Stains.

A mucormycosis may be diagnosed rapidly by examining tissue specimens or exudate from infected lesions in a calcofluor white or potassium hydroxide preparation. Branching, broad-diameter, predominantly nonseptate hyphae are observed (Figure 60-2). It is important that the laboratory notify the clinician of these findings, because mucorales grow rapidly, and vascular invasion occurs at a rapid rate.

Antigen-Protein.

Antigen-protein–based assays are not used for the diagnosis of mucormycosis.

Nucleic Acid Amplification.

Nucleic acid testing is not routinely used for the diagnosis of mucormycosis. These assays may be available in research settings.

Cultivation.

The colonial morphologic features of the mucorales allow immediate suspicion that an organism belongs to this group. Colonies characteristically produce a fluffy, white to gray or brown hyphal growth that resembles cotton candy and that diffusely covers the surface of the agar within 24 to 96 hours (Figure 60-3). The hyphae can grow very fast and may lift the lid of the agar plate (also known as a “lid lifter”). The hyphae appear to be coarse. The entire culture dish or tube rapidly fills with loose, grayish hyphae dotted with brown or black sporangia. The different genera and species of mucorales cannot be differentiated by colonial morphologic features, because most are identical.

Stains.

Calcofluor white or potassium hydroxide preparations reveal the presence of hyaline septate hyphae and/or arthroconidia (see Figures 59-4 and 60-6). Direct microscopic examination of infected hairs may reveal the hair shaft to be filled with masses of large arthroconidia (4 to 7 µm) in chains, characteristic of an endothrix type of invasion. In other instances, the hair shows external masses of spores that ensheath the hair shaft; this is characteristic of the ectothrix type of hair invasion. Hairs infected with Trichophyton schoenleinii reveal hyphae and air spaces within the shaft.

Antigen-Protein.

Antigen-protein—based assays are not useful for the detection or identification of dermatophytes.

Nucleic Acid Amplification.

Nucleic acid amplification assays for dermatophytes are not routine; these are available in research settings.

Cultivation.

Because the dermatophytes generally present a similar microscopic appearance in infected hair, skin, or nails, final identification typically is made by culture. A summary of the colonial and microscopic morphologic features of these fungi is presented in Table 60-1. Figure 60-7 presents an identification schema useful to the clinical laboratory for identification of commonly encountered dermatophytes. The schema begins with the microscopic features of the dermatophytes that may be visible in the initial examination of the culture. In many instances, the primary recovery medium fails to function as well as a sporulation medium. Often the initial growth must be subcultured onto cornmeal agar or potato dextrose agar to induce sporulation.

Trichophyton spp.

Microscopically, Trichophyton organisms are characterized by smooth, club-shaped, thin-walled macroconidia with three to eight septa ranging from 4 × 8 µm to 8 × 15 µm. The macroconidia are borne singly at the terminal ends of hyphae or on short conidiophores; the microconidia (which may be described as “birds on a fence”) predominate and are usually spherical, pyriform (teardrop shaped), or clavate (club shaped), and 2 to 4 µm (Figure 60-8). Only the common Trichophyton species are described here.

T. rubrum and T. mentagrophytes are the most common species recovered in the clinical laboratory. T. rubrum is a slow-growing organism that produces a flat or heaped-up colony, generally white to reddish, with a cottony or velvety surface. The characteristic cherry-red color is best observed on the reverse side of the colony; however, this is produced only after 3 to 4 weeks of incubation. Occasional strains may lack the deep red pigmentation on primary isolation. Two types of colonies may be produced: fluffy and granular. Microconidia are uncommon in most of the fluffy strains and more common in the granular strains; they occur as small, teardrop-shaped conidia often borne laterally along the sides of the hyphae (see Figure 60-8). Macroconidia are seen less commonly, although they are sometimes found in the granular strains, where they appear as thin-walled, smooth-walled, multicelled, cigar-shaped conidia with three to eight septa. T. rubrum has no specific nutritional requirements. It does not perforate hair in vitro or produce urease.

