兔镰刀菌和曲霉菌真菌性角膜炎的研究
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摘要
真菌是引起角膜化脓性感染的主要病原之一。综合不同国家的统计资料,真菌性角膜炎的发病人数占所有化脓性角膜炎的比率高达30～65%。临床上,真菌性角膜炎的诊断相对较困难,有效的治疗手段也较缺乏,预后差。
针对真菌性角膜炎,我们已经开展了下列研究:开发了重症真菌性角膜的手术治疗方法:建立了一种新的玻片培养系统宋鉴定从真菌性角膜炎病例中分离培养到的真菌菌株,并对各分离菌株进行抗真菌药物敏感性的测定,为临床药物的选用提供参考依据。在这一研究中,我们共鉴定了61株真菌病原,其中分离到镰刀菌33株,占所有分离菌株的54.1%,曲霉菌9株,占14.8%。由此可见,镰刀菌和曲霉菌是引起真菌性角膜炎最常见的两种真菌菌属,这一结果与其他一些国家如印度和美国南部的报道结果相类似。
临床实践中可以观察到镰刀菌和曲霉菌性角膜炎病例的临床表现不尽相同。镰刀菌性角膜炎早期就诊病例的临床特征为角膜浸润病灶,表现为密度浓淡不一,表面干燥,病灶周围出现羽毛状浸润;而曲霉菌性角膜炎早期就诊病例的临床特征为角膜化脓性浸润病灶,表现为密度较高,质地均匀一致。晚期就诊病例,两种角膜炎的特征差异不明显,均表现为大量的组织化脓性坏死,出现角膜脓疡或深层溃疡,甚至角膜穿孔。
为观察镰刀菌和曲霉菌所导致的真菌性角膜炎的临床发展过程和表现的差异,以及两种真菌性角膜炎发展过程中真菌病原的繁殖和炎症细胞在角膜组织内浸润的演化过程,以
Fungal keratitis is one of severe suppurative corneal infection. This entity accounts for 30~65% of all cases of culture-proven microbial keratitis, and is being increasingly recognized as an important cause of blindness because fungal keratitis can lead to corneal opacity, stromal melting or corneal perforation.In the previous study, we developed a new slide culture system for identifying the fungal species which were isolated from patients with fungal keratitis. Among 61 fungal isolates, fusarium(n=33, 54.1%) and aspergillus(n=9, 14.8%) are the two most common genera causing corneal infection. The result is close to other studies from India and Southern United States.Although it is well known that the typical corneal infiltrate of fungal keratitis is gray-white opacity with dry rough texture and irregular feathery margin, in clinical practice, the diversity of eye presentation can be observed in each case, especially between fusarium and aspergillus keratitis. In most cases of early stage of fusarium keratitis, the typical corneal infiltrate features are usually observed. But in aspergillus keratitis, the corneal infiltrate commonly presented as uniform density without feathery border. However, in later stage of fungal keratitis, the eye symptoms may progress to similar, such as large amount of necrotic slough, corneal abscess or deep ulcer, even corneal perforation could be found in both
fusarium and aspergillus keratitis.In this study, we focused on producing an experimental fusarium and aspergillus keratitis in rabbit model to clarify the natural course of fungal keratitis and its clinical and pathological features, and to elucidate the differences of clinical and pathological features of this two most common filamentous fungal keratitis.MethodsNewzeland white rabbits of either sex weighing 2 to 2.5 kg were used for animal model. Two wild-type strains of Fusarium.solani and Aspergillus.fumigatus in this study were isolated from two clinical cases of fungal keratitis through corneal scraping. The species identification was made by combining the macroscopic with microscopic criteria. Both strains that were cultured previously on Sabouraud's dextrose agar plate for 4~7 days were harvested in sterile saline with removal of hyphaes. The homogenate was quantified under microscopy using cell counter and adjusted the density to 0.5~1.0×10~7 CFU/ml. An inoculums of 100- μl, containing approximately 0.5~ 1.0×10~6 conidia were injected into the anterior and middle of corneal stroma using 27 gauge needle through three points around the pupil. Animals that served as negative control were mock inoculated with same volume sterile saline. After inoculation, all eyes were treated with 0.1% dexamethasone and 0.3% tobramycin ointment one time a day. The duration of topical steroids using is 10 days in all animals which were inoculated with Fusarium.solani, Aspergillus.fumigatus and negative controls.All rabbits were scored daily and photographed every two days for corneal involvements including corneal infiltrate, corneal opacity and edema, corneal ulceration, and inflammation reaction of the eye after infection such as conjunctival injections and hypopyon. The severity of keratomycosis in rabbits was visually scored with slit lamps and photographs. Groups of rabbits were killed and eyeballs were enucleated 3, 5, 10, 15, 20, 25, 30, 35 days after inoculation. The infected corneas were removed at the limbus and bisected longitudinally
