13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

Abstract

BackgroundPathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers.Methodology/Principal FindingsWe tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [13C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of δ13CO2 formation were determined. Samples obtained prior to inoculation served as control samples for background 13CO2 conversion in the rabbit model. 13CO2, from metabolic conversion of [13C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of 13CO2 formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of 13CO2 formation.Conclusions/SignificanceUrea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the δ13CO2 signal from urease-positive gastrointestinal organisms.

Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

“…Urease activity in the M. tuberculosis WT, mutant, and complemented strains was determined using a BD BBL Taxo differentiation disc urea kit (Becton Dickinson, Inc., Sparks, MD), as previously described (16). Briefly, the strains were grown to mid-log phase and concentrated to 1 ϫ 10 9 CFU/ml in Middlebrook 7H9 medium.…”

“…The ⌬ureC mutant was then complemented whereby the ureABC portion of the urease operon expressed under the control of its original promoter was introduced in the ⌬ureC mutant using the promoterless integrative plasmid pMV306 (36). A colorimetric assay was performed to assess the urease activity in the ⌬ureC mutant, WT, and complemented strains as described elsewhere (16). While both cultures of the WT and complemented strains turned pink upon incubation with a urea disc, the culture of the ⌬ureC mutant remained yellow, indicating the absence of urease activity in the mutant strain (Fig.…”

Detecting virulence and drug-resistance mycobacterial phenotypes in vivo

“…M. tuberculosis was tested as this is a bacterial pathogen that expresses the virulence factor urease [3, 4], an enzyme traditionally used in phenotypic culture identification [5]. Direct lung delivery in a rabbit model of tuberculosis was used and discrimination between infection and controls was possible soon after delivery [6]. Additionally, 13 CO 2 levels correlated with bacterial burden during antibiotic treatment and withdrawal, suggesting that the initial efficacy of a regimen (and hence drug sensitivity) might be rapidly and simply determined in a manner similar to early bactericidal activity studies [6].…”

“…Direct lung delivery in a rabbit model of tuberculosis was used and discrimination between infection and controls was possible soon after delivery [6]. Additionally, 13 CO 2 levels correlated with bacterial burden during antibiotic treatment and withdrawal, suggesting that the initial efficacy of a regimen (and hence drug sensitivity) might be rapidly and simply determined in a manner similar to early bactericidal activity studies [6]. However, urease is widely expressed by lung pathogens, and although it broadly allows bacterial presence and response to therapy measurements, identification of specific species requires additional approaches.…”

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

“…Commercial and experimental methods target various biomarkers in serum, urine, sputum, and exhaled breath. [3–6] However, performance has been found unsatisfactory. The most promising development has been the Gene-Xpert system [7], which applies an advanced molecular detection method in sputum samples with an automated system.…”