T. mentagrophytes produces two distinct colonial forms: the downy variety recovered from patients with tinea pedis and the granular variety recovered from lesions acquired by contact with animals. The rapidly growing colonies may appear as white to cream-colored or yellow, cottony or downy, and coarsely granular to powdery. They may produce a few spherical microconidia. The granular colonies may show evidence of red pigmentation. The reverse side of the colony is usually rose-brown, occasionally orange to deep red, and may be confused with T. rubrum. Granular colonies sporulate freely, with numerous small, spherical microconidia in grapelike clusters and thin-walled, smooth-walled, cigar-shaped macroconidia measuring 6 × 20 µm to 8 × 50 µm, with two to five septa (Figure 60-9). Macroconidia characteristically exhibit a definite narrow attachment to their base. Spiral hyphae may be found in one third of the isolates recovered.

T. mentagrophytes produces urease within 2 to 3 days after inoculation onto Christensen’s urea agar. Unlike T. rubrum, T. mentagrophytes perforates hair (Figure 60-10), a feature that may be used to distinguish between the two species when differentiation is difficult.

T. tonsurans is responsible for an epidemic form of tinea capitis that commonly occurs in children and occasionally in adults. It has displaced Microsporum audouinii as a primary cause of tinea capitis in most of the United States. The fungus causes a low-grade superficial lesion of varying severity and produces circular, scaly patches of alopecia (loss of hair). The stubs of hair remain in the epidermis of the scalp after the brittle hairs have broken off, which may give the typical “black dot” ringworm appearance. Because the infected hairs do not fluoresce under a Wood’s lamp, the physician should carefully search for the embedded stubs, using a bright light.

Cultures of T. tonsurans develop slowly and are typically buff to brown, wrinkled and suedelike in appearance. The colony surface shows radial folds and often develops a craterlike depression in the center with deep fissures. The reverse side of the colony is yellowish to reddish brown. Microscopically, numerous microconidia with flat bases are produced on the sides of hyphae. With age, the microconidia tend to become pleomorphic, are swollen to elongated, and are referred to as balloon forms (Figure 60-11). Chlamydoconidia are abundant in old cultures; swollen and fragmented hyphal cells resembling arthroconidia may be seen. T. tonsurans grows poorly on media lacking enrichments (casein agar); however, growth is greatly enhanced by the presence of thiamine or inositol in casein agar.

T. verrucosum causes a variety of lesions in cattle and in humans; it is most often seen in farmers, who acquire the infection from cattle. The lesions are found chiefly on the beard, neck, wrist, and back of the hands; they are deep, pustular, and inflammatory. With pressure, short stubs of hair may be recovered from the purulent lesion. Direct examination of the hair shaft reveals sheaths of isolated chains of large spores (5 to 10 µm in diameter) surrounding the hair shaft (ectothrix), and hyphae within the hair (endothrix). Masses of these conidia may also be seen in exudate from the lesions.

T. verrucosum grows slowly (14 to 30 days); growth is enhanced at 35° to 37°C and on media enriched with thiamine and inositol. T. verrucosum may be suspected when slowly growing colonies appear to embed themselves into the agar surface.

Kane and Smitka described a medium for the early detection and identification of T. verrucosum. The ingredients for this medium are 4% casein and 0.5% yeast extract. The organism is recognized by its early hydrolysis of casein and very slow growth rate. Chains of chlamydoconidia are formed regularly at 37°C. Early detection of hydrolysis, the formation of characteristic chains of chlamydoconidia, and the restrictive slow growth rate of T. verrucosum differentiate it from T. schoenleinii, another slowly growing organism. Colonies are small, heaped, and folded, occasionally flat and disk shaped. At first they are glabrous and waxy, with a short aerial mycelium. Colonies range from gray and waxlike to bright yellow. The reverse of the colony most often is nonpigmented but may be yellow.

Microscopically, chlamydoconidia in chains and antler hyphae may be the only structures observed microscopically in cultures of T. verrucosum (see Figures 59-10 and 59-16). Chlamydoconidia may be abundant at 35° to 37°C. Microconidia may be produced by some cultures if the medium is enriched with yeast extract or a vitamin (Figure 60-12). Conidia, when present, are borne laterally from the hyphae and are large and clavate. Macroconidia are rarely formed, vary considerably in size and shape, and are referred to as “rat tail” or “string bean” in appearance.

