prepared Endothelial Keratoplasty Tissue: Temperature-Dependent Risk Factors and Antifungal Supplementation of Optisol-Gentamicin and Streptomycin.

Importance: Fungal contamination and infection from donor tissues processed for endothelial keratoplasty is a growing concern, prompting analysis of donor tissues after processing. Objective: To determine whether eyebank-processed endothelial keratoplasty tissue is at higher risk of contamination than unprocessed tissue and to model eyebank processing with regard to room temperature exposure on Candida growth in optisol-gentamicin and streptomycin (GS) with and without antifungal supplementation. Design, Setting, and Participants: An examination of the 2013 Eversight Eyebank Study follow-up database for risk factors associated with post-keratoplasty infection identified an increased risk of positive fungal rim culture results in tissue processed for endothelial keratoplasty vs unprocessed tissue. Processing steps at room temperature were hypothesized as a potential risk factor for promotion of fungal growth between these 2 processes. Candida albicans, Candida glabrata, and Candida parapsilosis endophthalmitis isolates were each inoculated into optisol-GS and subjected to 2 different room temperature incubation regimens reflective of current corneal tissue handling protocols. Main Outcomes and Measures: Eversight Eyebank Study outcomes and measures were follow-up inquiries from 6592 corneal transplants. Efficacy study outcomes and measures were fungal colony-forming units from inoculated vials of optisol-GS taken at 2 different processing temperatures. Results: Donor rim culture results were 3 times more likely to be positive for fungi in endothelial keratoplasty-processed eyes (1.14%) than for other uses (0.37%) (difference, 0.77%; 95% CI, 0.17-.1.37) (P = .009). In vitro, increased room temperature incubation of optisol-GS increased growth of Candida species over time. The addition of caspofungin and voriconazole decreased growth of Candida in a species-dependent manner. Conclusions and Relevance: Detectable Candida growth in donor rim cultures, associated with a higher rate of post keratoplasty infection, is seen in endothelial keratoplasty tissue vs other uses at the time of transplantation, likely owing in part to eyebank preparation processes extending the time of tissue warming. Reduced room temperature incubation and the addition of antifungal agents decreased growth of Candida species in optisol-GS and should be further explored to reduce the risk of infection.

The Effect of Light Exposure on the Efficacy and Safety of Amphotericin B in Corneal Storage Media.

IMPORTANCE: The proportion of postkeratoplasty fungal infections is rising steadily. However, the most commonly used corneal storage medium in the United States, Optisol-GS, does not contain an antifungal additive. OBJECTIVES: To determine the lowest concentration of amphotericin B supplementation in Optisol-GS that will eliminate fungal contaminants effectively without resulting in toxic effects to the cornea and to determine what role light exposure plays in the efficacy and safety of amphotericin B supplementation. DESIGN, SETTING, AND MATERIALS: An in vitro laboratory efficacy study measured fungal colony growth in 10 vials of Optisol-GS supplemented with different concentrations of amphotericin B after inoculation with Candida albicans in light-exposed and light-protected conditions. Two vials each were supplemented with amphotericin B at concentrations of 0.06, 0.12, or 0.225 µg/mL; the remaining 2 vials received no C albicans inoculation and no antifungal supplementation (negative controls). After 24 hours, 1 vial from each pair was exposed to light for the remainder of the study. On days 2, 7, and 14, 1 mL of solution was removed from each vial and incubated at 36°C for 48 hours. In a separate safety study, 12 pairs of corneas were divided between amphotericin B supplementation and the control condition; 4 corneas each received the different amphotericin B concentrations. An additional 4 pairs of corneas were stored in the 0.225-µg/mL concentration, and 1 cornea from each pair was exposed to light for the duration of the study. Data were collected November 16, 2014, and analyzed from November 16 to 18, 2014, for the efficacy study; they were collected from April 14 to May 27, 2015, and analyzed from May 28 to 30, 2015, and on December 23, 2015, for the safety study. MAIN OUTCOMES AND MEASURES: Fungal colony growth was measured from the Optisol-GS vials. Corneal endothelial cell density, endothelial cell viability, and epithelial toxic effects were measured in stored corneas. RESULTS: In the efficacy study, Optisol-GS supplemented with concentrations of 0.06 and 0.12 µg/mL of amphotericin B eliminated all fungal contaminants by day 7 and reduced fungal growth on day 2 by a mean of 3.5 colony-forming units (95% CI, -6.19 to 13.20 colony-forming units; P = .34), a 77.8% decline compared with the postoperative controls. Optisol-GS supplemented with the 0.255-µg/mL concentration of amphotericin B eliminated all fungal contaminants by day 2. In the safety study, no evidence was found of toxic effects to the cornea in corneas stored in Optisol-GS supplemented with amphotericin B at any concentration compared with paired controls. No difference in the efficacy or safety of the light-exposed compared with light-protected amphotericin B-supplemented Optisol-GS was identified. CONCLUSIONS AND RELEVANCE: In this study, Optisol-GS supplemented with a 0.255-µg/mL concentration of amphotericin B effectively eliminated fungal contaminants within 48 hours and did not result in added toxic effects to the cornea. These results do not prove that amphotericin B should be added to Optisol-GS; larger-scale studies and cost-benefit analyses need to be completed. Given the increasing incidence of postkeratoplasty fungal infection, however, the addition of amphotericin B to Optisol-GS deserves further investigation.

