Serum neurofilament light chain associates with symptom burden in Lyme neuroborreliosis patients: a longitudinal cohort study from Norway.

OBJECTIVES: Serum neurofilament light chain (sNfL), an indicator of neuronal damage, is increasingly recognized as a potential biomarker for disease activity in neurodegenerative disorders. In this study, we wanted to investigate sNfL as a prognostic marker in a large, well-defined population of 90 patients with Lyme neuroborreliosis (LNB). In addition, we sought to explore associations between symptoms and sNfL levels during the acute phase of LNB. MATERIALS AND METHODS: Patients diagnosed with definite or possible LNB were recruited from a double-blinded, placebo-controlled, multi-center trial, in which the participants were randomly assigned to 2 or 6 weeks of oral doxycycline treatment. The sNfL levels were measured using a single molecule array assay at both diagnosis and 6-month follow-up, and analysed against clinical parameters, variations in symptom burden and long-term complaints as assessed by a composite clinical score. RESULTS: At the time of diagnosis, approximately 60% of the patients had elevated sNfL levels adjusted for age. Notably, mean sNfL levels were significantly higher at diagnosis (52 pg/ml) compared to 6 months after treatment (12 pg/ml, p < 0.001), when sNfL levels had normalized in the majority of patients. Patients with objective signs of spinal radiculitis had significantly higher baseline sNfL levels compared to patients without spinal radiculitis (p = 0.033). CONCLUSION: Our findings suggest that sNfL can serve as a biomarker for peripheral nerve tissue involvement in the acute phase of LNB. As found in an earlier study, we confirm normalization of sNfL levels in blood after treatment. We found no prognostic value of acute-phase sNfL levels on patient outcome.

Comparison of the Euroimmun Borrelia 'antibody index' with Virotech immunoblot-based detection of intrathecal Borrelia antibody production for the diagnosis of Lyme neuroborreliosis.

For diagnosis of neuroborreliosis, calculation of the antibody index, based on Euroimmun Anti-Borrelia plus VlsE ELISA was compared to Virotech Borrelia Europe plus TpN17 immunoblot-based detection of Borrelia-specific intrathecal antibody production. CXCL13 results in cerebrospinal fluid were used to evaluate discordant results. A total of 64 serum/CSF pairs were analysed. Patients were classified according to European Federation of Neurological Societies criteria incorporating Virotech results. For the Euroimmun assay, a sensitivity of 100% and specificity of 94% was found. Agreement between the both tests was almost perfect (κ 0.81). Both methods are appropriate for the detection of Borrelia-specific intrathecal antibody production.

Are other tick-borne infections overlooked in patients investigated for Lyme neuroborreliosis? A large retrospective study from South-eastern Sweden.

In Europe, the hard tick Ixodes ricinus is considered the most important vector of human zoonotic diseases. Human pathogenic agents spread by I. ricinus in Sweden include Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Rickettsia helvetica, the recently described Neoehrlichia mikurensis, Borrelia miyamotoi, tick-borne encephalitis virus (TBEV), and Babesia spp. (Babesia microti, Babesia venatorum and Babesia divergens). Since these pathogens share the same vector, co-infections with more than one tick-borne pathogen may occur and thus complicate the diagnosis and clinical management of the patient due to possibly altered symptomatology. Borrelia burgdorferi s.l., TBEV and B. miyamotoi are well-known to cause infections of the central nervous system (CNS), whereas the abilities of other tick-borne pathogens to invade the CNS are largely unknown. The aim of this study was to investigate the presence and clinical impact of tick-borne pathogens other than B. burgdorferi s.l. in the cerebrospinal fluid (CSF) and serum samples of patients who were under investigation for Lyme neuroborreliosis (LNB) in a tick-endemic region of South-eastern Sweden. CSF and serum samples from 600 patients, recruited from the Regions of Östergötland County, Jönköping County and Kalmar County in South-eastern Sweden and investigated for LNB during the period of 2009-2013, were retrospectively collected for analysis. The samples were analysed by real-time PCR for the presence of nucleic acid from B. burgdorferi s.l., B. miyamotoi, A. phagocytophilum, Rickettsia spp., N. mikurensis, TBEV and Babesia spp. Serological analyses were conducted in CSF and serum samples for all patients regarding B. burgdorferi s.l., and for the patients with CSF mononuclear pleocytosis, analyses of antibodies to B. miyamotoi, A. phagocytophilum, spotted fever group (SFG) rickettsiae, TBEV and B. microti in serum were performed. The medical charts of all the patients with CSF mononuclear pleocytosis and patients with positive PCR findings were reviewed. Of the 600 patients, 55 (9%) presented with CSF mononuclear pleocytosis, 13 (2%) of whom had Borrelia-specific antibodies in the CSF. One patient was PCR-positive for N. mikurensis, and another one was PCR-positive for Borrelia spp. in serum. No pathogens were detected by PCR in the CSF samples. Four patients had serum antibodies to B. miyamotoi, four patients to A. phagocytophilum, five patients to SFG rickettsiae, and six patients to TBEV. One patient, with antibodies to SFG rickettsiae, had both clinical and laboratory signs suggestive of a current infection. Nine patients had serum antibodies to more than one pathogen, although none of these was assessed as a current co-infection. We can conclude from this study that tick-borne co-infections are uncommon in patients who are being investigated for suspected LNB in South-eastern Sweden, an area endemic for borreliosis and TBE.