T. schoenleinii causes a severe type of infection called favus. It is characterized by the formation of yellowish cup-shaped crusts, or scutulae, on the scalp, considerable scarring of the scalp, and sometimes permanent alopecia. Infections are common among members of the same family. A distinctive invasion of the infected hair, the favic type, is demonstrated by the presence of large, inverted cones of hyphae and arthroconidia at the base of the hair follicle and branching hyphae throughout the length of the hair shaft. Longitudinal tunnels or empty spaces appear in the hair shaft where the hyphae have disintegrated. In calcofluor white or potassium hydroxide preparations, these tunnels are readily filled with fluid; air bubbles may also be seen in these tunnels.

T. schoenleinii is a slowly growing organism (30 days or longer) that produces a white to light gray colony with a waxy surface. Colonies have an irregular border consisting mostly of submerged hyphae, which tend to crack the agar. The surface of the colony is usually nonpigmented or tan, furrowed, and irregularly folded. The reverse side of the colony is usually tan or nonpigmented. Microscopically, conidia commonly are not formed. The hyphae tend to become knobby and club shaped at the terminal ends, with the production of many short lateral and terminal branches (Figure 60-13). Chlamydoconidia are generally numerous. All strains of T. schoenleinii may be grown in a vitamin-free medium and grow equally well at room temperature or at 35° to 37°C.

Trichophyton violaceum causes an infection of the scalp and body and is seen primarily in people living in the Mediterranean region, the Middle and Far East, and Africa. Hair invasion is of the endothrix type; the typical “black dot” type of tinea capitis is observed clinically. Direct microscopic examination of a calcofluor white or potassium hydroxide preparation of the nonfluorescing hairs shows dark, thick hairs filled with masses of arthroconidia arranged in chains, similar to those seen in T. tonsurans infections.

Colonies of T. violaceum are very slow growing, beginning as cone-shaped, cream-colored, glabrous colonies. Later these become heaped up, verrucous (warty), violet to purple, and waxy in consistency. Colonies may often be described as “port wine” in color. The reverse side of the colony is purple or nonpigmented. Older cultures may develop a velvety area of mycelium and sometimes lose their pigmentation. Microscopically, microconidia and macroconidia generally are not present; only sterile, distorted hyphae and chlamydoconidia are found. In some instances, however, swollen hyphae containing cytoplasmic granules may be seen. Growth of T. violaceum is enhanced on media containing thiamine.

Microsporum spp.

Species of the genus Microsporum are immediately recognized by the presence of large (8-15 × 35-150 µm), spindle-shaped, echinulate, rough-walled macroconidia with thick walls (up to 4 µm) containing four or more septa (Figure 60-14). The exception is Microsporum nanum, which characteristically produces macroconidia having two cells. Microconidia, when present, are small (3 to 7 µm) and club shaped and are borne on the hyphae, either laterally or on short conidiophores. Cultures of Microsporum spp. develop either rapidly or slowly (5 to 14 days) and produce aerial hyphae that may be velvety, powdery, glabrous, or cottony, varying in color from whitish, buff, to a cinnamon brown, with varying shades on the reverse side of the colony.

In past years, M. audouinii was the most important cause of epidemic tinea capitis among schoolchildren in the United States. This organism is anthropophilic and is spread directly by means of infected hairs on hats, caps, upholstery, combs, or barber clippers. Most infections are chronic; some heal spontaneously, whereas others may persist for several years. Infected hair shafts fluoresce yellow-green under a Wood’s lamp. Colonies of M. audouinii generally grow more slowly than other members of the genus Microsporum (10 to 21 days), and they produce a velvety aerial mycelium that is colorless to light gray to tan. The reverse side often appears salmon-pink to reddish brown. Colonies of M. audouinii do not usually sporulate in culture. The addition of yeast extract may stimulate growth and the production of macroconidia in some instances. Most commonly, atypical vegetative forms, such as terminal chlamydoconidia and antler and racquet hyphae, are the only clues to the identification of this organism. M. audouinii often is identified as a cause of infection by exclusion of all the other dermatophytes.