Interface infection after descemet stripping automated endothelial keratoplasty: outcomes of therapeutic keratoplasty.

PURPOSE: The aim of this study was to evaluate the visual outcomes and graft survival rate after therapeutic keratoplasty performed for interface infection after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This is a retrospective, interventional case series. The study population comprised 7 patients who developed unilateral post-DSAEK interface infection unresponsive to conservative treatment, with or without graft exchange, and were treated with penetrating keratoplasty (PK), 9 to 9.5 mm in diameter, with en bloc excision of the recipient cornea and DSAEK graft. The main outcome measures included best spectacle-corrected visual acuity, refractive error, histological examination, reinfection, and rejection and graft survival rates. RESULTS: Interface infection was diagnosed in 10 (0.92%) of 1088 eyes that underwent DSAEK at our institution between 2005 and 2013. Seven of 10 eyes (0.64% of the total) were unresponsive to conservative treatment and underwent therapeutic keratoplasty. Candida and Staphylococcus species were identified in 3 cases each, and Nocardia species was identified in 1 case. With a mean post-PK follow-up of 25.4 months (range 4-60 months), no recurrence of infection was seen in any eye, and 5 of 7 PK grafts remained clear. Best spectacle-corrected visual acuity was 20/20 in 2 eyes, better than 20/50 in 4 eyes, and 20/100 or worse in 3 eyes, in 2 of which the graft had failed within 1 year of performing the PK. CONCLUSIONS: Therapeutic keratoplasty is instrumental in eliminating interface infection after DSAEK, possibly leading to excellent visual outcomes with a relatively high graft survival rate.

Efficacy and safety of antifungal additives in Optisol-GS corneal storage medium.

IMPORTANCE: Optisol-GS, the most common corneal storage medium in the United States, contains antibacterial but no antifungal supplementation. Most postkeratoplasty endophthalmitis and keratitis cases are now of a fungal origin. OBJECTIVE: To assess the efficacy and safety of voriconazole and amphotericin B in reducing Candida species contamination of Optisol-GS under normal storage conditions. DESIGN, SETTING, AND PARTICIPANTS: In vitro laboratory study using 15 pairs of research-grade donor corneas and 20-mL vials of Optisol-GS. INTERVENTIONS: Twenty vials of Optisol-GS were supplemented with either voriconazole at 1×, 10×, 25×, or 50× minimum inhibitory concentration (MIC) or amphotericin B at 0.25×, 0.5×, 1×, or 10× MIC. Known concentrations of Candida albicans and Candida glabrata were each added to a set of vials. Safety studies were performed by separating 15 pairs of donor corneas into unsupplemented Optisol-GS or Optisol-GS plus an antifungal. MAIN OUTCOMES AND MEASURES: Efficacy outcomes were viable fungal colony counts determined from samples taken on days 2, 7, and 14 immediately after removal from refrigeration and after warming to room temperature for 2 hours. Safety outcomes included percentage of intact epithelium and endothelial cell density on days 0, 7, and 14, as well as percentage of nonviable endothelial cells by vital dye staining on day 14. RESULTS: Growth of C albicans and C glabrata was observed in all voriconazole-supplemented vials. In contrast, there was no growth of either organism in amphotericin B-supplemented vials, except at 0.25× and 0.5× MIC on day 2, when viable counts of C glabrata were reduced by 99% and 96%, respectively. Compared with paired controls, with the exception of Optisol-GS plus amphotericin B at 10× MIC, donor corneas in supplemented Optisol-GS appeared to have no difference in endothelial cell density reduction, percentage of intact epithelium, or percentage of nonviable endothelial cells. CONCLUSIONS AND RELEVANCE: The addition of amphotericin B to Optisol-GS may significantly improve activity against contamination with Candida species, the primary cause of fungal infection after corneal transplantation. This study found significant endothelial toxic effects at the maximal concentration of amphotericin B.

