ABSTRACT. Lyme disease is caused by the bacterium Borrelia burgdorferi, and several closely related genospecies. Over the past 25 years, the number of Lyme disease cases have increased ~ 1500% making it the number one reported vector-borne disease in the United States. Upon infection, patients have mild, flu-like symptoms that can go on to be more severe. In later stages, even after antibiotic therapy, patients can experience heart, joint, and neurological problems. Despite the dramatic increase, and potentially devastating consequences, diagnosing Lyme disease is still a major challenge. Current diagnostic testing for Lyme disease is indirect— measuring an antibody response to the most common epitope(s). Indirect testing reports on exposure, not active infection. Furthermore, producing detectable titers can take weeks, and even still, the false negative rate is estimated to be high. The lead investigator of this proposal discovered that B. burgdorferi sheds ~45% of its peptidoglycan (PG)—the essential component of the bacterial cell-wall—from inside the cell, into its environment. B. burgdorferi PG can be detected in the synovial fluid of Lyme arthritis patients’ months after oral and/or intravenous antibiotics. Virtually all bacteria possess PG, but, as it turns out, the PG of B. burgdorferi is extremely unique, but conserved among Lyme Borreliae. These findings led us to hypothesize that the detection of shed B. burgdorferi PG fragments can act as a biomarker for both acute and late stages of the disease. Using previously developed methods, in conjunction with a novel Immuno-PCR approach, we will test the sensitivity and specificity of our assays, in experimentally infected mice and in human samples, at different stages of disease and treatment. Our studies promise to provide the first direct diagnostic test for Lyme disease.