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对体液和组织中的病原体和/或其产物的直接检测。这种方法的灵敏度和检测速度有限。我们建议开发一种新的基于宿主的血液检测诊断检测平台，结合：(1)抗原特异性免疫反应的准确性，(2)荧光原位杂交（FISH）核酸检测的多功能性和稳健性，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淋巴细胞可诱导关键细胞因子（IL-2， IFN-γ和TNF-α） mRNA的表达，这些mRNA可通过基因特异性FISH探针和单参数和多参数流式细胞术检测到。我们现在提议与牛津免疫公司合作，通过临床前产品开发来诊断结核病（TB），牛津免疫公司是fda批准的基于宿主的结核病诊断的全球领导者。之所以选择结核病，是因为：（一）它是一个全球性的卫生问题；(2)

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-γ and TNF-α) 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.