Serum matrix metalloproteinase-9 (MMP-9) as a biomarker in paediatric and adult tick-borne encephalitis patients.

Matrix metalloproteinases (MMPs) play an important role in central nervous system infections. We analysed the levels of 8 different MMPs in the cerebrospinal fluid (CSF) of 89 adult patients infected with tick-borne encephalitis (TBE) virus and compared them with the levels in a control group. MMP-9 was the only MMP that showed significantly increased CSF levels in TBE patients. Serum MMP-9 levels were subsequently measured in 101 adult TBE patients at various time points during the neurological phase of TBE and at follow-up. In addition, serum MMP-9 was analysed in 37 paediatric TBE patients. Compared with control levels, both paediatric and adult TBE patients had significantly elevated serum MMP-9 levels. In most adult patients, serum MMP-9 levels peaked at hospital admission, with higher serum MMP-9 levels observed in patients with encephalitis than in patients with meningitis. Elevated serum MMP-9 levels were observed throughout hospitalisation but decreased to normal levels at follow-up. Serum MMP-9 levels correlated with clinical course, especially in patients heterozygous for the single-nucleotide polymorphism rs17576 (A/G; Gln279Arg) in the MMP9 gene. The results highlight the importance of MMP-9 in the pathogenesis of TBE and suggest that serum MMP-9 may serve as a promising bioindicator of TBE in both paediatric and adult TBE patients.

Selected Biomarkers of Tick-Borne Encephalitis: A Review.

Tick-borne encephalitis (TBE) is an acute disease caused by the tick-borne encephalitis virus. Due to the viral nature of the condition, there is no effective causal treatment for full-blown disease. Current and nonspecific TBE treatments only relieve symptoms. Unfortunately, the first phase of TBE is characterized by flu-like symptoms, making diagnosis difficult during this period. The second phase is referred to as the neurological phase as it involves structures in the central nervous system-most commonly the meninges and, in more severe cases, the brain and the spinal cord. Therefore, it is important that early markers of TBE that will guide clinical decision-making and the choice of treatment are established. In this review, we performed an extensive search of literature reports relevant to biomarkers associated with TBE using the MEDLINE/PubMed database. We observed that apart from routinely determined specific immunoglobulins, free light chains may also be useful in the evaluation of intrathecal synthesis in the central nervous system (CNS) during TBEV infection. Moreover, selected metalloproteinases, chemokines, or cytokines appear to play an important role in the pathogenesis of TBE as a consequence of inflammatory reactions and recruitment of white blood cells into the CNS. Furthermore, we reported promising findings on tau protein or Toll-like receptors. It was also observed that some people may be predisposed to TBE. Therefore, to understand the role of selected tick-borne encephalitis biomarkers, we categorized these factors and discussed their potential application in the diagnosis, prognosis, monitoring, or management of TBE.

The increased intrathecal expression of the monocyte-attracting chemokines CCL7 and CXCL12 in tick-borne encephalitis.

Tick-borne encephalitis (TBE) is a relatively severe and clinically variable central nervous system (CNS) disease with a significant contribution of a secondary immunopathology. Monocytes/macrophages play an important role in the CNS inflammation, but their pathogenetic role and migration mechanisms in flavivirus encephalitis in humans are not well known. We have retrospectively analyzed blood and cerebrospinal fluid (CSF) monocyte counts in 240 patients with TBE presenting as meningitis (n = 110), meningoencephalitis (n = 114), or meningoencephalomyelitis (n = 16), searching for associations with other laboratory parameters, clinical presentation, and severity. We have measured concentrations of selected monocytes-attracting chemokines (CCL7, CXCL12, CCL20) in serum and CSF of the prospectively recruited patients with TBE (n = 15), with non-TBE aseptic meningitis (n = 6) and in non-infected controls (n = 8). The data were analyzed with non-parametric tests, p < 0.05 considered significant. Monocyte CSF count correlated with other CSF inflammatory parameters, but not with the peripheral monocytosis, consistent with an active recruitment into CNS. The monocyte count did not correlate with a clinical presentation. The median CSF concentration of CCL7 and CXCL12 was increased in TBE, and that of CCL7 was higher in TBE than in non-TBE meningitis. The comparison of serum and CSF concentrations pointed to the intrathecal synthesis of CCL7 and CXCL12, but with no evident concentration gradients toward CSF. In conclusion, the monocytes are recruited into the intrathecal compartment in concert with other leukocyte populations in TBE. CCL7 and CXCL12 have been found upregulated intrathecally but are not likely to be the main monocyte chemoattractants.

