A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s.

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B. burgdorferi surface lipoproteins of the OspEF-related protein family-termed ElpB and ElpQ-that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. In vitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using binding assays and complement component C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks complement component C4 cleavage by competing with C1s-C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp-C1s binding, we performed site-directed mutagenesis, which showed that ElpB and ElpQ binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

The FGF/FGFR system in the microglial neuroinflammation with Borrelia burgdorferi: likely intersectionality with other neurological conditions.

BACKGROUND: Lyme neuroborreliosis, caused by the bacterium Borrelia burgdorferi affects both the central and peripheral nervous systems (CNS, PNS). The CNS manifestations, especially at later stages, can mimic/cause many other neurological conditions including psychiatric disorders, dementia, and others, with a likely neuroinflammatory basis. The pathogenic mechanisms associated with Lyme neuroborreliosis, however, are not fully understood. METHODS: In this study, using cultures of primary rhesus microglia, we explored the roles of several fibroblast growth factor receptors (FGFRs) and fibroblast growth factors (FGFs) in neuroinflammation associated with live B. burgdorferi exposure. FGFR specific siRNA and inhibitors, custom antibody arrays, ELISAs, immunofluorescence and microscopy were used to comprehensively analyze the roles of these molecules in microglial neuroinflammation due to B. burgdorferi. RESULTS: FGFR1-3 expressions were upregulated in microglia in response to B. burgdorferi. Inhibition of FGFR 1, 2 and 3 signaling using siRNA and three different inhibitors showed that FGFR signaling is proinflammatory in response to the Lyme disease bacterium. FGFR1 activation also contributed to non-viable B. burgdorferi mediated neuroinflammation. Analysis of the B. burgdorferi conditioned microglial medium by a custom antibody array showed that several FGFs are induced by the live bacterium including FGF6, FGF10 and FGF12, which in turn induce IL-6 and/or CXCL8, indicating a proinflammatory nature. To our knowledge, this is also the first-ever described role for FGF6 and FGF12 in CNS neuroinflammation. FGF23 upregulation, in addition, was observed in response to the Lyme disease bacterium. B. burgdorferi exposure also downregulated many FGFs including FGF 5, 7, 9, 11, 13, 16, 20 and 21. Some of the upregulated FGFs have been implicated in major depressive disorder (MDD) or dementia development, while the downregulated ones have been demonstrated to have protective roles in epilepsy, Parkinson's disease, Alzheimer's disease, spinal cord injury, blood-brain barrier stability, and others. CONCLUSIONS: In this study we show that FGFRs and FGFs are novel inducers of inflammatory mediators in Lyme neuroborreliosis. It is likely that an unresolved, long-term (neuro)-Lyme infection can contribute to the development of other neurologic conditions in susceptible individuals either by augmenting pathogenic FGFs or by suppressing ameliorative FGFs or both.

Role of glutathione metabolism in host defense against Borrelia burgdorferi infection.

Pathogen-induced changes in host cell metabolism are known to be important for the immune response. In this study, we investigated how infection with the Lyme disease-causing bacterium Borrelia burgdorferi (Bb) affects host metabolic pathways and how these metabolic pathways may impact host defense. First, metabolome analysis was performed on human primary monocytes from healthy volunteers, stimulated for 24 h with Bb at low multiplicity of infection (MOI). Pathway analysis indicated that glutathione (GSH) metabolism was the pathway most significantly affected by Bb Specifically, intracellular levels of GSH increased on average 10-fold in response to Bb exposure. Furthermore, these changes were found to be specific, as they were not seen during stimulation with other pathogens. Next, metabolome analysis was performed on serum samples from patients with early-onset Lyme disease in comparison with patients with other infections. Supporting the in vitro analysis, we identified a cluster of GSH-related metabolites, the γ-glutamyl amino acids, specifically altered in patients with Lyme disease, and not in other infections. Lastly, we performed in vitro experiments to validate the role for GSH metabolism in host response against Bb. We found that the GSH pathway is essential for Bb-induced cytokine production and identified glutathionylation as a potential mediating mechanism. Taken together, these data indicate a central role for the GSH pathway in the host response to Bb GSH metabolism and glutathionylation may therefore be important factors in the pathogenesis of Lyme disease and potentially other inflammatory diseases as well.

