荧光染色在鼻窦真菌快速诊断中的应用评价

Application and evaluation of fluorescent staining in rapid diagnosis of sinus fungus

目的评价荧光染色在快速诊断真菌性鼻-鼻窦炎中的应用效果。方法对96例2017年7月至2018年4月首都医科大学附属北京同仁医院临床诊断为真菌性鼻-鼻窦炎的患者术中留取少量真菌团块组织,拉片制成薄层涂片,滴适量荧光染料,加盖玻片,室温放置5 min,在荧光显微镜下从低倍至高倍观察真菌结构。常规进行乳酸酚棉蓝染色及1.79 mol/L KOH湿片处理,以真菌培养结果作为金标准。真菌培养阴性、镜检阳性的样本提取核酸通过ITS序列扩增,进行真菌成分的分子检测。结果荧光染色、乳酸酚棉蓝染色、1.79 mol/L KOH湿片标本一次镜检阳性率分别为95.83%(92/96),93.75%(90/96)及91.67%(88/96),差异无统计学意义;涂片阴性标本再次涂片染色镜检,除1.79 mol/L KOH漏检2例外,荧光及乳酸酚棉蓝染色均可捕捉到真菌结构。培养阳性的65例标本,荧光着色理想,可见清晰的真菌结构,尤其对曲霉菌属与暗色真菌属菌丝区分良好,直接荧光染色拟诊与培养结果一致。有3例经乳酸酚棉蓝染色及1.79 mol/L KOH湿片观察到特征性顶囊结构及分生孢子判断为烟曲霉及链格孢属,结果与培养一致,而荧光染色此类结构着色不清。31例镜检阳性、培养阴性的样本分子检测结果显示,黄曲霉9例、烟曲霉3例、杂色曲霉1例、产黄青霉1例、链格孢霉属2例、尖端赛多孢1例、近平滑假丝酵母菌1例,13例样本为阴性结果。结论荧光染色法可用于鼻窦真菌的快速拟诊,但在观察真菌特征性产孢及产色素结构上,与常规方法相比并无显著优势。

Objective To evaluate the application of fluorescence staining in the rapid diagnosis of fungal rhino-sinusitis. Methods Ninety six samples from the patients with suspected fungal rhino-sinusitis in Beijing Tongren Hospital affiliated to Capital Medical University were collected. A small amount of the mildew blocks from each patient was taken to make a thin layer of smear. Moderate fluorescent dye was dripped on slips and covered to perform fluorescence staining. The slide was placed at room temperature for 5 minutes.The mycelium and spore were observed under fluorescence microscope. The results were compared to those of culture and conventional direct examination which was wet slides treated with 1.79 mol/L KOH or stained with lactate gossypol. The results of fungal culture were regarded as the gold standard for diagnosis. The nucleic acids were extracted from the samples which were positive results of examination under the microscope while negative in culturing,and were amplified by ITS sequence to perform molecular detection of fungal components. Results The positive rates of fluorescence staining,lactate gossypol staining and 1.79 mol/L KOH wet slide method were95.83%（ 92/96）,93.75%（ 90/96） and 91.67%（ 88/96） for the first time,respectively,and the differences were not statistically significant. The negative samples of smear slide were smeared again and stained for microscopic examination. The fungal structure of mildew blocks could be captured by fluorescence and lactate gossypol staining either except for 2 cases missed in 1. 79 mol/L KOH method. The 65 positive specimens in culturing showed satisfactory fluorescent staining especially to distinguish the hypha of Aspergillus and demalaceous fungi with clearly visible fungal structure. The results of direct fluorescence staining were consistent with those of the culture. The characteristic apical cyst structure of Aspergillus fumigatus and conidia of Alternaria in three case were observed under the microscope by lactate gossypol staining and 1.79 mol/L KOH wet slide method,and the results were consistent with those of culture,but fluorescent staining could not distinguish the colors of such structures. The molecular detection results for 31 positive cases under the microscope while negative in culturing showed that 9 belonged to Aspergillus flavus,3 to Aspergillus fumigatus,1 to Aspergillus versicolor,1 to Penicillium chrysogenum,2 to Alternaria,1 to Scedosporium apiospermum,1 to Candida parapsilosis,and 13 were negative.Conclusion Fluorescence staining can be used for rapid diagnosis of nasal sinus fungi,but there should be no significant advantages compared with conventional methods in observation of characteristic structures of fungal sporulation and pigment.