13[C]-urea breath test as a novel point-of-care biomarker for tuberculosis treatment and diagnosis

Abstract

Background: 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.

Methodology/principal findings: We 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. [(13)C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of delta(13)CO(2) formation were determined. Samples obtained prior to inoculation served as control samples for background (13)CO(2) conversion in the rabbit model. (13)CO(2), from metabolic conversion of [(13)C]-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 (13)CO(2) formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of (13)CO(2) formation.

Conclusions/significance: Urea 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 delta(13)CO(2) signal from urease-positive gastrointestinal organisms.

Figure 1. Mycobacterial urease assay by conversion of [¹³C] urea to ¹³CO₂ reports upon CFU over a wide range.

M. bovis BCG was incubated for 30 minutes with 5 mg ml⁻¹ [¹³C]-urea and headspace gas sampled and analysed for ¹³CO₂ enrichment (reported as δ¹³CO₂).

Figure 2. Gross pathology of selected lung specimens at necropsy.

(A) M. tuberculosis H37Rv T1. (B) M. tuberculosis H37Rv T2. (C) M. bovis B1. (D) M. bovis B2. (E) M. bovis B3. (F) M. bovis B4.

Figure 3. Exhaled δ¹³CO₂ increased linearly after urea instillation.

Breath samples were obtained prior to, and 1, 5, 10 and 20 minutes after urea instillation. Fig 3 shows typical data from a heavily infected rabbit.

Figure 4. Conversion rate of [¹³C]-urea prior and after treatment with INH.

Baseline level of [¹³C]-urea conversion was obtained on all animals prior to bronchoscopic infection. Urea conversion rates were obtained at a single time point after 4–8 weeks post-infection. INH 20mg/kg daily for a 2-week interval was initiated after 8 weeks from the time of infection. Conversion rates were ascertained either during or within 10 days of treatment cessation. Urease conversion were lastly determined at single time points after 2–4 weeks and 6–8 post-treatment in selected rabbits.

Figure 5. Mean conversion rates of [¹³C]-urea per minute among M. tuberculosis H37Rv and M. bovis-infected rabbits.

Repeated measurements had occurred of each breath test sample. Significant conversion rate differences are noted after both infection and treatment with 25mg/kg of INH. Rates were obtained prior to infection, 4–8 weeks after bronchoscopic infection and within two weeks post-INH treatment.