Rapid Detection of Multiple Drug resistant TB from Sputum

从痰中快速检测多重耐药结核病

• 批准号: 9140204
• 负责人: John F Davidson
• 金额: $ 21.6万
• 依托单位: TANGEN BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9140204
• 关键词: AIDS/HIV problem; Acceleration; Adverse effects; Alleles; Biological Assay; Caring; Cessation of life; Clinic; Clinical; Communicable Diseases; Complex; Cytolysis; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Discrimination; Disease; Disinfection; Drug resistance; Drug resistance in tuberculosis; Dyes; Early Diagnosis; Failure; Filtration; Genes; Genetic; Genomics; Hand; Health; Heating; Human; Incidence; Individual; Infection; Injection of therapeutic agent; Location; Mediating; Molecular Diagnosis; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Mycobacterium tuberculosis; Patient-Focused Outcomes; Patients; Peripheral; Pharmaceutical Preparations; Point-of-Care Systems; Preparation; Process; Reaction; Reagent; Reporting; Reproducibility; Resistance; Rifampicin resistance; Rifampin; Sampling; Sensitivity and Specificity; Sonication; Sputum; Staging; System; Technology; Temperature; Testing; Time; Tuberculosis; World Health Organization; base; cost; design; drug sensitivity; effective therapy; evaluation/testing; instrument; isoniazid; mortality; mutant; mycobacterial; non-tuberculosis mycobacteria; point of care; public health relevance; rapid detection; resistance allele; synthetic construct; touchscreen

 DESCRIPTION (provided by applicant): Tuberculosis (TB) is a leading cause of mortality from an infectious disease in the world, second only to HIV/AIDS. The WHO reports that in 2013 there were 1.5M deaths from TB. When a case of TB does not respond to two or more of the first-line drugs available, the infection is considered to be multidrug-resistant (MDR-TB). The rising incidence of MDR-TB is leading to poor patient outcomes since the second line drugs are introduced at a late stage of the disease. These drugs are expensive, require injection and have serious side effects, therefore cannot be administered before a diagnosis of MDR-TB occurs. The majority of TB cases are seen at peripheral health clinics, with no access to current diagnosis technologies. Early diagnosis, before the failure of the first line drugs, is needed for effective treatment, as well as for isolation of infected individuals. We propose to develop a test that will detect the 15 known Mycobacteria tuberculosis alleles that are found in 96-98% of resistance to the main first line drugs, Rifampicin and Isoniazid, using our hand-held, battery powered TangenDx Point of Care (POC) diagnosis system. TangenDx consists of a sample preparation device, which is followed by mycobacterial capture via filtration, at which point, lysing and amplification solutions are introduced. Lysis is performed by sonication, after which the sample is introduced into a disposable assay disk for DNA extraction and amplification, using isothermal amplification and real time detection via fluorescent intercalating dyes. The assay amplifies the hspx gene, which is unique to MTB. A built-in precise temperature controller enables the use of the highly robust and versatile loop-mediated isothermal amplification (LAMP). STEM primers are used with the LAMP technology to accelerate reactions. We were able to detect 8-40 CFUs of TB per disk assay or 20 genomic copies with >95% confidence. We propose to add DST to our TangenDx TB Assay to enable detection of 96-98% of all known MDR-TB strains by following these specific aims: In Specific Aim 1, LAMP primer sets specific to the three genetic locations associated with resistance to rifampicin and isoniazid will be designed and screened. STEM primers to each of the mutant alleles will be designed and tested for their ability to selectively accelerate LAMP reactions using synthetic DNA targets. Finally, we will determine the conditions that allow amplification and multiplexing of the three MDR-TB genetic regions (rpoB, katG and inhA) in the central chamber of the Tangen Dx Disk prior to STEM specific acceleration of specific LAMP reactions in the detection chambers. In Specific Aim 2, the optimized primer sets and conditions of Aim 1 will be tested with the whole TangenDx TB Assay system using characterized drug resistant TB strains spiked into healthy human sputum samples. The proposed system would make quick MDR-TB diagnosis accessible and feasible in regions of the world where such technologies are lacking, and where TB is most widely spread. A timely diagnosis, with an accurate DST evaluation, could significantly lower TB associated mortality and costs of care worldwide.

 描述（由适用提供）：结核病（TB）是世界上传染病死亡率的主要原因，仅次于艾滋病毒/艾滋病。世卫组织报道说，2013年，结核病死亡150万。当结核病病例对两种或多种一线药物无反应时，该感染被认为是耐多药物（MDR-TB）。 MDR-TB的上升事件导致患者的结果差，因为第二行药物是在疾病的后期引入的。这些药物很昂贵，需要注射并具有严重的副作用，因此在诊断为MDR-TB之前不能给药。大多数结核病病例都在外围健康诊所看到，无法获得当前的诊断技术。在第一行药物失败之前，早期诊断是有效治疗以及被感染个体的隔离所需的。我们建议开发测试 这将检测到15个已知的结核细菌性结核病等位基因，这些等位基因在96-98％的对主要第一线药物的耐药性，利福平和异尼氏二氮二氮的耐药性中，使用我们的手持电池供电的Tangendx护理点（POC）诊断系统。 Tangendx由样品制备装置组成，然后通过过滤进行分枝杆菌捕获，此时引入了裂解和放大溶液。裂解是通过超声处理进行的，然后将样品引入一次性测定磁盘中，以进行DNA提取和放大，并使用等温扩增和通过荧光插入式染料进行实时检测。测定放大器HSPX基因是MTB所独有的。内置的精确温度控制器可实现高度稳健和多功能环的等温放大（LAMP）的使用。 STEM引物与LAMP技术一起加速反应。我们能够检测每个磁盘测定法的8-40个TB或20个基因组拷贝，置信度> 95％。我们建议将DST添加到我们的Tangendx TB分析中，以实现所有已知的MDR-TB菌株的96-98％，通过遵循以下特定目的：在特定的AIM 1中，将设计和筛选与对利福平抗性和抗性相关的三个遗传位置的灯底漆集。将对每个突变等位基因的茎底漆进行设计和测试，以选择其使用合成DNA靶标有选择性加速灯反应的能力。最后，我们 将确定允许在检测室中特定灯反应的茎特异性加速之前，允许三个MDR-TB遗传区域（RPOB，KATG和INHA）扩增和多路复用。在特定的目标2中，将使用整个Tangendx结核病测定系统测试优化的AIM 1的底漆集和条件。拟议的系统将使缺乏此类技术的世界以及结核病最广泛传播的世界各地的MDR-TB诊断能够访问和可行。具有准确的DST评估的及时诊断可能会显着降低与全球的结核病相关死亡率和护理成本。