Multiplex PCR to Streptococcus pneumoniae serotype identification directly in cerebrospinal fluid samples

Area of Science:

• Clinical Microbiology
• Molecular Diagnostics
• Epidemiology

Background:

• Streptococcus pneumoniae causes serious invasive diseases like meningitis.
• Cerebrospinal fluid (CSF) culture, the standard diagnostic, often yields negative results, hindering surveillance and immunization strategies.
• Genotyping pneumococcus directly from CSF could overcome limitations of culture-based methods.

Purpose of the Study:

• To standardize a multiplex PCR (mPCR) method for pneumococcal genotyping directly from CSF samples.
• To evaluate the predictive performance of this mPCR assay.
• To mitigate information loss due to absent bacterial isolates in meningitis diagnostics.

Main Methods:

• Eight multiplex PCR (mPCR) assays were applied to CSF samples.
• mPCR results were compared against the Quellung reaction on isolated strains.
• Different master mix kits were evaluated for mPCR efficiency; a retrospective study analyzed CSF samples positive for the lytA gene.

Main Results:

• mPCR genotyping showed 100% correlation with the Quellung reaction, with performance dependent on the master mix used.
• In a retrospective analysis (2014-2020), 73.4% of CSF samples were successfully genotyped.
• A lytA gene cycle threshold (Ct value) around 30 predicted a 75% chance of successful genotyping, while Ct > 35 predicted only 12.5%.

Conclusions:

• Standardized mPCR of CSF samples is a reliable method for pneumococcal genotyping, especially when isolates are absent.
• The assay effectively predicts Streptococcus pneumoniae serotypes, providing crucial data for surveillance.
• Genotype 19A was identified as prevalent (12%), information previously unavailable, highlighting the assay's value for public health insights.