Rapid characterization of mycobacteria and drug resistance in paucibacillary TB

描述(申请人提供)：结核病(TB)控制的全球挑战是多药耐药(MDR-TB；NIAID生物防御议程下的C类优先病原体)以及老年人、艾滋病毒感染、2型糖尿病和其他免疫抑制慢性病等人群的易感性增加。我们建议利用我们在南得克萨斯州/墨西哥边境两侧和哥伦比亚麦德林建立的研究地点，那里耐多药结核病和2型糖尿病的发病率很高。我们将使用我们最近开发的提取/实时聚合酶链式反应(QPCR)技术来研究结核病的发病机制和耐多药结核病在这些人群中的演变。我们最初的重点将放在少菌病上，如肺外结核病(EPTB)和涂阳肺结核(PTB)。在目标1中，我们研究了我们的高灵敏度和特异性的结核DNA定量聚合酶链式反应的潜力，以改进对结核杆菌的检测，并对其在几个不同的细胞和生理间隔中的分布进行定量。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)。它也足够灵敏地检测包括耐多药结核在内的少杆菌结核标本中的分枝杆菌DNA，在这些样本中，诊断和发病机制是主要挑战。在目标2中，我们采用我们的使用分子信标探针检测突变的qPCR方法来检测利福平(RIF)，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qPCR检测耐药突变的可行性，从目前定性的“存在”或“不存在”，到量化RIF耐药与敏感细菌的比率。这一方法将为研究可能感染耐药和敏感结核杆菌混合人群的患者的耐药性演变提供一种手段。我们对糖尿病患者在这方面特别感兴趣，我们已经证明他们特别容易患耐多药结核病的复发性疾病。PI是一名助理教授，在一个医疗服务不足的社区工作，该社区的特点是艾滋病毒合并感染率低，但2型糖尿病的发病率高。R21的资金对于产生初步数据至关重要，这些数据将加强未来对耐多药结核病在高危人群中的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.