Endotelitis y trabeculitis herpética con afectación corneal tardía / Herpetic endotheliitis and trabeculitis with delayed corneal involvement

Caso clínico: Varón de 62 años de edad con trasplante renal y tratamiento inmunosupresor que acude por disminución de la visión (20/100) en ojo izquierdo, edema corneal y presión intraocular de 46mmHg. Un mes después aparece queratitis dendrítica marginal inferior. El raspado corneal y la proteína C reactiva demuestran la presencia de un virus del herpes simple (HVS). Discusión: La epiteliopatía corneal autoinmunitaria o endotelitis idiopática corneal se caracteriza por edema corneal y precipitados queráticos. Se cree que el HVS podría ser secretado desde el trabeculum, inervado por el nervio trigémino, apoyado clínicamente por la progresión del edema estromal desde la periferia(AU)

Case report: A 62-year-old man with previous renal transplant and immunosuppressive treatment presented with decreased visual acuity (20/100) in his left eye, corneal oedema and intraocular pressure of 46mmHg. One month later an inferior marginal dendritic keratitis appeared. Corneal scraping and real-time polymerase chain reaction showed herpes simplex virus (HSV). Discussion: The autoimmune corneal endotheliopathy or acute idiopathic corneal endotheliitis is characterised by corneal stromal oedema and keratic precipitates. HSV might be secreted from the trabeculum, innervated by the trigeminal nerve. This hypothesis is supported by the clinical observation that the corneal stromal oedema usually starts from the periphery(AU)

Efficacy and safety of voriconazole as an additive in Optisol GS: a preservation medium for corneal donor tissue.

PURPOSE: To assess the endothelial toxicity and the microbiological efficacy of voriconazole (100 microg/mL) as an antimicrobial additive to Optisol GS. METHODS: A total of 533 donor rims were studied. One half of each donor rim was placed in standard Optisol GS and the other half rim in Optisol GS fortified with voriconazole (100 microg/mL). All rims were refrigerated for 24 hours at 3 degrees C and placed in thioglycolate broth and incubated at 37 degrees C for 7 days. A pair of donor buttons not used in transplantation was stored for 2 days in each solution and examined for endothelial changes with electron microscopy (EM). A second pair of cornea buttons was examined for toxicity by endothelial staining with 0.3% trypan blue and 0.2% alizarin red. RESULTS: Seven of 533 corneal rim cultures were positive for fungal organisms in the Optisol GS group. No rims were positive for fungal growth in the voriconazole-fortified Optisol GS medium. The difference was statistically significant (P = 0.015; Fisher exact test). There was no difference in the cellular morphology of the button stored in voriconazole fortified Optisol GS compared with Optisol GS using EM. In the bioassay, the percentage of nonviable cells in the voriconazole-fortified medium compared with the control medium was nonsignificant (P < 0.05, Student t test). CONCLUSIONS: Voriconazole seems to be safe as a fortifying agent for cornea storage medium. It significantly reduces the rate of positive fungal rim cultures and shows no signs of endothelial cytotoxicity as viewed by EM and by a bioassay of trypan blue and alizarin red.

