Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene.
Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene.
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DOI：
10.1038/s41598-022-15718-1
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发表时间：
2022-07-06
影响因子：
4.6
通讯作者：
Cappello, Michael
中科院分区：
文献类型：
--
作者：
George, Santosh;Suwondo, Peter;Akorli, Jewelna;Otchere, Joseph;Harrison, Lisa M.;Bilguvar, Kaya;Knight, James R.;Humphries, Debbie;Wilson, Michael D.;Caccone, Adalgisa;Cappello, Michael
钩虫感染的全球控制依赖于对高风险群体定期进行苯并咪唑类药物的大规模药物管理，无论感染状况如何。已在兽医线虫中鉴定出同种型-1 β-微管蛋白基因突变，导致结构变化和药物结合减少。在加纳，先前的研究表明，阿苯达唑在感染美洲钩虫的人群中的有效性存在显著差异，尽管驱虫反应的机制尚未确定。使用来自加纳横断面研究的钩虫卵样本，我们开发了一种多重扩增子深度测序（MAD-seq）方法来筛选N. americanus同种型-1 β-微管蛋白基因。使用MAD-seq在30个匹配的治疗前和治疗后样本中对同种型-1 β-微管蛋白基因编码区内与耐药相关的突变（F167 Y、E198 A、F200 Y）对应的3个单核苷酸多态性（SNP）进行了表征，这些样本来自治疗后持续感染的个体。测序后分析表明，每个PCR扩增子的最高平均替代等位基因为0.034%（167个扩增子）和0.025%（198/200个扩增子），表明等位基因变异最小。没有样品包含F167 Y SNP，而一个样品包含与E198 A（3.15%）和F200 Y（3.13%）相关的低频读取。这种MAD-seq方法提供了一种高度灵敏的工具，可以在个体和群体水平上监测三种推定的苯并咪唑抗性标记。需要进一步的工作来了解这些多态性与治疗反应的关联。
Global control of hookworm infections relies on periodic Mass Drug Administration of benzimidazole drugs to high-risk groups, regardless of infection status. Mutations in the isotype-1 β-tubulin gene have been identified in veterinary nematodes, resulting in structural changes and reduced drug-binding. In Ghana, previous studies have demonstrated significant variability in albendazole effectiveness among people infected with the hookworm Necator americanus, although the mechanisms underlying deworming response have not been defined. Using hookworm egg samples from a cross-sectional study in Ghana, we developed a multiplex amplicon deep sequencing (MAD-seq) method to screen genomic regions encapsulating putative drug-resistance markers in N. americanus isotype-1 β-tubulin gene. Three single nucleotide polymorphisms (SNPs) corresponding to resistance-associated mutations (F167Y, E198A, F200Y) within the coding region of the isotype-1 β-tubulin gene were characterized using MAD-seq in 30 matched pre- and post-treatment samples from individuals with persistent infection following therapy. Post-sequence analysis showed that the highest mean alternative nucleotide allele at each PCR amplicon was 0.034% (167amplicon) and 0.025% (198/200amplicon), suggesting minimal allelic variation. No samples contained the F167Y SNP, while one contained low-frequency reads associated with E198A (3.15%) and F200Y (3.13%). This MAD-seq method provides a highly sensitive tool to monitor the three putative benzimidazole resistance markers at individual and community levels. Further work is required to understand the association of these polymorphisms to treatment response.
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