多基因核 DNA 条码鉴定尼日利亚青蒿琥酯/甲氟喹治疗前后疟原虫的遗传相关性。
A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria.
机构信息
Medical Research Council Unit of The London School of Hygiene and Tropical Medicine, Bakau, The Gambia.
Parasitology and Bioinformatics, Department of Zoology, Faculty of Science, University of Lagos, Lagos, Nigeria.
出版信息
BMC Infect Dis. 2018 Aug 13;18(1):392. doi: 10.1186/s12879-018-3314-3.
BACKGROUND
The decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains.
METHODS
We observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1-86 and mdr1-184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments.
RESULTS
By varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (H) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1-86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed.
CONCLUSIONS
Persistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria.
背景
青蒿素类复方疗法（ACT）在一些流行地区的疗效下降，威胁到全球消除疟疾的进展。在疟疾流行地区进行耐药性分子监测对于发现和传播突变株至关重要。
方法
我们观察了尼日利亚拉各斯 89 名患有无并发症恶性疟原虫感染的青蒿琥酯-咯萘啶治疗的疗效，并确定了人群中耐药株的流行率。通过显微镜检查和高度敏感的 VAR 基因酸性末端序列（VARATS）聚合酶链反应，对 65 名患者在治疗开始后第 0、1、3、7、14、21 和 28 天的寄生虫清除率进行了测定。使用 24 个核单核苷酸多态性（SNP）条码对疟原虫 DNA 的基因组指纹图谱进行了测定。用高分辨率熔解分析聚合酶链反应（PCR）片段对氯喹耐药转运蛋白基因（crt-76）、多药耐药基因（mdr1-86 和 mdr1-184）、二氢叶酸合成酶（dhps-540）、二氢叶酸还原酶（dhfr-108）和kelch 结构域（K-13580）的耐药相关等位基因进行了基因分型。
结果
通过 VARATS qPCR，12 名（18.5%）参与者在治疗后 3 天血液中检测到可检测的寄生虫 DNA，而 8 名（12.3%）个体在第 28 天出现可基因分型的寄生虫血症。感染复杂性（CoI）在第 0 天为 1.30，第 28 天为 1.34，所有条码的平均预期杂合度（H）值分别为 0.50±0.05 和 0.56±0.05。条码（π）成对比较显示，在 8 名重新出现感染的个体中，有 3 名（37.5%）的第 0 天和第 28 天寄生虫分离物之间存在高度遗传相关性。crt-76 突变等位基因存在于 38 株（58.5%）分离株中。mdr1-86 突变等位基因存在于 56 株（86.2%）分离株中。未观察到 K-13580 突变。
结论
在超过 18%的病例中，治疗后仍可检测到 DNA 寄生虫血症，并且在治疗前和治疗后感染之间存在遗传相关性的迹象，这表明尼日利亚青蒿素类复方疗法的疗效可能有所下降，需要对更大的人群进行进一步调查。
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