through the focus of corneal infiltrate. One half was processed for histopathological examination; the other was cut into pieces for fungal culture. Hematoxylin-erosin and periodic acid-Schiff staining were applied in histological testing to evaluate inflammations cell infiltration, corneal structural destruction and hyphal invasion. Lastly, we compared the clinical and histopathological findings of Fusarium.solani and Aspergillus.fumigatus keratitis.ResultsAll rabbits that were infected with Fusarium.solani and Aspergillus.fumigatus progressed to severe keratomycosis and persisted for more than 1 mo
针对真菌性角膜炎,我们已经开展了下列研究:开发了重症真菌性角膜的手术治疗方法:建立了一种新的玻片培养系统宋鉴定从真菌性角膜炎病例中分离培养到的真菌菌株,并对各分离菌株进行抗真菌药物敏感性的测定,为临床药物的选用提供参考依据。在这一研究中,我们共鉴定了61株真菌病原,其中分离到镰刀菌33株,占所有分离菌株的54.1%,曲霉菌9株,占14.8%。由此可见,镰刀菌和曲霉菌是引起真菌性角膜炎最常见的两种真菌菌属,这一结果与其他一些国家如印度和美国南部的报道结果相类似。
临床实践中可以观察到镰刀菌和曲霉菌性角膜炎病例的临床表现不尽相同。镰刀菌性角膜炎早期就诊病例的临床特征为角膜浸润病灶,表现为密度浓淡不一,表面干燥,病灶周围出现羽毛状浸润;而曲霉菌性角膜炎早期就诊病例的临床特征为角膜化脓性浸润病灶,表现为密度较高,质地均匀一致。晚期就诊病例,两种角膜炎的特征差异不明显,均表现为大量的组织化脓性坏死,出现角膜脓疡或深层溃疡,甚至角膜穿孔。
为观察镰刀菌和曲霉菌所导致的真菌性角膜炎的临床发展过程和表现的差异,以及两种真菌性角膜炎发展过程中真菌病原的繁殖和炎症细胞在角膜组织内浸润的演化过程,以
Fungal keratitis is one of severe suppurative corneal infection. This entity accounts for 30~65% of all cases of culture-proven microbial keratitis, and is being increasingly recognized as an important cause of blindness because fungal keratitis can lead to corneal opacity, stromal melting or corneal perforation.In the previous study, we developed a new slide culture system for identifying the fungal species which were isolated from patients with fungal keratitis. Among 61 fungal isolates, fusarium(n=33, 54.1%) and aspergillus(n=9, 14.8%) are the two most common genera causing corneal infection. The result is close to other studies from India and Southern United States.Although it is well known that the typical corneal infiltrate of fungal keratitis is gray-white opacity with dry rough texture and irregular feathery margin, in clinical practice, the diversity of eye presentation can be observed in each case, especially between fusarium and aspergillus keratitis. In most cases of early stage of fusarium keratitis, the typical corneal infiltrate features are usually observed. But in aspergillus keratitis, the corneal infiltrate commonly presented as uniform density without feathery border. However, in later stage of fungal keratitis, the eye symptoms may progress to similar, such as large amount of necrotic slough, corneal abscess or deep ulcer, even corneal perforation could be found in both
fusarium and aspergillus keratitis.In this study, we focused on producing an experimental fusarium and aspergillus keratitis in rabbit model to clarify the natural course of fungal keratitis and its clinical and pathological features, and to elucidate the differences of clinical and pathological features of this two most common filamentous fungal keratitis.MethodsNewzeland white rabbits of either sex weighing 2 to 2.5 kg were used for animal model. Two wild-type strains of Fusarium.solani and Aspergillus.fumigatus in this study were isolated from two clinical cases of fungal keratitis through corneal scraping. The species identification was made by combining the macroscopic with microscopic criteria. Both strains that were cultured previously on Sabouraud's dextrose agar plate for 4~7 days were harvested in sterile saline with removal of hyphaes. The homogenate was quantified under microscopy using cell counter and adjusted the density to 0.5~1.0×10~7 CFU/ml. An inoculums of 100- μl, containing approximately 