云南省卵形疟原虫的实验室诊断研究

Laboratory diagnosis of Plasmodium ovale in Yunnan Province

目的通过实验室综合诊断一例卵形疟原虫感染,为云南省鉴别当地罕见疟原虫虫种提供借鉴。方法对一例尼日利亚务工回国疟疾病例采血,制作厚、薄血涂片,经Giemsa染色后在油镜下观察薄血膜疟原虫形态,鉴定虫种。针对P.v、P.f、P.m、P.o 4种疟原虫的18SrRNA基因进行巢式PCR扩增并测序,在GenBank分别与P.f的M19172和P.o的KC866363、HQ697267、KF192072、KF192073、KJ871672、AF145337、KC633223、L48987参比虫株进行Blast比对,应用MEGA5.1软件进行DNA序列同源性比较并绘制系统进化树。结果显微镜下可观察到成熟度各异的卵形疟原虫滋养体和裂殖体,薄血膜中被寄生的红细胞大小正常或稍胀大,变形,边缘呈伞矢状或锯齿状,并有凹凸、拖尾、毛刺等卵形疟特征性形态,经巢式PCR扩增出约800bp的卵形疟特异性基因片段,经Blast比对与GenBank中已知卵形疟部分参比虫株18SrRNA的同源性为89%～91%。结论经镜检、巢式PCR、测序及同源性分析,基本证实该例自尼日利亚务工回国的疟疾病例为卵形疟原虫感染。

Objective To provide a reference for identification of rare infections with Plasmodium ovale infection through comprehensive laboratory diagnosis. Methods One venous blood sample was collected from a patient who was infected with P. ovale while in Nigeria. The Plasrnodiurn species was identified using a Giemsa stained smear under a microscope with a magnification of 100x. DNA of the 18S rRNA Plasrnodium gene was amplified with nested PCR using ge nus and species-specific primers and the PCR product was then sequenced. Pairwise nucleotide differences and the genetic distance between sequences were calculated using MEGAS. 1. A phylogenetic tree was constructed using the neighbor joining method and MEGA5. 1. Reference sequences from GenBank included P. falciparum （M19172） and P. ovale （KC866363, HQ697267, KF192072, KF192073, KJ871672, AF145337, KC633223, andL48987）. Results Different stages of developing trophozoites and schizonts were observed microscopically. Infected erythrocytes were normal or slightly enlarged in size, they had an irregular shape, and their edges were pointed or jagged. Nested PCR amplified a gene fragment specific to P. ovale 18S rRNA that was about 800 bp in size. Blast results revealed that the 18S rRNA was 89%- 91% similar to the corresponding portion of P. ovale registered in GenBank. The phylogenetic tree indicated that P. ovale was in the same clade with the reference strains of P. ovale even though P. ovale and P. falciparurn were in two separate clades. Conclusion The patient who was infected with P. ovale while in Nigeria was confirmed to be infected with P. ovale by a comprehensive diagnosis including microscopy, nested PCR, sequencing, and Blast analysis.