Subacute transverse myelitis with optic symptoms in neuroborreliosis: a case report.

BACKGROUND: Subacute transverse myelitis is one of the late manifestations of neuroborreliosis with only a few cases described to the present day. CASE PRESENTATION: We present magnetic resonance imaging, cerebrospinal fluid, and electroneurography findings of a young female patient suffering from neuroborreliosis-associated transverse myelitis with a wide constellation of symptoms including papilloedema. Magnetic resonance imaging of the cervical spine has shown an enlargement of the spinal cord in the mid-cervical region. Cerebrospinal fluid findings included lymphocytic pleocytosis, increased levels of anti - Borrelia antibodies, and increased intrathecal anti -Borrelia antibody index. Following the 28-day course of intravenous ceftriaxone, the patient attained complete recovery. CONCLUSIONS: Subacute transverse myelitis in the course of neuroborreliosis should be considered in the differential diagnosis of patients with abnormal magnetic resonance scans of the spinal cord, lymphocytic pleocytosis, and intrathecal antibody production, especially in the tick-endemic areas, even if the tick bite was not reported. Infrequent accompanying symptoms such as papilloedema are diagnostically challenging and cannot be treated as clinching evidence.

The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization.

The Lyme disease-causing organism Borrelia burgdorferi is transmitted into the mammalian host by an infected-tick bite. Successful infection relies on the ability of this extracellular pathogen to persist and colonize different tissues. B. burgdorferi encodes a large number of adhesins that are able to interact with host ligands to facilitate adherence and tissue colonization. Multiple glycosaminoglycan binding proteins present in B. burgdorferi offer a degree of redundancy of function during infection, and this highlights the importance of glycosaminoglycans as host cell receptors for spirochete adherence. Of particular interest in this study is Borrelia glycosaminoglycan binding protein (Bgp), which binds to heparin-related glycosaminoglycans. The properties of a bgp transposon mutant and a trans-complemented derivative were compared to those of the wild-type B. burgdorferi in the in vitro binding assays and in infection studies using a C3H/HeJ mouse infection model. We determined that the loss of Bgp impairs spirochete adherence, infectivity, and tissue colonization, resulting in a reduction of inflammatory manifestations of Lyme disease. Although Bgp is not essential for infectivity, it is an important virulence factor of B. burgdorferi that allows adherence and tissue colonization and contributes to disease severity.

Laboratory diagnosis of Lyme borreliosis: Current state of the art and future perspectives.

This review is directed at physicians and laboratory personnel in private practice and clinics who treat and diagnose Lyme borreliosis (LB) in patients as part of their daily work. A major objective of this paper is to bring together background information on Borrelia (B.) burgdorferi sensu lato (s.l.) and basic clinical knowledge of LB, which is one of the most frequently reported vector-borne diseases in the Northern Hemisphere. The goal is to provide practical guidance for clinicians and for laboratory physicians, and scientists for a better understanding of current achievements and ongoing obstacles in the laboratory diagnosis of LB, an infectious disease that still remains one of the diagnostic chameleons of modern clinical medicine. Moreover, in bringing together current scientific information from guidelines, reviews, and original papers, this review provides recommendations for selecting the appropriate tests in relation to the patient's stage of disease to achieve effective, stage-related application of current direct and indirect laboratory methods for the detection of B. burgdorferi s.l. Additionally, the review aims to discuss the current state of the art concerning the diagnostic potential and limitations of the assays and test methods currently in use to optimize LB patient management and provide insight into the possible future prospects of this rapidly changing area of laboratory medicine.

Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

BACKGROUND: Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. RESULTS: We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). CONCLUSIONS: Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of only about 0.65% that of single nucleotide changes, making rearrangement-derived novel junctions (mosaic boundaries) ideal phylogenetic markers in the study of B. burgdorferi population structure and plasmid evolution and exchange.

Receptor tyrosine kinases play a significant role in human oligodendrocyte inflammation and cell death associated with the Lyme disease bacterium Borrelia burgdorferi.

