The aim of the proposed research is to develop an enabling technology that will allow development of a molecular diagnostic for identification and discrimination among closely related bacterial species that cause significant disease at low microorganism numbers. The technology will be advanced through the development of a diagnostic system for detection of species of Borrelia with special emphasis on the species Borrelia lonestari, a bacterium which has been found in about 1.2% of the Lone Star ticks removed from humans in nine southeastern US states. Lone Star ticks are associated with a condition called "southern tick associated rash illness" (STARI), which is similar to Lyme disease. However, B. lonestari has not been found in many patients suffering from STARI, perhaps because of the lack of diagnostic tests. In the proposed diagnostic, DNA from a patient's blood (or other specimen) is digested using a restriction enzyme to generate small DNA fragments carrying one or more species specific signature sequences. Heat is then used to separate the two strands of DNA. This is followed by a reaction that uses an enzyme to specifically join the ends of single stranded DNA fragments that have a specific sequence at each end to form a single "stranded DNA circle containing the pathogen" specific sequence. A reaction called rolling circle amplification is then performed to make a long DNA strand that is a tandem repeat of the circular DNA's complement. A second section of DNA is used to initiate a hyper branched strand displacement amplification reaction, yielding a 109 fold increase in the specific nucleic acid fragment in less than 90 minutes. In Phase II, we will extend this diagnostic to additional Borrelia species and have a certified laboratory perform the test procedure that was developed in Phase I on samples from human patients to determine the source of STARI. A fringe benefit of this work will be a sensitive diagnostic for Lyme disease.
Public Health Relevance: The proposed work would develop an enabling molecular technology for diagnosis of extremely low copy-number DNA from microorganisms that cause disease in humans. Some bacteria such as species of the genus Borrelia are able to cause significant disease at low copy" number. The proposed research will produce a diagnostic system that can simultaneously test for Lyme disease and all other Borrelia species suspected of causing disease in humans. The proposed research also seeks to discover the cause of "southern tick associated rash illness" (STARI). Lyme borreliosis is the most common vector borne disease in North America with more than 200,000 cases reported since 1982. In 2002, the incidence rate was 8.2 cases per 100,000 with approximately 95% of the cases occurring in the northeast, north central and mid Atlantic states. In the southern United States an additional species, Borrelia lonestari, has been shown to cause Lyme disease like symptoms (STARI). The incidence rate of STARI is currently unknown, because there is no molecular or serological diagnostic test to identify infection with B. lonestari and related Borrelia species. If Borrelia species other that the causative agent of Lyme disease are the cause of STARI, patients in the southeastern US that have a persistent infection currently remain undiagnosed, and untreated.
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