Evaluation of a sample-to-answer real-time PCR assay for enterovirus detection in cerebrospinal fluid

Enteroviruses frequently cause aseptic meningitis, necessitating differentiation from bacterial meningitis to avoid unnecessary antimicrobial treatment and improve patient management. Polymerase chain reaction (PCR) remains the gold standard for enterovirus detection in cerebrospinal fluid (CSF) due to its high sensitivity and rapid turnaround time. The discontinuation of the Cepheid Xpert EV assay has created a gap in diagnostics. This study aimed to optimize two laboratory-developed real-time PCR assays using Diasorin direct amplification disc (DAD) and universal disc (UD) methods compared to the previously standard Cepheid Xpert EV assay. A total of 87 clinical CSF specimens were tested to assess sensitivity, specificity, and overall performance. There was no significant difference between the performance of the three assays compared to standard of care results. The Xpert EV and DAD methods had a 79.6% positive agreement, while the UD method had an 86.4% positive agreement. All methods achieved 100% negative agreement. Specificity testing against non-enteroviral pathogens confirmed no cross-reactivity. The estimated limit of detection was 100 copies/mL for Xpert EV, 250 copies/mL for UD, and 1,000 copies/mL for DAD, though clinical sensitivity remained high. Blood contamination affected the DAD assay but not the UD method due to its RNA extraction step. This study demonstrates the potential of DAD and UD methods as viable alternatives for rapid enterovirus detection in CSF, essential for patient management following the discontinuation of the Xpert EV assay. Further validation and comparison are needed for broader clinical applications.