摘要
真菌性角膜炎是一种世界范围内广泛流行、较顽固、可致盲的真菌感染，常伴有角膜溃疡、化脓性感染及视力受损，严重影响患者的健康和生活质量。近年来，真菌性角膜炎发病率显著增加，其原因主要与植物性角膜外伤、慢性眼表性疾病、眼科手术及角膜移植有关，广谱抗生素、糖皮质激素的滥用及隐形眼镜的使用不当，及AIDS、糖尿病、系统性免疫缺陷病等免疫低下人群的增加也提高了真菌性角膜炎的发病率。
真菌性角膜炎发病隐匿，感染进展迅速，早期诊断困难。临床上，眼科常用抗真菌治疗药物较少，病原菌耐药现象严重，有效治疗困难。因此，了解真菌性角膜炎的常见临床表现、易感因素、高发年龄及多发季节，掌握常见病原真菌种类及其药物敏感性特点，建立快速、特异的鉴定技术，对该病的早期准确诊断、及时有效治疗、控制感染及预后恢复具有重要意义。
眼部感染常用的诊断方法包括直接涂片镜检、真菌培养、共聚焦显微镜成像、血清学诊断及组织病理学检查等。由于角膜刮片取材较困难、标本量少，真菌生长缓慢，导致检出率较低、耗时长，且易造成角膜损伤，难以满足临床的需求。
近年来，以核酸为基础的PCR及DNA测序技术逐步发展起来。因其反应迅速、敏感性强、特异性好，在临床检测方面应用前景广阔。目前，用于真菌性角膜炎的鉴定诊断及流行病学研究的PCR技术有巢式PCR、RFLP等。但由于尚缺乏合适的微量DNA的有效提取方法和重要病原真菌的特异性引物，使得临床检测仍多依赖于真菌培养。复合PCR技术作为一种新型PCR技术，因其可利用一个反应同时鉴定多种病原菌，且操作简便、反应迅速、检出率高，被广泛应用于微生物和寄生虫感染的诊断中。目前，国内外利用该技术诊断真菌性角膜炎的报道甚少。
本研究在掌握吉林省真菌性角膜炎病原学特点及流行病学规律的基础上，针对吉林省的优势病原菌设计种特异性引物，并结合改良的感染角膜组织DNA提取方法，利用复合PCR技术对临床患者角膜刮片标本中的病原菌进行了快速鉴定。
1.真菌性角膜炎病原学及流行病学分析
本研究对2004年10月至2011年12月，采集吉林大学附属医院眼科疑似真菌性角膜炎患者的角膜刮片标本225例，进行了KOH湿片法、真菌分离培养、鉴定、药物敏感性分析及流行病学调查。有208例患者确诊为真菌性角膜炎。共分离获得15属、28种、168株病原真菌。以镰刀菌属最多见，其次是曲霉属和假丝酵母属；优势病原菌种为茄病镰刀菌、烟曲霉及光滑假丝酵母。
统计结果显示，吉林省真菌性角膜炎在秋冬季发病率较高。患者来自吉林省各个地区，男女患者比例为1.5:1，平均年龄为50.1±12.5岁，农民占大多数，。角膜外伤是最常见的易感因素。半数以上的患者有局部抗生素、糖皮质激素、抗真菌药治疗史。药物敏感性试验表明，28株受试的茄病镰刀菌对纳他霉素（敏感性达100%）、伏立康唑及两性霉素B的敏感性较高，而对伊曲康唑不敏感；12株烟曲霉和10株光滑假丝酵母对伊曲康唑、伏立康唑及纳他霉素的敏感性均可达100%，而对两性霉素B的敏感性分别为50%和80%。提示纳他霉素可作为临床预防和治疗真菌性角膜炎的首选药物。
2.优势病原菌种的复合PCR鉴定技术
本研究利用真菌线粒体细胞色素b基因通用引物，扩增获得了真菌性角膜炎常见病原菌及环境真菌的DNA序列，同源性比对分析，寻找3种吉林省真菌性角膜炎优势病原菌种茄病镰刀菌、烟曲霉及光滑假丝酵母的特异性位点，设计了3对种特异性引物，建立并优化了复合PCR鉴定技术。茄病镰刀菌、烟曲霉及光滑假丝酵母可分别产生约320bp、270bp及230bp的特异性条带，其它种属真菌、常见病原细菌及正常角膜组织均未见扩增，表明设计的引物具有良好的种内通用性和种间特异性。该反应体系的敏感性可达到50pg级。利用该技术，对研究中一些形态学鉴定困难或鉴定有误的菌株进行了重新鉴定和分类。
3.真菌性角膜炎患者标本的快速检测
本研究利用角膜基质注射法，建立了豚鼠镰刀菌性角膜炎模型，经真菌培养和组织病理学检查验证，有90%建模成功。建立了改良的感染角膜组织DNA的提取方法。并利用该方法提取了42例疑似真菌性角膜炎患者的角膜刮片标本DNA，进行了PCR检测。PCR检测阳性率（88%）高于真菌培养法，具有统计学意义。利用建立的复合PCR技术对主要病原菌种进行了鉴定。与传统真菌培养（需要1~2周）相比，该非培养技术具有快速、特异性高的特点，可在4~8h内迅速判断有无真菌感染、单一或混合感染及何种真菌感染。
综上所述，本研究分析了吉林省真菌性角膜炎的病原学特点及流行病学规律，为临床的诊治积累了有用资料；建立了改良的豚鼠镰刀菌性角膜炎角膜刮片DNA提取方法，针对吉林省的优势病原菌种自主设计了3对种特异性引物，利用复合PCR技术对临床患者角膜刮片标本中的病原菌进行了快速鉴定，并证明其特异性高、敏感性好、检出率高，实现了真菌性角膜炎的非培养快速诊断，为临床推广应用奠定了理论基础和技术支撑。
