Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations
Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations
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DOI：
10.1016/j.ijpara.2018.10.005
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发表时间：
2019-01-01
影响因子：
4
通讯作者：
Gilleard, John S.
中科院分区：
文献类型：
--
作者：
Avramenko, Russell W.;Redman, Elizabeth M.;Gilleard, John S.
寄生性胃肠道线虫导致严重的人类发病率，每年造成数十亿美元的农业生产损失。控制依赖于驱虫药的使用，而在牲畜寄生虫的情况下，由于抗药性的广泛发展，驱虫药的使用受到严重影响。现在人们担心，由于大规模药物管理计划造成的选择压力，人类寄生线虫中出现了驱虫剂耐药性。因此，迫切需要灵敏、可扩展和准确的诊断工具来检测驱虫剂耐药性的出现。检测和测量寄生虫种群中耐药相关突变的频率有可能从早期阶段提供敏感和定量的耐药性评估。在这里，我们描述了深度扩增子测序的开发和验证，作为一种强大的新方法来检测和量化与苯并咪唑耐药相关的单核苷酸多态性的频率。我们使用绵羊体内的寄生虫群落对这种方法进行了概念验证研究。绵羊提供了一个极好的宿主系统，因为有多种共感染的圆线虫，每种都可能具有不同的苯并咪唑耐药性。我们证明，该方法提供了耐药等位基因频率的准确测量，并且可以可靠地检测频率低至0.1%的耐药等位基因，这使得它对于在耐药早期阶段筛查突变特别有价值。我们通过在一次MiSeq测序运行中筛选来自164个种群（95只来自母羊，69只来自羔羊）的7种不同寄生虫物种中β-微管蛋白基因3个不同密码子处的苯并咪唑抗性相关单核苷酸多态性，说明了该技术的强大功能。这种方法提供了一个强大的新工具，筛选驱虫剂抗性突变的出现在动物和人类的寄生线虫种群。(C)2018年由Elsevier Ltd代表澳大利亚寄生虫学会出版。
Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the beta-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans. (C) 2018 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.