PMC3320381_01
Clinical aspects of the lesion when the patient sought treatment at the University Hospital. The figure depicts the wide extension of the lesion in a frontal (A) and in depth (B) medial views.
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F1
At admission, the physical examination showed a tibial ulcer 15 cm in diameter with an infiltrating and nodular proximal border (Figure 1).
Biopsied section of the lesion: A) branch and broad hyphal fragment of Pythium insidiosum (Gomori methemine silver, magnification x200); B) hyphal fragment surrounded by inflammatory cells (hematoxylin and eosin [HE], magnification x200); C) hyphal fragment surrounded by eosinophilic material, which suggests a Splendore-Hoeppli phenomenon (HE, magnification x200).
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F2
Results of a second biopsy from the border of the ulcer again suggested zygomycosis (Figure 2).
Slide culture illustrating the broad, branched, and sparsely septate hyphae of Pythium insidiosum (lactophenol cotton-blue, magnification x40).
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F3
On microscopic examination, slide cultures stained with lactophenol cotton-blue showed broad, branched, and sparsely septated hyphae, without fruiting bodies (Figure 3), which later were identified as colonies of P. insidiosum.
Polymerase chain reaction with ITS4/ITS5 primers: lane 1, ladder 100 bp; lane 2, positive control showing Pythium brasiliensis band at position 634 bp; lanes 3 and 4, P. insidiosum isolate B01 showing band at position  850 bp.
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F4
The first round of amplification with the generic primers for fungi (ITS4/ITS5) produced weak bands; in a second round, double PCR, defined and sharp bands of approximately 850 bp were seen (Figure 4).