The Effect of Candida albicans and Dexamethasone on the Secretion of Tumor Necrosis Factor-alpha from Cultured Human Keratocytes

PURPOSE: To measure the secretion of TNF-alpha from cultured human keratocytes after inoculation of Candida albicans, and to evaluate the change of secretion of TNF-alpha following application of dexamethasone. METHODS: Human corneal keratocytes were cultured independently in vitro. The specimens were divided into 4 groups: Group I with only pure culture as control, Group II with C. albicans, Group III with C. albicans and dexamethasone, and Group IV with only dexamethasone. RESULTS: As a whole, Group II showed the highest secretion of TNF-alpha, followed by Group III, Group I, and Group IV respectively (P< 0.05). CONCLUSION: Keratocytes with the addition of both C. albicans and dexamethasone, secreted much lower level of TNF-alpha , but showed more proliferation in comparison to keratocytes with addition of C. albicans alone. Therefore, the author may conclude that in fungal keratitis, the early administration of dexamethasone may be beneficial in reducing inflammatory responses induced by increased TNF-alpha secretion. However, because steroids may stimulate the proliferation of fungus leading to tissue damages, more cautious use might be needed.

A Comparison of Anti-Pseudomonal effects by Multi-purpose Contact Lens Solutions

PURPOSE: To compare the antimicrobial effects of various kinds of multi-purpose solutions, study effective ways of washing contact lens (CLs), and suggest the most effective lens care system using P. aeruginosa-contaminated CLs. METHODS: Each disposable soft CL was incubated in 0.1 ml of diluted solution of P. aeruginosa standardstrain (10(8)CFU / mm(3)) and 0.9 ml tryptic soy broth in cell culture wells for 24 hours. In the first experimental group, to find the most antimicrobial solution, 40 CLs contaminated by P. aeruginosa were divided into four subgroups washed with four different kinds of multi-purpose solutions. Ten CLs were used in each subgroup and in the control group; Ten CLs contaminated by P. aeruginosa were washed with phosphate buffered saline (PBS) instead of cleaning solution. In the second experiment groups to find the most effective way of washing, using only Renu Multiplus multi-purpose solutionR we compared the antimicrobial effect of four different ways of washing including 30 seconds rubbing, 30 minutes soaking, 4 hours soaking, 30 seconds rubbing and 4 hours soaking. RESULTS: There was no significant difference of antimicrobial effect between four kinds of multi-purpose solutions. All methods have a significant antimicrobial effect over control group (P<0.001) and the most effective method of washing CLs was 4 hours soaking. CONCLUSIONS: we should advise all the soft CL users that they should have their CLs soaked over 4 hours everyday after wearing it.

Serratia Marcescens Keratitis

PURPOSE: Serratia marcescens keratitis is relatively common among the various bacterial corneal infections and has been known to be associated with contact lens wear. The authors performed a study to identify the risk factors, clinical features and treatment results. METHODS: Epidemiologic study was prospectively performed under the identical protocol from May 1995 to April 2000 in 11 hospitals. RESULTS: Twenty cases of Serratia marcescens keratitis were diagnosed. Contact lens wear was involved in Serratia marcescens keratitis in 90%. The highest age prevalence was in the thirties(45%). The majority of them were students(30%) and office workers(30%). CONCLUSIONS: The principle risk factor of Serratia marcescens keratitis in this study was contact lens wear. The proper use of contact lens and education on it's complications may play an important role in reducing the incidence rate of Serratia marcescens keratitis.

Epidemiology of Staphylococcus epidermidis Keratitis

PURPOSES: Staphylococcus epidermidis is a causative agent of bacterial keratitis and the authors performed an epidemiologic study to identify the risk factors, clinical features and treatment results. METHODS: Epidemiologic study on Staphylococcus epidermidis keratitis, which were confirmed by cultures in 6 university hospitals, was performed prospectively under the identical protocol. RESULTS: Seventeen cases of Staphylococcus epidermidis keratitis were diagnosed by culture with corneal scraping and comprised 4.1% of 401 cases of confirmed bacterial keratitis. Male patients were affected more than female patients and the highest prevalent age was in the seventies. Most of their occupation was farming. The most prevalent season was fall and it mostly occurred after corneal trauma. CONCLUSIONS: Staphylococcus epidermidis keratitis usually occurred in a relatively old age group followed by corneal trauma. Its prevention along with early and proper antimicrobial therapy based on sensitivity results is important.

