目的：将荧光染色技术应用于组织病理诊断棘阿米巴性角膜炎(AK)，并将阅片结果与HE染色和PAS染色的阅片结果进行比较，分析这3种染色方法的敏感性与特异性，探究荧光染色技术在角膜组织活检中检测阿米巴病原体的应用价值。方法：回顾性研究。收集2015-05/2020-06于山东第一医科大学附属眼科医院就诊的感染性角膜炎患者的手术切除标本74例75眼。根据角膜刮片、培养和组织病理诊断结果，将感染性角膜标本分为AK组和非棘阿米巴性角膜炎(NAK)组。将连续切片的组织分别行HE染色、PAS染色和荧光染色，分析3种染色方法对AK诊断的敏感性与特异性，使用受试者工作特征(ROC)曲线计算曲线下面积(AUC)，并进一步分析在同一部位相同倍率视野下，计数三种染色方法找到棘阿米巴病原体的数量，明确荧光染色法对AK的诊断价值。结果：HE染色的敏感性为69%(27/39)、特异性为92%，PAS染色的敏感性为62%(24/39)、特异性为97%，荧光染色的敏感性为95%(37/39)、特异性为97%，3种染色方法对AK诊断的敏感性存在差异(χ2=19.857，P<0.001)； 进行两两比较发现，HE染色、PAS染色与荧光染色诊断AK的敏感性有差异(P=0.003、<0.001)，HE染色与PAS染色诊断AK的敏感性没有差异(P=0.978)； AUC最大为荧光染色(0.960)，其次分别为HE染色(0.804)，PAS染色(0.794)。计数在同一部位相同倍率视野下，HE染色、PAS染色和荧光染色查见棘阿米巴包囊数量的中位数分别为4(0，11)、2(0，9)、12(3，33)个(χ2=56.561，P<0.001)； 进行两两比较发现，HE染色和荧光染色、PAS染色和荧光染色找到棘阿米巴包囊的数量有差异(P<0.001)，HE和PAS两种染色方法找到棘阿米巴包囊的数量没有差异(P=0.210)，荧光染色的组织病理切片更容易辨别阿米巴病原体。结论：荧光染色技术应用于组织病理诊断AK的敏感性比HE染色和PAS染色高，可显著提高阿米巴病原体检测的阳性率。

AIM:To investigate the application value of fluorescent staining technique in the detection of amoebic pathogens in corneal tissue biopsy, and to apply fluorescent staining technique in the histopathological diagnosis of Acanthamoeba keratitis(AK), comparing the results with those of hemotoxyiln-eosin staining(HE staining)and periodic acid-schiff staining(PAS staining), and analyzing the sensitivity and specificity of these three staining methods.METHODS:Specimens of infected corneal tissue were collected from 74 cases(75 eyes), and then they were divided into an AK group and a non-Acanthamoeba keratitis(NAK)group based on the results of corneal scraping, culture and histopathological diagnosis. The tissues of consecutive sections were stained with HE staining, PAS staining and fluorescence respectively, and the sensitivity and specificity of the three staining methods for the diagnosis of AK were analyzed. Area under the curve(AUC)was calculated using the receiver operating characteristic(ROC)curve. Further analysis was performed to count the number of Acanthamoeba pathogens found by the three staining methods under the same magnification field of view at the same site, and to clarify the diagnostic value of fluorescent staining technique for AK.RESULTS: The sensitivity of HE staining was 69%(27/39)with a specificity of 92%; the sensitivity of PAS staining was 62%(24/39)with a specificity of 97%, and the sensitivity of fluorescent staining was 95%(37/39)with a specificity of 97%. There were differences in the sensitivity of the three staining methods for the diagnosis of AK(χ2=19.857, P<0.001), and pairwise comparison revealed that the differences between HE staining and fluorescent staining, PAS staining and fluorescent staining for the diagnosis of AK were statistically significant(P=0.003,<0.001), while the difference in sensitivity between HE staining and PAS staining for the diagnosis of AK was not statistically significant(P=0.978). The maximum AUC was 0.960 for fluorescence staining, followed by 0.804 for HE staining and 0.794 for PAS staining, respectively. The median number of amoeba cysts detected by HE staining, PAS staining and fluorescent staining at the same site under the same magnification field of view was 4(0, 11), 2(0, 9)and 12(3, 33), respectively(χ2=56.561, P<0.001). Pairwise comparison revealed that the differences in the number of amoeba cysts found by HE staining and fluorescence staining, PAS staining and fluorescence staining were statistically significant(P<0.001), while the difference in the number of amoeba cysts found by HE staining and PAS staining was not statistically significant(P=0.210). Fluorescently stained histopathological sections make it easier to identify amoebic pathogens.CONCLUSION:Fluorescent staining technique is more sensitive to histopathological diagnosis of AK than HE staining and PAS staining, which can significantly improve the positive rate of detection of amoebic pathogens.

国家自然科学基金项目(No.81900907)