Tick-Borne Encephalitis: A Differential Pattern of Intrathecal Humoral Immune Response and Inflammatory Cell Composition Compared with Other Viral CNS Infections.

To investigate whether and how cerebrospinal fluid (CSF) findings can contribute to distinguish tick-borne encephalitis (TBE) from herpes simplex virus (HSV) and varicella zoster virus (VZV) induced central nervous system (CNS) infections (HSV-I, VZV-I). Chart review and identification of TBE, HSV- I, and VZV-I was carried out, fulfilling the following criteria: (1) clinical signs of encephalitis and/or meningitis, (2) complete CSF analysis and confirmed viral etiology by either PCR or antibody testing in CSF, (3) hospitalized patients, and (4) available brain magnetic resonance imaging (MRI). Fifty-nine patients with 118 CSF/serum pairs were included. These comprised 21 with TBE (35 CSF/serum pairs), 20 (40 CSF/serum pairs) with HSV-I, and 18 (43 CSF/serum pairs) with VZV-I. In contrast to HSV-I and VZV-I, CSF cell differentiation in TBE showed more often an increased (>20%) proportion of granulocytes (p < 0.01) and a more frequent quantitative intrathecal IgM synthesis (p = 0.001 and p < 0.01, respectively), while the second was even more pronounced when follow-up CSF analyses were included (p < 0.001). CSF findings help to distinguish TBE from other viral infections. In cases with CSF pleocytosis and a positive history for a stay in or near an endemic area, TBE antibodies in CSF and serum should be determined, especially if granulocytes in CSF cell differentiation and/or an intrathecal IgM synthesis is present.

The Cerebrospinal Fluid Interleukin-6/Interleukin-10 Ratio Differentiates Pediatric Tick-borne Infections.

BACKGROUND: Borrelia burgdorferi and tick-borne encephalitis (TBE) virus are 2 types of tick-borne pathogens that can cause central nervous system infection. Routine diagnostics have so far included analysis of cerebrospinal fluid (CSF) cell numbers, CSF serology for Borrelia burgdorferi and serum serology for TBE virus. However, early diagnosis may be difficult based on antibody detection which takes time to analyze, and with the possibility of false negative results, thus delaying treatment. Cytokine analyses are becoming increasingly available in clinical routine care and may offer important information. METHODS: Fifteen cytokines and chemokines were measured in the CSF from the diagnostic lumbar puncture of 37 children with TBE, 34 children with neuroborreliosis and 19 children without evidence of central nervous system infection, using Luminex technology. RESULTS: Significantly higher levels of proinflammatory interleukin-6 were detected in the samples from TBE-infected children, when compared with neuroborreliosis or controls. In comparison, children with neuroborreliosis had significantly higher levels of interleukin-7, interleukin-8, interleukin-10, and interleukin-13 when compared with TBE infected or controls. Furthermore, the ratio between interleukin-6 and interleukin-10 was significantly different between the 2 types of tick-borne infections. CONCLUSIONS: The interleukin-6/interleukin-10 ratio can be used as a rapid diagnostic cue upon suspected tick-borne infection, enabling fast and correct treatment. Also, in serology-negative results, such information may strengthen a clinical suspicion.

Evaluation of cerebrospinal fluid CXCL13 concentrations and lymphocyte subsets in tick-borne encephalitis.

OBJECTIVES: Recent studies suggest that the clinical presentation of tick-borne encephalitis (TBE) is determined by the host immune responses to the tick-borne encephalitis virus (TBEV). The aim of the study was to characterize immune responses in TBE to give a better insight into the immunopathogenesis of this disease. METHODS: Anti-TBEV antibody levels, cerebrospinal fluid (CSF) and blood lymphoid populations, and concentrations of CXCL13 (a potent B-cell and T-cell chemoattractant), were analyzed in 35 patients with TBE (20 adults and 15 children). RESULTS: When compared with the blood, the CSF lymphoid population was significantly enriched in CD4+ T-cells and relatively depleted in natural killer (NK) cells and B lymphocytes. In comparison with TBE meningitis, patients suffering from TBE meningoencephalitis (n = 11, 31%) had a 3.5-fold higher median CSF CXCL13 concentration, 1.8-fold higher CSF/serum ratio of anti-TBEV IgG antibodies, and 1.8-fold higher median CSF cell count. CSF CXCL13 levels did not change significantly in children with TBE meningitis receiving supportive treatment, but decreased in children with TBE meningoencephalitis who received intravenous steroids. CONCLUSIONS: CD4+ cells are abundant in the CSF of patients with TBE. CXCL13 may be involved in the neuropathology of TBE by attracting different subsets of lymphocytes to the CSF.

