Mitigating the global impact of fasciolosis: developing diagnostics for triclabendazole resistance in Fasciola species

摘要：片吸虫和一种健康片吸虫感染家畜，作为一种人畜共患寄生虫，可导致人感染。对感染的估计表明，全世界有240万至1700万人受到感染，通常在LMIC流行地区。世卫组织承认筋膜炎是一种被忽视的热带疾病。三氯苯达唑是治疗家畜感染的首选药物，因为它针对所有生命周期阶段，也是唯一获得许可的人类药物。片吸虫对三氯苯达唑的耐药性最初是由于密集的治疗方案而在牲畜物种中报告的，这不可避免地导致人类感染三氯苯达唑耐药寄生虫，从而无法有效地治疗感染。在牲畜和人类中检测抗药性寄生虫的能力是有效控制的关键；对牲畜进行更好的控制可以降低向人类传播的风险，如果人类被感染，抗药性检测可以为非许可使用替代疗法提供信息。我们需要一种在LMIC环境下可用于家畜和人身上的三氯苯达唑耐药性的分子诊断。项目目的和目的肝片吸虫和巨大片吸虫两种寄生虫引起的肝片吸虫。肝片吸虫具有复杂的人口学特征，它们要么以物种特有的感染形式存在(例如秘鲁的肝片吸虫)，要么在它们分布重叠的地区以杂交形式存在(例如在印度和巴基斯坦)，并已有报道在物种之间引入遗传位点。我们已经在确定肝片吸虫对三氯苯达唑耐药的基因组位点方面取得了很大进展，并将其定位于七个基因簇；其中有两个候选基因，ADP核糖化因子(ARF)和ABC基因(ABCB1)，这两个基因都具有非同义的SNPs，处于三氯苯达唑的选择之下。在家畜中，感染的检测是通过粪便中的辅原抗原/虫卵的存在或血清中的抗体检测来进行的。类似地，人类粪便样本可以用来检测卵子或片吸虫DNA。该项目旨在建立在我们对家畜中三氯苯达唑耐药性的现有了解的基础上，以减轻其对人类健康的影响。我们将：1.对来自代表不同人口统计数字的国家(秘鲁、印度、巴基斯坦、喀麦隆和塞内加尔)的多个家畜和人类分离株进行全基因组重新测序2.开展比较基因组学，以定位巨形吸虫、肝片吸虫和杂交/导入物种之间的基因交换，特别是对三氯苯达唑耐药基因的研究。确定在家畜和人的多个已知抗药性的肝片吸虫和巨大肝片吸虫分离株中，是否对ARF和ABCB1基因中的SNPs进行了三氯苯达唑的选择。4.与印度和秘鲁的临床医生合作，优化诊断分子方法，对人类粪便样本和十二指肠/胆汁抽吸物中的片状吸入物进行物种特异性检测。建立分子诊断检测方法，如Lec-LAMP，用于检测肝片吸虫和巨型肝片吸虫三氯苯达唑耐药基因中的SNPs，用于LMIC环境中的护理点使用。本项目的目标是生成新的基因组序列数据，将其映射到当前参考基因组并询问来自多个肝片吸虫分离株的基因注释，以确定抗药性基因是否在不同种类和杂交形式的肝片吸虫中保守。将询问已知耐药株和敏感株之间的候选基因序列变异，以确定与耐药相关的基因变化(耐药标记)。最后，将开发一种定制的分子诊断工具，在牲畜和人类的样本中检测这些耐药性标记。该项目旨在将发现科学与应用诊断学相结合，对牲畜和人类的健康产生积极影响。

Summary:Fasciolosis and one health Fasciola spp infect livestock and, as a zoonotic parasite, this leads to infection in humans. Estimates of infection suggest between 2.4 and 17 million people are infected worldwide, typically in LMIC endemic regions. WHO recognises fasciolosis as a neglected tropical disease. Triclabendazole is the drug of choice for treating infection in livestock as it targets all life cycle stages and it is the only drug licenced for humans. Triclabendazole resistance in Fasciola species was initially reported in livestock species due to intensive treatment regimes, inevitably this led to infection of humans with triclabendazole resistant parasites and failure to effectively treat infections. The ability to detect drug resistant parasites in livestock and humans is key to effective control; better control in livestock reduces the risk of transmission to humans and if humans get infected, testing for resistance can inform on the off-licence use of alternative therapeutics. We need a molecular diagnostic for triclabendazole resistance in Fasciola spp that can be used on both livestock and humans in LMIC settings.Project aims and objectivesFasciolosis is caused by two parasites, Fasciola hepatica and F. gigantica. Fasciola spp have a complex demography, they are either present as species-specific infections (e.g Fasciola hepatica in Peru) or as hybrid forms in regions where their distributions overlap (e.g in India and Pakistan) and introgression of genetic loci between the species has been reported. We have made substantial inroads into identifying a genomic locus that confers triclabendazole resistance in F. hepatica and have localised it to a cluster of seven genes; amongst which are two candidate genes, an ADP ribosylation factor (ARF) and ABC genes (ABCB1), both of which have non-synonymous SNPs that are under selection by triclabendazole. In livestock detection of infection is by coproantigen/presence of eggs in faeces or by antibody detection in serum. Similarly, human faecal samples can be used to detect eggs or Fasciola spp DNA. This project aims to build on our existing understanding of triclabendazole resistance in livestock to mitigate its impact on human health. We will: 1. Conduct whole genome re-sequencing from multiple isolates from both livestock and humans from countries representing different demographics (Peru, India, Pakistan, Cameroon and Senegal)2. Carry out comparative genomics to localise genetic exchange within F. gigantica, F. hepatica and hybrids/introgressed species, with a particular focus on triclabendazole-resistance genes.3. Determine if triclabendazole selection is placed on SNPs within ARF and ABCB1 genes in multiple F. hepatica and F. gigantica isolates of known resistance status from both livestock and humans. 4. Optimise diagnostic molecular methods for species-specific detection of Fasciola spp in faecal samples and duodenal/bile aspirates in humans, in collaboration with clinicians in India and Peru.5. Establish proof-of-principal methodology for molecular diagnostic detection e.g Lec-lamp, to detect SNPs in triclabendazole resistance genes in F. hepatica and F. gigantica isolates for point-of-care use in LMIC settings.The goal of this project is to generate novel genomic sequence data, mapping this to current reference genomes and interrogate gene annotations from multiple liver fluke isolates in order to determine whether resistance genes are conserved across the different species and hybrid forms of Fasciola. Candidate gene sequence variation between known drug resistant and susceptible isolates will be interrogated in order to identify gene changes linked to resistance (resistance markers). Finally, a bespoke molecular diagnostic tool will be developed to detect these resistance markers in samples from livestock and humans. The project aims to combine discovery science with applied diagnostics to positively impact on livestock and human heal