BACKGROUND: In previous studies, human oligodendrocytes were demonstrated to undergo apoptosis in the presence of Borrelia burgdorferi under an inflammatory milieu. Subsequently, we determined that the MEK/ERK pathway played a significant role in triggering downstream inflammation as well as apoptosis. However, the identity of receptors triggered by exposure to B. burgdorferi and initiating signaling events was unknown. METHODS: In this study, we explored the role of several TLR and EGFR/FGFR/PDGFR tyrosine kinase pathways in inducing inflammation in the presence of B. burgdorferi, using siRNA and/or inhibitors, in MO3.13 human oligodendrocytes. Cell death and apoptosis assays were also carried out in the presence or absence of specific receptor inhibitors along with the bacteria to determine the role of these receptors in apoptosis induction. The expression pattern of specific receptors with or without B. burgdorferi was also determined. RESULTS: TLRs 2 and 5 had a minimal role in inducing inflammation, particularly IL-6 production. Rather, their effect was mostly inhibitory, with TLR2 downregulation significantly upregulating CXCL8, and CXCL (1,2,3) levels, and TLR5 likely having a similar role in CXCL8, CXCL(1,2,3), and CCL5 levels. TLR4 contributed mostly towards CCL5 production. On the other hand, inhibition of all three EGF/FGF/PDGF receptors significantly downregulated all five of the inflammatory mediators tested even in the presence of B. burgdorferi. Their inhibition also downregulated overall cell death and apoptosis levels. The expression pattern of these receptors, as assessed by immunohistochemistry indicated that the PDGFRß receptor was the most predominantly expressed receptor, followed by FGFR, although no significant differences were discernible between presence and absence of bacteria. Interestingly, inhibition of individual EGFR, FGFR, or PDGFR receptors did not indicate an individual role for any of these receptors in the overall downregulation of pathogenesis. Contrarily, suppression of FGFR signaling alone in the presence of bacteria significantly upregulated inflammatory mediator levels indicating that it might control an inhibitory pathway when triggered individually. CONCLUSIONS: Unlike TLRs, EGF/FGF/PDGF receptors collectively play a significant role in the inflammation and apoptosis of human oligodendrocytes as mediated by B. burgdorferi. It is likely that these three receptors need to be triggered simultaneously to achieve this effect.

Antibody profile to Borrelia burgdorferi in veterinarians from Nuevo León, Mexico, a non-endemic area of this zoonosis.

OBJECTIVES: Lyme disease is a tick-borne disease caused by infections with Borrelia. Persons infected with Borrelia can be asymptomatic or can develop disseminated disease. Diagnosis and recognition of groups at risk of infection with Borrelia burgdorferi is of great interest to contemporary rheumatology. There are a few reports about Borrelia infection in Mexico, including lymphocytoma cases positive to B. burgdorferi sensu stricto by PCR and a patient with acrodermatitis chronica atrophicans. Veterinarians have an occupational risk due to high rates of tick contact. The aim of this work was to investigate antibodies to Borrelia in students at the Faculty of Veterinary Medicine and Zootechnics, at Nuevo León, Mexico, and determine the antibody profile to B. burgdorferi antigens. MATERIAL AND METHODS: Sera were screened using a C6 ELISA, IgG and IgM ELISA using recombinant proteins from B. burgdorferi, B. garinii and B. afzelii. Sera with positive or grey-zone values were tested by IgG Western blot to B. burgdorferi sensu stricto. RESULTS: All volunteers reported tick exposures and 72.5% remembered tick bites. Only nine persons described mild Lyme disease related symptoms, including headaches, paresthesias, myalgias and arthralgias. None of the volunteers reported erythema migrans. Nine samples were confirmed by IgG Western blot. The profile showed 89% reactivity to OspA, 67% to p83, and 45% to BmpA. CONCLUSIONS: Positive sera samples shared antibody reactivity to the markers of late immune response p83 and BmpA, even if individuals did not present symptoms of Lyme arthritis or post-Lyme disease. The best criterion to diagnose Lyme disease in our country remains to be established, because it is probable that different strains coexist in Mexico. This is the first report of antibodies to B. burgdorferi in Latin American veterinarians. Veterinarians and high-risk people should be alert to take precautionary measures to prevent tick-borne diseases.

