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.
Highlights
After a long hiatus in the development of new drugs for the treatment of tuberculosis (TB), exciting compounds such as TMC207/ R207910, PA824 and OPC6768 are coming out of preclinical stages and entering into clinical trials, with the promise of other new compounds to follow [1,2,3]
Detection was possible at levels of 104 colony forming units (CFU) ml21, significantly lower than most observed levels in sputum, which are observed in the range of 106 to 108 CFU ml21 in early bactericidal activity (EBA) studies so that there is the potential for great sensitivity (17)
The results of this study suggest that delivery of [13C]-urea into the lungs of rabbits infected with urease-producing mycobacteria could be used as a diagnostic modality to estimate lung burdens of disease and their response to therapy
Summary
After a long hiatus in the development of new drugs for the treatment of tuberculosis (TB), exciting compounds such as TMC207/ R207910, PA824 and OPC6768 are coming out of preclinical stages and entering into clinical trials, with the promise of other new compounds to follow [1,2,3]. It has become clear that there is a critical need for new biomarkers of the response of TB to drug therapy, that can provide surrogates of drug efficacy to guide design and conduct of these clinical trials [4,5,6] Without such biomarkers, these trials could be longer, more complicated and costlier, thereby delaying the availability of new drugs and perhaps even discouraging trials of others. Surrogate biomarkers are especially important in Phase IIa/b design, where issues such as drug doses and regimen design are often complex, they would be useful throughout a trial, and beyond into treatment monitoring The need for these new biomarkers derives from inadequacies in current endpoints. Pathogen-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
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