**6. Breath tests in TB diagnosis**

Volatile organic compounds (VOCs) in breath have shown to contain biomarkers of active pulmonary tuberculosis derived from the infectious organism (metabolites of *M. tuberculosis*) and from the infected host (products of oxidative stress).

#### **6.1. Breath sample collection**

According to the method described by Braden and colleague, [66], a portable breath collection apparatus (BCA) is used to capture the VOCs in 1.0 L breath and 1.0 L room air on to separate sorbent traps. The geometry of the breath reservoir of the BCA is set to ensure that the sample comprises >99% alveolar breath. Subjects wear a nose-clip and respire normally for 2.0 min through a disposable valve mouthpiece with a bacterial filter to prevent mycobacterial contamination of the instrument. The mouthpiece and filter present low-resistance to respiration ensuring that samples are collected without causing any discomfort to patients.

#### **6.2. Breath sample analysis**

The VOCs captured in the sorbent traps are then analyzed in the laboratory according to the method described by Phillips where an automated thermal desorption, gas chromatography, and mass spectroscopy (ATD/GC/MS) is employed. To quantify peak areas and control for drift in instrument performance, an internal standard is normally run with every chromatographic assay of breath and air (0.25 mL 2 ppm 1-bromo-4-fluorobenzene, Supelco, Bellefonte, and PA).

Chromatographic data collected are then converted into a series of data points by segmenting them into a series of time slices. The alveolar gradient of each time slice is then determined (i.e. abundance in alveolar breath minus abundance in ambient room air) [67, 68].

For each of the time slice, the alveolar gradient is calculated by taking: ¼ Vb /Ib Va/Ia.

Where, Vb is the integrated abundance of analytes detected by mass spectroscopy in a breath, and Ib is the area under the curve (AUC) of the chromatographic peak associated with the internal standard. Va and Ia denote corresponding values derived from the associated sample of room air.

#### **6.3. Identification of biomarker time slices**

In a study by Cannas and colleagues [61], traces of mycobacteria DNA were detected in the urine of TB patients at 79% while the controls were negative. In addition, proteins produced in lung lesions and excreted in urine were also recognized by immunoglobulin G (IgG) from active TB patients [62]. Other TB protein biomarkers in the urine of TB patients have been reported by Young and colleagues [63]. In a similar study, Lim and friends [64] tested urine for TB by using a colorimetric sensor array (CSA). Urine headspace analysis showed discrimination between TB and control patients with 85.5% sensitivity and 79.5% specificity. Analysis of volatile organic compounds using headspace gas chromatography/mass spectrometry (GC/MS) showed increased levels of o-xylene and isopropyl acetate and decreased levels of 3-pentanol, dimethylstyrene, and cymol in the urine of TB patients compared to controls with

Urine-based TB diagnostic procedures can only detect biomarkers related to active TB and not latent TB. However, the promising feature is that it can detect extra pulmonary TB. Nevertheless, more researches are required to unravel the practicability of the tests.

Volatile organic compounds (VOCs) in breath have shown to contain biomarkers of active pulmonary tuberculosis derived from the infectious organism (metabolites of *M. tuberculosis*)

According to the method described by Braden and colleague, [66], a portable breath collection apparatus (BCA) is used to capture the VOCs in 1.0 L breath and 1.0 L room air on to separate sorbent traps. The geometry of the breath reservoir of the BCA is set to ensure that the sample comprises >99% alveolar breath. Subjects wear a nose-clip and respire normally for 2.0 min through a disposable valve mouthpiece with a bacterial filter to prevent mycobacterial contamination of the instrument. The mouthpiece and filter present low-resistance to respiration

The VOCs captured in the sorbent traps are then analyzed in the laboratory according to the method described by Phillips where an automated thermal desorption, gas chromatography, and mass spectroscopy (ATD/GC/MS) is employed. To quantify peak areas and control for drift in instrument performance, an internal standard is normally run with every chromatographic assay of breath and air (0.25 mL 2 ppm 1-bromo-4-fluorobenzene, Supelco,

Chromatographic data collected are then converted into a series of data points by segmenting them into a series of time slices. The alveolar gradient of each time slice is then determined

(i.e. abundance in alveolar breath minus abundance in ambient room air) [67, 68].

ensuring that samples are collected without causing any discomfort to patients.

respiratory diseases other than TB [65].

12 Basic Biology and Applications of Actinobacteria

**6. Breath tests in TB diagnosis**

**6.1. Breath sample collection**

**6.2. Breath sample analysis**

Bellefonte, and PA).

and from the infected host (products of oxidative stress).

The alveolar gradients are identified by comparing the patients positive or negative for active pulmonary TB and rank them as candidate biomarkers according to the value of the C-statistic that is, the AUC of the receiver operating characteristic (ROC) curve [69]. The Kovats Index windows for active TB are clearly distinct from controls in a test that takes 6 min. The detection of TB biomarkers is 80% accurate with 71.2% sensitivity and 72% specificity [70].

TB testing using breath provides hope for a future non-invasive diagnostic procedure. Phillips et al. have put effort to assess the presence of volatile organic compounds in the breath of active TB patients. A special device collected and concentrated breath from TB patients and controls at point-of-care centers. However, the procedure requires special set up and equipment and may not be suitable in low-income endemic settings.
