Patients
This was a retrospective study that enrolled patients treated at the University Medical Centre Freiburg in Germany between 2005 and 2016, using an electronic database search. Lumbar puncture (LP) had already been performed in all patients for clinical purposes after obtaining written consent. Paired CSF and serum samples were collected on the same day and stored according to the consensus protocol for the standardization of CSF collection and biobanking [
18]. Haemolytic CSF samples were excluded.
Patients with RDwCNS had been diagnosed by board certified rheumatologists of the University Medical - Centre Freiburg. Systemic lupus erythematosus (SLE) was identified according to the 2007 revised criteria for the classification of SLE [
19]; ANCA associated vasculitides, both, MPO-associated MPA and PR3-associated GPA (Wegener’s granulomatosis) according to the revised international Chapel Hill consensus conference nomenclature of vasculitides [
20,
21], and Behçet’s disease according to the International Study Group for Behçet’s disease [
22]. CNS involvement of RDwCNS was defined on the basis of clinical signs such as headache, neuropsychological disturbances or focal neurological disturbances (all without better explanation), and the presence of either of the following two paraclinical findings: (1) inflammatory changes in the CSF, such as elevated cell count, intrathecal immunoglobulin synthesis, positive oligoclonal bands (OCB), or significant disturbance in the blood-CSF barrier indicated by an age-related elevation in the albumin quotient or (2) inflammatory signs in brain or spinal MRI compatible with RDwCNS as assessed by neuroradiologists of the University Medical - Centre Freiburg. Patients with suspected RDwCNS were excluded if they fulfilled the 2010 revised McDonald criteria for MS [
23]. All patients with RDwCNS who fulfilled these criteria were enrolled in this study.
Diagnosis of MS was established according to the 2010 revised McDonald criteria with particularly careful exclusion of the relevant differential diagnoses [
23]. MS patients were drawn from a cohort of MS patients (comprising 103 patients with PPMS and 100 with RRMS) from an earlier study [
11]. A previously established in-house matching software was used to select the MS patients for this study who best matched for age and sex with the RDwCNS patients in a 2:1 (MS:RDwCNS) ratio [
24].
As the third study group, forty-eight patients with OIND, for whom MRZR test results were available from previous research [
17], were enrolled. Data concerning the ethnicity and immunization status of study patients were not available. This study was approved by the ethics committee of the University Medical Centre Freiburg (EK-Fr 489/14).
Materials and methods
MRZR was analysed at the Department of Virology of the University of Freiburg. All routine CSF measurements were carried out in the CSF laboratory of the University Medical Centre Freiburg. Total immunoglobulin concentrations in the serum and the CSF were detected nephelometrically (ProSpect System, Siemens, Germany), while measles-, rubella- and varicella-IgG (IgGspec) levels in the CSF and the serum were measured using enzyme-linked immunosorbent assay (Serion
classic ELISA, Germany). MRZR was determined from the three respective virus-specific AIs that were calculated as follows: AI (antibody index) = QIgG[spec]/QIgG[total], if QIgG[total] < Qlim, and AI = QIgG[spec]/Qlim, if QIgG[total] > Qlim [
25]. The upper reference range of QIgG, Qlim, was calculated according to Reiber’s formula [
25]. For a positive AI result indicative of intrathecal IgG production against the respective pathogen, a threshold of ≥1.5 was applied. Most previous studies have varied as to how many positive AIs are required for a positive MRZR [
11]. In this study, MRZR-2 was defined as that with two or three positive AIs, and MRZR-1 as that requiring only one or more positive AIs. In cases where an AI could not be calculated because no antibodies were detected in the CSF, the AI was considered as 1.0 (negative).
As the focus in this study was on RDwCNS, additional data of these patients regarding the following were obtained: (1) results of brain/spinal MRI performed and routinely assessed at the Department of Neuroradiology of the University Medical - Centre Freiburg from medical records, (2) results of the CSF routine test (including the parameters of total cell count, age-related albumin quotient, quantitative intrathecal antibody synthesis, and OCB) from medical records, and (3) the AIs of four characteristic autoantibodies (dsDNA, Cardiolipin, PR3, and MPO). The serum samples of RDwCNS patients were therefore initially screened for the presence of autoantibodies associated with rheumatologic disorders if sufficient serum sample was available after the routine examinations and the MRZR measurements. Anti-nuclear antibody (ANA) staining pattern was assessed using indirect immunofluorescence (IIF) on 2100-Ro HEp-2000® cells (Fluorescent ANA-RoTest System, Immuno Concepts N.A. Ltd., Sacramento, CA, USA). RDwCNS Patients with positive IIF were screened for antibodies using enzyme-linked immunosorbent assay (ELISA) directed against extractable nuclear antigens (ENA) using ANA Profil 3 (DL1590–3 G, EUROIMMUN AG, Luebeck, Germany). Antibodies against double stranded deoxyribonucleic acid (DNA) were detected using dsDNA IgG ELISA (212,196, Euro Diagnostica AB, Malmö, Sweden). Phospholipid antibodies were measured using Cardiolipin IgG ELISA (212,796, Euro Diagnostica AB, Malmö, Sweden). ANCA specificity for PR3 (Orgentec Diagnostika GmbH, Mainz, Germany) or myeloperoxidase (MPO) (Euroimmun, Medizinische Labordiagnostika AG, Luebeck, Germany) was measured using ELISA as well. Assessment was done according to the manufacturers’ reference ranges with the upper normal limit of 40 U/mL for dsDNA, 14 U/mL for Cardiolipin, 10 U/mL for PR3, and 20 U/mL for MPO. In case of an elevated concentration of antibodies against dsDNA, Cardiolipin, PR3, or MPO in serum, the CSF titre was additionally measured for calculating the respective AI, as described for MRZR above.
The detection of OCB was performed using an isoelectric focusing technique on agarose gel followed by immunofixation (Hydragel Isofocusing, Sebia, France). A positive OCB result was defined as two or more OCB [
26].