Prevalence and clinical manifestation of Borrelia miyamotoi in Ixodes ticks and humans in the northern hemisphere: a systematic review and meta-analysis.

BACKGROUND: Various studies have evaluated the infection of Ixodes ticks and humans with the relapsing fever spirochaete Borrelia miyamotoi. However, to our knowledge, the prevalence of infection and disease has not been assessed systematically. We aimed to examine the prevalence of B miyamotoi in Ixodes ticks and humans, and the disease it can cause, in the northern hemisphere. METHODS: For this systematic review and meta-analysis, we searched PubMed and Web of Science up to March 1, 2021. Studies assessing Ixodes tick infection published since Jan 1, 2011 were eligible, whereas no time limitation was placed on reports of human infection and disease. We extracted B miyamotoi test positivity ratios and used a random-effects model to calculate estimated proportions of infected ticks, infected humans, and human disease with 95% CI. This study was registered with PROSPERO, CRD42021268996. FINDINGS: We identified 730 studies through database searches and 316 additional studies that referenced two seminal articles on B miyamotoi. Of these 1046 studies, 157 were included in the review, reporting on 165 637 questing ticks, 45 608 unique individuals, and 504 well described cases of B miyamotoi disease in humans. In ticks, the highest prevalence of B miyamotoi was observed in Ixodes persulcatus (2·8%, 95% CI 2·4-3·1) and the lowest in Ixodes pacificus (0·7%, 0·6-0·8). The overall seroprevalence in humans was 4·4% (2·8-6·3), with significantly (p<0·0001) higher seroprevalences in the high-risk group (4·6%, 2·6-7·1), participants with confirmed or suspected Lyme borreliosis (4·8%, 1·8-8·8), and individuals suspected of having a different tick-borne disease (11·9%, 5·6-19·9) than in healthy controls (1·3%, 0·4-2·8). Participants suspected of having a different tick-borne disease tested positive for B miyamotoi by PCR significantly more often than did the high-risk group (p=0·025), with individuals in Asia more likely to test positive than those in the USA (odds ratio 14·63 [95% CI 2·80-76·41]). INTERPRETATION: B miyamotoi disease should be considered an emerging infectious disease, especially in North America and Asia. Prospective studies and increased awareness are required to obtain further insights into the burden of disease. FUNDING: ZonMW and the European Regional Development Fund (Interreg).

Initial Severe Acute Respiratory Syndrome Coronavirus 2 Viral Load Is Associated With Disease Severity: A Retrospective Cohort Study.

Background: We assessed the association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and hospital admission, intensive care unit (ICU) admission, and in-hospital mortality. Methods: All SARS-CoV-2-positive persons with a combined nasopharyngeal and oropharyngeal swab that was collected between 17 March 2020 and 31 March 2021 in public health testing facilities were included. Results: From 20 207 SARS-CoV-2-positive persons, 310 (1.5%) were hospitalized within 30 days. High viral loads (crossing point [Cp] <25) were associated with an increased risk of hospitalization as compared to low viral loads (Cp >30), adjusted for age and sex (adjusted odds ratio [aOR], 1.57 [95% confidence interval {CI}, 1.11-2.26]). The same association was seen for ICU admission (aOR, 7.06 [95% CI, 2.15-43.57]). The median [interquartile range] Cp value of the 17 patients who died in hospital was significantly lower compared to the 226 survivors (22.7 [3.4] vs 25.0 [5.2]). Conclusions: Higher initial SARS-CoV-2 viral load is associated with an increased risk of hospital admission, ICU admission, and in-hospital mortality. Our findings emphasize the added value of reporting SARS-CoV-2 viral load or cycle threshold/Cp values to identify persons who are at the highest risk of adverse outcomes such as hospital or ICU admission and who therefore may benefit from more intensive monitoring or early initiation of antiviral therapy.

The Effect of Varying Interval Definitions on the Prevalence of SARS-CoV-2 Reinfections: A Retrospective Cross-Sectional Cohort Study.