The chemotactic cytokines in the cerebrospinal fluid of patients with neuroborreliosis.

BACKGROUND: The outcome of neuroborreliosis (NB) is variable and may partially depend on host-related immune factors. In NB, the cerebrospinal fluid (CSF) contains a large population of T lymphocytes, but the mechanisms and consequences of their recruitment have not been fully elucidated. We have studied expression of T lymphocyte chemoattractant cytokines in association with CSF cytometric parameters and clinical data in NB patients. METHODS: The blood and CSF of 17 patients with NB and blood of 12 patients with erythema migrans (EM) were obtained before the antibiotic administration, and in fraction of NB patients during and/or after antibiotic treatment. The control samples came from blood donors (blood) and patients in whom neuroinfection was excluded by a lumbar puncture (CSF). Concentrations of IL-16, CXCL9, CXCL10, CXCL11, CCL2 and CCL5 in serum and CSF were measured with commercial ELISA. Data were analyzed with non-parametric tests, p < 0.05 considered significant. RESULTS: The serum concentrations of IL-16, CXCL9, CXCL10 and CCL5 were increased, higher in NB than in EM. In CSF all the cytokines were upregulated, CXCL10, CXCL9 and IL-16 over ten-fold. The CSF concentration index favored the intrathecal synthesis of all the cytokines except CCL5, for which it could not be reliably estimated. CCL2, CXCL10 and CXCL9 created concentration gradients towards CSF. The intrathecal expression of IL-16, CCL5 and CXCL9 correlated with CSF lymphocyte counts, of IL-16, CXCL9 and CXCL10 - with a blood-brain barrier disruption, and of CXCL9 and CXCL10 with intrathecal specific IgG synthesis. The expression of CCL2, CXCL10 and CXCL11 peaked early after NB onset and decreased naturally afterwards. High initial CSF CXCL9, CXCL10 and CXCL11 levels associated with a persistent CSF pleocytosis and BBB disruption after treatment, but no cytokine was predictive of clinical outcome. In follow up (post-treatment) examinations, CSF CXCL10 and CCL5 associated positively and CCL2 negatively with a protracted lymphocytic pleocytosis. CONCLUSIONS: Several cytokines chemotactic for T lymphocytes are upregulated intrathecally in NB, with different dynamics and relation to other inflammatory parameters, suggesting their distinct pathogenetic roles. CXCL10 and CXCL9 are vividly upregulated and seem deeply involved in the pathogenesis of the intrathecal inflammation. IL-16 and CCL5 may directly drive T lymphocyte migration from periphery, but their ability to create an adequate chemotactic gradient remains to be confirmed. A delayed normalization of pleocytosis is accompanied by higher intrathecal expression of Th1-related and lower of Th2-related chemokines, in agreement with the protective role of Th1 to Th2 transition in the course of NB.

Lyme neuroborreliosis in Swedish children-PCR as a complementary diagnostic method for detection of Borrelia burgdorferi sensu lato in cerebrospinal fluid.

