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Article: Comparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

TitleComparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens
Authors
Issue Date2013
PublisherAmerican Society for Microbiology. The Journal's web site is located at http://jcm.asm.org/
Citation
Journal of Clinical Microbiology, 2013, v. 51, p. 2592-2598 How to Cite?
AbstractMacrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy. Copyright © 2013, American Society for Microbiology.
Persistent Identifierhttp://hdl.handle.net/10722/186087
ISSN
2015 Impact Factor: 3.631
2015 SCImago Journal Rankings: 2.151
PubMed Central ID
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorChiu, SSS-
dc.contributor.authorChan, KH-
dc.contributor.authorTo, KKW-
dc.contributor.authorLi, PY-
dc.contributor.authorYuen, KY-
dc.contributor.authorHo, PL-
dc.contributor.authorChan, WK-
dc.date.accessioned2013-08-20T11:53:34Z-
dc.date.available2013-08-20T11:53:34Z-
dc.date.issued2013-
dc.identifier.citationJournal of Clinical Microbiology, 2013, v. 51, p. 2592-2598-
dc.identifier.issn0095-1137-
dc.identifier.urihttp://hdl.handle.net/10722/186087-
dc.description.abstractMacrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy. Copyright © 2013, American Society for Microbiology.-
dc.languageeng-
dc.publisherAmerican Society for Microbiology. The Journal's web site is located at http://jcm.asm.org/-
dc.relation.ispartofJournal of Clinical Microbiology-
dc.rightsJournal of Clinical Microbiology. Copyright © American Society for Microbiology.-
dc.titleComparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens-
dc.typeArticle-
dc.identifier.emailChiu, SSS: ssschiu@hku.hk-
dc.identifier.emailChan, KH: chankh2@hkucc.hku.hk-
dc.identifier.emailTo, KKW: kelvinto@hkucc.hku.hk-
dc.identifier.emailLi, PY: coloryan@hku.hk-
dc.identifier.emailYuen, KY: kyyuen@hkucc.hku.hk-
dc.identifier.emailHo, PL: plho@hkucc.hku.hk-
dc.identifier.authorityChiu, SSS=rp00421-
dc.identifier.authorityChan, KH=rp01921-
dc.identifier.authorityTo, KKW=rp01384-
dc.identifier.authorityYuen, KY=rp00366-
dc.identifier.authorityHo, PL=rp00406-
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1128/JCM.00785-13-
dc.identifier.pmid23720793-
dc.identifier.pmcidPMC3719661-
dc.identifier.scopuseid_2-s2.0-84880589798-
dc.identifier.hkuros219559-
dc.identifier.volume51-
dc.identifier.spage2592-
dc.identifier.epage2598-
dc.identifier.isiWOS:000321951800018-
dc.publisher.placeUnited States-

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