Analysis of CCL-4, CCL-17, CCL-20 and IL-8 concentrations in the serum of patients with tick-borne encephalitis and anaplasmosis.

PURPOSE: Tick-borne co-infections are a serious epidemiological and clinical problem. Only a few studies aimed to investigate the effect of tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) co-infection in the course of the inflammatory process and the participation of chemokines in the pathomechanism of these diseases. The aim of the study was to evaluate CCL-4, CCL-17, CCL-20, and IL-8 serum concentrations in patients with HGA, TBE and HGA + TBE co-infection. METHODS: Eighty-seven patients with HGA (n = 20), TBE (n = 49) and HGA + TBE (n = 18) were included to the study. The control group (CG) consisted of 20 healthy people. Concentrations of cytokines were measured in serum using commercial ELISA assays. In patients with TBE and HGA + TBE inflammatory markers were assessed during the acute and convalescent period. The results were analyzed using non-parametric tests with p < 0.05 considered as significant. RESULTS: Before treatment, significantly higher concentrations of IL-8, CCL-4 and CCL-20 were observed in HGA patients. CCL-4 and CCL-20 concentrations were significantly higher in TBE patients compared to CG. Concentrations of IL-8, CCL-4, and CCL-20 were significantly higher in HGA + TBE than in CG. After treatment, a significant reduction of IL-8, CCL-4, and CCL-20 concentrations in TBE patients and IL-8 in HGA + TBE co-infection was observed. CCL-4 concentration was higher in HGA + TBE co-infection in comparison to patients with TBE after treatment. CONCLUSIONS: Our study confirms that concentrations of IL-8, CCL-4, and CCL-20 are increased in the course of HGA and TBE. Their concentrations in serum may be used to monitor the course of TBE and HGA, as well as possibly detect co-infections with the diseases.

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.

Intrathecal complement activation by the classical pathway in tick-borne encephalitis.

Tick-borne encephalitis (TBE) is one of the most prevalent viral central nervous system (CNS) infections in Eurasia and neurological sequelae are common. The immune responses are considered crucial for the pathogenesis. The aim of this study was to explore the activation of the complement system in TBE. The complement system is a part of the innate immune response in the CNS, which previously has been reported to be activated in other flavivirus infections. We analyzed complement factors in 44 paired cerebrospinal fluid (CSF) and serum samples from 20 cases of TBE in the acute and later stages, as well as in serum and CSF from 32 healthy controls. The concentrations of complement factors C1q, C3a, C3b, and C5a were determined with commercially available ELISA kits. Clinical data to categorize the severity of disease and outcome was retrieved from the medical records of the TBE patients. We found significantly higher concentrations of all of the analyzed complement factors in the CSF from TBE patients compared to the healthy controls. In particular, the marked increment of C1q concentrations in the CSF (p < 0,001 as compared to controls) indicated an intrathecal activation by the classical pathway. There was no correlation between complement factor concentrations in the CSF and severity of the disease in the acute phase or with sequelae at 6 months follow-up. We have found an intrathecal complement activation in TBE, and the marked increase of complement factor C1q indicated an activation by the classical pathway.

Assessment of the tau protein concentration in patients with tick-borne encephalitis.

