Histopathologic findings (hematoxylin & eosin, Giemsa) in 36 biopsy specimens taken from 30 patients with CL were studied to identify patterns of infiltrates, types of granulomas, accompanying epidermal changes, and composition of the infiltrate around granulomas as well as incidental findings such as vasculitis, necrosis of adnexal structures, and changes in the connective tissue. In 21 specimens in which intracytoplasmatic inclusions were few, the diagnosis of CL was confirmed by polymerase chain reaction (PCR) studies specific for leishmania performed on the very same paraffin-embedded tissue studied microscopically.

Total DNA was isolated (QIAamp DNA Mini Kit; QIAGEN) from 15 sections (5 mm). Leishmania-specific PCR fragments (about 250 bp) from Old World species were amplified according to Minodier et al.[7] PCR reactions were performed in 12.5-ml reaction mixtures (1x reaction buffer, HotStarTaq QIAGEN; 3.5 mM MgCl2; 1.2 mM of each primer; about 40–60 ng total DNA; 200 M of each dNTP; 2.5 units HotStarTaq DNA polymerase QIAGEN). The thermal profile was as follows: 1. 95ºC/15 min (1 cycle); 2. 94ºC/20 sec; 54ºC/30 sec; 72ºC/45 sec (35 cycles); 3. 72ºC/5 min (1 cycle); 4. 94ºC/20 sec; 52ºC/45 sec; 72ºC/30 sec (20 cycles); 72ºC/15 min (1 cycle). Paraffin blocks without tissue samples served as negative as well as cross contamination controls (every second sample). PCR products were analyzed in 2% agarose gels (Fig. 1). Leishmania-specific PCR products (about 250 bp) were purified by gel electrophoresis and subsequently sequenced (BigDye Terminator Cycle Sequencing Mix; Applied Biosystems; ABI 377 automatic sequencer). Sequences were further processed using BioEdit version 5.0.9 and identified using the NCBI BLAST search.