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Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis

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. (2012). Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis. PLoS ONE, 7(3), article no. e32836.

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URL: http://www.plosone.org/article/info%3Adoi%2F10.137...
DOI (Digital Object Identifier) Link: http://dx.doi.org/10.1371/journal.pone.0032836
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Abstract

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.

Item Type: Journal Article
Copyright Holders: 2012 O’Sullivan et al.
ISSN: 1932-6203
Project Funding Details:
Funded Project NameProject IDFunding Body
Not Set083597Wellcome Trust
Not SetF/00 094/BLThe Leverhulme Trust
Not SetNot SetDepartment for International Development, UK.
Keywords: tuberculosis, GC-MS, thermochemolysis, sputum, Mycobacterium tuberculosis, PDIMs, methyl mycoserosates
Academic Unit/Department: Science > Physical Sciences
Interdisciplinary Research Centre: Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR)
Item ID: 33438
Depositing User: Geraint Morgan
Date Deposited: 16 Apr 2012 08:33
Last Modified: 13 Aug 2013 01:56
URI: http://oro.open.ac.uk/id/eprint/33438
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