Specific induction of fibronectin binding activity by hemoglobin in Candida albicans grown in defined media.

Fibronectin (FN) is a major component of host extracellular matrix that may play an important role in the initiation and dissemination of Candida albicans infections. Expression of FN binding requires growth of C albicans blastoconidia in complex medium, and the regulation of FN receptor expression is poorly understood. We now demonstrate that hemoglobin is a potent and specific inducer of FN receptor expression and describe a defined medium supplemented with hemoglobin that greatly and stably enhances the binding activity of C. albicans for soluble FN. Enhancement of FN binding by hemoglobin in strain 44807 was concentration dependent and was maximal at 0.1% hemoglobin with 20- to 80-fold enhancement. The hemoglobin-induced FN binding to C. albicans was saturable, with a Kd of 2.7 X 10(-8) M. Enhancement required growth of C. albicans in hemoglobin-containing medium, since simply exposing blastoconidia to hemoglobin in a nongrowing status did not enhance binding. Induction was reversible following removal of hemoglobin from the growth medium and not associated with germination. Inorganic or protein-bound iron was not sufficient for the induction, since other iron-containing proteins or inorganic iron salts were inactive. Growth in the simple medium yeast nitrogen base supplemented with hemoglobin increased cell adhesion to immobilized FN and to cultured monolayers of bovine corneal endothelial cells. These data suggest that hemoglobin may be an important regulator of FN binding activity in C. albicans and thus may play a role in its pathogenesis.

Use of Merocyanine 540 and Hoechst 33258 for the selective killing of contaminating mycoplasmas in cell cultures.

Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans, M. hyorhinis and M. arginini, from experimentally infected cell cultures. This approach is based on the differential binding of the lipophilic fluorescent probe Merocyanine 540 followed by illumination with visible light. The efficiency of the procedure depends on the Merocyanine 540 concentration, the intensity of illumination, and the presence of oxygen in the medium. When A. laidlawii contaminated corneal endothelial cell cultures were treated simultaneously with Merocyanine 540 and DNA-binding fluorochrome Hoechst 33258 and then illuminated, a significant degree of eradication was observed, even after one cycle of treatment. This combined treatment is therefore recommended as an effective method of purging mycoplasmas from contaminated cultures.

Expression of SV40 virus large T antigen by recombinant adenoviruses activates proliferation of corneal endothelium in vitro.

Infection with the Ad5-SVR4 virus was used to introduce the large T antigen encoding region of the SV40 virus into bovine and human corneal endothelial cells. Expression of large T antigen occurred in 40% of bovine corneal endothelial cells after a 24-h incubation time versus 12% after 8 h of incubation. By 48 h after infection, almost all (92.8%) bovine corneal endothelial cells expressed large T antigen. Bovine and human corneal endothelial cells which expressed large T antigen proliferated and the characteristic morphologic features of corneal endothelium were maintained. This method may enable growth of enough corneal endothelium to perform studies to elucidate the biochemical mechanisms involved in regulating endothelial cell function.

Demonstration of herpes simplex virus DNA in idiopathic corneal endotheliopathy.

A 56-year-old man developed idiopathic corneal endotheliopathy. The lesion consisted of severe stromal edema at the lower half of the cornea along with a number of associated keratic precipitates and steadily progressed to the upper half of the cornea. By polymerase chain reaction, herpes simplex virus DNA was demonstrated in the aqueous humor of this patient. Corneal stromal edema was resolved in response to treatment with topically applied and systemic acyclovir. Herpes simplex virus DNA was repeatedly demonstrated in the aqueous humor when the endothelial lesion recurred later. This evidence strongly indicates that this unique endothelial disorder is of viral origin.

[An experimental study on the susceptibility of human corneal cells to infection by herpes simplex virus type 1].

The degree of susceptibility of various human corneal cells to infection by the Mckrae strain of herpes simplex type 1 at different time-points in vitro was found to be the epithelial cells, the endothelial cells, and the keratocytes in increasing order. This finding was in agreement with the natural resistance of the three types of cells, and explained the mechanism of clinical manifestations of herpes simplex keratitis.