Epidemiologic study of Alternaria Keratitis

PURPOSE: Alternaria spp. is opportunistic fungus and the most prevalent cause of asthma and allergic disease. Keratitis caused by the fungus Alternariaspp. is quite rare. The authors performed an epidemiological study to identify the risk factor and to evaluate clinical manifestations, methods and results of treatment in Alternaria keratitis. METHODS: Epidemiological study of Alternaria keratitis was performed prospectively under the identical protocol from April 1995 to May 2000 in 5 hospitals. RESULTS: Seven cases of Alternaria keratitis were identified by culture with corneal scraping or corneal biopsy. They were 8.5% of the 82 cases of fungal keratitis. Of the 7 patients, 5 were men (71.4%). All patients were over 50 years and 5 of them (71.4%) were farmers. In terms of trauma history, 6 out of 7 patients had previous history of corneal traumas, and most of these corneal traumas were vegetable matter-related. CONCLUSIONS: The most common (83.3%) predisposing factor for Alternaria keratitis was vegetable matter-related corneal trauma. The prompt referral for diagnostic studies and specific antifungal management, or surgical therapy might be helpful to reduce the incidence rate of complication of keratomycoses.

A Case of Fusarium solani Keratitis mixed by Acremonium falciforme

Although more reports on the fungal keratitis are recently coming out, cases of mixed infection by two or more fungal agents have not yet beenreported in the literature. The authors experienced a case of fungal keratitis caused by a combination of Fusarium solani and Acremonium falciforme in which central corneal ulcer with severe suppurative infilteration and hypopyon were observed. Since the conditions were resistant to conservative treatment and corneal perforation occurred despite of medical treatment, penetrating keratoplasty was performed along with postoperative topical and systemic antifungal therapies for several weeks and good results were obtained with no evidence of recurrence.

Secretion of Interleukin-8 from Human Keratocyte Stimulated by Aspergillus fumigatus and Effect of Amphotericin B and Dexamethasone on The Secretion

PURPOSE: To measure the secretion of IL-8 from cultured human keratocytes after inoculation of conidia of A. fumigatus, and to compare the change of secretion of IL-8 following application of amphotericin B and dexamethasone. METHODS: Human corneal keraoctytes were cultured independently in vitro. The specimens were divided into 4 groups : Group I with only pure culture as control, Group II with conidia of A. fumigatus, Group III with conidia of A. fumigatus and amphotericin B, and Group IV with conidia of A. fumigatus and dexamethasone. The supernatants were aspirated from each group at different time intervals, and then were assayed for IL-8. RESULTS: Group II showed increased secretion of IL-8, at all selected time intervals except 12-h, in comparison with other three groups. Group III secreted IL-8 significantly less than the other groups(p<0.01). Group IV secreted IL-8 less than Group I and II at all selected time points(p<0.01), but more than Group III at the other time points except for 72-h(p<0.01). CONCLUSIONS: The secretion of IL-8 increased in the early stage of fungal keratitis but decreased in case of the administration of amphotericin B or dexamethasone. Amphotericin B was more potent than dexamethasone in decreasing the secretion of IL-8.

Epidemiology of Infectious Keratitis(II): A Multi-center Study

To identify risk factors and causative organisms, and to evaluate clinical manifestations, methods and results of treatment in infectious keratitis, an epidemiological study was performed prospectively under the identical protocol from April 1995 to March 2000.Logistic regression analysis was used to evaluate possible risk factors. The 1474 cases of infectious keratitis reported from 22 hospitals were studied. Five hundred forty-four organisms(442 bacteria, 82 fungi, 20 A c a n t h a m o e b a)were detected in 1320 eyes with infectious keratitis excluding 154 herpetic keratitis. The Pseudomonas aeruginosa was the most common organism in bacterial keratitis, and Fusariumspp. was the major isolate in fungal keratitis. Contact lens wear and occupation(industry, forester, miner, fisherman)were the risk factors for bacterial keratitis. Risk factors in fungal keratitis were fifth decade of age, farmer, and systemic diseases(diabetes mellitus etc.). Risk factors in herpetic keratitis were male and occupation(office worker, service, student, housewife). Risk factors in Acanthamoeba keratitis was contact lens wear.

Complications after Excimer Laser Photorefractive Keratectomy in Myopia

No Abstract Available.

