Occupation-Associated Fatal Limbic Encephalitis Caused by Variegated Squirrel Bornavirus 1, Germany, 2013.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.

Performance Assessment of the New EUROLINE Neurologic Syndrome 15 Ag (IgG) for the Determination of Autoantibodies Associated with Neurological Disorders.

OBJECTIVE: Here, we assess the performance of the new EUROLINE Neurologic Syndrome 15 Ag (IgG) which expands the EUROLINE Paraneoplastic Neurologic Syndrome 12 Ag by adding CDR2L (together with CDR2 targeted by anti-Yo), AK5, and Neurochondrin (NCDN). BACKGROUND: Many paraneoplastic as well as non-paraneoplastic autoantibodies (AAbs) have been described in neurological disorders in the last decade. By integrating the associated antigens into existing assays, the diagnostic work-up of patients is being improved and diagnostic gaps reduced. DESIGN/METHODS: Sensitivity of each AAb was analyzed using a total of 194 clinically and diagnostically pre-characterized samples (Table 1). Specificity of each AAb was investigated using a minimum of 100 sera from healthy blood donors. RESULTS: Using the EUROLINE Neurologic Syndrome 15 Ag, autoantibody positivity was confirmed in 89-100% of samples. In particular, all samples for which clinical and tissue-based indirect immunofluorescence assay pre-characterization indicated anti-Yo positivity were anti-CDR2 and -CDR2L double positive. Anti-AK5 was determined in serum and cerebrospinal fluid (CSF) with a sensitivity of 90 and 100%, respectively, and anti-NCDN with a sensitivity of 100%. The individual specificities were ≥99%. CONCLUSIONS: This kit is a tool for the qualitative in vitro determination of AAbs against a large panel of 15 different neuronal autoantigens to support the diagnosis of neurologic syndromes. The parallel detection of anti-CDR2 and anti-CDR2L (both anti-Yo) increases the diagnostic significance, as double positivity is strongly related to paraneoplastic cerebellar degeneration.[Table: see text].

CDR2 and CDR2L line blot performance in PCA-1/anti-Yo paraneoplastic autoimmunity.

Background: Purkinje cytoplasmic autoantibody type 1 (PCA-1)/anti-Yo autoimmunity is a common high-risk paraneoplastic neurological disorder, traditionally attributed antigenically to cerebellar degeneration-related protein 2 (CDR2), predominantly affecting women with gynecologic or breast adenocarcinoma. Single-modality CDR2 testing may produce false-positive results. We assessed the performance characteristics of the more recently purported major PCA-1/Yo antigen, CDR2-like (CDR2L), side by side with CDR2, in a line blot format. Methods: CDR2 and CDR2L were tested in six specimen groups (serum and cerebrospinal fluid (CSF)). Group 1, PCA-1/Yo mouse brain indirect immunofluorescence assay (IFA) positives; Group 2, PCA-1/Yo IFA mimics; Group 3, suspected CDR2 line blot false positives; Group 4, consecutive patient samples tested for neural antibodies over 1 year; Group 5, healthy subject serums; and Group 6, polyclonal (non-specific) immunoglobulin G (IgG)-positive serums. Results: Group 1: Of 64 samples tested, all but two were CDR2 positive (both CSF samples) and all were CDR2L positive. In individual patients, CDR2L values were always higher than CDR2. The two "CDR2L-only" positives were CSF samples with low titer PCA-1/Yo by IFA with serum negativity but with typical clinical phenotype. Group 2: All 51 PCA-1/Yo mimics were CDR2/CDR2L negative. Group 3: Nine samples [six of 1289 (0.47%) serums and three of 700 CSF samples (0.43%) were PCA-1/Yo IFA negative/CDR2 positive; two of the six available (serums from the same patient) were also CDR2L positive; the other four CDR2L negative had low CDR2 values (17-22). Group 4: Twenty-two patients had unexpected CDR2 or CDR2L positivity; none had tissue IFA positivity. Eleven of the 2,132 serum (0.5%) and three of the 677 CSF (0.4%) samples were CDR2 positive; median value was 19 (range, 11-48). Seven of the 2,132 serum (0.3%) and three of the 677 CSF (0.4%) samples were CDR2L positive; median value was 18 (range, 11-96). Group 5: All 151 healthy serum samples were negative. Group 6: One of the 46 polyclonal serum samples was CDR2L positive. Optimum overall performance was accomplished by requiring both CDR2 and CDR2L positivity in serum (sensitivity, 100%; and specificity, 99.9%) and positivity for CDR2L in CSF (sensitivity, 100%; and specificity, 99.6%). Conclusion: CDR2L provides additional PCA-1/anti-Yo sensitivity in CSF, and dual positivity with CDR2 provides additional specificity assurance in serum. Combining antigen-specific and tissue-based assays optimizes PCA-1/anti-Yo testing.

Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome.

OBJECTIVE: To describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma. METHODS: Sera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein. RESULTS: The sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years). CONCLUSION: Our results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma. CLASSIFICATION OF EVIDENCE: This study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.

Effects of IVIg treatment on autoantibody testing in neurological patients: marked reduction in sensitivity but reliable specificity.

BACKGROUND: Therapy of autoimmune diseases of the central and peripheral nervous system with intravenous IgG immunoglobulin (IVIg) is well established. Since IVIg is produced from pooled human plasma, autoantibodies can be found in IVIg products and, accordingly, in patient sera after transfusion. The de novo evidence or disappearance of anti-neural autoantibodies after IVIg treatment has so far not been systematically examined. METHODS: We screened 50 neurological patients before and after IVIg treatment for classical onconeural and the most common neurological surface autoantibodies as well as for ganglioside autoantibodies and 23 different antinuclear autoantibodies using immunoblot or cell-based indirect immunofluorescence assays. Furthermore, we screened 31 neurological patients with previously known seropositivity for disappearance of the corresponding antibody after treatment. RESULTS: After IVIg treatment, 90% of all sera were de novo positive for antinuclear antibodies, especially for Ro-52. In contrast, 94% of all sera did not show any de novo-positive anti-neural antibodies. In the remaining three cases, titers were very low. Importantly, 12.9% of all tested sera of patients with known antibody positivity turned false negative after IVIg treatment and titers were falsely low in 37% of the remaining sera. CONCLUSIONS: Here, we present for the first time results of a broad screening for clinically relevant autoantibodies before and after IVIg treatment in neurological patients. We identified a high specificity but reduced sensitivity for anti-neural antibody testing after IVIg transfusion. In contrast, antinuclear antibody testing is not reliable after IVIg treatment. These results are of high practical importance for diagnostic of neuroimmunological diseases.

N-terminal disulfide-bridging of Borrelia outer surface protein C increases its diagnostic and vaccine potentials.

OspC is the main target for IgM in early-stage Lyme disease. As such it is employed as its native or recombinant form in routine immunoassays for the determination of Borrelia-specific antibodies. However, recombinant OspC has so far not shown the antigenicity of the native protein. The latter contains an intrinsic signal sequence and an adjacent cysteine residue, the attachment site of the lipid membrane anchor which has been discussed to have an adjuvant effect on the immune reaction. In expression experiments, we have found a recombinant variant, an OspC covalently homodimerized via an N-terminal disulfide bridge, that shows a remarkably enhanced antigenicity without lipid attachment. Three such OspCs derived from different Borrelia strains were subsequently expressed in E. coli and purified under non-reducing conditions. In non-reducing SDS-PAGE, OspC(Δ1-18) exhibited a 48-kDa band of dimeric OspC. When incubated with IgM-OspC-positive human sera, the reaction at 48kDa was always stronger than at 24kDa of monomeric OspC(Δ1-18, C19G). A lineblot with OspC(Δ1-18) also showed a higher diagnostic accuracy than that obtained with OspC(Δ1-18, C19G) based on a higher affinity of IgM for the dimeric form. When used for the immunization of mice, dimeric OspC(Δ1-18) induced consistent high-titre antibodies against OspC, whereas OspC(Δ1-18, C19G) failed to provoke significant titres in some animals. We conclude that the disulfide-bridging of 2 OspC molecules via their N termini forms a complex that is more suitable for the determination of IgM-OspC and is a promising candidate for a monovalent vaccine.