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.

[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.

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.

[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.

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.

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.

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.

Reactivity of neuroborreliosis patients (Lyme disease) to cardiolipin and gangliosides.

A subset of patients (50%) with neuroborreliosis (Lyme disease) showed IgG reactivity to cardiolipin in solid phase ELISA. In addition, a subset of patients with neuroborreliosis (29%) and syphilis (59%) had IgM reactivity to gangliosides with a Gal(beta 1-3) GalNac terminal sequence (GM1, GD1b, and asialo GM1). Anti-ganglioside IgM antibodies were significantly more frequent in these two groups of patients compared to patients with cutaneous and articular Lyme disease, primary antiphospholipid syndrome, systemic lupus erythematosus and normal controls. Correlative evidence and adsorption experiments indicated that antibodies to cardiolipin had separate specificities from those directed against the gangliosides. IgM antibodies to Gal(beta 1-3) GalNac gangliosides appeared to have similar specificities since these were positively correlated and inhibitable by cross adsorption assays. Given the clinical associations of patients with neuroborreliosis and syphilis with IgM reactivity to gangliosides sharing the Gal(beta 1-3) GalNac terminus, we suggest that these antibodies could represent a response to injury in neurological disease or a cross reactive event caused by spirochetes.

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.

[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.

[Anti-Borrelia burgdorferi antibodies in chronic erythema migrans, benign lymphadenosis cutis, scleroderma and scleroatrophic lichen]. / Anticorpi anti-Borrelia burgdorferi nell'eritema cronico migrante, nella lymphadenosis cutis benigna, nella sclerodermia e nel lichen scleroatrofico.

Fiftyone patients with a diagnosis of erythema chronicum migrans (ECM), lymphadenosis cutis benigna (LABC), systemic progressive sclerosis, localized scleroderma and lichen sclerosus et atrophicans were investigated in order to obtain serological evaluation of Borrelia burgdorferi circulating antibodies. In addition sera from 9 patients with pellagroid erythema, prurigo and panniculitis were performed: in these dermatoses there was no evidence of a possible borrelia relationship. Indirect immunofluorescence assay was used for serologic testing. Elevated IgG antibody titers were detected in one female patient with localized scleroderma (1:128) and in one male patient with LABC (1:64). Serologic testing was not positive in the other 58 patients. The very small number of patients with positive antibodies in our study would indicate that Borrelia burgdorferi infection is sporadic in Lazio.

[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.

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.

[Determination of antibodies against Borrelia burgdorferi in patients with morphea, lichen sclerosus et atrophicus and erythema chronicum migrans]. / Determinación de anticuerpos frente a Borrelia burgdorferi en pacientes con morfea, liquen escleroso y atrófico y eritema crónico migrans.

Several cutaneous entities described in Europe as Chronic Migrans Erythema (CME), Mild Cutis Lymphadenosis (MCL) and Chronic Atrophyc Acrodermatitis (CAA) constitute clinical manifestations of a Borrellia Burgdorferi. The presence of clinical and hystologic lesions similar to those of liquen esclerosus and atrophyc (LEA) and localized esclerodermia (morphea) in patients with CAA has driven to several authors to demonstrate the aethiologic participation of B. Burgdorferi in patients carrying those cutaneous lesions with contradictory results. A serologic study with indirect immunofluorescence (IIF) and FIAX test was performed in 16 patients (9 with morphea, 6 with LEA and 1 with CME) in order to evaluate the role of this microorganism in our environment. Five reactive sera were obtained of which only one presented IgG antibodies titrated at 1/256. The IgM antibodies by IIF and IgG by FIAX test turned out to be negative. A specific relationship between B. Burgdorferi and the studied entities could not be established.

Ovalbumin blocking improves sensitivity and specificity of immunoglobulin M immunoblotting for serodiagnosis of patients with erythema migrans.

To improve the serodiagnosis of erythema migrans, we evaluated how sensitivity and specificity of immunoblotting are influenced by antigen concentration and blocking conditions. We found that an antigen concentration of 0.5 micrograms per lane in concert with ovalbumin blocking of the nitrocellulose provided the best results. In this case, 81% of the erythema migrans had positive immunoglobulin M tests, whereas only 33% were positive in a flagellum enzyme-linked immunosorbent assay (ELISA) and 28% were positive in a sonicate ELISA.

Liver function test abnormalities in early Lyme disease.

OBJECTIVE: Lyme disease is a widespread, tick-borne, spirochetal infection with multiple organ system involvement Hepatic dysfunction has not been emphasized in the literature. We report clinical findings and laboratory abnormalities in 73 patients with the pathognomonic erythema migrans rash early in the course of the illness. DESIGN: Case series. SETTING: Offices of family physicians in private practice and the model offices of a family practice residency program in the lower Connecticut River valley, an area to which Lyme disease is endemic. PATIENTS: Thirty-seven female and 36 male patients with erythema migrans who had not yet been treated with antimicrobial agents. MAIN OUTCOME MEASURE: Liver function tests. RESULTS: Twenty patients (27%) had liver function abnormalities. Elevation of gamma-glutamyltransferase was the most common finding. Only seven patients (9%) had a positive titer in response to the enzyme-linked immunosorbent assay for Lyme disease. Other laboratory and clinical findings are described. CONCLUSION: Subclinical hepatitis is a common finding in early Lyme disease.