The regional decline and rise of tick-borne encephalitis incidence do not correlate with Lyme borreliosis, Austria, 2005 to 2018.

BackgroundTick-borne encephalitis (TBE) virus is a human pathogen that is expanding its endemic zones in Europe, emerging in previously unaffected regions. In Austria, increasing incidence in alpine regions in the west has been countered by a decline in traditional endemic areas to the east of the country.AimTo shed light on the cause of this disparity, we compared the temporal changes of human TBE incidences in all federal provinces of Austria with those of Lyme borreliosis (LB), which has the same tick vector and rodent reservoir.MethodsThis comparative analysis was based on the surveillance of hospitalised TBE cases by the National Reference Center for TBE and on the analysis of hospitalised LB cases from hospital discharge records across all of Austria from 2005 to 2018.ResultsThe incidences of the two diseases and their annual fluctuations were not geographically concordant. Neither the decline in TBE in the eastern lowlands nor the increase in western alpine regions is paralleled by similar changes in the incidence of LB.ConclusionThe discrepancy between changes in incidence of TBE and LB support the contributions of virus-specific factors beyond the mere availability of tick vectors and/or human outdoor activity, which are a prerequisite for the transmission of both diseases. A better understanding of parameters controlling human pathogenicity and the maintenance of TBE virus in its natural vector-host cycle will generate further insights into the focal nature of TBE and can potentially improve forecasts of TBE risk on smaller regional scales.

Lyme Borreliosis with Scalp Eschar Mimicking Rickettsial Infection, Austria.

We report on a patient in Austria with scalp eschar and neck lymphadenopathy. Rickettsial etiology was excluded by culture, PCR, and serologic tests. Borrelia afzelii was identified from the eschar swab by PCR. Lyme borreliosis can mimic rickettsiosis; appropriate tests should be included in the diagnostic workup of patients with eschars.

Human Borrelia miyamotoi Infection, Austria.

We report a human case of Borrelia miyamotoi infection diagnosed in Austria. Spirochetes were detected in Giemsa-stained blood smears. The presence of B. miyamotoi in the patient's blood was confirmed by PCR, and phylogenetic analysis identified an infection with a strain from Europe.

Francisella tularensis as the cause of protracted fever.

BACKGROUND: Tularemia, a re-emerging, potential life threatening infectious disease, can present itself with nonspecific clinical symptoms including fever, chills and malaise. Taking a detailed history of exposure and a highly raised index of clinical suspicion are necessary to take the appropriate diagnostic and therapeutic steps in this setting. Here, a case report of typhoid tularaemia is presented. CASE PRESENTATION: A 53-year old male forester and farmer with protracted fever, abdominal pain, diarrhoea and loss of weight, who experienced productive cough and a pulmonary infiltrate later in the course of disease, was admitted for further investigation. Tularaemia was suspected only owing to history and confirmed by serologic testing more than three weeks after the beginning of the symptoms. The initial antibiotic therapy with ceftriaxone/doxycycline was switched to ciprofloxacin, resulting in the resolution of fever and symptoms. CONCLUSION: Tularaemia has to be considered as a differential diagnosis in febrile patients, even more in cases with protracted fever. Since tularaemia is expanding geographically, involving more animal hosts and causing larger outbreaks, clinicians have to be aware of this potentially fatal disease.

Molecular diagnosis of autochthonous human anaplasmosis in Austria - an infectious diseases case report.

BACKGROUND: The diagnosis of human anaplasmosis remains elusive and is probably often missed. This case report highlights the efficacy of molecular diagnostic techniques. CASE PRESENTATION: We would like to report the case of a 74-year-old man who was admitted to hospital because of a high fever, marked chills, transient diplopic images and vertigo, 6 weeks after multiple tick bites. The laboratory results showed mild anemia, marked thrombocytopenia and leukopenia and a moderately elevated C-reactive protein. The initial serology seemed to indicate an active infection with Borrelia spp., and Anaplasma phagocytophilum was detected in peripheral blood by polymerase chain reaction (PCR) and subsequent sequencing. The patient received intravenous ceftriaxone for 14 days and oral doxycycline for 4 weeks and made a fast and complete recovery. CONCLUSIONS: While human anaplasmosis has been reported very rarely in Austria, it should be considered as a differential diagnosis in febrile patients with low leukocyte and platelet counts with elevated levels of C-reactive protein after exposure to tick bites. Molecular detection of A. phagocytophilum is the technique of choice allowing rapid and reliable diagnosis.

