Justyna Dunaj

Evaluation of CXCL10, CXCL11, CXCL12 and CXCL13 chemokines in serum and cerebrospinal fluid in patients with tick borne encephalitis (TBE)

PURPOSE The aim of the study was to assess the concentration of chemokines: CXCL10, XCL11, CXCL12, CXCL13 in serum and cerebrospinal fluid (CSF) in patients with tick-borne encephalitis (TBE) before and after treatment. We evaluated also the usefulness of these molecules in diagnosis and monitoring of inflammation in TBE. METHODS Twenty three patients hospitalized in The Department of Infectious Diseases and Neuroinfections of Medical University in Białystok, Poland were included in the study. Patients were divided into 2 groups: TBE group-patients with confirmed TBE and control group (CG): patients with excluded TBE and other inflammatory diseases of CNS. Concentration of CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1α, CXCL13/BLC/BCA-1 in serum and CSF were measured with ELISA kits (R&D Systems, USA) according to the protocols. RESULTS The analysis of chemokines concentration in TBE patients before treatment and control group using ROC showed that serum CXCL10 and CXCL13 and CSF CXCL10, CXCL11, CXCL12 and CXCL13 differentiate both groups (p<0.05). The analysis of CXCL10, CXCL11, CXCL12 and CXCL13 before and after treatment showed that CXCL10 and CXCL11 in CSF and CXCL13 in serum differentiates both groups with p<0.05. CONCLUSIONS Concentration of CSF CXCL10, CXCL11, CXCL12, CXCL13 and serum CXCL10, CXCL13 may be good biomarkers of CNS inflammation caused by TBEV. Moreover concentration of CXCL10 in CSF and CXCL13 in serum may be used as indicators of patients recovery.

Infection with Babesia microti in humans with non-specific symptoms in North East Poland

Abstract Aim: The aim of the study was to evaluate the clinical course and effectiveness of diagnostics tools for Babesia spp. infection in patients bitten by ticks. Materials and methods: Five hundred and forty-eight patients hospitalised or seen in outpatients department because of various symptoms after a tick bite were included in the study. PCR, nucleotide sequencing of Babesia 18S rRNA gene fragment, blood smears and serological tests for Babesia spp., TBEV, A. phagocytophilum and B. burgdorferi were performed in all patients. Six patients infected with Babesia were included in the final analysis. They had PCR, Babesia 18S rRNA gene fragment nucleotide sequencing, blood smears and serological tests for Babesia spp., TBEV, A. phagocytophilum and B. burgdorferi performed twice. Results: Tick-borne infection with Babesia microti in six immunocompetent patients with non-specific symptoms was confirmed for the first time in Poland. No severe course of the disease was seen. No piroplasm forms were noticed within erythrocytes on blood smear. Three patients developed a serological response. Conclusions: Immunocompetent patients may be unaware of infection with Babesia microti after a tick bite. It must be included in the differential diagnosis after the tick bite. In patients with low parasitaemia PCR and serology seem useful when blood smear is negative. Self-elimination of Babesia spp. is possible, especially in cases with low parasitaemia.

Increased concentration of interferon lambda-3, interferon beta and interleukin-10 in the cerebrospinal fluid of patients with tick-borne encephalitis

Tick-borne encephalitis (TBE) has a wide clinical spectrum, from asymptomatic to severe encephalitis, and host-dependent factors determining the outcome remain elusive. We have measured concentrations of pro-inflammatory/Th1 interferon-γ (IFNγ), immunomodulatory/Th2 interleukin-10 (IL-10), anti-viral type I (IFNβ) and type III (IFNλ3) interferons in cerebrospinal fluid (csf) and serum of 18 TBE patients, simultaneously genotyped for polymorphisms associated with the expression of genes IFNL3 (coding IFNλ3), IL10, CD209 and CCR5. IL-10, IFNβ and IFNλ3 were up-regulated in csf, with IFNλ3 level higher in patients with the milder clinical presentation (meningitis) than in meningoencephalitis. There was an increased serum IFNβ and a tendency for increased serum IL-10 in meningitis patients. Genotype in rs12979860 locus upstream of IFNL3 was associated with IFNλ3 expression and in rs287886 (CD209) - IL-10 expression. IL-10, IFNβ and IFNλ3 are expressed and play a protective role in TBE and their expression in TBE patients is associated with genetic polymorphisms.

