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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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Thermochemolytic-gas chromatography-electron impact/mass spectrometry (THM-GC-EI/MS) of purified phthiocerol dimycocerosate (PDIM) waxes.The selected ion monitored EI/MS chromatogram shows the target fragment ions m/z 101 and m/z 88, resulting from the major trimethyl C29 and tetramethyl C30, and C32 mycocerosates released by thermochemolysis of the PDIMs. A minor proportion of a tetramethyl C31 mycocerosate is also indicated. The characteristic doublet peaks are due to racemisation during the alkaline hydrolysis. The method relied on recognizing comparable proportions of the combined C29/C30 peak and the C32 peak, as was the case in all the positive specimens. Some minor variation in the ratios of the individual peaks was observed, but this did not affect the diagnostic ratios.
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pone-0032836-g002: Thermochemolytic-gas chromatography-electron impact/mass spectrometry (THM-GC-EI/MS) of purified phthiocerol dimycocerosate (PDIM) waxes.The selected ion monitored EI/MS chromatogram shows the target fragment ions m/z 101 and m/z 88, resulting from the major trimethyl C29 and tetramethyl C30, and C32 mycocerosates released by thermochemolysis of the PDIMs. A minor proportion of a tetramethyl C31 mycocerosate is also indicated. The characteristic doublet peaks are due to racemisation during the alkaline hydrolysis. The method relied on recognizing comparable proportions of the combined C29/C30 peak and the C32 peak, as was the case in all the positive specimens. Some minor variation in the ratios of the individual peaks was observed, but this did not affect the diagnostic ratios.

Mentions: Thermochemolytic-gas chromatography-electron impact mass spectrometry (THM-GC-EI/MS) of standard phthiocerol dimycocerosate (PDIM) waxes generated profiles characteristic of mycocerosic acid methyl esters from M. tuberculosis. The diagnostic mycocerosates are trimethyl C29 and tetramethyl C30 and C32 acids [4], [9]. As shown in Figure 2, each mycocerosate is represented as a characteristic double peak due to racemisation during the alkaline hydrolysis [4]. As explained in the discussion section, the earlier eluting double peak represents overlapping trimethyl C29 and tetramethyl C30 mycocerosates; the additional methyl branch in the larger C30 mycocerosate increases its volatility so that it co-elutes with the smaller C29 component. The later eluting double peak corresponds to the two diastereoisomers of tetramethyl C32 mycocerosate.


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)

Thermochemolytic-gas chromatography-electron impact/mass spectrometry (THM-GC-EI/MS) of purified phthiocerol dimycocerosate (PDIM) waxes.The selected ion monitored EI/MS chromatogram shows the target fragment ions m/z 101 and m/z 88, resulting from the major trimethyl C29 and tetramethyl C30, and C32 mycocerosates released by thermochemolysis of the PDIMs. A minor proportion of a tetramethyl C31 mycocerosate is also indicated. The characteristic doublet peaks are due to racemisation during the alkaline hydrolysis. The method relied on recognizing comparable proportions of the combined C29/C30 peak and the C32 peak, as was the case in all the positive specimens. Some minor variation in the ratios of the individual peaks was observed, but this did not affect the diagnostic ratios.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293907&req=5

pone-0032836-g002: Thermochemolytic-gas chromatography-electron impact/mass spectrometry (THM-GC-EI/MS) of purified phthiocerol dimycocerosate (PDIM) waxes.The selected ion monitored EI/MS chromatogram shows the target fragment ions m/z 101 and m/z 88, resulting from the major trimethyl C29 and tetramethyl C30, and C32 mycocerosates released by thermochemolysis of the PDIMs. A minor proportion of a tetramethyl C31 mycocerosate is also indicated. The characteristic doublet peaks are due to racemisation during the alkaline hydrolysis. The method relied on recognizing comparable proportions of the combined C29/C30 peak and the C32 peak, as was the case in all the positive specimens. Some minor variation in the ratios of the individual peaks was observed, but this did not affect the diagnostic ratios.
Mentions: Thermochemolytic-gas chromatography-electron impact mass spectrometry (THM-GC-EI/MS) of standard phthiocerol dimycocerosate (PDIM) waxes generated profiles characteristic of mycocerosic acid methyl esters from M. tuberculosis. The diagnostic mycocerosates are trimethyl C29 and tetramethyl C30 and C32 acids [4], [9]. As shown in Figure 2, each mycocerosate is represented as a characteristic double peak due to racemisation during the alkaline hydrolysis [4]. As explained in the discussion section, the earlier eluting double peak represents overlapping trimethyl C29 and tetramethyl C30 mycocerosates; the additional methyl branch in the larger C30 mycocerosate increases its volatility so that it co-elutes with the smaller C29 component. The later eluting double peak corresponds to the two diastereoisomers of tetramethyl C32 mycocerosate.

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