Fibroblast growth factor receptor inhibitors mitigate the neuropathogenicity of Borrelia burgdorferi or its remnants ex vivo.

In previous studies, we showed that fibroblast growth factor receptors (FGFRs) contribute to inflammatory mediator output from primary rhesus microglia in response to live Borrelia burgdorferi. We also demonstrated that non-viable B. burgdorferi can be as pathogenic as live bacteria, if not more so, in both CNS and PNS tissues. In this study we assessed the effect of live and non-viable B. burgdorferi in inducing FGFR expression from rhesus frontal cortex (FC) and dorsal root ganglion (DRG) tissue explants as well as their neuronal/astrocyte localization. Specific FGFR inhibitors were also tested for their ability to attenuate inflammatory output and apoptosis in response to either live or non-viable organisms. Results show that in the FC, FGFR2 was the most abundantly expressed receptor followed by FGFR3 and FGFR1. Non-viable B. burgdorferi significantly upregulated FGFR3 more often than live bacteria, while the latter had a similar effect on FGFR1, although both treatments did affect the expressions of both receptors. FGFR2 was the least modulated in the FC tissues by the two treatments. FGFR1 expression was more prevalent in astrocytes while FGFR2 and FGFR3 showed higher expression in neurons. In the DRG, all three receptor expressions were also seen, but could not be distinguished from medium controls by immunofluorescence. Inhibition of FGFR1 by PD166866 downregulated both inflammation and apoptosis in both FC and DRG in response to either treatment in all the tissues tested. Inhibition of FGFR1-3 by AZD4547 similarly downregulated both inflammation and apoptosis in both FC and DRG in response to live bacteria, while with sonicated remnants, this effect was seen in one of the two FC tissues and 2 of 3 DRG tissues tested. CCL2 and IL-6 were the most downregulated mediators in the FC, while in the DRG it was CXCL8 and IL-6 in response to FGFR inhibition. Downregulation of at least two of these three mediators was observed to downregulate apoptosis levels in general. We show here that FGFR inhibition can be an effective anti-inflammatory treatment in antibiotic refractive neurological Lyme. Alternatively, two biologics may be needed to effectively curb neuroinflammation and pathology in the CNS and PNS.

Preparation of Borrelia-Infected Mammalian Cells for Helium Ion Microscopy.

Preparation of mammalian cells for a Borrelia burgdorferi infection can be cumbersome especially if investigating possible cell entry processes. The initial steps of infection or entry into cells by a pathogen often involve attachment to the cell surface and plasma membrane changes. To topologically investigate with great resolution and detail these interactions of the pathogen and the mammalian cell, helium ion microscopy (HIM) can be employed. Here we describe a protocol used to define a specific multiplicity of infection (MOI) of Borrelia burgdorferi on a human chondrosarcoma cell line (SW1353) so that fine detail structures on the mammalian cell can be observed and quantified by HIM.

Effects of Regulatory T Cell Depletion in BALB/c Mice Infected with Low Doses of Borrelia burgdorferi.

We previously demonstrated that a depletion of regulatory T (Treg) cells in Lyme arthritis-resistant C57BL/6 mice leads to pathological changes in the tibiotarsal joints following infection with Borrelia burgdorferi. Here, we assessed the effects of Treg cells on the response to B. burgdorferi infection in BALB/c mice, which exhibit infection-dose-dependent disease and a different sequence of immune events than C57BL/6 mice. The depletion of Treg cells prior to infection with 1 × 102, but not 5 × 103, organisms led to increased swelling of the tibiotarsal joints. However, Treg cell depletion did not significantly affect the development of histopathology at these low doses of infection. BALB/c mice depleted of Treg cells before infection with 1 × 103 spirochetes harbored a higher borrelial load in the hearts and exhibited higher levels of serum interleukin-10 five weeks later. These results indicate that Treg cells regulate certain aspects of the response to B. burgdorferi in a mouse strain that may display a range of disease severities. As the presentation of Lyme disease may vary among humans, it is necessary to consider multiple animal models to obtain a complete picture of the various means by which Treg cells affect the host response to B. burgdorferi.

