改良瑞氏-吉氏染色法检测人芽囊原虫的建立与评价

doi:10.12140/j.issn.1000-7423.2025.05.021

摘要/Abstract

摘要：

收集2021年4月至2024年12月于中山大学附属第八医院就诊患者的粪样，将经PCR基因鉴定检测确认的人芽囊原虫感染阳性者131例、阴性者50例按照数字表法随机分为实验组（含阳性92例、阴性35例）和验证组（含阳性39例、阴性15例）。实验组每份粪样制成悬液，分别吸取50、100、150 μl制成薄涂片，相比常规瑞氏-吉氏染色法增加干燥步骤，分别于25、30、35、40、45、50、55、60、65、70、75、80 ℃干燥，瑞氏-吉氏染色5~10 min，置光学显微镜下观察鉴定。以人芽囊原虫特异性引物BhRDr/RD5的PCR扩增及基因分型结果为参考标准，评价改良瑞氏-吉氏染色法的诊断效能。验证组用改良瑞氏-吉氏染色法和PCR法同时进行人芽囊原虫检测，以灵敏度、特异度、约登指数、预测值（阳性/阴性）、kappa一致性系数和符合率验证改良方法的诊断效能。结果显示，筛选最佳粪样涂片用量为50 μl，最适干片温度为（70 ± 5）℃。PCR扩增在约600 bp处出现特异条带，基因分型以ST3为主，其他依次为ST7、̖̖ST1、ST6、ST2。实验组中，改良瑞氏-吉氏染色法的灵敏度为98.9%（95% CI：94.0~100.0），特异度为97.1%（95% CI: 85.3~99.9），kappa系数为0.961（95% CI：0.906~1.000），检出符合率为98.4%（125/127），与PCR法高度一致。验证组中，McNemar精确检验比较两种方法的检测结果，差异无统计学意义（P > 0.05）。改良瑞氏-吉氏染色法的灵敏度为97.4%（95% CI：86.2~99.9），特异度为86.7%（95% CI：59.5~98.3），阳性预测值为95.0%（95% CI：83.8~99.5），阴性预测值为92.9%（95% CI：69.8~99.8），约登指数为0.841（95% CI：0.662~1.000），kappa系数为0.858（95% CI：0.703~1.000），检出符合率为94.4%（51/54）。本研究建立的改良瑞氏-吉氏染色法操作简便、结果稳定，与PCR法高度一致，适用于基层医疗机构人芽囊原虫快速筛查及现场流行病学调查。

关键词: 人芽囊原虫, 改良瑞氏-吉氏染色法, 诊断效能

Abstract:

Fecal samples were collected from patients admitted to The Eighth Affiliated Hospital of Sun Yat-sen University from April 2021 to December 2024, and a total of 131 cases tested positive for Blastocystis hominis infections using PCR assay and 50 cases tested negative were randomly divided into the experimental group (92 cases tested positive, 35 cases tested negative) and validation group (39 cases tested positive, 15 cases tested negative) using a random number table. Each fecal sample of experimental group was prepared into suspensions, and 50, 100, and 150 μl of suspensions were sampled for preparation of thin smears. Compared to conventional Wright-Giemsa staining, which involves an additional drying step, they dried at 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 ℃. The smears were stained with Wright-Giemsa stains for 5 to 10 minutes and then observed under an optical microscope. The diagnostic performance of the modified Wright-Giemsa staining method was evaluated using PCR amplification and genotyping results with B. hominis-specific primers BhRDr/RD5 as a reference standard. The validation group used both the modified Wright-Giemsa staining method and PCR method for simultaneous detection of B. hominis. The sensitivity, specificity, Youden index, predictive values (positive/negative), kappa coefficient, and concordance rate were as reference standards to evaluate the diagnostic performance of the modified method. The optimal fecal smear volume was 50 μl, and the most suitable drying temperature was (70 ± 5) ℃. PCR amplification produced a specific band at approximately 600 bp, with ST3 as the predominant genotype, followed by ST7, ST1, ST6, and ST2. The diagnostic sensitivity, specificity, kappa coefficient and concordance rate of the modified Wright-Giemsa staining method were 98.9% (95% CI: 94.0% to 100.0%), 97.1% (95% CI: 85.3% to 99.9%), 0.961 (95% CI: 0.906 to 1.000), and 98.4% (125/127) in the experimental group, showing high consistency with PCR assay. McNemar’s test was used to compare the differences in detection results between the two methods in the validation group, and the differences were not statistically significant (P > 0.05). The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, Youden index, kappa coefficient and concordance rate of the modified Wright-Giemsa staining method were 97.4% (95% CI: 86.2% to 99.9%), 86.7% (95% CI: 59.5% to 98.3%), 95.0% (95% CI: 83.8% to 99.5%), 92.9% (95% CI: 69.8% to 99.8%), 0.841 (95% CI: 0.662 to 1.000), 0.858 (95% CI: 0.703 to 1.000), and 94.4% (51/54), respectively. The modified Wright-Giemsa staining method established in this study is simple to operate, yields stable results, and shows high consistency with PCR assay, which is suitable for rapid screening of B. hominis in grassroots medical institutions and field epidemiological investigations.

Key words: Blastocystis hominis, Modified Wright-Giemsa staining, Diagnostic performance

中图分类号:

R382

刘小晴, 邹小红, 王慧婷, 周晨, 李振华, 石亮. 改良瑞氏-吉氏染色法检测人芽囊原虫的建立与评价[J]. 中国寄生虫学与寄生虫病杂志, 2025, 43(5): 731-735.

LIU Xiaoqing, ZOU Xiaohong, WANG Huiting, ZHOU Chen, LI Zhenhua, SHI Liang. Establishment and evaluation of modified Wright-Giemsa staining for detection of Blastocystis hominis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2025, 43(5): 731-735.

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