0.5~ 1.0×10~6 conidia were injected into the anterior and middle of corneal stroma using 27 gauge needle through three points around the pupil. Animals that served as negative control were mock inoculated with same volume sterile saline. After inoculation, all eyes were treated with 0.1% dexamethasone and 0.3% tobramycin ointment one time a day. The duration of topical steroids using is 10 days in all animals which were inoculated with Fusarium.solani, Aspergillus.fumigatus and negative controls.All rabbits were scored daily and photographed every two days for corneal involvements including corneal infiltrate, corneal opacity and edema, corneal ulceration, and inflammation reaction of the eye after infection such as conjunctival injections and hypopyon. The severity of keratomycosis in rabbits was visually scored with slit lamps and photographs. Groups of rabbits were killed and eyeballs were enucleated 3, 5, 10, 15, 20, 25, 30, 35 days after inoculation. The infected corneas were removed at the limbus and bisected longitudinally
through the focus of corneal infiltrate. One half was processed for histopathological examination; the other was cut into pieces for fungal culture. Hematoxylin-erosin and periodic acid-Schiff staining were applied in histological testing to evaluate inflammations cell infiltration, corneal structural destruction and hyphal invasion. Lastly, we compared the clinical and histopathological findings of Fusarium.solani and Aspergillus.fumigatus keratitis.ResultsAll rabbits that were infected with Fusarium.solani and Aspergillus.fumigatus progressed to severe keratomycosis and persisted for more than 1 mo
引文
1. Upadhyay MP, Karmacharya PCD, Koirala S, et al. Epidemiologic characteristics, predisposing factor, and etiologic diagnosis of corneal ulceration in Nepal. Am J Ophthalmol 1991; 111:92-99.
2. Tanure MAG, Gohen EJ, Sudesh S, et al. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000; 19:307-312.
3. Thomas PA. Current perspective on ophthalmic mycoses. Clinical Microbiology Review. 2003; 16: 730-797.
4. Yao YF, Zhang YM, Zhou P, Zhang B, Qiu WY, Tseng SC. Therapeutic penetrating keratoplasty in severe fungal keratitis using cryopreserved donor corneas. Br J Ophthalmol. 2003;87:543-7.
5.周萍,姚玉峰,裘文亚等.重症真菌性角膜炎经治疗性角膜移植术后再次光学性角膜移植的临床疗效评价.中华眼科杂志(in press).
6. Qiu WY, Yao YF, Zhu YF, et al. Fungal Spectrum Identified by a New Slide Culture and in Vitro Drug Susceptibility Using Etest in Fungal Keratitis. Current Eye Research. 2005 (in press).
7. Chowdhary A, Singh K.Spectrum of fungal keratitis in North India Cornea. 2005;24:8-15.
8. Rosa RH Jr, Miller D, Alfonso EC.The changing spectrum of fungal keratitis in south Florida. Ophthalmology. 1994 Jun; 101 (6): 1005-13.
9. Fons A, Garcia-de-Lomas J, Nogueira JM, et al. Histopathology of experimental Aspergillus fumigatus keratitis. Mycopathologia. 1988; 101: 129-31.
10. Dignani MC, Anaissie E. Human fusariosis. Clin Microbiol Infect. 2004;10 Suppl 1:67-75.
11. Denis M. O'Day, W. SH,et al. Differences in Virulence between Two Candida albicans Strains in Experimental Keratitis. Invest Ophthalmol Vis Sci. 2000; 41: 1116-1121.
12. Wolfram S, Antje O, Christian K, et al. Combined Topical Fluconazole and Corticosteroid Treatment for Experimental Candida albicans Keratomycosis. Invest Ophthalmol Vis Sci. 2003; 44: 2634-2643.
13. Wu TG, Wilhelmus KR, Mitchell BM. Experimental Keratomycosis in a Mouse Model. Invest Ophthalmol Vis Sci. 2003; 44: 210-216.
14. Wu TG, Keasler VV, Mitchell BM, Wilhelmus KR. Immunosuppression affects the severity of experimental Fusarium solani keratitis.Infect Dis. 2004; 190: 192-8.
15. Vemuganti GK, Garg P, Rao GN, et al. Evaluation of agent and host factors in progression of mycotic keratitis: a histologic and microbiologic study of 167 corneal buttons. Ophthalmology 2002; 109:1538-1546.
