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Evidence is emerging for differential pathogenicity among Borrelia burgdorferi genotypes in the United States. By using two linked genotyping systems, ribosomal RNA intergenic spacer type (RST) and outer surface protein C (OspC), we studied the inflammatory potential of B. burgdorferi genotypes in cells and patients with erythema migrans or Lyme arthritis. When macrophages were stimulated with 10 isolates of each RST1, RST2, or RST3 strain, RST1 (OspC type A)-stimulated cells expressed significantly higher levels of IL-6, IL-8, chemokine ligand (CCL) 3, CCL4, tumor necrosis factor, and IL-1β, factors associated with innate immune responses. In peripheral blood mononuclear cells, RST1 strains again stimulated significantly higher levels of these mediators. Moreover, compared with RST2, RST1 isolates induced significantly more interferon (IFN)-α, IFN-γ, and CXCL10, which are needed for adaptive immune responses; however, OspC type I (RST3) approached RST1 (OspC type A) in stimulating these adaptive immune mediators. Similarly, serum samples from patients with erythema migrans who were infected with the RST1 genotype had significantly higher levels of almost all of these mediators, including exceptionally high levels of IFN-γ-inducible chemokines, CCL2, CXCL9, and CXCL10; and this pronounced inflammatory response was associated with more symptomatic infection. Differences among genotypes were not as great in patients with Lyme arthritis, but those infected with RST1 strains more often had antibiotic-refractory arthritis. Thus, the B. burgdorferi RST1 (OspC type A) genotype, followed by the RST3 (OspC type I) genotype, causes greater inflammation and more severe disease, establishing a link between spirochetal virulence and host inflammation., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A case of Ixodes tick-borne borreliosis is described characterized by a rare form of skin lesion (panniculitis) that made difficult definitive diagnosis.

Borrelia burgdorferi sensu lato (s.l.) complex, the etiological factor of Lyme disease, includes over a dozen species of bacteria and 3 pathogenic within it. According to many authors, the clinical symptoms of borreliosis depend on the species that cause the disease. The most frequent symptom of early localized borreliosis is erythema migrans (EM). The aim of the research was to determine species of B. burgdorferi s.l. in 32 patients from the Western Pomerania region in whom EM has been recognized. Blood samples of patients were investigated by PCR-RFLP method, with the use of enzyme differentiating species. The DNA of spirochetes was detected in 25 patients (25/32, 78.1%), compared with 23/32 (71.8%) of ELISA positive patients. Among 25 positive samples, 10 contained the DNA of B. garinii (10/25, 40%), 5 the DNA of B. afzelii (5/25, 20%), 4 the DNA of B. burgdorferi sensu stricto (s.s.) (4/25, 16%) and in 6 samples (6/25, 24%) the DNA of both B. garinii and B. afzelii was found. The DNA of B. burgdorferi s.l. spirochetes may be detected in patients with EM after antibiotic treatment. The most frequent species in patients with EM from the Western Pomerania region is B. garinii. Infections with more than one species of B. burgdorferi s.l. may occur in patients with EM.

The three skin disorders of Lyme borreliosis in Europe include erythema migrans, an acute, self-limited lesion; borrelial lymphocytoma, a subacute lesion; and acrodermatitis chronica atrophicans, a chronic lesion. Using quantitative reverse transcription-PCR, we determined mRNA expression of selected chemokines, cytokines, and leukocyte markers in skin samples from 100 patients with erythema migrans, borrelial lymphocytoma, or acrodermatitis chronica atrophicans and from 25 control subjects. Chemokine patterns in lesional skin in each of the three skin disorders included low but significant mRNA levels of the neutrophil chemoattractant CXCL1 and the dendritic cell chemoattractant CCL20 and intermediate levels of the macrophage chemoattractant CCL2. Erythema migrans and particularly acrodermatitis lesions had high mRNA expression of the T-cell-active chemokines CXCL9 and CXCL10 and low levels of the B-cell-active chemokine CXCL13, whereas lymphocytoma lesions had high levels of CXCL13 and lower levels of CXCL9 and CXCL10. This pattern of chemokine expression was consistent with leukocyte marker mRNA in lesional skin. Moreover, using immunohistologic methods, CD3(+) T cells and CXCL9 were visualized in erythema migrans and acrodermatitis lesions, and CD20(+) B cells and CXCL13 were seen in lymphocytoma lesions. Thus, erythema migrans and acrodermatitis chronica atrophicans have high levels of the T-cell-active chemokines CXCL9 and CXCL10, whereas borrelial lymphocytoma has high levels of the B-cell-active chemokine CXCL13.

