The persistence of spirochetal nucleic acids in active Lyme arthritis

JF Bradley, RC Johnson, JL Goodman - Annals of internal medicine, 1994 - acpjournals.org
JF Bradley, RC Johnson, JL Goodman
Annals of internal medicine, 1994acpjournals.org
Methods Samples from patients and controls were obtained over a 10-month period from
both the university hospital and from clinicians in Minnesota and Wisconsin, areas where the
background seroprevalence of B. burgdorferi among healthy individuals is 1% to 2%. Lyme
arthritis was suspected in patients with mono-or oligoarticular large joint involvement,
seropositivity for B. burgdorferi, and no other known underlying disease. Controls were from
the same areas. They presented with various arthritic processes (see Results) and were …
Methods
Samples from patients and controls were obtained over a 10-month period from both the university hospital and from clinicians in Minnesota and Wisconsin, areas where the background seroprevalence of B. burgdorferi among healthy individuals is 1% to 2%. Lyme arthritis was suspected in patients with mono-or oligoarticular large joint involvement, seropositivity for B. burgdorferi, and no other known underlying disease. Controls were from the same areas. They presented with various arthritic processes (see Results) and were having arthrocentesis of involved joints. Synovial fluid was processed in a building where B. burgdorferi had never been cultivated. Synovial fluid (0.5 to 1.0 mL) was centrifuged at 16 000 g for 15 minutes, and the pellet was resuspended in 200 µL of supernatant fluid. One half was used for PCR, and the other was cultured as described previously [13] but with 0.1% agar. Nucleic acids were guanidium isothiocyanate-extracted and subjected to PCR [10] using primers 991 and 992 corresponding to nucleotides 16-43 and 222-247, respectively, of a chromosomal sequence that amplifies 231 bp of B. burgdorferi DNA [10]. The PCR products were subjected to electrophoresis in agarose and transferred to nylon membranes. A 167-bp digoxigenin-11-deoxyuridine triphosphate probe (nucleotides 81-247, reference [10]) was used for Southern hybridization as specified by the manufacturer (GENIUS kit, Boehringer-Mannheim, Indianapolis, Indiana). Confirmation studies used two different primer pairs (A2, A4 and A149, A319) and internal probes for the plasmid-encoded B. burgdorferi outer surface protein A gene [11]. Study personnel were blinded to the suspected disease for all but one patient sample. Immunoglobulin G antibody against B. burgdorferi was measured by enzyme immunoassay with seropositivity defined as optical density values≥ 3 standard deviations above the mean for 200 blood donors.
Results
Synovial fluid from six of seven (86%; 95% CI, 42% to 100%) persons thought to have Lyme arthritis was positive by PCR for B. burgdorferi DNA. Results from three of these (samples 5, 8, and 12) are shown (Figure 1). Results of the following tests were negative for all seven patients: rheumatoid factor, antinuclear antibodies, examinations for crystals, and aerobic and anaerobic cultures. All cultures for B. burgdorferi were negative.
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