Optimizing evaluation of Lissamine Green parameters for ocular surface staining.

PURPOSE: The recently published seminal dry eye workshop proceedings defined Lissamine Green (LG), an organic dye, as a gold standard for demonstrating ocular surface staining. The purpose of the current study was to determine the optimal parameters of 1% LG instillation for the ocular surface examination in dry eye patients. DESIGN: Prospective and observational quality improvement study. METHODS: A quality improvement study evaluated 16 eyes from eight dry eye patients with different levels of severity. LG (1%), in three volumes (5, 10, and 20 µl) was instilled into the conjunctival cul-de-sac, and four masked observers with different levels of clinical expertise examined the patients with and without red filter. The staining pattern of the conjunctiva and cornea was documented with the Oxford scale within 4 min of LG instillation. Optimal volume and inter-observer reliability were assessed. RESULTS: All dye volumes were tolerated well by all patients. Experienced observers preferred 10 µl volume because of the ease of examination and accuracy. Although instillation of 20 µl yielded similar scores as 10 µl, it resulted in overflow of the lid and facial skin staining. The use of red filter significantly improved reading scores (P<0.01). Inter-observer reliability was higher for conjunctival scores than for corneal scores for all patients. The highest reliability was demonstrated with 10 µl volume and increased with greater experience of the observer. CONCLUSIONS: Ocular surface examination with instillation of 10 µl 1% LG has good inter-observer reliability and is well tolerated. Observation through a red filter facilitates the examination.

Localized conjunctival argyrosis: a late sequela of strabismus surgery.

We describe a case of focal argyrosis of the conjunctiva clinically simulating a melanoma. An 82-year-old woman was referred for an asymptomatic pigmented conjunctival lesion. Her only significant past ocular history was strabismus surgery 76 years earlier. Biopsy of the conjunctiva and lateral rectus muscle revealed the discoloration was pigment granules. Energy-dispersive x-ray microanalysis revealed the pigmentation to be silver deposits. The patient had strabismus surgery probably using a silver clip. Argyrosis should be considered in the differential diagnosis of focal pigmented conjunctival lesions.

Evaluation of the effect of lissamine green and rose bengal on human corneal epithelial cells.

PURPOSE: To examine the effects of lissamine green and rose bengal on proliferating human corneal epithelial (HCE) cells in vitro. METHODS: HCE cells derived from explants of discarded corneoscleral rims were cultured by the standard technique. Experimental cells were exposed to 1, 0.5, or 0.1% of either lissamine green or rose bengal for 10 min while control cells were exposed to a phosphate buffer solution (PBS). RESULT: Cell viability was 92% greater for 1% lissamine green (p = 0.013) and 81.2% greater for 0.5% lissamine green (p = 0.006) compared to 1 and 0.5% rose bengal, respectively. The difference between the effect of 0.1% rose bengal and 0.1% lissamine green on cell viability was not statistically significant (p = 0.83). Rose bengal staining of HCE cells was immediate and readily detectable with unaided eyes at all three concentrations, whereas no observable staining of healthy HCE cells was noted with lissamine green. CONCLUSION: Rose bengal adversely affects HCE cell viability and stains normal proliferating HCE cells in contrast to lissamine green, which exhibited neither of these characteristics. Therefore, we recommend the use of lissamine green over rose bengal in evaluating ocular surface disorders.

Patient tolerance and ocular surface staining characteristics of lissamine green versus rose bengal.

