Protein biomarker profiles in serum and CSF in 158 patients with PTLDS or persistent symptoms after presumed tick-bite exposure compared to those in patients with confirmed acute neuroborreliosis.

BACKGROUND: Current diagnostics for patients with lingering symptoms categorized as post-treatment Lyme disease syndrome (PTLDS) have their limitations and may be difficult to interpret. The aim of this exploratory study was to evaluate the feasibility of protein biomarker profiling as a diagnostic platform for this category of patients and to compare these results with similarly obtained results from a group of patients with acute neuroborreliosis. METHODS AND FINDINGS: Two groups of patient cohorts (Cohort 1 and 2) were analyzed for biomarkers in serum and cerebrospinal fluid (CSF); the results were used for group-level comparison. Cohort 1 comprised 158 adult patients selected from 224 previously diagnosed patients, who between October 2015 and December 2018, after referral, were enrolled and structurally investigated based on defined inclusion criteria. They displayed similar lingering symptoms, with a duration of at least 6 months, after presumed previous tick-borne infection (TBI) and are fully described in a previously published study originating from the Center for Vector-borne Infections (CVI), Uppsala University Hospital, Sweden. Cohort 2, comprised 30 patients diagnosed at Uppsala University Hospital between 2016 and 2019 with laboratory-confirmed acute neuroborreliosis. Their proteomic results, based on serum and CSF analyses, were compared with the 158 patients in Cohort 1. The expression and the concentration of potential biomarkers in each patient's serum and CSF samples were measured based on two multiplex protein panels enabling simultaneous analysis of 92 inflammatory and neurology biomarkers. The PTLDS patient subgroup showed no nominally significant proteins compared to the other CVI patients in Cohort 1. However, CVI patients with signs of inflammation, which were evenly distributed in Cohort 1, showed 16 significantly (p <0.05) different proteins in both CSF and serum, but no association was seen with laboratory-confirmed exposure to Borrelia spp or other TBIs. When comparing the two cohorts, different protein profiles were observed, with 125/148 significantly different proteins in CSF and 93/174 in serum, in patients with laboratory confirmed acute neuroborreliosis, of which 6 in CSF and 6 in serum were significant at the p <0.001 level. CONCLUSIONS: In this first comprehensive inflammatory and neurological biomarker profile study no differences in biomarker profiles were detected between patients with PTLDS and patients with similar persisting symptoms but who did not meet the PTLDS criteria, regardless of whether laboratory verified previous exposure to Borrelia or other TBI's were present. However, the expressed markers differed from those found in patients with confirmed acute neuroborreliosis, which does not support the view that PTLDS reflects an ongoing Borrelia infection. Further studies are needed to understand and assess the usefulness of biosignatures of patients with PTLDS before they can be applied in a clinical setting.

Cytokine responses of immunosuppressed and immunocompetent patients with Neoehrlichia mikurensis infection.

PURPOSE: The tick-borne bacterium Neoehrlichia mikurensis causes the infectious disease neoehrlichiosis in humans. Vascular endothelium is one of the target cells of the infection. Neoehrlichiosis patients with compromised B cell immunity present with more severe inflammation than immunocompetent patients. The aim of this study was to compare the cytokine profiles of immunocompetent and immunosuppressed patients with neoehrlichiosis. METHODS: Blood samples from Swedish and Norwegian immunosuppressed (N = 30) and immunocompetent (N = 16) patients with neoehrlichiosis were analyzed for the levels of 30 cytokines, using a multiplex cytokine assay and ELISA. A gender-matched healthy control group (N = 14) was analyzed in parallel. Data were analyzed using the multivariate method OPLS-DA. RESULTS: The multiplex cytokine analyses generated more cytokine results than did the uniplex ELISA analyses. Multivariate analysis of the multiplex cytokine results established that increased levels of FGF2, GM-CSF, CXCL10, and IFN-γ were associated with immunosuppressed patients, whereas increased levels of IL-15 and VEGF were associated with immunocompetent neoehrlichiosis patients. When multivariate analysis findings were confirmed with uniplex ELISA, it was found that both groups of patients had similarly elevated levels of VEGF, FGF2 and IFN-γ. In contrast, the immunosuppressed patients had clearly elevated levels of CXCL10, CXCL13 and BAFF, whereas the immunocompetent patients had the same levels as healthy controls. CONCLUSION: Pro-angiogenic and type 1 cytokines were produced as part of the host response of neoehrlichiosis independent of immune status, whereas immunosuppressed neoehrlichiosis patients produced cytokines required for B cell-mediated defense.

