**3. The epidemiologic triad of histoplasmosis**

Like other infectious diseases, histoplasmosis results from the complex interaction between the pathogen and the susceptible host in a favorable environment that supports the transmission of the agent from the source to that host.

## **3.1 Agent**

Histoplasmosis is a worldwide distributed non-contagious fungal infection caused by *H. capsulatum*.

### *3.1.1 Characteristics of* H. capsulatum

*H. capsulatum* is a thermally dimorphic, primary systemic, and endemic fungal pathogen. *Thermal dimorphism* implies the existence of the pathogen in two different forms, depending on the temperatures. *H. capsulatum* presents itself, either in a hyaline mold in the environment or in the laboratory at 25–35°C, or in an intracellular budding yeast form in mammalian tissues or when grown on enriched medium in the laboratory at 37°C [41]. Thermal dimorphism is not restricted to the fungus morphology, but also implies the shift between the saprophytic, avirulent mycelial form, and the parasitic, pathogenic yeast form [42].

The fungus is characterized as a *primary pathogen* because of its ability to cause infection both in previous healthy individuals and immunocompromised hosts and *systemic* for its tendency to involve deep viscera after dissemination from the lungs [17, 41].

In 95% of immunocompetent individuals, the infection with *H. capsulatum* evolves usually benign and asymptomatic. Histoplasmosis is life-threatening particularly in immunocompromised patients, clinical manifestations, and the prognosis of the disease depending on the size inoculum and virulence of the infecting strain [17]. Since the beginning of HIV pandemic in 1980s, histoplasmosis has also been considered an AIDS-defining illness starting with 1987 [43–45].

*H. capsulatum* is also known as an *endemic* fungus, its natural habitat being delimitated to specific geographic regions and infection is acquired by inhalation of the spores from that specific environment or geographic area. Endemic areas for histoplasmosis include Mid-western and South-eastern parts of the United States (especially Ohio, Mississippi, and Missouri river valleys), Central and South America, sub-Saharan Africa, Eastern Asia, and Australia [46].

In the kingdom of fungi, *H. capsulatum* specie belongs to the phylum Ascomycota, family Onygenaceae [47, 48].

In fact, *H. capsulatum* is the asexual (anamorph) state of the fungus, which has a heterothallic form, designated Ajellomyces capsulatus or Emmonsiella capsulata (teleomorph or sexual state). The last is the perfect state of the fungus, being capable of producing sexual spores. When encountered and combined onto a sporulating medium, mating types (+) and (−) produce fruiting bodies containing asci. Although the mating type ratio in soil is 1:1, in isolates from patients the (−) mating type is found two to seven times more frequently than the opposite mating type [34, 49–52].

## *3.1.2 Classifications and mycology of* H. capsulatum

*H. capsulatum* was historically classified, according to geographic distribution, morphology, host-association, and clinical manifestations, into three varieties: (i) *H. capsulatum var. capsulatum*, responsible for classic histoplasmosis, causing pulmonary and disseminated infection worldwide; (ii) *H. capsulatum var. duboisii*, causing predominantly skin and bone lesions, mostly in the African continent; and (iii) *H. capsulatum var. farciminosum*, predominantly responsible for epizootic lymphangitis in equines. Morphologically, the two variants of *H. capsulatum* pathogenic for humans, cultured at 25°C, are macroscopically and microscopically indistinguishable. Growth is obtained on blood agar, chocolate agar, or Sabouraud's agar. Macroscopically, the mold is slowly growing as white (A type) or tan to brown (B type) colonies, usually in 2 to 6 weeks. The A type grows faster, is nonpigmented, and loses its ability to produce spores when subcultured. Also, yeast cells produced from the A type are less virulent in mice than those obtained from B type [49]. Microscopic evaluation of the mold reveals the mycelium, with septate, hyaline hyphae producing two types of conidia (**Figure 3**). Macroconidia are large (8–15 μm in diameter), spherical, thickwalled bodies formed on short, hyaline conidiospores. Their surface is decorated with spike-like or finger-like projections, hence the name tuberculate macroconidia, which represents the typical microscopic structure for presumptive diagnostic. Microconidia are small (2–4 μm in diameter), smooth-walled, oval to pyriform bodies, sessile or attached on short stalks, at right angles, on the sides of the hyphae. These conidia are considered infectious forms due to the small size that allows them to penetrate up to the level of the alveoli.