PURPOSE: To determine patient tolerance and ocular surface staining characteristics of 1% lissamine green versus 1% rose bengal solutions in patients with dry eye and in those with normal ocular surfaces by comparison of subjective sensation and objective staining scores. METHODS: Twelve patients with keratoconjunctivitis sicca and eight subjects with no ocular surface disease recorded their symptoms on a graded scale (0-5) and the duration of symptoms after instillation of one drop of 1% lissamine green and 1% rose bengal on two different occasions in different order. Lissamine green and rose bengal staining patterns were recorded on a graded scale (0-4) by one of the authors. RESULTS: For patients with keratoconjunctivitis sicca mean sensation score with lissamine green (2.42) was significantly lower (P = 0.00006) than with rose bengal (4.58). The duration of symptoms was significantly longer (P = 0.0007) after rose bengal instillation as well. For subjects with no ocular surface disease, mean sensation score with lissamine green (1.375) was significantly lower (P = 0.01) than with rose bengal (2.5). In this group, duration of symptoms also was significantly longer (P = 0.001) after rose bengal instillation. In both groups, there was no difference in objective staining scores. CONCLUSION: Lissamine green is better tolerated than rose bengal by patients and is equally as effective as rose bengal in evaluating the ocular surface in keratoconjunctivitis sicca.

Cytomegalovirus keratitis after penetrating keratoplasty.

We report the development of cytomegalovirus (CMV) keratitis in the penetrating keratoplasty of a 59-year-old human immunodeficiency virus-negative woman after uncomplicated corneal transplantation. Immunosuppression with topical cyclosporine A 2% in corn oil and topical prednisolone acetate 1% suspension was used postoperatively. The 15-month postoperative course was complicated by multiple episodes of endothelial rejection, medically controlled elevated intraocular pressure, polymicrobial bacterial (coagulase-negative staphlococcus and alpha-hemolytic streptococcus) keratitis, and endothelial plaque formation with associated hypopyon and epithelial defect. The graft failed and penetrating keratoplasty was repeated. Cytomegalovirus infection of superficial keratocytes in a region of scarring was identified in histological sections stained with hematoxylin and eosin and confirmed using mouse monoclonal anti-cytomegalovirus antibodies. Excision of the diseased corneal button with no additional treatment appears to have been curative. Low-grade keratitis was the only manifestation of the CMV infection, and it has not recurred 6 months postoperatively.

Decreased corneal sensation as an initial feature of Acanthamoeba keratitis.

BACKGROUND: Herpes simplex keratitis is the most common misdiagnosis in patients with Acanthamoeba keratitis, which is increasing in frequency and is associated with daily wear soft contact lenses. Both entities usually present as unilateral keratitis. The manifestations of superficial Acanthamoeba keratitis (i.e., unilaterality, dendriform appearance, positive response to antivirals, and decreased corneal sensation) increase the opportunity for misdiagnosis as herpes simplex keratitis. The authors have encountered six patients with Acanthamoeba keratitis in whom the correct diagnosis was delayed from 2 weeks to 3 months. METHODS: All six patients underwent testing with the Cochet-Bonnet esthesiometer and extensive pharmacologic treatment for herpes simplex keratitis. Corneal scrapings were taken between 2 and 6 weeks after the initial examination. RESULTS: In all six patients, corneal sensation was decreased significantly. Drug therapy was ineffective. Cultures were positive for Acanthamoeba. Five of six patients underwent penetrating keratoplasty. CONCLUSIONS: Decreased corneal sensation has contributed to the misdiagnosis of Acanthamoeba as herpes simplex keratitis. Misdiagnosis results in delayed treatment and worse outcome. The authors found that significantly decreased corneal sensation is a frequent finding in early Acanthamoeba keratitis. Therefore, physicians should consider Acanthamoeba keratitis as an alternative diagnosis in patients with presumed herpes simplex keratitis with decreased corneal sensation.

Immunohistochemical analysis of the pathogenesis of posterior polymorphous dystrophy.

The pathogenesis of posterior polymorphous dystrophy was analyzed by immunohistologic methods. Sections of corneal buttons from two patients undergoing transplantation owing to posterior polymorphous dystrophy were stained with 2B4.14.1, a monoclonal antibody that reacts with human corneal endothelium, and with a cocktail of antihuman cytokeratin monoclonal antibodies that do not react with normal corneal endothelium. Single-stained sections revealed a variegated, intermittent staining pattern of antibody reactive and nonreactive cells. Double-stained sections revealed some cells that stained with only one of the antibodies and many cells that stained with both antibodies. The presence of cells staining positively for both 2B4.14.1 antigen and cytokeratins supports the hypothesis that the cytokeratin-expressing epithelial-like cells found in corneas with posterior polymorphous dystrophy arise via a metaplastic process in which the phenotype of endothelial cells becomes progressively abnormal.

