Development and validation of a novel multiplex digital PCR assay for identification of pathogens in cerebrospinal fluid of children with bacterial meningitis

Background and aims: Identifying the pathogens of bacterial meningitis (BM) is crucial for its diagnosis and treatment. The aim of this study is to develop and validate a novel method for detecting pathogens in cerebrospinal fluid (CSF) of children with BM using a digital polymerase chain reaction (dPCR) assay. Materials and methods: A novel multiplex dPCR assay method has been developed and validated. The diagnostic performance of the dPCR assay was compared with that of synchronous CSF culture, and the factors affecting its performance were analyzed. Results: A total of 69 children with BM were enrolled prospectively. The sensitivity of the dPCR assay was 94.44 %, specificity was 100 %, coincidence rate was 98.55 %, Kappa value was 0.959, and net reclassification improvement was 61.11 %. Compared with the CSF culture assay, the dPCR assay had higher sensitivity in different bacterial groups. Multiple factors affected its performance, including previous use of antibiotics, sampling time, BM complications, and levels of inflammatory biomarkers in CSF and blood (all P < 0.05). Patients who required intensive care and died had a higher bacterial DNA loads identified by dPCR assay (both P < 0.05). Conclusion: This novel assay has better pathogen detection ability than CSF culture. Its performance was influenced by sampling time, previous use of antibiotics, and disease severity.