Blastomyces dermatitidis. Microscopically, hyphae of the mold form of B. dermatitidis are septate and delicate and measure approximately 2 µm in diameter. Commonly, ropelike strands of hyphae are seen; however, these are found with most of the dimorphic fungi. The characteristic microscopic morphologic features are single, circular to pyriform conidia produced on short conidiophores that resemble lollipops (Figure 1); less commonly, the conidiophores may be elongated. The production of conidia in some isolates is minimal or absent, particularly on a medium containing blood enrichment.
Fig1. Mycelial form of Blastomyces dermatitidis shows oval conidia borne laterally on branching hyphae (×1000).
When incubated at 37°C, colonies of the yeast form develop within 7 days and appear waxy and wrinkled and cream to tan. Microscopically, large, thick-walled yeast cells (8 to 15 µm in diameter) with buds attached by a broad base are seen. Some strains may produce yeast cells as small as 2 to 5 µm, called micro forms. These small forms may resemble C. neoformans var. neoformans or H. capsulatum. Although these micro forms may be present, a thorough search should reveal more typical yeast forms. During conversion, swollen hyphal forms and immature cells with rudimentary buds are also likely to be present. Because the conversion of B. dermatitidis is easily accomplished, this is feasible in the clinical laboratory; however, this is the most appropriate instance in which mold to yeast conversion should be attempted. B. dermatitidis may also be identified by the presence of a specific band (i.e., A band) in the exoantigen test or by nucleic acid probe testing. In some instances, H. capsulatum, P. boydii, or T. rubrum might be confused microscopically with B. dermatitidis. The site of infection and the relatively slow growth rate of B. dermatitidis and careful examination of the microscopic morphologic features usually differentiates it from these fungi.
Coccidioides immitis. Microscopically some C. immitis cultures show small, septate hyphae that often exhibit rightangle branches and racquet forms. With age, the hyphae form arthroconidia that are characteristically rectangular to barrel shaped. The arthroconidia are larger than the hyphae from which they were produced and stain darkly with lactophenol cotton or aniline blue. The arthroconidia are separated by clear or lighter staining, nonviable cells (disjunctor cells). These types of conidia are referred to as alternate arthroconidia. Arthroconidia have been reported to range from 1.5 to 7.5 µm in width and 1.5 to 30 µm in length, whereas most are 3 to 4.5 µm in width and 3 µm in length. Variation has been reported in the shape of arthroconidia, ranging from rounded to square or rectangular to curved; however, most are barrel shaped. Even if alternate arthroconidia are observed microscopically, definitive identification should be made using nucleic acid probe testing. If a culture is suspected of being C. immitis, it should be sealed with tape to prevent chances of laboratoryacquired infection. Because C. immitis is the most infectious of all the fungi, extreme caution should be used in handling cultures of this organism. Safety precautions include the following:
1. If culture dishes are used, they should be handled only in a Level 3 BSC. Cultures should be sealed with tape if the specimen is suspected to contain C. immitis.
2. The use of cotton plug test tubes is discouraged, and screwcapped tubes should be used if culture tubes are preferred. All handling of cultures of C. immitis in screwcapped tubes should be performed inside a BSC.
3. All microscopic preparations for examination should be performed in a Level 3 BSC.
4. Cultures should be autoclaved as soon as final identification of C. immitis is made.
Other, usually nonvirulent fungi that resemble C. immitis microscopically may be found in the environment. Some molds, such as Malbranchea sp., also produce alternate arthroconidia, although these tend to be more rectangular; however, such species must be considered when making the identification. G. candidum and Trichosporon spp. produce hyphae that disassociate into contiguous arthroconidia; these should not be confused with C. immitis (Figure 2). The colonial morphologic features of older cultures of these fungi may resemble C. immitis, but as noted, the arthroconidia are not alternate. It is also important to remember that if confusion in identification does arise, or when occasional strains of C. immitis that fail to sporulate are encountered, identification by exoantigen or nucleic acid probe testing may be performed.
Fig2. Trichosporon spp. produce arthroconidia (A) and an occasional blastoconidium (B).
Histoplasma capsulatum. Microscopically the hyphae of H. capsulatum are small (approximately 2 µm in diameter) and are often intertwined to form ropelike strands. Commonly, large (8 to 14 µm in diameter) spherical or pyriform, smooth-walled macroconidia are seen in young cultures. With age, the macroconidia become roughened or tuberculate and provide enough evidence to make a tentative identification (Figure 3). The macroconidia are produced either on short or long conidiospores. Some isolates produce round to pyriform, smooth micro conidia (2 to 4 µm in diameter), in addition to the characteristic tuberculate macroconidia. Some isolates of H. capsulatum fail to sporulate despite numerous attempts to induce sporulation.
