中缅边境地区恶性疟原虫dhfr和dhps基因多态性分析

Polymorphisms of Plasmodium falciparum dhfr and dhps gene in China-Myanmar border

目的 分析中缅边境地区恶性疟原虫dhfr和dhps基因多态性。方法 收集2001-2012年中缅边境中国云南西双版纳州景洪市、缅甸克钦邦拉咱市和缅甸掸邦第二特区勐冒县恶性疟患者滤纸血样本175份,采用巢式PCR法分别扩增恶性疟原虫二氢叶酸还原酶基因(Plasmodium falciparum dihydrofolate reductase gene,pfdhfr)第51-164位点片段和二氢蝶酸合成酶基因(Plasmodium falciparum dihydropteroate synthase gene,pfdhps)第436-613位点片段,并对扩增产物进行测序分析。结果 共检测到恶性疟原虫pfdhfr基因4个突变位点(N51I、C59R、S108N和I164L)和7种基因型,3个地区pfdhfr基因均存在四重突变型(IRNL),其中云南景洪市2001年和2006年IRNL分别占26.7%和28%,缅甸拉咱市2001年、2007年和2012年分别占35%、40%和60%,缅甸勐冒县2009年占比为40%。pfdhfr基因野生型,云南景洪市2001年占比23.3%,2006年未检测到野生型,以二重突变型(NRNI)为主,占44%;缅甸拉咱市2001年占比10%,单重突变型率(NRSI)5%,双重突变型率(IRSI,NRNI)10%,三重突变型率(IRNI,NRNL)40%,2007年除四重突变外(40%),三重突变型率(NRNL)和二重突变型率(NRNI)均为30%,2012年三重突变以IRNI型为主占25%;缅甸勐冒县2009年pfdhfr基因野生型比例较高,占45%,三重突变型率(NRNL和IRNI)占12.5%。共检测到pfdhps基因4个突变位点(S436A/F,A437G,K540E/N和A581G)和14种基因型,其中云南景洪市2001年样本中pfdhps基因未发现三重突变,以二重突变型(SAEG)为主,占53.3%,2006年出现了三重突变型(FAEG),占8%,单突变型率(AAKA)40%;缅甸拉咱市pfdhps基因三重突变型率(FAEG)2001年、2007年和2012年分别为5%、35%和37.5%,单突变型(SGKA,SAEA,SAKG)仅在2001年样本中检测到,占比30%,2007年(SAEG,FGKA,FAEA,AAEA)及2012年(SAEG,FAKG,FGKA,AAEA)中双重突变型率分别占60%和55%;缅甸勐冒县2009年样本中pfdhps基因三重突变型率(FAEG)为27.5%,无野生型及单突变型,二重突变型率(AAEA和FGKA)为50%。结论 中缅边境3个地区pfdhfr基因和pfdhps基因突变程度均较高,提示该地区恶性疟原虫对磺胺多辛-乙胺嘧啶药物存在抗性,可为中缅边境地区恶性疟的防治提供参考。

Objective To analyze the polymorphisms of dhfr and dhps genes of Plasmodium falciparum in China-Myanmar border.Methods Totally 175 Plasmodium falciparum filter paper blood samples were collected in Jinghong City,Xishuangbanna Prefecture of Yunnan Province,China and Lazan City of Kachin State and Mengmao County,the Second Special Zone of Shan State in Myanmar from 2001 to 2012.The fragments of Plasmodium falciparum dihydrofolate reductase gene(pfdhfr)(positions 51 to 164) and Plasmodium falciparum dihydroponic synthase(pfdhps) gene(positions 436 to 613) were amplified by Nested PCR,and then products were sequenced and analyzed.Results Four mutation sites(N51I,C59 R,S108N and I164L) and seven haplotypes were detected in pfdhfr gene,of which the quadruple mutants(IRNL) were the major haplotype in above three areas.The percentage of haplotype(IRNL) was 26.7% in 2001 and 28% in 2006 in Jinghong City.In Lazan City,the percentage of haplotype(IRNL) were 35%,40% and 60% in 2001,2007 and 2012,respectively,and it accounted for 40% in Mengmao County in 2009.For pfdhfr gene wild type,the proportion of the wild type was 23.3% in 2001 in Jinghong City.In 2006,no wild type was detected,while the double mutants(NRNI) accounted for 44%.In Lazan City,the proportions of wild type was 10%.Single mutants(NRSI),double mutants(IRSI,NRNI) and triple mutants(IRNI,NRNL) were 5%,10% and 40% in 2001,respectively.Except for quadruple mutations(40%) in 2007,the triple mutants(NRNL) and double mutants(NRNI) each accounted for 30%.However,the major triple mutants were IRNI,which accounted for 25% in 2012.In Mengmao County,the proportion of wild-type in 2009 was 45%,and the triple mutants(NRNL,IRNI) accounted for 12.5%.Totally four mutation sites(S436A/F,A437G,K540E/N and A581G) and fourteen haplotypes were detected in pfdhps gene.In Jinghong City,no triple mutants was detected in 2001,the proportion of double mutants(SAEG) was 53.3%.While the proportions of triple mutants(FAEG) and single mutants(AAKA) were 8% and 40% in 2006,respectively.In Lazan City,the rates of triple mutants(FAEG) of pfdhps gene were 5%,35% and 37.5% in 2001,2007 and 2012,respectively.In addition,single mutants(SGKA,SAEA,SAKG) were only detected in 2001,with the proportions of 30%.And double mutants accounted for 60% in 2007(SAEG,FGKA,FAEA,AAEA) and 55% in 2012(SAEG,FAKG,FGKA,AAEA).In Mengmao County,the proportion of triple mutants(FAEG) of pfdhps gene was 27.5%,double mutants(AAEA,FGKA) accounted for 50%,while no wild type and single mutants were found.Conclusion The high mutation rates of pfdhfr and pfdhps genes suggest that the P.falciparum parasites were high resistant to SP drugs in three areas along the China-Myanmar border,which also provide guidance for the treatment of Plasmodium falciparum in China-Myanmar border.