Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping

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dc.contributor.authorDube, Felix Sen_ZA
dc.contributor.authorvan Mens, Suzan Pen_ZA
dc.contributor.authorRobberts, Lourensen_ZA
dc.contributor.authorWolter, Nicoleen_ZA
dc.contributor.authorNicol, Paulen_ZA
dc.contributor.authorMafofo, Josephen_ZA
dc.contributor.authorAfrica, Samanthaen_ZA
dc.contributor.authorZar, Heather Jen_ZA
dc.contributor.authorNicol, Mark Pen_ZA
dc.date.accessioned2015-12-28T06:51:40Z
dc.date.available2015-12-28T06:51:40Z
dc.date.issued2015en_ZA
dc.description.abstractBACKGROUND: Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. METHODS: A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. RESULTS: Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A. CONCLUSION: The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.en_ZA
dc.identifier.apacitationDube, F. S., van Mens, S. P., Robberts, L., Wolter, N., Nicol, P., Mafofo, J., ... Nicol, M. P. (2015). Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping. <i>PLoS One</i>, http://hdl.handle.net/11427/16067en_ZA
dc.identifier.chicagocitationDube, Felix S, Suzan P van Mens, Lourens Robberts, Nicole Wolter, Paul Nicol, Joseph Mafofo, Samantha Africa, Heather J Zar, and Mark P Nicol "Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping." <i>PLoS One</i> (2015) http://hdl.handle.net/11427/16067en_ZA
dc.identifier.citationDube, F. S., van Mens, S. P., Robberts, L., Wolter, N., Nicol, P., Mafofo, J., ... & Nicol, M. P. (2015). Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping. PloS one, 10(9), e0137349. doi:10.1371/journal.pone.0137349en_ZA
dc.identifier.ris TY - Journal Article AU - Dube, Felix S AU - van Mens, Suzan P AU - Robberts, Lourens AU - Wolter, Nicole AU - Nicol, Paul AU - Mafofo, Joseph AU - Africa, Samantha AU - Zar, Heather J AU - Nicol, Mark P AB - BACKGROUND: Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. METHODS: A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. RESULTS: Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A. CONCLUSION: The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable. DA - 2015 DB - OpenUCT DO - 10.1371/journal.pone.0137349 DP - University of Cape Town J1 - PLoS One LK - https://open.uct.ac.za PB - University of Cape Town PY - 2015 T1 - Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping TI - Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping UR - http://hdl.handle.net/11427/16067 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/16067
dc.identifier.urihttp://dx.doi.org/10.1371/journal.pone.0137349
dc.identifier.vancouvercitationDube FS, van Mens SP, Robberts L, Wolter N, Nicol P, Mafofo J, et al. Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping. PLoS One. 2015; http://hdl.handle.net/11427/16067.en_ZA
dc.language.isoengen_ZA
dc.publisherPublic Library of Scienceen_ZA
dc.publisher.departmentDivision of Medical Biochemistryen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_ZA
dc.rights.holder© 2015 Dube et alen_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourcePLoS Oneen_ZA
dc.source.urihttp://journals.plos.org/plosoneen_ZA
dc.subject.otherPolymerase chain reactionen_ZA
dc.subject.otherSequence databasesen_ZA
dc.subject.otherGenome analysisen_ZA
dc.subject.otherInfantsen_ZA
dc.subject.otherSequence analysisen_ZA
dc.subject.otherSequence assembly toolsen_ZA
dc.subject.otherChild healthen_ZA
dc.subject.otherVaccinesen_ZA
dc.titleComparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotypingen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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