The aim of this study was to evaluate polymerase chain reaction (PCR) as a diagnostic method for the detection of Borrelia burgdorferi s.l. in CSF of Swedish children with LNB. This study was performed retrospectively on CSF and serum samples collected from children evaluated for LNB (n = 233) and controls with other specific neurological disorders (n = 59) in a Swedish Lyme endemic area. For anti-Borrelia antibody index, the IDEIA Lyme Neuroborreliosis kit (Oxoid) was used. Two in-house real-time PCR assays targeting the 16S rRNA gene were evaluated (TaqMan® and LUX™). Among patients classified as LNB cases (n = 102), five children (5%) were Borrelia PCR-positive in CSF with the TaqMan® assay. In the Non-LNB group (n = 131), one patient was Borrelia PCR positive with the TaqMan® assay. Among controls (n = 59), all CSF samples were PCR negative. When amplifying and sequencing ospA, we found B. garinii (n = 2), B. afzelii (n = 2), B. bavariensis (n = 1), and one untypable (n = 1). With the LUX™ technology, all CSF samples were PCR negative. The TaqMan® assay could detect only few cases (n = 6) of B. burgdorferi s.l. in CSF among children with LNB and the sensitivity was very low (5%). However, using larger CSF volumes and centrifugation of samples, the PCR technique could still be useful as a complementary diagnostic method when evaluating LNB. Furthermore, detection of spirochete DNA in clinical matrices, including CSF, is the method of choice for studying epidemiological aspects of LNB, a tick-borne emerging disease.

Quantitative serological antibody testing for suspected neuroborreliosis.

OBJECTIVE: To assess the importance of serum IgG/IgM antibody titers for the differentiation of Lyme neuroborreliosis (LNB) from its mimics. METHOD: This was a retrospective, cross-sectional study conducted at two German neurological centers. Serological parameters (ELISA or CLIA analysis) and clinical presentation of 28 patients with definite LNB were compared to those of 36 patients with neurological symptoms mimicking LNB (mimics). Analysis was performed using receiver operating characteristic (ROC) and binary logistic regression. RESULTS: Elevated IgG-titers had a high sensitivity for neuroborreliosis in both centers (0.95 and 1.0). The optimal cutoff-values were set to 26.35 in center A (ELISA), and 64.0 in center B (CLIA). Diagnostic specificity was 0.41 and 0.89 in this constellation. Elevated IgM-titers showed a high diagnostic specificity for a cutoff at 68.10 (A) and 47.95 (B) (0.93 and 0.89). Sensitivity was 0.45 and 0.5. Overall diagnostic accuracy was low in both centers (A: IgG AUC = 0.665, IgM AUC = 0.629; B: IgG AUC = 0.917, IgM AUC = 0.556). In logistic regression of antibody titers and clinical measures, prediction of LNB was significantly better than the "null hypothesis". Clinical measures showed the highest odds ratio. CONCLUSION: Data show that in addition to the clinical presentation of patients with symptoms suggesting central or peripheral nervous system manifestation, serum IgG- and IgM-titers help to identify LNB-patients. The results should guide physicians counseling patients with suspected LNB about further diagnostic steps and treatment.

A Neurologist's View of Lyme Disease and Other Tick-Borne Infections.

Tick-borne infections-including tick-borne encephalitis viruses, represented in the United States by rare infections with Powassan and deer tick viruses, and more often Lyme disease-are of increasing importance to neurologists. Lyme neuroborreliosis (LNB) causes all or part of a triad including meningitis, radiculoneuritis, and cranial neuritis. Rarely, parenchymal brain and spinal cord involvement occur, with focal findings on examination and magnetic resonance imaging (MRI). LNB diagnosis requires plausible exposure, objective evidence of nervous system involvement, and, generally, positive two-tier serology. Central nervous system (CNS) LNB is almost always accompanied by abnormal cerebrospinal fluid (CSF) (cells, protein), often with intrathecal antibody production, which is determined by concentration-adjusted comparison of serum and CSF antibody. Measuring CSF antibody in isolation and nucleic acid-based testing of CSF are not useful in LNB and should be avoided. LNB treatment is highly effective with a 2- to 3-week course of antibiotics. Increasing evidence suggests that LNB not involving the CNS parenchyma can be treated successfully with oral doxycycline.

