Genome-wide DNA Methylation Profiling in Lyme Neuroborreliosis Reveals Altered Methylation Patterns of HLA Genes.

Lyme neuroborreliosis (LNB) is a complex neuroinflammatory disorder caused by Borrelia burgdorferi, which is transmitted through tick bites. Epigenetic alterations, specifically DNA methylation (DNAm), could play a role in the host immune response during infection. In this study, we present the first genome-wide analysis of DNAm in peripheral blood mononuclear cells from patients with LNB and those without LNB. Using a network-based approach, we highlighted HLA genes at the core of these DNAm changes, which were found to be enriched in immune-related pathways. These findings shed light on the role of epigenetic modifications in the LNB pathogenesis that should be confirmed and further expanded upon in future studies.

Spiroplasma ixodetis Infections in Immunocompetent and Immunosuppressed Patients after Tick Exposure, Sweden.

We report 2 cases of Spiroplasma ixodetis infection in an immunocompetent patient and an immunocompromised patient who had frequent tick exposure. Fever, thrombocytopenia, and increased liver aminotransferase levels raised the suspicion of anaplasmosis, but 16S rRNA PCR and Sanger sequencing yielded a diagnosis of spiroplasmosis. Both patients recovered after doxycycline treatment.

Brain damage markers neuron-specific enolase (NSE) and S100B in serum in children with Lyme neuroborreliosis-detection and evaluation as prognostic biomarkers for clinical outcome.

Lyme borreliosis (LB) is the most common tick-borne infection in Europe, with Lyme neuroborreliosis (LNB) its second most frequent clinical manifestation. Prognostic factors for clinical outcomes in LNB have not been identified. Elevated serum levels of the brain damage markers neuron-specific enolase (NSE) and S100 calcium-binding protein B (S100B) have been associated with poor clinical outcomes in other disorders of the central nervous system. The aim of this study is to assess NSE and S100B in serum as prognostic biomarkers for clinical outcomes in paediatric LNB patients. Children evaluated for LNB (n = 121) in Sweden were prospectively included during 2010-2014, serum samples were collected on admission, and all children underwent a 2-month follow-up. Patients with pleocytosis and anti-Borrelia antibodies in cerebrospinal fluid (CSF) were classified as having LNB (n = 61). Controls were age- and gender-matched non-LNB patients (n = 60). NSE was elevated in 38/61 (62%) LNB patients and in 31/60 (52%) controls. S100B was elevated in 3/60 (5%) LNB patients and 0/59 (0%) controls. NSE and S100B concentrations did not differ significantly when comparing LNB patients with controls. No differences were found in the concentrations when comparing the clinical recovery of LNB patients at the 2-month follow-up. NSE was detectable in the majority of LNB patients and controls, whereas S100B was detectable in only a few LNB patients and no controls. NSE and S100B in serum cannot be recommended as prognostic biomarkers for clinical outcomes in children with LNB.

CXCL13 in laboratory diagnosis of Lyme neuroborreliosis-the performance of the recomBead and ReaScan CXCL13 assays in human cerebrospinal fluid samples.

The chemokine CXCL13 is used as complement to serology in the diagnostics of Lyme neuroborreliosis (LNB). We evaluated and compared the semi-quantitative, cassette-based ReaScan CXCL13 assay with the quantitative recomBead CXCL13 assay using a collection of 209 cerebrospinal fluid samples. The categorical agreement between results interpreted as negative, grey zone, and positive by the two methods was 87%. The diagnostic sensitivity was higher using the recomBead assay, whereas specificity was higher using ReaScan. Few manual steps, and a short turn-around time with no batching of samples makes the ReaScan CXCL13 assay an attractive complement to serology in the diagnostics of LNB.

Cerebrospinal fluid markers of inflammation and brain injury in Lyme neuroborreliosis - a prospective follow-up study.

