Lack of quadruple and quintuple mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax isolates from Brazilian endemic areas

BACKGROUND AND OBJECTIVE Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated. METHODS Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs). FINDINGS All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%). CONCLUSION No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

研究背景与目的 美洲地区的间日疟原虫（Plasmodium vivax）感染病例中，巴西所占比例极高。鉴于耐氯喹间日疟原虫虫株的出现、抗叶酸类药物用于间日疟治疗的有效性，以及间日疟原虫二氢叶酸还原酶（dihydrofolate reductase, dhfr）与二氢蝶酸合酶（dihydropteroate synthase, dhps）基因的突变与磺胺多辛-乙胺嘧啶（sulfadoxine-pyrimethamine，SP）治疗失败存在相关性，本研究针对上述两类基因的点突变展开了检测分析。
研究方法 本研究纳入亚马逊地区54例出现症状性间日疟发作的患者，采集其血液样本开展检测。采用QIAGEN™基因组DNA提取试剂盒提取基因组DNA，通过嵌套聚合酶链式反应（Nested PCR）进行扩增，随后采用桑格测序（Sanger sequencing）检测单核苷酸多态性（SNPs）。
研究结果 所有受试疟原虫分离株均在间日疟原虫二氢叶酸还原酶基因（pvdhfr）中检出非同义突变：117N突变检出率为100%（54/54），58R突变检出率为46%（25/54）。其中最常见的双突变等位基因为58R/117N（FRTNI，占比28%），随后依次为三突变等位基因（58R/117N/173L，FRTNL，11%；58R/61M/117N，FRMNI，5%；117N/173L，FSTNL，4%）以及四突变等位基因58R/61M/117N/173L（FRMNL，占比2%）。在间日疟原虫二氢蝶酸合酶基因（pvdhps）的C383G密码子位点，仅检出单一位点突变（SGKAV，占比48%）。
研究结论 本研究的巴西样本中未检出与间日疟原虫对SP耐药相关的分子标志物。