The association of lymphocytotoxic antibodies with corneal allograft rejection in high risk patients. The Collaborative Corneal Transplantation Studies Research Group.

The Collaborative Corneal Transplantation Studies are a pair of multicenter prospective clinical trials evaluating the effectiveness of histocompatibility matching in high risk keratoplasty patients. The antigen matching study (AMS) evaluated HLA matching in patients without circulating lymphocytotoxic antibody to HLA antigens and the cross-match study (CS) evaluated the effect of using cross-match-negative donors in patients with identified circulating lymphocytotoxic antibodies to HLA antigens. Sera from 510 patients considered for enrollment in the studies were screened preoperatively for the presence of anti-class I lymphocytotoxic antibodies (LA). The 42 patients (8%) found to have detectable LA entered the CS. The 468 patients found not to have detectable LA preoperatively entered the AMS. Fifteen of the 37 transplanted CS patients were found to have donor-specific anti-class I antibody (before or after surgery). These patients were also screened for anti-class II LA and 25 had anti-class II panel reactive antibody > or = 5%. Forty-nine of the 419 transplanted AMS patients (12%) were found to have produced anti-class I LA after surgery, and in 19 patients, antibody specificities were those of donor HLA antigens. There was a significant association between the number of mismatched class I antigens and the number of donor-specific LA produced. The production of LA by AMS patients was significantly associated with reaction episodes; eighty-two percent of patients (40 of 49) with LA had reaction, compared with 63% of patients (230 of 365) without LA (P = 0.02). Likewise, production of donor-specific LA was significantly associated with immune-mediated graft failure (P = 0.025). For CS patients, there was no correlation between the production of donor-specific anti-class I or nonspecific anti-class II antibodies and graft outcome. However, the CS patients had poorer graft survival than did AMS patients at 3 years (57% vs. 66%, P = 0.01). These data demonstrate that LA, especially directed against donor class I HLA antigens following corneal transplantation in high risk patients, are associated with immune graft rejection and can be an indicator of allograft rejection.

Rapid assay of lactoferrin in keratoconjunctivitis sicca.

We compared measurement of lactoferrin concentration by the LactoCard solid phase ELISA assay with the LactoPlate radial immunodiffusion assay in tears of normal patients and those with keratoconjunctivitis sicca. The LactoCard, a new lactoferrin assay, allows rapid determination of tear lactoferrin concentration in 10-15 min, a notable improvement over the 3 days required of the LactoPlate. There was no statistically significant difference between the accuracy of the two assays in normal patients nor in those patients with a diagnosis of keratoconjunctivitis sicca. Both assays showed a significant decrease in tear lactoferrin concentration in patients with severe keratoconjunctivitis sicca when compared to normal patients. The LactoCard is a rapid and reliable means of measuring tear lactoferrin concentration in a clinical setting.

The effect of poor fixation on computer-assisted topographic corneal analysis. Pseudokeratoconus.

INTRODUCTION: The effect of changing the alignment of the corneal surface in relation to the plane of the videokeratoscope mires in computer-assisted topographic corneal analysis was evaluated. METHODS: The corneas of five subjects were analyzed with the visual axis at 0 degrees, 2.5 degrees, 5 degrees, and 10 degrees below the central axis of the videokeratoscope. The relative steepness of the inferior and superior cornea was compared using inferior-superior (I-S) values at each deviation from the central axis. A similar analysis was performed on three aspheric test surfaces with different central curvatures. RESULTS: The difference in the relative steepness of the inferior and superior cornea was found to increase in proportion to the deviation from alignment with the central axis in both the test subjects and the aspheric test surfaces. CONCLUSION: This change in relative steepness produces a pattern that mimics keratoconus and can be seen at deviations of less than 5 degrees in some patients. The increase in the I-S value was significant at 5 degrees of deviation (P < 0.05). It also was found that this effect increased as the central curvature of the aspheric surfaces increased.