M. canis is primarily a pathogen of animals (zoophilic); it is the most common cause of ringworm infection in dogs and cats in the United States. Children and adults acquire the disease through contact with infected animals, particularly puppies and kittens, although human-to-human transfer has been reported. Hairs infected with M. canis fluoresce a bright yellow-green under a Wood’s lamp, which is a useful tool for screening pets as possible sources of human infection. Direct examination of a calcofluor white or potassium hydroxide preparation of infected hairs reveals small spores (2 to 3 µm) outside the hair. Culture must be performed to provide the specific identification.

Colonies of M. canis grow rapidly, are granular or fluffy with a feathery border, white to buff, and characteristically have a lemon-yellow or yellow-orange fringe at the periphery. On aging, the colony becomes dense and cottony and a deeper brownish-yellow or orange and frequently shows an area of heavy growth in the center. The reverse side of the colony is bright yellow, becoming orange or reddish-brown with age. In rare cases, strains are recovered that show no reverse side pigment. Microscopically, M. canis shows an abundance of large (15-20 × 60-125 µm), spindle-shaped, multisegmented (four to eight) macroconidia with curved ends (see Figure 60-14). These are thick walled with spiny (echinulate) projections on their surfaces. Microconidia are usually few in number, but large numbers occasionally may be seen.

Microsporum gypseum, a free-living organism of the soil (geophilic) that only rarely causes human or animal infection, occasionally may be seen in the clinical laboratory. Infected hairs generally do not fluoresce under a Wood’s lamp. However, microscopic examination of the infected hairs shows them to be irregularly covered with clusters of spores (5 to 8 µm), some in chains. These arthroconidia of the ectothrix type are considerably larger than those of other Microsporum species.

M. gypseum grows rapidly as a flat, irregularly fringed colony with a coarse, powdery surface that appears to be buff or cinnamon color. The underside of the colony is conspicuously orange to brownish. Microscopically, macroconidia are seen in large numbers and are characteristically large, ellipsoidal, have rounded ends, and are multisegmented (three to nine) with echinulated surfaces (Figure 60-15). Although they are spindle shaped, these macroconidia are not as pointed at the distal ends as those of M. canis. The appearance of the colonial and microscopic morphologic features is sufficient to make the distinction between M. gypseum and M. canis.

Epidermophyton sp.

E. floccosum, the only member of the genus Epidermophyton, is a common cause of tinea cruris and tinea pedis. Because this organism is susceptible to cold, specimens submitted for dermatophyte culture should not be refrigerated before culture, and cultures should not be stored at 4°C. In direct examination of skin scrapings using the calcofluor white or potassium hydroxide preparation, the fungus is seen as fine branching hyphae. E. floccosum grows slowly; the growth appears olive green to khaki, with the periphery surrounded by a dull orange-brown. After several weeks, colonies develop a cottony white aerial mycelium that completely overgrows the colony; the mycelium is sterile and remains so even after subculture. Microscopically, numerous smooth, thin-walled, club-shaped, multiseptate (2 to 4 µm) macroconidia are seen (Figure 60-16). They are rounded at the tip and are borne singly on a conidiophore or in groups of two or three. Microconidia are absent, spiral hyphae are rare, and chlamydoconidia are usually numerous. The absence of microconidia is useful for differentiating this organism from Trichophyton spp.; the morphology of the macroconidia (smooth, thin walled) is useful for differentiating it from Microsporum spp.

Stains.

Specimens submitted for direct microscopic examination containing organisms in this group demonstrate septate hyphae that usually show evidence of dichotomous branching, often of 45 degrees (Figure 60-17). In addition, some hyphae may have rounded, thick-walled cells. Although often considered to represent an Aspergillus species, these cannot be reliably distinguished from hyphae of Fusarium spp., Pseudallescheria boydii, or other hyaline molds.

Antigen-Protein.

Antigen-protein–based assays are beginning to be used to monitor patients at high risk for developing invasive fungal infections. One of these assays, the galactomannan assay, specifically targets antigens of Aspergillus spp., the most common source of invasive fungal infections caused by the hyaline septate molds (i.e., hyalohyphomycosis). The beta-glucan assay is designed to detect antigens common to all clinically important fungi. The ways these tests will be used in the future and how they will compare with nucleic acid amplification tests remain to be determined.

