Retrospective Evaluation of Various Serological Assays and Multiple Parameters for Optimal Diagnosis of Lyme Neuroborreliosis in a Routine Clinical Setting.

Laboratory diagnosis of Lyme neuroborreliosis (LNB) is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Random forest (RF) modeling was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine cerebrospinal fluid (CSF) parameters (i.e., leukocyte count, total protein, blood-CSF barrier functionality, and intrathecal total antibody synthesis), two-tier serology on serum, the CSF level of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13), and a Borrelia species PCR on CSF. In total, 156 patients were included who were classified as definite LNB (n = 10), possible LNB (n = 7), or non-LNB patient (n = 139) according to the criteria of the European Federation of Neurological Societies using a consensus strategy for intrathecal Borrelia-specific antibody synthesis. The seven antibody assays showed sensitivities ranging from 47.1% to 100% and specificities ranging from 95.7% to 100%. RF modeling demonstrated that the sensitivities of most antibody assays could be improved by including other parameters to the diagnostic repertoire for diagnosing LNB (range: 94.1% to 100%), although with slightly lower specificities (range: 92.8% to 96.4%). The most important parameters for LNB diagnosis are the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. In conclusion, this study shows that LNB diagnosis is best supported using multiparameter analysis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed for improved LNB diagnosis. IMPORTANCE The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Multiparameter analysis was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine (CSF) parameters. The results of this study show that LNB diagnosis is best supported using the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, we propose a collaborative prospective study to investigate the potential role of constructing a diagnostic algorithm using multiparameter analysis for improved LNB diagnosis.

The Usefulness of Two CXCL13 Assays on Cerebrospinal Fluid for the Diagnosis of Lyme Neuroborreliosis: a Retrospective Study in a Routine Clinical Setting.

Recent studies have shown elevated levels of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13) in the cerebrospinal fluid (CSF) of patients with early Lyme neuroborreliosis (LNB). In this retrospective study, we evaluated the diagnostic performance of the Quantikine CXCL13 enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., MN, USA) and the recomBead CXCL13 assay (Mikrogen, Neuried, Germany) for the detection of CXCL13 in CSF. All consecutive patients from whom a CSF and a serum sample had been collected between August 2013 and June 2016 were eligible for inclusion. Patients suspected of LNB were classified as definite, possible, or non-LNB according to the guidelines of the European Federation of Neurological Societies (EFNS). Due to the limited number of LNB patients in the predefined study period, additional LNB patients were included from outside this period. In total, 156 patients (150 consecutive patients and 6 additional LNB patients) were included. Seven (4.5%) were classified as definite, eight (5.1%) as possible, and 141 (90.4%) as non-LNB patients. Receiver operating characteristic (ROC) curve analysis comparing definite-LNB patients with non-LNB patients showed a cutoff value of 85.9 pg/ml for the Quantikine CXCL13 ELISA and 252.2 pg/ml for the recomBead CXCL13 assay. The corresponding sensitivity was 100% (95% confidence interval [CI], 100% to 100%) for both, and the corresponding specificities were 98.6% (95% CI, 96.5% to 100%) for the CXCL13 ELISA and 97.2% (95% CI, 93.6% to 100%) for the recomBead CXCL13 assay. This study showed that CXCL13 in CSF can be of additional value for the diagnosis of LNB.

Consequences of the Edge Effect in a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Lyme Neuroborreliosis.

The diagnosis of Lyme neuroborreliosis (LNB) is based on neurological symptoms, cerebrospinal fluid (CSF) pleocytosis, and intrathecally produced Borrelia-specific antibodies. In most cases, the presence of intrathecally produced Borrelia-specific antibodies is determined by using an enzyme-linked immunosorbent assay (ELISA). The edge effect is a known phenomenon in ELISAs and can negatively influence the assay reproducibility and repeatability, as well as index calculations of sample pairs which are tested in the same run. For LNB diagnostics, an index calculation is used for which the relative amounts of Borrelia-specific antibodies in CSF and serum are measured to calculate a CSF/serum quotient, which is needed to calculate the Borrelia-specific antibody index (AI). The presence of an edge effect in an ELISA used for LNB diagnostics may thus have implications. In this study, we investigated the intra-assay variation of the commercial Enzygnost Lyme link VlsE/IgG ELISA used for LNB diagnostics and showed the presence of an edge effect. Minor adaptations in the ELISA protocol decreased this effect. The adapted protocol was subsequently used to test 149 CSF-serum pairs of consecutive patients received in a routine diagnostic laboratory. By simulation, we showed that, if the standard protocol would have been used, then the edge effect for this study population could have resulted in 15 (10.1%) false-pathological and two (1.3%) false-normal Borrelia-specific IgG AIs. Thus, the observed edge effect can lead to inaccurate LNB diagnoses. Our study underlines that the edge effect should be investigated when ELISAs are implemented in routine diagnostics, as this phenomenon can occur in any ELISA.