BACKGROUND: We assessed the SARS-CoV-2 reinfection rate in a large patient cohort, and evaluated the effect of varying time intervals between two positive tests on assumed reinfection rates using viral load data. METHODS: All positive SARS-CoV-2 samples collected between 1 March 2020 and 1 August 2021 from a laboratory in the region Kennemerland, the Netherlands, were included. The reinfection rate was analyzed using different time intervals between two positive tests varying between 2 and 16 weeks. SARS-CoV-2 PCR crossing point (Cp) values were used to estimate viral loads. RESULTS: In total, 679,513 samples were analyzed, of which 53,366 tests (7.9%) were SARS-CoV-2 positive. The number of reinfections varied between 260 (0.52%) for an interval of 2 weeks, 89 (0.19%) for 4 weeks, 52 (0.11%) for 8 weeks, and 37 (0.09%) for a minimum interval of 16 weeks between positive tests. The median Cp-value (IQR) in the second positive samples decreased when a longer interval was chosen, but stabilized from week 8 onwards. CONCLUSIONS: Although the calculated reinfection prevalence was relatively low (0.11% for the 8-week time interval), choosing a different minimum interval between two positive tests resulted in major differences in reinfection rates. As reinfection Cp-values stabilized after 8 weeks, we hypothesize this interval to best reflect novel infection rather than persistent shedding.

The impact of the updated EORTC/MSG criteria on the classification of hematological patients with suspected invasive pulmonary aspergillosis.

OBJECTIVES: Our aim was to evaluate the effect of the updated European Organization for Research and Treatment of Cancer (EORTC) and Mycoses Study Group 2019 definitions for invasive pulmonary aspergillosis (IPA) on patient classification and the related all-cause 12-week mortality. METHODS: In this retrospective cohort study from our tertiary care centre, we reclassified patients with haematological malignancy who underwent bronchoalveolar lavage between 2014 and 2019 for suspected IPA using the novel EORTC 2019 criteria. We performed receiver operating characteristic curve analysis to define the optimal cut-off for positive PCR and galactomannan and present survival analyses and their possible association with these diagnostic criteria through post hoc comparisons with log rank and Cox regression. RESULTS: From 323 episodes of suspected IPA in 282 patients, 73 were reclassified: 31 (42.5%) from possible to probable IPA, 5 (6.8%) from EORTC criteria not met to probable IPA, and 37 (50.7%) from EORTC criteria not met to possible IPA. Probable IPA increased therefore 11.1% (64/323, 19.8% to 100/323, 30.9%), mostly due to positive PCR (31/36, 86.1%). There was no difference in mortality between newly defined possible and probable IPA (log rank p = 0.950). Mortality was higher in probable cases with lower cycle thresholds (Ct values) versus higher Ct values (p = 0.004). Receiver operating characteristic curve analysis showed an optimal Ct value cut-off of 36.8 with a sensitivity of 75% (95% CI 64.9%-85.1%) and a specificity of 61.7% (95% CI 53.5-69.9) for 12-week mortality. DISCUSSION: The new EORTC criteria led to 11.1% more probable IPA diagnoses, mostly due to Aspergillus PCR. Restricting positive PCR to below a certain threshold might improve the discrimination of the new EORTC IPA categories for mortality.

SARS-CoV-2 viral-load distribution reveals that viral loads increase with age: a retrospective cross-sectional cohort study.

BACKGROUND: Describing the SARS-CoV-2 viral-load distribution in different patient groups and age categories. METHODS: All results from first nasopharyngeal (NP) and oropharyngeal (OP) swabs from unique patients tested via SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) collected between 1 January and 1 December 2020 predominantly in the Public Health Services regions Kennemerland and Hollands Noorden, province of North Holland, the Netherlands, were included in this study. SARS-CoV-2 PCR crossing-point (Cp)-values were used to estimate viral loads. RESULTS: In total, 278 455 unique patients were tested, of whom 9.1% (n = 25.374) were SARS-CoV-2-positive. PCRs performed by Public Health Services (n = 211 914), in which sampling and inclusion were uniform, revealed a clear relation between age and SARS-CoV-2 viral load, with especially children aged <12 years showing lower viral loads than adults (ß: -0.03, 95% confidence interval: -0.03 to -0.02, p < 0.001), independently of sex and/or symptom duration. Interestingly, the median Cp-values between the >79- and <12-year-old populations differed by more than four PCR cycles, suggesting an ∼16-fold difference in viral load. In addition, the proportion of children aged <12 years with a low load (Cp-value >30) was higher compared with other patients (31.1% vs 17.2%, p-value < 0.001). CONCLUSIONS: In patients tested by Public Health Services, SARS-CoV-2 viral load increases with age. Further studies should elucidate whether the lower viral load in children is indeed related to their suggested limited role in SARS-CoV-2 transmission. Moreover, as rapid antigen tests are less sensitive than PCR, these results suggest that SARS-CoV-2 antigen tests have lower sensitivity in children than in adults.