Fungal keratitis is a world-wide, refractory and potentially blinding fungal infection,with corneal ulceration, suppurative infection and visual loss, which seriously affect thehealth and life of patients. In recent years, the incidence of fungal keratitis increasesignificantly in association with previous vegetative corneal injury, chronic ocularsurface disease, corneal surgery or transplantation, inadequate use of topical antibioticsand steroids or wearing contact lenses, host hypoimmunity with AIDS, diabetes andsystemic immunodeficiency diseases and so on.
Fungal keratitis is an insidious, rapidly progressive disease that is difficult diagnosis.Treatment remains limite by scarcity of effective antifungals and serious resistance withpathogenic fungi. Therefore, it is significant to understand the common clinicalmanifestations, risk factors, age and season of fungal keratitis; grasp the predominantpathogenic fungi and the characteristics of their drug sensitivity; establish a rapid,specific method to identify the pathogens of fungal keratitis. These would be useful forrapid accurately diagnosis, accelerating early antifungal therapy, control the infection,increasing the cure rate, and saving the diseased eye.
The commonly used diagnostic methods of ocular infection are direct smearexamination, fungal culture, in vivo confocal microscopy, serological approaches andhistopathological examination. These methods are difficult to meet needs of patientswith lower isolating rate, time-consuming and easily lead to corneal damage by thelimited corneal scraping size and slow growing of pathogenic fungi.
In recent years, the DNA-based PCR and DNA sequencing techniques developedgradually. It has broad prospects for application in clinical detection of fungal keratitisdue to theirs high sensitivity and specificity. PCR techniques for diagnostic andepidemiological studies of fungal keratitis include nested PCR, RFLP and others. Butthe clinical detection is still more dependent on fungal culture owing to the shortage ofeffective extraction method with microamounts of DNA from corneal scrapings andspecific primers of important pathogenic fungi. Recently, multiplex PCR as a novelPCR technique has been wildly used in the diagnosis of microbial and parasitic infections, because it can detect a variety of pathogens at the same time and is a simple,rapid method with high isolating rate. It has rarely been reported using this method todiagnose fungal keratitis currently.
