In vitro and in vivo studies of a rapid and selective breath test for tuberculosis based upon mycobacterial CO dehydrogenase

Abstract

One of the major hurdles in treating tuberculosis (TB) is the time-consuming and difficult methodology for diagnosis. Stable-isotope breath tests hold great potential for rapidly diagnosing an infectious disease, monitoring therapy, and determining a bacterial phenotype in a rapid, point-of-care manner that does not require invasive sampling. Here we describe the preclinical development of a potentially highly selective TB diagnostic breath test based upon the organism's CO dehydrogenase activity. After development of the test in vitro, we were able to use the breath test to discriminate between infected and control rabbits, demonstrating that a diagnosis can potentially be made and also that a complex bacterial phenotype can be noninvasively and rapidly studied in the host. IMPORTANCE Tuberculosis (TB) remains a major infectious cause of disease and death worldwide, and effective diagnosis and then treatment are the tools with which we fight TB. The more quickly and more specific the diagnosis can be made, the better, and this is also true of diagnosis being as close to the patient (point of care) as possible. Here we report our preclinical development of breath tests based upon specific mycobacterial metabolism that could, with development, allow rapid point-of-care diagnosis through measuring the mycobacterial conversion of labeled CO to labeled CO2.

FIG 1

Dose, CFU, and time dependence of CODH activity using ¹³CO. (A) M. bovis BCG (3 ml, 1 × 10⁷ CFU/ml) was incubated with increasing doses of ¹³CO at 37°C for 4 h. Headspace δ-¹³CO₂ was determined by IRMS. Numbers are means ± SD (n = 4) of results from separate biological replicates. By one-way ANOVA with the Tukey test as a post hoc test, P was <0.001 (0 ml versus 2 or 4 ml). (B) Different dilutions of M. bovis BCG (3 ml) were incubated with 2 ml ¹³CO for 4 h. Headspace δ-¹³CO₂ was determined by IRMS. Numbers are means ± SD of results from separate biological replicates with 1 × 10⁵, 1 × 10⁷, or 1 × 10⁸ CFU (n = 3) or 0 or e6 CFU (n = 2). By one-way ANOVA with the Tukey test as a post hoc test, P was <0.05 (1 × 10⁵ versus 1 × 10⁷ and 1 × 10⁸; the significance for 1 × 10⁶ cannot be determined). (C) M. bovis BCG (3 ml, 1 × 10⁷ CFU/ml) was incubated with 2 ml ¹³CO for differing time points. Headspace δ-¹³CO₂ was determined by IRMS. Numbers are means ± SD (n = 4) of results from separate biological replicates. By one-way ANOVA with the Tukey test as a post hoc test, P was <0.05 (0 h versus 0.5, 1, 3, or 5 h).

FIG 2

¹³C¹⁸O dose curve. M. bovis BCG (3 ml, 1 × 10⁷ CFU/ml) was incubated with ¹³C¹⁸O gas for 22 h. Headspace gas was analyzed by isotope ratio mass spectrometry (IRMS) to determine Δ47. Data represent means ± SD (n = 4) of results from separate biological replicates. By one-way ANOVA with the Tukey test as a post hoc test (ANOVA between groups), P was <0.001 (*) versus results with a 0-ml CO dose.

FIG 3

Cell density-dependent conversion of ¹³C¹⁸O to ¹³C¹⁸O¹⁶O. M. bovis BCG culture (6 ml) was incubated with 1 ml of ¹³C¹⁸O gas for 0.5 h. Headspace gas was measured to determine Δ47. Data represent means ± SD (n = 4) of results from separate biological replicates. By one-way ANOVA with the Tukey test as a post hoc test, P was <0.001 (*) versus 0 CFU/ml.

FIG 4

Time-dependent conversion of ¹³C¹⁸O to mass 47 (¹³C¹⁸OO). M. bovis BCG or M. tuberculosis H37Rv (6 ml, 3 × 10⁷ CFU/ml) was incubated with 1 ml of ¹³C¹⁸O gas; M. bovis Ravenel (3 ml of 1 × 10⁸ CFU/ml) was incubated with 3 ml ¹³C¹⁸O. Headspace gas Δ47 was measured, and Δ47 per 1 × 10⁸ CFU per ml added CO was calculated and plotted. Data represent means ± SD (n = 4) of results from separate biological replicates. By one-way ANOVA with the Tukey test as a post hoc test, for 0 h versus 0.5 h, P was <0.001 for all groups; for 0 h versus 4 h, P was <0.01 for M. tuberculosis H37Rv only; and differences were not significant for M. bovis BCG and M. bovis Ravenel.

FIG 5

Conversion of ¹³C¹⁸O to mass 47 (¹³C¹⁸OO gas) by M. bovis Ravenel-infected rabbits. Detailed methods are described in Materials and Methods. Exhaled breath was measured to determine δ47. ●, infected rabbits; □, uninfected rabbits. Data represent individual data for separate animals, with the bar representing the mean. An ANOVA split plot was used as a measure of between-subjects effects (uninfected versus infected), and the slopes were significantly different (P = 0.018). Individual time points were compared between infected and control animals using one-way ANOVA with the Holm-Šidák test as a post hoc test.