Assessment of the YKL-40 concentration in patients with tick-borne encephalitis

Background Tick-Borne Encephalitis (TBE) is a viral infection of the Central Nervous System (CNS) caused by TickBorne Encephalitis Virus (TBEV). It might take several clinical courses such as: meningitis, meningoencephalitis or meningoencephalomyelitis. The aim this study was to compare the YKL-40 concentration in cerebrospinal uid (CSF) of patients with different clinical presentations of TBE and patients with excluded meningitis (control group). Methods in CSF was determined using tests in 32 patients with TBE: group I—patients with meningoencephalitis (n = 16); group II—patients with meningitis (n = 16). The control group whom system


Introduction
Tick-Borne Encephalitis (TBE) is a viral infection of the Central Nervous System (CNS) caused by Tick-Borne Encephalitis Virus (TBEV) transmitted by Ixodide tick bite that might lead to severe sequelae [1,2]. The annual incidence of TBE in Poland is 200-300 with 90% of them are from two regions of the two northeastern provinces of the country [3]. In years 2000-2015, 3662 cases were reported in Poland at country level where 45% of them were from the Podlaskie province(the place where this study is conducted) [4].
The clinical features of TBE elapse from asymptomatic to cognitive and neurologic impairments. It could appear as meningitis, meningoencephalitis, and meningoencephalomyelitis [5]. According to various reports 20-58% of patients may develop sequelae after TBE (mild, moderate or severe) while others completely recover from their illness without indications of sequelae [6,7].
Is has been proven that some neurodegenerative diseases such as Alzheimer's Disease (AD) might be induced by infectious diseases [8,9]. In two previous studies conducted in the same institution we have observed that Neuron Speci c Enolase (NSE) and tau protein were increased in cerebrospinal uid (CSF) [10,11] which suggested that TBE may lead to neurodegeneration.
Therefore we wanted to assess whether other markers of neurodegeneration also take part in the pathogenesis of TBE. We decided to choose YKL-40 (chitinase-3 like-1 [CHI3L1]) a member of the family 18 glycosyl hydrolases, secreted by activated neutrophils and macrophages. In the central nervous system (CNS), YKL-40 is mostly expressed by microglia, especially when responding to acute and chronic in ammation [12,13,14].
The aim this study was to assess of the YKL-40 concentration in CSF of patients with different encephalitis and meningitis associated with TBE.
This study has the following speci c objectives: Group I: patients with menigoencephalitis (n = 16; 7 women, 9 men ,mean age 54.5 ± 8.93 years).
Meningoencephalitis was diagnosed when the in ammatory parameters in CSF, altered consciousness and focal neurological symptoms were present.
Meningitis was diagnosed on the basis of in ammatory parameters in CSF with no focal neurological symptoms.
During this study, no patient was hospitalized due to meningoencephalomyelitis.
None of the participants were vaccinated for TBE. TBE was diagnosed based on the clinical picture, presence of in ammatory parameters in the CSF, and speci c antibodies present in serum and CSF. TBE antibody titer was measured with Enzygnost Anti-TBE/FSME Virus (IgG, IgM) Siemens test. A case of TBE was diagnosed according to EAN rules [15]. No patient had a history of any neurodegenerative disorder that could have effect to the result of the study. Patients were treated with 0.25 g/kg 15% Mannitol per dose 2-4 times a day and, if needed, NSAIDs (ketoprofenum) was also administered. The treatment lasted for 4-7 days and the effect of treatment on the study was considered by collecting sample 1 before treatment initiated and sample 2 taken after seven days after drug withdrawal.
The control group (CG) consisted of 17 patients (6 women, 11 men, mean age 50.1 ± 10.39 years). These group of patients were admitted to the hospital with a compliant of headache with a ruled out CSF in ammatory process (mean cytosis − 3,4 ± 1.804 cells/µL; protein concentration − 36,67 ± 10.66 mg/dL). They also had no history of neurodegenerative disorders.
The concentration of YKL-40 (ng/ml) in CSF was determined using Fujirebio test (Ghent, Belgium). YKL-40 concentration in CSF was measured on admission (Sample 1) and 14 days after admission or seven days after drug withdrawal (Sample 2). The ratio of sample 2 against sample 1(YKL 2/YKL 1) was also calculated to identify the effect of treatment on YKL-40. As a follow up, patients were examined for the presence of sequelae in the outpatients department.
Patients voluntarily agreed to participate in the study and gave their written informed consent. The study was approved by the Local Bioethics Committee of the Medical University of Bialystok.
The statistical analysis was performed using STATISTICA 10. Groups were compared with Mann-Whitney U test, Wilcoxon-matched pair test and receiver operating characteristic curve (ROC) tests. Correlations were measured by the Spearman rank test.
Patients diagnosed with meningitis had no focal neurological symptoms. All patients diagnosed with meningoencephalitis presented with consciousness disturbances. The main neurological symptoms in this group were: ataxia 6/16 patients, tremor 5/16 patients, paresis 4/16 patients, pyramidal symptoms 1/16 patients.
The laboratory parameters of patients are presented in Table 1. Mean YKL-40 concentration in CSF in sample 1 was signi cantly higher in both examined groups (Group I and II) than in CG (p < 0.05). It was also signi cantly higher in Group II than in Group I (p < 0.05) ( Table 2). ROC curve analysis indicates that YKL-40 protein concentration in CSF differentiates TBE patients from CG. At the cut-off at 783.87 ng/ml speci city is 100% and sensitivity 75%. AUC = 0.915, p < 0.05 (Fig. 1).
Serum concentration of YKL-40 did not differ in Sample 1 and 2 neither in meningitis nor in meningoencephalitis group.
CSF concentration of YKL-40 measured in sample 2 was signi cantly lower (p < 0.05) than in sample 1 in meningitis group while in meningoencephalitis group the result was nonsigni cant.
The serum YKL-40 ratio in Group I was 1.23 ± 0.62 ng/ml and in Group II 1.11 ± 0.4 ng/ml. The result was non-signi cant.
Comparison of sequelae group and other TBE patients did not show any statistically signi cant difference as far as YKL-40 concentrations in serum and CSF are concerned.

