Although the Lyme disease spirochete, Borrelia burgdorferi, is difficult to recover from infected human tissues and fluids, we believe it is likely that persistent live spirochetes or spirochetal fragments are driving the illness throughout much of its course. Evidence for this view includes the responsiveness of many patients to antibiotics even after long-term infection, the occasional sightings of spirochetes in affected tissues, the antigen-specific proliferation of lymphocytes from these patients, and time-dependent variations in the antibody responses to spirochetal antigens in some individuals. However, the recovery of spirochetes from infected tissues by culture is only occasionally possible, and in direct methods based on a strong, specific immunological response have so far demonstrated a fundamental lack of sensitivity and specificity. We propose to use the polymerase chain reaction (PCR) as a diagnostic tool for the detection of the Lyme disease spirochete. This ingenious method uses repeated cycles of oligonucleotide-directed DNA synthesis to amplify small numbers of target sequences to detectable levels in a matter of hours. PCR can be used to search for a genetic needle in a genomic DNA haystack, precisely the mission required for the direct detection of B. burgdorferi in human clinical samples. However, with new and powerful methods come problems of an equal magnitude. The greatest asset of PCR, high sensitivity, is tempered by a dangerous predilection for producing false positives, made worse because the products of the reaction itself (amplicons) may serve as carried-over substrates for future reactions. In addition to employing recommended procedures for reducing amplicon carryover, we have addressed this problem by: 1) having results verified in two outside laboratories, 2) employing amplicon sterilization techniques on a regular basis, and 3) using multiple genetic target loci for detection of the organism. Aside from addressing false positives, we feel the latter """"""""multi-locus"""""""" approach will be extremely useful for highlighting genetic differences between spirochetal isolates from different geographic areas, and even more importantly, for avoiding false negative results due to such differences when these methods are used on infected tissues. Thus, having set the stage with sterilization techniques and multiple genetic targets, and armed with preliminary data from old ticks, old (and young) mice, and human clinical specimens, we are equipped to proceed with large-scale, multi- center clinical and prospective trials of this diagnostic technology for the detection of B. burgdorferi in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032403-04
Application #
2067271
Study Section
Special Emphasis Panel (SRC (70))
Project Start
1991-09-30
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
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