The diagnostic material was pre-treated with N-acetyl-l -cysteine in combination with 1% NaOH (NALC–NaOH) and inoculated into Loewenstein–Jensen (LJ) and Finn II egg-based media as recommended in previous works [29 ,30 ,31 ]. Primary identification of mycobacteria was carried out based on visual registration of the culture’s growth (not earlier than 3–4 weeks of incubation) and its features (colonies of characteristic morphology and color). The primary identification of strains included microscopic confirmation of the Ziehl–Neelsen-stained bacteria and assessment of the cord factor.
An immunochromatographic test was used to detect a fraction of the mycobacterial protein MPT64, which is isolated from MBT cells during cultivation on liquid and solid nutrient media [25 (link)]. SD BIOLINE TB Ag MPT64 Rapid tests (Standard Diagnostics, Korea) were used for identification. The test was applied to both samples cultured on solid and liquid nutrient media. The presence of two colored bands “T” and “C” in the result window indicated a positive result.
To differentiate individual species within the M. tuberculosis complex, we used the main biochemical tests to determine nicotinic acid and nitrate reductase, and an additional test for colony growth on nutrient media containing thiophene-2-carboxylic acid hydrazide (2 μg/mL). Unlike M. tuberculosis, only M. bovis is sensitive to low concentrations of thiophene-2-carboxylic acid hydrazide and is characterized by negative results of biochemical tests. No M. bovis isolates were detected in this study.
To differentiate M. tuberculosis from non-tuberculous acid-fast mycobacteria, the following basic biochemical tests were used: nitrate reductase test, thermostable catalase test, salicylic sodium test, and niacin test. We additionally assessed the growth on nutrient media containing Tween 80 and thiophene-2-carboxylic acid hydrazide.
An immunochromatographic test was used to detect a fraction of the mycobacterial protein MPT64, which is isolated from MBT cells during cultivation on liquid and solid nutrient media [25 (link)]. SD BIOLINE TB Ag MPT64 Rapid tests (Standard Diagnostics, Korea) were used for identification. The test was applied to both samples cultured on solid and liquid nutrient media. The presence of two colored bands “T” and “C” in the result window indicated a positive result.
To differentiate individual species within the M. tuberculosis complex, we used the main biochemical tests to determine nicotinic acid and nitrate reductase, and an additional test for colony growth on nutrient media containing thiophene-2-carboxylic acid hydrazide (2 μg/mL). Unlike M. tuberculosis, only M. bovis is sensitive to low concentrations of thiophene-2-carboxylic acid hydrazide and is characterized by negative results of biochemical tests. No M. bovis isolates were detected in this study.
To differentiate M. tuberculosis from non-tuberculous acid-fast mycobacteria, the following basic biochemical tests were used: nitrate reductase test, thermostable catalase test, salicylic sodium test, and niacin test. We additionally assessed the growth on nutrient media containing Tween 80 and thiophene-2-carboxylic acid hydrazide.
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