Lactoferrin is a natural inhibitor of plasminogen activation.

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

Severe Myasthenic Manifestation of Leptospirosis Associated with New Sequence Type of Leptospira interrogans.

We report the rapid development of a myasthenic crisis as the first-time manifestation of myasthenia gravis. The symptoms developed in the course of acute leptospirosis associated with a new sequence type of Leptospira interrogans. Antibiotic treatment led to rapid amelioration of myasthenia.

Adult Hyalomma marginatum tick positive for Rickettsia aeschlimannii in Austria, October 2018.

We report on a non-indigenous adult Hyalomma marginatum tick in Austria carrying the human pathogenic Rickettsia aeschlimannii; presumably introduced as a nymph via migratory birds and completed the moulting within the same year. It was negative for Crimean-Congo haemorrhagic fever virus, but the finding of R. aeschlimannii represents a potential threat for humans due to its zoonotic character. Awareness of invasive tick species and carried pathogens should be improved in central and northern Europe.

Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/Ehrlichia (including "Candidatus Neoehrlichia"), Babesia, and Coxiella The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferisensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferisensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis, Borrelia lusitaniae, and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. "Candidatus Neoehrlichia mikurensis," Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and "Candidatus Neoehrlichia mikurensis" showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferisensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.

Oligoarthritis caused by Borrelia bavariensis, Austria, 2014.

A case of Lyme oligoarthritis occurred in an 11-year-old boy in Vienna, Austria. DNA of Borrelia bavariensis was detected by PCR in 2 aspirates obtained from different joints. Complete recovery was achieved after a 4-week course with amoxicillin. Lyme arthritis must be considered in patients from Europe who have persisting joint effusions.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae.

BACKGROUND: Intracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS. RESULTS: Monocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1ß, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1ß, and CXCL-8, which correlated with TNF-α secretion. CONCLUSION: Infection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.

Intrathecally produced IgG and IgM antibodies to recombinant VlsE, VlsE peptide, recombinant OspC and whole cell extracts in the diagnosis of Lyme neuroborreliosis.

Detection of intrathecally produced specific antibodies (AI) is essential in the diagnosis of Lyme neuroborreliosis (LNB); however, the performance of various newer AI detection methods has not been systematically assessed. Here we assessed and compared advanced test systems for detecting borrelia IgG-AI and IgM-AI. Serum and cerebrospinal fluid (CSF) samples from well-defined LNB and tick-borne encephalitis (TBE) patients, 25 each, were tested with three antibody detection systems, one based on chemiluminescence (CLA) and two based on enzyme-linked immunosorbent assays (ELISA), employing different antigens for detection of IgG and IgM antibodies. In samples from patients with LNB, IgG-AI was detected in 20 samples by CLA, 19 by ELISA1, and 22 by ELISA2, and IgM-AI was detected in 16 samples by CLA, six by ELISA1, and 11 by ELISA2. In samples from TBE patients, IgG-AI was positive in one case by CLA and ELISA2, and in 7 cases by ELISA1, whereas IgM-AI was positive in one case by CLA and in none by ELISA. IgG-AI and IgM-AI were not detected within the first week of disease. Duration of disease correlated with IgG-AI while IgM-AI results were heterogeneous for each test assay. Moreover, the levels of IgG-AI, but not IgM-AI, correlated with protein concentration in CSF. IgG is the relevant immunoglobulin isotype for detecting intrathecal synthesis of borrelia antibodies. The highest sensitivity and specificity were achieved by the antibody detection assay using VlsE IR6 peptide. Detection of IgM-AI yielded heterogenous results and did not support the laboratory diagnosis of LNB.

Utility of serodiagnostics designed for use in the United States for detection of Lyme borreliosis acquired in Europe and vice versa.