Assessment of TLR-2 concentration in tick-borne encephalitis and neuroborreliosis.

The aim of the study was to check whether measurement of TLR-2 in serum or cerebrospinal fluid (CSF) can help differentiate between neuroborreliosis (NB) and tick-borne encephalitis (TBE). Eighty patients with meningitis and meningoencephalitis were divided into two groups: Group I - patients with NB (n = 40) and Group II - patients with TBE (n = 40). Diagnosis was based on the clinical picture, CSF examination and presence of specific antibodies in serum and CSF. The control group (CG) consisted of healthy blood donors (n = 25) and patients in whom inflammatory process in central nervous system was excluded (n = 25). Concentration of TLR-2 was measured using a commercial kit [TLR-2 Elisa Kit (EIAab, China)]. The serum and CSF TLR-2 concentration of NB patients was significantly higher than in CG. The serum and CSF TLR-2 concentration in TBE patients was significantly higher than in the CG. Receiver operating characteristic analysis of the serum TLR-2 concentration showed significant differences between the group of patients with NB and a group of patients with TBE. TLR-2 is involved in the development of inflammatory process in the CNS caused by both tick-borne pathogens: viral and bacterial as TLR-2 concentration in both CSF and serum differentiates these groups from healthy patients. Although TLR-2 cannot be used as a sole and reliable biomarker differentiating NB from TBE, results of our study are a step forward toward discovering such biomarker in the future.

Limitations and Confusing Aspects of Diagnostic Testing for Neurologic Lyme Disease in the United States.

In the United States, laboratories frequently offer multiple different assays for testing of cerebrospinal fluid (CSF) samples to provide laboratory support for the diagnosis of central nervous system Lyme disease (CNSLD). Often included among these diagnostic tests are the same enzyme immunoassays and immunoblots that are routinely used to detect the presence of antibodies to Borrelia burgdorferi in serum. However, performing these assays on CSF alone may yield positive results simply from passive diffusion of serum antibodies into the CSF. In addition, such tests are only U.S. Food and Drug Administration cleared and well validated for testing serum, not CSF. When performed using CSF, positive results from these assays do not establish the presence of intrathecal antibody production to B. burgdorferi and therefore should not be offered. The preferred test to detect intrathecal production of antibodies to B. burgdorferi is the antibody index assay, which corrects for passive diffusion of serum antibodies into CSF and requires testing of paired serum and CSF collected at approximately the same time. However, this assay also has limitations and should only be used to establish a diagnosis of CNSLD in conjunction with patient exposure history, clinical presentation, and other laboratory findings.

Serologic Evidence of Tick-Borne Encephalitis Virus Infection in a Patient with Suspected Lyme Disease in Japan.

Tick-borne encephalitis (TBE) is widely prevalent on the Eurasian continent, including Japan, but four cases of TBE have been reported in Japan. To inspect unconfirmed TBE cases in Japan, we conducted a retrospective seroepidemiological study of a total of 158 samples from 81 meningoencephalitis patients suspected as Lyme disease. Two serum samples from one patient showed neutralizing antibodies against TBE virus. The patient with severe and progressive encephalitis had a history of tick bite in Hokkaido in 2012. These results demonstrated that tick-borne encephalitis virus (TBEV) case was actually unconfirmed in Japan. Further seroepidemiological surveys are required to identify unconfirmed TBEV infections to consider the pros and cons of introducing specific countermeasures including vaccination in Japan.

Assessment of HMGB-1 concentration in tick-borne encephalitis and neuroborreliosis.

