We aim to develop the MPL-rPCR to detect all serotypes as the WHO suggests by designing specific primers and probes targeting individual serotypes.

Materials and methods

We designed the specific primers and probes targeting the specific sequences at the capsular synthesis locus and from that, developed a 10-multiplex assay, each detects 4 serotypes or serogroups. This MLP rPCR can be performed on the S. pneumonia2 isolates, as well as on various samples, including DNA extractions from blood, cerebrospinal fluid (CSF) or sputum. We subsequently analyzed the results and compared to the PCR method suggested by the WHO.

Results

We gathered 90 S. pneumoniae isolates from blood and CFS, along with 63 sputum samples. From the blood and CFS isolates, with our MPL-rPCR, we identified 12 serotypes or serogroups. The most prevalent were 19A, 19F, and 23F, each accounting for 18.9%, followed by 6A/B at 13.3%. Other serotypes were each detected in less than 10%, including 14 (7.8%), 15B/C (6.7%), 15A/F (4.4%), 9A/V (4.4%), 9N/L 3.3%), 4 (1.1%), 18 A/B/C/E (1.1%), và 23A (1.1%). In the 63 sputum samples collected from inpatients with pneumonia, 76.2% of the samples were detected the pneumococcal serotypes that were belonged to 16 serotypes or serogroups. In which the serotype 9N/L was dominated (23.8%), followed by 24 A/F at 7.9%, and 39, 10F/C/33C, each at 6.3%.  Our findings were comparable with the results obtained from the PCR recommended by the WHO and the agreement that we had received was 100%.

Conclusions

The MLP rPCR technique that we develop is proficient in detecting S. pneumoniae serotypes, aligning with WHO recommendations. If widely and routinely applied, this technique facilitates addressing antibiotic resistance, vaccine coverage, and advancing research on serotypes associated with various clinical conditions

Keywords: Pneumococcal serotypes, Multiplex Real-time PCR

Table 1: PCR-serotyping scheme for USA based on current serotype prevalence (recommended by WHO), and the S. pneumoniae serotype must be identified by agarose electrophoresis

Table 2: The MPL-rPCR for identification of the S. pneumoniae serotype is based on 10 MPL-rPCR mix

Figure 1: The MPL-rPCR procedure for identification of the S. pneumoniae serotype from the bacterial colonies