Deep-amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations. Deep-amplicon sequencing for benzimidazole resistance mutations

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, are severely compromised by the widespread development of drug resistance. More recently, there are concerns regarding the emergence of anthelmintic resistance in human parasitic nematodes in response to the selection pressure resulting from mass drug administration (MDA) 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. 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 (SNPs) 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 with 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 SNPs at three different codons of the β-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.

寄生性胃肠道线虫（Parasitic gastrointestinal nematodes）可造成严重的人体发病负担，同时每年造成数十亿美元的农业生产损失。目前此类寄生虫的防控依赖驱虫药（anthelmintic drugs），但针对畜禽寄生线虫的驱虫治疗已因广泛出现的抗药性而严重受限。近期，学界开始担忧在大规模药物给药（MDA）项目带来的选择压力下，人体寄生线虫也会出现驱虫药抗药性。因此，亟需开发灵敏、可规模化且精准的诊断工具，以检测抗药性的出现。通过检测并量化寄生虫种群中与抗药性相关的突变频率，有望从早期阶段实现对抗药性出现的灵敏定量评估。本研究阐述了深度扩增子测序（deep-amplicon sequencing）的开发与验证流程，该方法可作为一种全新的高效手段，用于检测并定量与苯并咪唑类药物抗药性（benzimidazole resistance）相关的单核苷酸多态性（SNPs）。我们以绵羊体内的寄生虫群落为研究对象，开展了该方法的概念验证研究。绵羊是极佳的宿主模型，其体内存在多种共感染的毛圆科线虫（trichostrongylid nematode），且各类线虫的苯并咪唑类药物抗药性流行率各不相同。研究结果证实，该方法可精准测定抗药性等位基因的频率，且能够可靠检测到频率低至0.1%的抗药性等位基因，尤其适用于抗药性早期阶段的突变筛查。我们通过单次MiSeq测序运行，对英国164个绵羊种群（其中95个来自母羊、69个来自羔羊）体内7种不同寄生虫的β微管蛋白基因（β-tubulin gene）的3个不同密码子处的苯并咪唑类药物抗药性相关SNPs进行了筛查，以此展示了该技术的应用效能。本方法可为筛查动物与人体寄生线虫种群中的驱虫药抗药性突变提供一款全新的高效工具。