Diagnosing Treponema pallidum in Secondary Syphilis by PCR and Immunohistochemistry Marc Buffet, Philippe A. Grange, Philippe Gerhardt, Agnès Carlotti,

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Diagnosing Treponema pallidum in Secondary Syphilis by PCR and Immunohistochemistry Marc Buffet, Philippe A. Grange, Philippe Gerhardt, Agnès Carlotti, Vincent Calvez, Anne Bianchi, Nicolas Dupin Journal of Investigative Dermatology Volume 127, Issue 10, Pages 2345-2350 (October 2007) DOI: 10.1038/sj.jid.5700888 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Epidermal detection of T. pallidum. Four-micrometer-thick sections of skin biopsy samples were cut from a paraffin block and incubated with the polyclonal antibody anti-T. pallidum as described in Materials and Methods. Several bacteria ranging from (a) low, (b) moderate, and (c) high were detected in the samples. (d) Negative control obtained from skin biopsy sample from syphilis-negative patient. a, epidermis; b, superficial dermis; c, deep dermis. Arrows indicate the location of bacteria (original magnification × 40). Journal of Investigative Dermatology 2007 127, 2345-2350DOI: (10.1038/sj.jid.5700888) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dermal detection of T. pallidum. Four-micrometer-thick sections of skin biopsy samples were cut from a paraffin block and incubated with the polyclonal antibody anti-T. pallidum as described in Materials and Methods. T. pallidum was detected in (a) the perivascular area (original magnification × 40); in (b) a blood vessel (original magnification × 100; in (c) the pilary infundibulum (original magnification × 100); and in (d) the arrector pili muscle (original magnification × 40). a, epidermis; b, superficial dermis; ec, epithelial cell; vl, vascular lumen; ip, pilary infundibulum. Arrows indicate the location of bacteria. Journal of Investigative Dermatology 2007 127, 2345-2350DOI: (10.1038/sj.jid.5700888) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Detection of T. pallidum tp47 gene by PCR in human skin representative samples. PCR products were separated by 1% agarose gel and detected under UV in the presence of Bet. (a) tp47 amplification using KO3A and KO4 primers and (c) human albumin amplification products. (b) Southern blot with the internal KO17 probe as described in Materials and Methods. Lane 1: molecular mass standards. Lane 2: DNA from rabbit testis inoculated with T. pallidum Nichols strand used as positive control. Lane 3: negative control, distilled water only. Lane 4: sample from nonsyphilitic patient. Lanes 5–9: samples from syphilitic patient. PCR amplification product sequencing. (d) Comparison between consensus sequence of tp47 (GenBank accession no. AAC65545) and sequences obtained after PCR amplification. (e and f) Electrophoregrams obtained with KO3A and KO4 primers, respectively. Journal of Investigative Dermatology 2007 127, 2345-2350DOI: (10.1038/sj.jid.5700888) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions