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


CONCLUSIONS
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.
Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Felix S
FS
Dube
Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; Department of Medical Microbiology and Immunology, St Antonius Hospital, Nieuwegein, the Netherlands.
Suzan P
SP
van Mens
Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Lourens
L
Robberts
Centre for Respiratory Diseases and Meningitis (CRDM), National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa; School of Pathology, Faculty of Health Sciences, University of the Witswatersrand, Johannesburg, South Africa.
Nicole
N
Wolter
The State Agricultural Biotechnology Centre, Murdoch University, Murdoch, Australia.
Paul
P
Nicol
Centre for Proteomic and Genomic Research (CPGR), Cape Town, South Africa.
Joseph
J
Mafofo
Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Samantha
S
Africa
Department of Paediatrics and Child Health, Red Cross War Memorial Children's Hospital, Cape Town, South Africa; MRC Unit on Child and Adolesscent Health, University of Cape Town, Cape Town, South Africa.
Heather J
HJ
Zar
Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa.
Mark P
MP
Nicol
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D002353:Carrier State / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D057166:High-Throughput Screening Assays; D006801:Humans; D007223:Infant; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009305:Nasopharynx / Q000382:microbiology; D011008:Pneumococcal Infections / Q000453:epidemiology / Q000382:microbiology / Q000517:prevention & control; D022242:Pneumococcal Vaccines; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D012703:Serotyping; D013019:South Africa / Q000453:epidemiology; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification
C538862:13-valent pneumococcal vaccine; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D022242:Pneumococcal Vaccines; C500440:CpsB protein, Streptococcus pneumoniae; D017027:Protein Tyrosine Phosphatases