2012年和2018年我国输入性恶性疟原虫氯喹抗性基因多态性分析

Analysis of chloroquine-resistant gene polymorphisms in Plasmodium falciparum imported into China in 2012 and 2018

目的对2012年和2018年我国输入性恶性疟原虫样本氯喹抗性分子标记位点基因多态性进行检测,分析恶性疟原虫氯喹抗性转运蛋白基因(Plasmodium falciparum chloroquine re sistant transporter,Pfcrt)第72~76位密码子抗性相关位点突变类型,并分析不同输入来源样本的特异性。方法收集2012年和2018年国家疟疾诊断参比实验室674例输入性恶性疟病例滤纸血样本,以样本中恶性疟原虫7号染色体上Pfcrt基因第72~76位点为扩增片段,采用巢式PCR法进行扩增并测序,对目的产物片段测序结果、地理分布等特征进行统计分析。结果2012年和2018年我国674例输入性恶性疟病例中,95.5%(644/674)来自非洲,其余4.5%(30/674)来自东南亚和大洋洲(巴布亚新几内亚);非洲又以西非和中非为主(占非洲样本的80.4%,518/644)。共检测到C72S、M74I、N75E、K76T 4个位点突变和5种单体型类型(CVMNK、CVIET、SVMNT和两种混合型),其中CVMNK与CVIET为非洲和东南亚地区恶性疟原虫共有的单体型类型,SVMNT仅在东南亚(缅甸)和巴布亚新几内亚输入样本中检测出;2种混合型为CVMNK/CVIET和CVMNK/SVMNT,前者在非洲和东南亚输入样本中分布,后者仅在东南亚缅甸来源样本中检出。自非洲输入的样本野生型较多,占77.7%(478/615);而自东南亚和巴布亚新几内亚输入的样本中,抗性分子标记样本占68.0%(17/25),两者差异有统计学意义(χ^2=28.5,P<0.05)。非洲不同地区来源样本中,抗性基因比例和野生型构成差异有统计学意义(P<0.01),西非野生型所占比例最低。结论2012年和2018年我国674例输入性恶性疟病例样本中,自东南亚输入的恶性疟原虫Pfcrt基因第72~76位点抗性基因比例和分子多态性均较非洲来源样本高。

Objective To detect the chloroquine-resistant molecular marker polymorphisms in Plasmodium falciparum imported into China,investigate the mutation types of P.falciparum chloroquine resistant transporter(Pfcrt)gene at positions 72 to76,and analyze the specificity of the P.falciparum specimens with different origins.Methods A total of 674 filter paper blood samples were collected from the National Malaria Diagnosis Reference Laboratory of China in 2012 and 2018.The amino acid positions 72 to 76 of the Pfcrt gene on chromosome 7 were amplified using nested PCR assay and sequenced,and the sequencing results of the target gene fragment and the geographical region-specific prevalence of the mutations in the Pfcrt gene were analyzed.Results Among the 674 imported P.falciparum malaria cases in China in 2012 and 2018,99.5%(644/674)were from Africa,which were predominantly from western and central Africa(80.4%,518/644),and 4.5%(30/674)from Southeast Asia and Oceania(Papua New Guinea).A total of 4 site mutations(C72 S,M74 I,N75 E and K76 T)and 5 haplotypes(CVMNK,CVIET and SVMNT and two mixed types)were identified,with haplotypes CVMNK and CVIET present in parasites of both African and Southeast Asian origins,SVMNT detected in Southeast Asia(Myanmar)and Papua New Guinea isolates,the mixed type of haplotypes CVMNK/CVIET detected in P.falciparum of African and Southeast Asian origins,and the mixed type of haplotypes CVMNK/SVMNT detected only in the Myanmar isolate.Most P.falciparum parasites of the African origin carried the wild-type Pfcrt allele(77.7%,478/615),and 68.0%(17/25)of the P.falciparum parasites of the Southeast Asian and Papua New Guinea origins harbored chloroquine resistant molecular markers(χ^2=28.5,P<0.05).The constituent ratio of the wild-and mutant-type Pfcrt allele varied in different geographical regions of Africa(P<0.01),and the lowest prevalence of the wild-type Pfcrt allele was seen in western Africa.Conclusion Among the 674 imported malaria cases in China in 2012 and 2018,the P.falciparum imported from Sotheast Asia habors a higher proportion of resistance to chloroquine and a higher molecular polymophism at amino acid positions 72 to 76 of the Pfcrt gene than the parasite of the African origin.