**6. Histopathology and cytology**

The presence of yeast cells consistent with *H. capsulatum* in the tissues allows a presumptive diagnosis of HPM. The organism is sufficiently characteristic to allow diagnosis of proven HPM according to the European Organization for Research and Treatment of Cancer (EORTC) and Mycoses Study Group Education and Research Consortium (MSGERC) consensus guidelines [36]. *H*. *capsulatum* is a non-encapsulated organism. The "capsule" noted in the original initial description report that gave the species its name was an artifact of tissue processing.

*H*. *capsulatum* var. *capsulatum* appears as 2–4 μm narrow, based budding yeast with thin, non-refractile cell walls, stained with Gomori methenamine silver (GMS) or periodic acid-Schiff stains (PAS). Yeasts are typically found intracellularly, phagocytosed in macrophages and histiocytes, often in clusters of many organisms, but can also be found in extracellular spaces, free in tissues. In bone marrow samples, Giemsa staining helps visualize the yeast forms. Wright-Giemsa staining is also applied to peripheral blood and smears to identify intracellular clusters of budding yeasts in patients with disseminated disease. Hematoxylin-eosin (H&E) staining detects *H*. *capsulatum* when the organism load is very high (otherwise too insensitive).

*H*. *capsulatum* var. *duboisii*, the causative agent of African histoplasmosis, is larger (6–12 μm) and easily distinguished; it can be seen as short chains in tissues.

Some organisms can mimic the appearance of *H*. *capsulatum* in tissues. However, the clinical picture and specific histochemical stains that show a different appearance on histopathological examination help to distinguish *H*. *capsulatum* from other organisms, such as *Cryptococcu*s spp., *Balstomyces* spp., *Candida glabrata*, *Pneumocistis* spp., *Coccidioides* spp., *Talaromyces* spp., *Leishmania* spp., *Toxoplasma gondi*, and *Trypanosoma cruzi*.

In an appropriate clinical context (e.g., acute pneumonia), the presence of *H. capsulatum* yeasts in certain tissues or sterile body fluids (e.g., skin lesions) is indicative of acute infection.

The histopathologic examination requires invasive procedures such as bronchoscopy or biopsies. It is more useful in disseminated HPM than in localized pulmonary HPM and is more likely to be positive in the subacute or chronic form than in the acute [6].

Sometimes non-viable organisms can be found in mediastinal or pulmonary granuloma tissue years after initial infection. Pathology may show incomplete granulomas and/or fibrosis rather than a well-formed pyogranulomatous reaction. Complementary tests such as negative cultures, antigenemia, and luck in symptoms can help distinguish between healed, old disease, and active infection.

Patients with severe disease with diffuse pulmonary infiltrates are likely to have organisms detected on lung biopsy. In patients with granulomatous mediastinitis, the caseous specimen collected from necrotic nodules may contain some yeast-like *H*. *capsulatum*. In biopsies from patients with fibrosing mediastinitis, organisms are not usually detected in the fibrotic tissue.

Examination of fluids or tissue aspirates for individual cells can provide narrowbased evidence for HPM. Cytologic specimens stained with GMS or PAS show closely spaced budding yeast cells, mainly in macrophages.

*Histoplasmosis: Laboratory Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.112411*

It is uncommon to find *H*. *capsulatum* on cytologic examination of sputum unless it is a burden infection. Cytopathologic examination of BAL has a sensitivity of 50% for acute pulmonary HPM [37]. When cytopathologic examination BAL is combined with fluid antigen testing, the sensitivity increases to 97% [15].

Fine needle aspiration is a method that can provide a cytodiagnosis of HPM when performed on lymph nodes, adrenal glands, or other tissue.
