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 1×10(4) 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.
Analytical Chemistry & Forensic Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands. Electronic address: ngocadang@gmail.com.
Ngoc A
NA
Dang
Analytical Chemistry & Forensic Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Marta
M
Mourão
Analytical Chemistry & Forensic Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Sjoukje
S
Kuijper
Desmond Tutu TB Centre, Stellenbosch University, P.O. Box 19063, Tygerberg 7505, South Africa.
Elisabetta
E
Walters
Analytical Chemistry & Forensic Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands; Unilever Research and Development, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands.
Hans-Gerd
HG
Janssen
Analytical Chemistry & Forensic Science, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Arend H J
AH
Kolk
D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D013183:Sputum / Q000737:chemistry / Q000382:microbiology*
Direct detection; Gas chromatography–mass spectrometry; Mycobacterium tuberculosis; Solid phase extraction; Sputum