Rapid High-Throughput Mycobacterium Tuberculosis Genotyping

快速高通量结核分枝杆菌基因分型

• 批准号: 7483737
• 负责人: CHRISTIAN MASSIRE
• 金额: $ 37.08万
• 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/7483737
• 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; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium tuberculosis; New York City; Nucleotides; Patients; Pharmaceutical Preparations; Phase; Phenotype; Physicians; Polymerase Chain Reaction; Preparation; Process; Protocols documentation; Public Health; Reaction; Resistance; Resistance profile; Resolution; Rest; Rifampin; Risk; Sampling; Screening procedure; Solutions; Spectrometry, Mass, Electrospray Ionization; Standards of Weights and Measures; Technology; Testing; Tigers; Time; Treatment Protocols; Tuberculosis; Validation; base; cost effective; design; isoniazid; pathogen; prevent; programs; prospective; resistance mechanism; 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(由申请人提供)：防止耐多药和极端耐多药结核分枝杆菌(MDR/XDR)菌株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，目前不可能在做出阳性诊断后的几个月内及时和准确地获得抗生素敏感性数据。这种延误大大增加了疾病传播的风险。具有讽刺意味的是，治疗结核分枝杆菌最重要的药物的耐药性机制已经被很好地理解了，应该可以用快速的分子方法来确定。然而，发展一种快速而准确的结核分枝杆菌耐药性分子检测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。2.)抗药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“抗药性空间”。新出现的耐药突变发生的速度比分子方法开发、验证、批准和上市更快。在我们的第一阶段计划中，我们成功地证明了使用一种强大的新方法可以克服这些障碍，该方法将多重PCR反应与电喷雾电离质谱仪(PCR/ESI-MS)和高分辨率的扩增碱基组成分析相结合。这项技术以前被称为TIGER，目前在商业上以IBIS T5000的名称出售，它为跟上不断进化的微生物的耐药性这一难题提供了一种高产量和高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，我们成功地识别了大约95%已知的与耐药性相关的突变。在第二阶段的工作中，我们建议扩展这一范例，以达到确定所有重要结核病药物耐药性的分子方法的理论最大灵敏度。我们采取了“内部/外部”的策略，解决了跟上不断演变的突变的难题。在基础检测中，我们全面覆盖热点内的区域，以便自动检测这些区域内的新突变，而不必预测它们和改变检测。发生在热点之外的新的耐药突变将通过在检测中留出扩展空间来覆盖，以便可以快速添加新的引物对，而不会干扰检测的其余部分。我们还建议开发一种廉价和简单的样本制备方案，并将通过从纽约市卫生局筛选大约一年的结核病样本(约750株)来验证扩大的检测方法，在那里，耐药性将通过传统的培养方法来确认。这一第二阶段应用程序的交付将是一种经过验证的产品，可以在适合公共卫生行动的水平上识别耐药和耐多药/广泛耐药结核病，并为预期的人类临床试验做好准备。公共卫生相关性：防止耐多药和极端耐多药结核分枝杆菌菌株(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几个小时内确定结核分枝杆菌菌株的耐药性，而不是使用目前的技术数月。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，并将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进行适当的抗生素治疗。