Measurement of Organ-Specific and Acute-Phase Blood Protein Levels in Early Lyme Disease.

Lyme disease results from infection of humans with the spirochete Borrelia burgdorferi. The first and most common clinical manifestation is the circular, inflamed skin lesion referred to as erythema migrans; later manifestations result from infections of other body sites. Laboratory diagnosis of Lyme disease can be challenging in patients with erythema migrans because of the time delay in the development of specific diagnostic antibodies against Borrelia. Reliable blood biomarkers for the early diagnosis of Lyme disease in patients with erythema migrans are needed. Here, we performed selected reaction monitoring, a targeted mass spectrometry-based approach, to measure selected proteins that (1) are known to be predominantly expressed in one organ (i.e., organ-specific blood proteins) and whose blood concentrations may change as a result of Lyme disease, or (2) are involved in acute immune responses. In a longitudinal cohort of 40 Lyme disease patients and 20 healthy controls, we identified 10 proteins with significantly altered serum levels in patients at the time of diagnosis, and we also developed a 10-protein panel identified through multivariate analysis. In an independent cohort of patients with erythema migrans, six of these proteins, APOA4, C9, CRP, CST6, PGLYRP2, and S100A9, were confirmed to show significantly altered serum levels in patients at time of presentation. Nine of the 10 proteins from the multivariate panel were also verified in the second cohort. These proteins, primarily innate immune response proteins or proteins specific to liver, skin, or white blood cells, may serve as candidate blood biomarkers requiring further validation to aid in the laboratory diagnosis of early Lyme disease.

Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans.

The most common tick-borne human disease in Norway is Lyme borreliosis. Ticks in Norway also harbour less known disease-causing agents such as Candidatus Neoehrlichia mikurensis, Borrelia miyamotoi and Rickettsia helvetica. However, human infections caused by these pathogens have never been described in Norway. The main aims of the study were to evaluate the contribution of several tick-borne bacterial agents, other than Borrelia burgdorferi sensu lato, to zoonotic diseases in Norway and to determine their clinical pictures. Blood samples from 70 symptomatic tick-bitten adults from the Agder counties in southern Norway were screened for seven tick-borne pathogens by using a commercial multiplex PCR-based method and by singleplex real-time PCR protocols. Most patients (65/70) presented with a rash clinically diagnosed as erythema migrans (EM). The most frequently detected pathogen DNA was from Ca. N. mikurensis and was found in the blood of 10% (7/70) of the patients. The Ca. N. mikurensis-infected patients presented with an EM-like rash as the only symptom. B. burgdorferi s.l. DNA was present in the blood of 4% (3/70) of the study participants. None had detectable Anaplasma phagocytophilum, B. miyamotoi, Rickettsia typhus group or spotted fever group, Francisella tularensis, Coxiella burnetii or Bartonella spp. DNA in the blood. The commercially available multiplex PCR bacteria flow chip system failed to identify half of the infected patients detected by corresponding real-time PCR protocols. The recovery of Ca. N. mikurensis DNA was higher in the pellet/plasma fraction of blood than from whole blood. To conclude, Ca. N. mikurensis appeared to be the etiological agent in patients with EM in a surprisingly large fraction of tick-bitten persons in the southern part of Norway.

Decreased Th1-type inflammatory cytokine expression in the skin is associated with persisting symptoms after treatment of erythema migrans.

