Distribution of Borrelia burgdorferi s.l. and Borrelia miyamotoi in Ixodes tick populations in Northern Germany, co-infections with Rickettsiales and assessment of potential influencing factors.

To determine Borrelia spp. (Spirochaetales: Spirochaetaceae) prevalence and species distribution in Northern Germany, Ixodes ticks were sampled from April to October in 2018 and 2019 by the flagging method at three locations each in five regions. Analysis by quantitative real-time PCR of 3150 individual ticks revealed an overall prevalence of 30.6%, without significant differences between tick stages (31.7% positive adults, 28.6% positive nymphs). Significant differences were observed in seasonal infection rates, but not between regions, landscape types or sampling years. Analysis of co-infections with Rickettsiales indicated a negative association between Borrelia and Anaplasma phagocytophilum infection. The most frequent Borrelia species differentiated by Reverse Line Blot were B. afzelii and B. garinii/B. bavariensis, followed by B. valaisiana, B. burgdorferi sensu stricto, B. spielmanii and B. lusitaniae. Furthermore, B. miyamotoi was identified in 12.9% of differentiable samples. No effect of region nor landscape type on species composition was found, but significant variations in the distribution at the different sampling sites within a region were observed. The detected monthly fluctuations in prevalence and the differences in intra-regional Borrelia species distribution underline the importance of long-term and multi-location monitoring of Borrelia spp. in ticks as an essential part of public health assessment.

Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi.

BACKGROUND: Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology. RESULTS: Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids. CONCLUSION: Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.

PURPOSE: For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. METHODS: Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. RESULTS: The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. CONCLUSION: The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis.

BACKGROUND: Interpretation of serological assays in Lyme borreliosis requires an understanding of the clinical indications and the limitations of the currently available tests. We therefore systematically reviewed the accuracy of serological tests for the diagnosis of Lyme borreliosis in Europe. METHODS: We searched EMBASE en MEDLINE and contacted experts. Studies evaluating the diagnostic accuracy of serological assays for Lyme borreliosis in Europe were eligible. Study selection and data-extraction were done by two authors independently. We assessed study quality using the QUADAS-2 checklist. We used a hierarchical summary ROC meta-regression method for the meta-analyses. Potential sources of heterogeneity were test-type, commercial or in-house, Ig-type, antigen type and study quality. These were added as covariates to the model, to assess their effect on test accuracy. RESULTS: Seventy-eight studies evaluating an Enzyme-Linked ImmunoSorbent assay (ELISA) or an immunoblot assay against a reference standard of clinical criteria were included. None of the studies had low risk of bias for all QUADAS-2 domains. Sensitivity was highly heterogeneous, with summary estimates: erythema migrans 50% (95% CI 40% to 61%); neuroborreliosis 77% (95% CI 67% to 85%); acrodermatitis chronica atrophicans 97% (95% CI 94% to 99%); unspecified Lyme borreliosis 73% (95% CI 53% to 87%). Specificity was around 95% in studies with healthy controls, but around 80% in cross-sectional studies. Two-tiered algorithms or antibody indices did not outperform single test approaches. CONCLUSIONS: The observed heterogeneity and risk of bias complicate the extrapolation of our results to clinical practice. The usefulness of the serological tests for Lyme disease depends on the pre-test probability and subsequent predictive values in the setting where the tests are being used. Future diagnostic accuracy studies should be prospectively planned cross-sectional studies, done in settings where the test will be used in practice.

[Challenge of neuroborreliosis]. / Herausforderung Neuroborreliose.

Lyme borreliosis is the most frequent tick-borne infection in Europe. It is a multisystemic disease affecting the skin, joints, heart, in rare cases the eyes and regularly the nervous system. Taking current clinical and microbiological guidelines into account, neuroborreliosis can in general be diagnosed and treated successfully. An appropriate guideline-conform antibiotic treatment is effective and in most cases recovery from acute neuroborreliosis is complete. Nevertheless, the evidence base regarding pharmacological treatment needs reform and improvement. Contrary to this scientifically based medical opinion, divergent opinions presented in the media cause uncertainty and confusion among patients and also some physicians. The currently available scientific data on epidemiology, treatment and performance of microbiological testing reveals gaps and therefore a large scope for interpretation. In clinical practice, diagnostics and therapeutic methods that do not fulfill the criteria of evidence-based medicine are widely used due to the uncertain data situation. The Clinical Network Neuroborreliosis (KNN) is a publicly funded network of clinicians and clinical laboratory physicians with the goal to improve knowledge for disease-oriented evaluation of diagnostic and therapeutic methods. In addition, the Robert Koch Institute (RKI) and the KNN are performing an epidemiological study for collation and estimation of the number of neuroborreliosis cases in Germany (aNBorD study).

