**5. Culture and microbiology stains**

The gold standard for diagnosis of HPM is isolation of the fungus in culture and observation of characteristic intracellular yeasts in histopathology.

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

Culture sensitivity is low, the fungus requires several weeks to grow in a standard culture, and the laboratory should have safety level 3 to handle it.

In the microbiology laboratory, *H. capsulatum* can be detected by staining and direct microscopy of body fluids or tissue samples. *H. capsulatum* stains poorly with Gram stains, so it is rarely detected by this method. Fluorescent staining: calcofluor white, that binds chitin in the cell wall of the fungus, is useful for identifying H*. capsulatum* in clinical specimens.

*H. capsulatum* can be identified in culture after specimens are inoculated on appropriate medium: Sabouraud dextrose agar, and incubated at 25°C, allowing the fungus to grow.

Growth of the mycelial phase occurs at 25 to 30°C, and colonies usually appear in 2–3 weeks but can take up to 8 weeks. The colony is white to tan in color. After identification of a colony on solid medium, a lactophenol cotton blue test can be performed to determine the morphology of the mold. Initially, septate hyphae are seen, followed by smooth-walled (or less commonly spiny) microconidia (size 2–5 μm), and finally tuberculate macroconidia (size 7–15 μm), which are characteristic of *H. capsulatum* and have a distinct projection on their surface; this development depends on the maturity of the mycelia. Identification of the tuberculate macroconidia strongly suggests *H. capsulatum*, but the fungus belonging to the genus *Sepedonium* may also have such a structure. Therefore, a more definitive, specific test is needed to verify that the mold is *H. capsulatum* before a definitive diagnosis of HPM can be made. There are commercially available, highly specific molecular tests that allow rapid identification when applied to the isolate. There are also more complicated and time-consuming methods, such as the exoantigen test, which is less practical and requires a biosafety level 3 laboratory. They are being replaced by molecular tests.

Yeast-like colonies appear when plates are originally incubated at 37°C, and at microscopy will appear small round narrow-budding yeast. In vitro, the colony is cream-colored and becomes gray with age.

Incubation of the mold at 37°C will transform the mycelia phase into a yeast phase. This has been used in the past as a method to confirm *H. capsulatum* due to its dimorphic nature, but the conversion rate is low and laborious, and therefore, it cannot be used as a diagnostic tool.

There are some factors that influence the sensitivity of cultures to detect *H. capsulatum*: clinical manifestation (the highest positivity (74%) occurs in patients with disseminated chronic cavitary pulmonary HPM followed by acute disseminated pulmonary HPM (42%)) [19], host immunity and disease burden, exposure to a large inoculum for the organism. In other forms of HPM such as mild or moderate acute pulmonary HPM, granulomatous mediastinitis, mediastinal fibrosis, and chronic meningitis, cultures are usually negative. In PLWHA, up to 90% of respiratory cultures and 50% of blood cultures may be positive [25].

In disseminated histoplasmosis, specimens collected from the blood, as well as from affected organs such as bone marrow, liver, skin, and mucosal lesions, may result in isolation of *H. capsulatum*. The average growth time for *H. capsulatum* in blood cultures is between 12 and 15 days [34] and is rarely observed in conventional blood culture systems (where blood culture bottles are incubated only for 5 days). The lysis centrifugation system (isolator tubes) was more sensitive than automated systems for growing *H. capsulatum* from blood [35]. Hyphal forms and large, bizarre yeast shapes are seen on smears of blood cultures, rather than typical small, oval yeasts. If sputum or BAL is sent for culture, the laboratory should be informed of the suspected diagnosis to use selective medium (which adds ammonium hydroxide to the agar surface to

increase pH, which is helpful, decreases commensal fungal growth, and increases *H. capsulatum* growth).