BACKGROUND: Despite the good prognosis of erythema migrans (EM), some patients have persisting symptoms of various character and duration post-treatment. Several factors may affect the clinical outcome of EM, e.g. the early interaction between Borrelia (B.) burgdorferi and the host immune response, the B. burgdorferi genotype, antibiotic treatment as well as other clinical circumstances. Our study was designed to determine whether early cytokine expression in the skin and in peripheral blood in patients with EM is associated with the clinical outcome. METHODS: A prospective follow-up study of 109 patients with EM was conducted at the Åland Islands, Finland. Symptoms were evaluated at 3, 6, 12 and 24 months post-treatment. Skin biopsies from the EM and healthy skin were immunohistochemically analysed for expression of interleukin (IL)-4, IL-10, IL-12p70 and interferon (IFN)-γ, as well as for B. burgdorferi DNA. Blood samples were analysed for B. burgdorferi antibodies, allergic predisposition and levels of systemic cytokines. FINDINGS: None of the patients developed late manifestations of Lyme borreliosis. However, at the 6-month follow-up, 7 of 88 patients reported persisting symptoms of diverse character. Compared to asymptomatic patients, these 7 patients showed decreased expression of the Th1-associated cytokine IFN-γ in the EM biopsies (p=0.003). B. afzelii DNA was found in 48%, B. garinii in 15% and B. burgdorferi sensu stricto in 1% of the EM biopsies, and species distribution was the same in patients with and without post-treatment symptoms. The two groups did not differ regarding baseline patient characteristics, B. burgdorferi antibodies, allergic predisposition or systemic cytokine levels. CONCLUSION: Patients with persisting symptoms following an EM show a decreased Th1-type inflammatory response in infected skin early during the infection, which might reflect a dysregulation of the early immune response. This finding supports the importance of an early, local Th1-type response for optimal resolution of LB.

Identification of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in patients with erythema migrans.

Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.

[Seronegative Lyme arthritis]. / Seronegatywne boreliozowe zapalenie stawów.

Serologic confirmation is required for diagnosis of Lyme borreliosis in all the patients except those with well confirmed and typical erythema migrans (EM) lesion. In patients with articular symptoms, the primary cause of seronegativity is performing a test at a very early stage of the disease, before detectable levels of anti-Borrelia burgdorferi s.l. antibodies build up. However, recent epidemiologic history and presence of primary skin lesion facilitate diagnosis in most patients at this stage of the infection. Secondly, insufficient antibiotic treatment of EM may halt development of humoral response without total eradication of the pathogen, which may result in the relapse of arthritis without detectable anti-B. burgdorferi s.l. antibodies for up to several months later--a rather rare phenomenon, which, however, must be taken into consideration in patients with a history of treated EM. A fe cases of seronegative Lyme arthritis not related to previous antibiotherapy have been reported, but i seems an extremely rare condition, and, if suspected, should be confirmed by histopathologic or molecular examination of the material from the affected joint.

[Clinical forms of neuroborreliosis among hospitalized patients in the years 2000-2005]. / Kliniczne postacie neuroboreliozy u chorych hospitalizowanych w latach 2000-2005.

THE AIM OF THE STUDY: To evaluate the frequency of clinical forms as well as laboratory and neuroimaging results of patients with diagnosed neuroborreliosis in the years 2000-2005 due to neuroborreliosis. MATERIAL AND METHODS: The records of 125 patients at the age of 21-83 (mean 49 years) treated in the years 2000-2005 in the Department of Infectious Diseases and Neuroinfections, Medical University, Bialystok were subject to retrospective analysis. Diagnosis was based on case history along with a clinical picture and presence of antibodies against Borrelia burgdorferi, using ELISA test (Borrelia IgM and Borrelia IgG recombinant Biomedica). The subject of the detailed analysis was demographic data, clinical symptoms as well as subjective complaints, results of neurological examinations, the results of cerebrospinal fluid (CSF) parameters and results of serologic tests. RESULTS: The most frequent clinical symptoms observed were: headaches 71%, vertigo 44%, meningeal symptoms 22% and neurological paresis 27% (including facial palsy--23%). Inflammatory changes in CSF in the form of increased proteins concentration and pleocytosis were present among 34% of patients. In all cases the antibodies against B. burgdorferi were present in CSF in diagnostically significant titer. Serum presence of antibodies antiborrelia IgM was found with 55% of patients and anibodies antiborrelia IgG with 76% of patients. 17% of patients suffering from neuroborreliosis were also coinfected with tick-borne encephalitis virus. Along with the neurological symptoms, which were crucial to diagnosis, general symptoms coexisted, such as: weakness 35%, arthralgia 54% and nausea 17%. In the analyzed period of time neuroborreliosis was diagnosed in a 13% of hospitalized patient suffering from borreliosis. CONCLUSIONS: Absence of erythema migrans does not exclude existence of neuroborreliosis. Symptoms that may suggest presence of neuroborreliosis are not only neurological symptoms such as facial palsy, but also memory and concentration disorders and general symptoms.

