Estimation of D-Arabinose by Gas Chromatography/Mass Spectrometry as Surrogate for Mycobacterial Lipoarabinomannan in Human Urine

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.

Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity

“…The carbohydrate structure of LAM( Fig. 1 ) ( 3 ) poses unsolved challenges in terms of identifying adequate probes for affinity isolation in urine in the presence of a vast excess of interfering urinary proteins and other biomolecules ( 7 , 21 , 22 ). For this study, 37 different dye chemistries (table S1) were screened to identify a molecular bait that would sequester LAM from urine with high affinity, deplete the supernatant, dissociate LAM-binding proteins, and permit a high-yield quantitative recovery ( Fig.…”

“…This study indicates that it is possible to detect urinary LAM during active pulmonary TB, that detection does not require physiologic or immunologic consequences of HIV infection ( 8 , 9 ), and that detection is not limited to patients with TB colonization of the kidney ( 10 ). Instead, as others have suspected ( 7 , 29 ), the LAM antigen concentration in HIV-negative patients is below the level of sensitivity of previous immunoassays, and the urinary LAM may be obscured by interfering LAMbinding substances in the urine protein matrix ( 7 ). The high-affinity copper dye bait for LAM introduced in this study effectively sequesters LAM away from any potential urine-binding protein and concentrates the LAM by a large factor, depending on the input volume of the urine and the output total volume of the nanocages.…”

“…These tests can detect urinary LAM in patients with pulmonary TB who are coinfected with HIV ( 5 , 6 ) but not in those who are HIV-negative ( 5 , 6 ). Quantitative gas chromatography–mass spectrometry has been used to identify D-arabinose as a proxy for LAM in TB patients irrespective of HIV status, but the sensitivity is limited to 10 to 40 ng/ml ( 7 ). Unfortunately, the failure to detectLAMin the urine ofHIV-negative patients limits the applicability of these assays, because most of the TB patients areHIV-negative (85% of the 9.6million patients worldwide) ( 1 ).…”

“…The second hypothesis is that LAM is shed into the urine of patients with active TB only when there is extrapulmonary renal tract involvement, such that the antigen can enter the urine directly from infected tissue ( 10 ). The third hypothesis is that the concentration of LAM in patients with active pulmonary TB is below the concentration detection limits of existing assays and may be masked by formation of immune complexes, excess non-LAM proteins, or other inhibitors present in the urine ( 7 ). Here, we apply a new class of analytical nanocage technology to definitively address these hypotheses and solve this dilemma.…”

Mycobacteria-derived biomarkers for tuberculosis diagnosis

“…None of the currently available microbiological, serologic or molecular tests designed for the diagnosis of active TB is good enough for clinical use in children 15 16 . A gas chromatography/mass spectrometry (GC/MS) analysis aimed at identification of D-arabinose and tuberculostearic acid as surrogates for mycobacterial LAM in the human urine substantiated the antigen as a powerful biomarker for active TB 17 . However, the concentration of excreted LAM depends on the clinical manifestation of TB and varies between 1 ng/ml and several 100 ng/ml 18 19 20 .…”