**4. Diagnosis**

Histoplasmosis differential diagnosis usually begins with clinical presentation in a patient; however, these clinical signs and symptoms are not always specific because they are similar to those of other diseases like pulmonary tuberculosis, mistaking it for classical histoplasmosis. Unlike classical histoplasmosis which normally affects the lungs and at dissemination, affects the central nervous system (brain and the spinal cord); Osteoarticular, ganglionic, and very infrequently pulmonary sites are affected by African histoplasmosis [8, 20]. Rare incidences of urogenital skin injury often involve secondary skin invasions in people with widespread cases [21, 22]. African histoplasmosis also presents clinically as a "localized skin, bone, lymph node infections or as disseminated with multiple cutaneous lesions present all over the body, subcutaneous abscesses, enlarged lymph nodes, liver and spleen, and visceral organ enlargement [20, 23, 24]. Cutaneous manifestations are clearly isolated, and sometimes present with nodules, papules, or ulcers [20, 25]. Most frequent symptoms of Hcd include lesions mainly on the limbs, trunk, and face; osteoarticular infections in the spine, thorax, and bones of the upper and lower limbs; superficial and deep-seated lymphadenopathies. Rarely does Hcd cause infection in the Lungs, Adrenal glands, and nasal or buccal cavity, gastrointestinal tract (gastric or duodenal lesions), peritoneal cavity [7]. In general, Hcd manifestation is different from classical histoplasmosis, usually affecting bones and the skin and infrequently the lungs [4], and it has been observed in HIV infections less frequently than Hcc [26–29]. Microscopy and culture are the only readily available diagnostic tools in Africa; serological, immunological, radiological, and molecular methods are still lacking, and are therefore unable to be used to diagnose histoplasmosis (for example, by detecting the *H. capsulatum* circulating antigen in bodily fluids using an enzyme immunoassay methods) [30].

Histoplasmosis is diagnosed with microscopic histopathologic investigations of bone marrow aspirate or biopsy material, bronchoalveolar lavage fluid or lung biopsy material, sputum, urine, white blood cells in peripheral blood, and skin lesions [31–35]. Microscopically, Hcd is identified as an ovoid budding yeast with thick cell walls, bigger (6–12 m in diameter), and intracellular fat droplets [36]. On the histology

## *Epidemiology and Knowledge Gap of Histoplasmosis in Africa DOI: http://dx.doi.org/10.5772/intechopen.112084*

of a tissue specimen, Hcc however, shows up as 2–4um narrow-based budding yeast [36]. Other yeasts can be distinguished from Histoplasma yeasts by their dominant cellular location (intracellular for *H. capsulatum* and extracellular for *C*. *glabrata*), size and form variation (uniform versus heterogeneous), and histopathologic response (granulomatous versus suppurative). It is possible to distinguish between these infections by using particular histochemical stains, such as periodic acid-Schiff, Gomori methenamine silver, hematoxylin and eosin, and Giemsa [36]. A conclusive diagnosis of invasive infection is made by histopathologic analysis of the bone marrow [37]. These microscopic examinations are important in alerting the laboratory about a suspectible pathogen [38]. However, due to the *H. capsulatum* yeasts similar structure to other yeasts such as *Candida glabrata*, *Penicillium marneffei, Pneumocystis (carinii) jeroveci, Toxoplasma gondii, Leishmania donovani and Cryptococcus neoformans* many diagnostic techniques lack sensitivity and specificity which can lead to misidiagnosis [39–41].

