Comparison of PCR methods and culture for the detection of Borrelia spp. in patients with erythema migrans.
The sensitivities of two PCR assays and culture were compared for the detection of Borrelia spp. in skin specimens of 150 patients with typical erythema migrans. In addition, the accuracy of the methods for the identification of Borrelia spp. was compared by analysing culture isolates and material obtained directly from skin biopsy specimens. Borrelia burgdorferi sensu lato was isolated from 73 (49%) of 150 skin biopsy specimens. Using a nested PCR targeting the rrf-rrl region and a PCR targeting the flagellin gene, 107 (71%) and 36 (24%) specimens, respectively, were positive. With both PCRs, positive results were more frequent with culture-positive samples (67/73 (92%) and 24/73 (33%) for the nested and flagellin PCRs, respectively) than with culture-negative samples (40/77 (52%) and 12/77 (16%) for nested and flagellin PCR, respectively). Pulsed-field gel electrophoresis after MluI restriction identified 69/73 (95%) isolates, of which 58/69 (84%) were Borrelia afzelii and 11/69 (16%) were Borrelia garinii. After MseI restriction of PCR products amplified from the intergenic rrf-rrl region, B. afzelii was identified in 73/107 (68%) samples, B. garinii in 22/107 (21%) samples, and both species in 11/107 (10%) samples. The corresponding results for culture-positive specimens were 41/69 (59%), 14/69 (20%), and 7/69 (10%). Comparison of the results for specimens positive according to both approaches revealed complete uniformity in 80% of the cases. Overall, nested PCR was the most sensitive method for the demonstration of Borrelia spp. in erythema migrans skin lesions, followed by culture and PCR targeting the flagellin gene. The congruence of identification results obtained by analyzing culture isolates and material obtained directly from skin biopsies was relatively high but incomplete.
Cerar
T
T
Institute of Microbiology and Immunology, Faculty of Medicin, University of Ljubljana, Slovenia. tjasa.cerar@mf.uni-lj.si
Ruzić-Sabljić
E
E
Glinsek
U
U
Zore
A
A
Strle
F
F
D015373:Bacterial Typing Techniques; D001706:Biopsy; D015748:Borrelia burgdorferi Group / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000302:isolation & purification; D021901:DNA, Intergenic / Q000032:analysis; D016521:Electrophoresis, Gel, Pulsed-Field; D015787:Erythema Chronicum Migrans / Q000382:microbiology*; D005408:Flagellin / Q000032:analysis; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology*; D046509:Tissue Culture Techniques
D004269:DNA, Bacterial; D021901:DNA, Intergenic; D005408:Flagellin