There have been suggestions that tick-borne encephalitis (TBE) may cause neurodenenerative changes in the brain. The aim of this study was the assessment of the tau protein concentration in cerebrospinal fluid (CSF) of patients with different clinical forms of TBE. The concentration of tau protein in CSF was determined using Fujirebio tests (Ghent, Belgium) in 35 patients with TBE: group I-patients with meningitis (n = 16); group II-patients with meningoencephalitis (n = 19). None of the patients reported any neurodegenerative disorder that could affect the results of the study. The control group (CG) consisted of 10 patients in whom inflammatory process in central nervous system was excluded. Tau protein concentration in CSF before treatment did not differ significantly between the examined groups, while its concentration was significantly higher in encephalitis group than in CG after 14 days of treatment. Significant increase in tau protein concentration after treatment was observed in both examined groups. The comparison between the group of patients who fully recovered and patients who presented with persistent symptoms on discharge showed significant differences in tau protein concentration before and after treatment. ROC curve analysis indicates that CSF tau protein concentration before treatment may predict complicated course of the disease with 90.9% specificity and 80% sensitivity, while after treatment, specificity became 72.7% and 71.4% for sensitivity. Correlation analysis showed that in TBE patients (both meningoencephalitis and meningitis groups), CSF pleocytosis before treatment correlated negatively with tau protein concentration in CSF. (1) Neurodegeneration process is present in TBE encephalitis. (2) Tau protein concentration may be used as a predictor of complicated course of TBE.

Evaluation of NSE and S100B in patients with tick-borne encephalitis.

INTRODUCTION: The aim of this study was the assessment of neuron-specific enolase (NSE) and S-100 concentration in serum and cerebrospinal fluid (CSF) in patients with different clinical forms of tick-borne encephalitis (TBE). MATERIAL AND METHODS: The serum and CFS concentrations of S100B and NSE of 43 patients with TBE were measured with ELISA method using commercial kits: NSE and S100B Elisa Kit (DRG, Germany). Subjects were divided into: Group I-patients with meningoencephalitis (n = 17) and Group II-patients with meningitis (n = 26). None of the patients reported any neurodegenerative disorder that could affect the results of the study. The control group (CG) consisted of 13 patients. These patients were admitted to the hospital because of headache, and the CSF examination excluded inflammatory process. Samples were collected on admission (sample 1) and after treatment (sample 2). RESULTS: Neuron-specific enolase concentration in CSF was higher in group I than in group II (p = 0.0002) and controls (p = 0.04). NSE concentration was higher in the second serum and CSF sample in both groups. S100B concentration did not differ between TBE patients and controls. NSE concentration in serum after 14 days was higher in the sequelae group (34.3 ± 9.7 vs. 16.7 ± 15, p = 0.04). Also, NSE serum sample 2/serum sample 1 ratio was significantly higher in the sequelae group (3.57 ± 0.92 vs. 1.53 ± 1.99, p = 0.04). Receiver Operating Characteristic curve analysis indicated that NSE concentration in serum II differentiates sequelae group from other meningoencephalitis patients (p = 0.0001). S100B serum sample 2/CSF sample 2 ratio was lower in the sequelae group (0.05 ± 0.1 vs. 0.37 ± 0.28, p = 0.02). CONCLUSIONS: (a) Neurodegeneration process is present in TBE encephalitis. (b) NSE concentration correlates with inflammatory parameters in CSF in TBE. (c) Neurodegeneration is present even after clinical recovery of TBE. (d) NSE could be used in the prediction of TBE course. (e) S-100 did not differ between TBE patients and controls.

The intrathecal expression and pathogenetic role of Th17 cytokines and CXCR2-binding chemokines in tick-borne encephalitis.

