涂片真菌荧光染色法对真菌性角膜炎诊断价值的研究

摘要

目的 分析真菌荧光染色技术在真菌性角膜炎诊断中的敏感性及特异性,探讨其临床应用价值.方法 前瞻性病例对照研究.连续收集2017年8月至2018年4月就诊于首都医科大学附属北京同仁医院确诊为感染性角膜炎的患者105例(105只眼),其中男性69例,女性36例,年龄(45.1±20.1)岁.根据裂隙灯显微镜、角膜激光共聚焦显微镜(IVCM)检查,以及病灶处角膜刮片吉姆萨染色、荧光染色和病原学培养的结果,将感染性角膜炎患者分为真菌性角膜炎(FK)组以及非真菌性角膜炎(NFK)组,分析上述检查方法对真菌性角膜炎诊断的敏感性和特异性,并通过受试者工作特征(ROC)曲线计算曲线下面积(AUC),明确真菌荧光染色方法对真菌性角膜炎的诊断价值.结果 105例感染性角膜炎患者中,真菌性角膜炎66例,非真菌性角膜炎39例(其中细菌性角膜炎29例,阿米巴角膜炎10例).FK组真菌培养阳性者46例(69.7％),其中以镰刀菌感染为主(20/46,43.5％),其次为链隔孢属(10/46,21.7％)以及曲霉属(9/46,19.6％)等.真菌涂片荧光染色后,组织背景呈均质的黑色或弱蓝色荧光,真菌细胞壁成分呈亮蓝紫色或蓝色的明亮荧光,形态、结构、菌丝密度等均容易识别.不同检查方法对角膜真菌感染诊断的敏感性分别为涂片荧光染色(97.0％)、IVCM(87.9％)、吉姆萨染色(86.7％)、真菌培养(69.7％);特异性方面,真菌培养最高(100.0％),其次为IVCM和吉姆萨染色(94.9％)和荧光染色(87.2％).AUC值由低至高依次为真菌培养(0.848),涂片吉姆萨染色(0.906),IVCM(0.914)和涂片荧光染色(0.921).结论 真菌荧光染色是一种快速、敏感的真菌镜检筛查手段,对真菌性角膜炎辅助诊断的敏感性及特异性较高,尤其适用于菌量少及抗真菌治疗后患者的诊断.%Objective To analyze the sensitivity and specificity of fungal fluorescent staining in the diagnosis of fungal keratitis,and to compare it with conventional fungal culture,in vivo confocal microscopy (IVCM) and Giemsa staining.To explore its value of clinical application.Methods Prospective case-control study.A total of 105 consecutive patients (105 eyes) diagnosed with infectious keratitis at Beijing Tongren Hospital from August 2017 to April 2018 were included.Patients with infectious keratitis were divided into fungal keratitis (FK) group and non-fungal keratitis (NFK) group by slit lamp microscopy,corneal in vivo confocal microscopy (IVCM) examination,and the results of Giemsa staining,fluorescent staining and pathogenic culture of corneal scraping from ulcer.The sensitivity and specificity of the above-mentioned examination methods for the diagnosis of fungal keratitis were analyzed.The receiver operating characteristic curve (ROC curve) and Area Under Curve (AUC) values were calculated to determine the diagnostic value of fungal fluorescent staining for fungal keratitis.Results Among the 105 patients with infectious keratitis,66 were fungal keratitis,39 were non-fungal keratitis (29 cases of bacterial keratitis and 10 cases of acanthamoeba keratitis).Isolation from fungal keratitis were mainly Fusarium spp.(43.5％),followed by Alternaria spp.(21.7％) and Aspergillus spp.(19.6％).After fluorescent staining of the ulcer smear,the background of tissue demonstrated homogeneous black or weak blue fluorescence.The cell wall of fungi showed bright blue-violet to blue fluorescence,and the morphology,structure and hyphal density were easily recognized.The sensitivity of different methods for the diagnosis of corneal fungal infection were smear fluorescence staining (97.0％),IVCM(87.9％),Giemsa staining (86.7％),and fungal culture (69.7％);the specificity of fungal culture was the highest (100％),followed by IVCM and Giemsa staining (94.9％),and fluorescent staining (87.2％).The ascending order of AUC values was:fungal culture (0.848) ＜Giemsa staining (0.906)＜IVCM (0.914)＜fluorescence staining (0.921).Conclusion Fungal fluorescent staining is a rapid and sensitive screening method under microscope with high sensitivity and specificity for the diagnosis of fungal keratitis.It is especially suitable for the diagnosis of patients with low load of hypha or after antifungal therapy.