荧光染色与过碘酸希夫染色对真菌性角膜炎诊断效果的比较

目的比较荧光染色和过碘酸希夫染色对真菌性角膜炎(FK)的诊断效果。方法收集2017年1月至2019年5月于山东第一医科大学附属眼科医院就诊且角膜刮片或真菌培养阳性的FK患者147例角膜标本147份,其中行穿透角膜移植术(PKP)患者84例,板层角膜移植术(LKP)患者42例,病灶切除患者21例;选取11例单纯疱疹病毒性角膜炎活检组织作为阴性对照。对角膜组织标本分别行真菌荧光染色和过碘酸希夫染色。将染色完成的切片分别置于荧光显微镜和光学显微镜下查找真菌菌丝或孢子。比较2种染色方法的阳性率、不同手术方式获得的FK角膜组织以及不同真菌菌株标本间2种染色方法的阳性例数。结果过碘酸希夫染色的阳性率为60.5%(89/147),真菌荧光染色的阳性率为79.6%(117/147),荧光染色法诊断FK的阳性率明显高于过碘酸希夫染色法,差异有统计学意义(χ2=28.00,P<0.01),2种染色方法的特异性均为100%。PKP手术切除标本荧光染色的阳性率为85.7%(72/84),明显高于过碘酸希夫染色的阳性率65.5%(55/84),差异有统计学意义(χ2=17.00,P<0.01);LKP手术切除标本荧光染色的阳性率为71.4%(30/42),明显高于过碘酸希夫染色的52.4%(22/42),差异有统计学意义(χ2=8.00,P<0.01);病灶切除标本分别采用荧光染色和过碘酸希夫染色法进行检查,阳性率分别为71.4%(15/21)和57.1%(12/21),差异无统计学意义(χ2=1.30,P=0.25)。不同真菌菌株标本间2种染色的阳性例数不同,其中茄病镰刀菌复合群、谲诈腐霉菌、烟曲霉复合群、季也蒙假丝酵母、木霉和铺叶沼兰褐莺真菌行荧光染色的阳性例数分别为19、5、5、1、1和1例,行过碘酸希夫染色的阳性例数分别为11、0、3、0、0和0例。11例阴性对照均为阴性结果。结论荧光染色技术应用于石蜡包埋角膜组织检查真菌成分较过碘酸希夫染色法敏感性高,可显著提高真菌检测的阳性率。

Comparison of fluorescence staining and periodic acid-Schiff staining in the diagnosis of fungal keratitis

Objective To compare the effect of fluorescence staining and periodic acid-Schiff staining in the diagnosis of fungal keratitis(FK).Methods A total of 147 corneal specimens from 147 FK patients treated in Eye Hospital of Shandong First Medical University from January 2017 to May 2019 with positive corneal scraping or fungal culture were collected.Among them,there were 84 cases with penetrating keratoplasty(PKP),42 cases with lamellar keratoplasty(LKP)and 21 cases with lesion resection.Another 11 cases with herpes simplex virus keratitis served as negative control.The corneal tissue specimens were performed with fungal fluorescence staining and periodic acid-Schiff staining,respectively.The stained sections were placed under fluorescence microscope and optical microscope to observe fungal hyphae or spores,respectively.The positive rates of the two staining methods were compared,and the positive cases of the diagnosis of FK in corneal tissue samples obtained by different surgical methods and corneal infection caused by different strains of the two staining methods were compared.Written informed consent was obtained from each patient.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital(No.SDSYKYY-2016012).Results The positive rate of periodic acid-Schiff staining and fungal fluorescence staining was 60.5%(89/147)and 79.6%(117/147),respectively.The positive rate of fluorescence staining in the diagnosis of fungal keratitis was significantly higher than that of periodic acid-Schiff staining(χ2=28.00,P<0.01),and both the specificity of the two staining methods was 100%.The positive rate of specimens obtained by PKP with fluorescent staining was 85.7%(72/84),and the positive rate with periodic acid-Schiff staining was 65.5%(55/84),and the difference was statistically significant(χ2=17.00,P<0.01).The positive rate of specimens obtained by LKP with fluorescent staining was 71.4%(30/42),and the positive rate with periodic acid-Schiff staining was 52.4%(22/42),and the difference was statistically significant(χ2=8.00,P<0.01).The positive rate of resected foci specimens with fluorescent staining was 71.4%(15/21),and the positive rate with periodic acid-Schiff staining was 57.1%(12/21),and the difference was not statistically significant(χ2=1.30,P=0.25).The positive cases of two kinds of staining were different among different fungal strains.Among them,the positive cases of Fusarium solani complex,Pythium insidiosum,Aspergillus fumigatus complex,Candida guilliermondii,Trichoderma and Nigrograna mackinnonii with fluorescence staining were 19,5,5,1,1 and 1,and the positive cases of periodic acid-Schiff staining were 11,0,3,0,0 and 0,respectively.The staining results of the 11 negative controls were negative.Conclusions Fluorescence staining is more sensitive than periodic acid-Schiff staining in the detection of fungal components in paraffin-embedded corneal tissues,and it can significantly improve the fungal detection rates.