BACKGROUND: Tick-borne encephalitis (TBE) is a clinically variable but potentially severe Flavivirus infection, with the outcome strongly dependent on secondary immunopathology. Neutrophils are present in cerebrospinal fluid (CSF) of TBE patients, but their pathogenetic role remains unknown. In animal models, neutrophils contributed both to the Flavivirus entry into central nervous system (CNS) and to the control of the encephalitis, which we attempted to evaluate in human TBE. METHODS: We analyzed records of 240 patients with TBE presenting as meningitis (n = 110), meningoencephalitis (n = 114) or meningoencephalomyelitis (n = 16) assessing CSF neutrophil count on admission and at follow-up 2 weeks later, and their associations with other laboratory and clinical parameters. We measured serum and CSF concentrations of Th17-type cytokines (interleukin-17A, IL-17F, IL-22) and chemokines attracting neutrophils (IL-8, CXCL1, CXCL2) in patients with TBE (n = 36 for IL-8, n = 15 for other), with non-TBE aseptic meningitis (n = 6) and in non-meningitis controls (n = 7), using commercial ELISA assays. The results were analyzed with non-parametric tests with p < 0.05 considered as significant. RESULTS: On admission, neutrophils were universally present in CSF constituting 25% (median) of total pleocytosis, but on follow-up, they were absent in most of patients (58%) and scarce (< 10%) in 36%. CSF neutrophil count did not correlate with lymphocyte count and blood-brain barrier integrity, did not differ between meningitis and meningoencephalitis, but was higher in meningoencephalomyelitis patients. Prolonged presence of neutrophils in follow-up CSF was associated with encephalitis and neurologic sequelae. All the studied cytokines were expressed intrathecally, with IL-8 having the highest CSF concentration index. Additionally, IL-17A concentration was significantly increased in serum. IL-17F and CXCL1 CSF concentrations correlated with neutrophil count and CXCL1 concentration was higher in patients with encephalitis. CONCLUSIONS: The neutrophil CNS infiltrate does not correlate directly with TBE severity, but is associated with clinical features like myelitis, possibly being involved in its pathogenesis. Th17 cytokine response is present in TBE, especially intrathecally, and contributes to the CNS neutrophilic inflammation. IL-8 and CXCL1 may be chemokines directly responsible for the neutrophil migration.

Intrathecal expression of IL-5 and humoral response in patients with tick-borne encephalitis.

AIM: The aim of the study was to assess the role of an early specific humoral response in human infection with a tick-borne encephalitis virus (TBEV) and the role of IL-5 as its potential mediator and marker. MATERIALS AND METHODS: The retrospective study involved a cohort of 199 patients diagnosed with TBE, in whom anti-TBEV IgM and IgG antibody titers were analyzed on admission and compared with clinical presentation and basic laboratory parameters. The prospective study included 50 TBE patients in whom IL-5 serum and CSF concentration was measured with ELISA on admission in the TBE neurologic phase and in selected patients before discharge, at follow-up or in samples obtained before the neurologic phase onset. RESULTS: The serum anti-TBEV IgM correlated with good clinical outcome and the CSF anti-TBEV IgM with more pronounced CSF inflammation on admission, but also with its more complete resolution on follow-up. The serum anti-TBEV IgG correlated with milder presentation and better outcome. Concentration of IL-5 was increased in CSF but not in the serum of TBE patients. IL-5 concentration index on admission favored its intrathecal synthesis. IL-5 did not correlate significantly with clinical presentation and specific IgM and IgG titers. CONCLUSIONS: Specific anti-TBEV IgM systemic and intrathecal response and IgG systemic response are protective, together favoring milder presentation, better outcome and resolution of central nervous system (CNS) inflammation. IL-5 is expressed intrathecally in TBE, but its pathogenetic role remains unclear.

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.

Clinical outcome and cerebrospinal fluid profiles in patients with tick-borne encephalitis and prior vaccination history.

BACKGROUND: Tick-borne encephalitis (TBE) is endemic in southern and eastern districts of Germany. Approximately 10-14% of the infected individuals suffer from long-term disability and in 1.5-3.6% the course is fatal. Two well-tolerated vaccines are available, which provide high protection and which have been confirmed in several field studies. Here we investigate clinical course, long-term outcome and cerebrospinal fluid (CSF) characteristics of TBE cases with a prior history of any vaccination as well as real vaccination breakthrough (VBT). METHODS: A case series of 11 patients with a prior history of vaccination, part of a recently published lager cohort of 111 TBE cases. Evaluation included clinical data, degree of disability (modified RANKIN scale, mRS) and analysis of CSF and serum samples. Furthermore, metadata for extended analysis on clinical outcome of TBE with VBT were analysed. RESULTS: One patient had a clear VBT and ten of them had irregular vaccinations schedules (IVS). Infection severity did not differ in patients with IVS as compared to a non-vaccinated control cohort (median mRS: both 3.0) but these patients showed a stronger cellular immune response as measured by CSF pleocytosis (IVS, 205 cells/µL versus non-vaccinated control, 114 cell/µL, P < 0.05) and by differential pattern of CSF (intrathecal) immunoglobulin synthesis. However, shift analysis of VBT metadata using linear-by-linear association revealed a more serious course of TBE in patients with VBT than in a non-vaccinated control cohort (χ2 = 9.95, P = 0.002). Furthermore, ordinal logistic regression analysis showed that VBT patients had an age-corrected, 2.65 fold (CI: 1.110-6.328; χ2 = 4.813; p = 0.028) significant higher risk to suffer from moderate or severe infections, respectively. CONCLUSION: A history of IVS surprisingly seems to have no impact on the clinical course of TBE but may leave marks in the specific brain immune response. VBT patients, however, carry an age-independent, significant risk to experience a severe infection.