Nucleic Acid Amplification.

Nucleic acid amplification assays are not commonly performed to detect or identify these fungi. However, a variety of both broad-range assays (those that detect all fungi) and species-specific assays have been developed and in specialized centers may be used for patient care.

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization).

MALDI-TOF is a biophysical method that significantly reduces the time required to specifically identify fungal isolates and is being implemented in some large reference laboratories. An overview of the technique is discussed in more detail in Chapter 7.

Cultivation.

Because aspergilli are recovered frequently, it is imperative that the organism be demonstrated in the direct microscopic examination of fresh clinical specimens and/or that it be recovered repeatedly from patients with a compatible clinical picture to ensure that the organism is clinically significant. Correlation with biopsy results is the best means of establishing the significance of an isolate. Most Aspergillus spp. are susceptible to cycloheximide. Therefore, specimens submitted for recovery or subculture of these species should be inoculated onto media that lack this ingredient.

A. fumigatus is the most commonly recovered species from immunocompromised patients; moreover, it is the species most often seen in the clinical laboratory. Aspergillus flavus sometimes is recovered from immunocompromised patients and represents a frequent isolate in the clinical microbiology laboratory. Recovery of A. fumigatus or A. flavus from surveillance (nasal) cultures has been correlated with subsequent invasive aspergillosis; however, the absence of a positive nasal culture does not preclude infection. Aspergillus niger is commonly seen in the clinical laboratory, but its association with clinical disease is somewhat limited; this organism is a cause of fungus ball and otitis externa. Aspergillus terreus is a significant cause of infection in immunocompromised patients, but its frequency of recovery is much lower than that of the previously mentioned species. However, correct identification of A. terreus is important, because it is innately resistant to ampicillin B.

Aspergillus spp.

A. fumigatus is a rapidly growing mold (2 to 6 days) that produces a fluffy to granular, white to blue-green colony. Mature sporulating colonies most often have a blue-green, powdery appearance. Microscopically, A. fumigatus is characterized by the presence of septate hyphae and short or long conidiophores with a characteristic “foot cell” at their base. The foot cell is T or L shaped at the base of the conidiophore, but it is not a separate cell. The tip of the conidiophore expands into a large, dome-shaped vesicle with bottle-shaped phialides covering the upper half or two thirds of its surface. Long chains of small (2 to 3 µm in diameter), spherical, rough-walled, green conidia form a columnar mass on the vesicle (Figure 60-18). Cultures of A. fumigatus are thermotolerant and able to withstand temperatures up to 45°C.

A. flavus is a somewhat more rapidly growing species (1 to 5 days) that produces a yellow-green colony. Microscopically, vesicles are globose, and phialides are produced directly from the vesicle surface (uniserate) or from a primary row of cells called metulae (biserate). The phialides give rise to short chains of yellow-orange elliptical or spherical conidia that become roughened on the surface with age (Figure 60-19). The conidiophore of A. flavus is also coarsely roughened near the vesicle.

A. niger produces darkly pigmented, roughened spores macroscopically, but microscopically its hyphae are hyaline and septate, as are those of other aspergilli (i.e., it is not melanized). A. niger produces mature colonies within 2 to 6 days. Growth begins initially as a yellow colony that soon develops a black, dotted surface as conidia are produced. With age, the colony becomes jet black and powdery but the reverse remains buff or cream colored; this occurs on any culture medium. Microscopically A. niger shows septate hyphae, long conidiophores supporting spherical vesicles giving rise to large metulae, and smaller phialides (biserate), from which long chains of brown to black, rough-walled conidia are produced (Figure 60-20). The entire surface of the vesicle is involved in sporulation.

A. terreus is less commonly seen in the clinical laboratory; it produces tan colonies that resemble cinnamon. Vesicles are hemispherical, as seen microscopically, and phialides cover the entire surface and are produced from a primary row of metulae (biserate). Phialides produce globose to elliptical conidia arranged in chains. This species produces larger cells, aleurioconidia, which are found on submerged hyphae (Figure 60-21).

