FISH-Flow platform for host-based tuberculosis diagnostics

描述（由申请人提供）：感染的诊断通常基于体液和组织中病原体和/或其产物的直接检测。这种方法具有有限的灵敏度和检测速度。我们建议开发一种新的基于宿主的诊断检测平台，用于基于血液的检测，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）流式细胞术单细胞分析的诊断和预后能力。我们的初步工作表明，体外刺激外周血T淋巴细胞诱导关键细胞因子（IL-2，IFN-？和TNF-？）通过与基因特异性FISH探针杂交以及单参数和多参数流式细胞术检测。我们现在建议通过临床前产品开发，与FDA批准的基于宿主的结核病诊断的世界领导者Oxford Immunotec合作，将该平台用于结核病（TB）诊断。选择结核病是因为：（i）它是一个全球性的健康问题;（ii） M.通过检测抗原特异性T细胞应答来诊断结核感染;和（iii）i具有从无症状潜伏感染（LTBI）到活动性肺结核（PTB）的复杂临床表现。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具有上级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相关。我们将优化关键的检测参数，并与现有FDA批准的检测方法相比，验证所得标准化检测方法的方案稳健性和重现性、检测持续时间以及诊断准确性的初步评估。还将探索未来套件的可制造性。第一个结果将是用于LTBI诊断的简单且准确的单细胞因子测定，其等于或超过现有LTBI诊断的临床准确性，并且在速度和易于执行方面超过它们。第二个结果将是评价用于区分LTBI和PTB的三种细胞因子读数。PI研究中心和Oxford Immunotec与结核病低流行率和高流行率地区的临床研究中心建立的合作将提供所有结核病诊断组的献血者血液样本。我们还表明，FISH流协议是适合于适应基于微流体的自动化平台。第三个成果将是流式细胞术检测上游的所有测定步骤（从样品制备到核酸染色）的自动化，以获得使用标准流式细胞术操作的半自动化装置。这项工作由我们在诊断设备商业化方面经验丰富的生物工程师合作伙伴进行，最终将导致一种具有样品输入-回答能力的手持式全自动设备。手动和自动版本的FISH-Flow平台将适用于检测许多感染性和非感染性病理中的受体介导的反应。

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.