Topical fluconazole for experimental candida keratitis in rabbits.

Using a reproducible model of Candida albicans keratitis in rabbits we studied the effect of topical fluconazole, a new triazole. Candida albicans DSM 70010 (2.5 X 10(5) cells) was injected into the corneal stroma of both eyes of 21 rabbits. All eyes developed a corneal ulcer. Forty-eight hours after inoculation the animals were divided into three groups: (1) 14 eyes, received fluconazole (2 mg/ml) and the epithelium subsequently removed; (2) 14 eyes, received only fluconazole drops; (3) 14 eyes, received 0.9% NaCl: half of this group was also debrided. We applied one drop of either substance 10 times a day for 24 days. A further six rabbits were used to judge if the drug penetrated into the cornea and aqueous humour. There was a highly significant difference between the fluconazole groups (1,2) and the control group (3) as to hypopyon and complications (descemetocele, corneal perforation) as well as recultivation of C. albicans from corneal tissue. The difference between the fluconazole groups with and without debridement was not significant. The drug penetrated into the cornea and aqueous humour of both uninflamed and inflamed eyes.     

明胶酶在兔真菌性角膜炎病理改变中的作用研究

目的探讨明胶酶包括基质金属蛋白酶（MMP）2与MMP-9在兔真菌性角膜炎病理改变中的作用。方法80只新西兰白兔随机分为4组,每组20只。其中3组为实验组,兔右眼分别注入100μl茄病镰刀菌、烟曲霉菌及白色念珠菌的悬液;另1组为对照组,兔右眼注入等量生理盐水。免疫组织化学方法观察MMP-2与MMP-9的来源,明胶酶谱法检测其活性。组织病理学方法观察炎性细胞的浸润、角膜细胞外基质（ECMs）的降解以及真菌菌丝在角膜内的生长方式与入侵深度。结果MMP-2主要由角膜基质细胞产生,真菌感染后5d检测出活性,8d活性升高。MMP-9主要来源于嗜中性粒细胞,接种后1d即检测到活性,3d活性升高,之后逐渐下降。茄病镰刀菌感染后3d,角膜内散在嗜中性粒细胞,浅层ECMs被降解,菌丝平行于角膜基质纤维生长。烟曲霉菌和白色念珠菌感染后3d,角膜内可见大量嗜中性粒细胞,周围ECMs降解明显,菌丝表现为垂直生长。接种后8d,茄病镰刀菌和白色念珠菌感染的角膜内炎性细胞和菌丝明显减少,而烟曲霉菌感染的角膜变化不明显。结论茄病镰刀菌、烟曲霉菌及白色念珠菌感染兔角膜后,产生的明胶酶活性明显不同;明胶酶对降解角膜ECMs发挥了重要作用;随着ECMs降解程度的不同,菌丝在角膜内的生长方式、入侵深度等病理改变出现差异。     

Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea

PURPOSE: To investigate the roles of adherence and matrix metalloproteinases (MMPs) in growth patterns of major fungal pathogens in cornea. METHODS: Ninety-six eyes in 96 rabbits were equally divided into four groups receiving inoculation of fungal conidia of Aspergillus fumigatus, Candida albicans, Fusarium solani, and Penicillium citreo-viride, respectively, to induce fungal keratitis. Corneas in each group were obtained at 2, 8, 16 hr, and 1, 2, 3, 5, and 8 days after inoculation and were subjected to scanning electron microscopy, histopathological examination, and gelatin zymography. Eight saline-inoculated eyes in another eight rabbits served as controls. RESULTS: All eyes in the fungus-inoculated groups developed fungal keratitis. The binding of conidia to corneal epithelial basement membrane was initiated earlier in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. Destruction of basement membrane began at 1 to 3 days. Histopathologically, infiltration of inflammatory cells was more evident in the A. fumigatus and C. albicans groups than the F. solani and P. citreo-viride groups at 3 days. The hyphae of A. fumigatus and C. albicans traversed the cornea in a plane perpendicular to the stromal lamellae, whereas the hyphae of F. solani and P. citreo-viride lay parallel to the corneal lamellae. MMP-9 and MMP-2 were found in all infected corneas. At 3 days, proteolysis was most active; the level of MMP-9 was higher in the A. fumigatus and C. albicans groups than in the F. solani and P. citreo-viride groups. There were positive correlations among the number of binding conidia, degree of inflammation, and level of MMP-9 (p < 0.05). CONCLUSIONS: The adherence ability, chemotaxis to neutrophils, and MMP-9 expression level differ in eyes with different fungal pathogens, which may contribute to the different growth patterns of fungi in cornea.     