Molecular and serological diagnosis of Borrelia burgdorferi infection among patients with diagnosed Erythema migrans.

The aim of the study was to assess the frequency of Borrelia burgdorferi DNA detection in the blood and urine of patients diagnosed with erythema migrans, and compare the results of PCR-based methods with ELISA methodology. The latter was used to detect serum antibodies against Borrelia burgdorferi of the IgM and IgG classes, before and after antibiotic therapy. The study included 86 patients hospitalized in the Department of Infectious Diseases and Neuroinfections in the Medical Academy in Bialystok, diagnosed with the erythema migrans phase of Lyme borreliosis. Examinations were carried out twice: the first at the moment of diagnosis (Trial 1), the second after 4 weeks of antibiotic therapy. The study showed that antibiotic therapy in the early phase of borreliosis does not decrease the sensitivity of PCR and that after 4 weeks of therapy (Trial 2), spirochete DNA is still detectable in most patients (45/86). There was no correlation between detectability of spirochete DNA and the presence of antibodies against B. burgdorferi s.l. (assessed by ELISA) during the course of erythema migrans. The largest percentage of positive results in the detection of B. burgdorferi s.l. DNA was observed in patients who simultaneously possessed IgM and IgG antibodies against B. burgdorferi, while the lowest percentage of PCR positive results was among patients with only IgM antibodies.

Hematogenous dissemination in early Lyme disease.

Hematogenous dissemination has long been considered an important pathogenetic event in Lyme borreliosis but has been hard to document and characterize due to insensitive blood culture methods. In a series of recent investigations involving adult patients from the United States with erythema migrans, it was concluded that plasma was a better source of culture material than serum or whole blood, and yield correlated directly with the volume of material cultured. The rate of recovery of Borrelia burgdorferi from 9 mL of plasma exceeded 40%. Both the genotype of the strain of B. burgdorferi introduced by the tick and host factors, such as having a first episode of Lyme borreliosis and being more than 55 years of age, appear to affect the risk of hematogenous dissemination. Although the majority of spirochetemic patients are symptomatic, spirochetemia can be a subclinical event. In summary, hematogenous dissemination is a common and important feature of early Lyme borreliosis in the United States. The high rate, early onset and prolonged duration of risk of spirochetemia probably explain why untreated patients with erythema migrans may develop objective clinical manifestations at a distant anatomic site.

Clinical relevance of different IgG and IgM serum antibody responses to Borrelia burgdorferi after antibiotic therapy for erythema migrans: long-term follow-up study of 113 patients.

OBJECTIVES: To investigate the kinetics of anti-Borrelia burgdorferi antibodies for a minimum of 1 year after antibiotic therapy in patients with erythema migrans (EM) and to correlate antibody titer kinetics with clinical variables. DESIGN: Retrospective study of serial anti-B burgdorferi antibodies in correlation to clinical variables. SETTING: University-based hospital. PATIENTS: One hundred thirteen patients with EM. INTERVENTIONS: Pretreatment and a median of 4 consecutive posttreatment serum samples from median follow-up of more than 400 days were simultaneously investigated for anti-B burgdorferi IgG and IgM antibodies. Semiquantitative titers were plotted to identify different groups of antibody kinetics. Individual patients were then stratified to those groups according to their antibody development. A statistical comparison of clinical and therapy-related characteristics among the serologic groups was performed. RESULTS: Anti-B burgdorferi IgG and IgM antibody titers developed in 3 distinct courses: persistent positivity across follow-up (IgG: 12 patients, 11%; IgM: 14, 12%), persistent negativity (IgG: 63, 56%; IgM: 47, 42%), and decrease of a positive pretreatment titer to a negative titer approximately 5 months after therapy (IgG: 34, 30%; IgM: 49, 43%). Statistics revealed significant correlations only between persistent positive IgG titers and long disease duration or large EM lesions before therapy. CONCLUSIONS: Long duration or large size of EM before therapy correlates with persistence of a positive anti- B burgdorferi IgG antibody titer after therapy. Serologic profiles do not depend on the type or duration of therapy or the clinical course thereafter. Thus, antibody testing in the follow-up of patients with EM is inappropriate for the assessment of therapeutic response.

