Methods for one– and two–dimensional gas chromatography with flame ionization detection for identification of Mycobacterium tuberculosis in sputum
Two simplified methods based on manual thermally–assisted hydrolysis and methylation (THM) GC and GC × GC with flame ionization detection (FID) were developed for the detection of mycobacteria and Mycobacterium tuberculosis (MTB) in sputum. A central composite design was employed to optimize the THM derivatization conditions. For the detection of MTB the known mycobacterial markers tuberculostearic acid (TBSA) and hexacosanoic acid (C26), as well as three MTB specific markers, the mycocerosates, were evaluated. We found that the optimum conditions for THM release of TBSA and C26 differ from those for maximum release of the mycocerosates. Higher reagent volumes, higher temperatures and longer incubation increase the mycocerosates yield. Application of these conditions unfortunately resulted in unacceptable safety hazards. A GC × GC–FID method was developed that allowed accurate detection of mycocerosates even at poor conversion yields of the derivatization reaction. Using spiked sputum samples from non–TB patients, the detection limit of the method based on TBSA and C26 was found to be comparable to that of microscopy, i.e. 104–105 bacteria/mL sputum. To validate the new test, we compared the results we found for fifteen sputum samples from patients from South Africa suspected of having tuberculosis with those of culture, the gold standard method. Based on the presence of TBSA and C26, all eight microscopy and culture positive samples, and even two microscopy negative but culture positive samples were positive by THM–GC–FID. All five microscopy and culture negative sputum samples were also negative for THM–GC–FID, giving a specificity of 100%. Using GC × GC–FID we could detect mycocerosates, the specific markers for MTB in seven out of ten MTB culture positive sputum samples. The five culture negative cases were also negative for mycocerosates in manual THM–GC × GC–FID giving again 100% specificity. The results obtained indicate that the new methods hold great potential for the early diagnosis of TB in developing countries.