Symptomatic reinfections with Borrelia burgdorferi sensu lato occur, but their clinical and microbiologic characteristics are insufficiently defined. It, therefore, remains unclear whether having had a primary infection influences the presentation or course of reinfection. We compared the clinical and microbiologic findings in patients with primary infection versus reinfection, with each episode presenting with erythema migrans (EM). The analysis included a cohort of patients diagnosed with EM between 1990 and 2014 at an outpatient clinic in Slovenia. A total of 11,169 patients with EM were included, comprising 635 reinfections and 10,534 primary infections. Patients with reinfection were older (56 vs 48 years; p < 0.001), less frequently had multiple EM lesions (4.4

We identified human Borrelia miyamotoi infections in Slovenia in 2 of 337 adults with undifferentiated fever tested positive by metagenomic sequencing and PCR. Both patients reported recent local tick bites. The illness was mild and self-limited. Our findings underscore the need to consider this pathogen in evaluating fever after tick bite.

OBJECTIVES:Febrile illness without clear localisation presents a significant diagnostic challenge due to non-specific symptoms and diverse aetiologies. Spiroplasma ixodetis, an emerging tick-associated pathogen previously linked mainly to congenital cataracts, has not been well characterised in adults. We investigated the prevalence and clinical features of S. ixodetis infection in adults with acute febrile illness without localisation. METHODS:Shotgun metagenomic sequencing identified S. ixodetis in the initial 209 patient cohort and the sequences were used to developed a novel real-time PCR assay targeting the 23S rRNA gene. Initial cohort screening was followed by testing 128 patients from an additionally selected targeted cohort. Positive results were confirmed by sequencing of 16S and 23S rRNA genes. RESULTS:S. ixodetis DNA was confirmed in 7.2% patients from the initial and in 35.2% patients from the additional cohort (60 in total). All were identified in the period from April to October and 57% reported a recent tick-bite. Clinical presentation was homogenous, characterised by fever, headache, bicytopenia and liver enzyme abnormalities. Outcomes were favourable, with 15% requiring hospitalisation. CONCLUSION:This study identifies S. ixodetis as a previously unrecognised cause of adult febrile illness without localisation, bridging the gap between previously published data between tick studies and isolated human case reports.

Tick-borne encephalitis (TBE) is a viral infectious disease that involves the nervous system. Frequently, there is a febrile illness phase 1-21 days before the onset of neurological symptoms. The most common neurological manifestations include meningitis, encephalitis, myelitis, radiculitis, or a combination thereof. Long-term sequelae are present in almost every second person with nervous system involvement in this vaccine-preventable disease.

Objectives: Borrelia burgdorferi sensu lato, a spirochete bacterium responsible for Lyme borreliosis—the most common tick-borne infection in North America and Europe—can trigger the production of antiphospholipid antibodies. These antibodies target host lipids such as cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), and phosphatidylserine (PS), which the spirochete incorporates into its membrane from the surrounding environment. Although antiphospholipid antibodies are typically associated with antiphospholipid syndrome (APS), they may also arise during infections, including Lyme borreliosis. This study aimed to develop and optimize several enzyme-linked immunosorbent assays (ELISAs) for measuring various antiphospholipid antibodies in patients with Lyme borreliosis. Methods: Thirty patients diagnosed with Lyme borreliosis were enrolled: ten with solitary erythema migrans (EM), ten with multiple EM (MEM), and ten with late manifestations known as acrodermatitis chronica atrophicans (ACA). Forty healthy blood donors served as controls. Four distinct antiphospholipid antibody ELISAs were developed, each using a different phospholipid coating: CL, PA, PC, and PS. Serum of APS patient was used as a positive control and for standard curve generation. Results: All four ELISAs were successfully established and demonstrated good measurement precision. Significant differences in antiphospholipid antibody levels and positivity rates were observed between Lyme borreliosis patients and healthy blood donors. Notably, levels of antibodies directed against PA (aPA), PC (aPC), and PS (aPS), both IgG and IgM, were significantly higher in patients with late Lyme borreliosis, manifested as ACA, compared to healthy blood donors. In contrast, anti-CL (aCL) levels did not differ significantly between groups. Patients with ACA also showed the highest frequency of multiple antiphospholipid antibody positivity, with 7 out of 10 patients testing positive for three or more antiphospholipid antibodies. Conclusions: Accurate and precise in-house ELISAs for the detection of aCL, aPA, aPC, and aPS using APS sera as standard material were developed and validated for the analysis of samples of patients with Lyme borreliosis. Our data suggest that antiphospholipid antibody levels—specifically aPA, aPC, and aPS—differ across clinical manifestations of Lyme borreliosis, with the greatest increases observed in patients with ACA.