Although two-tier testing is standard practice in both the United States and Europe for the serologic diagnosis of Lyme borreliosis (LB), the test kits generally differ. The purpose of this study was to determine if the testing used in the United States will detect LB acquired in Europe and vice versa. Testing was performed on a convenience sample of archived sera from 40 LB patients from Austria and 39 from the United States, using first- and second-tier test kits from both the United States and Europe. The sensitivity of four first-tier tests from Europe and two first-tier tests from the United States was similar. Thus, two-tier testing was compared to the C6 ELISA as the first-tier test, since it is licensed in both the United States and Europe. The sensitivity of C6 two-tier testing with US assays was 9/40 (22.5 % [95 % CI 10.8-38.5 %]) for detection of LB acquired in Europe, and just 20.0 % (95 % CI 2.5-55.6 %) in the ten European patients with neurologic involvement. These results differed significantly from the sensitivity of European C6 two-tier testing that was 70.0 % (95 % CI 53.5-83.4 %) overall (p < 0.001) and 90.0 % (95 % CI 55.5-99.7 %) for the European patients with neurologic manifestations specifically (p = 0.016). In contrast, the sensitivity of European and US C6 two-tier testing was similar for detection of LB acquired in the United States. Two-tier serologic testing with the US test kits may be unsatisfactory for detection of LB acquired in Europe. First-tier testing with an assay such as the C6 ELISA should be considered as a stand-alone diagnostic strategy in such cases.

Lyme borreliosis.

Lyme borreliosis (Lyme disease) is caused by spirochaetes of the Borrelia burgdorferi sensu lato species complex, which are transmitted by ticks. The most common clinical manifestation is erythema migrans, which eventually resolves, even without antibiotic treatment. However, the infecting pathogen can spread to other tissues and organs, causing more severe manifestations that can involve a patient's skin, nervous system, joints, or heart. The incidence of this disease is increasing in many countries. Laboratory evidence of infection, mainly serology, is essential for diagnosis, except in the case of typical erythema migrans. Diagnosed cases are usually treated with antibiotics for 2-4 weeks and most patients make an uneventful recovery. No convincing evidence exists to support the use of antibiotics for longer than 4 weeks, or for the persistence of spirochaetes in adequately treated patients. Prevention is mainly accomplished by protecting against tick bites. There is no vaccine available for human beings.

Bacteria and protozoa with pathogenic potential in Ixodes ricinus ticks in Viennese recreational areas.

Ixodes ricinus is the most relevant vector for tick-borne diseases in Austria and responsible for the transmission of Borrelia burgdorferi sensu lato (s. l.), which causes Lyme borreliosis in humans; however, also other bacteria and protozoa can be found in ticks and have the potential of infecting people and animals. In this study we collected ticks in popular recreational areas in the city of Vienna in the years 2019 and 2020 and analyzed them for the presence of such putative pathogenic microorganisms. By using reverse line blot (RLB) hybridization we detected DNA of B. burgdorferi s. l., Rickettsia spp., Babesia spp., Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum. Moreover, we also screened them for the relapsing fever spirochete Borrelia miyamotoi employing real-time PCR. The most frequently detected pathogens were B. burgdorferi s. l. in 28.6% of the ticks in 2019 and 21.3% of the ticks in 2020. The genus Rickettsia was detected in 13.8% of the ticks from 2019 and only in 4.6% from 2020. Babesia spp. were detected in 5.7% in 2019 and 4.2% in 2020. Furthermore, we detected CNM in 4.0% (2019) and 5.6% (2020), A. phagocytophilum in 0.5% (2019) and 1.3% (2020) and finally B. miyamotoi in 3.3% (2019) and 1.7% (2020). Collectively, we show that various microorganisms are prevalent in ticks collected in Vienna and identify hotspots for B. miyamotoi, which we have detected for the first time in the city.

Nonspecific symptoms following infection with Borrelia burgdorferi sensu lato: A retrospective cohort study.

Whether infection with Borrelia burgdorferi sensu lato (sl) can cause nonspecific symptoms is a matter of controversy. We investigated whether individuals infected with B. burgdorferi sl develop unspecific symptoms more frequently than non-infected controls. Eighty-nine persons having presented with B. burgdorferi sl infection between 2015 and 2019 were asked to participate. The infection was defined as erythema migrans diagnosed either in the course of a previous study, during medical visits in the outpatient department, or as seroconversion in asymptomatic subjects. The control group consisted of 85 seronegative individuals without erythema migrans in the past. About two and a half years later, participants were asked to fill out a questionnaire with a list of nonspecific symptoms. The data of 37 persons with previous Borrelia infection and 49 uninfected controls were available for analysis. Muscle pain was significantly (P = 0.040) more frequent in the control group. Fatigue occurred more often in the infected group, but this was not statistically significant (P = 0.109). Likewise, the distribution of other symptoms did not differ considerably. The analysis revealed no difference in the frequency of symptoms of persons who had EM or asymptomatic Borrelia infection 2 years prior assessment in comparison to persons without Borrelia infection.

