The novel DPO, LNA, and other assay-sets designed here, combined with previously published serogroup 6, 18, and 22 assay-sets effectively discriminated between current PCV-formulation (PCV10, PCV13) VT (6A and 6B, 18C), next-generation PCV-formulation (PCV15, PCV20) VT 22F and NVT (6C, 6D, 18A, 18B, 18F and 22A) within a nanofluidic molecular serotyping reaction-set. Serogroups 6, 18, and 22 have previously been discriminated using conventional PCR 15,26,27, or sequencing-based methods 12,13,28. Sequencing-based methods are expensive, not all laboratories have access to sequencing platforms, bioinformatics software and expertise. Further, some methods were unable to fully distinguish 6C/D or 18B/C 28. Microarray assays have been developed, but these are also costly and require initial time-consuming culture steps 29. No previous high-throughput real-time PCR assay has been reported to fully distinguish serogroup 6, 18, or 22 10,30−32 or made use of modified primer or probe strategies such as LNA-probes or DPO primers as utilised in this study. This is also the first study to describe the use of thermodynamically modified assay-sets within the Fluidigm platform.
Comprehensive accurate serotyping is essential where VTs may still be circulating and to assess the relative benefit of next-generation PCVs that will include serotype 22F. Current published molecular assays, including the serogroup 6 and 18 described here, now enable detection of PCV13-VT 1, 3, 4, 5, 6A, 6B, 14, 19A, 19F, and 23F individually 10,11. Our reaction-set is the first to distinguish all PCV13-serotypes except serogroup 9A/V and 7A/F to single serotypes. Relative to 9V, 9A has a frameshift deletion in a G-polymer region surrounded by an AT-rich region 20. Similarly, 7A lacks a side chain due to a frameshift mutation in the glycosyl transferase (wcwD) gene 20 located in a poly-T region within a GC region. These molecular features make the SNPs in 9V and 7F difficult to target and thermodynamically improbable to distinguish with molecular methods other than sequencing-based approaches.
The performance (sensitivity, specificity, and limit of detection) of the serogroup 6, 18, and 22 reaction-sets in the Fluidigm platform is comparable with other high-throughput strategies including the TaqMan Array Cards and the same Fluidigm platform including using different real-time PCR chemistry 10,32,33. The performance of this reaction-set demonstrates that modified primers (DPO) or probes (LNA) are easily adapted to high-throughput real-time PCR, including where specific target pre-amplification is undertaken in multiplex (up to 34 assay-sets per tube). The successful combination of a DPO primer with a LNA probe to target 18C/F is a novel strategy. The reaction-sets described here can be easily adapted to other real-time PCR platforms.
This study was limited in that non-pneumococcal Streptococcus species were not included in the validation of the assay-sets. Previous studies have validated the assay-sets for 6A/B/C/D, 6C/D, 18A/B/C, and 22AF against non-pneumococcal Streptococcus 10 and our algorithm included that samples must be detected with the serogroup 6 assay-set (6A/B/C/D), 18A/B/C, and 22AF to be assigned a serogroup 6, 18 or 22 type respectively, hence the designed assays are unlikely to detect other non-pneumococcal species. The methods described here are not currently aimed at detecting the hybrid serotypes 6F/G/H, as no reference specimens or clinical samples were available to validate the detection of these additional serotypes. Serotype 6B ‘sub-class II’ (6E) is a genetic variant of 6B, and the genetic regions targeted here are ubiquitous in 6B sub-classes so would be correctly assigned serotype 6B 23. Prospective studies should include surveillance for hybrid serotypes and conduct confirmatory sequencing for isolates typed as serogroup 6 where PCVs are in use and residual serogroup 6 carriage is observed. The Fluidigm could not be fully evaluated against the Quellung-method for detection of serotypes 6D, 18A/B/F or 22A in clinical isolates as these serotypes were not detected in the archived clinical samples by Quellung, most likely due to their low prevalence in Africa. Nevertheless, our algorithm correctly identified cultured control strains and would be able to correctly discern serotype 6D, 18A, 18F, 22A and 22F in clinical isolates. While the culture-based Quellung method is specific, PCR is more sensitive 34 and capable of detecting multiple co-colonising or culture non-typeable serotypes, even at a low density 35. Since the positive predictive value of Quellung is not perfect, discrepant serotype positive samples detected by PCR are resolved as ‘true positives’ by confirming the presence of pneumococcal reference genes 36. As Fluidigm PCR is compared to Quellung in this study, this may affect the measured concordance, for example serotype 6B where this serotype was detected in an additional 73 samples with Fluidigm. Further, the clinical samples that were re-analysed with Fluidigm as part of this study, had undergone multiple freeze-thaw cycles. This may have affected the sensitivity of Fluidigm compared with Quellung.
The DPO assay-sets targeting wciPα (6A/C) and wciX (18C/F) in conjunction with the designed wciX (18B/C/F), wcxM (18F), wcwA (22F), and published serogroup 6, 18 and 22 (6A/B/C/D; 18A/B/C; 22AF) 11 and 6C/D 37 assay-sets using the applied algorithms, can be used to correctly serotype circulating serogroup 6, 18 and 22 to individual serotypes using Fluidigm® real-time PCR in clinical samples.