lyme disease is the most prevalent vector-borne illness in the United States. The causative agent of the disease is the spirochete, B. burgdorferi. The spirochete is transmitted to humans via the bite of an infected deer tick, Ixodes dammini. The disease is characterized in its early stages by a characteristic skin rash which may be accompanied by flu- like symptoms. Left untreated, the disease can progress to later manifestations which may include neurological and cardiac abnormalities and arthritis. Treatment with appropriate antibiotics during the earliest stages of the disease is usually successful. Later disease stages require longer and more aggressive antibiotic therapies and these treatments are often not effective. It is, therefore, critical that a reliable diagnostic test for the early detection of Lyme disease be available. Current laboratory diagnosis of Lyme disease is based on the detection of circulating antibodies to B. burgdorferi. These antibodies do not appear until 3-4 weeks after initial infection, are prone to false-negative and false-positive findings and results are often variable between different tests and laboratories. In addition, these methods do not recognize the presence of the spirochete itself, but the infected host's response. Thus, no reliable, direct test is presently available which can confirm the early diagnosis of Lyme disease in patients. The studies proposed herein are designed to produce a sensitive, specific and reliable diagnostic test for detection of B. burgdorferi at any stage of infection. The DNA sequences for the ribosomal RNAs of B. burgdorferi will be determined and oligonucleotide probes specific for this spirochete will be developed. In addition, sequences will be identified which can serve as primers for the amplification of portions of the rRNA genes by the polymerase chain reaction (PCR). The goal is to design a diagnostic assay which will be sensitive enough to detect <10 spirochetes in a small volume of clinical specimen (serum, urine, synovial fluid) and also have absolute specificity for B. burgdorferi to the exclusion of other microorganisms. The advantages of employing rRNA or its genes as the detection target are its abundance, mutational stability and well-defined function. The diagnostic probes will first be tested on spirochete-seeded samples and subsequently on clinical specimens obtained from patients with clinically defined early or late Lyme disease, those with symptoms consistent with Lyme disease but lacking well-defined clinical or serological evidence of the disease and healthy controls. The successful conclusion of these studies will result in a reproducible and sensitive diagnostic test for all stages of Lyme disease.
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