To determine the frequency of confirmed Lyme neuroborreliosis (LNB) cases in adult patients with three different clinical presentations consistent with early LNB. Data were obtained through routine health care at the UMC Ljubljana, Slovenia from 2005 to 2022, using clinical pathways. The patients were classified into three groups: (i) radicular pain of new onset (N = 332); or (ii) involvement of cranial nerve(s) but without radicular pain (N = 997); or (iii) erythema migrans (EM) skin lesion(s) in conjunction with symptoms suggestive of nervous system involvement but without either cranial nerve palsy or radicular pain (N = 240). The diagnosis of LNB considered the following variables: the presence of: (1) neurologic symptoms consistent with LNB (with no other obvious explanation); (2) cerebrospinal fluid (CSF) pleocytosis (> 5 × 106 leukocytes/L); and (3) demonstration of intrathecal synthesis of borrelial antibodies, and/or cultivation of borrelia from CSF, and/or the presence of EM. Patients fulfilling only the first two criteria were interpreted as having possible LNB, while those who satisfied all three criteria were regarded as having confirmed LNB. Of 1569 adult patients, 348 (22.2

IntroductionBlood culture is the cornerstone of microbiological diagnostics for patients with acute undifferentiated fever and no obvious localization of infection; however, up to 50% of cases remain undiagnosed. Infections caused by arboviruses, fastidious or even uncultivable bacteria, or parasites often go undiagnosed without the use of target-specific molecular methods. These are typically performed in a stepwise manner, increasing cost and delaying results. Metagenomic next-generation sequencing (mNGS) has recently gained recognition as a potential universal pathogen detection tool for such cases. Our study aimed to develop a streamlined mNGS workflow for simultaneous detection of intracellular and cell-free pathogens within a single sequencing library.MethodsTotal nucleic acid was isolated separately from 200 EDTA blood samples. The plasma isolate was processed with DNase, followed by the depletion of host ribosomal and messenger RNA, reverse transcription, and sequence-independent single primer amplification (SISPA). The whole blood isolate was only reverse transcribed, with no other pre-processing manipulation. Finally, the two fractions were combined prior to library preparation and sequencing using either Oxford Nanopore Technologies or Illumina. Following established bioinformatics analysis, we developed a mathematical ranking approach (ClinSeq score) that enabled quick identification of relevant pathogens in approximately one hour.ResultsThe mNGS workflow reached 79.5% (159/200) overall sensitivity. For bacteria the sensitivity was 88.6% (70/79), DNA viruses, 66.7% (10/15) and for RNA viruses 73.8% (76/103). Pathogen detections by individual sequencing methods showed overall sensitivity of Illumina and ONT to be 80.0% (76/95) and 79.1% (83/105) respectively. The ClinSeq score correctly highlighted the pathogen in 126/200 (63.0%) samples effectively with a Cohen’s kappa (κ) agreement of 0.61 with manual analysis.ConclusionDeveloped comprehensive mNGS workflow detects a wide range of pathogens in patients with acute undifferentiated fever. The unified workflow improves sensitivity for intracellular bacteria and RNA viruses, reduces time, cost and complexity by eliminating the need for separate library preparations, enabling faster turnaround suitable for clinical settings. The ClinSeq score effectively differentiates true pathogen signals from background noise, reducing false positives and manual interpretation time. Overall, the workflow demonstrates flexible, and efficient pathogen detection, supporting its potential for clinical diagnostics and improved patient management.

