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

Abstract

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.

Fig 1. Flow chart showing the pneumococcal isolates included in the study.

*Of the 40 isolates that were tested by rmPCR, only 25 were included as part of the rmPCR targets.

Fig 2. Serotype distribution of nasopharyngeal pneumococcal isolates.

The figure includes serotypes detected from the Drakenstein Child Health Study, determined by Quellung reaction, excluding duplicate serotypes from the same infant. Blue = serotypes included in PCV13; Red = serotypes not included in PCV13. Green = non-typable isolates.

Fig 3. Similarity of 16F-like capsular polysaccharide (cps) gene loci.

Sequences from pneumococci serotyped as 16F Quellung but sequetyped as 9V was compared to reference 9V (CR931648) and 16F (CR931668) cps sequences. Artemis Comparison Tool (ACT) was used to generate and view gene homology. The top lines represent the forward and reverse strand of a serotype 9v reference, the middle lines represent the queried 16F strain and the bottom lines shows the 16F reference. The portion of the wzh gene that is amplified by the sequetyping assay is shown by the blue rectangle. The clear blocks below the blue box shows regions were the genes that are not similar. BLASTN matches are shown as red bands between sequences, indicating the degree of similarity between the sequences.

Fig 4. Comparative genome analysis of pneumococcal serotypes 16F and 9V genetic background.

When the sequence identities of all four genomes were compared using RAST(Rapid Annotation using Subsystem Technology), the genome backbone of all three 16F (103347 and 103385 from this study and a 16F control strain) were mostly identical but divergent from 9V. The colour codes represent how close or divergent the genomes are. Therefore, similar genome backgrounds will have similar colours.