Development of Gene-based Conventional PCR Assay for Diagnostic Application of Streptococcus pneumoniae Infections as an Alternative to Conventional Culture: A Cross-sectional Study

Introduction: Streptococcus pneumoniae (S. pneumoniae), an opportunistic pathogen, is the primary cause of pneumonia and is classified as a high-priority bacterial pathogen. However, it is often underdiagnosed by conventional microbiological tests. The present study was conducted to develop a simple, rapid and reliable molecular method for the early and accurate diagnosis of S. pneumoniae using the Duplex Polymerase Chain Reaction (PCR) Assay. Among various genes responsible for the pathogenesis of pneumococci, the LytA gene and the CpsA gene were targeted for the molecular identification of S. pneumoniae. Aim: To evaluate the developed duplex PCR assay alongside the microbial culture of the S. pneumoniae pathogen. Materials and Methods: This study was carried out at the microbiology department of Surat Municipal Institute of Medical Education and Research (SMIMER), Surat, Gujarat, India, from March 2020 to March 2022. A molecular method for the detection of S. pneumoniae present in various clinical samples by PCR was developed. The optimised Duplex PCR assay employs primers specific for the LytA and CpsA genes of S. pneumoniae. The method involves the extraction of bacterial genomic DNA, PCR amplification and agarose gel electrophoresis to analyse the amplification products. The performance of the newly developed Duplex PCR assay was evaluated and compared its results with previously described assays, such as monoplex PCR assays targeting a single gene and conventional microbiological tests. Different clinical samples (n=75) from patients with pneumonialike symptoms were used for PCR analysis and culture. The sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of the PCR assay were determined in comparison with the gold standard microbial culture test. The level of significance was assessed using Fisher’s exact test, with tests considered significant at a p-value <0.05. Results: The duplex PCR assay demonstrated the best performance in terms of sensitivity and specificity, with no misidentifications found among non-pneumococcal strains for the CpsA and LytA genes. The assay was capable of detecting 6 CFU/mL of S. pneumoniae American Type Culture Collection (ATCC)® 49619™. The specificity of the developed Duplex PCR assay was 100% for both LytA and CpsA-specific genes. In total, 20% (n=15/75) of different clinical samples yielded positive results using the culture method. S. pneumoniae was isolated from 7 (9.3%) of sputum samples, 4 (5.3%) of blood cultures, 3 (4%) of cerebrospinal fluid (CSF), and 1 (1.3%) of pleural fluid samples, respectively, by conventional microbiological culture methods. Using the S. pneumoniae-specific Duplex PCR assay, 23 (30.7%) of samples tested positive for S. pneumoniae, as these samples exhibited amplification for both the LytA and CpsA genes. Of these, S. pneumoniae was detected in 9 (12%) of nasopharyngeal/oropharyngeal (NP/OP) swabs, 6 (8%) of sputum samples, 4 (5.3%) of blood cultures, 3 (4%) of CSF samples, and 1 (1.3%) of pleural fluid samples, respectively. The sensitivity, specificity, PPV and NPV of PCR for detecting S. pneumoniae in comparison with the culture method were 100%, 96.7%, 88% and 100%, respectively. Conclusion: The developed and optimised S. pneumoniaespecific Duplex PCR assay targeting the CpsA and LytA genes, as described in this study, is a powerful strategy that resulted in no false identifications. This significantly increases the sensitivity and specificity of pneumococcal identification compared to standard microbial culture and monoplex PCR assays targeting only a single gene.