Superficial Infections.

H. werneckii, the causative agent of tinea nigra, may be recovered on common fungal media but grows very slowly. Initial colonies of H. werneckii may be olive to black, shiny, and yeastlike (Figure 61-2), and usually grow within 2 to 3 weeks. As the cultures age, colonies become filamentous, with velvety gray aerial hyphae. P. hortae, the causative agent of black piedra, is easily cultured on any fungal culture medium lacking cycloheximide. Colonies of this organism are also very slow growing, appear dark brown to black, and also produce aerial mycelium. Some isolates may produce a red to brown diffusible pigment.

Mycetoma

Chromoblastomycosis.

The fungi known to cause chromoblastomycosis, Cladosporium, Cladophialophora, Phialophora spp., and Fonsecaea spp., are all dematiaceous. These fungi are slow growing and produce heaped-up, slightly folded, darkly pigmented colonies with a gray to olive to black and velvety or suedelike appearance. The reverse side of the colonies is jet black. Microscopic examination is necessary to identify the pathogenic agent definitively.

Phaeohyphomycosis.

The colonies of many of the rapidly growing dematiaceous molds are similar; therefore, identification relies on microscopic examination. The colonies of Alternaria spp. are rapidly growing, fluffy, and gray to gray-brown or gray-green. Curvularia spp. produce rapidly growing colonies that resemble those of Alternaria spp. Bipolaris spp. produce colonies that are gray-green to dark brown and slightly powdery, as do Drechslera spp. and Exserohilum spp.

The colonies of many of the slow-growing dematiaceous molds are also similar to one another and require identification based on microscopic morphology. E. jeanselmei and E. dermatitidis grow slowly (7 to 21 days) and initially produce shiny black, yeastlike colonies. E. dermatitidis often is mucoid and may be brown, compared with E. jeanselmei, but the two organisms are very similar in appearance. Colonies become filamentous and velvety with age as a result of the production of mycelium. The colonial morphology of other slowly growing dematiaceous fungi (e.g., Fonsecaea spp.) was described in the previous section.

White Grain Mycetoma: Pseudallescheria boydii and Acremonium spp.

As previously mentioned, these fungi are hyaline molds that produce septate hyphae. The features described here are useful for identification regardless of the disease process (i.e., mycetoma or hyalohyphomycosis). P. boydii is also involved in causing a variety of infections elsewhere in the body. These include infections of the nasal sinuses and septum, meningitis, arthritis, endocarditis, mycotic keratitis, external otomycosis, brain abscess, and disseminated invasive infection. Most of these more serious infections occur primarily in immunocompromised patients.

P. boydii is an example of an organism that exhibits both asexual and sexual reproduction. The teleomorphic, or sexual, form of this fungus, which is evidenced by the production of cleistothecia, is P. boydii; if asexual reproductive structures alone are observed, the organism may be called Scedosporium apiospermum. The asexually produced conidia of P. boydii/S. apiospermum are golden brown, elliptical to pyriform, and single celled and are borne singly from the tips of long or short conidiophores (annellophores) (see Figure 59-2). This anamorph (a fungus that disseminates reproductive structures without meiosis) predominates in cultures from clinical specimens. Another anamorphic form, the Graphium stage of P. boydii, may be seen less commonly. It consists of clusters of conidiophores with conidia produced at the ends; it has also been referred to as coremia (see Figure 59-3). The teleomorphic (sexual) form of the organism produces brown to black cleistothecia, which are pseudoparenychatous, saclike structures containing asci and ascospores. When the latter are fully developed, the large (50 to 200 µm), thick-walled cleistothecia rupture, releasing the asci and ascospores (see Figure 59-1). Ascospores are oval and delicately pointed at each end. Isolates of P. boydii may be induced to form cleistothecia by culturing on plain water agar; however, they are seldom found on primary recovery of a culture from a clinical specimen. Recognition of P. boydii is important, because the organism is resistant to amphotericin B, an antifungal agent commonly used for systemic infections.

