深圳市1例黑热病病例实验室检测与分子鉴定  

Laboratory detection and molecular identification of a kala-azar case in Shenzhen

作　　者：黄达娜[1] 刘小莲 高世同[1] 李媛[1] 唐屹君[1] 张倩[1] 彭博[1] 阳帆[1] 牛丛 张仁利[1] HUANG Dana;LIU Xiaolian;GAO Shitong;LI yuan;TANG Yijun;ZHANG Qian;PENG Bo;YANG Fan;NIU Cong;ZHANG Renli(Shenzhen Center for Disease Control and Prevention,Shenzhen,Guangdong 518055,China)

机构地区：[1]深圳市疾病预防控制中心,广东深圳518055

出　　处：《中国热带医学》2024年第2期218-222,共5页China Tropical Medicine

基　　金：深圳市“新冠肺炎特异性抗病毒治疗性药物的研发”应急科研攻关项目(No.JSGG20200807171801013);清华大学医学院程功教授重大媒介传染病防控研究创新团队(No.SZSM202211023)。

摘　　要：目的对深圳市1例输入性黑热病病例进行实验室检测和分子溯源分析以确定感染虫株。方法收集2023年3月15日深圳市确诊1例黑热病病例的骨髓穿刺液和血液进行实验室检测。对患者骨髓穿刺液涂片姬姆萨染色后进行显微镜检查,对血液样品采用内脏利什曼原虫快速诊断试剂(rk39)进行血清抗体检查,并提取全血DNA,PCR扩增内转录间隔区Ⅰ(internal transcribed spacer-1,ITS-1)序列并测序比对,同时基于ITS-1序列构建系统进化树。结果对患者骨髓涂片显微镜检查查见大量利什曼原虫无鞭毛体,确诊为黑热病,患者血液采用rk39快速诊断试剂检测结果呈阳性,PCR扩增出ITS-1基因产物序列与预期大小一致,经NCBI数据库中比对,与婴儿利什曼原虫ITS-1基因序列相似度为100%,确定感染虫株为婴儿利什曼原虫。对扩增的ITS-1序列进行系统发育树构建发现与婴儿利什曼原虫聚到一个分支,且与所选的参比序列中的KC347299距离较近。结论深圳市1例黑热病病例是由婴儿利什曼原虫引起的,黑热病在我国仍时有发生,应加强非疫区医务人员诊断技术,积极配合使用新的诊断技术进行辅助诊断,同时应提高对利什曼原虫的防控能力。Objective To perform laboratory detection and molecular traceability analysis on a case of imported kalaazar in Shenzhen to determine the infection strain.Methods Bone marrow puncture fluid and blood samples from a case of kala-azar in Shenzhen were collected for laboratory tests.The patient's bone marrow puncture fluid smears were stained with Giemsa and examined under a microscope.Blood samples were examined for antibodies using the rk39 visceral leishmania rapid diagnostic reagent.Whole blood DNA was extracted,and the ITS-1 sequence was amplified by PCR,sequenced and aligned,and a phylogenetic tree was constructed based on the ITS-1 sequence.Results Microscopic examination of the patient's bone marrow smears revealed a large number of Leishmania amastigotes without flagella,confirming the diagnosis of kala-azar.The patient's blood was tested positive with the rk39 rapid diagnostic reagent,and PCR amplification yielded an ITS-1 gene product sequence that matched the expected size.Sequence alignment with the NCBI database showed 100%sequence similarity with the ITS-1 gene sequence of Leishmania infantum,confirming the infecting strain as Leishmania infantum.Phylogenetic tree construction of the amplified ITS-1 sequence revealed clustering into a clade with Leishmania infantum,and close to KC347299,one of the reference sequence selected.Conclusions The case of kala-azar in Shenzhen was caused by Leishmania infantum.Kala-azar still occurs in China,so the diagnostic technology of medical personnel in nonepidemic areas should be strengthened so that they can actively use new diagnostic technologies to assist in diagnosis,thus improving their prevention and control ability of Leishmania parasites.

关 键 词：黑热病 利什曼原虫 实验室检测 基因溯源 

分 类 号：R531.6[医药卫生—内科学]