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

Borrelia burgdorferi sensu lato and co-infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg, Germany.

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe-based quantitative real-time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co-infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co-infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia-positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.

[CXCL13: a biomarker for acute Lyme neuroborreliosis: investigation of the predictive value in the clinical routine]. / CXCL13 als Biomarker der akuten Neuroborreliose : Überprüfung des prädiktiven Wertes in der klinischen Routine.

BACKGROUND: The level of CXCL13 is a cerebrospinal fluid (CSF) biomarker for acute Lyme neuroborreliosis (LNB) with a high sensitivity. As the concentration rapidly declines during antibiotic therapy CXCL13 can also be used as a follow-up parameter. However, CXCL13 is not yet in use as a routine parameter due to concerns about the specificity. OBJECTIVES: The sensitivity, specificity and predictive value of CXCL13 in the clinical routine work-up of suspected LNB was analyzed. MATERIAL AND METHODS: Since July 2010 the CSF of all patients (n = 204) with suspected acute LNB was not only analyzed for the routine parameters (i.e. pleocytosis and intrathecal production of Borrelia-specific antibodies, AI) but also for CXCL13. In cases of incongruent findings, a follow-up puncture after antibiotic therapy was carried out. The cut-off level for acute LNB was set at 250 pg/ml. RESULTS: This study included 179 patients who were not pretreated with antibiotics. Of these patients 15 suffered from definite LNB, 3 had a probable LNB and all had a CXCL13 value above the cut-off level. Only 2 of the 161 patients with a non-LNB diagnosis (both with a lymphoma) had a CXCL13 value in the CSF higher than 250 pg/ml. Especially noteworthy were two patients without pleocytosis in the CSF but with CXCL13 levels above the cut-off level in whom LNB could be confirmed in the follow-up CSF analysis. CONCLUSIONS: The biomarker CXCL13 has a higher sensitivity (100 % vs. 87 %) with a specificity (99 %) comparable with the established diagnostic markers for LNB, e.g. CSF pleocytosis and Borrelia-AI in the investigated patient population. The negative predictive value of CXCL13 is 100 %. Therefore, a normal CXCL13 level virtually excludes LNB. In the clinical routine CXCL13 is a valuable and practical diagnostic marker for LNB and can even detect an acute LNB in patients without CSF pleocytosis.

[Threat of transmission of infectious pathogens by Ixodes ricinus ticks in Germany]. / Gefahren der Übertragung von Krankheitserregern durch Schildzecken in Deutschland.

Tick-transmitted diseases are of great importance for the general health of the German population. Several viruses, such as tick-borne encephalitis virus (TBEV), Uukuniemi virus, Tribec virus, Eyach virus or bacteria, such as Borrelia, Rickettsiae, Francisella tularensis, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis (CNM) and Coxiella burnetii were detected in the most prominent tick in Germany, the hard tick Ixodes ricinus. While infections, such as TBE and Lyme disease are well known, other infections are hardly known even among experts. Although there have been a few descriptions of isolated cases in Germany, a systematic investigation regarding the distribution and the pathogenic potential of these pathogens is still lacking. In particular elderly people and people with underlying diseases seem to be mostly affected. The importance of new infectious disease agents, such as Candidatus Neoehrlichia mikurensis but also of long known pathogens, such as Rickettsiae still remains unclear, while some of them could be detected in 20 % of investigated ticks. Whether climate change contributes to the further distribution of these infectious agents remains unclear and requires further investigation. The increasing initiatives to create natural environments and the trend towards spending more time in nature for recreational activities will increase the danger of coming into contact with ticks and the respective infectious agents. Considering these circumstances an increase of diseases caused by these pathogens is to be expected.