OBJECTIVES: The purpose of this study was to evaluate levels and kinetics of cerebrospinal fluid (CSF) markers of inflammation and brain injury in patients with Lyme neuroborreliosis (LNB). METHODS: Adult patients with clinically suspected LNB were enrolled, in a prospective clinical study in the South East of Sweden. Patients were classified according to the European Federation of Neurological Societies' guidelines. Definite cases of LNB were re-examined one month later including a repeat CSF investigation. Routine laboratory parameters were investigated along with CSF levels of neurodegenerative markers glial fibrillary acidic protein (GFAp), total tau (t-tau) and neurofilament light protein (NFL), as well as neuroinflammatory markers soluble triggering receptor expressed on myeloid cells 2 (sTREM2), YKL-40 and CXCL13. Non-LNB served as controls. An additional comparison group consisted of spinal anesthesia subjects (SAS) without known central nervous system conditions. RESULTS: CSF levels of sTREM2 and CXCL13 were elevated in definite LNB patients at diagnosis compared with non-LNB patients (p<0.001) and SAS (p≤0.01). In addition, CSF levels of sTREM2, YKL-40 and CXCL13 rapidly declined in at follow-up after antibiotic treatment. In contrast, CSF levels of GFAp and t-tau did not differ across LNB groups, and did not change after treatment. CONCLUSIONS: Although in a limited number of LNB patients, the results indicate a predominance of microglial and neuroinflammatory involvement rather than parenchymal CNS injury in CSF at diagnosis of LNB with a prompt decline after antibiotic treatment. The findings provide pathogenetic insights and may be of value in differential diagnosis of CSF findings.

Serodiagnosis of Lyme borreliosis-is IgM in serum more harmful than helpful?

Interpretation of serological findings in suspected Lyme borreliosis (LB) may be challenging and IgM reactivities in serum are often unspecific (false positive). There is a risk for overdiagnosis of LB, inadequate use of antibiotics, and potential delay of proper diagnosis. In this study, we evaluated the diagnostic value of IgM analysis in serum and IgM antibody index (AI) in LB diagnosis. This was a retrospective observational study regarding Borrelia-specific antibodies in serum and Borrelia-specific AI in LB investigations being made during 2017 in Jönköping County, Sweden. Medical records of 610 patients with detectable anti-Borrelia antibodies in serum (IgM and/or IgG) and 15 patients with elevated Borrelia-specific AI were retrospectively scrutinized, and the compliance to current European recommendations was assessed. Among the 610 patients, only 30% were tested according to the European recommendations. Within this group of tests taken correctly, 50% of the LB diagnoses in patients with isolated IgM reactivity in serum were retrospectively assessed as incorrect (LB unlikely). Three pediatric patients with clinical and laboratory findings suggestive of Lyme neuroborreliosis (LNB) had elevated IgM AI alone. Serological testing without distinct clinical signs/symptoms consistent with LB contributes to most misdiagnoses. Isolated IgM positivity in serum shows limited clinical value and needs further assessment before being reported by the laboratory. Detection of IgM in combination with IgG antibodies in serum shows no clinical enhancement for correct LB diagnosis compared to isolated IgG positivity. However, Borrelia-specific IgM AI may be important for sensitivity in early LNB.

Cerebrospinal fluid cytokines and chemokines in children with Lyme neuroborreliosis; pattern and diagnostic utility.

BACKGROUND: Lyme neuroborreliosis (LNB) is characterized by cerebrospinal fluid (CSF) inflammation with several cytokines/chemokines and B-lymphocytes. Clinically, LNB in children may be difficult to discriminate from non-Lyme aseptic meningitis (NLAM). We aimed to identify CSF cytokine/chemokine patterns in children with LNB, NLAM and controls and elucidate the diagnostic value of these cytokines/chemokines alone or in combination to discriminate between LNB and NLAM. METHODS: Children with symptoms suggestive of LNB were included prospectively and categorized as LNB, NLAM or controls (no pleocytosis). Cytokines/chemokines in CSF were measured by multiplex bead assays and levels were compared between the three groups by nonparametric statistical tests. Previous results from the same children on the established biomarker, CXCL13, were included in the statistical analyses. The diagnostic properties of cytokines/chemokines to discriminate between LNB and NLAM were determined by receiver operating characteristic curve analyses with estimates of area under curve (AUC). To explore diagnostic properties of combinations of cytokines/chemokines, prediction models based on logistic regression were used. RESULTS: We included 195 children with LNB (n = 77), NLAM (n = 12) and controls (n = 106). Children with LNB had higher CSF levels of CCL19, CCL22 and CXCL13 compared to NLAM and controls, whereas INFγ was higher in NLAM than in LNB and controls. CXCL13 was the superior single cytokine/chemokine to discriminate LNB from NLAM (AUC 0.978). The combination CXCL13/CCL19 (AUC 0.992) may possibly improve the specificity for LNB, especially for children with moderate CXCL13 levels. CONCLUSIONS: The intrathecal immune reaction in LNB is characterized by B cell associated chemokines. Whether the combination CXCL13/CCL19 further improves discrimination between LNB and NLAM beyond the diagnostic improvements by CXCL13 alone needs to be tested in new studies.

