一种高灵敏度巢式聚合酶链反应的开发及其在斯里兰卡皮肤利什曼病诊断中的应用。
Development of a Highly Sensitive Nested PCR and Its Application for the Diagnosis of Cutaneous Leishmaniasis in Sri Lanka.
作者信息
De Silva Nirmitha Lalindi, De Silva Viraji Nefertiti Hiromel, Deerasinghe Arachchige Theja Hemapala, Rathnapala Upeksha Lakmini, Itoh Makoto, Takagi Hidekazu, Weerasooriya Mirani Vasanthamala, Kato Hirotomo, Yahathugoda Thishan Channa
机构信息
Department of Parasitology, Faculty of Medicine, University of Ruhuna, Galle 80000, Sri Lanka.
Base Hospital Tangalle, Tangalle 82200, Sri Lanka.
出版信息
Microorganisms. 2022 May 9;10(5):990. doi: 10.3390/microorganisms10050990.
The recent surge in cutaneous leishmaniasis (CL) in Sri Lanka has rendered clinical diagnosis difficult; thus, laboratory confirmation is indispensable. A modified (two novel inner primers to detect CL caused by Leishmania donovani) nested Internal Transcribed Spacer-1 (ITS1) PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed and tested. The sensitivity of the modified nested PCR was tested using serial dilutions (103 to 10−2) of the DNA extract of a cultured L. donovani DD8 strain. Patients (n = 194) from Southern Sri Lanka were examined clinically, microscopically (Slit Skin Smear-SSS) and using the modified nested PCR. The modified nested PCR detected 2.55 fg of parasite DNA compared to ITS1 PCR (25 fg) and detected more cases than SSS (94.3% vs. 77.3%; p < 0.01). The RFLP pattern was L. donovani in all cases. The modified nested PCR performed well in clinically doubtful lesions (95% by PCR vs. 60% by SSS; p < 0.01), ulcerated nodules (91% vs. 71.8%; p < 0.01) and plaques (100% vs. 66.7%; p < 0.01). SSS demonstrated sensitivity (80.9%), specificity (81.8%), PPV (98.7%) and NPV (20.5%) against modified PCR. Low parasite loads and atypical lesions can be diagnosed by the proposed method with higher accuracy.
最近，斯里兰卡皮肤利什曼病（CL）病例激增，导致临床诊断困难，因此实验室确诊必不可少。我们开发并测试了一种改良的巢式内转录间隔区1（ITS1）聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法（使用两种新型内部引物检测杜氏利什曼原虫引起的CL）。使用培养的杜氏利什曼原虫DD8株DNA提取物的系列稀释液（10³至10⁻²）测试改良巢式PCR的灵敏度。对来自斯里兰卡南部的194名患者进行了临床、显微镜检查（ slit skin smear-SSS）以及改良巢式PCR检测。与ITS1 PCR（25 fg）相比，改良巢式PCR可检测到2.55 fg的寄生虫DNA，且检测出的病例数比SSS更多（94.3%对77.3%；p<0.01）。所有病例的RFLP模式均为杜氏利什曼原虫。改良巢式PCR在临床可疑病变（PCR检测为95%，SSS检测为60%；p<0.01）、溃疡结节（91%对71.8%；p<0.01）和斑块（100%对66.7%；p<0.01）中表现良好。SSS针对改良PCR的灵敏度为80.9%，特异性为81.8%，阳性预测值为98.7%，阴性预测值为20.5%。所提出的方法能够更准确地诊断低寄生虫载量和非典型病变。
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