Bartonella spp. seroprevalence in tick-exposed Swedish patients with persistent symptoms.

BACKGROUND: Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months. METHODS: Serological testing for Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort. RESULTS: Seroprevalence was 7% for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups. CONCLUSIONS: Antibodies to Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure.

A comprehensive clinical and laboratory evaluation of 224 patients with persistent symptoms attributed to presumed tick-bite exposure.

BACKGROUND: Persistent symptoms attributed to presumed tick-bite exposure constitute an unresolved medical controversy. We evaluated whether Swedish adults who met the criteria for post-treatment Lyme disease syndrome (PTLDS) exhibited characteristics distinguishable from adults who did not, but who displayed similar symptoms and disease course after suspected previous tick-bite infection (TBI). METHODS AND FINDINGS: During 2015-2018, 255 patients-referred to the Centre for Vector-borne Infections, Uppsala University Hospital, Sweden with symptoms lasting longer than six months-were recruited. Of this group, 224 completed the study. Each patient was examined by an infectious disease specialist and, besides a full medical history, underwent a panel of blood and cerebrospinal fluid laboratory tests including hematological, biochemical, microbiological and immunological analyses, and the RAND-36 scale to measure quality of life. For analysis purposes, patients were divided into five subgroups, of which one represented PTLDS. According to serological results indicating TBI and documented/ reported objective signs of Lyme disease, 85 (38%) patients fulfilled the criteria for PTLDS and were compared with the other 139 (62%) serologically classified patients. In the PTLDS group, erythema chronicum migrans (ECM) was documented/reported in 86% of patients, previous neuroborreliosis in 15%, and acrodermatitis chronica atroficans (ACA) in 3.5%. However, there were no significant differences regarding symptoms, laboratory results or disease course between patients with PTLDS and those without laboratory evidence of Borrelia exposition. Most reported symptoms were fatigue-related (70%), musculoskeletal (79%), neurological (82%) and neurocognitive (57%). Tick bites were recalled by 74%. The RAND-36 score was significantly below that of the general Swedish population. Signs of immunological/inflammatory reactivity with myositis antibodies were detected in 20% of patients, fibrinogen levels were moderately increased in 21% and elevated rheumatoid factor in 6%. CONCLUSIONS: The PTLDS group did not differ exclusively in any respect from the other subgroups, which either lacked previously documented/reported evidence of borreliosis or even lacked detectable serological signs of exposure to Lyme disease. The results suggest that symptoms often categorized as Chronic-Lyme-Disease (CLD) in the general debate, cannot be uniquely linked to Lyme disease. However, approximately 20% of the total group of patients showed signs of autoimmunity. Further studies are needed to elucidate the underlying causes and mechanisms of PTLDS and there is reason to consider a multifactorial approach.

Vasculitis due to Candidatus Neoehrlichia mikurensis: A Cohort Study of 40 Swedish Patients.

BACKGROUND: Candidatus (Ca.) Neoehrlichia (N.) mikurensis is an emerging tick-borne pathogen of humans that is closely related to Ehrlichia and Anaplasma species. This strict intracellular bacterium escapes detection by routine microbiologic diagnostic methods such as blood culture, leading to considerable under-diagnosis of the infectious disease it causes, neoehrlichiosis. METHODS: Here, we describe the vascular and thromboembolic events afflicting a series of 40 patients diagnosed with neoehrlichiosis in Sweden during a 10-year period (2009-2019). RESULTS: The majority of the patients (60%) developed vascular events ranging from repeated thrombophlebitis, deep vein thrombosis, pulmonary embolism, transitory ischemic attacks, to arteritis. Younger age was a risk factor for vascular complications. In contrast, there was no difference in the incidence of vascular events between immunosuppressed and immunocompetent patients. However, there were qualitative differences, such that deep vein thrombosis exclusively afflicted the immunosuppressed patients, whereas arteritis was restricted to the immunocompetent persons. We also present the case histories of two patients who developed vasculitis mimicking polyarteritis nodosa and giant cell arteritis. Both were cured by doxycycline treatment. CONCLUSIONS: Ca. N. mikurensis infection should be considered in patients living in tick-endemic areas of Europe and northern Asia who present with atypical vascular and/or thromboembolic events. Early diagnosis and antibiotics targeting this emerging infectious agent can eradicate the infection and prevent the development of new vascular events.