Development of a Field Applicable Screening Tool (FAST) kit for detecting resista

描述(申请人提供)：长期目标是促进在疟疾和丝虫病控制运动中合理使用杀虫剂，其基础是对耐药性机制有更全面的了解。这项提议的具体目的是：i)识别导致冈比亚按蚊的基因。蚊子对用于疟疾和丝虫病的室内滞留喷洒(IRS)的一系列杀虫剂，即有机氯(DDT)、氨基甲酸酯(苯敌畏)和二类拟除虫菊酯(-氯氟氰菊酯)具有抵抗力。二)开发基于DNA的杀虫剂抗药性筛查工具，供受疟疾和丝虫病影响的撒哈拉以南非洲国家的病媒控制方案工作人员使用。这些常规耐药筛查将允许对突变进行常规监测，提供对预测IRS和ITN计划成功至关重要的信息。 这一目标将分三个具体阶段实现： 阶段1-使用候选基因关联图谱和全基因组微阵列发现抗药性相关基因座。在两个非洲重点国家，将使用两种互补的方法识别可能与三种主要红外线杀虫剂(滴滴涕、本敌畏、-氯氟氰菊酯)抗药性有关的基因：i)将使用定制候选基因1536plex SNP阵列研究DNA水平的变异；ii)将通过全基因组微阵列研究冈比亚按蚊抗药性和敏感性之间的差异基因表达。最有希望的基因或基因调节器内部和周围与抗药性相关的SNPs将被推进到第二阶段，以进行更广泛的田间验证。 阶段2-撒哈拉以南非洲的PMI和TDR网络国家的候选基因验证。将通过与现有的世界卫生组织/热带病研究(TDR)和总统疟疾倡议(PMI)方案合作，从撒哈拉以南非洲大片地区的更大样本中筛选出与第一阶段确定的假定耐药相关的SNPs与耐药性有关。其目的是筛选SNPs与群体内和群体间耐药性的关联，进行多点关联荟萃分析，以确定具有主要和/或一般效应的耐药性相关SNPs。 阶段3-将代谢抗性标记整合到现场适用的筛查工具中。该方案的最后阶段将涉及设计、测试和推出现场适用的筛查工具(FAST)，该工具将包括将在受疾病影响国家的实验室中使用的简单和可靠的诊断方法。快速诊断将包括对红外线杀虫剂的抗性最重要的遗传标记，以及任何关键的预先识别的标记(例如，用于物种/分子形式鉴定)。通过与现有的创新病媒控制联盟(IVCC)、TDR和PMI方案合作，将实现快速推广。 蚊子对杀虫剂产生抗药性是疟疾控制的主要威胁。评估耐药性的传统方法成本高昂、不敏感且不准确。我们建议开发检测方法，使疾病规划管理人员能够在抗药性仍然处于低频率和控制失败发生之前检测到抗药性。

DESCRIPTION (provided by applicant): The long term objective is to facilitate the rational use of insecticides in malaria and filaria control campaigns based by upon a more complete understanding of resistance mechanisms. The specific aims of this proposal are to: i) identify genes that render Anopheles gambiae s.l. mosquitoes resistant to a range of insecticides that are used for indoor residual spraying (IRS) for malaria and filariasis namely an organochloride (DDT), a carbamate (Bendiocarb) and a class II pyrethroid (?-cyhalothrin). ii) develop DNA-based insecticide resistance screening tools for use by the staff of vector control programmes in the malaria and filariasis affected countries of sub-Saharan Africa. These routine resistance screens will permit routine monitoring of mutations giving information vital for predicting the success of IRS and ITN programmes. The objective will be addressed in three specific phases: Phase 1 - The discovery of resistance-associated loci using candidate gene association mapping and whole genome microarrays. In two focal African countries, genes potentially involved in resistance to three major IRS insecticides (DDT, Bendiocarb, ?-cyhalothrin) will be identified using two complementary approaches:- i) Variation at the DNA level will be investigated using a custom candidate gene 1536plex SNP array and ii) differential gene expression between resistant and susceptible Anopheles gambiae will be investigated via whole genome microarrays. Putative resistance-associated SNPs within and around the most promising genes or gene regulators will be taken forward to phase 2 for wider field validation. Phase 2 - Candidate gene validation in the PMI and TDR network countries of sub-Saharan Africa. The putative resistance-associated SNPs identified in phase 1 will be screened for association with resistance in a much larger sample from a large section of sub-Saharan Africa via collaboration with extant World Health Organization/Tropical Disease Research (TDR) and President's Malaria Initiative (PMI) programmes. The aim is to screen for association of SNPs with resistance within and across populations producing a multi-site association meta-analysis to identify resistance-associated SNPs of major and/or generic effect. Phase 3 - Integrating metabolic resistance markers into a Field Applicable Screening Tool. The final phase of the programme will involve design, testing and roll-out of a field applicable screening tool (FAST), which will comprise straightforward and robust diagnostics to be used in laboratories in the disease-affected countries. The FAST diagnostics will include the most important genetic markers for resistance to the IRS insecticides, in addition to any critical pre-identified markers (e.g. for species/ molecular form identification). FAST roll-out will occur via collaboration with extant Innovative Vector Control Consortium (IVCC), TDR and PMI programmes. The development of insecticide resistance in Anopheles mosquitoes is a major threat to malaria control. Conventional means of assessing resistance are costly, insensitive and inaccurate. We propose to develop assays that will allow disease programme managers to detect resistance when it is still at a low frequency and before control failure has occurred.