Characterisation of drug resistance in field-collected schistosomes

大规模的吡喹酮（PZQ）单药治疗项目是控制 消灭血吸虫病微囊体中耐药性的潜力是药物治疗的关键领域。 研究，因为我们扩大治疗覆盖面。在实验室中，PZQ抗性（PZQ-R）易于选择， 导致药物反应的差异>368倍，遗传分析明确确定了一个区域， Chr. 3含有瞬时受体电位（TRP）通道。在肯尼亚西部， 在最近的一项研究中， （SCORE）我们观察了几个未能降低感染强度和流行率的“热点”村庄， 尽管每年都用PZQ进行大量治疗。一种假设是PZQ-R有助于 观察到大规模PZQ治疗未能降低感染。在目标1中，我们会问， “热点”村庄的感染率可以用耐药性来解释。我们建立了一个小说 该平台用于测试来自田间环境的个体成体染色体中PZQ-R的程度。这么做 我们建立了一个大型的蜗牛繁殖区和仓鼠繁殖设施。因此我们可以 通过（i）收获S.感染的曼氏菌卵 患者，（ii）用毛蚴感染蜗牛，（iii）用释放的尾蚴感染仓鼠，和（iv）灌注 仓鼠体内的蠕虫我们将筛选个体S的药物反应。曼氏蠕虫 在96孔板上，并使用简单的L-乳酸测定暴露于PZQ。通过这种方式，我们将直接 比较热点和非热点村寄生虫的耐药状况。我们还将比较游泳池 的PZQ-R和PZQ-S寄生虫分离的领域，以确定基因组区域的基础上， 观察到的差异，并测试假设，在TRP通道的遗传变异的chr。 是PZQ耐药性的基础。在aim 2中，我们将利用我们独特的平台来检查遗传 对新药物的耐药性变异。一组显示出强活性的化合物 现在可以使用了。最近的实地研究表明， 在东非，这种药物的使用最少， 自由生活的线虫C.秀丽线虫对一系列驱虫药显示出天然的抗性， 化合物.我们将批判性地检验这一假设，即抗性的“常设变异”是常见的， 一些药物正在开发中。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。获得的知识将是至关重要的- 通过大规模PZQ治疗消除血吸虫病的努力，将提供有价值的信息 用于开发新的抗肿瘤药物。该项目还将促进研究的发展 在肯尼亚，基因组测序、计算生物学和驱虫药理学能力得到加强。

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.