Borrelia-associated crystalline keratopathy with intracorneal detection of Borrelia garinii by electron microscopy and polymerase chain reaction.

PURPOSE: First report of a patient with Borrelia-associated crystalline keratopathy with intracorneal evidence of Borrelia garinii by polymerase chain reaction (PCR) and electron microscopy (EM). METHODS: Report of a 67-year-old patient with medical history of recurrent iridocyclitis and arthritis presented with a bilateral, progressive, asymmetric crystalline keratopathy, which was particularly pronounced in the peripheral temporal superior cornea. After penetrating keratoplasty, crystalline keratopathy with stromal haziness recurred. Corneal regrafting was performed. The corneal specimen from the penetrating keratoplasty was examined by light and EM as well as by PCR. RESULTS: In the explanted corneal graft, as well as retrospectively in the corneal specimen from the first keratoplasty, spirochetelike bodies and fragments were detected by light and EM. Borrelia burgdorferi sensu lato DNA was demonstrated by broad-range (16S rDNA) PCR. A more precise identification as Borrelia garinii serotype 5 was possible by analyses of the flaB and ospA gene sequences. Borrelia-specific serological tests showed borderline titers in immunofluorescence and weak reaction in immunoblot, respectively. CONCLUSIONS: This case illustrates that borreliae must be considered as a cause of crystalline keratopathy; Borrelia-specific serological tests can be false negative; explanted cornea specimens of etiologically unclear crystalline keratopathy should be analyzed by EM or PCR for detection of pathogens; and prolonged antibiotic treatment might be effective to prevent progression or recurrence of the disease.

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.

Ultrastructure of Borrelia burgdorferi after exposure to benzylpenicillin.

The aim of this study was to investigate the morphological changes of Borrelia burgdorferi associated with penicillin treatment. An isolate of B. burgdorferi from an erythema migrans lesion was cultivated in BSK II medium and exposed to increasing concentrations (0.0625 mg/l-2 mg/l) of penicillin G for 5 days. The in vitro minimal inhibitory concentration (MIC) was determined to be 0.5 mg/l by broth dilution method. The morphological structures of untreated spirochetes, as well as their characteristic ultrastructural changes when exposed to penicillin, were observed by electron microscopy. The following alterations were discovered: (i) Numerous outer sheath blebs at a penicillin concentration of 0.0625 mg/l. (ii) A characteristic irregular waveform of the borrelial cells and complete loss of the outer sheath at a penicillin concentration of 0.125 mg/l. (iii) The presence of "spheroplasts" at the same concentration. (iv) Structural changes of the protoplasmic cylinder complex which showed an irregular pattern at a penicillin concentration of 0.125 mg/l. (v) Disruption of the protoplasmic cylinder complex into several parts at penicillin concentrations of 0.25 mg/l and 0.5 mg/l. (vi) Severe cytolysis at penicillin concentrations of 1 mg/l and 2 mg/l.