Methods used to diagnose Lyme borreliosis (LB) vary according to clinical presentations. A very good basis to clarify this nosological and clinical entity is the study published by the "European Concerted Action on Lyme Borreliosis" (EUCALB). In fact, only few studies were performed on cohorts of patients including all clinical forms of LB. For Erythema migrans, serology sensitivity is low (20% to 50%), while the sensitivity of culture or PCR reaches 50%. In early-complicated forms, serology is more sensitive (70 to 90%) with the presence of concomitant IgG and IgM. Screening for antibodies in CSF is very useful for the diagnosis of neuroborreliosis. For this clinical form, culture or PCR sensitivity is disappointing (10 to 30%). In arthritis and acrodermatitis chronica atrophicans (ACA), IgG serology is 100% positive with very high titers; however IgM serology is only positive in 5 to 10% of the cases. In ACA, culture sensitivity ranges from 20 to 60% and PCR sensitivity from 60 to 90%. Specificity of antibodies, natural exposure to the etiologic agent, and cross-reactivity are critical for the final interpretation of serological assessment. Only the use of "serological profiles" allows the exploitation of detailed results (isotypes, intensity). In this approach, IgG avidity could be constructive. The western-blot is intended to confirm the specificity of antibodies found in screening methods (Elisa).

To determine whether recurrent episodes of appropriately treated Lyme disease are caused by reinfection or relapse, we monitored pertinent clinical manifestations and serology of residents of an endemic site each year for 14 years. Of 253 episodes of early Lyme disease recorded among 213 residents, we observed 40 recurrent episodes. Virtually all included an erythema migrans (EM) rash that appeared at body sites that differed from those of the initial rash, no subjects produced detectable levels of specific antibody between sequential episodes, all episodes occurred a year or more after the initial EM episode, and all occurred during late spring and early summer. People experiencing recurrent episodes tended to have frequent contact with vector ticks. Prompt administration of standard antibiotic therapy for early Lyme disease reliably eliminates persistent infection and prevents relapse.

Erythema migrans is the characteristic exanthem of Lyme disease. The rash initially occurs at the site of inoculation; subsequently satellite lesions can occur. We describe an adolescent girl in whom the rash appeared after the initiation of ceftriaxone therapy for aseptic meningitis. We suggest that the occurrence of rash in this patient was a result of liberated toxin from local bacterial lysis.

Lyme borreliosis often presents initially with erythema migrans. Borreliae may disseminate from the primary skin lesion, and different organs and systems could be affected. Borrelia strains were isolated from blood of 70 patients with Lyme borreliosis, including 10 patients from whom borreliae were also isolated from skin. The aim of the present study was to characterise the isolates with regard to their phenotypic and genotypic characteristics. Borreliae were cultivated in MKP medium. Species identification and plasmid profiles were determined by pulsed-field gel electrophoresis (PFGE) and protein profiles by SDS-PAGE. Digestion of Borrelia burgdorferi sensu lato DNA showed 63 (90%) B. afzelii Mla1 and 7 (10%) B. garinii Mlg2. No B. burgdorferi sensu stricto were isolated. Borreliae were isolated from both skin and blood of 10 patients, nine pairs of isolates were identical: seven B. afzelii and two B. garinii. B. afzelii was isolated from the skin and B. garinii from blood of the tenth patient. All but one isolate possessed at least one large plasmid and varying numbers of smaller plasmids. Eight (11.4%) of 70 isolates possessed an unusual plasmid profile (2 of 63 B. afzelii and 6 of 7 B. garinii). Borreliae differed in their protein profiles. OspA and OspB proteins were expressed by all B. afzelii isolates; 85.7% of B. garinii isolates expressed OspA and 71.4% expressed OspB. OspC was expressed by 65% of B. afzelii isolates and all B. garinii isolates. The ratios of B. afzelii and B. garinii isolated from blood and skin were similar. These results do not support the hypothesis that B. garinii has a higher propensity for haematogenous dissemination than B. afzelii. Antigen diversity as well as species and plasmid heterogeneity could play a role in the pathogenesis of the infection, suggesting distinctive strain organotropism.

Background: We assessed the isolation rate of Borrelia burgdorferi sensu lato from blood in European patients with typical erythema migrans and evaluated the course and outcome of their illness., Patients and Methods: Adult patients diagnosed with erythema migrans and from whom borreliae cultured from blood were included in this study., Results: Borreliae were isolated from the blood of 35/2,828 (1.2%) patients, on average 7 days (range 1-47 days) after the appearance of erythema migrans. Only seven (20%) patients reported constitutional symptoms. 24/35 isolates were typed of which 20 were Borrelia afzelii and four were Borrelia garinii. 31 (88.6%) patients were treated with oral antibiotics while four (11.4%) received ceftriaxone iv. The course and outcome of the illness were favorable in all patients., Conclusion: In European patients with erythema migrans the yield of blood culturing was low, spirochetemia was often clinically silent and the course and outcome of the illness were favorable; the predominantly isolated strain was B. afzelii.

