Research Project
10249227
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.
