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

摘要：片形吸虫病和一种健康片形吸虫感染牲畜，作为一种人畜共患寄生虫，可导致人类感染。据估计，全世界有240万至1700万人受到感染，特别是在低收入和中等收入国家流行区域。世卫组织承认片形吸虫病是一种被忽视的热带病。三氯苯达唑是治疗牲畜感染的首选药物，因为它适用于所有生命周期阶段，也是唯一获准用于人类的药物。由于强化治疗方案，最初在牲畜物种中报告了片形吸虫对三氯苯达唑的耐药性，这不可避免地导致人类感染对三氯苯达唑耐药的寄生虫，并且未能有效治疗感染。在牲畜和人类中发现耐药寄生虫的能力是有效控制的关键；在牲畜中进行更好的控制可以减少向人类传播的风险，如果人类受到感染，对耐药性的检测可以告知未经许可使用替代疗法的情况。我们需要一种可用于低收入和中等收入国家的牲畜和人类的片形吸虫对三苯达唑耐药的分子诊断方法。片形吸虫病是由肝片形吸虫和巨型片形吸虫两种寄生虫引起的。片形吸虫具有复杂的人口统计学特征，它们要么以物种特异性感染的形式存在（例如秘鲁的肝片形吸虫），要么在其分布重叠的地区（例如印度和巴基斯坦）以杂交形式存在，并且已经报道了物种之间的遗传位点渗入。我们已经取得了重大进展，确定了赋予肝梭菌三氯苯达唑耐药性的基因组位点，并将其定位为7个基因簇；其中有两个候选基因，ADP核糖基化因子（ARF）和ABC基因（ABCB1），这两个基因都具有非同义的snp，正在接受三氯苯达唑的选择。在牲畜中，通过粪便中的粪原抗原/卵或血清中的抗体检测来检测感染。同样，人类粪便样本可用于检测卵子或片形吸虫的DNA。该项目旨在建立我们对牲畜对三氯咪唑耐药性的现有认识，以减轻其对人类健康的影响。我们将：1；对来自不同人口统计学国家（秘鲁、印度、巴基斯坦、喀麦隆和塞内加尔）的牲畜和人的多个分离株进行全基因组重测序2。2 .开展比较基因组学，以定位巨型F.，肝F.和杂交/渐染物种之间的遗传交换，特别关注三氯苯达唑抗性基因。确定从牲畜和人类中分离出的已知耐药状态的多个肝f.s和巨f.s的ARF和ABCB1基因的snp中是否存在三氯苯达唑选择。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/胆汁抽吸物中的片形吸虫进行物种特异性检测。建立分子诊断检测的主要证明方法，例如Lec-lamp，以检测肝念珠菌和巨型念珠菌分离株中三氯苯达唑耐药基因的snp，供低收入和中等收入国家的医疗点使用。该项目的目标是生成新的基因组序列数据，将其映射到当前的参考基因组，并询问来自多个肝吸虫分离物的基因注释，以确定抗性基因是否在不同物种和杂交形式的片形虫中保守。将调查已知耐药菌株和敏感菌株之间的候选基因序列差异，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，以检测来自牲畜和人类样本中的这些耐药性标记。该项目旨在将发现科学与应用诊断相结合，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