Establishment of a digital PCR method for detection of Borrelia burgdorferi sensu lato complex DNA in cerebrospinal fluid

Direct detection of Borrelia burgdorferi sensu lato bacteria in patient samples for diagnosis of Lyme neuroborreliosis (LNB) is hampered by low diagnostic sensitivity, due to few bacteria in cerebrospinal fluids (CSF) samples. Evaluation of novel molecular methods, including digital PCR (dPCR), as future tools in diagnostics of LNB is desirable. This study aimed to establish a dPCR assay and validate pre-PCR procedures for detection of Borrelia in CSF. Synthetic DNA fragments and cultured Borrelia reference strains were used during optimisation experiments. In addition, 59 CSF specimens from patients examined for LNB were included for clinical validation. The results showed that the pre-PCR parameters with the highest impact on Borrelia -specific dPCR method performance were incubation of the PCR-plate at 4 °C for stabilization of droplets, centrifugation for target concentration, quick-spin for dPCR rain reduction, and PCR inhibition by matrix components. Borrelia DNA in CSF was detected in one out of nine patients with LNB. Diagnostic sensitivity was determined to be 11.1% and specificity 100%. In conclusion, this study reports an optimized Borrelia -specific dPCR method for direct detection of Borrelia in CSF samples. The present study does not support the use of Borrelia -specific dPCR as a routine method for diagnosing LNB.