Characterisation of drug resistance in field-collected schistosomes

使用吡喹酮(PZQ)单一疗法的大规模治疗方案是努力控制 消灭血吸虫病。血吸虫的耐药性潜力是一个关键领域 在我们扩大治疗覆盖面的同时进行研究。在实验室中，PZQ电阻(PZQ-R)很容易选择， 结果导致药物反应的368倍差异，遗传分析明确地识别了 Cr.3含有瞬时受体电位(Trp)通道。在肯尼亚西部，大约30%的 血吸虫感染患者在服用PZQ治疗后卵子呈阳性，最近的一项研究 (得分)我们观察到了几个未能降低感染强度和流行率的“热点”村庄， 尽管每年用PZQ进行多次集体治疗。一种假设是PZQ-R有助于 观察到大量PZQ治疗未能降低感染。在目标1中，我们会问，坚持不懈 “热点”村庄感染的可能性可以用耐药性来解释。我们已经建立了一部小说 用于现场测试单个成体血吸虫体内PZQ-R范围的平台。要做到这一点 我们已经建立了大型的蜗牛养殖基地，以及仓鼠养殖设施。因此，我们可以 通过(I)从受感染的曼氏血吸虫卵中获取大量田间衍生的成虫种群 病人，(Ii)用毛虫感染蜗牛，(Iii)用释放的尾蚴感染仓鼠，和(Iv)灌流 仓鼠身上的成虫。我们将对维持的曼氏链球菌个体的药物反应进行筛选 在96孔板上，用简单的L-乳酸测定法暴露在PZQ中。使用这种方法，我们将直接 比较热点村与非热点村寄生虫的抗药性状况。我们还将比较池 从野外分离的PZQ-R和PZQ-S寄生虫的基因组区域 观察到的差异，并检验CHR上Trp通道的遗传变异。3. 这是PZQ在田野中抵抗的基础。在目标2中，我们将利用我们独特的平台来检测基因 血吸虫新药耐药性的变异。一系列具有很强活性的化合物 抗血吸虫药物现已问世。最近的实地研究表明，高水平的自然发生 奥沙米昆在东非使用最少的地区的耐药性，以及类似的研究 对于自由生活的线虫，线虫对一系列驱虫剂表现出自然产生的抗性 化合物。我们将批判性地检验这一假说，即抗药性的“常态变异”普遍存在于 血吸虫药物正在研发中。然后，我们将比较药物极端情况下的单个蠕虫 反应谱以确定抗性的遗传基础。所获得的知识将对- 通过大规模PZQ治疗进行血吸虫病消灭工作，将提供有价值的信息 用于开发新的抗血吸虫药物。该项目还将刺激研究的发展 肯尼亚在基因组测序、计算生物学和驱虫药理学方面的能力。

Large-scale treatment programs with praziquantel (PZQ) monotherapy are the mainstay of efforts to control and eliminate schistosomiasis. The potential for drug resistance in schistosomes is a critical area of research as we scale up treatment coverage. In the laboratory, PZQ resistance (PZQ-R) is easily selected, results in a >368-fold difference in drug response, and genetic analyses unambiguously identify a region of chr. 3 containing a transient receptor potential (TRP) channel. In Western Kenya, approximately 30% of schistosome-infected patients are egg-positive following treatment with PZQ, and in a recent study (SCORE) we observed several “hotspot” villages that failed to lower infection intensities and prevalence, despite multiple annual mass treatments with PZQ. One hypothesis is that PZQ-R contributes to the observed failure of mass PZQ treatment to lower infection. In aim 1 we will ask whether the persistence of infection in “hotspot” villages can be explained by resistance. We have established a novel platform for testing for the extent of PZQ-R in individual adult schistosomes from a field setting. To do this we have established a large snail breeding colony, and hamster breeding facility. We can therefore generate large populations of field-derived adult worms, by (i) harvesting S. mansoni eggs from infected patients, (ii) infecting snails with miracidia, (iii) infecting hamsters with released cercariae, and (iv) perfusing adult worms from hamsters. We will screen the drug response of individual S. mansoni worms maintained on 96-well plates and exposed to PZQ using a simple L-Lactate assay. Using this approach, we will directly compare resistance status of parasites from hotspot and non-hotspot villages. We will also compare pools of PZQ-R and PZQ-S parasites isolated from the field to determine the genome regions that underlie the differences observed, and to test the hypothesis that genetic variation in the TRP channel on chr. 3 underlies PZQ resistance in the field. In aim 2 we will exploit our unique platform to examine genetic variation for resistance to new schistosome drugs. A suite of compounds showing strong activity against schistosomes is now available. Recent field studies have shown high levels of naturally occurring resistance to oxamniquine in East Africa where this drug has been minimally used, and comparable work with the free-living nematode C. elegans shows naturally occurring resistance to a range of anthelmintic compounds. We will critically test the hypothesis that “standing variation” for resistance is common against schistosome drugs under development. We will then compare individual worms at the extremes of the drug response spectrum to identify the genetic basis of resistance. The knowledge gained will be critical to on- going schistosomiasis elimination efforts through mass PZQ treatment and will provide valuable information for the development of new anti-schistosome drugs. The project will also stimulate development of research capacity in genome sequencing, computational biology and anthelminthic pharmacology in Kenya.