In this study, we designed the species-specific primers of the predominant pathogensbased on comprehending the etiological and epidemiological characteristics of fungalkeratitis in Jilin Province, combined with improved DNA extraction method ofinfectious corneal tissue, we used multiplex PCR to rapid identify the pathogens withcorneal scrapings of clinical patients.
1. Etiological and epidemiological analysis of fungal keratitis
The present retrospective study of225patients with suspected fungal keratitis wasconducted over a period from October2004to December2011in Jilin Province.Isolation, identification and drug sensitivity test of the pathogens, and analysis ofepidemiological characteristics was performed.208cases were diagnosed with fungalkeratitis by KOH wet mount and fungal culture. Fifteen genera,28species and168strains were isolated. Strains of the genus Fusarium were isolated most frequently,followed by Aspergillus and Candida. And F. solani, A. fumigatus and C. glabrata wasthe predominant causative pathogen caused fungal keratitis.
The study showed that the incidence fungal keratitis in Jilin Province was increasedduring autumn and winter. The proportion of male and female patients was1.5:1. Mostof the patients were farmers. The average age was50.1±12.5years. Corneal trauma wasthe most common predisposing factors. More than a half of patients were previouslytreated topically with antibiotics, antifungals and steroids. The drug sensitivity testshowed that28isolates of F. solani were sensitive to Natamycin (sensitivity100%),Voriconazole and Amphotericin B, but resistance to Itraconazole.12isolates of A.fumigatus and10isolates of C. glabrata were sensitive to the four antifungalsmentioned above. This indicated Natamycin might be first choice in prevention andtreatment of fungal keratitis in clinic.
2. Multiplex PCR of the predominant pathogenic fungi
DNA sequence of pathogenic and environmental fungi was amplified by using theuniversal primers of mitochondrial cytochrome b gene. Homology alignment analysis ofDNA sequence was perfromed to design three pairs of species-specific primers of thepredominant pathogen of fungal keratitis, and established and optimized multiplex PCR.F. solani, A. fumigatus and C. glabrata could be amplified with the specific fragment of ~320bp,270bp and230bp, individually, whereas other fungi, bacteria and normalcornea could not be amplified, which showed the designed primers had goodintraspecific universal and interspecific specificity. And the sensitivity of reactionsystem was50pg. With this method, we reclassified the isolates which were difficult toidentification with morphological characters.
3. Rapid detection of patients with fungal keratitis
Fusarium keratitis in guinea pig model was established by using corneal stromalinjection method, and was confirmed by fungal culture and histopathologicalexamination with the achievement ratio was90%. We established the extraction methodof infectious corneal tissue DNA with fungal keratitis. Using the improved DNAextraction and PCR with the universal fungal primers, corneal scraping specimens of42patients with suspected fungal keratitis was detected. The positive rate of PCR (88%)was higher than the fungal culture method, which had statistically significant. Andmultiplex PCR with species-specific primers was used to rapid and specific identify thepredominant pathogens. Conventional fungal culture often took1~2weeks to obtainthe positive identification. While this culture-independent method could rapidly andspecifically diagnose only in4~8hours, with or without fungal infection, single ormixed infection, and infected by which species of fungus.
In summary, we analyzed the etiological and epidemiological characteristics of fungalkeratitis in Jilin Province. These informations could be useful for clinical diagnosis andtreatment. And we established the improved DNA extraction method by using Fusariumkeratitis in guinea pig model, designed three pairs of species-specific primers of thepredominant pathogens in Jilin Province, and used multiplex PCR to rapid identify thepathogens in corneal scrapings of clinical patients. It comfirmed this method was a rapid,culture independent diagnostic technique with high specificity and sensitivity. These canbe the foundation for widespread application to the clinic.
引文
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