中缅边境48份间日疟原虫标本pvcrt-o及pvmdr1基因多态性分析

Polymorphisms of pvcrt-o and pvmdr1 genes of 48 Plasmodium vivax isolates from the China-Myanmar border

目的 检测中缅边境拉咱地区48份间日疟原虫标本耐药相关基因pvcrt-o及pvmdr1的多态性,为指导该地区间日疟的治疗方案提供基础数据。方法 收集2007年缅甸拉咱地区的间日疟原虫样本48份,采用PCR方法扩增pvcrt-o及pvmdr1基因片段并进行测序,与间日疟原虫Sal-I株标准序列进行序列比对和分析。结果 在48份间日疟原虫样本中,扩增出pvcrt-o及pvmdr1基因片段分别39份和40份,其中pvcrt-o基因在第10个氨基酸前有AAG碱基序列插入(K10插入)25份(占64.1%);pvmdr1基因共检测到3个位点存在非同义突变,分别为T958M、Y976F和F1076L,其突变频率分别为100.0%、22.5%与55.0%,且存在3种基因型,其中三重突变型T958M/Y976F/F1076L占22.5%(9/40),双重突变型T958M/F1076L占32.5%(13/40),单重突变型T958M占45.0%(18/40);同时存在pvcrt-o和pvmdr1基因变异的虫株比例为63.16%,与未发生pvcrt-o变异但存在pvmdr1变异的虫株存在较大的遗传进化差异。结论 48份间日疟原虫pvcrt-o及pvmdr1基因发生变异比例高,且两种基因变异存在一定程度的关联,需要对这两类分子标志加强监测,以评估该地区间日疟原虫的药物抗性变化情况。

Objective To detect the polymorphisms of drug resistance-related genes pvcrt-o and pvmdr1 of Plasmodium vivax in lazan city in the China-Myanmar border, in order to guide the treatment plan of Plasmodium vivax.Methods A total of 48 Plasmodium vivax samples were collected from Lazan in the China-Myanmar border in 2007, and fragments of pvcrt-o and pvmdr1 genes were amplified by PCR and sequenced. The sequences were aligned with the Salvador I(Sal-I) strain reference genome sequences to determine the presence of SNPs.Results The target fragments of pvcrt-o gene were amplified from 39 Plasmodium vivax samples, while pvmdr1 genes were amplified from 40 samples. Amongst them, 25 samples had AAG insertion before the 10th amino acid(K10 insertion) of pvcrt-o gene, accounting for 64.1%. Non-synonymous mutations were detected at three loci of pvmdr1 gene(T958M, Y976F, and F1076L), the mutation rates were 100%, 22.5%, and 55.0%,respectively. There were three haplotypes of pvmdr1 gene, of which the triple mutant 958M/976F/1076L accounted for 22.5%(9/40), the double mutant 958M/Y976/1076L accounted for 32.5%(13/40), and the single mutant 958M/Y976/F1076 accounted for 45.0%(18/40). The proportion of strains with pvcrt-o and pvmdr1 gene mutation is 63.16%, which is significantly different from those only with pvmdr1 mutation.Conclusions The proportion of pvcrt-o and pvmdr1 gene mutation of 48 Plasmodium vivax isolates is high in the China-Myanmar border, and there is a certain degree of correlation between the two gene mutations.To assess changes in Plasmodium vivax drug resistance in this region, it is required to improve the surveillance of these two molecular markers.