2018-2020年赤道几内亚Bioko岛恶性疟原虫青蒿素耐药相关基因Pfubp1和Pfap2mu的单核苷酸多态性分析

Single⁃nucleotide polymorphisms of artemisinin resistance⁃related Pfubp1 and Pfap2mu genes in Bioko Island,Equatorial Guinea from 2018 to 2020

目的了解赤道几内亚Bioko岛恶性疟原虫分离株青蒿素耐药相关基因Pfubp1和Pfap2mu的单核苷酸多态性(single nucleotide polymorphisms,SNPs)流行情况,为该地区疟疾防治方案的制定提供基线数据。方法采集2018—2020年赤道几内亚Bioko岛恶性疟患者临床血样184份,提取血样中恶性疟原虫基因组DNA。采用巢式PCR技术和Sanger测序法检测恶性疟原虫Pfubp1和Pfap2mu基因,并对基因序列进行比对分析。结果赤道几内亚Bioko岛分离株恶性疟原虫Pfubp1基因中,无突变虫株占88.83%(159/179)。20株突变虫株(11.17%,20/179)共分离出6个不同SNPs,该6个SNPs有4个非同义突变,分别为E1516G、K1520E、D1525E、E1528D;95.00%(19/20)的突变分离株中仅有1个基因突变位点,其中非同义突变占68.42%(13/19)。与以青蒿素为基础的联合疗法(artemisinin⁃based combination therapies,ACTs)耐药相关的Pfubp1基因中,D1525E和E1528D为已知主要流行突变位点;在第1525位点氨基酸,野生型虫株占99.44%(178/179)、突变型虫株占0.56%(1/179),该突变位点仅在2018年采集的样本中发现;在第1528位点,野生型虫株占93.30%(167/179)、突变型虫株占6.70%(12/179)。在Pfap2mu基因3个区域(Pfap2mu⁃inner1、Pfap2mu⁃inner2、Pfap2mu⁃inner3)的目标扩增片段中,野生型虫株所占比例分别为95.72%(134/140)、79.25%(126/159)和95.83%(161/168);在所有测序成功的样本中筛出16个不同SNPs,共有7个非同义突变,分别为S160N、K199T、A475V、S508G、I511M、L595F、Y603H;43株突变分离株中,有1个以上突变位点的占16.28%(7/43),其中非同义突变占28.57%(2/7)。与ACTs相关的已知延迟清除位点为S160N,野生型虫株占89.94%(143/159)、突变型虫株占10.06%(16/159)。结论赤道几内亚Bioko岛恶性疟原虫Pfubp1和Pfap2mu基因均发生不同水平突变,Pfubp1基因突变型比例低、Pfap2mu基因突变型比例较高,本研究还发现了Pfubp1 E1504E、K1520E和Pfap2mu A475V、S508G等新位点。

Objective To investigate the prevalence of single nucleotide polymorphisms(SNPs)of artemisinin resistance⁃re⁃lated Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island,Equatorial Guinea,so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island.Methods A total of 184 clinical blood samples were collected from patients with P.falciparum malaria in Bioko Island,Equatorial Guinea from 2018 to 2020,and genomic DNA was extracted.The Pfubp1 and Pfap2mu gene SNPs of P.falciparum were determined using a nested PCR assay and Sanger se⁃quencing,and the gene sequences were aligned.Results There were 159 wild⁃type P.falciparum isolates(88.83%)from Bioko Island,Equatorial Guinea,and 6 SNPs were identified in 20 Pfubp1⁃mutant P.falciparum isolates(11.17%),in which 4 non⁃syn⁃onymous mutations were detected,including E1516G,K1520E,D1525E,E1528D.There was only one Pfubp1gene mutation site in 19 Pfubp1⁃mutant P.falciparum isolates(95.00%),in which non⁃synonymous mutations accounted for 68.42%(13/19).D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to ar⁃temisinin⁃based combination therapies(ACTs).At amino acid position 1525,there were 178 wild⁃type P.falciparum isolates(99.44%)and 1 mutant isolate(0.56%),with such a mutation site identified in blood samples in 2018,and at amino acid position 1528,there were 167 wild⁃type P.falciparum isolates(93.30%)and 12 mutant isolates(6.70%).The proportions of wild⁃type P.falciparum isolates were 95.72%(134/140),79.25%(126/159)and 95.83%(161/168)in the target amplification fragments of the three regions in the Pfap2mu gene(Pfap2mu⁃inner1,Pfap2mu⁃inner2,Pfap2mu⁃inner3),respectively.There were 16 different SNPs identified in all successfully sequenced P.falciparum isolates),in which 7 non⁃synonymous mutations were detected,in⁃cluding S160N,K199T,A475V,S508G,I511M,L595F,and Y603H.There were 7 out of 43 Pfap2mu⁃mutant P.falciparum iso⁃lates(16.28%)that harbored only one gene mutation site,in which non⁃synonymous mutations accounted for 28.57%(2/7).For the known delayed clearance locus S160N associated with ACTs,there were 143 wild⁃type(89.94%)and 16 Pfap2mu⁃mutant P.falciparum isolates(10.06%).Conclusions Both Pfubp1 and Pfap2mu gene mutations were detected in P.falciparum isolates from Bioko Island,Equatorial Guinea from 2018 to 2020,with a low prevalence rate of Pfubp1 gene mutation and a high preva⁃lence rate of Pfap2mu gene mutation.In addition,new mutation sites were identified in the Pfubp1(E1504E and K1520E)and Pfap2mu genes(A475V and S508G).