LYME DISEASE DIAGNOSIS BY PCR/DNA PROBE SYSTEM

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Current methods used for the diagnosis of Lyme disease depend upon serologic testing and are both insufficiently sensitive for the detection of early disease and insufficiently specific in the later stages. Direct detection of the causative organism Borrelia burgdorferi using a sensitive and specific assay appears to be the only solution to these problems. A DNA probe test coupled to amplification procedures such as the polymerase chain reaction (PCR) could provide extreme specificity and sensitivity as well as providing valuable information about the organism itself, eg. variation between strains (epidemiological data). In Europe, late-stage Lyme disease has most often been associated with neurological sequelae or chronic skin disease whereas in N. America, the most usual association is with Lyme arthritis. This disparity could be due to important differences between strains. B. burgdorferi has shown itself to be a highly heterogeneous group of organisms by both serologic and DNA homology studies. Thus far, attempts to design PCR systems capable of detecting all isolates of B. burgdorferi have met with limited success due to this genomic variability. The use of highly conserved genes, such as ribosomal RNA genes, has failed to provide the necessary specificity. However, prior studies by the P.I. have shown that another highly conserved gene ie. the flagellin gene is a potential candidate for both specific and sensitive detection. These preliminary studies have shown that the system is capable of both detecting B. burgdorferi specifically and differentiating B. burgdorferi strains into at least three distinct classes. The principal aim of the proposed research will be to develop this system into a specific and sensitive assay for the direct detection of Lyme disease which is at the same time able to differentiate B. burgdorferi into epidemiologically significant groups. Experiments will be undertaken to demonstrate that the PCR/DNA probe system is capable of amplifying an appropriate segment of DNA from all pathogenic isolates of B. burgdorferi without amplifying a corresponding segment of DNA from closely related species of human pathogenic Borrelia. In addition, experiments will be undertaken to test the probe sequences that have been developed for the differentiation of B. burgdorferi into distinct classes. The sequences will be tested against a very large number of strains from North America, Japan and Europe in order to determine the maximum number of sub-groups present within B. burgdorferi. This will proceed simultaneously with the development of probe sequences. Any new and unusual strains will be investigated in a detailed by cloning and sequencing of the relevant section(s) of their flagellin gene. Attempts will be made to establish a correlation between the groups and disease manifestation ie. neurologic versus arthritic sequelae. Experiments will also be undertaken to exhaustively assess the sensitivity of the primer/probe system in its final configuration. In addition, the ability of the primer/probe system to detect minute quantities of spirochetes which have been added to varying quantities of extraneous contaminating nucleic acids will be assessed. The aim of this work will be to determine the theoretical level of sensitivity of the assay under conditions which mimic those of real patient specimens. The question of optimal sample preparation for a variety of patient specimens will also be addressed. Samples which will be considered will include:skin punch biopsies, myocardial biopsies, blood, cerebro-spinal fluid, synovial fluid and urine. Finally, experiments will be carried out to demonstrate the clinical utility of the primer/probe system for the rapid diagnosis of Lyme disease using PCR and real patient samples.