[Epidemiology and therapy of Lyme arthritis and other manifestations of Lyme borreliosis in Germany: results of a nation-wide survey]. / Epidemiologie und Therapie der Lyme-Arthritis und anderer Manifestationen der Lyme-Borreliose in Deutschland. Ergebnisse einer bundesweiten Arzteumfrage.

AIM OF THE STUDY: Only little is known about the epidemiology of Lyme borreliosis in Germany. As an example, it is still unclear if there are regional differences in the incidence of Lyme disease in general or of certain clinical manifestations like Lyme arthritis. Moreover, standardization of diagnostic or therapeutic procedures does not exist. Therefore, a Germany-wide questionnaire-based survey was conducted in order to achieve more epidemiological data and to obtain more information about the diagnostic and therapeutic approaches of general practitioners and specialists. METHODS: A self-designed questionnaire was distributed along with two editions of the journal "Deutsches Arzteblatt" (which is delivered to every physician in Germany) and additionally by a pharmaceutical company. During the collection period from March 1, 1998 to February 28, 1999, patients with Lyme disease were reported and information was given about site of infection, diagnostic procedures, clinical symptoms, treatment, and outcome. RESULTS: Altogether 3935 patients were reported. Their mean age was 43.4 years with the peak incidences around the ages of 10 and 60 years. 37.3% of the questionnaires were sent in by general practitioners, 17.6% by dermatologists, 15.7% by pediatricians, 9.7% by internists, and 2.7% by neurologists. 83% of the patients did not have a special infecion risk. The most frequent clinical Lyme manifestation was erythema migrans (EM), which occurred in 50.9% of the patients. 21.3% suffered from general symptoms. Of special interest, 24.5% of the patients had Lyme arthritis (14.7% mon- or oligoarthritis, 9.8% polyarthritis). Therefore, arthritis was more frequently reported than neuroborreliosis (18.4%). Only 16% of the neuroborreliosis patients and 32% of the arthritis patients remembered having had an EM. 189 patients (4.8%) with lymphadenosis cutis benigna and 100 patients (2.5%) with acrodermatitis chronica atrophicans were reported. In 80.4% of the patients, positive Lyme serology was detected. In a few cases, the diagnosis was established by isolation of borreliae, PCR or histology. 3754 patients were treated by antibiotics. The most frequently used compounds were doxycycline (50.4%), followed by ceftriaxone (22.4%), amoxicillin (13.6%), penicillin (7%), and erythromycin (4.2%) with differences depending on clinical manifestations and specialization of the prescribing physician. In less than 10% of the cases, not evaluated or recommended therapeutic procedures were performed. DISCUSSION: Lyme disease is endemic throughuot Germany. The most frequent manifestations are EM, followed by Lyme arthritis and neuroborreliosis. Less than one third of patients suffering from disseminated or chronic Lyme disease remembered an EM. Most of the physicians taking part in this survey follow treatment recommendations concerning choice of antibiotics and treatment durations.

Detection of Borrelia burgdorferi DNA in urine of patients with ocular Lyme borreliosis.

AIM: To evaluate the diagnostic value of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi DNA in patients with ocular Lyme borreliosis. METHODS: Of 256 consecutive uveitis patients six selected individuals with clinical evidence for Lyme borreliosis and 30 patients with non-Lyme uveitis were enrolled. Lyme serology was performed by ELISA and western blotting. Urine samples were examined by an optimised nested polymerase chain reaction (PCR) protocol. RESULTS: Only four of six uveitis patients suspected for Lyme borreliosis were ELISA positive, while all six subjects showed a positive western blot. B burgdorferi PCR was positive in all of these six patients. Whereas two of the 30 controls had a positive Lyme serology, B burgdorferi DNA was not detectable by PCR in any sample from these patients. CONCLUSIONS: PCR for the detection of B burgdorferi DNA in urine of uveitis patients is a valuable tool to support the diagnosis of ocular Lyme borreliosis. Moreover, these patients often show a weak humoral immune response which may more sensitively be detected by immunoblotting.

Rapid typing of Borrelia burgdorferi sensu lato species in specimens from patients with different manifestations of Lyme borreliosis.