The erythema migrans (EM) skin lesion is the most common clinical manifestation of Lyme borreliosis. Information about EM in Lyme borreliosis reinfection is limited. Of the 12,384 cases with diagnosed EM at an outpatient clinic during 1990-2014 in Slovenia, 1,962 (15.8%) cases occurred in patients who were treated previously for Lyme borreliosis, including 1,849 (94.2%) who had previously had EM. The percentage of reinfected patients who sought care with disseminated Lyme borreliosis at the time of reinfection, as manifested by multiple EM skin lesions, was significantly lower than for EM patients with no history of Lyme borreliosis (5.5% [108/1,962] vs. 7.4% [769/10,427]; p = 0.002). None of the clinical manifestations of Lyme borreliosis in Europe will completely protect against EM developing in patients in the future. The reoccurrence of Lyme borreliosis manifested by multiple EM lesions is significantly less likely than for patients with no history of Lyme borreliosis.

There is a lack of systematic data on cerebrospinal fluid (CSF) findings in the different clinical manifestations of tick-borne encephalitis (TBE) and according to the duration of neurological involvement. This study aims to evaluate and compare routine CSF parameters in TBE patients presenting with meningitis, meningoencephalitis, or meningoencephalomyelitis, and to assess temporal changes in CSF during the first 10 days after the onset of neurological symptoms. We analysed 15 CSF parameters in 717 consecutive adult patients hospitalized for TBE at the Department of Infectious Diseases, University Medical Centre Ljubljana, Slovenia. Among these patients, 230 (32

Tick-borne encephalitis (TBE) is a viral infection of the central nervous system, caused by the tick-borne encephalitis virus (TBEV) presenting clinically as meningitis, meningoencephalitis, and meningoencephalomyelitis. To investigate genetic susceptibility to TBE, and its severe forms, we conducted a genome-wide association study in the European population comprising 1,600 TBE cases and 9,699 controls. We identified several suggestive (p < 1 × 10-5) intronic and exonic variants in ABCG1, the only gene significantly associated with TBE susceptibility. These variants were shown to influence ABCG1 expression in peripheral blood, a finding corroborated by RNA expression analysis. In vitro inhibition or silencing of ABCG1 significantly reduced TBEV replication in both neuronal cells and macrophages, highlighting the potential role of ABCG1 in TBEV biology. Additionally, we detected a genome-wide significant variant within TEX41, located downstream of ZEB1, associated with severe forms of TBE. These findings provide novel insights into the genetic factors underlying TBE susceptibility and severity.

We report possible intraoperative transmission of Brucella melitensis in Slovenia, likely caused by aerosolized particles during wound irrigation. Whole-genome multilocus sequence typing revealed that isolates from the patient and the surgeon belonged to the same transmission cluster, differing by 1 allele. Our findings raise awareness of occupational risks faced by orthopedic surgeons.

Tick-borne encephalitis virus (TBEV) vaccine breakthrough (VBT) infections are not uncommon in endemic areas. The clinical and immunological outcomes have been poorly investigated. We assessed the magnitude and specificity of virus-specific antibody and T cell responses after TBE in previously vaccinated subjects and compared the results with those of unvaccinated TBE patients and study subjects that received vaccination without VBT infection. Symptomatic TBEV infection of unvaccinated study subjects induced virus-specific antibody responses to the E protein and non-structural protein 1 (NS1) as well as T cell responses to structural and other non-structural (NS) proteins. After VBT infections, significantly impaired NS1-specific antibody responses were observed, while the virus-specific T cell responses to the NS proteins were relatively strong. VBT infection caused predominantly moderate to severe disease during hospitalization. The level of TBEV EDIII- and NS1-specific antibodies in unvaccinated convalescent patients inversely correlated with TBE severity and neurological symptoms early after infection.

Erythema migrans (EM) is the initial and the most frequent clinical manifestation of Lyme borreliosis (LB). Herein, we report on the capacity of culture and serology for the demonstration of Borrelia infection in a cohort of 292 patients diagnosed with typical EM at a single medical center. The median duration of EM at diagnosis was 12 days, and the largest diameter was 16 cm; 252 (86.3%) patients presented with solitary EM, whereas 40 (13.7%) had multiple EM. A total of 95/292 (32.5%) patients had positive IgM, and 169 (57.9%) had positive IgG serum antibodies; the Borrelia isolation rate was 182/292 (62.3%). The most frequent species by far was B. afzelii (142/148, 95.9%) while B. garinii (2.7%) and B. burgdorferi s.s. (1.4%) were rare. IgM seropositivity was associated with a younger age, multiple EM and the absence of underlying chronic illness; IgG seropositivity was associated with the duration of EM at diagnosis, the diameter of the EM, having had a previous episode of LB and the absence of symptoms at the site of the EM. Furthermore, the Borrelia isolation rate was statistically significantly lower in patients with positive Borrelia IgM antibodies. Although microbiologic analyses are not needed for the diagnosis of typical EM, they enable insights into the etiology and dynamics of the immune response in the course of early LB.