Tick-Tattoo: DNA Vaccination Against B. burgdorferi or Ixodes scapularis Tick Proteins.

Introduction: Borrelia burgdorferi sensu lato (sl) is the causative agent of Lyme borreliosis. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines. DNA tattoo vaccination with B. afzelii strain PKo OspC in mice has proven to be fully protective against B. afzelii syringe challenge and induces a favorable humoral immunity compared to recombinant protein vaccination. Alternatively, several recombinant protein vaccines based on tick proteins have shown promising effect in tick-bite infection models. In this study, we evaluated the efficacy of DNA vaccines against Borrelia OspC or tick antigens in a tick-bite infection model. Method: We vaccinated C3H/HeN mice with OspC using a codon-optimized DNA vaccine or with recombinant protein. We challenged these mice with B. burgdorferi sensu stricto (ss)-infected Ixodes scapularis nymphs. Subsequently, we vaccinated C3H/HeN mice with DNA vaccines coding for tick proteins for which recombinant protein vaccines have previously resulted in interference with tick feeding and/or Borrelia transmission: Salp15, tHRF, TSLPI, and Tix-5. These mice were also challenged with B. burgdorferi ss infected Ixodes scapularis nymphs. Results: DNA tattoo and recombinant OspC vaccination both induced total IgG responses. Borrelia cultures and DNA loads of skin and bladder remained negative in the mice vaccinated with OspC DNA vaccination, except for one culture. DNA vaccines against tick antigens Salp15 and Tix-5 induced IgG responses, while those against tHRF and TSLPI barely induced any IgG response. In addition, Borrelia cultures, and DNA loads from mice tattooed with DNA vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 were all positive. Conclusion: A DNA tattoo vaccine against OspC induced high specific IgG titers and provided near total protection against B. burgdorferi ss infection by tick challenge. In contrast, DNA tattoo vaccines against tick proteins TSLPI, Salp15, tHRF, and Tix-5 induced low to moderate IgG titers and did not provide protection. Therefore, DNA tattoo vaccination does not seem a suitable vaccine strategy to identify, or screen for, tick antigens for anti-tick vaccines. However, DNA tattoo vaccination is a straightforward and effective vaccination platform to assess novel B. burgdorferi sl antigen candidates in a relevant tick challenge model.

Borrelia miyamotoi Activates Human Dendritic Cells and Elicits T Cell Responses.

The spirochete Borrelia miyamotoi has recently been shown to cause relapsing fever. Like the Lyme disease agent, Borrelia burgdorferi, B. miyamotoi is transmitted through the bite of infected ticks; however, little is known about the response of the immune system upon infection. Dendritic cells (DCs) play a central role in the early immune response against B. burgdorferi We investigated the response of DCs to two different strains of B. miyamotoi using in vitro and ex vivo models and compared this to the response elicited by B. burgdorferi. Our findings show that B. miyamotoi is phagocytosed by monocyte-derived DCs, causing upregulation of activation markers and production of proinflammatory cytokines in a similar manner to B. burgdorferi. Recognition of B. miyamotoi was demonstrated to be partially mediated by TLR2. DCs migrated out of human skin explants upon inoculation of the skin with B. miyamotoi. Finally, we showed that B. miyamotoi-stimulated DCs induced proliferation of naive CD4+ and CD8+ T cells to a larger extent than B. burgdorferi. In conclusion, we show in this study that DCs respond to and mount an immune response against B. miyamotoi that is similar to the response to B. burgdorferi and is able to induce T cell proliferation.

Case report: chronic relapsing cryptococcal meningitis in a patient with low mannose-binding lectin and a low naïve CD4 cell count.