16. Garg PM, Vemuganti GK,et al. Pigmented Plaque Presentation of Dematiaceous Fungal Keratitis: A Clinicopathologic Correlation. Cornea. 2004; 23:571-576.
17. Naiker S, Odhav B. Mycotic keratitis: profile of Fusarium species and their mycotoxins. Mycoses. 2004;47:50-6
18. Tsunawaki S, Yoshida LS, Nishida S, et al.Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase. Infect Immun. 2004;72:3373-82.
19. Yamada A, Kataoka T, Nagai K.The fungal metabolite gliotoxin: immunosuppressive activity on CTL-mediated cytotoxicity. Immunol Lett. 2000 Jan 10;71(l):27-32.
20. Thomas PA. Mycotic keratitis - an underestimated mycosis. J Med Vet Mycol 1994; 32: 235-56.
2. Tanure MAG, Gohen EJ, Sudesh S, et al. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000; 19:307-312.
3. Thomas PA. Current perspective on ophthalmic mycoses. Clinical Microbiology Review. 2003; 16: 730-797.
4. Yao YF, Zhang YM, Zhou P, Zhang B, Qiu WY, Tseng SC. Therapeutic penetrating keratoplasty in severe fungal keratitis using cryopreserved donor corneas. Br J Ophthalmol. 2003;87:543-7.
5.周萍,姚玉峰,裘文亚等.重症真菌性角膜炎经治疗性角膜移植术后再次光学性角膜移植的临床疗效评价.中华眼科杂志(in press).
6. Qiu WY, Yao YF, Zhu YF, et al. Fungal Spectrum Identified by a New Slide Culture and in Vitro Drug Susceptibility Using Etest in Fungal Keratitis. Current Eye Research. 2005 (in press).
7. Chowdhary A, Singh K.Spectrum of fungal keratitis in North India Cornea. 2005;24:8-15.
8. Rosa RH Jr, Miller D, Alfonso EC.The changing spectrum of fungal keratitis in south Florida. Ophthalmology. 1994 Jun; 101 (6): 1005-13.
9. Fons A, Garcia-de-Lomas J, Nogueira JM, et al. Histopathology of experimental Aspergillus fumigatus keratitis. Mycopathologia. 1988; 101: 129-31.
10. Dignani MC, Anaissie E. Human fusariosis. Clin Microbiol Infect. 2004;10 Suppl 1:67-75.
11. Denis M. O'Day, W. SH,et al. Differences in Virulence between Two Candida albicans Strains in Experimental Keratitis. Invest Ophthalmol Vis Sci. 2000; 41: 1116-1121.
12. Wolfram S, Antje O, Christian K, et al. Combined Topical Fluconazole and Corticosteroid Treatment for Experimental Candida albicans Keratomycosis. Invest Ophthalmol Vis Sci. 2003; 44: 2634-2643.
13. Wu TG, Wilhelmus KR, Mitchell BM. Experimental Keratomycosis in a Mouse Model. Invest Ophthalmol Vis Sci. 2003; 44: 210-216.
14. Wu TG, Keasler VV, Mitchell BM, Wilhelmus KR. Immunosuppression affects the severity of experimental Fusarium solani keratitis.Infect Dis. 2004; 190: 192-8.
15. Vemuganti GK, Garg P, Rao GN, et al. Evaluation of agent and host factors in progression of mycotic keratitis: a histologic and microbiologic study of 167 corneal buttons. Ophthalmology 2002; 109:1538-1546.
16. Garg PM, Vemuganti GK,et al. Pigmented Plaque Presentation of Dematiaceous Fungal Keratitis: A Clinicopathologic Correlation. Cornea. 2004; 23:571-576.
17. Naiker S, Odhav B. Mycotic keratitis: profile of Fusarium species and their mycotoxins. Mycoses. 2004;47:50-6
18. Tsunawaki S, Yoshida LS, Nishida S, et al.Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase. Infect Immun. 2004;72:3373-82.
19. Yamada A, Kataoka T, Nagai K.The fungal metabolite gliotoxin: immunosuppressive activity on CTL-mediated cytotoxicity. Immunol Lett. 2000 Jan 10;71(l):27-32.
20. Thomas PA. Mycotic keratitis - an underestimated mycosis. J Med Vet Mycol 1994; 32: 235-56.