Diagnosis

The diagnosis of PCM requires the identification of the fungus in any clinical specimen by direct mycological examination, fungal culture, and histopathology or by molecular detection.

For direct examination, better results are obtained when the sample is clarified with KOH (10%) demonstrating large yeast cells with thick, birefringent walls and multipolar budding. Isolation of the fungus may be obtained by culturing on Sabouraud glucose agar, brain-heart infusion agar, Mycosel®, or glucose-peptone- yeast extract (GPY) agar with incubation at 25 and 35 °C to confirm dimorphism.

Fine needle aspiration cytology is indicated for solid tissues, and the puncture should be guided by equipment that provides a high-resolution image. The aspirate may also be employed for culturing.

Histopathological demonstration of Paracoccidioides spp. is enhanced by staining with Gomori-Grocott or PAS. Special attention is required when the histo­logical sections show only some small yeast elements with single buddings, since small forms of P. brasiliensis exist. In this situation, culturing, immunohis­tochemistry, or molecular biology should be employed for differential diagnosis.

Serological tests are important, both for diagnosis and for monitoring treatment. The most employed techniques are double immunodiffusion in agar gel (ID) and immunoenzymatic assays, such as ELISA (enzyme-linked immunosorbent assay), which present high sensitivity and specificity. Cryptic species in Paracoccidioides may have an impact on immunodiagnosis of PCM. PCM patients from the southeast of Brazil were 100% positive in serology when tested with B339 antigen from P. brasiliensis. When the same sera were tested using an antigen preparation from P. lutzii 510B isolate, positivity decreased to 41%. Conversely, patients from the midwest of Brazil tested 92% positive with antigens from P. lutzii and 26% with P. brasiliensis antigen (Batista et al. 2010; Gegembauer et al. 2014).

Molecular techniques have high sensitivity and specificity and have a great potential for diagnosis and environmental detection. The most frequently used technique is nested PCR with primers derived from rDNA. Also panfungal primers ITS1-ITS4 or ITS4-ITS5 (White et al. 1990) or species-specific primers for P. brasiliensis and P. lutzii can be used (Theodoro et al. 2005; Arantes et al. 2013).

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