Tick feeding modulates the human skin immune landscape to facilitate tick-borne pathogen transmission.

During cutaneous tick attachment, the feeding cavity becomes a site of transmission for tick salivary compounds and tick-borne pathogens. However, the immunological consequences of tick feeding for human skin remain unclear. Here, we assessed human skin and blood samples upon tick bite and developed a human skin explant model mimicking Ixodes ricinus bites and tick-borne pathogen infection. Following tick attachment, we observed rapidly occurring patterns of immunomodulation, including increases in neutrophils and cutaneous B and T cells. T cells upregulated tissue residency markers, while lymphocytic cytokine production was impaired. In early stages of Borrelia burgdorferi model infections, we detected strain-specific immune responses and close spatial relationships between macrophages and spirochetes. Preincubation of spirochetes with tick salivary gland extracts hampered accumulation of immune cells and increased spirochete loads. Collectively, we showed that tick feeding exerts profound changes on the skin immune network that interfere with the primary response against tick-borne pathogens.

Persistent Anti-Borrelia IgM Antibodies without Lyme Borreliosis in the Clinical and Immunological Context.

The aim of the study was to investigate the etiology of persistent IgM antibodies against Borrelia burgdorferi sensu lato (sl) and to analyze their association with nonspecific symptoms. The study group comprised individuals with persistent IgM antibodies in the absence of IgG. The relation between ELISA values and time elapsed since past erythema migrans (EM) was analyzed. Previous antibiotic treatments were assessed. The association between persistent IgM and nonspecific symptoms was evaluated statistically. Specificity of IgM antibodies for outer surface protein C (OspC) of B. burgdorferi sl was examined by immunoblotting. Further, we investigated the cross-reactivity with Borrelia-unrelated proteins. Fifty-nine patients (46 women; 78%) were included in the study group. The mean IgM-ELISA values did not change significantly during follow-up (median 6.2 months). The mean ELISA value in the study group was dependent on time elapsed since past EM. Nonspecific symptoms improved significantly more often in patients with lower IgM ELISA results. Persistent IgM antibodies were specific for the C-terminal PKKP motif of OspC. Cross-reacting C-terminal PKKP antigens from both human and prokaryotic origins were identified. We demonstrate that the C-terminal PKKP motif plays a main role for the reactivity of persistent Borrelia IgM toward OspC. However, cross-reactivity to other eukaryotic and/or prokaryotic antigens may hamper the specificity of OspC in the serological diagnosis of Lyme borreliosis. Lack of improvement of nonspecific symptoms was associated with higher IgM ELISA values. IMPORTANCE The reactivity of human IgM with the outer surface protein C (OspC) of Borrelia burgdorferi sensu lato is frequently used to detect Borrelia specific IgM in commercial immunoassays, and such antibodies usually occur in the early phase of the infection. We identified a group of individuals with persistent Borrelia IgM without symptoms of Lyme borreliosis. We used their sera to demonstrate that the C-terminal epitope of OspC binds the IgM. Strikingly, we found that the same epitope occurs also in certain proteins of human and environmental origin; the latter include other bacteria and food plants. Our experimental data show that these Borrelia-unrelated proteins cross-react with the OpsC-specific IgM. This knowledge is important for the development of serologic assays for Lyme borreliosis and provides a cross-reactive explanation for the persistence of Borrelia-IgM.

Isolation of Francisella tularensis from Skin Ulcer after a Tick Bite, Austria, 2020.

Ulceroglandular tularemia is caused by the transmission of Francisella tularensis by arthropods to a human host. We report a case of tick-borne tularemia in Austria which was followed by an abscess formation in a lymph node, making drainage necessary. F. tularensis subsp. holarctica was identified by PCR and multilocus sequence typing.