BACKGROUND: Cryptococcal meningitis is most commonly found in HIV-infected patients. In HIV-negative patients, its low incidence can lead to prolonged time to diagnosis. Detailed case reports of chronic cryptococcal meningitis are scarce, but could provide clues for earlier diagnosis in this patient category. CASE PRESENTATION: A 60-year old man presented June 2015 with intermittent headaches for several months without any fever. Initial work-up showed a leukocytosis, raised CSF opening pressure and raised leukocytes and protein in the CSF. An MRI revealed leptomeningeal contrast enhancement and cerebellar oedema. While malignancy and various infectious causes were excluded, the patient had a spontaneous clinical and radiological recovery. One year later, the patient returned with complaints of headaches. Also, cerebellar oedema and leptomeningeal contrast enhancement had recurred. Eventually in March 2017, the novel cryptococcal antigen lateral flow assay (CrAg LFA) was positive on CSF, and one colony of Cryptococcus neoformans was cultured from CSF. The patient was treated with the standard antifungal regimen which resulted in resolution of his headaches. In retrospect, the cryptococcal antigen test was already positive on a serum sample from June 2015. Interestingly, post-treatment immunological analysis revealed both a low mannose-binding lectin (MBL) concentration and low naïve CD4 counts. CONCLUSIONS: We present a patient with cryptococcal meningitis in an HIV-negative patient with low MBL and low naïve CD4 count suffering a chronic relapsing meningo-encephalitis with relatively mild symptoms for around 2 years. In patients with an unexplained meningo-encephalitis such as this case, early performance of CrAg LFA on serum and/or CSF is an inexpensive and rapid method to reduce time-to diagnosis.

Author Correction: Immune evasion of Borrelia miyamotoi: CbiA, a novel outer surface protein exhibiting complement binding and inactivating properties.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The role of Mannose Binding Lectin in the immune response against Borrelia burgdorferi sensu lato.

The causative agents of Lyme borreliosis, spirochetes belonging to the Borrelia burgdorferi sensu lato group, have developed several ways to protect themselves against killing by the host complement system. In addition, it has been shown that serum sensitive isolates are (partially) protected by the Ixodes Tick Salivary Lectin Pathway Inhibitor (TSLPI) protein; a salivary gland protein that inhibits the function of Mannose Binding Lectin (MBL). MBL is a C-type lectin that recognizes oligosaccharides on pathogens and activates the complement system via the lectin pathway. MBL deficiency has been linked to a more severe course of several infectious diseases and humans with detectable antibodies against B. burgdorferi are significantly more often MBL deficient compared to humans without antibodies against B. burgdorferi. Here we set out to investigate the role of MBL in the immune response against B. burgdorferi in more detail. We demonstrate that B. burgdorferi N40 needle-infected C57BL/6 MBL deficient mice harbored significantly higher B. burgdorferi numbers in skin tissue during the early course of infection. In line with these findings they also developed higher anti-B. burgdorferi IgG serum antibodies compared to WT controls. In contrast, B. burgdorferi loads in distant tissue such as heart, joints or bladder at later time points were similar for both mouse strains. These in vivo findings were corroborated using a B. burgdorferi N40-infected I. scapularis infestation model. We showed that MBL is capable of binding B. burgdorferi through its carbohydrate recognition domains, but in vitro complement killing assays, peritoneal macrophage and whole blood stimulations, phagocytosis assays and an in vivo migration experiment did not reveal the mechanism by which MBL facilitates early clearance of B. burgdorferi. To conclude, we show a protective role of MBL in the early stages of B. burgdorferi infection, yet the underlying mechanism warrants further investigation.

Acute syphilitic meningitis in an HIV-infected patient.

We present a case of acute syphilitic meningitis in a 28-year old HIV-infected patient, presenting with a seizure. MRI revealed a meningeal lesion with cortical involvement, and the patient described previous treatment for syphilis. Final diagnosis was established by PCR and spirochete staining on a brain biopsy, upon which the patient was successfully treated with benzylpenicillin.

In Vitro Antimicrobial Susceptibility of Clinical Isolates of Borrelia miyamotoi.

Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is reported to cause human disease in regions in which Lyme borreliosis is endemic. We recently showed that B. miyamotoi tick isolates are resistant to amoxicillin in vitro; however, clinical isolates have not been studied. Therefore, our aim was to show the antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi A dilution series of various antibiotics was made in modified Kelly-Pettenkofer medium with 10% fetal calf serum. The susceptibilities of different B. miyamotoi clinical, B. miyamotoi tick, RF Borrelia, and Borrelia burgdorferisensu lato isolates were tested by measuring MICs through colorimetric changes and by counting motile spirochetes by dark-field microscopy after 72 h of incubation. The ceftriaxone and azithromycin MIC ranges of the six B. miyamotoi clinical isolates tested were 0.03 to 0.06 mg/liter and 0.0016 to 0.0032 mg/liter, respectively. These values are similar to MICs for RF Borrelia strains and B. miyamotoi tick isolates. All tested RF Borrelia strains were susceptible to doxycycline (microscopic MIC range, 0.0625 to 0.25 mg/liter). In contrast to the MICs of the tested B. burgdorferi sensu lato strains and in line with our previous findings, the amoxicillin MICs (range, 8 to 32 mg/liter) of all RF Borrelia strains, including B. miyamotoi clinical isolates, were above the clinical breakpoint for resistance (≤4 mg/liter). Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin, and ceftriaxone in vitro Interestingly, as described previously for tick isolates, amoxicillin shows poor in vitro activity against B. miyamotoi clinical isolates.