The Use of Natural Bioactive Nutraceuticals in the Management of Tick-Borne Illnesses.

The primary objective of this paper is to provide an evidence-based update of the literature on the use of bioactive phytochemicals, nutraceuticals, and micronutrients (dietary supplements that provide health benefits beyond their nutritional value) in the management of persistent cases of Borrelia burgdorferi infection (Lyme disease) and two other tick-borne pathogens, Babesia and Bartonella species. Recent studies have advanced our understanding of the pathophysiology and mechanisms of persistent infections. These advances have increasingly enabled clinicians and patients to utilize a wider set of options to manage these frequently disabling conditions. This broader toolkit holds the promise of simultaneously improving treatment outcomes and helping to decrease our reliance on the long-term use of pharmaceutical antimicrobials and antibiotics in the treatment of tick-borne pathogens such as Borrelia burgdorferi, Babesia, and Bartonella.

The Diagnostic Challenges and Clinical and Serological Outcome in Patients Hospitalized for Suspected Lyme Neuroborreliosis.

The aim of our study was to evaluate the differential diagnosis and clinical/serological outcome to antibiotic treatment in patients hospitalized for suspected Lyme neuroborreliosis (LNB). A prospective study included patients hospitalized in a Romanian hospital between March 2011 and October 2012 with neurological symptoms, positive laboratory tests for Borrelia burgdorferi, cerebrospinal fluid (CSF) analysis, and no previous treatment for LNB. A questionnaire was completed for each patient at admission, at the end of treatment, and 3 months later. Patients were treated with antibiotic therapy (ceftriaxone/cefotaxime), irrespective of CSF analysis results. A symptomatic scoring scale was used for the follow-up. Out of the 42 patients included, no patient fulfilled criteria for definite LNB; 7 patients were classified as possible LNB; and in 33 patients, LNB was excluded. Two patients could not be classified (insufficient amount of CSF). Clinical follow-up suggested a better response to therapy in the group of patients with possible LNB than in the group with LNB excluded. The patients' differential diagnosis and serological follow-up are presented. Patients investigated for suspected LNB present diverse clinical manifestations and comorbidities that complicate differential diagnosis. LNB may be misdiagnosed if CSF analysis is not performed.

Are other tick-borne infections overlooked in patients investigated for Lyme neuroborreliosis? A large retrospective study from South-eastern Sweden.

In Europe, the hard tick Ixodes ricinus is considered the most important vector of human zoonotic diseases. Human pathogenic agents spread by I. ricinus in Sweden include Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum, Rickettsia helvetica, the recently described Neoehrlichia mikurensis, Borrelia miyamotoi, tick-borne encephalitis virus (TBEV), and Babesia spp. (Babesia microti, Babesia venatorum and Babesia divergens). Since these pathogens share the same vector, co-infections with more than one tick-borne pathogen may occur and thus complicate the diagnosis and clinical management of the patient due to possibly altered symptomatology. Borrelia burgdorferi s.l., TBEV and B. miyamotoi are well-known to cause infections of the central nervous system (CNS), whereas the abilities of other tick-borne pathogens to invade the CNS are largely unknown. The aim of this study was to investigate the presence and clinical impact of tick-borne pathogens other than B. burgdorferi s.l. in the cerebrospinal fluid (CSF) and serum samples of patients who were under investigation for Lyme neuroborreliosis (LNB) in a tick-endemic region of South-eastern Sweden. CSF and serum samples from 600 patients, recruited from the Regions of Östergötland County, Jönköping County and Kalmar County in South-eastern Sweden and investigated for LNB during the period of 2009-2013, were retrospectively collected for analysis. The samples were analysed by real-time PCR for the presence of nucleic acid from B. burgdorferi s.l., B. miyamotoi, A. phagocytophilum, Rickettsia spp., N. mikurensis, TBEV and Babesia spp. Serological analyses were conducted in CSF and serum samples for all patients regarding B. burgdorferi s.l., and for the patients with CSF mononuclear pleocytosis, analyses of antibodies to B. miyamotoi, A. phagocytophilum, spotted fever group (SFG) rickettsiae, TBEV and B. microti in serum were performed. The medical charts of all the patients with CSF mononuclear pleocytosis and patients with positive PCR findings were reviewed. Of the 600 patients, 55 (9%) presented with CSF mononuclear pleocytosis, 13 (2%) of whom had Borrelia-specific antibodies in the CSF. One patient was PCR-positive for N. mikurensis, and another one was PCR-positive for Borrelia spp. in serum. No pathogens were detected by PCR in the CSF samples. Four patients had serum antibodies to B. miyamotoi, four patients to A. phagocytophilum, five patients to SFG rickettsiae, and six patients to TBEV. One patient, with antibodies to SFG rickettsiae, had both clinical and laboratory signs suggestive of a current infection. Nine patients had serum antibodies to more than one pathogen, although none of these was assessed as a current co-infection. We can conclude from this study that tick-borne co-infections are uncommon in patients who are being investigated for suspected LNB in South-eastern Sweden, an area endemic for borreliosis and TBE.

LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species.

BACKGROUND: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. METHODS: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system LoopTag for detection of Borrelia species. Advantages of the LoopTag system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. RESULTS: Using the LoopTag probe system we were able to detect all nine tested European species belonging to the Borrelia burgdorferi (sensu lato) complex and differentiated them from relapsing fever Borrelia species. As few as 10 copies of Borrelia in one PCR reaction were detectable. CONCLUSION: We established a novel multiplex probe real-time PCR system, designated LoopTag, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the Borrelia burgdorferi s.l. complex.

Borrelia burgdorferi hijacks cellular metabolism of immune cells: Consequences for host defense.

Changes in cellular metabolism have proven to be important factors in driving cell behavior. It has been shown that cellular metabolism of immune cells changes when exposed to or infected by several pathogens: while this is often an adaptation of the host cells to the infection, sometimes it represents a mechanism through which the pathogens evade immune activation. Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is a pathogen that highly depends on the host to survive, as the bacterium lacks many central metabolic pathways to generate its own nutrients. It is therefore quite likely that the bacterium interacts with host cells to obtain these metabolites and thereby affects metabolism in the host. Previously, several studies have assessed metabolic pathways in B. burgdorferi s.l. and how it adapts to its different host species. However, few studies have looked into how the interaction with the bacterium might affect the host cell metabolism. In this review we present the major metabolic pathways activated during Lyme borreliosis, viewed from both bacterium and host metabolism, and we discuss how these pathways interact with each other, and how they influence pathogenesis of Lyme borreliosis.

Assessment of Anaplasma phagocytophilum presence in early Lyme borreliosis manifested by erythema migrans skin lesions.

BACKGROUND: To investigate to what extent early Lyme borreliosis patients with erythema migrans are infected with Anaplasma phagocytophilum. METHODS: Three hundred ten patients from Poland with erythema migrans were included in the study. One hundred and eighty-three patients (59%) agreed to have both skin biopsy and blood samples analysed for Borrelia burgdorferi, A. phagocytophilum and 'Candidatus Neoehrlichia mikurensis', with PCR. Positive samples were confirmed with sequencing. RESULTS: B. burgdorferi DNA was detected in 49.7% of the skin samples and in 1.1% of the blood samples. A. phagocytophilum DNA was found in 7.1% blood samples, and in 8.2% of the skin biopsies. In four patients, A. phagocytophilum DNA was detected only in blood; in one case A. phagocytophilum DNA was found simultaneously in blood and skin, and additionally in this patients' blood Borrelia DNA was detected. In four skin samples B. burgdorferi DNA was detected simultaneously with A. phagocytophilum DNA, indicative of a co-infection. CONCLUSIONS: A. phagocytophilum may be present in early Lyme borreliosis characterized by erythema migrans and should always be considered as a differential diagnostic following a tick bite and considered in treatment schemes, as these differs (in early stage of Lyme borreliosis doxycycline, amoxicillin, cefuroxime axetil and azithromycin are recommended, while in anaplasmosis the most effective courses of treatment are doxycycline, rifampin and levofloxacin). Consequently, the role of A. phagocytophilum in erythema migrans should be further studied.