[Lyme borreliosis]. / Lyme-Borreliose.

Lyme borreliosis is a multisystem infectious disease affecting mainly the skin, nervous system, joints and heart. It is caused by spirochetes of the Borrelia burgdorferi sensu lato complex which are transmitted by ticks. The diagnosis of Lyme borreliosis is based primarily on typical clinical symptoms and signs with serological confirmation. Antibiotic therapy is beneficial for all manifestations and treatment refractory cases are rare. The diagnosis "chronic Lyme borreliosis" is increasingly being misused for all conceivable medically unexplained symptoms.

Spectrum of MRI findings in central nervous system affection in Lyme neuroborreliosis.

Affections of the central nervous system (CNS) rarely occur in Lyme neuroborreliosis (LNB). CNS manifestations can have residual neurological symptoms despite antibiotic treatment. We explored the spectrum of CNS affections in patients with LNB in a tertiary care center in a region endemic for Lyme borreliosis. We retrospectively included patients treated at a tertiary care center from January 2020-December 2021 fulfilling the case criteria for LNB as stated in the current German guideline on LNB. Clinical data, cerebrospinal fluid (CSF) findings and MRI imaging were collected. We included 35 patients with LNB, 24 with early manifestations and 11 with CNS-LNB. CNS-LNB patients had encephalomyelitis (n = 6) or cerebral vasculitis (n = 5). Patients with early LNB and CNS-LNB differed regarding albumin CSF/serum quotient and total protein in CSF. Duration from onset of symptoms until diagnosis was statistically significantly longer in patients with encephalomyelitis. MRI findings were heterogeneous and showed longitudinal extensive myelitis, perimedullar leptomeningeal enhancement, pontomesencephalic lesions or cerebral vasculitis. CNS-LNB can present with a variety of clinical syndromes and MRI changes. No clear pattern of MRI findings in CNS-LNB could be identified. The role of MRI consists in ruling out other causes of neurological symptoms.

Rational diagnostic strategies for Lyme borreliosis in children and adolescents: recommendations by the Committee for Infectious Diseases and Vaccinations of the German Academy for Pediatrics and Adolescent Health.

The varying clinical manifestations of Lyme borreliosis, transmitted by Ixodes ricinus and caused by Borrelia burgdorferi, frequently pose diagnostic problems. Diagnostic strategies vary between early and late disease manifestations and usually include serological methods. Erythema migrans is pathognomonic and does not require any further laboratory investigations. In contrast, the diagnosis of neuroborreliosis requires the assessment of serum and cerebrospinal fluid. Lyme arthritis is diagnosed in the presence of newly recognized arthritis and high-titer serum IgG antibodies against B. burgdorferi. The committee concludes the following recommendations: Borrelial serology should only be ordered in case of well-founded clinical suspicion for Lyme borreliosis, i.e., manifestations compatible with the diagnosis. Tests for borrelial genomic sequences in ticks or lymphocyte proliferation assays should not be ordered. When results of such tests or of serological investigations that were not indicated are available, they should not influence therapeutic decisions. Laboratories should be cautious when interpreting results of serological tests and abstain from giving therapeutic recommendations and from proposing retesting after some time without intimate knowledge of patient's history and disease manifestations.

EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis.

BACKGROUND: Lyme neuroborreliosis (LNB) is a nervous system infection caused by Borrelia burgdorferi sensu lato (Bb). OBJECTIVES: To present evidence-based recommendations for diagnosis and treatment. METHODS: Data were analysed according to levels of evidence as suggested by EFNS. RECOMMENDATIONS: The following three criteria should be fulfilled for definite LNB, and two of them for possible LNB: (i) neurological symptoms; (ii) cerebrospinal fluid (CSF) pleocytosis; (iii) Bb-specific antibodies produced intrathecally. PCR and CSF culture may be corroborative if symptom duration is <6 weeks, when Bb antibodies may be absent. PCR is otherwise not recommended. There is also not enough evidence to recommend the following tests for diagnostic purposes: microscope-based assays, chemokine CXCL13, antigen detection, immune complexes, lymphocyte transformation test, cyst formation, lymphocyte markers. Adult patients with definite or possible acute LNB (symptom duration <6 months) should be offered a single 14-day course of antibiotic treatment. Oral doxycycline (200 mg daily) and intravenous (IV) ceftriaxone (2 g daily) are equally effective in patients with symptoms confined to the peripheral nervous system, including meningitis (level A). Patients with CNS manifestations should be treated with IV ceftriaxone (2 g daily) for 14 days and late LNB (symptom duration >6 months) for 3 weeks (good practice points). Children should be treated as adults, except that doxycycline is contraindicated under 8 years of age (nine in some countries). If symptoms persist for more than 6 months after standard treatment, the condition is often termed post-Lyme disease syndrome (PLDS). Antibiotic therapy has no impact on PLDS (level A).