Inhibition of proliferating lens epithelium with antitransferrin receptor immunotoxin.

We investigated the effect of an antitransferrin receptor immunotoxin (454A12-rRA) on proliferating human and baboon lens epithelium in vitro. Human and baboon lens epithelial cells grown in modified TC-199 medium at 35 degrees Celsius in 7% CO2 were seeded in 24 well plates at a density of 17,500 cells/ml to 40,000 cells/ml. The cells were exposed to various concentrations of 454A12-rRA for seven days. The sensitivity of proliferating human lens epithelium to 454A12-rRA was dependent on the dose, with a 60% to 70% reduction in cell counts at immunotoxin concentrations of 100 ng/ml and above. The immunotoxin had no significant effect on baboon lens epithelium in vitro, which suggests that it is specific for human tissue. By preventing the proliferation of human lens epithelial cells, immunotoxin 454A12-rRA may be useful in the management of posterior capsule opacification after planned extracapsular cataract surgery.

Suppression of human corneal epithelial proliferation with breast carcinoma immunotoxin.

We examined the effects of the immunotoxin 260F9 Mab-recombinant ricin A (developed against human breast carcinoma) on proliferating and confluent human corneal epithelium (HCE) cells in vitro. HCE cells derived from explants of discarded human donor corneoscleral rims were established as proliferating and confluent cell cultures, and were exposed continuously for 7 days to immunotoxin. Final cell counts at day 7, and thymidine uptake measured at days 1 and 7 postexposure, showed > 95% suppression of proliferating cells at an immunotoxin concentration of 10 ng/ml, with confluent HCE cells relatively unaffected. This immunotoxin may prove useful in treatment of proliferative ocular epithelial diseases such as epithelial downgrowth or squamous cell carcinoma of the ocular surface.

Inhibition of human corneal epithelium with immunotoxin 454A12-rRA.

We examined the effects of the immunotoxin 454A12-rRa on proliferating and confluent human corneal epithelium (HCE) in vitro. Proliferating HCE was sensitive to 454A12-rRA in a dose-dependent fashion. At immunotoxin concentrations of 1,000 ng/ml for 7 days we observed an 86% reduction in cell counts. Confluent HCE was not sensitive to 454A12-rRA at equivalent concentrations of immunotoxin. These data confirm previous observations regarding selective sensitivity of proliferating ocular tissue to immunotoxin, but suggest that HCE is less sensitive to 454A12-rRA than other ocular cell types.

Inhibition of human subconjunctival fibroblast proliferation by immunotoxin.

The ability to target proliferating cells is important for agents used to modulate wound healing by decreasing the growth of fibroblasts. Proliferating cells are known to express increased numbers of transferrin receptors and have increased receptor turnover. 454A12 Mab-rRA, an immunotoxin containing anti-human transferrin receptor monoclonal antibody conjugated to recombinant ricin A chain, was shown to inhibit the proliferation of human subconjunctival fibroblasts in vitro. A dose-related reduction of cell counts was observed in proliferating cells. More than 90% inhibition was achieved with an immunotoxin concentration of 10 ng/ml per 20,000 cells plated. In contrast, confluent fibroblasts were markedly less sensitive to the immunotoxin at equivalent concentrations. Comparative experiments demonstrated that 5-fluorouracil has less specificity for proliferating cells, with significant death of confluent fibroblasts at high drug concentrations.

Fungal scleritis after cataract surgery. Successful outcome using itraconazole.

We report the development of fungal scleritis in a 53-year-old man after uncomplicated cataract surgery. Histopathology and culture identified the organism as Aspergillus flavus. Clinically, the patient worsened on treatment with oral ketoconazole and topical amphotericin B with progression of multifocal scleral nodules and necrosis. Resolution of inflammation was achieved using oral itraconazole, a new triazole antifungal agent. The patient achieved 20/15-2 visual acuity and remains free of symptoms and signs for greater than 2 years after discontinuation of all treatment.