Fusarium spp.

Colonies of Fusarium spp. grow rapidly, within 2 to 5 days, and are fluffy to cottony and may be pink, purple, yellow, green, or other colors, depending on the species. Microscopically the hyphae are small and septate and give rise to phialides producing either single-celled microconidia, usually borne in gelatinous heads similar to those seen in Acremonium spp. (see Figure 59-17) or large, multicelled macroconidia that are sickle or boat shaped and contain numerous septations (Figure 60-22). Some cultures of Fusarium spp. commonly produce numerous chlamydoconidia. The most common medium used to induce sporulation is cornmeal agar. The keys to identification of Fusarium spp. are based on growth on potato dextrose agar.

Geotrichum candidum.

G. candidum often initially appears as a white to cream-colored, yeastlike colony; some isolates may appear as white, powdery molds. Hyphae are septate and produce numerous rectangular to cylindrical to barrel-shaped arthroconidia (Figure 60-23). Arthroconidia do not alternate but are contiguous, in contrast to Coccidioides immitis (Figure 60-24). Blastoconidia are not produced.

Acremonium spp.

Colonies of Acremonium spp. are rapid growing and also may appear yeastlike when initial growth is observed. Mature colonies become white to gray to rose or reddish-orange. Microscopically, small septate hyphae that produce single, unbranched, tubelike phialides are observed. Phialides give rise to clusters of elliptical, single-celled conidia contained in a gelatinous cluster at the tip of the phialide (see Figure 59-17).

Penicillium spp.

Colonies of Penicillium spp. are most commonly shades of green or blue-green, but pink, white, or other colors may be seen. The surface of the colonies may be velvety to powdery because of the presence of conidia. Microscopically hyphae are hyaline and septate and produce brushlike conidiophores (i.e., penicilli). Conidiophores produce metulae from which flask-shaped phialides producing chains of conidia arise (Figure 60-25). P. marneffei, a particularly virulent species in this genus, is discussed in the section on hyaline, septate, dimorphic molds.

Paecilomyces spp.

Colonies of Paecilomyces spp. are often velvety, tan to olive brown, and somewhat powdery. Colonies of P. lilacinus exhibit shades of lavender to pink. Microscopically Paecilomyces spp. resemble Penicillium spp. in that a penicillus is formed. However, the phialides of Paecilomyces spp. are long, delicate, and tapering (Figure 60-26), in contrast to the more blunted phialides of Penicillium spp.. The penicillus produces numerous chains of small, oval conidia that are easily dislodged. Single phialides producing chains of conidia may also be present.

Scopulariopsis spp.

Scopulariopsis spp. have been associated with onychomycosis, pulmonary infection, fungus ball and, more recently, invasive fungal disease in the immunocompromised host. Colonies of Scopulariopsis spp. initially appear white but later become light brown and powdery. Colonies often resemble those of M. gypseum. Microscopically a Scopulariopsis organism resembles a large Penicillium organism at first glance, because a rudimentary penicillus is produced. Annellophores produce the flask-shaped annellides, which support the lemon-shaped conidia in chains. Conidia are large, have a flat base, and are rough walled (Figure 60-27). The hyaline and septate species is S. brevicaulis. S. brumptii is a dematiaceous species and is occasionally recovered in the clinical laboratory; it has been reported to have caused a brain abscess in a liver transplant recipient.

Stains.

The microscopic morphologic features of the tissue forms, or what has been termed the parasitic forms, of the dimorphic fungi vary with the genus and are described for each.

Blastomyces dermatitidis.

The diagnosis of blastomycosis may easily be made when a clinical specimen is observed by direct microscopy. B. dermatitidis appears as large, spherical, thick-walled yeast cells 8 to 15 µm in diameter, usually with a single bud that is connected to the parent cell by a broad base (Figures 60-28 to 60-30). A smaller form (2-8 µm) is seen in rare cases.

Coccidioides immitis.