Another Scedosporium species, S. prolificans, has been associated with infections other than mycetomas, such as arthritis or invasive disease in immunocompromised patients. S. prolificans differs from S. apiospermum in that it produces inflated, flask-shaped annellophores. The obsolete or previous name for S. prolificans was S. inflatum, which more accurately reflects the morphology of the conidiophore. Recognition of this organism also is important, because it is resistant to most if not all the commonly used antifungal agents.

Acremonium spp. develops hyaline hyphae and produces simple, unbranched, erect conidiophores. Single-celled conidia are produced loosely or in gelatinous masses at the tip of the conidiophore (see Figure 59-17). Intercalary and terminal chlamydoconidia may also be produced.

Black Grain Mycetoma: Exophiala jeanselmei, Curvularia spp., and Madurella mycetomatis.

Sterile hyphae are produced when M. mycetomatis is grown on rich fungal media. Nutritionally poor media may be used to induce sporulation. Long, tapering phialides with collarettes and sclerotia may be seen. Temperature tolerance, biochemical hydrolysis, and assimilation studies may be used to differentiate M. mycetomatis from Madurella grisea. (See Phaeohyphomycosis, later in the chapter, for the description of E. jeanselmei and Curvularia spp.)

Chromoblastomycosis: Cladosporium, Phialophora, and Fonsecaea spp.

The taxonomy of the organisms that cause chromoblastomycosis is complex. Their identification is based on somewhat distinct microscopic morphologic features. These are polymorphic fungi that may produce more than one type of conidiation. The genus Cladosporium includes species that produce long chains of budding, often fusiform, conidia (blastoconidia) that have a dark septal scar. Some of these organisms have been reclassified into the genus Cladophialophora, because they also produce phialides; however, for the most part, the genus name Cladosporium is used in this chapter.

The genus Phialophora includes species that produce short, flask-shaped to tubular phialides, each having a well-developed collarette. Clusters of conidia are produced by the phialides through an apical pore and often remain aggregated near the opening in a gelatinous mass. Phialophora spp. produce colonies that are wooly and olive-brown to brownish gray; some strains may appear to have concentric zones of color. Microscopically, hyphae are dematiaceous, and sporulation is common. Phialophora richardsiae produces phialides with distinct flattened or saucerlike collarettes (Figure 61-3). In contrast, Phialophora verrucosa produces deeper, more cup- or flask-shaped phialides. Pleomorphic phialides may also be seen with these species; however, all produce either or both hyaline elliptical conidia or brown elliptical conidia within the phialides.

The genus Fonsecaea includes organisms that exhibit a mixed type of sporulation. The genus produces a distinct Fonsecaea-type conidiophore, which somewhat resembles truncated Cladosporium-type sporulation. It may also produce a Rhinocladiella-type sporulation, in which single-celled conidia are produced on denticles that arise from all sides of conidiophores (sympodially). A mixture of the Fonsecaea, Rhinocladiella, and Cladosporium types may occur, and phialides with collarettes or Phialophora-type sporulation also may be present.

The diagnostic features of the Cladosporium, Phialophora, and Fonsecaea genera can be summarized as follows:

• Cladosporium (Cladosporium carrionii): Cladosporium type of sporulation with long chains of elliptical conidia (2-3 × 4-5 µm) borne from erect, tall, branching conidiophores (Figure 61-4).

• Phialophora spp.: P. verrucosa produces phialides, each with a distinct cup- or flask-shaped collarette (Figure 61-5); P. richardsiae produces phialides with a flattened collarette (see Figure 61-3). Conidia are produced endogenously and occur in clusters at the tip of the phialide.

• Fonsecaea spp.: Conidial heads with sympodial arrangement of conidia are seen, with primary conidia giving rise to secondary conidia (Figure 61-6). Cladosporium-type, Phialophora-type, and/or Rhinocladiella-type sporulation may also occur.

Phaeohyphomycosis: Alternaria, Bipolaris, Cladosporium, Curvularia, Drechslera, Exophiala, Exserohilum, and Phialophora spp.

