云南省疟疾疫情病例疟原虫镜检疑难形态的鉴定

发布时间：2018-05-22 10:28

  本文选题：云南 + 疟原虫　； 参考：《中国病原生物学杂志》2017年03期

【摘要】：目的采用分子生物学方法鉴定云南省疟疾参比实验室镜检疟原虫疑难血样。方法选取2012年8月-2015年4月云南省疟疾疫情报告病例中疟原虫形态学鉴定疑难血样,对其18srRNA基因进行巢式PCR扩增和产物测序,测序结果在NCBI和Mega6.06软件与4种疟原虫参比虫株序列(恶性疟原虫P.falciparum:KC906781;间日疟原虫P.vivax:X13926;三日疟原虫P.malariae:M54897;卵形疟原虫P.ovale:L48987)进行相似性和同源性分析。结果38份疑难血样中,采自缅甸感染、云南感染、非洲感染者的血样分别为81.58%(31/38)、15.79%(6/38)和2.63%(1/38)。12份P.malariae及感染红细胞的镜检形态特点为:疟原虫体积占红细胞的体积1/4,可见不同发育期的疟原虫形态,但同时可见"带状"滋养体、"梅花状"裂殖体典型形态的仅有4例,占33.33%(4/12),"带状"滋养体、"梅花状"裂殖体单独可见的比例分别为83.33%(10/12)和75.0%(9/12),100%(12/12)的疟原虫感染红细胞体积不胀大或略为缩小。5份P.ovale及感染红细胞的镜检形态为:疟原虫体积占红细胞的体积1/4,可见各期疟原虫,红细胞体积正常或缩小的发生率为100%(5/5),红细胞呈伞矢状、拖尾、红细胞边缘呈刺突变形的比例分别为80%(4/5)、100%(5/5)和80%(4/5)。11份可疑为P.vivax的形态仅剩疟色素样或疟原虫细胞核样物体。10份P.falciparum的形态不同于以往云南省常见的疟原虫胞浆、胞核纤细、致密的恶性疟原虫,胞浆粗大,虫体占红细胞体积的三分之一。上述血样经18S rRNA基因PCR扩增和DNA测序分析,阳性符合率为100%(38/38),4种疟原虫的18SrRNA基因DNA序列与4种参比序列的相似性在81%~100%之间,也与各自的参比序列聚类成彼此分离的进化分枝,即序列分类与形态识别结果吻合。结论云南省的输入性三日疟、卵形疟病例时有发生,对疑难形态疟原虫的虫种鉴定须借助分子生学技术加予验证。
[Abstract]:Objective to identify the difficult blood samples of Plasmodium falciparum in malaria reference laboratory of Yunnan Province by molecular biological method. Methods from August 2012 to April 2015, difficult blood samples of malaria parasite were identified in Yunnan Province. The 18srRNA gene was amplified by nested PCR and its products were sequenced. The results were compared with four Plasmodium falciparum: KC906781, P.vivax: X13926, P.malariae: M54897 and P.ovale1: L48987 by NCBI and Mega6.06 software. The results showed that P. falciparum: KC906781; P. vivax: X13926; P. malariae: M54897; P. ovale: L48987; P. falciparum: KC906781; P. vivax: X13926; P. malariae: M54897; P. Results 38 difficult blood samples were collected from Myanmar infection and Yunnan infection. The blood samples of the infected people in Africa were 81.58% and 15.79% (6 / 38) and 2.63% / 38%, respectively, and the microscopic morphological characteristics of 12 P.malariae samples and infected red blood cells were as follows: the volume of Plasmodium parasites accounts for 1 / 4 of the volume of red blood cells, and the morphology of malaria parasites at different stages of development can be seen. But at the same time, "banded" trophozoites were seen, and only 4 cases of "plum blossom" merozoite were typical. Accounting for 33.33% 4 / 12%, "banded" trophozoites, "plum blossom" splits were found separately in 83.33% 10 / 12) and 75.0% of 9 / 12% / 100% 12 / 12).) the erythrocyte volume of Plasmodium parasites infected by Plasmodium falciparum did not swell or decreased slightly, and the microscopic morphology of the infected red blood cells was as follows: Plasmodium parasites accounted for the volume of the infected erythrocytes, and the microscopic morphology of the infected erythrocytes was: Plasmodium plasmodium accounted for the volume of the parasite. The volume of red blood cells is 1 / 4, and you can see the malaria parasites at all stages. The incidence of normal or reduced red blood cell volume is 100%, 5 / 5%, red blood cell is sagittal and tail. The proportion of erythrocyte edge deformed by spines was 80 / 4 / 5 / 100 / 100 / 5) and 80 / 4 / 5 / 5, respectively. Of the 11 suspected forms of P.vivax, only malarial pigment or plasmodium nucleoloid body. 10 P.falciparum were different from the plasmodium cytoplasm and nucleus of Plasmodium that were common in Yunnan Province in the past. Compact Plasmodium falciparum with a large cytoplasm, accounting for 1/3 of erythrocyte volume. By PCR amplification of 18s rRNA gene and DNA sequencing, the positive coincidence rate was 100%. The similarity between the DNA sequence of 18SrRNA gene of four Plasmodium species and the four reference sequences was between 81% and 100%. That is, the result of sequence classification is consistent with that of morphological recognition. Conclusion the cases of imported 3-day malaria and oval malaria occur from time to time in Yunnan Province. The identification of difficult form malaria parasites should be verified by molecular technique.
【作者单位】： 云南省寄生虫病防治所云南省疟疾研究中心云南省虫媒传染病防控研究重点实验室;
【基金】：国家自然科学基金地区科学基金项目(No.81660559) 云南省科技厅面上项目(No.2011FB136)
【分类号】：R382.31
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