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Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragmen...

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Published in:	Journal of clinical microbiology 2015-12, Vol.53 (12), p.3779-3783
Main Authors:	Maningi, Nontuthuko E, Daum, Luke T, Rodriguez, John D, Mphahlele, Matsie, Peters, Remco P H, Fischer, Gerald W, Chambers, James P, Fourie, P Bernard
Format:	Article
Language:	English
Subjects:	Amidohydrolases - genetics Antitubercular Agents - pharmacology Drug Resistance, Bacterial Genotyping Techniques - methods High-Throughput Nucleotide Sequencing - methods Humans Microbial Sensitivity Tests - methods Mycobacteriology and Aerobic Actinomycetes Mycobacterium tuberculosis Mycobacterium tuberculosis - drug effects Mycobacterium tuberculosis - genetics Pyrazinamide - pharmacology Sensitivity and Specificity
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container_title	Journal of clinical microbiology
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creator	Maningi, Nontuthuko E Daum, Luke T Rodriguez, John D Mphahlele, Matsie Peters, Remco P H Fischer, Gerald W Chambers, James P Fourie, P Bernard
description	The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.
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A.</contributor><creatorcontrib>Maningi, Nontuthuko E ; Daum, Luke T ; Rodriguez, John D ; Mphahlele, Matsie ; Peters, Remco P H ; Fischer, Gerald W ; Chambers, James P ; Fourie, P Bernard ; Land, G. A.</creatorcontrib><description>The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. 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Daum, Luke T ; Rodriguez, John D ; Mphahlele, Matsie ; Peters, Remco P H ; Fischer, Gerald W ; Chambers, James P ; Fourie, P Bernard</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c460t-def2676f4e5d1c66f679a399662372777f41cf7b153a5980954936ea76e1100f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amidohydrolases - genetics</topic><topic>Antitubercular Agents - pharmacology</topic><topic>Drug Resistance, Bacterial</topic><topic>Genotyping Techniques - methods</topic><topic>High-Throughput Nucleotide Sequencing - methods</topic><topic>Humans</topic><topic>Microbial Sensitivity Tests - methods</topic><topic>Mycobacteriology and Aerobic Actinomycetes</topic><topic>Mycobacterium tuberculosis</topic><topic>Mycobacterium tuberculosis - drug effects</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Pyrazinamide - pharmacology</topic><topic>Sensitivity and Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Maningi, Nontuthuko E</creatorcontrib><creatorcontrib>Daum, Luke T</creatorcontrib><creatorcontrib>Rodriguez, John D</creatorcontrib><creatorcontrib>Mphahlele, Matsie</creatorcontrib><creatorcontrib>Peters, Remco P H</creatorcontrib><creatorcontrib>Fischer, Gerald W</creatorcontrib><creatorcontrib>Chambers, James P</creatorcontrib><creatorcontrib>Fourie, P Bernard</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</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>Maningi, Nontuthuko E</au><au>Daum, Luke T</au><au>Rodriguez, John D</au><au>Mphahlele, Matsie</au><au>Peters, Remco P H</au><au>Fischer, Gerald W</au><au>Chambers, James P</au><au>Fourie, P Bernard</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates</atitle><jtitle>Journal of clinical microbiology</jtitle><addtitle>J Clin Microbiol</addtitle><date>2015-12-01</date><risdate>2015</risdate><volume>53</volume><issue>12</issue><spage>3779</spage><epage>3783</epage><pages>3779-3783</pages><issn>0095-1137</issn><eissn>1098-660X</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><notes>Present address: Matsie Mphahlele, Jhpiego South Africa, Pretoria, South Africa.</notes><notes>Citation Maningi NE, Daum LT, Rodriguez JD, Mphahlele M, Peters RPH, Fischer GW, Chambers JP, Fourie PB. 2015. Improved detection by next-generation sequencing of pyrazinamide resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol 53:3779–3783. doi:10.1128/JCM.01179-15.</notes><abstract>The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>26378284</pmid><doi>10.1128/JCM.01179-15</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record>
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source	American Society for Microbiology; PubMed Central (Open access)
subjects	Amidohydrolases - genetics Antitubercular Agents - pharmacology Drug Resistance, Bacterial Genotyping Techniques - methods High-Throughput Nucleotide Sequencing - methods Humans Microbial Sensitivity Tests - methods Mycobacteriology and Aerobic Actinomycetes Mycobacterium tuberculosis Mycobacterium tuberculosis - drug effects Mycobacterium tuberculosis - genetics Pyrazinamide - pharmacology Sensitivity and Specificity
title	Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates
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