Comparison of efficacy of topical and oral fluconazole treatment in experimental Aspergillus keratitis

PURPOSE: To investigate the therapeutic role of topical and oral fluconazole treatment using a rabbit model of Aspergillus fumigatus keratitis. METHODS: Aspergillus fumigatus spores were injected into the corneal stroma of the right eye of 20 rabbits. Forty-eight hours later the rabbits were randomly divided into three groups. Group 1 rabbits (six) were treated with topical fluconazole (2 mg/ml, eight times daily), group 2 rabbits (seven) received oral fluconazole (37.5 mg/kg bid), and group 3 rabbits (seven) were untreated controls. The eyes were examined and photographed with a slit-lamp 2, 6, 10, 16, and 20 days after inoculation and an observer graded the corneas in a masked fashion. Corneal cultures were taken on days 2, 14, and 20 for fungus growth. RESULTS: At the beginning of treatment, the slit-lamp scores did not differ among three groups. However, on days 6, 10, 16, and 20 the treated groups had statistically significant lower scores compared to the untreated controls. No significant difference was observed between topical and oral treated groups at any examination point. All cultures were positive on day 2, but on days 16 and 20, there were significantly lower positive fungal cultures in both treatment groups compared to the control (p < 0.01, chi square test). CONCLUSION: Our study showed that both topical and oral fluconazole were effective in lessening the severity of fungal keratitis in a rabbit model and should be considered effective treatment alternatives in the therapy of Aspergillus fumigatus keratitis.     

The effect of topical steroid medication on experimental candida keratitis

An immunological study was performed to investigate the effect of topical steroid medication on candida keratitis. 15 albino rabbits were used in this study. Candida keratitis was induced by inoculating candida albicans into the corneal stroma on the animals. In the experimental group (9 rabbits), prednisolone ointment was given twice daily for 4 days prior to the inoculation. In the control group (6 rabbits) no medication was given. The clinical course of keratitis was observed and stimulation of peripheral lymphocytes was measured. Candida extract (soluble antigen) and formalin-fixed candida whole cells (insoluble antigen) were used as antigens to stimulate the lymphocytes. Clinical observation was continued up to 21 days after inoculation. Stimulation of peripheral lymphocytes was measured by the stimulation index (SI) at before 4,7,11,14 and 21 days after inoculation. Inflammation was suppressed clinically in the active phase of the experimental group, but the total course of inflammation was longer than in the control group. In the control group, SI against both soluble and insoluble antigens was elevated. The peak SI was reached at 11 days after inoculation. In the experimental group, SI was elevated against soluble antigen but no elevation was observed against insoluble antigen. These results show that cellular immunological response does exist in experimental candida keratitis in rabbits. Since local steroid medication prolonged inflammation and suppressed cellular immunity, cellular immunity seems to play a significant role in the development and recovery of candida keratitis.     

Incidence and outcomes of lasik with diffuse lamellar keratitis treated with topical and oral corticosteroids

PURPOSE: To analyze the incidence and clinical outcomes of patients developing diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK) treated with topical and oral corticosteroids. SETTING: Oregon Eye Institute, Eugene, Oregon, USA. METHODS: A retrospective analysis of the last consecutive 1000 LASIK cases was performed. Eyes developing stage 3 DLK or at risk for progressing to stage 3 were treated with a combination of high-dose prednisolone acetate 1% and oral prednisone and evaluated for corneal scarring, loss of best spectacle-corrected visual acuity (BSCVA), and deviation from the intended refractive outcome. RESULTS: Diffuse lamellar keratitis developed in 40 eyes (4%). It progressed to stage 3 in 7 eyes (17%). Oral and topical steroids were used in 22 eyes (55%). The mean variation from the desired refractive outcome was 0.14 diopter +/- 0.53 (SD). There were no instances of corneal scarring or permanent loss of BSCVA. No eye had interface irrigation. CONCLUSIONS: Treatment of severe DLK with high-dose topical and oral corticosteroids produced excellent results without flap lifting and interface irrigation.     