Lipopolysaccharide-induced Production of Interleukin-8 by Cultured Human Keratocyte

The authors performed an experiment to determine if human corneal keratocytes release IL-8 after stimulation with lipopolysaccharide (LPS). Human corneal keratocytes were isolated from human corneal buttoms and grown independently in vitro. Cultured keratocytes were treated with various concentrations of LPS (0.01, 0.1 1, 10 microgram/ml). At 6 hours, 12 hours, 24 hours, and 48 hours after the stimulation with LPS, culture supernatants were aspirated and frozen. Supernatants were assayed by enzyme-linked immunosorbent assay for IL-8 content. Exposure of corneal keratocytes to LPS induced IL-8 production. Initially, the secretion of IL-8 was detected at 6 hours and increased upto 48 hours. Between 12 and 24 hours, the IL-8 was increased rapidly. At 6 and 12 hours, keratocytes exposed to 10 microgram / ml LPS produced more IL-8 than those exposed to other concentrations of LPS. In this study, the ability of corneal keratocytes to produce IL-8 upto 48 hours suggests that these cells can play important roles in the induction of the inflammatory response in cornea.

Phagocytosis and Acid Phosphatase Activity against Candida Albicans in Cultured Rabbit Keratocyte

It has been reported that keratocytes endocytose foreign particles both in vitro and in vivo, suggesting the active participation of keratocytes in corneal wound healing and host defense mechanism. This study was conducted to investigate the phagocytosis of keratocytes against Candida albicans[C.albicans]and the intracellular response after phagocytosis. C.albicans were fixed with glutaraldehyde and then coated with fibronectin. After exposing these C.albicans to the cultured rabbit keratocytes, the phagocytosis of keratocytes against C.albicans was evaluated by light microscope[LM]and transmission electron microscope[TEM], while the intracellular response was evaluated by changes of acid phosphatase activity. Also the study about latex beads was performed at the same time to know even if keratocytes can phagocytose foreign particles, regardless of wheather or not the particles are biodegradable. After Wright staining, phagocytosed latex beads and C.albicans were observed on LM and these were recognized to be surrounded by limiting membranes inside the cytoplasm of keratocytes on TEM. The phagocytic rates of fibronectin-coated were increased to 1.5 times , as compared with that of non-coated group. Acid phosphatase activities were higher in C.albicans-exposed groups than in control[keratocytes cultured without C.albicans or latex beads]during the culture period of 24 hours and they also increased according to culture duration and reached to the plateau after 12 hours. In comparison with non-coated group, fibronectin-coated groups showed a increasing tendency of acid phosphatase activity. These results suggest that keratocytes can phagocytose not only foreign particles but also C.albicans and that fibronectin may act as effective opsonin on phagocytosis, and that keratocytes phagocytosing C.albicans increase acid phosphatase activity to digest engulfed C.albicans when corneal stroma was wounded or inflammed.

Contamination of Contact Lens or Contact Lens Storage Case in Contact Lens Related Infectious Keratitis

In 44 out of 218 cases of contact lens related infectious keratitis from 19 hospitals throughout the country, contact lenses or contact lens storge cases were cultured. Microorganism was detected in 40 cases[90.9%]. Two or more organisms were isolated in 31 cases[77.5%]. Pseudomonas was the most common organism isolated from contact lens or contact lens storage medium[31 out of 84, 45.2%], followed by Serratia[15 out of 84, 17.9%], fungi [4], and acanthamoeba[4]. Acanthamoeba was found only in one hospital. Antibiotic sensitivity test for isolated pseudomonas showed that 96%of cases was sensitive to ciprofloxacin and 88%to ceftazidime.

Morphology and Ultrastructure of Microorganisms on Soft Contact Lens :Scanning Electron Microscopic Study

In order to examine the morphology and ultrastructure of contaminated soft contact lens by scanning electron microscopy, soft contact lenses were contaminated with P.aeruginosa, S.aureus, S.epidermidis and C .albicans. After incubation of soft contact lens in P.aeruginosa suspension for 60 minutes, a number of P.aeruginosa, S.aureus and S.epidermidis were attached to soft contact lens and amorphous materials covering the some of the soft contact lens surface were observed. Also, C.albicans attached to soft contact lens, looking like healthy and the fine fibrillar material covered the surface of soft contact lens and interconnected between cell walls of C.albicans at 14 days after contamination. Soft contact lens may provide a suitable environment for microorganisms that they could attach and proliferate. And microoragisms themselves secreted amorphous materials to protect them from the outside and promote the proliferation. So, contaminated soft contact lens may play a role as pathogen to the cornea.

