FISH-Flow platform for host-based tuberculosis diagnostics

用于基于宿主的结核病诊断的 FISH-Flow 平台

• 批准号: 8721843
• 负责人: Maria Laura Gennaro
• 金额: $ 112.33万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8721843
• 关键词: Acids; Antigens; Automation; Bacteria; Biological Assay; Biomedical Engineering; Blood; Blood donor; Blood specimen; Cell Nucleus; Cell Separation; Cells; Centrifugation; Cessation of life; Characteristics; Chronic; Clinical; Clinical Management; Clinical Sensitivity; Collaborations; Collection; Color; Complex; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Dose; Evaluation; FDA approved; FarGo; Flow Cytometry; Fluorescent in Situ Hybridization; Future; Genes; Hand; Human; Immune; Immune response; Immunology; Infection; Interferons; Interleukin-2; Lead; Length; Liquid substance; Manuals; Measures; Mediating; Medical; Methodology; Microfluidics; Mycobacterium tuberculosis; Noise; Nucleic Acid Hybridization; Nucleic Acids; Organism; Outcome; Pathogen detection; Pathology; Patients; Performance; Peripheral Blood Mononuclear Cell; Persons; Pharmaceutical Preparations; Population; Preparation; Prevalence; Production; Protocols documentation; Pulmonary Tuberculosis; Quality Control; Reading; Reagent; Reproducibility; Research Personnel; Respiratory Tract Infections; Sampling; Sensitivity and Specificity; Signal Transduction; Site; Specimen; Speed; Staging; Staining method; Stains; Symptoms; T cell response; T-Cell Activation; T-Lymphocyte; TNF gene; Test Result; Testing; Time; Tissues; Transcript; Tuberculosis; Whole Blood; Work; assay development; base; clinical epidemiology; clinical research site; compliance behavior; cytokine; design; diagnostic accuracy; enzyme linked immunospot assay; experience; global health; improved; instrument; latent infection; mRNA Expression; microbial; next generation; novel; novel diagnostics; nucleic acid detection; peripheral blood; pre-clinical; product development; prognostic; programs; public health relevance; receptor; response; sample fixation; scale up; single cell analysis

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.

描述（由申请人提供）：感染的诊断通常基于对体液和组织中病原体和/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型的、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精确性，（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，离体刺激外周血 T 淋巴细胞会诱导关键细胞因子（IL-2、IFN-α 和 TNF-α）mRNA 的表达，通过与基因特异性 FISH 探针以及单和多标记杂交可检测到这一表达。参数流式细胞仪。我们现在提议与 Oxford Nutritionec 合作，通过临床前产品开发来推动该平台用于结核病 (TB) 诊断。Oxford Immunotec 是经 FDA 批准的基于宿主的结核病诊断领域的全球领导者。结核病被选定为病理学，因为： (i) 它是一个全球性健康问题； (二) 通过检测抗原特异性 T 细胞反应来诊断结核分枝杆菌感染； (iii) i 具有复杂的临床表现，从无症状潜伏感染 (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具有更高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，可以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。我们将优化关键检测参数，并与 FDA 批准的现有检测相比，验证最终的标准化检测方案的稳健性和重现性、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。第一个成果将是用于 LTBI 诊断的简单而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，并在执行速度和简便性方面超越它们。第二个结果是评估用于区分 LTBI 和 PTB 的三细胞因子读数。 PI 中心和 Oxford Immunotec 与结核病低流行和高流行地区的临床中心建立的合作将提供来自所有结核病诊断组的捐献者的血液样本。我们还表明，FISH-Flow 协议适合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设备。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，最终应该会产生一种具有样本输入-答案输出功能的手持式全自动设备。 FISH-Flow 平台的手动和自动版本将适用于检测许多感染性和非感染性病理中受体介导的反应。