DIMORPHIC FUNGI

Dimorphic fungi are those fungi that can exist in two different morphological forms under two different environmental conditions. These fungi exist as moulds in their natural habitat and in vitro at room temperature and as yeasts in vivo or in vitro at 37^oC. Most of the dimorphic fungi are responsible for systemic or deep mycoses. For definitive identification of the fungus, yeast-to-mould conversion should be demonstrated. All dimorphic Isolates known to cause systemic mycoses must be handled with caution in a biological safety cabinet.

The dimorphic fungi are Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidiodes brasiliensis, Penicillium marneffi and Sporothrix schenkii.

Histoplasma capsulatum:

• Named after histological appearance of yeast cells within macrophage. Unlike its name, it is not capsulated.

• Histoplasma capsulatum has two varieties: H.capsulatum var capsulatum and H.capsulatum var duboisii. The latter is responsible for African histoplasmosis.

• Isolated from soil with high nitrogenous content, especially due to droppings of starling, chicken and bats.

• Infection is acquired following inhalation of air-borne conidia.

• Clinical manifestations are classified i) based on site as pulmonary, extrapulmonary and disseminated, ii) based on duration as acute, sub-acute and chronic and iii) based on pattern of infection as primary and reactivation.

• In cases of dissemination of H.capsulatum var. capsulatum infection, reticuloendothelial system is most frequently involved.

• H.capsulatum var duboisii rarely involves the lungs but commonly involves the bones and skin.

• Dissemination and fatal course are more common in the immunocompromised and elderly.

• Specimens collected are blood, urine, bone marrow, sputum and biopsy of cutaneous lesions.

• At 25�C on Sabouraud dextrose agar (SDA), the colonies are slow growing and granular to cottony in appearance. The color is white initially and usually becomes buff brown with age. Hyphae are septate and hyaline. It has both macro- and microconidia. Macroconidia are tuberculate, thick-walled, round, unicellular, hyaline and large. Microconidia are unicellular, hyaline and round, with a smooth or rough wall.

• At 37�C on Brain Heart Infusion Agar with blood creamy, slowly growing, moist and yeast-like colonies are formed. Narrow-based, ovoid, budding yeast cells are formed. Yeasts of var. capsulatum are smaller than (2-4 �m) those of var. duboisii (12-15 �m)

• Other methods of identifying are conversion to a yeast phase at 37�C, specific exoantigen testing, and nucleic acid hybridization methods

Blastomyces dermatitidis:

• It is a saprobe of the soil, specifically inhabits decaying wood material. Endemic in America and Africa.

• Blastomycosis in general is acquired by inhalation and initially presents with a pulmonary infection, which may later disseminate to other organs and systems. Primary cutaneous infection occurs due to direct inoculation of the fungus into the skin.

• Hematogenous spread of the organism results in infection of skin, bones, kidneys and male urogenital system, central nervous system, eyes, adrenal glands, uterus, ovaries, gastrointestinal tract, liver and spleen. Rarely meningitis may develop.

• Blastomyces dermatitidis has now emerged as an opportunistic pathogen in immunocompromised patients.

• The specimens collected are sputum, bronchial lavage and lung biopsy. Skin biopsy and urine may be collected.

• Application of both Gomori's methenamine silver stain (GMS) and periodic acid-Schiff (PAS) stain enables the detection of both host tissue reaction and the fungal cells effectively in infected tissues.

• Polymorphonuclear infiltration predominates in early lesions, while a granulomatous reaction and multinucleated giant cells appear later. The yeast cells may be located free or inside the giant cells.

• At 25�C, white to beige, membranous to woolly colony is formed following incubation for 7 days on potato dextrose agar. Septate hyaline hyphae with unbranched short conidiophores that arise at right angles to the vegetative hyphae are seen. The conidia are hyaline, unicellular, solitary and pyriform to globose in shape. 

• At 37�C, typically creamy and yeast-like colonies form following incubation for 7 days on brain-heart infusion agar. The yeast cells (8-12 �m in diameter) typically have thick walled refractile walls and a broad base attaching the bud to the parent cell.

