Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis
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...
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| Veröffentlicht in: | PloS one 2012-03, Vol.7 (3), p.e32836-e32836 |
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| creator | 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 Morgan, Geraint H |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0032836 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0032836</identifier><identifier>PMID: 22403716</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Accuracy ; Analysis ; Bacilli ; Biology ; Biomarkers ; Biomedical research ; Chemistry ; Chromatography ; Classification ; Culture Techniques ; Data processing ; Diagnosis ; Diagnostic systems ; Diagnostic tests ; Disease transmission ; Esters ; Fatty Acids - analysis ; Fatty Acids - metabolism ; Gas chromatography ; Gas Chromatography-Mass Spectrometry - methods ; Gas Chromatography-Mass Spectrometry - standards ; Humans ; Hygiene ; Laboratories ; Leprosy ; Lipids ; Lipids - analysis ; Lung ; Mass spectrometry ; Mass spectroscopy ; Medical diagnosis ; Medicine ; Methods ; Molecular biology ; Mycobacterium ; Mycobacterium tuberculosis ; Mycobacterium tuberculosis - cytology ; Mycobacterium tuberculosis - growth & development ; Mycobacterium tuberculosis - isolation & purification ; Mycobacterium tuberculosis - metabolism ; Natural gas ; Pathogens ; Patients ; Petroleum ether ; Phthiocerol dimycocerosate ; Polarity ; Reference Standards ; Scientific imaging ; Sensitivity ; Sensitivity analysis ; Sensitivity enhancement ; Solvents ; Spectroscopy ; Sputum ; Sputum - microbiology ; Temperature ; Transesterification ; Tuberculosis</subject><ispartof>PloS one, 2012-03, Vol.7 (3), p.e32836-e32836</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012 O'Sullivan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>O'Sullivan et al. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c724t-e24c259ada51e7c3332b3267c30eb84104a3745ba1c93fbdf80dc9300069831e3</citedby><cites>FETCH-LOGICAL-c724t-e24c259ada51e7c3332b3267c30eb84104a3745ba1c93fbdf80dc9300069831e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293907/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293907/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22403716$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>O'Sullivan, Denise M</creatorcontrib><creatorcontrib>Nicoara, Simona C</creatorcontrib><creatorcontrib>Mutetwa, Reggie</creatorcontrib><creatorcontrib>Mungofa, Stanley</creatorcontrib><creatorcontrib>Lee, Oona Y-C</creatorcontrib><creatorcontrib>Minnikin, David E</creatorcontrib><creatorcontrib>Bardwell, Max W</creatorcontrib><creatorcontrib>Corbett, Elizabeth L</creatorcontrib><creatorcontrib>McNerney, Ruth</creatorcontrib><creatorcontrib>Morgan, Geraint H</creatorcontrib><title>Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Accuracy</subject><subject>Analysis</subject><subject>Bacilli</subject><subject>Biology</subject><subject>Biomarkers</subject><subject>Biomedical research</subject><subject>Chemistry</subject><subject>Chromatography</subject><subject>Classification</subject><subject>Culture Techniques</subject><subject>Data processing</subject><subject>Diagnosis</subject><subject>Diagnostic systems</subject><subject>Diagnostic tests</subject><subject>Disease transmission</subject><subject>Esters</subject><subject>Fatty Acids - analysis</subject><subject>Fatty Acids - metabolism</subject><subject>Gas chromatography</subject><subject>Gas Chromatography-Mass Spectrometry - methods</subject><subject>Gas Chromatography-Mass Spectrometry - standards</subject><subject>Humans</subject><subject>Hygiene</subject><subject>Laboratories</subject><subject>Leprosy</subject><subject>Lipids</subject><subject>Lipids - analysis</subject><subject>Lung</subject><subject>Mass spectrometry</subject><subject>Mass spectroscopy</subject><subject>Medical diagnosis</subject><subject>Medicine</subject><subject>Methods</subject><subject>Molecular biology</subject><subject>Mycobacterium</subject><subject>Mycobacterium tuberculosis</subject><subject>Mycobacterium tuberculosis - cytology</subject><subject>Mycobacterium tuberculosis - growth & development</subject><subject>Mycobacterium tuberculosis - isolation & purification</subject><subject>Mycobacterium tuberculosis - metabolism</subject><subject>Natural gas</subject><subject>Pathogens</subject><subject>Patients</subject><subject>Petroleum