Protozoan infections are under-recognized in Swedish patients with gastrointestinal symptoms.

In acute gastroenteritis (GE), identification of the infectious agent is important for patient management and surveillance. The prevalence of GE caused by protozoa may be underestimated in Swedish patients. The purpose was to compare the prevalence of E. histolytica, Cryptosporidium spp., G. intestinalis, and C. cayetanensis in samples from patients where the clinician had requested testing for gastrointestinal parasites only (n = 758) to where testing for bacterial GE only (n = 803) or where both parasite and bacterial testing (n = 1259) was requested and a healthy control group (n = 197). This prospective cohort study was conducted in Region Jönköping County, Sweden (October 2018-March 2019). Fecal samples were analyzed with microscopy and real-time PCR. Cryptosporidium spp. was detected in 16 patients in the bacterial GE group and in 13 in the both bacterial and parasite group; no cases were detected in the group were only parasite infection was suspected. C. cayetanensis was detected in two patients in the bacterial GE group. One case of E. histolytica was detected in the bacterial group and one in the both bacterial and parasite group. G. intestinalis was detected in 14 patients in the parasite only group, 12 in the both parasite and bacterial group, three in the bacterial GE group, and one in the control group. Diarrhea caused by protozoa, especially Cryptosporidium was under-recognized by clinicians and is likely more common than hitherto estimated in Sweden. A more symptom-based diagnostic algorithm may increase detection and knowledge about protozoan infections.

Two Cases of Borrelia miyamotoi Meningitis, Sweden, 2018.

We report 2 human cases of Borrelia miyamotoi disease diagnosed in Sweden, including 1 case of meningitis in an apparently immunocompetent patient. The diagnoses were confirmed by 3 different independent PCR assays and DNA sequencing from cerebrospinal fluid, supplemented by serologic analyses.

Serological diagnostics of Lyme borreliosis: comparison of assays in twelve clinical laboratories in Northern Europe.

Lyme borreliosis (LB), caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, is the most common tick-borne infection in Europe. Laboratory diagnosis of LB is mainly based on the patients' medical history, clinical signs and symptoms in combination with detection of Borrelia-specific antibodies where indirect enzyme-linked-immunosorbent assay (ELISA) is the most widely used technique. The objective of the study was to evaluate and compare the diagnostic accuracy (sensitivities and specificities) of serological tests that are currently in use for diagnosis of LB in clinical laboratories in Northern Europe, by use of a large serum panel. The panel consisted of 195 serum samples from well-characterized and classified patients under investigation for clinically suspected LB (n = 59) including patients with Lyme neuroborreliosis, Lyme arthritis, acrodermatitis chronica atrophicans, erythema migrans or other diseases (n = 112). A total of 201 serum samples from healthy blood donors were also included. The panel (396 serum samples altogether) was sent to 12 clinical laboratories (using five different ELISA methods) as blinded for group affiliation and the laboratories were asked to perform serological analysis according to their routine procedure. The results from the study demonstrated high diagnostic concordance between the laboratories using the same diagnostic assay and lower diagnostic concordance between laboratories using different diagnostic assays. For IgG, the results were in general rather homogenous and showed an average sensitivity of 88% (range 85-91%) compared to IgM which showed lower average sensitivity of 59% (range 50-67%) and more heterogeneous results between assays and laboratories.

Diagnostic performance of cerebrospinal fluid free light chains in Lyme neuroborreliosis - a pilot study.

