Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon.

Frontiers in parasitology Pub Date : 2025-02-05 eCollection Date: 2024-01-01 DOI:10.3389/fpara.2024.1509261
Jacob M Sadler, Alfred Simkin, Valery P K Tchuenkam, Isabela Gerdes Gyuricza, Abebe A Fola, Kevin Wamae, Ashenafi Assefa, Karamoko Niaré, Kyaw Thwai, Samuel J White, William J Moss, Rhoel R Dinglasan, Sandrine Eveline Nsango, Christopher B Tume, Jonathan B Parr, Innocent Mbulli Ali, Jeffrey A Bailey, Jonathan J Juliano
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Abstract

Background: Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing.

Methods: Plasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach.

Results: We evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed.

Conclusion: Overall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible.

应用一种新的高复用扩增子测序工具评估喀麦隆Dschang地区个体和汇总样本中恶性疟原虫抗疟药及其相关性
背景:对抗疟药物的耐药性仍然是消除疟疾的主要障碍。多路、靶向扩增子测序正被用于监测耐药性和剖析复杂疟疾感染的遗传学。此外,在资源有限的地区进行寄生虫的基因分型和分子标记的耐药性检测,需要使用开放源代码的协议来处理样本,使用可获得的试剂,以及快速处理大量样本的方法,包括集合测序。方法:恶性疟原虫Streamlined Multiplex anti - malaria Resistance and亲缘性检测(Pf-SMARRT)是一种基于pcr的扩增子面板,由15个靶向抗疟耐药突变的扩增子和9个靶向高变区的扩增子组成。该分析使用两个池中的寡核苷酸引物和非专有文库和条形码方法。结果:我们使用不同寄生虫血症水平的对照模拟干血点(DBS)和已知频率的3D7和Dd2菌株混合物对Pf-SMARRT进行了评估,显示出在低寄生虫密度下进行基因分型和回忆样本内等位基因频率的能力。然后,我们将Pf-SMARRT应用于从喀麦隆西部Dschang三个卫生机构的无并发症疟疾病例中收集的100株寄生虫分离物的基因分型。抗疟药基因分型显示高水平的磺胺多辛-乙胺嘧啶耐药突变,包括DHPS A613S突变的患病率为31%。未检测到K13候选或已证实的青蒿素部分耐药突变,但观察到一个低水平的非同义变化。与分子倒置探针(MIP)测序相比,Pf-SMARRT的高可变靶标显示出相似的水平和模式,用于评估感染的复杂性和寄生虫的多样性和相关性。虽然单个样本和样本池之间的抗疟药突变具有很强的一致性,但合并样本中的低频变异往往被遗漏。结论:总体而言,Pf-SMARRT是一种可靠的工具,可用于评估寄生虫亲缘性和来自个体和汇总样本的抗疟药物耐药性标记。对照样本支持每微升低至1个寄生虫的准确基因分型通常是可能的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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