To further investigate the pathogenic potential of different Borrelia burgdorferi genospecies, specimens from 27 patients with different manifestations of Lyme borreliosis were analyzed by PCR and reverse line blotting (RLB). In samples from Lyme arthritis patients, B. burgdorferi sensu stricto was predominantly identified, while in patients with neuroborreliosis or acrodermatitis, Borrelia garinii and Borrelia afzelii, respectively, were exclusively detected. The results demonstrate that PCR-RLB is a valuable tool for epidemiological and pathogenetic studies of Lyme borreliosis.

Insights from a novel three-dimensional in vitro model of lyme arthritis: standardized analysis of cellular and molecular interactions between Borrelia burgdorferi and synovial explants and fibroblasts.

OBJECTIVE: To develop a novel 3-dimensional (3-D) in vitro model of Lyme arthritis to use in the study of the interactions between Borrelia burgdorferi (Bb) and human synovial host cells with respect to phagocytosis and potential persistence of Bb as well as the induction of proinflammatory cytokines and chemokines. METHODS: Two distinct culture systems, consisting of synovial membrane explants or interactive synovial cells embedded in 3-D fibrin matrices, were chosen. Both systems were artificially infected with Bb, and the interactions between Bb and synovial tissue/cells were studied by histology, immunohistochemistry, and electron microscopy. Functional analyses included the induction/secretion of cytokines by Bb in the model system. RESULTS: Both culture systems proved to be stable and reproducible. The host cells and spirochetes showed high levels of viability and maintained their physiologic shape for >3 weeks. Bb invaded the synovial tissue and the artifical matrix in a time-dependent manner. Host cells were activated by Bb, as indicated by the induction of interleukin-1beta and tumor necrosis factor alpha. Electron microscopic analysis revealed Bb intracellularly within macrophages as well as synovial fibroblasts, suggesting that not only professional phagocytes, but also resident synovial cells are capable of phagocytosing Bb. Most interestingly, the uptake of the spirochetes appeared to cause severe damage of the synovial fibroblasts, since the majority of these cells displayed ultrastructural features of disintegration. CONCLUSION: A novel 3-D in vitro model has been established that allows the study of distinct aspects of Lyme arthritis under conditions that resemble the pathologic condition in humans. This reproducible, standardized model supplements animal studies and conventional 2-D cultures. The disintegration of synovial fibroblasts containing Bb or Bb fragments challenges the concept of an intracellular persistence of Bb and may instead reflect a mechanism that contributes to the inflammatory processes characteristic of Lyme arthritis.

Studies on the pathogenesis and treatment of Lyme arthritis.

Lyme arthritis is one of the most common clinical manifestations of Lyme borreliosis. It is caused by an intraarticular infection with Borrelia (B.) burgdorferi. A small number of bacteria are liable to provoke severe arthritis by inducing mechanisms (including the induction of cytokines and chemokines) that amplify the inflammatory response. The cellular immune response against B. burgdorferi is characterised by a predominant T helper cell type 1 (Th1) pattern that appears to be inadequate to overcome the infection. In most cases, Lyme arthritis may be cured by antibiotic therapy. A brief summary of current recommendations for the treatment of Lyme arthritis in adults and children is given in this article. However, about 10% of Lyme arthritis patients do not respond sufficiently to antibiotic treatment. Two not mutually exclusive pathogenetic concepts of these treatment-resistant cases will be discussed in the present study: persistent infection and infection-induced immunopathology.

Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy.

OBJECTIVES: To identify possible sites of bacterial persistence in patients with treatment resistant Lyme arthritis. It was determined whether Borrelia burgdorferi DNA may be detectable by polymerase chain reaction (PCR) in synovial membrane (SM) when PCR results from synovial fluid (SF) had become negative after antibiotic therapy. METHODS: Paired SF and SM specimens and urine samples from four patients with ongoing or recurring Lyme arthritis despite previous antibiotic therapy were investigated. A PCR for the detection of B burgdorferi DNA was carried out using primer sets specific for the ospA gene and a p66 gene of B burgdorferi. RESULTS: In all four cases, PCR with either primer set was negative in SF and urine, but was positive with at least one primer pair in the SM specimens. In all patients arthritis completely resolved after additional antibiotic treatment. CONCLUSIONS: These data suggest that in patients with treatment resistant Lyme arthritis negative PCR results in SF after antibiotic therapy do not rule out the intraarticular persistence of B burgdorferi DNA. Therefore, in these patients both SF and SM should be analysed for borrelial DNA by PCR as positive results in SM are strongly suggestive of ongoing infection.

Borrelia burgdorferi induces chemokines in human monocytes.

Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.

An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis.

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.