Diagnosis of Lyme Borreliosis With a Novel, Seminested Real-Time Polymerase Chain Reaction Targeting the 5S-23S Intergenic Spacer Region: Clinical Features, Histopathology, and Immunophenotype in 44 Patients

Abstract

:Lyme borreliosis (LB) is the most common tick-borne infection in Europe and North America. Polymerase chain reaction (PCR) is an important tool to confirm the diagnosis, but not always successful, especially when organisms are sparse. We developed a novel, seminested real-time PCR assay [target: 5S-23S intergenic spacer region (IGS)] and compared it with 3 well-established conventional PCR assays (IGS/OspA/real-time IGS) on 596 formalin-fixed, paraffin-embedded routine skin biopsies. The seminested real-time assay identified 46 cases of borreliosis while 25, 27, and 38 were identified by the 3 other assays, respectively (P 0.01, P 0.02, and P 0.42; significance P < 0.05). Clinicopathologic and immunophenotypic analysis of PCR-positive cases revealed 38 erythema migrans (EM), 6 Borrelia lymphocytomas, and 2 acrodermatitis chronica atrophicans (ACA). In the 44 PCR-confirmed cases, plasma cells were present in only a third of EM cases. By contrast, CD123-positive plasmacytoid dendritic cells were common (74%) and therefore are unlikely to be helpful in the differential diagnosis between EM and tumid lupus erythematosus. A loss of CD34 in a third of all LB specimens limits its diagnostic value in the differential diagnosis with morphea. Interstitial macrophages were common in cutaneous LB (42/43) forming interstitial granulomas in a third of all cases, and 3/38 EM, 3/6 Borrelia lymphocytomas, and 1/2 ACA were only identified by the new seminested real-time assay, suggesting that it is especially helpful in confirming the diagnosis of Borrelia lymphocytoma.

No evidence of Borrelia in cutaneous infiltrates of B‐cell lymphomas with a highly sensitive, semi‐nested real‐time polymerase chain reaction targeting the 5S‐23S intergenic spacer region

“…For the detection of Borrelia-specific DNA, we used two assays including a recently developed, highly sensitive semi-nested PCR assay, which we previously demonstrated to be especially helpful in diagnosing Borrelia lymphocytoma. 18 Both PCR assays were negative in all specimens of B-cell lymphomas. In contrast, a positive PCR result could be detected in all pseudolymphomatous infiltrates of Borreliosis in our control group, the rt assay detecting 12/15 cases and the semi-nested approach identifying all of them.…”

“…The semi‐nested real‐time PCR assay targeting the 5S‐23S intergenic spacer (IGS) of Borrelia spp. was performed according to Podbicanin‐Ziburt et al 18 …”

“…In addition, samples were also analysed by conventional IGS PCR (fragment size: 250–300 bp) 18 . PCR products were sequenced (BigDyeTerminator V. 1.1 Cycle Sequencing Kit; Applied Biosystems, Darmstadt, Germany; ABI 3130 genetic analyser) and identified using the BLAST algorithm, GenBank.…”

“…The semi-nested real-time PCR assay targeting the 5S-23S intergenic spacer (IGS) of Borrelia spp. was performed according to Podbicanin-Ziburt et al 18 : Amplification of the first Borrelia IGS fragment (83 bp) was performed using primers 5 0 TTCTTC GCCTTAAAGCTCCT-3 0 (for.) and 5 0 -TGGCAAAATAGAGAT GGAAGAT-3 0 (rev.).…”

“…16,17 We recently developed a new semi-nested PCR approach to detect Borrelia-specific DNA in Formalin-fixed, Paraffinembedded (FFPE) skin biopsies which proved to be successful in biopsies of cutaneous Borreliosis in which three other conventional PCR assays had been negative. 18 The semi-nested assay was specifically helpful in detecting pseudolymphomatous manifestations of Borreliosis. The objective of this retrospective study is to investigate whether Borrelia spp.…”