Tick-borne encephalitis virus (TBEV) infection may cause acute central nervous system inflammation varying in clinical manifestations and severity. A possible correlation of TBEV-specific antibody and cell-mediated immune responses, shortly after infection, with clinical manifestations, severity and long-term outcome has been poorly investigated. In a cohort of thirty early tick-borne encephalitis (TBE) patients, we assessed the magnitude, specificity and functional properties of TBEV-specific T-cell and antibody responses. These responses early during disease were assessed in view of clinical manifestations, severity and long-term outcome. TBEV-specific T-cell responses to C, E, NS1, and NS5 proteins were significantly lower in patients with severe acute illness than in patients with mild TBE. Lower T-cell responses to E, NS1, and NS5 proteins also correlated with the development of meningoencephalomyelitis. Virus-specific antibody titres early after infection did not correlate with disease severity, clinical manifestations, or long-term outcome in this study, possibly due to the small number of patients of which matching serum and peripheral blood mononuclear cells were available. The findings suggest that virus-specific T cells afford a certain degree of protection against the development of severe TBEV-induced disease.

PurposeSince some patients with tick-borne encephalitis (TBE) have pronounced myalgias, and since myositis is reported in Flavivirus diseases such as dengue, we performed systematic search for abnormalities of muscle enzymes in a group of patients in whom the presence of tick-borne encephalitis virus (TBEV) RNA in the first phase of the disease was demonstrated and who developed second phase of TBE.MethodsTotal leukocyte and platelet blood counts were determined routinely at the initial examination during the first and the second phase of TBE. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), myoglobin and troponin was determined from the available stored serum specimens; the first and second phase disease specimens were tested simultaneously.ResultsOf 24 patients with biphasic course of TBE, 83% had leukopenia, 65% thrombocytopenia, 83% elevated AST and 4% elevated ALT level. Furthermore, 33% had elevated serum CK, 26% myoglobin and 22% troponin activity; at least one of the muscle enzymes was elevated in 42% of patients. Leukopenia, thrombocytopenia, elevated liver enzymes and elevations of CK and myoglobin were present in the initial phase but resolve later, while troponin abnormalities were also found in the second phase of TBE.ConclusionsThe present study exposes that in addition to previously known leukopenia, thrombocytopenia and increased liver enzymes activity, the initial phase of TBE is relatively often associated also with elevated muscle enzymes. Clinical relevance of these findings remains to be determined.

Diagnosis of (European) Lyme neuroborreliosis has been based on clinical presentation, cerebrospinal fluid (CSF) pleocytosis and demonstration of intrathecal borrelial antibody synthesis (ITBAS) to document Borrelia burgdorferi s. l. infection. It is not known if other criteria to document Borrelia infection may contribute to the diagnosis. We compared the sensitivity of three individual criteria (ITBAS, CSF Borrelia culture, and the presence of erythema migrans [EM]) to confirm the diagnosis of early Lyme neuroborreliosis in 280 patients ≥ 15 years of age evaluated at a Lyme borreliosis outpatient clinic in Slovenia. The patients had either radicular pain of new onset or involvement of a cranial nerve but without radicular pain, each in conjunction with CSF pleocytosis. Evaluation was of patients who had each of the three confirmatory criteria assessed, and for whom at least one criterion was positive. Analysis of 280 patients, 120 women and 160 men, median age 57 (range 15–84) years, revealed that ITBAS was the most frequently observed positive criterion (85.4

