Jonathan H. Lass

Fusarium and Candida albicans Biofilms on Soft Contact Lenses: Model Development, Influence of Lens Type, and Susceptibility to Lens Care Solutions

ABSTRACT Fungal keratitis is commonly caused by Fusarium species and less commonly by Candida species. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with ReNu with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was proposed to play a role in this outbreak. However, no in vitro model for contact lens-associated fungal biofilm has been developed. In this study, we developed and characterized in vitro models of biofilm formation on various soft contact lenses using three species of Fusarium and Candida albicans. The contact lenses tested were etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A, and polymacon. Our results showed that clinical isolates of Fusarium and C. albicans formed biofilms on all types of lenses tested and that the biofilm architecture varied with the lens type. Moreover, differences in hyphal content and architecture were found between the biofilms formed by these fungi. We also found that two recently isolated keratitis-associated fusaria formed robust biofilms, while the reference ATCC 36031 strain (recommended by the International Organization for Standardization guidelines for testing of disinfectants) failed to form biofilm. Furthermore, using the developed in vitro biofilm model, we showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus. However, Fusarium biofilms exhibited reduced susceptibility against these solutions in a species- and time-dependent manner. This in vitro model should provide a better understanding of the biology and pathogenesis of lens-related fungal keratitis.

A New Method for Grading the Severity of Keratoconus: The Keratoconus Severity Score (KSS)

Purpose: To define a new method for grading severity of keratoconus, the Keratoconus Severity Score (KSS). Methods: A rationale for grading keratoconus severity was developed using common clinical markers plus 2 corneal topographic indices, creating a 0 to 5 severity score. An initial test set of 1012 eyes, including normal eyes, eyes with abnormal corneal and topographic findings but not keratoconus, and eyes with keratoconus having a wide range of severity, was used to determine cutpoints for the KSS. Validation set 1, comprising data from 128 eyes, was assigned a KSS and compared with a clinicians ranking of severity termed the “gold standard” to determine if the scale fairly represented how a clinician would grade disease severity. κ statistics, sensitivity, and specificity were calculated. A program was developed to automate the determination of the score. This was tested against a manual assignment of KSS in 2121 (validation set 2) eyes from the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study, as well as normal eyes and abnormal eyes without keratoconus. Ten percent of eyes underwent repeat manual assignment of KSS to determine the variability of manual assignment of a score. Results: From initial assessments, the KSS used 2 corneal topography indices: average corneal power and root mean square (RMS) error for higher-order Zernike terms derived from the first corneal surface wavefront. Clinical signs including Vogt striae, Fleischer rings, and corneal scarring were also included. Last, a manual interpretation of the map pattern was included. Validation set 1 yielded a κ statistic of 0.904, with sensitivities ranging from 0.64 to 1.00 and specificities ranging from 0.93 to 0.98. The sensitivity and specificity for determining nonkeratoconus from keratoconus were both 1.00. Validation set 2 showed κ statistics of 0.94 and 0.95 for right and left eyes, respectively. Test-retest analysis yielded κ statistics of 0.84 and 0.83 for right and left eyes, respectively. Conclusion: A simple and reliable grading system for keratoconus was developed that can be largely automated. Such a grading scheme could be useful in genetic studies for a complex trait such as keratoconus requiring a quantitative measure of disease presence and severity.

MyD88 Regulation of Fusarium Keratitis Is Dependent on TLR4 and IL-1R1 but Not TLR2

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88−/− mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2−/−, TLR4−/−, and TLR2/4−/− mice were similar to those of C57BL/6 mice; however, TLR4−/− and TLR2/4−/− mice had impaired antifungal responses. The phenotype of infected IL-1R1−/− mice was similar to that of MyD88−/− mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1−/− mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.

Acyclic antimetabolite therapy of experimental herpes simplex keratitis.

In a masked controlled study we compared 3% acycloguanosine, 0.5% idoxuridine, and 3% vidarabine ointments in therapy of experimental herpes simplex virus keratitis in rabbits. The results of the acycloguanosine group were significantly better than the control groups and both other treatment groups, while producing none of the toxic side effects of increasing iritis, conjunctivitis or stromal keratitis, with continued drug application.

Levobunolol: A Beta-adrenoceptor Antagonist Effective in the Long-term Treatment of Glaucoma

We compared the ocular-hypotensive efficacy and systemic and ocular safety of an ophthalmic solution of levobunolol (0.5% and 1%) twice daily, with timolol (0.5%) twice daily in a long-term double-masked study of 391 patients with open-angle glaucoma or ocular hypertension. Patients received the test medication in both eyes for up to two years. Over the two-year period, both concentrations of levobunolol reduced mean IOP by 27% (range, -6 to -8 mmHg). This ocular-hypotensive effect was sustained throughout the study and was similar to that produced by timolol. Slight decreases in mean heart rate and blood pressure were observed. No unexpected adverse ocular or systemic reactions were reported. The results of these studies indicate that levobunolol is an effective therapy for the long-term treatment of glaucoma.

Effect of incision width on graft survival and endothelial cell loss after Descemet stripping automated endothelial keratoplasty.

