Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

PLOS ONE, Dec 2019

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.

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Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

September Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping Felix S. Dube 0 1 Suzan P. van Mens 0 1 Lourens Robberts 0 1 Nicole Wolter 0 1 Paul Nicol 0 1 Joseph Mafofo 0 1 Samantha Africa 0 1 Heather J. Zar 0 1 Mark P. Nicol 0 1 0 1 Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town , Cape Town , South Africa , 2 Department of Medical Microbiology and Immunology, St Antonius Hospital, Nieuwegein, the Netherlands, 3 National Health Laboratory Service, Groote Schuur Hospital , Cape Town , South Africa , 4 Department of Paediatrics and Child Health, Red Cross War Memorial Children's Hospital , Cape Town, South Africa, 5 MRC Unit on Child and Adolesscent Health , University of Cape Town , Cape Town , South Africa , 6 Centre for Respiratory Diseases and Meningitis (CRDM), National Institute for Communicable Diseases of the National Health Laboratory Service , Johannesburg , South Africa , 7 School of Pathology, Faculty of Health Sciences, University of the Witswatersrand , Johannesburg , South Africa , 8 The State Agricultural Biotechnology Centre, Murdoch University , Murdoch , Australia , 9 Centre for Proteomic and Genomic Research (CPGR) , Cape Town , South Africa 1 Editor: Shamala Devi Sekaran, University of Malaya , MALAYSIA - Funding: This work was funded in part by grants from the Bill and Melinda Gates Foundation Global Health Grant (OPP1017641) and H3Africa (1U01 HG006961-01). Felix S. Dube is supported by the National Research Foundation of South Africa; S.P. van Mens received funding from the St Antonius Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. 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. 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 Competing Interests: The authors have declared that no competing interests exist. 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. 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. The pneumococcus (Streptococcus pneumoniae) is a common cause of invasive disease and respiratory tract infections including bloodstream infections, meningitis, pneumonia and otitis media [1–3]. Patients at risk include those at the extremes of age and the immunocompromised, particularly those affected by cell-mediated immune deficiencies. Colonisation of the nasopharynx with a homologous strain of pneumococci precedes the development of invasive and respiratory tract disease [2,4,5]. Serotyping of the pneumococcal polysaccharide capsule, the immunogenic component of current vaccines, remains the cornerstone of strain characterization. To date, more than 90 capsular serotypes have been described and new ones continue to be described [6,7]. Multiple pneumococcal se (...truncated)


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Felix S. Dube, Suzan P. van Mens, Lourens Robberts, Nicole Wolter, Paul Nicol, Joseph Mafofo, Samantha Africa, Heather J. Zar, Mark P. Nicol. Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping, PLOS ONE, 2015, Volume 10, Issue 9, DOI: 10.1371/journal.pone.0137349