Fig3. Mycelial form of Histoplasma capsulatum produces characteristic tuberculate macroconidia (×1000).
Conversion of the mold to the yeast form is usually difficult and is not recommended. Microscopically a mixture of swollen hyphae and small budding yeast cells 2 to 5 µm in diameter should be observed. These are similar to the intracellular yeast cells seen in mononuclear cells in infected tissue. The yeast form of H. capsulatum cannot be recognized unless the corresponding mold form is present on another culture or unless the yeast is converted directly to the mold form by incubation at 25° to 30°C after yeast cells have been observed. The exoantigen test can be used for identification, but nucleic acid probe testing is now recommended as a definitive means of rapidly identifying this organism. Sepedonium sp., an environmental organism that grows on mush rooms, is always mentioned as being confused with H. capsulatum, because it produces similar tuberculate macroconidia. However, this organism is almost never recovered from clinical specimens, does not have a yeast form, fails to produce characteristic bands in the exoantigen test with H. capsulatum antiserum, and does not react in nucleic acid probe tests.
Paracoccidioides brasiliensis. Microscopically the mold form is similar to that seen with B. dermatitidis. Small hyphae (approximately 2 µm in diameter) are seen, along with numerous chlamydoconidia. Small (3 to 4 µm), delicate, globose or pyriform conidia may be seen arising from the sides of the hyphae or on very short conidiophores (Figure 4). Most often cultures reveal only fine septate hyphae and numerous chlamydoconidia.
Fig4. Mycelial form of Paracoccidioides brasiliensis shows septate hyphae and pyriform conidia singly borne (arrow) (×430).
After temperature-based conversion on a blood enriched medium, the colonial morphology of the yeast form is characterized by smooth, soft-wrinkled, yeastlike colonies that are cream to tan. Microscopically the colonies are composed of yeast cells 10 to 40 µm in diameter surrounded by narrow-necked yeast cells around the periphery, as previously described. If in vitro conversion to the yeast form is unsuccessful, the exoantigen test should be used to make the definitive identification of P. brasiliensis. Nucleic acid probe testing is not available for this organism. However, P. brasiliensis is known to cross react with B. dermatitidis. This cross reaction, in conjunction with microscopic and colonial morphology, epidemiologic data, and clinical features, may be used for definitive identification of this fungus.
Penicillium marneffei. At 25°C, P. marneffei grows rapidly and produces blue-green to yellowish colonies. A soluble red to maroon pigment, which diffuses into the agar, is highly suggestive of P. marneffei. At 37°C, conversion of mycelium to the infective, yeastlike form occurs in approximately 2 weeks. Oval, yeastlike cells (2 to 6 µm in diameter) with septa are seen; abortive, extensively branched, and highly septate hyphae may also be present.
Sporothrix schenckii. Microscopically, hyphae are deli cate (approximately 2 µm in diameter), septate, and branching. Single-celled conidia 2 to 5 µm in diameter are borne in clusters from the tips of single conidiophores (flowerette arrangement). Each conidium is attached to the conidiophore by an individual, delicate, threadlike structure (denticle) that may require examination under oil immersion to be visible. As the culture ages, single-celled, thick-walled, black pigmented conidia may also be produced along the sides of the hyphae, simulating the arrangement of microconidia produced by T. rubrum (sleeve arrangement) (Figure 5).
Fig5. Mycelial form of Sporothrix schenckii shows pyriform to-ovoid microconidia in a flowerette morphology at the tip of the conidiophore (arrow) (×750).
Because of similar morphologic features, saprophytic species of the genus Sporotrichum may be confused with S. schenckii, and they must be differentiated. During incubation of a culture at 37°C, a colony of S. schenckii transforms to a soft, cream-colored to white, yeastlike appearance. Microscopically singly or multiply budding, spherical, oval, or elongate, cigar-shaped yeast cells are observed without difficulty (Figure 6). Conversion from the mold form to the yeast form is easily accomplished and usually occurs within 1 to 5 days after transfer of the culture to a medium containing blood enrichment; most isolates of S. schenckii are converted to the yeast form within 12 to 48 hours at 37°C. Sporotrichum spp. do not produce a yeast form.
Fig6. Yeast form of Sporothrix schenckii consists of cigar shaped and oval budding cells (×500).
Table 1 presents a summary of the colonial and microscopic morphologic features of the dimorphic fungi in addition to other organisms previously discussed.
Table1. Summary of the Characteristic Features of Fungi Known to Be Common Causes of Selected Fungal Infection in Humans
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