Studies on frequencies of serum antibodies to outer surface proteins (Osps) in Lyme disease have produced conflicting results. Osp antigens (A, B, and C) enriched by butanol extraction, which aids band identification in immunoblotting, were used to test sera for IgG antibody to Osp antigens from Borrelia burgdorferi isolates from each subspecies (sensu stricto, afzelii, and garinii). Individual isolates were selected to include all five known European OspA genotypes. Of arthritis sera, 83% (n=29), and of acrodermatitis chronica atrophicans sera, 81% (n=26), recognized OspA, B, and/or C. Of erythema migrans sera, 23% recognized OspA and/or B and a further 15% OspC alone. Only 5 (6%) of 86 sera (4 arthritis, 1 acrodermatitis chronica atrophicans, 0 erythema migrans) recognized all five OspA phenotypes tested. Marked differences in the reactions of individual sera to the various Osp antigens were seen, which helps reveal the causes of discrepancies between previous reports.

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.

In order to evaluate the virtual advantage of proving borrelial infection in dermatoses clinically diagnosed as erythema (chronicum) migrans (ECM), acrodermatitis chronica atrophicans (ACA) or lymphadenosis cutis benigna (LCB) by means of histological, cultural or serological trials, skin and serum samples obtained from altogether 99 patients suffering from these dermatoses were examined. Serum and--to some extent--skin specimens gained from healthy individuals (n = 36), patients with not tick bite associated disorders of skin, internal organs and nervous system (n = 121) were used as controls. In addition, serum specimens from patients enduring circumscribed scleroderma (morphea), n = 31, and sera of Bavarian forest workers (n = 211) were proven for the presence of borrelial antibodies. Using an indirect immunofluorescence assay with borrelial strains, propagated in inbred mice, as antigens, elevated IgM- and/or IgG antibody titers were found in 51 (84%) out of 61 ECM-, in 17 (100%) ACA- and in 2 (33%) out of 6 LCB-sera. Antibody titers decreased significantly after adequate antibiotic therapy. The groups in comparison yielded spirochetal serum antibodies as follows: Healthy individuals and patients with various diseases 7 out of 157 (4.5%), morphea patients 7 out of 31 (23%) and forest workers 71 out of 211 (34%). Borrelia burgdorferi was cultivated from 6 out of 32 skin and from 1 blood specimen obtained from ECM patients and from 1 out of 5 ACA skin specimens. Borreliae could also be detected in the blood of 3 out of 11 thymusaplastic nude mice after intraperitoneal implantation of ECM-tissue. More frequently than borreliae spindle-shaped bacteria resembling fusobacterium sp. were detected not only by in vitro cultivation of ECM and ACA skin samples, but also in the blood of thymusaplastic mice after implantation of ECM-tissue. By Warthin-Starry silver strain borrelia-like structures were detected in 9 out of 28 ECM, in 3 out of 12 ACA and in 1 out of 5 LCB skin specimens. Therefore in our experience serological examinations turned out to be the most productive diagnostic tool.

In attempts to produce experimental Lyme disease, 33 rabbits were inoculated with Lyme spirochetes by tick feeding or from tick organ homogenates or cultures. Two rabbits developed erythema chronicum migrans at the site of inoculation, in one instance 2 days after injection of a tick organ homogenate and in the other instance, 17 days after feeding of infected Ixodes dammini ticks. Spirochetes were seen in skin biopsy specimens of the second lesion with Warthin-Starry and immunoperoxidase stains. Spirochetes were also recovered from blood cultures of two additional rabbits 2 weeks post-inoculation. These findings are characteristic of early Lyme disease in humans and give additional support for the spirochetal etiology of Lyme disease.