Background The aim of this study was to evaluate the diagnostic performance of cerebrospinal fluid (CSF) free light chains (FLCs) in the diagnosis of Lyme neuroborreliosis (LNB). Methods Serum and CSF levels of κ- and λ-FLC, albumin and total concentration of immunoglobulin M (IgM) were determined together with CSF chemokine CXCL13 in 23 patients with definite LNB, 35 inflammatory neurological disease control (INDC) and 18 non-inflammatory control (NIC) patients. Indices and intrathecal fractions (IFs) of FLC and IgM were calculated. Results Significant differences in FLC indices and IFs were found between the LNB group and both control groups, p ≤ 0.007. Sensitivity of intrathecal κ- and λ-FLC synthesis reached 78%-87% in LNB patients with a specificity of 94%-100% in NIC patients, whereas specificity in INDC patients was 69%. The corresponding frequencies of positive results for IF and index of IgM and CSF CXCL13 in these three diagnostic groups were 74%-96% in LNB patients, 0% in NIC patients and 3%-6% in INDC patients at the chosen cut-off levels. Conclusions The findings of this study show a moderate to high sensitivity of CSF κ- and λ-FLC in LNB patients with a high specificity in NIC patients. However, overlap in CSF κ- and λ-FLC levels between LNB and INDC patients calls for caution in the interpretation and limits the diagnostic usefulness in the LNB diagnosis. CSF CXCL13 appears to be the most valuable additional biomarker of LNB aside from routine parameters such as CSF pleocytosis and anti-Borrelia antibody index.

The chemokine CXCL13 in cerebrospinal fluid in children with Lyme neuroborreliosis.

Anti-Borrelia antibodies in the cerebrospinal fluid (CSF) are required for definite diagnosis of Lyme neuroborreliosis (LNB). However, children often present with early LNB, and antibody production in the CSF may not be demonstrated. Recent studies have suggested the chemokine CXCL13 to be an early marker for LNB. The aim of the study was to evaluate CXCL13 for laboratory diagnosis in pediatric LNB patients and to evaluate the association with pleocytosis in CSF, clinical features, and recovery. CSF samples were collected from LNB patients, classified as definite LNB (n = 44) or possible LNB (n = 22), and controls classified as non-LNB (n = 102) or other specific diagnoses (n = 23). CSF samples were analyzed with the recomBead CXCL13 assay (Mikrogen Diagnostik, Germany), cut-off 160 pg/mL. CXCL13 was significantly higher in LNB patients compared to controls (p < 0.001). Among LNB patients, 58/66 had elevated CXCL13, and among controls, 111/125 had CXCL13 levels under cut-off (sensitivity 88%, specificity 89%). In LNB patients with pleocytosis but no detectable anti-Borrelia antibodies in CSF (possible LNB), CXCL13 was elevated in 16/22 (73%). A weak correlation between CXCL13 and pleocytosis in CSF was found in LNB patients (Rho = 0.46, p < 0.01), but no differences in CXCL13 levels in relation to specific clinical features. In conclusion, CXCL13 is elevated in CSF in children with LNB, showing acceptable sensitivity and specificity. In patients with possible LNB, CXCL13 was elevated in a majority of cases (73%) and is suggested as a complementary diagnostic tool in pediatric LNB patients. CXCL13 was not associated with specific clinical features or recovery.

Serological reactivity to Anaplasma phagocytophilum in neoehrlichiosis patients.

The tick-borne bacterium Candidatus (Ca.) Neoehrlichia (N.) mikurensis is a cause of "fever of unknown origin" because this strict intracellular pathogen escapes detection by routine blood cultures. Case reports suggest that neoehrlichiosis patients may display serological reactivity to Anaplasma (A.) phagocytophilum. Since Anaplasma serology is part of the diagnostic work-up of undetermined fever in European tick-exposed patients, we wanted to investigate (1) the prevalence of A. phagocytophilum seropositivity among neoehrlichiosis patients, (2) the frequency of misdiagnosed neoehrlichiosis patients among A. phagocytophilum seropositive patients, and (3) the frequency of A. phagocytophilum and Ca. N. mikurensis co-infections. Neoehrlichiosis patients (n = 18) were analyzed for A. phagocytophilum IgM and IgG serum antibodies by indirect immunofluorescence assay. Serum samples from suspected anaplasmosis patients (n = 101) were analyzed for bacterial DNA contents by singleplex PCR specific for A. phagocytophilum and Ca. N. mikurensis, respectively. One fifth of the neoehrlichiosis patients (4/18) were seropositive for IgM and/or IgG to A. phagocytophilum at the time of diagnosis. Among the patients with suspected anaplasmosis, 2% (2/101) were positive for Ca. N. mikurensis by PCR whereas none (0/101) had detectable A. phagocytophilum DNA in the serum. To conclude, patients with suspected anaplasmosis may in fact have neoehrlichiosis. We found no evidence of A. phagocytophilum and Ca. N. mikurensis co-infections in humans with suspected anaplasmosis or confirmed neoehrlichiosis.