Purpose: To assess the effect of incision width (5.0 and 3.2 mm) on graft survival and endothelial cell loss 6 months and 1 year after Descemet stripping automated endothelial keratoplasty (DSAEK). Methods: One hundred sixty-seven subjects with endothelial decompensation from a moderate-risk condition (principally Fuchs dystrophy or pseudophakic corneal edema) underwent DSAEK by 2 experienced surgeons. The donor was folded over and inserted with single-point fixation forceps. This retrospective analysis assessed graft survival, complications, and endothelial cell loss, which was calculated from baseline donor and 6-month and 1-year postoperative central endothelial images evaluated by an independent specular microscopy reading center. Results: No primary graft failures occurred in either group. One-year graft survival rates were comparable (98% vs 97%) in the 5.0- and 3.2-mm groups, respectively (P = 1.0). Complications included graft dislocation, graft rejection episodes, and elevated intraocular pressure and occurred at similar rates in both groups (P ≥ 0.28). Pupillary block glaucoma did not occur in either group. Mean baseline donor endothelial cell density did not differ: 2782 cells per square millimeter in the 5.0-mm (n = 64) and 2784 cells per square millimeter in the 3.2-mm (n = 103) groups. Percent endothelial cell loss was 27% ± 20% (n = 55) versus 40% ± 22% (n = 71; 6 months) and 31% ± 19% (n = 45) versus 44% ± 22% (n = 62; 12 months) in the 5.0- and 3.2-mm incision groups, respectively (both P < 0.001). Conclusions: One year after DSAEK, overall graft success was comparable for the 2 groups; however, the 5.0-mm incision width resulted in substantially lower endothelial cell loss at 6 and 12 months.

A multicenter study to map genes for Fuchs endothelial corneal dystrophy: Baseline characteristics and heritability

Purpose: To describe the methods for family and case–control recruitment for a multicenter genetic and associated heritability analyses of Fuchs endothelial corneal dystrophy (FECD). Methods: Twenty-nine enrolling sites with 62 trained investigators and coordinators gathered individual and family information, graded the phenotype, and collected blood and/or saliva for genetic analysis on all individuals with and without FECD. The degree of FECD was assessed in a 0 to 6 semiquantitative scale using standardized clinical methods with pathological verification of FECD on at least 1 member of each family. Central corneal thickness was measured by ultrasonic pachymetry. Results: Three hundred twenty-two families with 330 affected sibling pairs with FECD were enrolled and included a total of 650 sibling pairs of all disease grades. Using the entire 7-step FECD grading scale or a dichotomous definition of severe disease, heritability was assessed in families via sib–sib correlations. Both binary indicators of severe disease and semiquantitative measures of disease severity were significantly heritable, with heritability estimates of 30% for severe disease, 37% to 39% for FECD score, and 47% for central corneal thickness. Conclusions: Genetic risk factors have a strong role in the severity of the FECD phenotype and corneal thickness. Genotyping this cohort with high-density genetic markers followed by appropriate statistical analyses should lead to novel loci for disease susceptibility.

Infectious crystalline keratopathy associated with topical anesthetic abuse

Two patients with infectious crystalline keratopathy associated with topical anesthetic abuse are described. No previously reported predisposing factors existed, including topical corticosteroid use during active Herpes simplex or Acanthamoeba keratitis, or following penetrating keratoplasty. Cultures from corneal biopsies of both patients grew Streptococcus viridans. Both infections resulted in corneal scarring with vascularization. Ultimately, corneal transplantation was performed in one case.

AN IN VITRO METHOD FOR MEASURING OPHTHALMIC PRESERVATIVE CYTOTOXICITY

AbstractUsing an in vitro model we studied the cytotoxicity of the topical ophthalmic preservatives benzalkonium chloride (BAC), thimerosal (TMS), and chlorobutanol (CHB) on confluent rabbit corneal epithelial cell cultures. Concentrations of the preservatives were BAC 0.0004–0.04%, TMS 0.0001–0.04%, and CHB 0.1–0.5%. Drug exposure times for all agents were 5, 30, and 60 min. Inhibition of 12-hr [3H]thymidine incorporation and phase contrast inverted light microscopy were performed to assess cytotoxic effect.In a concentration- and time-dependent manner all preservatives produced significant (p < 0.05) inhibition of [3H]thymidine incorporation within the range of their clinically used concentrations. This effect was associated with changes in cellular morphology as seen with light microscopy. The correlation of our in vitro data with the findings of previous in vivo studies suggests that our model is a rapid, objective, and quantitative screening method to study the cytotoxic effects of topical agents on ...

Disease severity and family history in keratoconus

Background/aims: To determine if disease severity is associated with a family history of keratoconus. Methods: Markers of disease severity in the CLEK Study cohort were assessed to determine if they could discriminate individuals with and without family history. Logistic regression was used to examine association between corneal scarring, average corneal power, flat and steep keratometry readings, and higher-order root mean square (RMS) wavefront error with family history. Results: In univariate analyses, none of the severity indices had any significant associations with family history; however, contact lens use, gender, and Caucasian race were found to be significant predictors. After controlling for these confounders, there were no significant associations between any severity indices and family history. Conclusions: Presence or absence of family history is not associated with more severe clinical disease, at least when each marker for severity is considered independently. The results of this analysis are important for genetic studies of keratoconus in that it will allow recruitment of keratoconus patients across all stages of disease severity because it does not influence familial aggregation.