Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions

Abstract

Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorfieri, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorfieri in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.

Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis

“…The positions of primers P A , P42, and IS2 are shown, and the sequences for P A and P42 are indicated. The sequence of IS2 has been described previously (30). ular characterization of uncultured B. burgdorferi, lysates of several I. scapularis ticks collected in New York State were tested.…”

Genetic Diversity of Borrelia burgdorferi in Lyme Disease Patients as Determined by Culture versus Direct PCR with Clinical Specimens

“…Skin biopsy and culture. Skin biopsy samples (2 mm) were obtained from the advancing border of primary erythema migrans lesions from patients enrolled in a prospective study at the Lyme Disease Diagnostic Center of the Westchester Medical Center, as previously described (25). Biopsy specimens were placed in a transport medium for later processing in the laboratory.…”

Induction of indoleamine 2,3-dioxygenase by Borrelia burgdorferi in human immune cells correlates with pathogenic potential

“…To simulate the temperature shifts that occur during tick-to-mammal transmission and that affect the expression of virulence factors, spirochetes were grown at 25°C, subcultured, and incubated at 37°C until spirochetes reached the mid-log phase of growth (4 3 10 7 -1 3 10 8 /ml). Spirochetes were enumerated and assessed for motility by dark-field microscopy [37]. Before coincubation with PBMCs, spirochetes were centrifuged for 10 min at 8000 g, washed twice with HBSS, and resuspended at a concentration of 5 3 10 8 /ml in RPMI 1640 containing 10% FBS.…”

“…Lysate was prepared from 50 ml cultures of B. burgdorferi B515 by use of glass bead-based lysis, as described previously [10]. Spirochete membrane disruption was confirmed by dark-field microscopy [37]. Lysate protein concentration was measured by the Bradford assay and adjusted to 1.0 mg/ml.…”