Intrathecal Th17- and B cell-associated cytokine and chemokine responses in relation to clinical outcome in Lyme neuroborreliosis: a large retrospective study.

BACKGROUND: B cell immunity, including the chemokine CXCL13, has an established role in Lyme neuroborreliosis, and also, T helper (Th) 17 immunity, including IL-17A, has recently been implicated. METHODS: We analysed a set of cytokines and chemokines associated with B cell and Th17 immunity in cerebrospinal fluid and serum from clinically well-characterized patients with definite Lyme neuroborreliosis (group 1, n = 49), defined by both cerebrospinal fluid pleocytosis and Borrelia-specific antibodies in cerebrospinal fluid and from two groups with possible Lyme neuroborreliosis, showing either pleocytosis (group 2, n = 14) or Borrelia-specific antibodies in cerebrospinal fluid (group 3, n = 14). A non-Lyme neuroborreliosis reference group consisted of 88 patients lacking pleocytosis and Borrelia-specific antibodies in serum and cerebrospinal fluid. RESULTS: Cerebrospinal fluid levels of B cell-associated markers (CXCL13, APRIL and BAFF) were significantly elevated in groups 1, 2 and 3 compared with the reference group, except for BAFF, which was not elevated in group 3. Regarding Th17-associated markers (IL-17A, CXCL1 and CCL20), CCL20 in cerebrospinal fluid was significantly elevated in groups 1, 2 and 3 compared with the reference group, while IL-17A and CXCL1 were elevated in group 1. Patients with time of recovery <3 months had lower cerebrospinal fluid levels of IL-17A, APRIL and BAFF compared to patients with recovery >3 months. CONCLUSIONS: By using a set of markers in addition to CXCL13 and IL-17A, we confirm that B cell- and Th17-associated immune responses are involved in Lyme neuroborreliosis pathogenesis with different patterns in subgroups. Furthermore, IL-17A, APRIL and BAFF may be associated with time to recovery after treatment.

Human Tick-Borne Encephalitis and Characterization of Virus from Biting Tick.

We report a case of human tick-borne encephalitis (TBE) in which the TBE virus was isolated from the biting tick. Viral growth and sequence were characterized and compared with those of a reference strain. Virus isolation from ticks from patients with TBE may offer a new approach for studies of epidemiology and pathogenicity.

Detection of Anaplasma phagocytophilum in Ixodes ricinus ticks from Norway using a realtime PCR assay targeting the Anaplasma citrate synthase gene gltA.

BACKGROUND: A TaqMan real-time PCR assay targeting the Anaplasma citrate synthase gene, gltA, was developed and used for detection of Anaplasma phagocytophilum in 765 Ixodes ricinus ticks collected from dogs and cats in northern Norway (n = 669) and Telemark county in southern Norway (n = 96). RESULTS: Among the ticks from northern Norway the prevalence of A. phagocytophilum was 3.0 %, while the prevalence in southern Norway was 2.1 % (p = 0.63). The gltA PCR assay showed a high analytical sensitivity (30 genomic units) and efficiency (98.5 %), and its utility in clinical diagnostics should be evaluated in future studies. CONCLUSION: This is the first report of A. phagocytophilum occurrence in ticks collected north of the Arctic Circle in Norway. The prevalence is comparable to that found in Telemark county in southern Norway.

Diagnostic patterns of serum inflammatory protein markers in children with Lyme neuroborreliosis.

