Direct detection of Mycobacterium tuberculosis in sputum using combined solid phase extraction-gas chromatography-mass spectrometry

Recently, thermally-assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry (THM-GC-MS) in combination with chemometrics has been used to develop a 20-compound model for fast differentiation of Mycobacterium tuberculosis (MTB) from Non-tuberculous mycobacteria (NTM) in bacterial cultures. This model provided better than 95% accuracy. In our current work a hexane/methanol/water extraction followed by a solid phase extraction (SPE) clean-up procedure was developed for use before THM-GC-MS, to make the test suitable for the identification of mycobacteria in sputum. The 20 biomarker model had to be adapted since many compounds were also found in the sputum of non-tuberculosis patients. An algorithm was established based on tuberculostearic acid, hexacosanoic acid and mycoserosates. The detection limit of the method was approximately 1 × 104 bacteria/mL sputum. Sputum specimens from 32 patients from South Africa who were suspected of having tuberculosis were blindly tested using the new method. Eight of the nine culture-positive sputum specimens were detected by the new SPE-THM-GC-MS method, resulting in a sensitivity of 89%. The specimen that was missed by the new method was also microscopy negative. The specificity of the test was 100%; all 23 microscopy- and culture-negative specimens were correctly identified as negative by SPE-THM-GC-MS.