Study of suppressive effect of intravenous fluconazole on endogenous Candida endophthalmitis in rabbits.

The effect of intravenous fluconazole on endogenous Candida endophthalmitis in rabbits was investigated. Preventive and therapeutic experiments were carried out. In the preventive series, rabbits were injected intravenously with 5 mg/kg of fluconazole at 30 minutes, 1 day and 2 days after intravenous inoculation with Candida albicans spores. The control group received no medication. No treated rabbits developed ocular lesions and no Candida spores were isolated from the treated eyes. On the other hand, all control rabbits developed bilateral chorioretinitis and C. albicans was isolated invariably from the control eyes. In the therapeutic series, intravenous fluconazole (5 mg/kg body weight) was administered from 3 to 6 days after inoculation. All rabbits developed chorioretinitis and Candida spores were isolated from all eyes. Therefore, the results of this study prove that intravenous fluconazole is more effective in preventive use than in therapeutic use against endogenous Candida endophthalmitis in rabbits.     

Confocal microscopy of Aspergillus fumigatus keratitis

AIM: To use a confocal microscope to characterise the treated and untreated courses of fungal keratitis. METHODS: In the first experiment, Aspergillus fumigatus stromal keratitis was produced in both eyes of seven New Zealand white rabbits. In the second experiment, keratitis was induced in right eyes of 20 rabbits. Group 1 rabbits were treated with topical fluconazole, group 2 rabbits received oral fluconazole, and group 3 rabbits were used as controls. The rabbits were examined with a slit lamp and confocal microscope 2, 6, 10, 14, and 20 days after inoculation. The corneal cultures were taken on days 2, 14, and 20 and biopsies were taken on days 2 and 22. RESULTS: On days 14 and 22 confocal microscopy was more sensitive than culture technique in both treated and untreated animals, since not all cases of fungal keratitis can be cultured. CONCLUSION: This study indicates that confocal microscopy is a rapid and sensitive diagnostic tool for both the early diagnosis and non-invasive follow up of fungal keratitis     

Comparison of micafungin and fluconazole for experimental Candida keratitis in rabbits

PURPOSE: To evaluate the efficacy of subconjunctival injection of micafungin in the treatment of experimental Candida albicans keratitis in rabbits compared with fluconazole. METHODS: In 1 eye of 24 New Zealand white rabbits, C. albicans (5 x 10 yeast cells) was inoculated in the corneal stroma. The animals were randomly assigned to 3 groups and received subconjunctival injection of 0.5 mL of 0.1% micafungin, 0.2% fluconazole, or physiologic saline once a day for 3 weeks. The eyes were examined slit-lamp biomicroscopically and histopathologically. The clinical course of fungal keratitis was compared among the 3 groups. In another 36 rabbits, a microbiological examination was performed using a quantitative isolate recovery technique, and the numbers of colony-forming units were compared among groups. RESULTS: The clinical scores were significantly lower in the micafungin group than in the other 2 groups throughout the study period (P < 0.0001 approximately P = 0.0027, Bonferroni multiple comparison). The fluconazole group showed significantly lower clinical scores than the saline group on day 18 (P = 0.0343). At the end of the study period, there were significant differences between the saline and micafungin groups (P < 0.0001), the saline and fluconazole groups (P = 0.0072), and the fluconazole and micafungin groups (P = 0.0013). Histopathologically, similar results were obtained. Moreover, the results of the microbiological examination nearly matched the clinical and histopathologic findings. CONCLUSIONS: Subconjunctival administration of micafungin was effective in the treatment of experimental Candida keratitis. Local application of micafungin to the eye would be a feasible treatment option for clinical fungal keratitis.     