Spirochetes were cultivated from 17% of 114 Ixodes ricinus ticks in the Stockholm area. Three strains of these spirochetes were selected for studies by electron microscopy. These three strains had definite morphological similarities to spirochetes of the genus Borrelia, as judged by the number of flagella, absence of cytoplasmic tubules, and dimensions. The three strains were not identical, but seemed to consist of two different kinds of cells, one with eight and one with eleven flagella. The three strains were also shown to react with a monoclonal antibody that reacts with Lyme disease spirochetes (Borrelia burgdorferi), but not with strains of other Borreliae, Treponemes, or Leptospiras. These results indicate the possibility of transmission of Borrelia spirochetes from ticks to humans in Sweden. The antibody response to one of the spirochetal strains isolated from Swedish I. ricinus was studied in 37 patients with the typical clinical picture of erythema chronicum migrans (ECM), in 45 patients with chronic meningitis (CMe) cured by high-dose intravenous penicillin, in 298 patients with post-infectious arthritis, and in controls. The antibody response was estimated by indirect immunofluorescence assay (IFA) enzyme-linked immunosorbent assay (ELISA). The antibody levels differed significantly between patients with CMe and healthy individuals (p less than 0.001), both with respect to serum antibody levels and CSF-antibody levels. The antibody levels also differed significantly between patients with ECM and healthy controls as measured by ELISA (p less than 0.05), whereas the difference was not significant as measured by IFA. Five of 298 patients with post-infectious arthritis had higher titers than any of the controls, and two of these five patients had titers higher than any patient with CMe or ECM. These results indicate spirochetal aetiology of ECM, and in some patents with CMe or postinfectious arthritis. As a diagnostic test for ECM, both IFA and ELISA were of limited value, since only 5/37 (14%) ECM patients were positive by IFA, and 14/37 (38%) by ELISA. Regarding patients with CMe, 23/45 (51%) were seropositive by IFA and 30/45 (67%) by ELISA. However, measurement of CSF-antibodies were found to be a more sensitive method than measurement of serum antibodies both by IFA and ELISA, since 38/45 (84%) CMe patients were positive by IFA, and 41/45 (91%) by ELISA. In addition, estimation of CSF antibodies was also found to be a more specific method than estimation of serum antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

In a study on 121 consecutive patients with erythema migrans, 65 patients obtained oral penicillin, 36 tetracyclines, and 20 amoxicillin-clavulanic-acid. Follow-up was carried out for a median of 29, 17, and 7 months, respectively. In another limited trial on 29 patients with acrodermatitis chronica atrophicans (ACA), 14 patients received oral penicillin, 9 parenteral penicillin, and 6 tetracyclines. There was no statistically significant difference among treatment groups in both therapeutic trials, with the exception of different follow-ups due to the nonrandomized study design and different occurrence of the Jarisch-Herxheimer reaction in patients with erythema migrans. Later extracutaneous manifestations developed in 27% of the patients with erythema migrans and in 47% of the patients with ACA despite antibiotic therapy. We could not prove the superiority of any antibiotic tested in either early or late European Lyme borreliosis.

Lyme disease typically begins with a unique skin lesion, erythema chronicum migrans (ECM) (stage 1). Patients with this lesion may also have headache, meningeal irritation, mild encephalopathy, multiple annular secondary lesions, malar or urticarial rash, generalized lymphadenopathy and splenomegaly, migratory musculoskeletal pain, hepatitis, sore throat, non-productive cough, conjunctivitis, periorbital edema, or testicular swelling. After a few weeks to months (stage 2), about 15% of patients develop frank neurologic abnormalities, including meningitis, encephalitis, cranial neuritis (including bilateral facial palsy), motor or sensory radiculoneuritis, mononeuritis multiplex, or myelitis. At this time, about 8% of patients develop cardiac involvement--AV block, acute myopericarditis, cardiomegaly, or pancarditis. Throughout this stage, many patients continue to experience migratory musculoskeletal pain in joints, tendons, bursae, muscle, or bone. Months to years after disease onset (stage 3), about 60% of patients develop frank arthritis, which may be intermittent or chronic. Recently evidence suggests that Lyme disease may also be associated with chronic neurologic or skin involvement. Thus, Lyme disease occurs in stages with different clinical manifestations at each stage, but the course of the illness in each patient is highly variable.

Previous studies on cells of strains B31 isolated in the U.S.A. from Ixodes dammini and strain G25 isolated in Sweden from Ixodes ricinus, showed that their ultrastructure was similar, but not identical. For this reason the studies were extended to spirochetes isolated directly from patients with Lyme disease and related disorders. Included in the present study were three strains isolated from skin, blood and spinal fluid, respectively, from patients with Lyme disease, two strains from patients with erythema chronicum migrans and one strain from a patient with acrodermatitis chronica atrophicans. Three additional strains isolated in Sweden from Ixodes ricinus were also studied. All spirochetes were examined after negative straining with 1% ammonium molybdate. The cells of each individual strain were identical except for one strain isolated from a tick. This isolate was found to consist of two morphologically different spirochetes. Comparison of morphological features of cells from various isolates revealed certain differences. The cells of the different strains could be divided into at least four groups for which cell size and shape as well as number of flagella varied. By morphological criteria, all cells were found to belong to the genus Borrelia.

In Lyme's disease (LD) as well as in the European form of erythema chronicum migrans (ECM), the etiologic agents are spirochetes. As fas as we know by now, these microbes are closely related but not identical. Consequently, LD and ECM should be regarded as closely related but not as identical diseases. The sera of our 21 patients suffering from acrodermatitis chronica atrophicans (ACA) contained elevated antibody titers directed against the etiological agent found in ECM. These findings strongly suggest that ACA is also induced by spirochetes-possibly by the same microbes found in ECM.