Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones
After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates...
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| Veröffentlicht in: | Journal of Clinical Microbiology 2009-12, Vol.47 (12), p.3985-3990 |
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| container_title | Journal of Clinical Microbiology |
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| creator | Bravo, Lulette Tricia C Tuohy, Marion J Ang, Concepcion Destura, Raul V Mendoza, Myrna Procop, Gary W Gordon, Steven M Hall, Geraldine S Shrestha, Nabin K |
| description | After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. |
| doi_str_mv | 10.1128/JCM.01229-09 |
| format | Article |
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Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.</description><identifier>ISSN: 0095-1137</identifier><identifier>EISSN: 1098-660X</identifier><identifier>DOI: 10.1128/JCM.01229-09</identifier><identifier>PMID: 19846642</identifier><identifier>CODEN: JCMIDW</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Antitubercular Agents - pharmacology ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacteriology ; Biological and medical sciences ; Drug Resistance, Bacterial - genetics ; Fluoroquinolones - pharmacology ; Fundamental and applied biological sciences. Psychology ; Genotype ; Humans ; Isoniazid - pharmacology ; Microbial Sensitivity Tests ; Microbiology ; Miscellaneous ; Mutation ; Mycobacteriology and Aerobic Actinomycetes ; Mycobacterium tuberculosis ; Mycobacterium tuberculosis - drug effects ; Mycobacterium tuberculosis - genetics ; Ofloxacin - pharmacology ; Phenotype ; Rifampin - pharmacology ; Sequence Analysis, DNA - methods ; Time Factors</subject><ispartof>Journal of Clinical Microbiology, 2009-12, Vol.47 (12), p.3985-3990</ispartof><rights>2015 INIST-CNRS</rights><rights>Copyright © 2009, American Society for Microbiology 2009</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c495t-43ce8c6b45cf1fe13f2ac39c48b851ddf24b5c726b207c5a7073519053a900df3</citedby><cites>FETCH-LOGICAL-c495t-43ce8c6b45cf1fe13f2ac39c48b851ddf24b5c726b207c5a7073519053a900df3</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/PMC2786679/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786679/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,3175,3176,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=22208770$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19846642$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bravo, Lulette Tricia C</creatorcontrib><creatorcontrib>Tuohy, Marion J</creatorcontrib><creatorcontrib>Ang, Concepcion</creatorcontrib><creatorcontrib>Destura, Raul V</creatorcontrib><creatorcontrib>Mendoza, Myrna</creatorcontrib><creatorcontrib>Procop, Gary W</creatorcontrib><creatorcontrib>Gordon, Steven M</creatorcontrib><creatorcontrib>Hall, Geraldine S</creatorcontrib><creatorcontrib>Shrestha, Nabin K</creatorcontrib><title>Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones</title><title>Journal of Clinical Microbiology</title><addtitle>J Clin Microbiol</addtitle><description>After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.</description><subject>Antitubercular Agents - pharmacology</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Drug Resistance, Bacterial - genetics</subject><subject>Fluoroquinolones - pharmacology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genotype</subject><subject>Humans</subject><subject>Isoniazid - pharmacology</subject><subject>Microbial Sensitivity Tests</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Mutation</subject><subject>Mycobacteriology and Aerobic Actinomycetes</subject><subject>Mycobacterium tuberculosis</subject><subject>Mycobacterium tuberculosis - drug effects</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>Ofloxacin - pharmacology</subject><subject>Phenotype</subject><subject>Rifampin - pharmacology</subject><subject>Sequence Analysis, DNA - methods</subject><subject>Time