Rapid High-Throughput Mycobacterium Tuberculosis Genotyping

描述（由申请人提供）：预防结核分枝杆菌耐多药和极端耐多药菌株（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，目前不可能在作出阳性诊断后的几个月内获得及时和准确的抗生素敏感性数据。这种延误大大增加了疾病传播的风险。具有讽刺意味的是，对治疗结核分枝杆菌的最重要药物的耐药机制已经很清楚，应该可以通过快速分子方法确定。然而，开发一种快速准确的结核分枝杆菌耐药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。2)。2 .耐药分子测定必须达到接近95%的准确度才能发挥作用，因此必须覆盖整个“耐药空间”。新出现的耐药突变比分子方法的开发、验证、批准和上市要快得多。在我们的I期项目中，我们成功地证明了这些障碍可以通过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，目前以Ibis T5000商业化销售，为跟上不断发展的微生物的耐药性难题提供了高通量和经济有效的解决方案。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，我们成功地确定了大约95%的已知与耐药性相关的突变。在第二阶段的研究中，我们建议扩展这一模式，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。我们通过采用“内/外”策略解决了跟上进化突变的难题。在碱基分析中，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，而无需预测它们并更改分析。在热点之外发生的新的耐药突变将通过在分析中留下扩展空间来覆盖，以便可以快速添加新的引物对，而不会干扰分析的其余部分。我们还建议开发一种廉价和简单的样品制备方案，并将通过筛选纽约市卫生部大约一年的结核病样品（约750株）来验证扩大的分析方法，在那里将通过常规培养方法确认耐药性。II期申请的交付成果将是一种经过验证的产品，该产品可识别耐药和耐多药/广泛耐药结核病，其水平适合采取公共卫生行动，并准备进行前瞻性人体临床试验。公共卫生相关性：预防多重耐药和极端多重耐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。我们建议开发并验证一种产品，该产品将在获得培养物后的数小时内确定结核分枝杆菌菌株的耐药谱，而目前的技术需要数月时间。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对结核病患者进行适当的抗生素治疗。

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.