No Evidence for Borrelia burgdorferi-Specific DNA in Lesions of Localized Scleroderma

Abstract

A possible association of Borrelia burgdorferi with localized scleroderma is currently the focus of intense research and discussion. Skin biopsies from 30 patients with localized scleroderma (28 of the plaque type/morphea; two linear scleroderma) were analyzed for the presence of Borrelia burgdorferi using three different polymerase chain reaction systems for amplification of segments of borrelial genes. Formalin-fixed, paraffin-embedded biopsies of 14 patients and fresh-frozen, cryo-conserved biopsies of 16 patients with localized scleroderma were obtained. Lesions of all patients showed clear signs of scleroderma and disease progression at the time of biopsy. Fresh-frozen as well as formalin-fixed biopsies from patients with erythema migrans or acrodermatitis chronica atrophicans were used as positive controls. With all three polymerase chain reaction systems, borrelial DNA was detected in none of the 30 specimens of localized scleroderma. In contrast, with one polymerase chain reaction system, Borrelia burgdorferi-specific DNA was found in 24 of 27 frozen biopsies from patients with erythema migrans and in all 5 analyzed frozen biopsies of patients with acrodermatitis chronica atrophicans. In approximately half of the paraffin-embedded biopsies from patients with erythema migrans (nine of 23) and acrodermatitis chronica atrophicans (13 of 27), Borrelia burgdorferi-specific DNA was identified. These results question the association of localized scleroderma with known subtypes of Borrelia burgdorferi.

Methodological Aspects

“…However, nonspecific amplification products were common, and these strains isolated in Finland failed to hybridize with a probe specific for an American strain (143). The detection rate of B. burgdorferi DNA by PCR was higher in frozen skin tissues (90%) than in paraffin-embedded skin specimens (44%) from patients with EM (186).…”

“…PCR sensitivity was higher when fresh frozen skin specimens were used than when formalin-fixed, paraffin-embedded tissues were as specimens (186). Methods for DNA preparation from skin biopsy specimens or histological specimens have been reviewed recently (55).…”

“…Direct comparison of PCR results with different primers are given in a few studies with SF (22,78,107,123,134), CSF (32,101), urine (32,156), and skin biopsy (35,186) specimens. PCR results with chromosomal primers in SF were less sensitive than were results with plasmid primers, despite a similar analytical sensitivity (123,134).…”

“…No statistically significant differences between primer pairs were seen in CSF and skin specimens. However, with primers generating long amplicons (730 bp) compared to short ones (164 bp), reactivity in formalinfixed, paraffin-embedded tissue samples was reduced, probably because of extensive degradation of DNA during the process of fixation in formalin (186).…”

“…Here, culture-negative but PCR-positive patients are tested by a second PCR targeting a different gene. However, only a few studies have compared PCR with different genes (32,35,78,101,107,123,156,186), and the results are not always in accord. Sampling errors and DNA preparation methods do affect an individual assay when amplifying specimens with low numbers of target molecules, i.e., Ͻ20.…”