First detection of tick-borne encephalitis virus RNA in clinical specimens of acutely ill patients in Hungary.

Tick-borne encephalitis virus (TBEV) is one of the endemic flaviviruses in Hungary, which is responsible for human infections every year. Neurological involvement in the disease is characterized by meningitis, encephalitis or meningoencephalitis which can result in long-term neurological and neuropsychiatric sequelae. Microbiological diagnosis of acute cases is predominantly based on serological tests due to the limited duration of viremia and long incubation period, however, the application of molecular methods can also supplement the serological diagnosis and provides epidemiological data. The aim of this study was to determine how viral RNA could successfully be detected from different body fluids of serologically confirmed acute cases. Serum, whole blood, cerebrospinal fluid and urine samples of 18 patients from the total of the 19 serologically diagnosed cases were investigated by using the RT-PCR method. Two sera and one urine sample of three patients tested positive and the European subtype of TBEV could be identified. As far as we know this was the first time that TBEV RNA could be detected from human clinical samples in Hungary. Our finding highlights that the application of molecular methods besides serological tests can be a valuable tool in differential diagnosis especially in areas like Hungary, where two or more flaviviruses are co-circulating.

The increased concentration of macrophage migration inhibitory factor in serum and cerebrospinal fluid of patients with tick-borne encephalitis.

BACKGROUND: Host factors determining the clinical presentation of tick-borne encephalitis (TBE) are not fully elucidated. The peripheral inflammatory response to TBE virus is hypothesized to facilitate its entry into central nervous system by disrupting the blood-brain barrier with the involvement of a signaling route including Toll-like receptor 3 (TLR3) and pro-inflammatory cytokines macrophage migration inhibitory factor (MIF), tumor necrosis factor-α (TNFα), and interleukin-1 beta (IL-1ß). METHODS: Concentrations of MIF, TNFα, and IL-1ß were measured with commercial ELISA in serum and cerebrospinal fluid (CSF) from 36 hospitalized TBE patients, 7 patients with non-TBE meningitis, and 6 controls. The CSF albumin quotient (AQ) was used as a marker of blood-brain barrier permeability. Single nucleotide polymorphisms rs3775291, rs5743305 (associated with TLR3 expression), and rs755622 (associated with MIF expression) were assessed in blood samples from 108 TBE patients and 72 non-TBE controls. The data were analyzed with non-parametric tests, and p < 0.05 was considered significant. RESULTS: The median serum and CSF concentrations of MIF and IL-1ß were significantly increased in TBE group compared to controls. MIF concentration in serum tended to correlate with AQ in TBE, but not in non-TBE meningitis. The serum concentration of TNFα was increased in TBE patients bearing a high-expression TLR3 rs5743305 TT genotype, which also associated with the increased risk of TBE. The low-expression rs3775291 TLR3 genotype TT associated with a prolonged increase of CSF protein concentration. The high-expression MIF rs755622 genotype CC tended to correlate with an increased risk of TBE, and within TBE group, it was associated with a mild presentation. CONCLUSIONS: The results point to the signaling route involving TLR3, MIF, and TNFα being active in TBE virus infection and contributing to the risk of an overt neuroinvasive disease. The same factors may play a protective role intrathecally contributing to the milder course of neuroinfection. This suggests that the individual variability of the risk and clinical presentation of TBE might be traced to the variable peripheral and intrathecal expression of the mediators of the inflammatory response, which in turn associates with the host genetic background.

Biomarkers in Cerebrospinal Fluid of Children With Tick-borne Encephalitis: Association With Long-term Outcome.