Topical ketoconazole for experimental Candida keratitis in rabbits

Topical ketoconazole (2% ointment three times per day for three weeks) markedly reduced the severity of keratitis produced with Candida albicans inoculation in the right eyes of 20 rabbits. Clinical scores of affected eyes were significantly lower in the treated eyes than in the controls, which consisted of the 20 inoculated but untreated left eyes of the same rabbits. All cultures of corneal scrapings from the treated eyes were negative on the 12th day after inoculation, while two cultures were still positive at the end of the experiment (day 21) in the control eyes. Histopathologic examination showed less severe inflammatory changes in the treated eyes than in the control eyes.     

Comparison between corneal cross-linking,topical antibiotic and combined therapy in experimental bacterial keratitis model

Purpose

This study was conducted to investigate the effects of an experimental bacterial keratitis model on the corneal collagen cross-linking treatment (CXL), and also to compare topical antibiotic treatment with the combined treatment.

The effect of propolis in experimental Acanthamoeba keratitis

PURPOSE: To examine the effect of propolis in a rat model of Acanthamoeba keratitis and to determine its in vitro cytotoxicity in cultured corneal epithelial cells. METHODS: Eighteen Wistar albino rats were used. Cultured corneal epithelial cells obtained from two healthy rats for in vitro cytotoxicity of propolis. Corneal stromal inoculation was performed in 16 rats with amoebic culture containing 1 x 10(6) amoeba/mL. Rats with Acanthamoeba keratitis 5 days later after the inoculation were divided randomly into four groups, and eight eyes of each group were treated with study drugs. The propolis, chlorhexidine (CHX), propolis plus CHX and control eyes were treated with topical propolis, 0.002% CHX, propolis plus 0.002% CHX and lubricant eye drops, respectively. The study drugs were instilled every one hour for 10 days. All eyes were examined and keratitis graded by slit-lamp biomicroscopy on days 2, 5 and 10 during the administration of the study drugs. After the completion of keratitis grading, all the 16 rats were humanely killed and their corneas were excised and used for Acanthamoeba culture to evaluate presence of Acanthamoeba growth after treatment 14 days later. RESULTS: Concentrations of propolis higher than 7.81 mg/mL cause damage to corneal epithelial cells in the experiment of in vitro cytotoxicity of propolis on corneal epithelial cells. The keratitis grade on day 2 in the CHX eyes was significantly lower than that in the control eyes (P < 0.05). The keratitis grades on days 5 and 10 in the propolis, CHX and propolis plus CHX eyes were significantly lower compared with those on days 5 and 10 in the control eyes (P < 0.05). In the propolis eyes, the keratitis grade on day 5 was significantly lower than that on day 2 (P < 0.05), and it was significantly lower on day 10 compared with that on day 5 (P < 0.05). In the CHX and propolis plus CHX eyes, the keratitis grade on day 10 was significantly lower compared with that on days 2 and 5 (P < 0.05). In the control eyes, there was no significant difference in the keratitis grades on days 2, 5 and 10 (P > 0.05). The culture positivity at Acanthamoeba growth after treatment experiment in the propolis, CHX and propolis plus CHX eyes was significantly lower than that in the control eyes (P < 0.05). CONCLUSIONS: We suggest that propolis had amoebicidal properties in this rat model of Acanthamoeba keratitis. Further investigations to evaluate the antimicrobial activity of the individual fractions of the resin could yield more information about its mechanism of action in treating this disease.     

