Comparison of in vitro culture, immunohistochemical staining, and PCR for detection of Borrelia burgdorferi in tissue from experimentally infected animals

Abstract

An avidin-biotin-amplified immunophosphatase staining method with a purified polyclonal rabbit anti-Borrelia burgdorferi hyperimmune serum was developed for identification of B. burgdorferi in tissue specimens. The diagnostic efficacy was compared with those of in vitro culture and PCR with fresh and fixed, paraffin-embedded tissues. A nested PCR assay was developed for identification of a 276-bp fragment of the B. burgdorferi flagellin gene. The diagnostic sensitivities of the different techniques were evaluated with spleen, renal, and urinary bladder tissues from eight experimentally infected gerbils. A systemic infection was verified by positivity of 23 of 24 (96%) organ cultures. B. burgdorferi was visualized immunohistochemically in 9 of 23 (39%) of the specimens. Among these nine specimens, an average of 33% of the 15 sections examined were positive. The spirochetes accumulated in discrete clusters and were associated with focal lymphocytic infiltration. The diagnostic sensitivity obtained by PCR with fixed, paraffin-embedded tissue was 21%, considerably lower than that with fresh tissue (71%). Thus, the reliable demonstration of B. burgdorferi by immunohistochemical staining is possible but extremely laborious, and considering the fact that the density of B. burgdorferi in human tissue is even lower than that in experimentally infected animals, the method is not useful in a clinical setting. It may, however, still be valuable in pathogenetic research. Detection of B. burgdorferi DNA by PCR should be performed with fresh tissue specimens and not with fixed, paraffin-embedded specimens.