The expression of the chemokine receptor CCR5 in tick-borne encephalitis

BackgroundChemokine receptor 5 (CCR5) is hypothesized to drive the lymphocyte migration to central nervous system in flavivirus encephalitis, and the non-functional CCR5Δ32 genetic variant was identified as a risk factor of a West Nile virus infection and of tick-borne encephalitis (TBE). We have attempted to investigate how CCR5 expression corresponds to the clinical course and severity of TBE.MethodsWe have repeatedly studied CCR5 expression in 76 patients during encephalitic and convalescent TBE phase, analyzing its association with clinical features, cerebrospinal fluid (csf) pleocytosis, and concentrations of CCR5 ligands (chemokines CCL3, CCL4, and CCL5) and CCR5 genotype. Fifteen patients with neuroborreliosis, 7 with aseptic meningitis, 17 in whom meningitis/encephalitis had been excluded, and 18 healthy blood donors were studied as controls. Expression of CCR5 was measured cytometrically in blood and csf-activated Th lymphocytes (CD3+CD4+CD45RO+). Concentrations of chemokines in serum and csf were measured immunoenzymatically, and CCR5Δ32 was detected with sequence-specific primers. Data were analyzed with non-parametric tests, and p < 0.05 was considered significant.ResultsThe blood expression of CCR5 did neither differ between the groups nor change in the course of TBE. The CCR5 expression in the inflammatory csf was several-fold increased in comparison with blood but lower in TBE than in neuroborreliosis. The csf concentration of CCL5 was increased in TBE, the highest in the most severe presentation (meningoencephalomyelitis) and correlated with pleocytosis. The CCR5Δ32/wt genotype present in 7 TBE patients was associated with a decreased CCR5 expression, but enrichment of csf Th population in CCR5-positive cells and the intrathecal inflammatory response were preserved, without a compensatory increase of CCL5 expression.ConclusionsWe infer CCR5 and CCL5 participate in the response to TBE virus, as well as to other neurotropic pathogens. The intrathecal response to TBE is not hampered in the bearers of a single copy of CCR5Δ32 allele, suggesting that the association of CCR5Δ32 with TBE may be mediated in the periphery at the earlier stage of the infection. Otherwise, a variability of the CCR5 expression in the peripheral blood lymphocytes seems not to be associated with a variable susceptibility to TBE.

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

BackgroundHost 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-1β).MethodsConcentrations of MIF, TNFα, and IL-1β 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.ResultsThe median serum and CSF concentrations of MIF and IL-1β 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.ConclusionsThe 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.

MRI and planimetric CT follow-up study of patients with severe tick-borne encephalitis.

Background: The aim of the study was to evaluate the magnetic resonance imaging (MRI) and planimetric computed tomography (CT) of brain lesions in patients with a history of tick-borne encephalitis (TBE); to assess the influence of steroid treatment on the brain and whether lesions were age-dependent. Methods: A total of 19 patients with abnormal initial imaging in the acute stage of the disease had a follow-up MRI after 1 year; 34 patients hospitalized for TBE encephalitis/encephalomyelitis had planimetric CT after 10 years. Results: On MRI cortico-subcortical atrophy with widening of anterior horns of the lateral ventricles and vascular changes was more marked on follow-up examination. Virchow–Robin spaces dilatation, widening of the lateral ventricles, periventricular lesions, and cortico-subcortical atrophy correlated with age. Results of planimetric CT study showed increased percentage of tracings, widened anterior horns, lateral ventricles, and III ventricle, which suggest new non-age-related atrophic lesions. Conclusions: Radiological lesions in the acute phase of TBE and after recovery are non-specific. Cortico-subcortical atrophy with widening of the anterior horns of the lateral ventricles and vascular changes are most common. Long-term follow-up confirms the formation of new non-age-related cerebral atrophic lesions due to TBE. The logit model may serve as a background for the hypothesis concerning an accelerated local atrophy of the brain tissues in patients with a history of severe TBE.

Tick-borne infections and co-infections in patients with non-specific symptoms in Poland

AIM The aim of the study was the evaluation of the frequency of infections and co-infections among patients hospitalized because of non-specific symptoms after a tick bite. MATERIALS AND METHODS Whole blood, serum and cerebrospinal fluid samples from 118 patients hospitalised for non-specific symptoms up to 8 weeks after tick bite from 2010 to 2013 were examined for tick-borne infections. ELISA, Western blot and/or molecular biology (PCR; fla gene; 16S rRNA; sequencing) and thin blood smears (MDD) were used. Control group included 50 healthy blood donors. All controls were tested with PCR and serology according to the same procedure as in patients. RESULTS Out of 118 patients 85 (72%) experienced headaches, 15 (13%) vertigo, 32 (27%) nausea, 17 (14%) vomiting, 37 (31%) muscle pain, 73 (62%) fever and 26 (22%) meningeal signs. 47.5% were infected with at least one tick-borne pathogen. Borrelia burgdorferi sensu lato infection was confirmed with ELISA, Western blot in serum and/or (PCR (fla gene) in whole blood in 29.7% cases. In blood of 11.9% patients Anaplasma phagocytophilum DNA (16S rRNA gene) was detected; in 0.9% patients 1/118 Babesia spp. DNA (18S rRNA gene) was also detected. Co-infections were observed in 5.1% of patients with non-specific symptoms. B. burgdorferi s.l. - A. phagocytophilum co-infection (5/118; 4.2%) was most common. In 1/118 (0.8%) A. phagocytophilum - Babesia spp. co-infection was detected. All controls were negative for examined pathogens. CONCLUSIONS Non-specific symptoms after tick bite may be caused by uncommon pathogens or co-infection, therefore it should be considered in differential diagnosis after tick bite.