Factors</subject><issn>0095-1137</issn><issn>1098-660X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp90k9vFCEcBmBiNHat3jwraaJedip_h-FiYlarNW00q028EYaBXZoZ2MKMun56WXdT9eIFDjx584MXAB5jdIoxaV5-WFyeIkyIrJC8A2YYyaaqa_T1LpghJHmFMRVH4EHO1whhxji_D46wbFhdMzIDPz5tU8z2ZrLB-LCCLia41BvfwTd2tGb0McDo4OXWxFab0SY_DXCcWpvM1MfsM1zaso46GAvHCJfe6WHjwxye5xi8_um7OdShg2f9FFO8mXyIfQw2PwT3nO6zfXTYj8HV2dsvi_fVxcd354vXF5Vhko8Vo8Y2pm4ZNw47i6kj2lBpWNM2HHedI6zlRpC6JUgYrgUSlGOJONUSoc7RY_Bqn7uZ2sF2xoYx6V5tkh902qqovfr3JPi1WsVvioimroUsAS8OAbvxbR7V4LOxfa-DjVNWglJMMW9Ekc__KwkmtIy3i5zvoSlvn5N1t-NgpHalqlKq-l2qQjv-5O8r_MGHFgt4dgA6G927VNrw-dYRQlAjBCruZO_WfrX-7pNVOg_q2gyKCYWJorLhBT3dI6ej0qtUgq4-E4QpwmL3uTj9BbQmwXs</recordid><startdate>20091201</startdate><enddate>20091201</enddate><creator>Bravo, Lulette Tricia C</creator><creator>Tuohy, Marion J</creator><creator>Ang, Concepcion</creator><creator>Destura, Raul V</creator><creator>Mendoza, Myrna</creator><creator>Procop, Gary W</creator><creator>Gordon, Steven M</creator><creator>Hall, Geraldine S</creator><creator>Shrestha, Nabin K</creator><general>American Society for Microbiology</general><general>American Society for Microbiology (ASM)</general><scope>FBQ</scope><scope>IQODW</scope><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>7QL</scope><scope>C1K</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20091201</creationdate><title>Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones</title><author>Bravo, Lulette Tricia C ; Tuohy, Marion J ; Ang, Concepcion ; Destura, Raul V ; Mendoza, Myrna ; Procop, Gary W ; Gordon, Steven M ; Hall, Geraldine S ; Shrestha, Nabin K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c495t-43ce8c6b45cf1fe13f2ac39c48b851ddf24b5c726b207c5a7073519053a900df3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Antitubercular Agents - pharmacology</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Drug Resistance, Bacterial - genetics</topic><topic>Fluoroquinolones - pharmacology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genotype</topic><topic>Humans</topic><topic>Isoniazid - pharmacology</topic><topic>Microbial Sensitivity Tests</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Mutation</topic><topic>Mycobacteriology and Aerobic Actinomycetes</topic><topic>Mycobacterium tuberculosis</topic><topic>Mycobacterium tuberculosis - drug effects</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Ofloxacin - pharmacology</topic><topic>Phenotype</topic><topic>Rifampin - pharmacology</topic><topic>Sequence Analysis, DNA - methods</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bravo, Lulette Tricia C</creatorcontrib><creatorcontrib>Tuohy, Marion J</creatorcontrib><creatorcontrib>Ang, Concepcion</creatorcontrib><creatorcontrib>Destura, Raul V</creatorcontrib><creatorcontrib>Mendoza, Myrna</creatorcontrib><creatorcontrib>Procop, Gary W</creatorcontrib><creatorcontrib>Gordon, Steven M</creatorcontrib><creatorcontrib>Hall, Geraldine S</creatorcontrib><creatorcontrib>Shrestha, Nabin K</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of Clinical Microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bravo, Lulette Tricia C</au><au>Tuohy, Marion J</au><au>Ang, Concepcion</au><au>Destura, Raul V</au><au>Mendoza, Myrna</au><au>Procop, Gary W</au><au>Gordon, Steven M</au><au>Hall, Geraldine S</au><au>Shrestha, Nabin K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones</atitle><jtitle>Journal of Clinical Microbiology</jtitle><addtitle>J Clin Microbiol</addtitle><date>2009-12-01</date><risdate>2009</risdate><volume>47</volume><issue>12</issue><spage>3985</spage><epage>3990</epage><pages>3985-3990</pages><issn>0095-1137</issn><eissn>1098-660X</eissn><coden>JCMIDW</coden><abstract>After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>19846642</pmid><doi>10.1128/JCM.01229-09</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Antitubercular Agents - pharmacology Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacteriology Biological and medical sciences Drug Resistance, Bacterial - genetics Fluoroquinolones - pharmacology Fundamental and applied biological sciences. Psychology Genotype Humans Isoniazid - pharmacology Microbial Sensitivity Tests Microbiology Miscellaneous Mutation Mycobacteriology and Aerobic Actinomycetes Mycobacterium tuberculosis Mycobacterium tuberculosis - drug effects Mycobacterium tuberculosis - genetics Ofloxacin - pharmacology Phenotype Rifampin - pharmacology Sequence Analysis, DNA - methods Time Factors |
| title | Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones |
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