Detection of spirochaetal DNA simultaneously in skin biopsies, peripheral blood and urine from patients with erythema migrans.

Lyme borreliosis is an emerging zoonosis transmitted by infected hard-bodied ticks. The disease is multisystemic. In the initial stage its typical manifestation is the erythema migrans, a cutaneous lesion that occurs in up to 90% of patients. In order to investigate the presence of the specific agent, Borrelia burgdorferi, in the early stages of the disease, DNA from skin biopsies, urine and peripheral blood of 30 patients with clinically documented erythema migrans and without apparent systemic involvement was analysed by polymerase chain reaction. Borrelia DNA in both blood and skin biopsies was detected in 23 patients, while in 9 patients it was discovered in urine and skin biopsies. These results demonstrate that Borrelia DNA is detectable systemically also in patients with early Lyme borreliosis and strongly suggest a possible dissemination of the causative agents even when only a local infection is assumed.

Borrelia-specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome.

The Borrelia-specific interferon (IFN)- gamma and interleukin (IL)-4 responses of 113 patients and control subjects were analyzed using the sensitive enzyme-linked immunospot method. Cerebrospinal fluid (CSF) and blood samples were obtained, during the course of disease, from patients with chronic or nonchronic neuroborreliosis (NB) and from control subjects without NB. Blood samples were obtained from patients with Lyme skin manifestations and from healthy blood donors. Early increased secretion of Borrelia-specific IFN- gamma (P<.05) and subsequent up-regulation of IL-4 (P<.05) were detected in the CSF cells of patients with nonchronic NB. In contrast, persistent Borrelia-specific IFN- gamma responses were observed in the CSF cells of patients with chronic NB (P<.05). In patients with erythema migrans, increased IFN- gamma (P<.001) was observed in blood samples obtained early during the course of disease, whereas increased IL-4 (P<.05) was observed after clearance. On the contrary, patients with acrodermatitis chronica atrophicans had Borrelia-specific IFN- gamma (P<.001), but not IL-4, detected in blood samples. The present data suggest that an initial IFN- gamma response, followed by up-regulation of IL-4, is associated with nonchronic manifestations, whereas a persistent IFN- gamma response may lead to chronic Lyme borreliosis.

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans.

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.

[Concentration of sVAP in serum of patients with different forms of Lyme borreliosis]. / Stezenie sVAP-1 w surowicy krwi w wybranych postaciach klinicznych boreliozy z Lyme.

OBJECTIVES: The aim of our work was to assess the serum concentration of soluble VAP-1 (sVAP-1) in patients with arthral type of borreliosis and erythema migrans. METHODS: We included in our study 30 patients in the age of 17-53 years who were treated for erythema migrans and 30 patients in the age of 25-63 years who were diagnosed with arthral type of borreliosis. The control group consisted of 30 healthy blood donors. The diagnosis of borreliosis was made on the basis of anamnesis, physical examination and additional tests, one of which proved the existence of IgM and IgG antibodies against Borrelia burgdorferi antigen present in the serum of patients. All samples for investigations were taken in a single collection manner. The serum concentration of sVAP-1 protein was assessed with ELISA method. All data were analysed statistically. We assumed 5% risk of conclusion error. CONCLUSIONS: 1. We did not observe an increase in serum sVAP-1 concentration in patients with erythema migrans compared to the control group. All observed differences in the serum level of this protein seemed to be random ones. 2. We showed a statistically important increase in soluble sVAP-1 serum concentration in patients with arthral type of borreliosis compared to the control group.