A useful approach to identification of the dematiaceous molds is first to determine whether single-celled or multicelled conidia are produced. If conidia are produced singly, the laboratorian should determine whether they are produced individually or in chains (e.g., Cladosporium spp.). In cellophane tape preparations, the chains of conidia produced by Cladosporium spp. are easily disrupted. If multicellular conidia are produced, examining the septations within the conidium is useful. Multicellular conidia with septations in the horizontal axis of the conidium (i.e., the axis perpendicular to the longitudinal axis of the conidium) are characteristic of certain organisms, such as in Bipolaris, Curvularia, and Drechslera spp.; conidia with septations in both the longitudinal and horizontal axes of the conidium are characteristic of other fungi, such as Alternaria spp.

Alternaria spp.

Microscopically hyphae are septate and golden brown pigmented; conidiophores are simple but sometimes branched. Conidiophores bear a chain of large, brown conidia resembling a drumstick and contain both horizontal and longitudinal septa (Figure 61-7). Observing chains of conidia sometimes is difficult, because they may be dislodged as the culture mount is prepared.

Bipolaris spp.

Hyphae are dematiaceous and septate. Conidiophores, however, are characteristically bent (geniculate) at the locations where conidia are attached; conidia are arranged sympodially and are oblong to fusoid. The hilum protrudes slightly (Figure 61-8). Germ tubes are formed at one or both ends, parallel to the long axis of the conidium, when the fungus is incubated in water at 25°C for up to 24 hours (i.e., from both poles, thus the name Bipolaris).

Cladosporium spp.

Microscopically hyphae are septate and brown. Conidiophores are long, branched, and give rise to branching chains of darkly pigmented, budding conidia. Conidia usually are single celled and exhibit prominent attachment scars (dysjunctors). The cells that produce the branch points are often referred to as shield cells (Figure 61-9). This organism frequently fails to reveal chains of conidia on wet mounts, because conidia are so easily dislodged.

Curvularia spp.

Microscopically hyphae are dematiaceous and septate. Conidiophores are geniculate (i.e., bent where conidia are attached). Conidia are arranged sympodially and are golden brown, multicelled, and curved, with a central swollen cell (Figure 61-10). The end cells are lighter in color than the swollen cell.

Drechslera spp.

Microscopically the hyphae are septate and darkly pigmented, and conidiophores are geniculate. Conidia are produced sympodially (Figure 61-11). However, sporulation is generally sparse with this organism and is not commonly seen. The conidia of Drechslera spp. are impossible to differentiate from those of Bipolaris spp. based on morphologic criteria alone. The germ tube test may be used to differentiate these organisms. Conidia are placed in a drop of water, a coverslip is applied, and the organism is observed after an incubation period of at least 24 hours. Any of the cells of the conidia belonging to Drechslera spp. may germinate, and these may grow perpendicular to the long axis of the conidium. In contrast, only the end cells of the conidia belonging to Bipolaris spp. germinate, and these grow predominantly parallel to the longitudinal axis of the conidium.

Exophiala spp.

Only the Exophiala species E. jeanselmei and E. dermatitidis are considered here; although other species exist, they are recovered far less commonly in the clinical laboratory. The microscopic features of young colonies of Exophilia sp. exhibit dematiaceous, yeastlike cells (Figure 61-12). Although these may appear to be budding, close inspection may disclose that the daughter cells are produced by annellides rather than true buds. The microscopic features of young colonies of Exophilia sp. exhibit dematiaceous, yeastlike cells. Felt-like, filamentous colonies produce dematiaceous hyphae and conidiophores that are cylindrical and have a tapered tip. Annellations may be visible at the tip, and clusters of oval to round conidia are apparent (Figure 61-13). Potassium nitrate is utilized by E. jeanselmei but not by E. dermatitidis. Temperature studies are also useful for differentiating the most common Exophiala species. Both E. jeanselmei and E. dermatitidis grow at 37°C, but only E. dermatitidis can grow at 40° to 42°C.

Exserohilum spp.

Hyphae are septate and dematiaceous. Conidiophores are geniculate, and conidia are produced sympodially. Conidia are elongate, ellipsoid to fusoid, and exhibit a prominent hilum that is truncated and protruding (Figure 61-14). The conidia are multicellular, have perpendicular septa, and usually contain five to nine septa.