Rapid High-Throughput Mycobacterium Tuberculosis Genotyping

快速高通量结核分枝杆菌基因分型

• 批准号: 7616235
• 负责人: CHRISTIAN MASSIRE
• 金额: $ 37.92万
• 依托单位: IBIS BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616235
• 关键词: Achievement; Adopted; Antibiotic Therapy; Antibiotic susceptibility; Area; Base Composition; Biological Assay; Clinical Trials; Collection; Couples; DNA; Data; Development; Diagnosis; Disease; Drug resistance; Genes; Genotype; Gold; Growth; Health; Hot Spot; Hour; Human; Laboratories; Left; Location; Marketing; Mediating; Methodology; Methods; Microbe; Molecular; Molecular Diagnostic Techniques; Multi-Drug Resistance; Mutation; Mycobacterium tuberculosis; New York City; Nucleotides; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physicians; Preparation; Process; Protocols documentation; Public Health; Reaction; Resistance; Resistance profile; Resolution; Rest; Rifampin; Risk; Sampling; Screening procedure; Solutions; Spectrometry, Mass, Electrospray Ionization; Technology; Testing; Tigers; Time; Treatment Protocols; Tuberculosis; Validation; base; cost effective; design; isoniazid; pathogen; prevent; programs; prospective; public health relevance; resistance mechanism; resistance mutation; resistant strain; tuberculosis drugs

DESCRIPTION (provided by applicant): Preventing the spread of multidrug-resistant and extreme multidrug-resistant strains of Mycobacterium tuberculosis (MDR/XDR) is an important public health problem. Because of the slow growth of Mycobacterium tuberculosis, it is currently impossible to obtain timely and accurate antibiotic susceptibility data for up to several months after a positive diagnosis has been made. This delay substantially increases the risk of spreading the disease. Ironically, the mechanisms of resistance to the most important drugs to treat Mycobacterium tuberculosis are well understood and should be amenable to determination by rapid molecular methods. However, development of a rapid and accurate molecular test for drug resistance in Mycobacterium tuberculosis has proven to be elusive because 1.) Mutations that mediate drug resistance are dispersed across several regions of over a dozen genes. 2.) Molecular determination of drug resistance must be nearly 95% accurate in order to be useful, therefore the entire "resistance space" must be covered, and 3.) Emerging new drug resistance mutations occur more quickly than molecular methods can be developed, validated, approved, and marketed. In our Phase I program, we successfully demonstrated that these hurdles can be overcome using a powerful new methodology that couples multiplexed PCR reactions with electrospray ionization mass spectrometry (PCR/ESI-MS) and high resolution base composition analysis of the amplicons. This technology, previously known as TIGER and currently sold commercially as the Ibis T5000, offers a high throughput and cost effective solution to the conundrum of keeping up with the drug resistance of an evolving microbe. In Phase I we proved this principle by analyzing several key "hot spots" in drug-resistance genes. In this analysis we successfully identified approximately 95% of the mutations known to correlate with drug resistance. In the Phase II effort we propose to expand this paradigm to achieve the theoretical maximum sensitivity for a molecular method for determining the drug-resistance for all important TB drugs. We solved the conundrum of keeping up with evolving mutations by adopting an "inside/outside" strategy. In the base assay, we comprehensively cover the area inside the hot spots so that new mutations within these regions are automatically detected without having to anticipate them and change the assay. New drug-resistance mutations that occur outside the hot spots will be covered by leaving expansion space in the assay so that new primer pairs can be rapidly added without disturbing the rest of the assay. We also propose to develop an inexpensive and simple sample preparation protocol and will validate the expanded assay by screening approximately one year of TB samples (about 750 isolates) from the New York City Department of Health, where drug resistance will be confirmed by conventional culture methods. The deliverable of this Phase II application will be a validated product that identifies drug resistant and MDR/XDR TB at a level suitable for public health action and ready for a prospective human clinical trial. PUBLIC HEALTH RELEVANCE: Preventing the spread of multidrug-resistant and extreme multidrug-resistant strains of Mycobacterium tuberculosis (MDR/XDR) is an important public health problem. We propose to develop and validate a product that will determine the drug-resistance profile of a Mycobacterium tuberculosis strain within hours of obtaining the culture, as opposed to months with the current technology. The product will be validated at a level suitable for immediate use by public health officials, and it will be ready to be tested in a human clinical trial to prove that physicians can reliably use this product to direct appropriate antibiotic treatment for TB patients.

描述（申请人提供）：预防结核分枝杆菌多重耐药和极端多重耐药菌株（MDR/XDR）的传播是一个重要的公共卫生问题。由于结核分枝杆菌生长缓慢，目前不可能在阳性诊断后长达几个月的时间内获得及时准确的抗生素敏感性数据。这种拖延大大增加了疾病传播的风险。具有讽刺意味的是，对治疗结核分枝杆菌的最重要药物的耐药机制已经很好地理解，并且应该能够通过快速分子方法来确定。然而，开发一种快速准确的结核分枝杆菌耐药性分子检测方法已被证明是难以捉摸的，因为1。介导耐药性的突变分散在十几个基因的几个区域中。2.）的情况。药物抗性的分子测定必须接近95%的准确度才能有用，因此必须覆盖整个“抗性空间”，以及3.）新出现的耐药突变比分子方法的开发、验证、批准和上市更快。在我们的I期项目中，我们成功地证明了这些障碍可以使用一种强大的新方法来克服，该方法将多重PCR反应与电喷雾电离质谱（PCR/ESI-MS）和扩增子的高分辨率碱基组成分析相结合。这项技术以前被称为TIGER，目前作为Ibis T5000在商业上销售，为跟上不断发展的微生物的耐药性难题提供了高通量和成本效益的解决方案。在第一阶段，我们通过分析耐药基因中的几个关键“热点”来证明这一原理。在这项分析中，我们成功地鉴定了大约95%的已知与耐药性相关的突变。在第二阶段的努力中，我们建议扩大这一范例，以达到理论上的最大灵敏度的分子方法，确定所有重要的结核病药物的耐药性。我们通过采用“内部/外部”策略解决了跟上进化突变的难题。在基础检测中，我们全面覆盖了热点内的区域，以便自动检测这些区域内的新突变，而无需预测它们并改变检测。在热点之外发生的新的耐药突变将通过在测定中留下扩展空间来覆盖，以便可以快速添加新的引物对，而不会干扰测定的其余部分。我们还建议制定一个廉价和简单的样品制备方案，并将通过筛选来自纽约市卫生部的大约一年的结核病样品（约750个分离株）来验证扩展的检测方法，其中将通过常规培养方法确认耐药性。第二阶段申请的可交付成果将是一种经过验证的产品，可在适合公共卫生行动的水平上识别耐药和MDR/XDR结核病，并为前瞻性人体临床试验做好准备。 公共卫生关系：预防结核分枝杆菌多重耐药和极端多重耐药菌株（MDR/XDR）的传播是一个重要的公共卫生问题。我们建议开发和验证一种产品，该产品将在获得培养物后数小时内确定结核分枝杆菌菌株的耐药性，而不是目前技术的数月。该产品将在适合公共卫生官员立即使用的水平上进行验证，并准备在人体临床试验中进行测试，以证明医生可以可靠地使用该产品指导结核病患者的适当抗生素治疗。