Information on asplenic Lyme borreliosis (LB) patients with erythema migrans (EM) is lacking. We compared the course and outcome of 26 EM episodes in 24 post-trauma splenectomized patients (median age 51 years) diagnosed at a single clinical center in Slovenia during 1994–2023 with those of 52 age- and sex-matched patients with EM but with no history of splenectomy. All patients were followed for one year. A comparison of pre-treatment characteristics revealed that EM in splenectomized patients was of shorter duration before diagnosis (4 vs. 8 days, p = 0.034) with a smaller EM diameter (10.5 vs. 14 cm, p = 0.046), and more frequently fulfilled criteria for disseminated LB (3/26, 11.5% vs. 0%, p = 0.034). Treatment failure occurred in 5/26 (19.2%) EM episodes in splenectomized patients versus 0/52 in non-splenectomized patients (p = 0.003). The five treatment failure cases were retreated with antibiotic regimens used to treat EM and had complete resolution of all symptoms/signs. In conclusion, our study showed that splenectomized adult patients with EM differ somewhat in presentation and more often have treatment failure compared with non-splenectomized patients with EM.

In prior studies, the skin lesion erythema migrans (EM) was present for a longer time period before diagnosis of concomitant borrelial meningoradiculoneuritis (Bannwarth’s syndrome) compared to EM patients without neurologic symptoms. To determine if this observation pertains to other manifestations of Lyme neuroborreliosis (LNB), we compared EM characteristics in patients with borrelial meningoradiculoneuritis (n = 122) to those with aseptic meningitis without radicular pain (n = 72 patients), and to patients with EM but without neurologic involvement (n = 12,384). We also assessed factors that might impact duration. We found that the duration of EM at diagnosis in patients with borrelial meningoradiculoneuritis was not significantly different compared with those with LNB without radicular pain (34 vs. 26 days; p = 0.227). The duration of EM for each of these clinical presentations of LNB, however, was significantly longer than in patients with EM without LNB (10 days; p < 0.001). Contributing factors to this difference might have been that patients with LNB failed to recognize that they had EM or were unaware of the importance of not delaying antibiotic treatment for EM. In conclusion, the duration of the EM skin lesion in EM patients with LNB is longer than in patients with just EM, irrespective of the type of LNB.

Introduction: Corticosteroids are widely used for the treatment of coronavirus disease (COVID)-19. Genetic polymorphisms of the glucocorticoid receptor, metabolizing enzymes, or transporters may affect treatment response to dexamethasone. This study aimed to evaluate the association of the glucocorticoid pathway polymorphisms with the treatment response and short-term outcomes in patients with severe COVID-19.Methods: Our pilot study included 107 hospitalized patients with COVID-19 treated with dexamethasone and/or methylprednisolone, genotyped for 14 polymorphisms in the glucocorticoid pathway.Results: In total, 83% of patients had severe disease, 15.1% had critical disease and only 1.9% had moderate disease. CYP3A4 rs35599367 was the major genetic determinant of COVID-19 severity as carriers of this polymorphism had higher risk of critical disease (OR = 6.538; 95% confidence interval = 1.19-35.914: p = 0.031) and needed intensive care unit treatment more frequently (OR = 10; 95% CI = 1.754-57.021: p = 0.01). This polymorphism was also associated with worse disease outcomes, as those patients had to switch from dexamethasone to methylprednisolone more often (OR = 6.609; 95% CI = 1.137-38.424: p = 0.036), had longer hospitalization (p = 0.022) and needed longer oxygen supplementation (p = 0.040). Carriers of NR3C1 rs6198 polymorphic allele required shorter dexamethasone treatment (p = 0.043), but had higher odds for switching therapy with methylprednisolone (OR = 2.711; 95% CI = 1.018-7.22: p = 0.046). Furthermore, rs6198 was also associated with longer duration of hospitalization (p = 0.001) and longer oxygen supplementation (p = 0.001). NR3C1 rs33388 polymorphic allele was associated with shorter hospitalization (p = 0.025) and lower odds for ICU treatment (OR = 0.144; 95% CI = 0.027-0.769: p = 0.023). GSTP1 rs1695 was associated with duration of hospitalization (p = 0.015), oxygen supplementation and (p = 0.047) dexamethasone treatment (p = 0.022).Conclusion: Our pathway-based approach enabled us to identify novel candidate polymorphisms that can be used as predictive biomarkers associated with response to glucocorticoid treatment in COVID-19. This could contribute to the patient's stratification and personalized treatment approach.