Urine lipoarabinomannan as a marker for low-risk of NTM infection in the CF airway

Abstract

Background: Individuals with Cystic fibrosis (CF) are the most vulnerable population for pulmonary infection with nontuberculous mycobacteria (NTM). Screening, diagnosis, and assessment of treatment response currently depend on traditional culture techniques, but sputum analysis for NTM in CF is challenging, and associated with a low sensitivity. The cell wall lipoarabinomannan (LAM), a lipoglycan found in all mycobacterial species, and has been validated as a biomarker in urine for active Mycobacterium tuberculosis infection.

Methods: Urine from a CF cohort (n = 44) well-characterized for NTM infection status by airway cultures was analyzed for LAM by gas chromatography/mass spectrometry. All subjects with positive sputum cultures for NTM had varying amounts of LAM in their urine. No LAM was detected in subjects who never had a positive culture (14/45). One individual initially classified as NTM sputum negative subsequently developed NTM disease 657 days after the initial urine LAM testing. Repeat urine LAM testing turned positive, correlating to her positive NTM status. Subjects infected with subspecies of M. abscessus had greater LAM quantities than those infected with M. avium complex (MAC). There was no correlation with disease activity or treatment status and LAM quantity. A TB Capture ELISA using anti-LAM antibodies demonstrated very poor sensitivity in identifying individuals with positive NTM sputum cultures.

Conclusion: These findings support the conclusion that urine LAM related to NTM infection may be a useful screening test to determine patients at low risk for having a positive NTM sputum culture, as part of a lifetime screening strategy in the CF population.

Keywords: Cystic fibrosis (CF); Gas chromatography/mass spectrometry (GC/MS); Lipoarabinomannan (LAM); Non-tuberculous mycobacteria (NTM).

Conflict of interest statement

Declaration of Competing Interest Authors have no competing interests.

Copyright © 2020. Published by Elsevier B.V.

Figures

Figure 1:

Urine LAM analysis of subjects with negative NTM sputum cultures. Subjects (n=14) with no evidence for NTM airway infection with >5 cultures, and no history by chart review. Urine LAM assay collected at day 0 correlated completely with previous negative sputum culture for all subjects. Subject BP011 (top row) developed NTM disease 1.8 years following initial urine LAM (red circles). A urine LAM analysis at that time changed to positive (red arrow). The subject was initiated on treatment for M. avium, with subsequent conversion to negative sputum cultures (green circles).

Figure 2:. Quantity of urine LAM in persons with CF culture positive for MABSC and MAC.

Panel A: TBSA LAM (ng/ml) and Panel B: D-ara LAM (ng/ml) detected by GC/MS for subjects whose most recent sputum NTM culture was identified as MAC or MABSC. Mean TBSA and D-ara quantities were greater in subjects whose most recent sputum NTM species was within MABSC (*p=0.007 and **p=0.03 respectively, by one-way ANOVA). Individuals who have cleared their sputum cultures for > 1 year are depicted with open squares, while subjects who have had a positive NTM culture within a year are depicted with a red circle. Box and whiskers plot represent median, 25th-75th quartile, median and range

Figure 3:

Representative GC/MS chromatograms showing the absence and/or presence of urinary LAM related to NTM-negativity and/or positivity. A) D-Arabinose (D-ara) MS/MS method monitoring m/z 420.9-m/z 192.9): Four characteristic peaks of Internal Standard (13C5-D-Arabinose; top panel); Sequentially (top to bottom), 309 and 308 NTM negative, D-ara negative; BP201 and 303 are positive, LAM positive, NTM positive. B) Tuberculostearic acid (TBSA) Single ion monitoring (SIM) at m/z 297.3: TBSA standard (C:19; top panel); Sequentially, 309 and 308 NTM negative, TBSA negative; BP201 and 303 are TBSA positive, LAM positive, NTM positive.

Figure 4.. Direct ELISA for selection of antibody for NTMLAM analysis in clinical samples.

LAM samples were prepared and purified as described (14) except LepLAM was purified from M. leprae cells isolated from infected armadillo liver and spleen (18). A194–01 is a human monoclonal derived from a tuberculosis patient against TBLAM and CS35 and CHCS9–08 were raised against M. leprae whole cells. Reactivity and specificity of the mAbs have been described in our previous published work (8, 19).