Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples following pre-enrichment culture.

Molecular methods for diagnosing Lyme neuroborreliosis (LNB) have shown suboptimal diagnostic sensitivities. The objective of this study was to improve the clinical sensitivity of PCR detection of Borrelia burgdorferi sensu lato spirochetes by inoculating cerebrospinal fluid (CSF) from patients suspected of LNB directly into culture medium at the time of lumbar puncture, with this pursuing enrichment of Borrelia spirochetes before PCR analysis. Adult patients with symptoms suggestive of LNB were prospectively enrolled at two hospitals in the Region of Southern Denmark. The CSF-culture samples were incubated for at least eight weeks. During this period, culture sample aliquots were analysed for the presence of Borrelia DNA by separate PCR protocols in two independent clinical laboratories. The included patients were diagnosed with definite (n=12) or possible (n=2) LNB, and non-LNB (n=171) based on clinical and paraclinical findings. Patients in the LNB and the non-LNB group had a median duration from symptom onset to lumbar puncture of 40 days (IQR [23-90] days) and 120 days (IQR [32-365] days), respectively. Pre-enrichment growth of Borrelia spirochetes was accomplished from three patients (21 %) in the LNB group. The positive culture samples were confirmed by both the digital droplet PCR and the real-time PCR methods employed. All CSF samples were PCR negative in the non-LNB group. The results of this study do not support the use of Borrelia-specific PCR as a general routine diagnostic tool in adults. Still, they suggest it may prove of additional value in selected patients with a limited time from symptom onset to sample collection.

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.

Discriminating between Lyme neuroborreliosis and other central nervous system infections by use of biomarkers CXCL13 and IL-6.

CXCL13 in cerebrospinal fluid has gradually become an established biomarker for Lyme neuroborreliosis (LNB), however the diagnostic performance of CXCL13 may be improved by the addition of IL-6, a non-specific infection biomarker. The aim of this study was to measure the concentrations of CXCL13 and IL-6 in cerebrospinal fluid, in the attempt to evaluate the diagnostic performance of these two biomarkers, in the differentiation between definite and possible LNB, as well as between LNB and other neuroinfections. This study used a cross-sectional design to quantify the levels of CXCL13 and IL-6 in cerebrospinal fluid (CSF) specimens from consecutive patients examined for central nervous system (CNS) infections at Lillebaelt Hospital in the Region of Southern Denmark. CXCL13 and IL-6 were measured simultaneously using the Bio-Plex 200 multiplex Cytokine Immunoassay System (Bio-Rad). Based on clinical and paraclinical findings, we grouped patients into six separate groups: definite LNB, possible LNB, Viral CNS infection, non-Borrelia Bacterial CNS infection, Other CNS disease (with pleocytosis) and Negative (without pleocytosis). A combined interpretation of four variables (leukocyte cell counts, protein concentration, CXCL13 and IL-6 concentrations in CSF) is presented using principal component cluster analysis. We included by chart review 390 patients discharged with definite LNB (n = 31), possible LNB (n = 10), confirmed Viral or non-Borrelia Bacterial CNS infection (n = 34), Other CNS disease (n = 58), and Negative (n = 257) for CXCL13 and IL-6 analysis. Principal component analysis (PCA) revealed three distinct clusters based on leukocyte cell counts, protein concentration, CXCL13 and IL-6 concentrations in CSF from 380 included patients (10 possible LNB patients excluded). The clusters clearly differentiate the groups: definite LNB, non-Borrelia Bacterial CNS infection and Negative (without pleocytosis). A receiver operating characteristic (ROC) curve comparing LNB patients (n = 31) and all non-LNB conditions with CSF pleocytosis (n = 99) indicated an optimal CXCL13 cut-off value of 50.7 pg/mL, resulting in a sensitivity and a specificity of 93.6 and 91.1%, respectively. The ROC analysis comparing patients with confirmed non-LNB CNS infection (n = 34) and all others with CSF pleocytosis (n = 97) resulted in an optimal IL-6 cut-off value of 111.5 pg/mL, yielding a sensitivity and a specificity of 78.8% and 82.5% respectively. Of the ten possible LNB patients, three cases (with CXCL13 levels above cut-off) fall within the LNB cluster, and one case is just outside, providing some laboratory support for the diagnosis of LNB. The remaining six possible LNB patients (with CXCL13 levels below the 50.7 cut-off) had little support for the diagnosis of LNB in the PCA-plot. The results of this study confirm that CXCL13 is a valuable supplement for diagnosis of LNB, and that the combination of CXCL13 and IL-6 may be used to differentiate cases of LNB from other CNS infections. Furthermore, IL-6 can be of differential diagnostic value when evaluating patients with possible LNB.