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

O'Sullivan DM, Nicoara SC, Mutetwa R, Mungofa S, Lee OY, Minnikin DE, Bardwell MW, Corbett EL, McNerney R, Morgan GH - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, United Kingdom.

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.

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Structures of the M. tuberculosis C29, C30 and C32 mycocerosic acid methyl esters.The ions at m/z 452, 466 and 494 correspond to the molecular weights of these esters. Ions at m/z 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.
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pone-0032836-g001: Structures of the M. tuberculosis C29, C30 and C32 mycocerosic acid methyl esters.The ions at m/z 452, 466 and 494 correspond to the molecular weights of these esters. Ions at m/z 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.

Mentions: Detection of lipid moieties, which are essential components of the cell envelope of Mycobacterium tuberculosis, offers an alternative approach for diagnosis. Chemical analysis has the advantage over culture methods in that it can be rapid and does not require live organisms. The mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of complex lipids are established diagnostic markers for tuberculosis [4], [5]. Fatty acids of this type are found in M. tuberculosis, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium gastri, Mycobacterium haemophilum and Mycobacterium ulcerans[6], [7], [8]. Members of the M. tuberculosis complex have a characteristic profile composed of trimethyl C29 and tetramethyl C30 and C32 mycocerosates (Figure 1), while the other related mycobacteria mentioned have distinct profiles [4], [9]. Analysis of mycocerosates has previously been performed using gas chromatography (GC) followed by electron capture detection or negative ion chemical ionisation MS [4], [5], [9], [10], including detection in sputum [5], [10].


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

O'Sullivan DM, Nicoara SC, Mutetwa R, Mungofa S, Lee OY, Minnikin DE, Bardwell MW, Corbett EL, McNerney R, Morgan GH - PLoS ONE (2012)

Structures of the M. tuberculosis C29, C30 and C32 mycocerosic acid methyl esters.The ions at m/z 452, 466 and 494 correspond to the molecular weights of these esters. Ions at m/z 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293907&req=5

pone-0032836-g001: Structures of the M. tuberculosis C29, C30 and C32 mycocerosic acid methyl esters.The ions at m/z 452, 466 and 494 correspond to the molecular weights of these esters. Ions at m/z 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.
Mentions: Detection of lipid moieties, which are essential components of the cell envelope of Mycobacterium tuberculosis, offers an alternative approach for diagnosis. Chemical analysis has the advantage over culture methods in that it can be rapid and does not require live organisms. The mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of complex lipids are established diagnostic markers for tuberculosis [4], [5]. Fatty acids of this type are found in M. tuberculosis, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium gastri, Mycobacterium haemophilum and Mycobacterium ulcerans[6], [7], [8]. Members of the M. tuberculosis complex have a characteristic profile composed of trimethyl C29 and tetramethyl C30 and C32 mycocerosates (Figure 1), while the other related mycobacteria mentioned have distinct profiles [4], [9]. Analysis of mycocerosates has previously been performed using gas chromatography (GC) followed by electron capture detection or negative ion chemical ionisation MS [4], [5], [9], [10], including detection in sputum [5], [10].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus