A nested PCR-based point-of-care multiplex test for detection of bacterial pathogens in cerebrospinal fluids

Area of Science:

• Clinical microbiology
• Molecular diagnostics
• Infectious disease diagnostics

Background:

• Bacterial meningitis caused by Streptococcus pneumoniae (SPN), Streptococcus agalactiae (GBS), and Streptococcus pyogenes (GAS) requires rapid and accurate diagnosis for timely treatment.
• Current diagnostic methods, such as culture, are time-consuming, while existing molecular assays may lack the sensitivity or automation for point-of-care settings.

Purpose of the Study:

• To develop and validate a multiplex one-tube nested real-time polymerase chain reaction (mOTN-PCR) assay integrated with a point-of-care testing (POCT) instrument.
• To achieve an automated "sample-in, result-out" workflow for the rapid, sensitive detection of SPN, GBS, and GAS in cerebrospinal fluid (CSF).

Main Methods:

• Sensitivity and specificity of mOTN-PCR were assessed using recombinant plasmids and common pathogens.
• Simulated CSF samples underwent manual and automated nucleic acid extraction, followed by detection using mOTN-PCR and conventional real-time PCR.
• Clinical performance was evaluated using 109 clinical samples (36 CSF, 73 non-CSF) and compared against conventional real-time PCR with manual extraction.

Main Results:

• mOTN-PCR demonstrated high sensitivity with detection limits of 5-10 copies/μL for plasmids and 10-20 CFU/mL for simulated CSF samples.
• The integrated mOTN-PCR-POCT assay reduced total testing time to approximately 2 hours, compared to 4 hours for manual extraction.
• The mOTN-PCR-POCT assay identified three additional positive cases in clinical samples compared to conventional real-time PCR, indicating superior detection capability.

Conclusions:

• The mOTN-PCR-POCT assay provides a simple, rapid, and highly sensitive method for detecting SPN, GBS, and GAS directly from CSF.
• This automated workflow eliminates the need for bacterial culture and streamlines the diagnostic process for meningitis pathogens.
• The developed assay shows significant potential for improving the diagnosis of bacterial meningitis at the point-of-care.