Having access to a level 3 biosafety facility is necessary for culture since it poses a risk to laboratory staff and is not commonly found in African healthcare settings [17]. The culture, isolation, and confirmation of H. capsulatum from clinical and biological materials on selective media, such as Sabouraud agar, and incubation at 25°C for 6 to 12 weeks still serve as the basis for the final diagnosis. When incubated at 35–37°C, *H. capsulatum* molds are microscopically made up of hyaline septated hyphae with micro and tuberculate. Although the rate of conversion is modest and makes the procedure unusable as a diagnostic tool, it has been used to demonstrate that *H. capsulatum* is dimorphic. After conversion, smooth white to brown yeast colonies are seen and on microscopic examination, small round narrow budding yeasts are observed [38]. Gram-stained *H. capsulatum* exhibits poor staining compared to yeast cells from Candida and Cryptococcus, which are primarily extracellular macroconidia. Through culturing on enriched media like blood agar or Brain-Heart Infusion Agar (BHI) containing cysteine, *H. caspulatum* can be converted from to yeast phase. The majority of patients with an asymptomatic or moderate form of histoplasmosis have negative cultures since culture diagnosis is 100% specific but its sensitivity relies on the number of fungi present [41]. The most recent BACTEC type [42] is more successful than both lysis centrifugation and traditional biphasic blood systems [43, 44]. However, lysis centrifugation has more sensitivity than conventional and Bactec MYCO/F Lytic blood cultures for the recovery of *H. capsulatum* [45].

There are currently no molecular assays for *H. capsulatum* accepted by the FDA that are directly applicable to clinical specimens; however, laboratory-developed PCR assays using a range of target genes have been developed and show more sensitivity than cultures, such as between 59 and 100% [46] and 33 and 87% [47–49]. There is no recorded molecular method that has been used to detect histoplasmosis in Africa. The general lack of expertise in Africa to skillfully diagnose histoplasmosis makes it difficult to accurately diagnose histoplasmosis. Academic tertiary institutions provide theoretical knowledge but cannot provide practical high-end standard methods for diagnosing histoplasmosis. In situations where there is a minimal fungal burden, such as in asymptomatic or chronic pulmonary histoplasmosis, serology tests for anti-Histoplasma antibodies are incredibly helpful [1, 17]. Immunocompromised HIV-infected patients have lower sensitivity to antibody detection by immuno-diffusion or complement fixation than immunocompetent patients [50, 51]. Anti-H and anti-M antibody detection is the main focus of serologic diagnosis. Histoplasmin (HMIN) can be used to identify such antibodies. The antigenic extract of H. capsulatum mycelial culture is identified as HMIN. Centrifugation at 1050× g for 10 min is used to remove the cells, and the supernatant is then filtered through a 0.45 m membrane, concentrated and dialyzed

using phosphate-buffered saline (PBS) [52–57]. The M antigen is a catalase [58, 59] while the H antigen is a β-glucosidase [55]. Antibodies against the M and H antigens can be especially helpful in diagnosis due to their increased specificity to *H. capsulatum* [60–62]. Precipitating antibodies (H and M precipitin lines or bands) are qualitatively measured by the immunodiffusion (ID) test [63, 64]. Due to a noted rise in falsenegative results, these approaches should not be utilized in patients with the disseminated type of histoplasmosis. As Histoplasma-like antigens are present in patients with other prevalent pathogens such TB, lymphoma, sarcoidosis, and other fungal diseases [65], serologic cross-reactions may happen. Western blot test strips have shown high sensitivity and specificity rates [66]. However, Miravista Histoplasma antibody enzyme immunoassay (EIA) has proved to be more sensitive than other antibody assays, detecting both immunoglobulin G (IgG) and IgM antibodies, and complements antigen detection [67]. Thus becoming the best method to diagnose acute pulmonary histoplasmosis by combining antigen and EIA antibody tests. The invention of Histoplasma antigen testing has greatly aided in the diagnosis of disseminated histoplasmosis. A variety of EIA techniques have been used to find Hcc circulating antigens. Patients with immunosuppressed are more sensitive to disseminated histoplasmosis antigen testing in blood or urine, and individuals with a more severe illness have greater titers [68, 69]. The extent of the disease is proportional to the antigen level [70]. However, one of the gold standards for identifying histoplasmosis in immunocompromised patients remains Histoplasma antigen testing. The Histoplasma antigen does not interact with *C. neoformans* [71, 72]. Serology was only identified as being used to make a diagnosis in five African countries (Tanzania, Benin, South Africa, Egypt, and Uganda), and in three of those instances, the samples were processed in Western countries. Histology and culture were used to diagnose most reported cases from Africa [3].