BACKGROUND: The aim of this study was to determine the concentration of HMGB-1 (high mobility group box 1) in the serum and cerebrospinal fluid (CSF) of patients suffering from tick-borne encephalitis (TBE) and neuroborreliosis (NB). Focus was placed on HMGB-1 measurement in the CSF or serum in order to establish whether this could help to differentiate between NB and TBE. METHODS: Eighty patients with meningitis and meningoencephalitis were enrolled in the study. The patients were divided into two groups: group I comprised patients with NB (n=40) and group II comprised patients with TBE (n=40). The diagnosis was made based on the clinical picture, CSF examination, and the presence of specific antibodies in the serum and CSF. The control group for the evaluation of the parameters in serum were healthy blood donors (n=25), while the control group for the evaluation of parameters in the CSF were patients in whom a central nervous system (CNS) inflammatory process was excluded. The concentrations of HMGB-1 were measured by ELISA method using a commercial kit (HMGB-1 ELISA Kit; EIAab, China). The results were analyzed using Statistica 10, Gretl, receiver operating characteristics curve (ROC), and the Pearson correlation coefficient. RESULTS AND CONCLUSIONS: HMGB-1 is associated with the development of inflammatory processes in the CNS caused by both tick-borne pathogens: viral (TBE) and bacterial (Lyme borreliosis). Measurement of the serum HMGB-1 concentration in the early stages of both diseases of the CNS may contribute to the differentiation between TBE and NB, which may have a clinical impact for patients bitten by ticks.

[Diagnostic relevance of the chemokine CXCL13 and anti-C6 peptide antibodies in patients with neuroborreliosis]. / Diagnostický význam chemokinu CXCL13 a protilátek proti C6 peptidu u pacientu s neuroborreliózou.

AIM OF THE STUDY: The study was focused on testing the diagnostic value of detection of the chemokine CXCL13 (B lymphocyte chemoattractant) and anti-C6 peptide (synthetic peptide derived from B. burdorferi VlsE protein) antibodies in patients with neuroborreliosis (NB). MATERIAL AND METHODS: One hundred and twenty-nine patients with clinical suspicion of neuroinfection were included in the study. Eighty patients with NB (positive for antibodies in serum and CSF) were subdivided into four groups (A1-A4) based on positivity/negativity of the antibody index (AI) and pleocytosis. The control group was composed of 49 patients with a negative AI and absence of CSF pleocytosis. Chemokine CXCL13 and anti-C6 antibodies were examined by commercial kits (Human CXCL13/BLC/BCA-1 Immunoassay, R&D Systems, INC, USA and C6 B. burgdorferi (Lyme) ELISA, Immunetics Inc. USA). The CXCL13 cut-off values were set to 130 pg/ml for the CSF and 62 pg/ml for the serum. RESULTS: The highest CSF levels of CXCL13 chemokine were found in group A1 (pleocytosis, AI positive), and they were significantly higher (p < 0.001) comparing with other groups except A3 (pleocytosis, AI negative; p = 0.04). Group A3 also showed significantly higher levels of CXCL13 than groups A2 (without pleocytosis, AI positive; p = 0.005), A4 (without pleocytosis, AI negative), and B (p < 0.001). The differences in the serum CXCL13 levels between groups were non-significant. The serum anti-C6 antibodies were detected in all NB groups and the positivity rates did not differ between groups (92%) except for A3 where 55% of the patients were positive. In the CSF, the highest anti-C6 sensitivity was found in the patients with a positive AI (A1 88.6%; A2 76.9%) while in the groups with a negative AI, it was low (A3 25%; A4 0%). In group B, anti-C6 antibodies were not detected. CONCLUSION: The highest CSF CXCL13 levels were found in early stage NB. Elevated CXCL13 concentrations correlate better with pleocytosis than with AI positivity; however, there exist some patients with a positive AI who have low CXCL13 levels. These patients are most probably those in the late - subacute stage of neuroinfection. The CXCL13 testing seems to be the most diagnostically helpful in the acute stage of NB where AI is still negative. The clinical sensitivity of the C6 ELISA test appears to be insufficient for CSF examination under our conditions. On the contrary, the specificity of this test was proven high, because none of the controls tested positive.

[CXCL-13 as a biomarker in the diagnostics of neuroborreliosis]. / CXCL-13 als Biomarker in der Diagnostik der Neuroborreliose.

