# A novel real-time PCR assay for the simultaneous detection of the four main causes of bacterial meningitis

## TL;DR

This paper introduces a new real-time PCR test that can detect four major bacterial causes of meningitis at once, offering improved diagnostic accuracy and potential for use in low-resource settings.

## Contribution

The development of the first multiplex PCR assay for simultaneous detection of four WHO-priority meningitis pathogens with no loss in sensitivity.

## Key findings

- The multiplex PCR assay achieved 100% sensitivity for all four target pathogens.
- The assay demonstrated high specificity and consistent amplification across all targets.
- The assay's limit of detection ranged from 24 to 66 genome copies per microliter.

## Abstract

•First multiplex PCR for the four WHO priority bacterial meningitis pathogens.•Assay demonstrates 100% sensitivity for all targets with no performance loss from multiplexing.•A robust tool for improving meningitis diagnosis, particularly for Streptococcus agalactiae (GBS) in LMICs.

First multiplex PCR for the four WHO priority bacterial meningitis pathogens.

Assay demonstrates 100% sensitivity for all targets with no performance loss from multiplexing.

A robust tool for improving meningitis diagnosis, particularly for Streptococcus agalactiae (GBS) in LMICs.

Current multiplex assays cannot detect all four World Health Organization priority pathogens for meningitis diagnosis (Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Streptococcus agalactiae). This work developed a novel real-time polymerase chain reaction (PCR) assay capable of simultaneously detecting these pathogens.

Forty-four DNA samples, including type cultures (NCTC and ATCC), were tested. Specific primers and probes targeting sodC, to detect N. meningitidis, dmsA for H. influenzae, SP2020 for S. pneumoniae, and cfb for S. agalactiae were evaluated in monoplex and multiplex. Standard curves were generated, and limits of detection (LLD), slope, intercept, and R² were determined. Sensitivity, specificity, and predictive values (positive predictive values/negative predictive values [PPV/NPV]) were assessed for both monoplex and multiplex assays.

Monoplex and multiplex assays showed equivalent performance. Sensitivities were 100% for all targets; specificities ranged 91.7% to 100%, PPVs 72.7% to 100%, and NPVs 100%. The multiplex assay showed high efficiency and consistent amplification for each target gene. LLDs ranged from 24 (S. pneumoniae) to 66 (H. influenzae) genome copies/µl.

The multiplex PCR assay showed good performance for rapid and accurate detection of meningitis-associated bacteria. This test has applications for improved diagnosis of meningitis in low- and middle-income countries, where laboratory confirmation of major pathogens such as H. influenzae, S. pneumoniae, N. meningitidis, and Group B Streptococcus remains limited. Field validation with clinical specimens is required before implementation.