Polymicrobial keratomycosis in a three-year-old child

PURPOSE: To describe a previously unreported case of polymicrobial mycotic keratitis caused by an association between Candida lusitaniae, C. parapsilosis, and Geotrichum candidum. METHODS: A three-year-old child with an antecedent trauma with vegetable matter and a prolonged use of corticosteroid eyedrops developed fungal keratitis. RESULTS: The isolates of the corneal scraping using Sabaraud dextrose agar grew C. lusitaniae, C. parapsilosis, and G. candidum. After topical 0.2% and systemic fluconazole treatment, the corneal lesion resolved with no recurrence. CONCLUSIONS: Corneal trauma with vegetables and the indiscriminate use of corticosteroids are important risk factors for mycotic keratitis. A combination of topical 0.2% and systemic fluconazole therapy was effective in the treatment of this mycotic association. This is the first report of fungal keratitis caused by C. lusitaniae and G. candidum.     

Th1/Th2细胞因子在小鼠真菌性角膜炎中的表达

目的 通过检测Th1/Th2细胞因子在小鼠真菌性角膜炎中的表达水平,探讨机体免疫在该病发展中的作用. 方法应用角膜表层镜法建立Balb/c小鼠茄病镰刀菌性角膜炎模型,在感染后第1、3、5、7天,裂隙灯显微镜观察角膜炎特点;HE染色观察角膜病理变化;半定量RT-PCR和ELISA检测角膜中Thl细胞因子(IFN-γL-12)及Th2细胞因子(IL-4、IL-10)基因mRNA和蛋白的表达,半定量RT-PCR检测T-bet基因mRNA的表达;直线相关分析检测T-bet mRNA与IFN-比值的相关性. 结果角膜接种菌液后,早期角膜浸润混浊进行性加重,第5天后新生血管大量生长;HE染色可见第1、3天在角膜缘、角膜基质及前房中有大量的炎症细胞浸润,第5天后炎症细胞减少,角膜基质中纤维细胞逐渐增多;RT-PCR与ELISA检测结果表明,Thl细胞因子(IFN-γL-12)和Th2细胞因子(IL-4、IL-10)基因mRNA及蛋白在角膜接种菌液后均出现表达,且IFN-γL-12的表达显著强于IL-4、IL-10;IFN-γ/IL-4比值在第3天达最高值,而后逐渐降低;T-bet mRNA在第3天达最高值;T-bet mRNA的表达与IFN-γ/IL-4比值呈正相关(P＜0.05). 结论在真菌性角膜炎中,Thl/Th2型免疫应答共同参与调节机体的抗真菌免疫,但以Th1型应答为主;角膜感染真菌后第3天机体的免疫力达最强.     

Diffuse lamellar keratitis associated with recurrent corneal erosions after laser in situ keratomileusis

PURPOSE: Diffuse lamellar keratitis (DLK) is marked by the presence of diffuse or multifocal infiltrates confined to the laser in situ keratomileusis (LASIK) interface. These infiltrates are culture-negative, and the etiology is thought to be noninfectious. Most cases of DLK occur within the first week or two following surgery. METHODS: We describe one case of diffuse lamellar keratitis that occurred 3 months after LASIK. The patient developed a spontaneous corneal erosion in one eye. Over the next 2 days while the erosion was being treated, there was rapid development of DLK. Slit-lamp biomicroscopy and in vivo scanning slit confocal microscopy were performed. The patient was treated with intensive topical corticosteroids. RESULTS: Scanning slit confocal microscopy revealed numerous, highly-reflective round bodies consistent with a polymorphonuclear infiltrate located at the flap interface. Treatment with topical 1.0% prednisolone acetate was instituted, with rapid improvement in patient symptoms, visual acuity, and slit-lamp biomicroscopy. CONCLUSIONS: Diffuse lamellar keratitis may occur months after LASIK as a result of a spontaneous recurrent corneal epithelial erosion.     

