Sensitive transrenal Mycobacterium tuberculosis nucleic acid detection

灵敏经肾结核分枝杆菌核酸检测

• 批准号: 10249227
• 负责人: Alessandra Luchini
• 金额: $ 19.63万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249227
• 关键词: Adopted; Adult; Affinity; Aftercare; Antigens; Biological; Biological Markers; CLIA certified; Cells; Chemistry; Chest; Childhood; Clinical; Clinical Research; Collection; Consumption; Country; DNA; Devices; Diabetes Mellitus; Diagnostic; Disease; Drug resistance in tuberculosis; Elderly; Genes; Genomic DNA; Geography; Goals; Grant; Guinea-Bissau; HIV; HIV Seronegativity; HIV Seropositivity; HIV/TB; Harvest; Home; Hospitals; Human; Hydrogels; Image; India; Individual; Laboratories; Link; Liquid substance; Measurement; Measures; Methods; Microbiology; Microscopy; Mycobacterium tuberculosis; Nanotechnology; Nucleic Acid Amplification Tests; Nucleic Acids; Patients; Peptides; Performance; Peru; Phase; Proteins; Proteomics; Protocols documentation; Publishing; Pulmonary Tuberculosis; RNA; Refrigeration; Sampling; Sensitivity and Specificity; Severity of illness; Ships; Specificity; Sputum; Technology; Testing; Time; Tuberculosis; Ultracentrifugation; Universities; Urine; Validation; Venezuela; Virus; base; case control; cell free DNA; clinically relevant; co-infection; cohort; comorbidity; cost; detection sensitivity; digital; drug-sensitive; exosome; extracellular vesicles; geographic population; lipoarabinomannan; nanocage; nanoparticle; novel; nucleic acid detection; pathogen; pediatric patients; preservation; transcriptome sequencing; urinary

The goal of this proposal is to maximize the utility of Mycobacterium tuberculosis (Mtb) transrenal urinary nucleic acid measurement as a diagnostic approach valuable for non-sputum producing individuals, including pediatric, elderly, and extrapulmonary tuberculosis patients. Although a promising approach, urinary Mtb cell free DNA (cfDNA) measurement has suboptimal sensitivity in previously published studies. Recently, a clinical study provided evidence that Mtb RNA can be detected in exosomes isolated from the urine of patients with tuberculosis infection. Unfortunately, this second promising nucleic acid TB marker also lacked sufficient sensitivity. We propose to optimize a novel nanotechnology based enrichment and preservation technology to achieve very high sensitivity for Mtb urinary nucleic acid biomarkers. Validation of sensitivity and specificity will utilize our comprehensive urine sample bank donated by patients with active culture positive tuberculosis HIV negative and HIV positive, derived from diverse geographic populations. We have created hydrogel nanocage affinity bait biomarker harvesting technology and have used the technology detect, for the first time, very low abundance (picogram/mL) TB pathogen shed antigens lipoarabinomannan (LAM) and ESAT6, in the urine of HIV-negative patients hospitalized for sputum culture-proven active pulmonary TB, achieving a high sensitivity (95%) and specificity (80%), compared to diseased and healthy controls, revealing a significant correlation of the urinary concentration of LAM with disease severity. We confirmed the presence of urinary LAM in a larger (N=419 patients) and geographically diverse (5 countries) cohort of tuberculosis patients, including extrapulmonary adult and pediatric patients, in the presence or absence of HIV coinfection and in the presence of diabetes co-morbidities. Nanoparticle pre-concentrated urine revealed hundreds of Mtb peptides. Affinity nanoparticles were successfully applied to concentrate solution phase pathogen DNA and RNA and to preserve the captured nucleic acid from degradation. We demonstrated that nucleic acid amplification can be completed directly in the capturing nanoparticles, without the need of time consuming or costly nucleic acid extractions. Importantly, bait loaded nanoparticles capture and concentrate corpuscular bodies including exosomes, extracellular vesicles and viruses from biological fluids. Here we will answer the following relevant clinical questions: Is it possible to apply a high yield, fast and quantitative pre-analytical sample processing technology to achieve WHO recommended sensitivity and specificity thresholds using the widely adopted GeneXpert platform to analyze urine instead of sputum? Is Mtb RNA, packaged in extracellular vesicles, detectable in pulmonary TB patients? Do the urinary RNA levels correlate with urinary Mtb specific peptidome levels? Can we demonstrate correlation between urinary Mtb RNA and disease severity? We will also prove the feasibility of a low cost urine collection device optimized for TB testing that can be used in the field, in underdeveloped global regions.

本提案的目的是最大限度地利用结核分枝杆菌（Mtb）经肾尿核酸 酸测量作为一种诊断方法，对于不产生痰的个体，包括儿童， 老年人和肺外结核患者。虽然这是一种很有前途的方法，但尿Mtb细胞游离DNA 在先前发表的研究中，cfDNA测量具有次优的灵敏度。最近，一项临床研究 提供的证据表明，Mtb RNA可以在从患有以下疾病的患者的尿液中分离的外泌体中检测到： 肺结核感染。不幸的是，这第二个有希望的核酸结核病标记物也缺乏足够的 灵敏度我们建议优化一种新的基于纳米技术的富集和保存技术， 实现了对Mtb尿核酸生物标志物的非常高的灵敏度。灵敏度和特异性验证将 利用我们的综合尿液样本库捐赠的活动性培养阳性结核病艾滋病毒 阴性和艾滋病毒阳性，来自不同的地理群体。我们创造了水凝胶纳米笼 亲和诱饵生物标志物收获技术，并已使用该技术检测，第一次， 在100名受试者的尿液中，TB病原体的丰度（皮克/mL）为脂阿拉伯甘露聚糖（LAM）和ESAT 6， 因痰培养证实的活动性肺结核而住院的HIV阴性患者，实现了高灵敏度 (95%）和特异性（80%），与患病和健康对照相比，揭示了 尿LAM浓度与疾病严重程度的关系。我们证实了尿LAM的存在，在一个较大的 （N=419例患者）和地理分布多样化（5个国家）的结核病患者队列，包括 肺外成人和儿童患者，在存在或不存在HIV合并感染的情况下， 糖尿病合并症的风险纳米颗粒预浓缩的尿液揭示了数百个Mtb肽。亲和力 纳米颗粒成功地应用于浓缩溶液相病原体DNA和RNA， 捕获的核酸免于降解。我们证明，核酸扩增可以完成 直接在捕获纳米颗粒中，而不需要耗时或昂贵的核酸提取。 重要的是，装载诱饵的纳米颗粒捕获并浓缩包括外来体的微粒体， 细胞外囊泡和病毒。在这里，我们将回答以下相关的临床 问题：是否有可能应用高产、快速和定量的分析前样品处理技术 使用广泛采用的GeneXpert达到WHO推荐的灵敏度和特异性阈值 分析尿液而不是痰液的平台？包装在细胞外囊泡中的结核分枝杆菌RNA是否可在 肺结核患者？尿RNA水平与尿Mtb特异性肽组水平相关吗？我们能 证明尿结核分枝杆菌RNA与疾病严重程度之间的相关性？我们还将证明一个 低成本的尿液收集装置，优化用于结核病检测，可用于该领域，在欠发达的全球 地区