BACKGROUND: Tick-borne encephalitis (TBE) is a major cause of meningoencephalitis in children in endemic areas, and long-term residual problems are not uncommon. Currently, no predictive markers in the acute phase are available that identify children at risk of incomplete recovery. We measured cytokines, chemokines and markers of neuronal damage in cerebrospinal fluid (CSF) in children with TBE and central nervous system (CNS) involvement. METHODS: CSF from 37 children with TBE with CNS involvement was analyzed. Concentrations of 16 cytokines, chemokines and 5 markers of neuronal damage were assessed in CSF, using a multiplex assay, and correlated with clinical findings in the acute phase (n = 37), and with long-term outcome (n=22). RESULTS: Significantly higher levels of CSF interferon (IFN)-γ, interleukin (IL)-4, IL-6 and IL-8 were detected in the acute phase from children who later developed sequelae. Although most of the studied markers of neuronal damage displayed no significant differences between children with sequelae and those with good outcome, neuron-specific enolase correlated inversely. The grade of CSF pleocytosis correlated positively with the levels of IFN-γ, IL-4 and IL-6; however, pleocytosis alone did not predict sequelae. Increasing age correlated positively with IL-4, IL-6 and IL-8 values. CONCLUSIONS: The mechanism underlying the CNS pathology causing sequelae in TBE appears related to the grade of inflammation in CNS, rather than to direct neuronal destruction. High concentration of IFN-γ, IL-4, IL-6 and IL-8 in CSF might indicate a risk for incomplete recovery in childhood TBE.

Presence of herpesvirus DNA in cerebrospinal fluid of patients with tick-borne encephalitis and enteroviral meningoencephalitis.

Reactivation of HHVs in the CNS due to inflammation has not been well described yet. The primary aim of this study was to investigate the frequency of HHV DNA detection in the cerebrospinal fluid (CSF) of immunocompetent patients with meningoencephalitis of other than HHV origin. The secondary aim of this study was to evaluate the impact of herpesvirus co-infection on the clinical course and patient outcome. Ninety-six patients with clinically and laboratory proven tick-borne encephalitis (TBE) and 77 patients with a confirmed diagnosis of enteroviral meningitis (EVM), along with a control group of 107 patients without evidence of inflammation in the CSF were retrospectively tested by nested PCR for the presence of DNA of the neurotropic herpesviruses HSV1, HSV2, VZV, and HHV6 in the CSF. The clinical course, laboratory tests, antiviral treatment, and neurological complications in a 6-month follow-up were compared between the groups positive or negative for HHV DNA in the CSF. HHV DNA was found in the CSF of 12 (6.9%) patients (6.3% and 7.8% in the TBE and EVM groups, respectively) and in 1 (0.9%) control patient. None of the patients had recent blisters or rash. The clinical course was comparably mild in all patients. No permanent neurological sequelae were observed. Only the CSF total protein level was significantly higher in HHV DNA-positive than in HHV-negative patients.

Clinical course and sequelae for tick-borne encephalitis among children in South Moravia (Czech Republic).

UNLABELLED: This study of 170 children in the Czech Republic examines the clinical course and sequelae for tick-borne encephalitis. Evaluated were demographic and epidemiological data, signs and symptoms at admission, clinical course during hospital stay and laboratory findings. Cerebrospinal fluid was analysed for white blood cells, protein, impairment of blood-cerebrospinal fluid (CSF) barrier and tick-borne encephalitis virus (TBEV)-specific antibodies. Subjective complaints and objective neurological deficits were investigated. Tick bites were reported in 74 % of the children. The illness had a biphasic clinical course in 58 % of cases. The second phase was characterized by headache in 98 %, high fever in 86 % (more than 38.5 °C), vomiting in 64 % and meningeal signs in 92 % of children. Meningitis (77 %) dominated over meningoencephalitis (13 %). Inflammatory changes in CSF were found in 90 % of children. Immunoglobulin M (IgM) antibodies against TBEV in serum were found early in the infection in 99 %. IgM positivity lasted up to 1,126 days. Neurocognitive abnormalities were found in 19 (11 %) of children. Acquired aphasia, lasting tremor of the upper extremities, speech impairment, inversion of sleep and wakefulness, abnormal hyperkinetic movements and vertigo were found to be permanent but not progressing. Severe sequelae persisted in two children (1 %) while in three (2 %) were classified as mild or moderate. CONCLUSION: Tick-borne encephalitis in children has a benign course with minimal sequelae. Meningitis with biphasic course is the prevalent involvement and the duration of IgM antibodies in serum and index of positivity are not decisive for postencephalitic disorders.