O'Sullivan, Denise M.; Nicoara, Simona C.; Mutetwa, Reggie; Mungofa, Stanley; Lee, Oona Y-C.; Minnikin, David E.; Bardwell, Max W.; Corbett, Elizabeth L.; McNerney, Ruth and Morgan, Geraint H.
Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis.
PLoS ONE, 7(3),
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Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test.
||2012 O’Sullivan et al.
||This work was supported by the Wellcome Trust project grant 083597 and The Leverhulme Trust through an Emeritus Fellowship to D.E.M. and Project
||tuberculosis, GC-MS, thermochemolysis, sputum, Mycobacterium tuberculosis, PDIMs, methyl mycoserosates
||Science > Physical Sciences
|Interdisciplinary Research Centre:
||Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR)
||16 Apr 2012 08:33
||22 Nov 2012 12:13
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