A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry.
A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB.
Kaal
Erwin
E
Polymer-Analysis Group, Van 't Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 166 1018 WV Amsterdam, The Netherlands. kaal@atasgl.com
Kolk
Arend H J
AH
Kuijper
Sjoukje
S
Janssen
Hans-Gerd
HG
D053000:Analytic Sample Preparation Methods; D001419:Bacteria / Q000737:chemistry; D015415:Biomarkers; D003666:Decontamination; D005227:Fatty Acids / Q000032:analysis / Q000302:isolation & purification*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D008853:Microscopy / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000302:isolation & purification*; D000644:Quaternary Ammonium Compounds; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013183:Sputum / Q000737:chemistry* / Q000382:microbiology*; D013229:Stearic Acids / Q000032:analysis / Q000302:isolation & purification*; D014376:Tuberculosis / Q000175:diagnosis; D014783:Viscosity
D015415:Biomarkers; D005227:Fatty Acids; D000644:Quaternary Ammonium Compounds; D013229:Stearic Acids; C026082:tuberculostearic acid; C017364:hexacosanoic acid; C027917:tetramethylammonium