Comparison of Antimicrobial Efficacy of Topical Antibiotics in Rabbit Keratitis Model with Ciprofl oxacin, Methicillin-Resistant Staphylococcus Aureus

To compare the antimicrobial efficacy of several antibiotics in the treatment of ciprofloxacin and methicillin-resistant staphylococcus aureus(MCRSA)keratitis, we established a rabbit keratitis model by using MCRSA isolated from keratitis patient. A strain of MCRSA, approroximately 100 colony forming units in 10 microliterof phosphate buffered saline was injected intrastromally into the each cornea of New Zealand white rabbits. 30 rabbtits(30 eyes)were randomly divided into 5 groups and treated with either 0.3%ciprofloxacin, 0.3%trovafloxacin, 5%vancomycin, 5%cefazolin, or sterile deionized water.Topical antibiotics were administered every 15 min for 6 hours(750microliter). Rabbits were sacrified 1hour after the last eyedrop, and excised corneas were homogenized, diluted, and plated on BAP medium.Bacterial colonyforming units per cornea were quantified after 48-hour incubation. Aqueous humor were collected from each eye and drug concentration was counted with HPLC. The log1 0 value of colony forming units(CFUs)of vancomycin treated group was 4.85+/-0.64;ciprofloxacin, 6.16+/-0.46;trovafloxacin, 6.58+/-0.29;cefazolin, 6.25+/-0.25;and sterile deionized water, 6.57+/-0.33.In view of mean CFU, vancomycin showed greater antimicrobial activity and it was statistically significant(p<0.05). However, all of these treatment did not completely sterilize any corneas with 6-hour treatment. From these results, only topical vancomycin(5%)represented effective antimicrobial activity with short-term treatment in rabbit keratitis with MCRSA.

Epidemiology of Fungal Keratitis in Korea: A Multi-center Study

Fungal keratitis, one of devastating microbial keratitis, is difficult to diagnose and treat successfully. The authors performed prospectively an epidemiological study in Korea to identify the risk factors and to evaluate clinical manifestations, methods and results of treatment in fungal keratitis under the identical protocol from April 1995 to September 1999. Logistic regression analysis was used to evaluate possible risk factors. Sixty eight cases of fungal keratitis, which were identified by culture with corneal scraping or corneal biopsy and reported in 22 hospitals, were studied. The major causative organisms were Fusarium spp.(20 cases), Aspergillus spp.(17 cases), and Candida spp.(11 cases). The principal risk factors of fungal keratitis identified in this study were farmer, use of long-standing topical corticosteroids, and diabetes mellitus. Old age and corneal trauma with vegetable matters were the associated factors in fungal keratitis. Further evaluation of the risk factors and the establishment of the prevention in fungal keratitis are required.

The Minimal Inhibitory Concentrations of Antifungal Agents for Clinical Isolates in Fungal Keratitis

This study was performed for the standardization and proper selection of effective antifungal agents by measuring the minimal inhibitory concentra-tions[MICs]of antifungals to fungi, separated from keratitis patients.Two strains of A.fumigatus and single strain of F.solani, A.falciforme, and A . alternata were used for this test.Standard powders of miconazole, itraconazole, clotrimazole, ketoconazole, and amphotericin B were used as antifungal agents. Microscopic and macroscopic measurements of MIC after 24, 48 and 72 hours of inoculation[105 conidia /ml]into YNB broth with culture temperature of 25 degrees C were performed by use of broth microdilution method.The results are as follows : itraconazole, amphotericin B, and clotrimazole were effective against A.fumigatus.F.solani showed resistance to all kinds of antifungal agents.A.falciforme and A.alternata were sensitive to amphotericin B and itraconazole, respectively. Further studies may be needed for the standardized measurement of MIC against filamentous fungi and also for in Vitro-in Vivo correlations for the treatment of fungal keratitis.

Antibiotic Susceptibilities of Ocular Isolates from Patients with Bacterial Ke ratitis: A Multi-center Study

This multi-center study was performed prospectively to evaluate the causative organisms and antibiotic sensitivity of isolates in bacterial keratitis from April 1995 to December 1998. Total number of infectious keratitis was 1002 cases. Among them,279 cases were confirmed with bacterial keratitis and 314 strains were identified. Major causative organisms of bacterial keratitis were Pseudomonas aeruginosa 121 strains (38.54%), coagulase negative staphylococcus (CNS) 34 strains (10.83%), Streptococcus pneumoniae 19 strains (6.81%), Staphylococcus aureus 18 strains (5.73%), and Serratia marscence 16 strains (5.10%). P.aeruginosa was highly sensitive to ciprofloxacin, ceftazidime, piperacillin, cefoperazone, imipenem,tobramicin,and gentamycin. Coagulase negative streptococcus showed high sensitivity to vancomycin, teicoplanin, clindamycin, and ciplofloxacin, but low sensitivity to penicillin and gentamycin. In conclusion, the choice of the effective antibiotics in the treatment of bacterial keratitis were essential to decrease resistant bacterial strains.