When cultured at 37°C on enriched media (such BHIA—brain heart infusion agar containing blood), both *H. capsulatum* var. capsulatum and *H. capsulatum* var. duboisii develop smooth, creamy, moist, and yeast-like colonies. Initially, the colony

#### **Figure 3.**

*H. capsulatum grows in the soil as a saprophytic mold (left). After inhalation, triggered by the temperature of the mammalian host, converts to the pathogenic yeast form (center) capable of intracellular replication within host macrophages (right) [53]. https://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-11-216.*

appears white or cream-colored and becomes gray with age. Microscopically, there are differences in yeast-like colonies between the two varieties of Histoplasma. Numerous round to oval budding, uninucleate yeast-like cells are revealed, small (2–4 μm) and thin-walled for *H. capsulatum* var. capsulatum, and large (8–15 μm) and thick-walled for *H. capsulatum* var. duboisii. The yeast cell reproduces by polar budding, resulting in the characteristic narrow bridge appearance (**Figure 3**) between the mother cell and the daughter cell [48, 49, 54].

The cultures of *H. capsulatum* are associated with high individual risk and require Class II Biological Safety (BSCII) precautions [49].

Between 1986 and 1992, Vincent, Spitzer, Keath, and their colleagues performed genetic studies by comparing soil, human or veterinary isolates and finally classified *H. capsulatum* into six genotypes or classes, correlated with a peculiar geographic distribution [55–57].

Using DNA sequence variations of protein-coding genes of isolates from six continents, in 2003, Kasuga and colleagues defined eight phylogenetic clades grouped in seven phylogenetic species, as follows: (i) North American class 1 clade (NAm 1); (ii) North American class 2 clade (NAm 2); (iii) Latin American group A clade (LAm A); (iv) Latin American group B clade (LAm B); (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade—harboring isolates from China, India, Thailand, Egypt, and England; (viii) African clade. Each clade represents a genetically isolated specie, with only one exception, the Eurasian clade, which originated from LAm A specie. Thus, the original classification of Histoplasma became obsolete, *H. capsulatum* var. capsulatum being found in all phylogenetic species, *H. capsulatum* var. duboisii belonging to African phylogenetic species, and *H. capsulatum* var. farciminosum being placed in NAm 2 and African phylogenetic species and, mainly, in the Eurasian clade [36].

The studies carried out by Teixeira and colleagues refined this classification, and the number of phylogenetic species was increased to 11. He split LAm A and LAm B phylogenetic species into LAm A1 and LAm A2, respectively LAm B1 and LAm B2. Also, he added to the classification two new phylogenetic species, Rio de Janeiro—RJ, and a bat-associated clade—BAC1 (from Mexico), the last one being renamed NAm 3 for his similarities with NAm 2 [58].

In 2019, another improvement in phylogenetic classification of histoplasma was made due to Damasceno's et al. research on HIV-positive patient isolates from the northeastern part of Brazil. Two new monophyletic species were added to the previous classification of *H. capsulatum*, named Northeast BR 1 and Northeast BR 2 [45].

Using whole-genome data, Sepúlveda advanced a more robust analysis, dividing 30 isolates into 5 independently evolving lineages, which he considered separate phylogenetic species. He proposed another classification of the genus Histoplasma as follows: *H. capsulatum* sensu stricto (referring to the Panamanian lineage—H81), *H. mississippiense* (formerly known as NAm 1), *H. ohiense* (formerly known as NAm 2), *H. suramericanum* (formerly known as LAm A), and African clade [59].

Depending on the chemical differences of the wall, the yeast form of *H. capsulatum* is classified as chemotype I, when the α-(1,3)-glucan layer is absent and the fibers are entirely β-linked or chemotype II, when the wall contains a mixture of an α and β-(1,3)-glucans [60].

Chemotype I appears to be more virulent, being accountable for most infections in immunocompetent individuals in North America. Additionally, in mouse models, same chemotype causes more severe forms of disease than chemotype II [61].