• Conversion from the mycelial form to yeast form is possible only for few isolates pathogenic to humans. Other ways of verification of the identification are by use of exoantigen test, direct fluorescent antibody and nucleic acid probes.

Coccidioides immitis:

• The fungus is endemic only in regions of the Western Hemisphere that includes North, Central and South America.

•  Coccidioidomycosis is acquired from inhalation of the spores (arthroconidia). Once in the lungs, the arthroconidia transform into spherical cells called "spherules". Granulomatous inflammation usually occurs around developing spherules. Endospores are released when the wall of the spherule ruptures.

• The disease may be asymptomatic, acute pulmonary, chronic pulmonary or extrapulmonary. 50% of infected individuals don't develop any symptom. An acute respiratory infection occurs 7 to 21 days after exposure and typically resolves rapidly. In immunocompromised patients chronic pulmonary form presents as pulmonary nodules, peripheral thin-walled cavities, progressive pneumonia or pyopneumothorax. 

• Dissemination to extrapulmonary sites such as skin, soft tissue, osteomyelitis, arthritis and meningitis is known to occur.

• Specimens collected are sputum, tissue, CSF. Direct examination of clinical specimens in 10% KOH may show spherules (30-60 �m in diameter) with a thick wall (up to 2 �m) and endospores 2-5 �m in diameter.

• Isolation involves inoculating the clinical material onto BHI agar with 10% sheep blood and incubating at 30�C. Cultures should be kept 4 weeks before discarding as negative. 

• The fungus is fast growing and readily produces barrel-shaped arthroconidia with a disjunctor cell between each arthroconidium. Useful in vitro identification procedures include special conversion media, exoantigen tests, and DNA probes. 

• Slide cultures should not be set up when Coccidioides immitis is suspected due to its dangerous nature. It should be handled in Class II or III biological safety cabinet.

Paracoccidioides brasiliensis:

• Paracoccidioides has been isolated from soil and digestive tract of some animals. Paracoccidioides have been reported from Central and South America, particularly from Brazil, Venezuela, and Colombia. 

• It is the causative agent of a true systemic mycosis called paracoccidioidomycosis, which is also called South American blastomycosis.

• Inhalation of conidia is presumably the route of acquisition. The infection may become apparent several years after exposure to Paracoccidioides brasiliensis, suggesting the possibility of a long latent period. 

• The spectrum of the disease is wide, varying from an asymptomatic infection to a subclinical, symptomatic or chronic infection. In addition to the primary pulmonary form of the disease, acute pulmonary, chronic pulmonary, and disseminated forms may also be observed. In cases of disseminated paracoccidioidomycosis, reticuloendothelial system, skin and mucous membranes are frequently involved.

• Areas of granulomatous inflammation containing a focal area of central caseation mixed with pyogenic abscesses are usually present. Many giant cells are present in the granulomata, which contain the organisms.

• Direct examination of sputum, biopsy material or pus from suppurative draining lymph nodes using 10% KOH typically contain the yeast form. 

• Clinical material is inoculated onto Sabouraud dextrose agar and incubated at 30�C. Cultures should not be discarded as negative until 4 weeks.

• AT 25�C, the colonies are white cream, tan or brown, filamentous, slow growing, leathery, flat to wrinkled, woolly, cottony or glabrous to velvety. Microscopy shows hyaline, septate hyphae that are often sterile and do not sporulate. When present, conidia are oval, unicellular and with a broad base. Arthroconidia and intercalary chlamydospores may also be formed. 

• AT 37�C, white, heaped, wrinkled or folded yeast-like colonies are formed on enriched medium, such as brain heart infusion agar following 10 to 20 days of incubation. Typical multiple buds surrounding the whole surface of the mother yeast cell is seen. This appearance is variously described as "mariner's wheel" or "pilot wheel" or "Mickey mouse" appearance. The daughter cell (bud) is attached to the mother cell by a narrow neck portion.