Acute and chronic rhinosinusitis and allergic rhinitis in relation to comorbidity, ethnicity and environment.

BACKGROUND: This study was conducted to assess the effect of comorbidity, ethnicity, occupation, smoking and place of residence on allergic rhinitis (AR), acute rhinosinusitis (ARS) and chronic rhinosinusitis (CRS). METHODS: A GA2LEN (The Global Allergy and Asthma European Network) screening questionnaire was sent to a random sample of the Dutch population (n = 16700) in three different areas of the Netherlands. RESULTS: Fifty percent (8347) of the questionnaires sent were returned. A total of 29% respondents (27-31% in different areas) met the criteria for AR, 18% (17-21%) for ARS and 16% (13-18%) for CRS. Risk factors for AR were itchy rash, eczema, adverse response after taking a painkiller, asthma, CRS and ARS. Moreover, the risk of AR was twice as low for full-time housewives/househusbands than for people with jobs. The risk of ARS or CRS was significantly higher in respondents with a doctor's diagnosis of CRS, AR, itchy rash or smoking. The risk of CRS was also significantly higher in respondents with an adverse response after taking painkillers, active smoking or asthma. Caucasians are generally less likely to have AR or CRS than Latin-Americans, Hindustani and African-Creoles, and more likely to have ARS than Asian, Hindustani, Mediterranean and African-Creoles. CONCLUSIONS: This study found shared and distinct risk factors for AR, ARS and CRS and therefore provides support for the belief that they have shared symptoms but are different diseases with different aetiologies.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents.

Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B.miyamotoi A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B.miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferisensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B.miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study.

Rapid DNA vaccination against Burkholderia pseudomallei flagellin by tattoo or intranasal application.

Melioidosis is a severe infectious disease with a high mortality that is endemic in South-East Asia and Northern Australia. The causative pathogen, Burkholderia pseudomallei, is listed as potential bioterror weapon due to its high virulence and potential for easy dissemination. Currently, there is no licensed vaccine for prevention of melioidosis. Here, we explore the use of rapid plasmid DNA vaccination against B. pseudomallei flagellin for protection against respiratory challenge. We tested three flagellin DNA vaccines with different subcellular targeting designs. C57BL/6 mice were vaccinated via skin tattoo on day 0, 3 and 6 before intranasal challenge with B. pseudomallei on day 21. Next, the most effective construct was used as single vaccination on day 0 by tattoo or intranasal formulation. Mice were sacrificed 72 hours post-challenge to assess bacterial loads, cytokine responses, inflammation and microscopic lesions. A construct encoding a cellular secretion signal resulted in the most effective protection against melioidosis via tattooing, with a 10-fold reduction in bacterial loads in lungs and distant organs compared to the empty vector. Strikingly, a single intranasal administration of the same vaccine resulted in >1000-fold lower bacterial loads and increased survival. Pro-inflammatory cytokine responses were significantly diminished and strong reductions in markers for distant organ damage were observed. A rapid vaccination scheme using flagellin DNA tattoo provides significant protection against intranasal challenge with B. pseudomallei, markedly improved by a single administration via airway mucosa. Hence intranasal vaccination with flagellin-encoding DNA may be applicable when acute mass vaccination is indicated and warrants further testing.

Immune evasion of Borrelia miyamotoi: CbiA, a novel outer surface protein exhibiting complement binding and inactivating properties.

