FISH-Flow platform for host-based tuberculosis diagnostics

描述(申请人提供)：感染的诊断通常基于在体液和组织中直接检测病原体和/或其产物。这种方法的灵敏度和检测速度都很有限。我们建议开发一种新的、基于宿主的血液检测诊断分析平台，该平台结合了(1)抗原特异性免疫反应的精确度，(2)荧光原位杂交(FISH)核酸检测的通用性和稳健性，以及(3)单细胞分析的流式细胞术的诊断和预后能力。我们的初步工作表明，体外刺激外周血T淋巴细胞可以诱导关键细胞因子(IL-2、干扰素-1)的mRNA表达。和肿瘤坏死因子？)这是通过与基因特异的FISH探针以及单参数和多参数流式细胞术杂交来检测的。我们现在建议与牛津免疫技术公司合作，通过临床前结核病诊断产品开发这一平台，牛津免疫技术公司是FDA批准的基于宿主的结核病诊断的世界领先者。结核病之所以被选中是因为：(1)它是一个全球健康问题；(2) 结核分枝杆菌感染是通过检测抗原特异性T细胞反应来诊断的；以及(Iii)I有复杂的临床表现，从无症状的潜伏感染(LTBI)到活动性肺结核(PTB)。将我们的FISH-FLOW平台应用于结核病诊断，预计将产生比现有测试更高的诊断准确性和速度的测试。此外，该平台具有无与伦比的潜力，通过其多参数能力区分LTBI和PTB，因为感染的阶段/进展与不同的细胞因子谱有关。我们将优化关键的检测参数，并与现有FDA批准的检测进行比较，验证得到的标准化检测的协议稳健性和重复性、检测持续时间和诊断准确性的初步评估。还将探索未来的成套件可制造性。第一个结果将是一种简单而准确的LTBI诊断单细胞因子分析，它等于或超过现有LTBI诊断的临床准确性，并在速度和执行容易方面超过它们。第二个结果将是对用于区分LTBI和PTB的三种细胞因子读数的评估。国际结核病防治中心和牛津免疫技术中心与结核病患病率低和高的地区的临床地点建立的合作将提供所有结核病诊断小组的捐赠者的血液样本。我们还表明，鱼流协议适用于基于微流控的自动化平台。第三个结果将是实现流式细胞仪检测上游的所有分析步骤的自动化(从样品制备到核酸染色)，以获得与标准流式细胞仪一起运行的半自动设备。这项工作由我们的生物工程合作伙伴进行，他在诊断设备商业化方面经验丰富，最终应该会产生一种具有样本输入-回答能力的手持式、全自动设备。人工和自动版本的Flow-Flow平台将适用于许多感染性和非感染性病理中受体介导的反应的检测。

DESCRIPTION (provided by applicant): Diagnosis of infection is typically based on direct detection of the pathogen and/or its products in bodily fluids and tissues. This approach has limited sensitivity and speed of detection. We propose to develop a novel, host-based diagnostic assay platform for blood-based assays combining: (1) the precision of antigen-specific immune responses, (2) the versatility and robustness of nucleic acid detection by fluorescence in situ hybridization (FISH), and (3) the diagnostic and prognostic power of single-cell analysis by flow cytometry. Our preliminary work shows that stimulation of peripheral blood T lymphocytes ex vivo induces expression of mRNA for key cytokines (IL-2, IFN-? and TNF-?) that is detected by hybridization with gene-specific FISH probes and single- and multi-parameter flow cytometry. We now propose to move this platform through preclinical product development for diagnosis of tuberculosis (TB) in partnership with Oxford Immunotec, a world leader in FDA-approved, host-based TB diagnostics. TB is the selected pathology because: (i) it is a global health problem; (ii) M. tuberculosis infection is diagnosed by detecting antigen- specific T cell responses; and (iii) i has a complex clinical presentation ranging from asymptomatic, latent infection (LTBI) to active pulmonary tuberculosis (PTB). Application of our FISH-Flow platform to TB diagnosis is expected to yield a test with superior diagnostic accuracy and speed than the existing tests. Moreover, the platform has the unparalleled potential to distinguish between LTBI and PTB through its multi-parameter capacity, since stages/progression of infection are associated with different cytokine profiles. We will optimize key assay parameters and verify the resulting standardized assay for protocol robustness and reproducibility, assay duration, and initial assessment of diagnostic accuracy in comparison with an existing FDA-approved test. Future kit manufacturability will also be explored. The first outcome will be a simple and accurate single- cytokine assay for LTBI diagnosis that equals or exceeds the clinical accuracy of the existing LTBI diagnostics and surpasses them in speed and ease of execution. The second outcome will be the evaluation of the three- cytokine readout for distinguishing LTBI from PTB. Established collaborations at the PI site and at Oxford Immunotec with clinical sites in regions with low and high TB prevalence will provide blood samples from donors in all TB diagnostic groups. We have also shown that the FISH-Flow protocol is amenable to adaption to microfluidics-based, automated platforms. A third outcome will be automation of all assay steps upstream of flow-cytometry detection (from sample preparation to nuclei acid staining) to obtain a semi-automated device operating with standard flow cytometry. This work, conducted by our bioengineer partner who is experienced in commercializing diagnostic devices, should eventually lead to a hand-held, fully automated device with sample-in-answer-out capability. The FISH-Flow platform in its manual and automated versions will be applicable to detection of receptor-mediated responses in many infectious and non-infectious pathologies.