Epicentre Mbarara Research Centre, Mbarara, Uganda.
Department of Microbiology, Mbarara University of Science and Technology, Mbarara, Uganda.
Malar J. 2021 Feb 25;20(1):114. doi: 10.1186/s12936-021-03657-7.
Chloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries.
Quantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda.
The sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10 ng and the ability to identify mixed haplotypes as low as 10 % was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66 %, 162/244); however, there was a large regional variation between the sample groups (94 % CVIET in Group 1 and 44 % CVIET in Group 2).
The HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.
氯喹（CQ）耐药性是由恶性疟原虫 CQ 耐药转运蛋白（pfcrt）中的突变引起的。在停止使用 CQ 进行抗疟治疗后，在疟疾流行国家进行的研究显示出了一系列的反应。在一些地区，CQ 敏感寄生虫重新出现，而在其他地区，突变单倍型仍然存在。对临床寄生虫的耐药突变进行主动监测对于告知治疗方案至关重要；这项工作需要快速、可靠且具有成本效益的方法，能够在疟疾流行国家使用各种类型的样本和试剂。
在野外环境中，采用实时荧光定量 PCR 后高分辨率熔解（HRM）分析，评估了来自乌干达西南部的两组临床样本中的 pfcrt 突变。第 1 组样本（共 119 个）于 2010 年采集，主要为吉姆萨染色载玻片；第 2 组样本（共 125 个）于 2015 年采集，为滤纸血斑。利用 Rotor-Gene Q 仪器评估了不同的 PCR-HRM 试剂混合物的影响以及临床样本中存在的混合单倍型的检测。最后，在乌干达西南部这一研究不足的地区评估了野生型（CVMNK）和耐药 pfcrt 单倍型（CVIET 和 SVMNT）的流行率。
样本来源（即吉姆萨染色载玻片或血斑）和 LCGreen 为基础的试剂混合物的类型并不影响 PCR-HRM 的成功。10ng 的检测限和识别低至 10%的混合单倍型的能力与其他 HRM 平台相似。CVIET 单倍型在临床样本中占主导地位（66%，162/244）；然而，两组样本之间存在很大的区域差异（第 1 组 94%为 CVIET，第 2 组 44%为 CVIET）。
基于 HRM 的方法具有从疟疾临床管理过程中产生的各种样本类型中进行可靠的耐药等位基因评估所需的灵活性。乌干达西南部 CQ 耐药性单倍型的巨大区域差异强调了继续进行当地寄生虫基因型评估以告知抗疟治疗政策的必要性。
Zotero 插件