Borrelia (B.) miyamotoi, an emerging tick-borne relapsing fever spirochete, resists complement-mediated killing. To decipher the molecular principles of immune evasion, we sought to identify determinants contributing to complement resistance. Employing bioinformatics, we identified a gene encoding for a putative Factor H-binding protein, termed CbiA (complement binding and inhibitory protein A). Functional analyses revealed that CbiA interacted with complement regulator Factor H (FH), C3, C3b, C4b, C5, and C9. Upon binding to CbiA, FH retained its cofactor activity for Factor I-mediated inactivation of C3b. The Factor H-binding site within CbiA was mapped to domain 20 whereby the C-terminus of CbiA was involved in FH binding. Additionally, CbiA directly inhibited the activation of the classical pathway and the assembly of the terminal complement complex. Of importance, CbiA displayed inhibitory activity when ectopically produced in serum-sensitive B. garinii G1, rendering this surrogate strain resistant to human serum. In addition, long-term in vitro cultivation lead to an incremental loss of the cbiA gene accompanied by an increase in serum susceptibility. In conclusion, our data revealed a dual strategy of B. miyamotoi to efficiently evade complement via CbiA, which possesses complement binding and inhibitory activities.

Borrelia miyamotoi in vectors and hosts in The Netherlands.

Ixodes ticks transmit Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis (LB). These tick species also transmit Borrelia miyamotoi, which was recently found to cause infections in humans. We were interested in the prevalence of B. miyamotoi infection in ticks and natural hosts in The Netherlands, and to what extent ticks are co-infected with B. burgdorferi. In addition, erythema migrans has been sporadically described in B. miyamotoi-infected patients, but these skin lesions might as well represent co-infections with B. burgdorferi s.l. We therefore investigated whether B. miyamotoi was present in LB-suspected skin lesions of patients referred to our tertiary Lyme disease clinic. 3360 questing Ixodes ricinus nymphs as well as spleen tissue of 74 rodents, 26 birds and 10 deer were tested by PCR for the presence of B. miyamotoi. Tick lysates were also tested for the presence of B. burgdorferi s.l. Next, we performed a PCR for B. miyamotoi in 31 biopsies from LB-suspected skin lesions in patients visiting our tertiary Lyme center. These biopsies had been initially tested for B. burgdorferi s.l. by PCR, and the skin lesions had been investigated by specialized dermatologists. Out of 3360 unfed (or questing) nymphs, 313 (9.3%) were infected with B. burgdorferi s.l., 70 (2.1%) were infected with B. miyamotoi, and 14 (0.4%) were co-infected with B. burgdorferi s.l. and B. miyamotoi. Co-infection of B. burgdorferi s.l. with B. miyamotoi occurred more often than expected from single infection prevalences (p=0.03). Both rodents (9%) and birds (8%) were found positive for B. miyamotoi by PCR, whereas the roe deer samples were negative. Out of 31 LB-suspected skin biopsies, 10 (32%) were positive for B. burgdorferi s.l. while none were positive for B. miyamotoi. The significant association of B. burgdorferi s.l. with B. miyamotoi in nymphs implies the existence of mutual reservoir hosts. Indeed, the presence of B. miyamotoi DNA indicates systemic infections in birds as well as rodents. However, their relative contributions to the enzootic cycle of B. miyamotoi requires further investigation. We could not retrospectively diagnose B. miyamotoi infection using biopsies of LB-suspected skin lesions, supporting the hypothesis that B. miyamotoi is not associated with LB-associated skin manifestations. However, this warrants further studies in larger sets of skin biopsies. A prospective study focused on acute febrile illness after a tick bite could provide insight into the incidence and clinical manifestations of B. miyamotoi infection in The Netherlands.

Borrelia burgdorferi Induces TLR2-Mediated Migration of Activated Dendritic Cells in an Ex Vivo Human Skin Model.

Borrelia burgdorferi is transmitted into the skin of the host where it encounters and interacts with two dendritic cell (DC) subsets; Langerhans cells (LCs) and dermal DCs (DDCs). These cells recognize pathogens via pattern recognition receptors, mature and migrate out of the skin into draining lymph nodes, where they orchestrate adaptive immune responses. In order to investigate the response of skin DCs during the early immunopathogenesis of Lyme borreliosis, we injected B. burgdorferi intradermally into full-thickness human skin and studied the migration of DCs out of the skin, the activation profile and phenotype of migrated cells. We found a significant increase in the migration of LCs and DDCs in response to B. burgdorferi. Notably, migration was prevented by blocking TLR2. DCs migrated from skin inoculated with higher numbers of spirochetes expressed significantly higher levels of CD83 and produced pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR, CD86, CD38, or CCR7. To conclude, we have established an ex vivo human skin model to study DC-B. burgdorferi interactions. Using this model, we have demonstrated that B. burgdorferi-induced DC migration is mediated by TLR2. Our findings underscore the utility of this model as a valuable tool to study immunity to spirochetal infections.