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

•A Thermally assisted Hydrolysis and Methylation GC–MS method for biomarkers.•An algorithm for the detection of Mycobacterium tuberculosis in sputum.•A selective extraction procedure for Mycobacterium tuberculosis in sputum.•Solid phase extraction procedure for isolation of biomarkers of M. tuberculosis in sputum.•We report a detection limit of 1×104Mycobacterium tuberculosis/mL sputum using SPE–THM–GC–MS. 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.