Discussion
In our previous studies we reported that TBE may lead to neurodegeneration. First of all we observed that patients with a history of TBE in MRI present with cerebral atrophic lesions that cannot be explained by age [16]. Also we found out that several neurodegeneration markers are increased in TBE patients. Neuron-speci c enolase (NSE) concentration in CSF of patients with meningoencephalitis was signi cantly higher than in controls and in patients with meningitis. What is more NSE concentration in the CSF was higher in sample 2 than in sample 1 which implied that neurodegenerative processes were ongoing even after clinical recovery [11].
The same phenomenon was stated in our another study concerning Tau protein in the CSF of TBE patients. Additionally we observed that Tau protein concentration might be a predictor of sequelae development in the course of TBE [ 10].
In the current study we evaluated the usefulness of another well-known neurodegeneration biomarker -YKL-40 protein. Contrary to NSE and Tau protein, YKL-40 concentration was the highest at the beginning of the disease and decreased in the recovery phase. What is even more interesting it was signi cantly higher in the meningitis group, usually associated with mild course of the disease.
YKL-40 seems not very useful for prognosis of sequelae development as both serum and CSF concentrations were comparable in patients who recovered completely and those who presented with persisting symptoms. This is in disagreement to most reports that associate YKL-40 concentration increase with unfavorable course of many diseases.
According to the literature YKL-40 proved to be a promising biomarker in various diseases.
High concentrations of YKL-40 in serum are correlated to morbidity of such different diseases as community-acquired pneumonia, active rheumatoid arthritis, and ongoing hepatic brosis, as well as to mortality of recurrent breast cancer and colorectal cancer [12,17,18,19,20,21]. It has been proven that this protein takes part in Alzheimer disease development and can be used in prediction of dementia development risk among cognitively normal subjects [13] and for prognosis in NMDR encephalitis [14].
Østergaard et al. reported that Increased concentrations of YKL-40 were found in patients with purulent meningitis [12]. The authors did not nd any clear correlations with severe outcome or mortality. However they observed that YKL-40 CSF concentration correlated with neutrophils and lymphocyte count. This is in accordance with our study. In TBE neutrophils number in CSF is usually elevated at the early stage of CNS infection while at the later stage of the disease lymphocytes predominate.
Neutrophils migrate into CSF of patients with TBE independently of the lymphocyte in ux and BBB disruption. Of chemokines attracting neutrophils at least IL-8 and CXCL1 create chemotactic gradients towards CSF and may be responsible for neutrophil recruitment, and CXCL1 concentration correlates with CSF neutrophil count and with encephalitic presentation [22]. This would partially explain the increased concentration of YKL-40 in CSF Sample 1. However as the neutrophil count was higher in meningoencephalitis group, some other mechanisms of YKL-40 release to CSF have to be involved. Also the connection between lymphocytes and YKL-40 is unknown.
The results of our study should be interpreted with special care. The examined group was relatively small and no patients with the most severe form of TBE -meningoencephalomyelitis were hospitalized at the time of the study. Also limited funding of the study did not allow us to correlate YKL-40 concentrations with other markers of neurodegeneration.

Conclusions
YKL-40 takes part in TBE pathogenesis, its concentration is the highest at the early stage of Central Nervous System involvement and decreases in the convalescent period.
As YKL-40 is signi cantly higher in meningitis than in meningoencephalitis, it might be used as biomarker in differentiation of these clinical forms of TBE.

Declarations
Ethical Approval and Consent to participate: Patients voluntarily agreed to participate in the study and gave their written informed consent. The study was approved by Local Ethical Committee.
Consent for publication: All authors declare their consent for publication.

Availability of supporting data: Yes
Competing interests: Authors declare no con ict of interests.
Authors' contributions: Piotr Czupryna: collection of samples, analysis of results, writing a manuscript, funding of analyses Agnieszka Kulczyńska-Przybik: performance of analyses