BACKGROUND: The chemokine CXCL-13 is a potential intrathecal biomarker for neuroborreliosis (NB). According to the literature the sensitivity of CXCL-13 in the diagnostics of NB varies between 88% and 100% and the specificity between 63% and 99.7%. The objective of this study was to analyze the sensitivity and specificity of CXCL-13 in the diagnosis of NB in an endemic area of Borrelia burgdorferi. MATERIAL AND METHODS: In a retrospective analysis of data from August 2014 to August 2016, 63 patients with clinically suspected NB were identified. The diagnosis of NB was based on the guidelines of the German Society of Neurology (DGN). RESULTS: In 10 patients a definitive diagnosis of NB could be established (CXCL-13 min. 254 pg/ml /max. >900 pg/ml). The criteria for a probable NB were fulfilled by 2 patients (CXCL-13 concentration 8 pg/ml and 69 pg/ml, respectively), 9 patients had a chronic inflammatory demyelinating disease (CXCL-13 min. 10 pg/ml/max. 649 pg/ml) and 42 patients had other neurological diagnoses. Out of these, elevated intrathecal CXCL-13 concentrations were detected in 8 patients (e. g. tuberculosis, syphilis and anti-RI antibody positive paraneoplastic syndrome). CONCLUSION: By increasing the CXCL-13 cut-off level from 20 pg/ml to 200 pg/ml, the diagnostic sensitivity for NB remains 100% and consequently the specificity increases from 69.8% to 92.4%. Moreover, a CXCL-13 cut-off set at 200 pg/ml would exclude NB in the 2 patients with probable NB. We conclude from these results that CXCL-13 represents a valuable biomarker for the exclusion of untreated NB, although with limited specificity.

Evaluation of NF-κB concentration in patients with tick-borne encephalitis, neuroborreliosis, anaplasmosis and Anaplasma phagocythophilum with tick-borne encephalitis virus co-infection.

OBJECTIVES: The aim of the study was the evaluation of NF-κB concentration in serum and cerebrospinal fluid (CSF) of patients with diagnosis of tick-borne diseases: tick-borne encephalitis (TBE), neuroborreliosis (NB), anaplasmosis (ANA) and patients co-infected with tick-borne encephalitis virus and Anaplasma phagocythophilum (TBE+ANA). Additionally NF-κB concentration during acute and convalescent period was compared. METHODS: Sixty-seven patients with diagnosis of tick-borne diseases were included in the study. The control group (CG) consisted of 18 patients hospitalized because of headaches and had lumbar puncture performed. The NF-κB was measured by human inhibitory subunit of NF-κB ELISA Kit during acute and convalescent period. RESULTS: In serum the significant differences were observed only in patients with TBE+ANA co-infection. In CSF the concentration of NF-κB was significantly higher in patients with TBE, TBE+ANA co-infection, and patients with NB than in CG. Receiver operating characteristic (ROC) curves analysis showed that NF-κB concentration in CSF differentiated patients with NB with CG; patients co-infected with TBE and ANA with CG and patients with TBE with CG. NF-κB concentration in serum differentiated patients co-infected with TBE and ANA with NB and with ANA, with TBE and with CG. In TBE group the serum NF-κB concentration significantly decreased in convalescent period, while in NB and TBE groups significant CSF decrease of NF-κB concentration was observed.

Immunogenetic Markers Definition in Latvian Patients with Lyme Borreliosis and Lyme Neuroborreliosis.

The aim of this study was to determine the human leukocyte antigen (HLA)-DRB1 alleles in two groups of patients in Latvia: patients with Lyme borreliosis and patients with Lyme neuroborreliosis. The study included 216 patients with Lyme borreliosis, 29 patients with Lyme neuroborreliosis and 282 control persons. All surveyed persons were residents of Latvia. The HLA-DR genotyping was performed by polymerase chain reaction- sequence specific primer (PCR-SSP). The predisposition to the Lyme borreliosis is associated with the HLA-DRB1*07, -DRB1*17(03), -DRB1*04, -DRB1*15(02) alleles. The allele -DRB1*11(05), -DRB1*14(06) and -DRB1*13(06) were significantly more frequent in controls. In-group with Lyme neuroborreliosis differences were found for the -DRB1*07 and -DRB1*04 alleles, but only HLA-DRB1*07 allele was statistically significant after Bonferroni correction and associated with Lyme neuroborreliosis in Latvian patients.

Prospective study on the chemokine CXCL13 in neuroborreliosis and other aseptic neuroinfections.