人羊膜匀浆提取液抑制大鼠角膜碱烧伤后新生血管生成

目的:研究人羊膜匀浆提取液对角膜碱烧伤后新生血管形成过程中色素上皮衍生因子(pigment epithelium-derived fac-tor,PEDF)和血管内皮生长因子(vascular-endothelial growthfactor,VEGF)的表达影响及对角膜新生血管的抑制作用。方法:SD大鼠54只建立双眼角膜碱烧伤模型,随机分为3组:A组为3g/L氧氟沙星滴眼液组,B组为10g/L泼尼松龙滴眼液组,C组为新鲜人羊膜匀浆提取液组,并分别于治疗后不同时间点进行角膜新生血管增生情况观察,HE染色病理学观察,免疫组化行VEGF和PEDF的表达检测。结果:B,C组比A组新生血管增生少,多形核白细胞(poly-morphonuclear leukocyte,PMN)浸润程度轻,VEDF表达水平显著降低,但B,C两组之间VEDF表达无显著性差异(P>0.05)。C组PEDF表达水平显著高于A,B组(P<0.01),但A,B之间PEDF表达无显著性差异(P>0.05)。结论:新鲜人羊膜匀浆提取液能减少碱烧伤后角膜组织中PMN浸润,增强角膜组织PEDF的表达水平。     

Polymicrobial Keratomycosis in a Three-Year-Old Child

Purpose: To describe a previously unreported case of polymicrobial mycotic keratitis caused by an association between Candida lusitaniae, C. parapsilosis, and Geotrichum candidum. Methods: A three-year-old child with an antecedent trauma with vegetable matter and a prolonged use of corticosteroid eyedrops developed fungal keratitis. Results: The isolates of the corneal scraping using Sabaraud dextrose agar grew C. lusitaniae, C. parapsilosis, and G. candidum. After topical 0.2% and systemic fluconazole treatment, the corneal lesion resolved with no recurrence. Conclusions: Corneal trauma with vegetables and the indiscriminate use of corticosteroids are important risk factors for mycotic keratitis. A combination of topical 0.2% and systemic fluconazole therapy was effective in the treatment of this mycotic association. This is the first report of fungal keratitis caused by C. lusitaniae and G. candidum.     

外伤所致真菌性角膜溃疡的氟康唑综合治疗

目的 研究氟康唑综合治疗外伤所致真菌性角膜溃疡的疗效。方法 对38例真菌性角膜溃疡应用氟康唑、角膜碘酊烧灼、口服消炎痛等药物，观察综合用药的疗效。结果 30例在10～40天内愈合，6例好转，视力均有明显提高。2例未愈。结论 应用氟康唑综合治疗真菌性角膜溃疡疗效显著。     

Treatment of HSV-1 stromal keratitis with topical cyclosporin A: a pilot study

· Background: In stromal keratitis induced by herpes simplex virus (HSV) the host’s immune response contributes to corneal scarring and neovascularization. The purpose of this study was to analyze the efficacy of topically applied cyclosporin A (CsA) in patients with HSV keratitis. · Methods: The authors performed a prospective pilot study in patients with HSV stromal keratitis (n=18). Eyes were treated with CsA eyedrops and acyclovir ointment. The drugs were tapered off gradually. Visual acuity, slit-lamp appearance, intraocular pressure and corneal sensitivity were evaluated monthly (follow-up 5.2±0.28 months, mean±SEM. · Results: Keratitis resolved with CsA treatment in 10 of 14 patients with non-necrotizing keratitis and in 2 of 4 with necrotizing keratitis. As CsA was used topically, the corticosteroids could be withdrawn in all patients with non-necrotizing keratitis and in 1 of 3 with necrotizing keratitis. Under CsA therapy, persistent or progressive inflammation was noted in 6 of the 18 patients. These 6 patients with keratitis improved only with combined CsA/corticosteroids. Corneal ulcers healed in 4 patients with topical CsA, and corneal neovascularization improved in a further 8. Except for toxic epitheliopathy, no further CsA complications were noted. · Conclusions: The findings in this pilot study suggest that HSV stromal keratitis can be treated successfully with CsA eyedrops, especially in non-necrotizing disease. CsA may be particularly helpful in the presence of steroid glaucoma, herpetic corneal ulcers, and to taper off topical corticosteroids. Additional use of acyclovir may aid in suppressing the recurrence of epithelial HSV keratitis. A randomized study should be performed to evaluate the role of topical CsA in more detail. Received: 23 April 1998 Revised version received: 12 August 1998 Accepted: 14 September 1998