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

描述（由申请方提供）：长期目标是在更全面了解抗药性机制的基础上，促进在疟疾和丝虫控制活动中合理使用杀虫剂。该提案的具体目标是：i）鉴定使冈比亚按蚊s.l.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（苯敌威）和II类拟除虫菊酯（？氯氟氰菊酯）。ii）开发基于DNA的杀虫剂抗药性筛查工具，供撒哈拉以南非洲受疟疾和丝虫病影响国家的病媒控制方案工作人员使用。这些例行的抗药性筛选将允许对突变进行例行监测，提供对预测IRS和ITN方案成功与否至关重要的信息。 将分三个具体阶段实现这一目标： 第一阶段：利用候选基因关联图谱和全基因组微阵列发现耐药相关基因座。在两个重点非洲国家，基因可能涉及对三种主要的室内滞留杀虫剂（滴滴涕、苯敌威、？氯氟氰菊酯）将使用两种互补的方法来鉴定：i）使用定制的候选基因1536重SNP阵列来研究DNA水平上的变异，和ii）通过全基因组微阵列来研究抗性冈比亚按蚊和易感冈比亚按蚊之间的差异基因表达。在最有希望的基因或基因调节子内部和周围的推定的抗性相关SNP将被推进到第2阶段，以进行更广泛的田间验证。 第2阶段-在撒哈拉以南非洲的PMI和TDR网络国家进行候选基因验证。通过与现存的世界卫生组织/热带疾病研究（TDR）和总统疟疾倡议（PMI）计划合作，将在来自撒哈拉以南非洲大部分地区的更大样本中筛选第1阶段确定的推定耐药相关SNP与耐药性的关联。目的是筛选SNP与群体内和群体间耐药性的关联，产生多位点关联荟萃分析，以鉴定具有主要和/或一般效应的耐药性相关SNP。 第3阶段-将代谢抗性标记物整合到现场适用的筛选工具中。该方案的最后阶段将涉及设计、测试和推出一种适用于实地的筛查工具，其中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。FAST诊断将包括对IRS杀虫剂抗性的最重要的遗传标记，以及任何关键的预先确定的标记（例如，用于物种/分子形式鉴定）。FAST的推广将通过与现有的创新病媒控制联盟、TDR和PMI方案合作进行。 按蚊抗药性的发展是疟疾控制的主要威胁。评估抗性的传统手段成本高、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耐药性仍然处于低频率时和发生控制失败之前检测到耐药性。

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.