ether</subject><subject>Phthiocerol dimycocerosate</subject><subject>Polarity</subject><subject>Reference Standards</subject><subject>Scientific imaging</subject><subject>Sensitivity</subject><subject>Sensitivity analysis</subject><subject>Sensitivity enhancement</subject><subject>Solvents</subject><subject>Spectroscopy</subject><subject>Sputum</subject><subject>Sputum - microbiology</subject><subject>Temperature</subject><subject>Transesterification</subject><subject>Tuberculosis</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk1uL1DAUx4so7jr6DUQLgpeHGXPpNM2LsKy3gZUFb68hTU-nGdJmNknFfgk_s6nTXaayiOQhh9Pf-Z_mXJLkMUYrTBl-vbO966RZ7W0HK4QoKWh-JznFnJJlThC9e2SfJA-83yG0pkWe309OCMlQ1MhPk19vIYAK2naprdNPg7KlVAGc7ts09CU41RvrtU91l_p9H6K7HNKt9KlqnG1lsFsn982wbKX3kYha0Q3BDaNeNJrBpG2UVeCslwF86sCA9FCNQqEB11rVQGvNENM8TO7V0nh4NN2L5Nv7d1_PPy4vLj9szs8uloqRLCyBZIqsuazkGgNTlFJSUpJHC0FZZBhlkrJsXUqsOK3Lqi5QFS2EUM4LioEukqcH3X18nZhK6QWmhHLG81jLRbI5EJWVO7F3upVuEFZq8cdh3VZIF7QyIHDO8krWjAEmGWWEq7quYo-KAhHFZRm13kzZ-rKFSkEXnDQz0fmXTjdia38ISjjliEWBF5OAs1c9-CBa7RUYIzuwvRecsLxgKBvJl_8kMcKYoCzjOKLP_kJvr8NEbWV8qu5qG_9QjaLiLGMMrxnmY9rVLVQ8FbRaxQmtdfTPAl7NAiIT4GfYyt57sfny-f_Zy-9z9vkR24A0ofHW9OOE-zmYHUAV59I7qG_agZEYF-y6GmJcMDEtWAx7ctzKm6DrjaK_ARoVI3o</recordid><startdate>20120305</startdate><enddate>20120305</enddate><creator>O'Sullivan, Denise M</creator><creator>Nicoara, Simona C</creator><creator>Mutetwa, Reggie</creator><creator>Mungofa, Stanley</creator><creator>Lee, Oona Y-C</creator><creator>Minnikin, David E</creator><creator>Bardwell, Max W</creator><creator>Corbett, Elizabeth L</creator><creator>McNerney, Ruth</creator><creator>Morgan, Geraint H</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20120305</creationdate><title>Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis</title><author>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 ; Morgan, Geraint H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c724t-e24c259ada51e7c3332b3267c30eb84104a3745ba1c93fbdf80dc9300069831e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Accuracy</topic><topic>Analysis</topic><topic>Bacilli</topic><topic>Biology</topic><topic>Biomarkers</topic><topic>Biomedical research</topic><topic>Chemistry</topic><topic>Chromatography</topic><topic>Classification</topic><topic>Culture Techniques</topic><topic>Data processing</topic><topic>Diagnosis</topic><topic>Diagnostic systems</topic><topic>Diagnostic tests</topic><topic>Disease transmission</topic><topic>Esters</topic><topic>Fatty Acids - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>O'Sullivan, Denise M</au><au>Nicoara, Simona C</au><au>Mutetwa, Reggie</au><au>Mungofa, Stanley</au><au>Lee, Oona Y-C</au><au>Minnikin, David E</au><au>Bardwell, Max W</au><au>Corbett, Elizabeth L</au><au>McNerney, Ruth</au><au>Morgan, Geraint H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-03-05</date><risdate>2012</risdate><volume>7</volume><issue>3</issue><spage>e32836</spage><epage>e32836</epage><pages>e32836-e32836</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22403716</pmid><doi>10.1371/journal.pone.0032836</doi><tpages>e32836</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2012-03, Vol.7 (3), p.e32836-e32836 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1323979628 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Accuracy Analysis Bacilli Biology Biomarkers Biomedical research Chemistry Chromatography Classification Culture Techniques Data processing Diagnosis Diagnostic systems Diagnostic tests Disease transmission Esters Fatty Acids - analysis Fatty Acids - metabolism Gas chromatography Gas Chromatography-Mass Spectrometry - methods Gas Chromatography-Mass Spectrometry - standards Humans Hygiene Laboratories Leprosy Lipids Lipids - analysis Lung Mass spectrometry Mass spectroscopy Medical diagnosis Medicine Methods Molecular biology Mycobacterium Mycobacterium tuberculosis Mycobacterium tuberculosis - cytology Mycobacterium tuberculosis - growth & development Mycobacterium tuberculosis - isolation & purification Mycobacterium tuberculosis - metabolism Natural gas Pathogens Patients Petroleum ether Phthiocerol dimycocerosate Polarity Reference Standards Scientific imaging Sensitivity Sensitivity analysis Sensitivity enhancement Solvents Spectroscopy Sputum Sputum - microbiology Temperature Transesterification Tuberculosis |
| title | Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis |
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