Definite diagnosis of Lyme neuroborreliosis (LNB) requires investigation of serum and cerebrospinal fluid (CSF). Thus, lumbar puncture is necessary, and requires administration of sedating drugs in children. This study aimed to investigate if a pattern of different inflammatory biomarkers in serum could contribute to the selection of children for lumbar puncture in suspected LNB. Patients were included from a cohort of children who was previously investigated for LNB including serum and CSF sampling during the years 2010-2014. The multiplex proximity extension assay (PEA) inflammation panel Target 96 (Olink Bioscience, Uppsala, Sweden) was used to examine 92 biomarkers in serum. Based on the presence of CSF pleocytosis and Borrelia-specific antibodies, patients were divided into a definite LNB group (n=61) and a non-LNB control group (n=58). Following PEA and statistical analysis with multivariate logistic regression, five biomarkers remained significant (p < 0.001), which were included in a calculation of protein index. The index biomarkers were CST5, IL-15RA, CXCL10, DNER and CX3CL1. A receiver operating characteristic curve was constructed from the index, which showed an 80 % sensitivity and 81 % specificity. Area under the curve was 0.889. We offer evidence that, with further refinements, patterns of serum biomarkers might help identify those children more or less likely to have LNB, perhaps ultimately decreasing the need for lumbar punctures.

Tick-borne diseases in the North Sea region-A comprehensive overview and recommendations for diagnostics and treatment.

As part of the NorthTick project, co-funded by the European Union through the European Regional Development Fund and the North Sea Region Programme, specialists in the field of tick-borne diseases from seven North Sea countries co-operated with patient organisations and governmental health care institutions to provide this comprehensive overview of diagnostics and treatment recommendations in the region for Lyme borreliosis, Borrelia miyamotoi infection, tick-borne encephalitis, human granulocytic anaplasmosis, rickettsiosis, neoehrlichiosis and babesiosis. The main conclusion is that the recommendations in these northern countries are essentially the same, with very few differences. This overview presents the current diagnostics and provides useful clinical guidance.

Intrathecal Th17-driven inflammation is associated with prolonged post-treatment convalescence for patients with Lyme neuroborreliosis.

Lyme neuroborreliosis (LNB) is associated with increased levels of pro-inflammatory cytokines and chemokines in the cerebrospinal fluid (CSF). Residual symptoms after antibiotic treatment can have deleterious effects on patients and knowledge regarding the pathogenesis linked to prolonged recovery is lacking. In this prospective follow-up study, we investigated the B cell-associated and T helper (Th) cell-associated immune responses in well-characterized patients with LNB and controls. The aims were to assess the kinetics of selected cytokines and chemokines involved in the inflammatory response and to identify potential prognostic markers. We investigated 13 patients with LNB according to a standardized clinical protocol before antibiotic treatment and after 1, 6 and 12 months of follow-up. CSF and blood samples were obtained at baseline and after 1 month. As controls, we used CSF samples from 37 patients who received spinal anesthesia during orthopedic surgery. The CSF samples were analyzed for CXCL10 (Th1-related), CCL22 (Th2-related) and IL-17A, CXCL1 and CCL20 (Th17-related), as well as for the B cell-related cytokines of a proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF) and CXCL13. The CSF levels of all the cytokines and chemokines, with the exception of APRIL, were significantly higher at baseline in patients with LNB compared with controls. All the cytokines and chemokines, except for IL-17A were significantly reduced at 1-month follow-up. Patients with quick recovery (< 1 month, n = 3) had significantly lower levels of CCL20 at baseline and lower levels of IL-17A at 1-month follow-up. Patients with time of recovery > 6 months (n = 7) had significantly higher levels of IL-17A at the one-month follow-up. No other cytokines or chemokines were associated with prolonged recovery. Dominating residual symptoms were fatigue, myalgia, radiculitis and/or arthralgia. In this prospective follow-up study of patients with LNB, we found significantly lower levels of CCL20 in those who recovered rapidly, and increased levels of IL-17A in patients with delayed recovery post-treatment. Our findings indicate persistent Th17-driven inflammation in the CSF, possibly contributing to a longer convalescence, and suggest IL-17A and CCL20 as potential biomarker candidates for patients with LNB.