Sensitive transrenal Mycobacterium tuberculosis nucleic acid detection

灵敏经肾结核分枝杆菌核酸检测

• 批准号: 10043479
• 负责人: Alessandra Luchini
• 金额: $ 23.55万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043479
• 关键词: Adopted; Adult; Affinity; Aftercare; Antigens; Biological; Biological Markers; CLIA certified; Cells; Chemistry; Chest; Childhood; Clinical; Clinical Research; Collection; Consumption; Country; DNA; Detection; 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 environment; 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; 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.

这项提议的目标是最大限度地利用结核分枝杆菌(Mtb)经肾尿核。 酸度测量作为一种诊断方法对不产生痰的个体很有价值，包括儿科， 老年人和肺外结核病患者。尽管这是一种很有希望的方法，但尿液结核分枝杆菌细胞游离DNA 在先前发表的研究中，(CfDNA)测量的灵敏度低于最佳。最近，一项临床研究 提供的证据表明，从慢性支气管炎患者的尿液中分离的外切体中可以检测到结核分枝杆菌RNA 肺结核感染。不幸的是，这第二个有希望的核酸TB标记也缺乏足够的 敏感度。我们建议优化一种基于纳米技术的新型浓缩和保存技术，以 实现对结核分枝杆菌尿核酸生物标志物的非常高的灵敏度。验证敏感度和特异度将 利用我们的综合尿样库为活动性培养阳性的结核病HIV患者捐献 阴性和艾滋病毒阳性，来自不同的地理人群。我们已经创造了水凝胶纳米笼子 亲和力诱饵生物标记物采集技术，并已使用该技术首次检测到非常低的 大量(皮克/毫升)结核病原体在尿液中脱落抗原脂阿拉伯甘露聚糖(LAM)和ESAT6 HIV阴性患者因痰培养证实为活动性肺结核而住院，实现了高敏感性 (95%)和特异性(80%)，与疾病和健康对照组相比，显示出显著的相关性 LAM的尿药浓度与病情严重程度相关。我们确认尿液LAM在较大的 (n=419名患者)和不同地域(5个国家)的结核病患者队列，包括 肺外成人和儿童患者，在有或没有艾滋病毒合并感染和存在的情况下 糖尿病的合并症。纳米颗粒预浓缩尿检出数百个结核分枝杆菌多肽。亲和力 纳米粒子被成功地应用于浓缩溶液相病原体DNA和RNA并保存 从降解过程中捕获的核酸。我们证明了核酸扩增是可以完成的。 直接在捕获纳米粒子，而不需要耗时或昂贵的核酸提取。 重要的是，装有诱饵的纳米颗粒捕获并浓缩了包括外周小体在内的微粒体， 来自生物体液的细胞外小泡和病毒。在这里，我们将回答以下相关临床问题 问题：是否有可能应用一种高产量、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推荐的敏感性和特异性阈值 分析尿液而不是痰的平台？Mtb RNA，包装在细胞外小泡中，可在 肺结核患者？尿RNA水平与尿Mtb特异性多肽水平相关吗？我们能不能 尿结核分枝杆菌核糖核酸与疾病严重程度的相关性？我们还将证明一项 低成本尿液采集设备，针对结核病检测进行了优化，可在全球欠发达地区现场使用 地区。