Abstract
Globally, tuberculosis is slowly declining each year and it is estimated that 37 million lives were saved between 2000 and 2013 through effective diagnosis and treatment. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis (Mtb), in clinical specimens by serial sputum microscopy, culture and molecular testing. Commercial immunoassay lateral flow kits developed to detect Mtb lipoglycan lipoarabinomannan (LAM) in urine as a marker of active TB exhibit poor sensitivity, especially in immunocompetent individuals, perhaps due to low abundance of the analyte. Our present study was designed to develop methods to validate the presence of LAM in a quantitative fashion in human urine samples obtained from culture-confirmed TB patients. Herein we describe, a consolidated approach for isolating LAM from the urine and quantifying D-arabinose as a proxy for LAM, using Gas Chromatography/Mass Spectrometry. 298 urine samples obtained from a repository were rigorously analyzed and shown to contain varying amounts of LAM-equivalent ranging between ~10–40 ng/mL. To further substantiate that D-arabinose detected in the samples originated from LAM, tuberculostearic acid, the unique 10-methyloctadecanoic acid present at the phosphatidylinositol end of LAM was also analyzed in a set of samples and found to be present confirming that the D-arabinose was indeed derived from LAM. Among the 144 samples from culture-negative TB suspects, 30 showed presence of D-arabinose suggesting another source of the analyte, such as disseminated TB or from non-tuberculosis mycobacterium. Our work validates that LAM is present in the urine samples of culture-positive patients in small but readily detectable amounts. The study further substantiates LAM in urine as a powerful biomarker for active tuberculosis.
Highlights
Tuberculosis (TB) is a sub-acute or chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), the bacillus that infects approximately one-third of the world’s population
The report notes that “TB remains unique among the major infectious diseases in lacking accurate and rapid point-of-care tests, largely due to insufficient progress in biomarker discovery. . .the most pressing priority in TB diagnostics research today is the development of a simple, low-cost, instrument-free rapid test.”
In order to validate the presence of LAM in the human clinical samples, we have developed a chemical approach utilizing specific derivatization and GC/MS analyses with the conviction that D-arabinose is only present in the mycobacteria and is absent among eukaryotes [24]
Summary
Tuberculosis (TB) is a sub-acute or chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), the bacillus that infects approximately one-third of the world’s population. A specific biomarker that could reduce the size and duration of clinical trials for new drug candidates through better identification of treatment efficacy, disease activity, cure and relapse would have a huge impact on the cost of new drug development. Biomarkers such as Interferon-γ-inducible protein 10 (IP10) have been shown to be non-specific for TB [7] and transrenal DNA has been used for extrapulmonary-TB diagnosis [8,9]. Urinary LAM has been detected with 82% sensitivity and 100% specificity only after using a laborious magnetic nanoparticle based concentration step [14]
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