2015年河南省输入性恶性疟病例中Pfcrt基因多态性分析

[Analysis of Pfcrt Gene Polymorphism in Imported Falciparum Malaria Cases in Henan Province in 2015].

作者信息

Zhou Rui-min, Wang Yi-nan, Qian Dan, Li Su-hua, Liu Ying, Yang Cheng-yun, Zhao Yu-ling, Xu Bian-li, Zhang Hong-wei

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2016 Oct;34(5):399-404.

Abstract

OBJECTIVE

To analyze Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene polymorphism in imported falciparum malaria cases in Henan Province in 2015.

METHODS

Blood samples were collected from 132 cases of imported falciparum malaria in Henan Province in 2015. DNA was extracted from the samples, and the Pfcrt was amplified by nested PCR using specific primers. The PCR products were digested by restriction endonuclease enzyme Apol I and sequenced. Pfcrt gene polymorphism and distribution were analyzed.

RESULTS

Most of the 132 cases of imported malaria were young male adults returning from the Africa, with the highest percentage in those from West Africa（38.6%, 51/132）, then Central Africa（26.5%, 35/132）, South Africa（25.0%, 33/132）, East Africa（8.3%, 11/132）, and North Africa（1.5%, 2/132）. The nested PCR yielded a 145-bp product for each sample, and 66.7%（88/132） of the products were completely digested by Apol I, resulting in two fragments of 114 bp and 31 bp; 32.6%（43/132） could not been digested and only a single fragment of 145 bp was shown; and 0.8%（1/132） were incompletely digested, yielding three fragments of 145 bp, 114 bp and 31 bp. By blasting against chloroquine sensitive strain 3D7, we found mutations of Pfcrt at sites correspondig to residues 74, 75 and 76 from ATG, AAT and AAA to ATT, GAA and ACA （i.e. M74I, N75E and K76T） in 43 of the 132 blood samples, and mixed type mutations into ATG/T, A/GAA/T and AA/CA at sites correspondig to residues 74, 75 and 76（CVM/I, N/E/D/K, T/K） in one blood sample. The other 88 blood samples showed a wild type with no mutation (CVMNK). Mutations occurred mainly in cases from West Africa（41.2%, 21/51）, then East Africa（36.4%, 4/11）, South Africa（30.3%, 10/33）, and Central Africa（22.9%, 8/27）（χ2=4.07, P＞0.05）. The 2 cases from the North Africa both had wild type Pfcrt; the one with mixed type mutation was from West Africa.

CONCLUSION

Three haplotypes of Pfcrt have been found, including wild type (CVMNK), mutation type (CVIET) and mixed type (CVM/I, N/E/D/K, K/T) in the imported malaria cases. The wild type occupies the highest proportion （66.7%）, while the mutation type possesses a high proportion of 41.2% in cases from West Africa.

摘要

目的

分析2015年河南省输入性恶性疟病例中恶性疟原虫氯喹抗性转运蛋白（Pfcrt）基因多态性。

方法

采集2015年河南省132例输入性恶性疟病例的血样。从样本中提取DNA，使用特异性引物通过巢式PCR扩增Pfcrt。PCR产物用限制性内切酶Apol I消化并测序。分析Pfcrt基因多态性及分布情况。

结果

132例输入性疟疾病例中多数为从非洲返回的年轻男性成年人，其中来自西非的比例最高（38.6%，51/132），其次是中非（26.5%，35/132）、南非（25.0%，33/132）、东非（8.3%，11/132）和北非（1.5%，2/132）。巢式PCR对每个样本均扩增出一条145 bp的产物，其中66.7%（88/132）的产物被Apol I完全消化，产生114 bp和31 bp两条片段；32.6%（43/132）未被消化，仅显示一条145 bp的片段；0.8%（1/132）被不完全消化，产生145 bp、114 bp和31 bp三条片段。与氯喹敏感株3D7比对发现，132份血样中有43份在对应于ATG、AAT和AAA的第74、75和76位残基处发生Pfcrt突变，变为ATT、GAA和ACA（即M74I、N75E和K76T），1份血样在对应于第74、75和76位残基处发生混合型突变，变为ATG/T、A/GAA/T和AA/CA（CVM/I、N/E/D/K、T/K）。其余88份血样显示为无突变的野生型（CVMNK）。突变主要发生在来自西非的病例中（41.2%，21/51），其次是东非（36.4%，4/11）、南非（30.3%，10/33）和中非（22.9%，8/27）（χ2 = 4.07，P＞0.05）。来自北非的2例均为Pfcrt野生型；发生混合型突变的1例来自西非。