[Neuroborreliosis]. / Neuroborreliose.

Neuroborreliosis is easily diagnosed by means of clinical symptoms and laboratory findings. Guiding symptoms are radicular pain and pareses of the extremities and the facial nerve. There is a great number of further less frequently occurring neurological symptoms, which can be attributed to a borrelial infection only by appropriate investigations of the CSF. Radiculitis is cured adequately by oral doxycycline while symptoms of the central nervous system are probably better treated intravenously by ceftriaxone, cefotaxime or penicillin G. Post-Lyme syndrome is a diffuse description of non-specific complaints, which are not the explicit result of a former infection with B. burgdorferi. As further antibiotics do not help and the CSF is unremarkable in most patients, a persistent infection with B. burgdorferi s.l. in all probability can be excluded.

[Lyme borreliosis: research gaps and research approaches. Results from an interdisciplinary expert meeting at the Robert Koch Institute]. / Lyme-Borreliose: Forschungsbedarf und Forschungsansätze. Ergebnisse eines interdisziplinären Expertentreffens am Robert Koch-Institut.

Lyme borreliosis is currently the most frequent tick-transmitted zoonosis in the northern hemisphere. Germany and other European countries are regarded as highly endemic areas; therefore the burden of disease and consequently the costs for the health systems are considered to be high. This report summarises the results of an interdisciplinary workshop on Lyme borreliosis which aimed to identify research deficits and to prioritise areas which need to be addressed. Research needs have been recognised for different areas: diagnosis, epidemiology, immunology, clinics, ecology and health services research. Examples of research areas which have priority are the standardisation of diagnostic tests, the development of markers to detect an active infection, the improvement of the epidemiological database and the analysis of the burden of disease.

Diagnostic value of cerebrospinal fluid CXCL13 for acute Lyme neuroborreliosis. A systematic review and meta-analysis.

OBJECTIVES: The utility of cerebrospinal fluid (CSF) CXCL13 for diagnosis of acute Lyme neuroborreliosis (LNB) has been debated and the test is not yet routinely performed. This study's aim was to evaluate its overall diagnostic accuracy through meta-analysis. METHODS: Electronic searches in PubMed MEDLINE and Web of Science were performed to identify relevant articles published before January 2018. A summary receiver operating characteristic curve and an optimal cut-off were estimated modelling multiple cut-offs. Publication bias was evaluated using a funnel plot and the associated regression test. RESULTS: A total of 18 studies involving 618 individuals with acute LNB and 2326 individuals with other neurological disorders meeting the eligibility criteria were identified. The pooled sensitivity for CSF CXCL13 was 89% (95% CI 85%-93%) and the pooled specificity was 96% (95% CI 92%-98%), using the identified optimal cut-off value of 162 pg/mL. There was marked heterogeneity between studies, caused by differences in the designs of the study populations and age distribution. The optimal cut-off in the seven studies with a cross-sectional design was 91 pg/mL (sensitivity 96%, 95% CI 92%-98%; specificity 94%, 95% CI 86%-97%) and in the 11 case-control studies it was 164 pg/mL (sensitivity 85%, 95% CI 78%-91%; specificity 95%, 95% CI 90%-98%). CSF CXCL13 values above the optimal cut-off level (determined in this meta-analysis) were also detectable in some other central nervous system disorders, namely neurosyphilis and central nervous system lymphoma. CONCLUSIONS: Our meta-analysis shows that CSF CXCL13 has the potential to become a useful adjunct in the diagnosis of acute LNB.