Rapid characterization of mycobacteria and drug resistance in paucibacillary TB

描述（由申请人提供）：结核病（TB）控制面临的全球挑战是多重耐药性（MDR-TB; NIAID生物防御议程下的C类优先病原体）以及老年人、HIV感染、2型糖尿病和其他免疫抑制性慢性疾病等人群易感性增加。我们建议利用我们在南德克萨斯州/墨西哥边境两侧和哥伦比亚麦德林的现有研究中心，那里的耐多药结核病和2型糖尿病发病率很高。我们将使用我们最近开发的提取/真实的时间PCR（qPCR）技术来研究这些人群中结核病的发病机制和耐多药结核病的演变。我们最初的重点将是少杆菌病，如肺外结核（EPTB）和涂片阴性肺结核（PTB）。在目标1中，我们检查了我们的TB DNA的高灵敏度和特异性qPCR的潜力，以改善MTB的检测以及定量其在几个不同的细胞和生理区室中的分布。这种非侵入性技术能够确定肺和肺外标本中分枝杆菌的基因拷贝数、细胞位置和性质（完整或裂解）。它还具有足够的灵敏度来检测少杆菌结核病（包括耐多药结核病）标本中的分枝杆菌DNA，其中诊断和发病机制是主要挑战。在目标2中，我们调整了我们的qPCR方法，使用分子信标探针检测利福平（RIF）突变，作为开发肺和肺外标本中MDR-TB快速直接指示剂的第一步。最后，我们将探索通过qPCR改进耐药突变检测的可行性，从目前的定性“存在”或“不存在”耐药性，到定量RIF耐药细菌与敏感细菌的比例。这种方法将提供一种手段来研究可能感染耐药和敏感MTB混合群体的患者的耐药性演变。我们对糖尿病患者的这方面特别感兴趣，我们已经证明他们特别容易患耐多药结核病复发。PI是一名助理教授，在医疗服务不足的社区工作，其特点是艾滋病毒合并感染率低，但2型糖尿病的发病率高。对R21的资助对于产生初步数据至关重要，这些数据将加强未来对高危人群中耐多药结核病发病机制和演变的更复杂研究。

DESCRIPTION (provided by applicant): Global challenges to tuberculosis (TB) control are multidrug-resistance (MDR-TB; a NIAID Category C Priority Pathogen under Biodefense Agenda) and the increased susceptibility of populations such as elderely, HIV infection, type 2 diabetes and other immunosuppressive chronic diseases. We propose to exploit our established study sites both sides of the south Texas/Mexico border and in Medellin, Colombia, where rates of MDR-TB and type 2 diabetes are high. We will use our recently developed extraction/real time PCR (qPCR) techniques to study the pathogenesis of TB and evolution of MDR-TB in these populations. Our initial emphasis will be on paucibacillary disease such as extrapulmonary TB (EPTB) and smear negative pulmonary TB (PTB). In aim 1 we examine the potential of our highly sensitive and specific qPCR for TB DNA to improve the detection of MTB as well as to quantitate its distribution in several different cellular and physiologic compartments. This non-invasive technique has the ability to determine the number of gene copies, cellular location and nature (intact or lysed) of mycobacteria in pulmonary and extra- pulmonary specimens. It is also sufficiently sensitive to detect mycobacterial DNA in specimens from paucibacillary TB, including MDR-TB, where diagnosis and pathogenesis are major challenges. In Aim 2 we adapt our qPCR method using molecular beacon probes for detection of mutations to rifampicin (RIF) as the first step in developing a rapid direct indicator of MDR-TB in pulmonary and extrapulmonary specimens. Finally we will explore the feasibility of refining the detection of resistance mutations by qPCR from the current qualitative "presence" or "absence" of resistance, to quantification of the ratio of RIF-resistant to sensitive bacteria. This approach will provide a means to study the evolution of resistance in patients who may be infected with mixed populations of resistant and sensitive MTB. We are particularly interested in this respect in patients with diabetes who we have shown to be particularly susceptible to recurrent disease with MDR-TB. The PI is an Assistant Professor working in a medically underserved community, characterized by low rates of HIV-co-infection, but high rates of type 2 diabetes. Funding of this R21 is critical to generate preliminary data that will strengthen future, more complex studies of the pathogenesis and evolution of MDR- TB in high-risk populations.