Epidemiological survey of human borreliosis diagnosed in Eastern Slovakia.

439 sera of patients from eastern Slovakia suspected of Lyme borreliosis were examined for anti-Borrelia IgG and IgM antibodies by ELISA (Ezygnost, Dade Behring, Germany). Out of the total number of 64 sera, e.g. in 14.6% found anti-Borrelia antibodies were. Among seropositive patients, 54.7% were women and 45.3% men. The highest incidence of the disease was diagnosed in the group of women aged 55-64 and men aged 45-54. Out of 29 positive sera of men, 55.2% had IgG antibodies, 27.6% IgM antibodies and 17.2% both types of antibodies. Out of 35 positive sera of women, 48.6% had IgG antibodies, 40.0% IgM antibodies and 11.4% both types of antibodies. Erythema Chronicum Migrans--ECM (31.3%) and arthritis (25.0%) prevailed among clinical symptoms, in contrast with only 7.8% of neurological cases. In men, arthritis most frequently occurred (27.6%), while in women erythema migrans forms (37.1%). Other manifestations of the disease appeared in 13 patients (20.3%), and 10 patients (15.6%) had no record of clinical manifestations. As shown by patients' records, 32.8% reported attachment of tick, 20.3% insect bites and 29.7% were not aware of being bitten by vectors. Seasonal dynamics of diagnosed cases reached three peaks with the highest numbers in February, May and October.

Cellular and humoral immune responses to Borrelia burgdorferi antigens in patients with culture-positive early Lyme disease.

We determined cellular and humoral immune responses to Borrelia burgdorferi lysate and to recombinant flagellin (FlaB), OspC, and OspA in acute- and convalescent-phase samples from 39 culture-positive patients with erythema migrans and in 20 healthy control subjects. During the acute illness, a median of 4 days after the onset of erythema migrans, 51% of the patients had proliferative cellular responses and 72% had antibody responses to at least one of the borrelial antigens tested. During convalescence, at the conclusion of antibiotic therapy, 64% of the patients had proliferative cellular reactivity and 95% had antibody reactivity with at least one of the spirochetal antigens tested. In both acute- and convalescent-phase samples, cellular immune responses were found as frequently to OspA as to OspC and FlaB. Although antibody responses were also frequently seen to OspC and FlaB, only a few patients had marginal antibody reactivity with OspA. The percentage of patients with proliferative responses was similar in those with clinical evidence of localized or disseminated infection, whereas humoral reactivity was found more often in those with disseminated disease. We conclude that cellular and humoral responses to B. burgdorferi antigens are often found among patients with early Lyme disease. In contrast with the other antigens tested, cellular but not humoral reactivity was often found with OspA.

Eritema migratorio necrolítico como manifestación inicial de glucagonoma normoglucémico / Necrolytic migratory erythema assocaited normoglycemic glucagonoma

No disponible

[Estimation of platelet counts and their morphological parameters in patients infected by borrelia burgdorferi]. / Ocena liczby plytek i ich parametrów morfologicznych u chorych zakazonych Borrelia burgdorferi.

Platelet counts and their morphologic parameters in patients with Lyme borreliosis before and after antibiotic therapy (4 weeks of treatment) were estimated. 30 patients aged 17-60, x = 41 were evaluated: 7 with Erythema migrans, 3 patients with neuroborreliosis in the from Lymphocytic meningitis and 20 ones with Lyme arthritis. Control group consisted of 19 healthy persons aged 34-52, x = 43. Hematologic analyser Coulter MAXM was used for testing PLT, MPV, PCT and PDW. The results indicated that patients with Lyme boreliosis have decreasing platelet count with simultaneously increasing their volume in comparison with healthy control. It may result from the involement of platelets in defense mechanisms of infected host. The decrease of platelet count after the antibiotic treatment in comparison with the control group may be the reflection of influence of antibiotic treatment on thrombopoesis.