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

Abstract

BackgroundPneumococcal 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.MethodsA 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.ResultsOf 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.ConclusionThe 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.

Longitudinal characterization of nasopharyngeal colonization with Streptococcus pneumoniae in a South African birth cohort post 13-valent pneumococcal conjugate vaccine implementation

“…The collected NP swabs were immediately placed into 1 ml skim milk-tryptone-glucose-glycerol (STGG), transported at 4 °C to the laboratory within 2 hours of collection and frozen at −80 °C for later batch culture. Presumptive pneumococcal isolates were identified by colony morphology, α-hemolysis, optochin disk susceptibility (Oxoid, Basingstoke, UK) and confirmed using lyt A PCR 19 . Serotyping was performed on a single morphologically distinct pneumococcal colony per sample by sequetyping 20 and confirmation using the Quellung method.…”

Distribution of capsular types and drug resistance patterns of invasive pediatric Streptococcus pneumoniae isolates in Teheran, Iran

“…9 Pneumococci are divided into more than 90 capsular types based on the structural characterization of the antigenic capsular polysaccharide. 10 The polysaccharide capsule, which delivers resistance to phagocytosis in the absence of type-specific antibody and helps the bacteria to evade the host's defenses, plays a major role in the invasion of pneumococcus into the systemic blood system. 11 Serotype-specific pneumococcal polysaccharide vaccines (PPVs) and pneumococcal conjugate vaccines (PCVs) have been designed according to the pathogenic mechanism of S. pneumoniae, which combine the capsular polysaccharides of the most prevalent invasive serotypes.…”

Development of a TaqMan Array Card for Pneumococcal Serotyping on Isolates and Nasopharyngeal Samples

“…After elucidation of the capsular biosynthetic locus ( 13 ), PCR assays for the capsular polysaccharide synthesis gene clusters have been devised. Sequencing-based assays of the cps and wzh genes ( 14 , 15 ) have been published, as have real-time PCR assays to detect 21 serotypes/serogroups ( 16 , 17 ). Nanofluidic, microarray, and Luminex-based systems have also been developed ( 18 – 21 ).…”