The study evaluates the clinical significance of CXCL13 (leukocyte chemoattractant synthesized in CSF ) in Lyme neuroborreliosis (LNB) and other aseptic CNS infections. 244 patients with symptoms of neuroinfection and/or LNB were divided into groups: A - patients with LNB-positive antibodies in serum and CSF (96) or CSF only (14); B - patients with aseptic non-borrelial neuroinfections (82); C - negative controls (52). Group A was divided into A1-A4 according to pleocytosis in CSF and AIIgG positivity. The highest CSF CXCL13 concentrations (max. 81,287.60pg/ml; median 1766.90pg/ml) were in A1 (positive AI, pleocytosis) and A3 (negative AIIgG, pleocytosis; max. 7201,60pg/ml, median 56.22pg/ml). A2 (positive AI without pleocytosis) and A4 (negative AI without pleocytosis) had low CXCL13 levels - A2 max. 650.50pg/ml (median<7.80pg/ml); A4 max. 118.56pg/ml (median<7.8pg/ml). In B the median was 28.10pg/ml (max. 595.87pg/ml). In C the CXCL13 concentrations were the lowest (max. 83.83pg/ml; median<7.80pg/ml). The lowest cut-off was 29pg/ml (sensitivity 90.0%, specificity 72.2%), the highest one 400pg/ml (sensitivity 59.6%, specificity 94.0%). The group differences of serum CXCL13 were insignificant. The highest concentrations were at the beginning of the disease. In LNB CXCL13 correlates better with the CSF pleocytosis than AI positivity.

Lipid peroxidation in the pathogenesis of neuroborreliosis.

This study analyzed the onset of lipid peroxidation (LPO) in neuroborreliosis and the effects of ceftriaxone therapy on LPO. Twenty-two patients with early neuroborreliosis and 22 healthy subjects were studied. LPO in the cerebrospinal fluid (CSF), as well as the plasma and urine was estimated by the levels of reactive aldehydes: 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal, malondialdehyde, and 4-oxononenal, F2-isoprostanes and A4/J4-neuroprostanes (NPs). The plasma level of 4-HNE-protein adducts arachidonic acid (AA), docosahexaenoic acid (DHA) and vitamin E was determined. Additionally, enzymatic activities of phospholipase A2 (PLA2), platelet-activating factor acetylhydrolase (PAF-AH) and glutathione peroxidase (GSH-Px) were determined. A decrease of AA, DHA levels and GSH-Px activity in plasma was associated with a significant increase of aldehydes in the CSF, plasma and urine. Similarly, the increase of F2-isoprostanes and NPs in the CSF and plasma was associated with the decreased activity of PLA2 and PAF-AH. Ceftriaxone therapy cured patients and reduced the levels of F2-isoprostanes, NPs and reactive aldehydes. However, the activities of PLA2 and PAF-AH increased. Pathophysiological association of neuroborreliosis with systemic LPO was revealed. Effective antibiotic therapy attenuated LPO. Biomarkers of LPO could be useful to monitor the onset of neuroborreliosis and show the effectiveness of pharmacotherapy.

Establishment of N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC) modified biochip enabling concurrent detection of serum infectious antibodies in neuroborreliosis.

In this study, we developed a novel protein biochip that was modified with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) and specialized for concurrent detection of serum IgG and IgM antibodies against Borrelia burgdorferi antigens, flagellin, outer surface protein C (OspC) and variable major protein-like sequence (VlsE) in the patients with neuroborreliosis (NB), respectively. Surface chemical characteristics of the biochips were validated with atomic force microscope (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The visualized detection limit for IgG antibodies against flagellin, OspC and VlsE antigens on the biochip were 0.78 µg/ml, 0.78 µg/ml and 1.56 µg/ml, respectively. Finally, serum IgG and IgM antibodies in 72 patients with NB and 188 healthy individuals were tested on the biochip. The seroimmunological outcome by the biochip were evaluated in comparison with enzyme linked immunosorbent assay (ELISA) assay. The results demonstrated that the prevalences of IgG and IgM antibodies in the cases were 41.7%, 63.9% to flagellin; 20.8% and 51.4% to OspC and 76.4%, 62.5% to VlsE, respectively. Utilization of the biochip in detection IgM antibody against flagellin was compatible with ELISA assay (R(2)=0.849). Thus, the protein biochip would provide a potential platform not only for enabling detection of corresponding antibodies directed against B. burgdorferi antigens, but also for monitoring course of the disease.