Borrelia burgdorferi genospecies detection by RLB hybridization in Ixodes ricinus ticks from different sites of North-Eastern Poland.

INTRODUCTION RLB (Reverse Line Blot Hybridization) is a molecular biology technique that might be used for Borrelia burgdorferi sensu lato (sl) DNA detection with genospecies specification. Among B. burgdorferi sl genospecies at least 7 are regarded as pathogenic in Europe. OBJECTIVE The aim of the study was to evaluate the frequency of different Borrelia genospecies DNA detection in Ixodes ricinus ticks in the endemic area of North-Eastern Poland by using RLB. MATERIALS AND METHOD Ixodes ricinus ticks were collected in May - June, from 6 different sites in North-Eastern Poland (Jakubin, Kolno, Grajewo, Suwałki, Siemiatycze, Białowieża) by flagging. Extracted DNA was amplified by polymerase chain reaction (PCR) targeting the intergenic spacer 5S 23S of B. burgdorferi sl. PCR products were hybridised to 15 different oligonucleotide probes for 9 different Borrelia genospecies (B. burgdorferi sl, B. burgdorferi ss, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B. spielmanii, B. bissettii and B. relapsing fever-like spirochetes (B. myamotoi)) by RLB. RESULTS Borrelia genospecies DNA was detected in 205 Ixodes ricinus ticks. Among 14 infected with Borrelia ticks, 4 were identified as B. garinii and 10 as B. afzelii. Higher numbers of infected ticks were noticed in the eastern part of the research area, where large forest complexes dominate. Nymphs appeared to be the most frequently infected tick stage, which has an epidemiological meaning in the incidence of Lyme borreliosis. CONCLUSIONS The study demonstrated that RLB might be easily used in Borrelia DNA detection with genospecies-identification, and indicated the domination of B. afzelii and B. garinii in ticks from North-Eastern Poland.

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‐&kgr;B 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‐&kgr;B 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‐&kgr;B was measured by human inhibitory subunit of NF‐&kgr;B 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‐&kgr;B 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‐&kgr;B 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‐&kgr;B 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‐&kgr;B concentration significantly decreased in convalescent period, while in NB and TBE groups significant CSF decrease of NF‐&kgr;B concentration was observed. ConclusionsInflammatory process in the CNS results in the increase of NF‐&kgr;B concentration in CSF, due to the damage of blood‐brain barrier.Co‐infection of TBE + ANA increases NF‐&kgr;B concentration in serum.NF‐&kgr;B concentration in serum may be useful in the differentiation of TBE + ANA co‐infection with TBE and NB.NF‐&kgr;B concentration may be used to monitor TBE and NB treatment effectiveness. HighlightsNF‐&kgr;B increases in cerebrospinal fluid (CSF) during nervous system inflammation.Tick‐borne encephalitis with anaplasmosis (TBE + ANA) increases NF‐&kgr;B in serum.Serum NF‐&kgr;B concentration differentiates TBE + ANA with TBE and neuroborreliosis.Serum and CSF NF‐&kgr;B concentration may monitor tick‐borne encephalitis treatment.Cerebrospinal fluid NF‐&kgr;B concentration may monitor neuroborreliosis treatment.

Comparison of detection of Borrelia burgdorferi DNA and anti-Borrelia burgdorferi antibodies in patients with erythema migrans in north-eastern Poland

Introduction Diagnostic methods in erythema migrans are still not standardized. Aim To evaluate the frequency of Borrelia burgdorferi s.l. DNA presence in patients with erythema migrans (EM); to assess the polymerase chain reaction (PCR) procedure for detecting B. burgdorferi s.l. DNA in patients with the skin form of Lyme borreliosis; and to compare the results of the PCR-based method with the traditional ELISA method. Material and methods Skin biopsy and blood samples from 93 patients with EM were examined for B. burgdorferi s.l. DNA detection (PCR). Seventy-one of these patients were examined for the presence of anti-B. burgdorferi s.l. antibodies (ELISA). Results Borrelia burgdorferi s.l. DNA was detected in 48% of the skin biopsy specimens and in 2% of blood samples. Only 1 patient was PCR positive in both blood and skin samples. Seventy percent of patients whose PCR results were positive were bitten by a tick less than 14 days before. IgM anti-B. burgdorferi s.l – specific antibodies were present in the serum of 35% of patients and IgG antibodies – in 30% of patients. Seventeen percent were positive in both IgM and IgG. Conclusions Polymerase chain reaction of skin biopsy specimens seems to be currently the most sensitive and specific test for the diagnosis of patients with EM, especially in patients with a short duration of the disease (< 14 days) but still its effectiveness is much lower than expected. Polymerase chain reaction of blood samples cannot be recommended at the present time for the routine diagnostic of patients with EM.