In Vitro Amoebicidal Efficacy of Hexamidine, Polyexamethylene Biguanide and Chlorhexidine on Acanthamoeba Ccular Isolates

To evaluate amoebicidal efficacy of various disinfectants, we treated ten ocular isolates of Acanthamoeba with polyhexamethylene biguanide(PHMB), chlorhexidine and hexamidine. Acanthamoeba cysts were collected from axenic nonnutrient agar plates from 4 week culture. Cyst suspension(100microliter) was inoculated on 96-well microplate with concentration of 105 cys/ml. Each well was treated with two-fold diluted hexamidine, PHMB and chlorhexidine(100microliter each, 8 and 48hrs). Each well was examined under the inverted microscope until 7 days after treatment. Minimal cysticidal concentration(MCC) of each disinfectant qas measured after five experiments. MCC of hexamidine, chlorhexidine and PHMB against ten ocular isolates ranged 6.07-500microgram/ml, 3.12-12.50microgram/ml, 1.17-4.68microgram/ml respectively in 48hr treatment. In 8hr treatment, MCC of PHMB and chlorhexidine ranged 4.42-12.50microgram/ml and 4.67-17.71microgram/ml, but hexamidine did not demonstrate cysticidal effect. For proper disinfection of contact lens, disinfectants having cysticidal efficacy should be included in the composition of contact lens solution. Among the currently used topical amoebicidal agents, PHMB showed the greatest cysticidal activity on ten ocular strains of Acanthamoeba in invitro study.

Central Corneal Thickness before and after Excimer Laser Photore fractive Keratectomy in Myopia

This prospective study was done to investigate the correlation between central corneal thickness before excimer laser photorefractive keratectomy in myopic eyes and clinical factors including age, sex, left or right eye, myopic degree, and preoperative contact lens wearings, and also the relationship between the changes in postoperative central corneal thickness and age, sex, and myopic degrees. The average values of preoperative groups of less than -6.0D and over -6.0D were 519.78+/-29.87micrometer and 514.63+/-3 0.5 3 micrometerrespectively, showing much thinner value in high degree myopic eyes with statistical significance(p=0.02). Female patient's value was much thinner than male patient's value with statistical significance(p=0.016). The postoperative changes according to time process in central corneal thickness in 79 eyes which were followed up for more than six months were statistically significant(p=0.046). Among the changes in postoperative central corneal thickness, the recovery pace had no relations with age but female patient and over -6.0D in myopic degree showed faster recovery paces than male patient(p=0.035) and under -6.0D in myopic degree respectively during follow up period(p=0.001).

Distribution of Adhesion Molecules in Rabiit Cornea with Aspergillus fumigatus Keratitis

In order to help define the possible role of adhesion molecules in corneal inflammation, we investigated the distribution of adhesion molecules in normal and Aspergillus fumigatus keratitis-induced corneas of rabbits in process if time. Each 4 corneas were resected at 3, 12, 24, and 72 hours after intracorneal injection with A. fumigatus. Normal corneas (4 eyes) were used as a control. Monoclonal antibodies to beta 1 subunit of VLAs, alpha 1 subunit of VLA-1, LFA-1, ICAM-1,and ELAM-1 were used for immunohistochemical staining of 20 corneas.The results were as follows: In normal cornea, beta 1 subunit of VLAs was expressed on all parts of the cornea, and ICAM-1 was expressed on corneal stroma and endothelium. Vascular endothelium showed expression of beta 1 subunit of VLAs and ICAM-1 after 12 hours of intracorneal injection, and alpha1 subunit of VLA-1 and ELAM-1 at 72 hours after injection. In inflamed cornea, beta 1 subunit of VLAs was expressed strongly at 72 hours after injection. Alpha1 subunit of VLA-1 was detected on corneal stroma after 12 hours of injection, and on corneal endothelium at 72hours after injection. Expression of beta 2 subunit of LFA-1 was weak on corneal stroma after 3 hours injection, and on corneal endothelium at 72 hours after injection. ICAM-1 expression was detected weakly on corneal epithelium, and increased on corneal stroma and endothelium at 72 hours after injection. ELAM-1 was expressed weakly on corneal stroma after 3 hours of injection, and on corneal endothelium at 72 hours after injection.In this study, it was found that the invasion of A. fumigatusinto the cornea causes localized inflammatory reaction that results in activation of corneal cells (keratocytes, corneal endothelial cells and epithelial cells), and subsequent expression of adhesion molecules in the cornea. Expression of adhesion molecules facilitates the inflammatory cells to be migrated into the cornea with inflammmation.