In direct microscopic examinations of sputum or other body fluids, C. immitis appears as a nonbudding, thick-walled spherule, 20 to 200 µm in diameter, that contains either granular material or numerous small (2 to 5 µm in diameter), nonbudding endospores (Figures 60-31 to 60-33). The endospores are freed by rupture of the spherule wall; therefore, empty and collapsed “ghost” spherules may also be present. Small, immature spherules measuring 5 to 20 µm may be confused with H. capsulatum or B. dermatitidis. Two endospores or immature spherules lying adjacent to one another may give the appearance that budding yeast is present. When identification of C. immitis is questionable, a wet preparation of the clinical specimen may be made using sterile saline, and the edges of the coverglass may be sealed with petrolatum and incubated overnight. When spherules are present, the endospores produce multiple hyphal strands.

Histoplasma capsulatum.

Direct microscopic examination of respiratory tract specimens and other similar specimens often fails to reveal the presence of H. capsulatum. However, an astute laboratorian may detect the organism when examining Wright- or Giemsa-stained specimens of bone marrow and, in rare cases, peripheral blood. H. capsulatum is found intracellularly in mononuclear cells as small, round to oval yeast cells 2 to 5 µm in diameter (Figure 60-34; also see Figure 60-6).

Paracoccidioides brasiliensis.

Specimens submitted for direct microscopic examination are important for the diagnosis of paracoccidioidomycosis. Large, round or oval, multiple budding yeast cells (8 to 40 µm in diameter) are usually recognized in sputum, mucosal biopsy specimens, and other exudates. Characteristic multiply budding yeast forms resemble a “mariner’s wheel” (Figure 60-35). The yeast cells surrounding the periphery of the parent cell range from 8 to 15 µm in diameter. Some cells may be as small as 2 to 5 µm but still exhibit multiple buds.

Penicillium marneffei.

Direct examination of infected tissues and exudates reveals that P. marneffei produces small, yeastlike cells (2 to 6 µm) that have internal cross-walls; no budding cells are produced (Figure 60-36). Like H. capsulatum, P. marneffei may be detected in peripheral blood smears with disseminated disease.

Sporothrix schenckii.

Exudate aspirated from unopened subcutaneous nodules or from open draining lesions often is submitted for culture and direct microscopic examination. Direct examination of this material usually has little diagnostic value, because demonstrating the rare characteristic yeast forms is difficult. S. schenckii usually appears as small (2 to 5 µm in diameter), round to oval, to cigar-shaped yeast cells (Figure 60-37). If stained using the periodic acid-Schiff (PAS) method in histologic section, an amorphous pink material may be seen surrounding the yeast cells (Figure 60-38).

Antigen-Protein.

Immunodiffusion methods (the exoantigen test) may be used to identify isolates of these organisms based on precipitation bands of identity between specific antibodies and fungal antigen extracts. However, these assays have been largely replaced by the more rapid nucleic acid hybridization reactions.

Nucleic Acid Amplification.

Nucleic acid amplification assays are not routinely performed but are available in some reference laboratories and in research settings. Real-time or homogeneous, rapid-cycle polymerase chain reaction (PCR) assays have been described for H. capsulatum and C. immitis. These assays have proved suitable for isolate identification. Perhaps the most significant advance in clinical mycology in the past few decades was the development of specific nucleic acid probes for identifying some of the dimorphic fungi (see Procedure 60-2 on the Evolve site). DNA probes (Gen-Probe, San Diego, California) are commercially available that are complementary to species-specific ribosomal RNA. Fungal cells are heat killed and disrupted by a lysing agent and sonication, and the nucleic acid is exposed to a species-specific DNA probe, which has been labeled with a chemiluminescent tag (acridinium ester). The labeled DNA probe combines with a ribosomal RNA of the organism to form a stable DNA : RNA hybrid. All unbound DNA probes are “quenched,” and light generated from the DNA : RNA hybrids is measured in a luminometer. The total testing time is less than 1 hour.

Nucleic acid probe identification is sensitive, specific, and rapid. Colonies contaminated with bacteria or other fungi may be tested; however, results from colonies recovered on a blood-enriched media must be interpreted with caution, because hemin may cause false-positive chemiluminescence. Nonetheless, nucleic acid probes should be used whenever possible to confirm the identification of an organism suspected of being H. capsulatum, B. dermatitidis, or C. immitis.

Cultivation.

The dimorphic fungi are regarded as slow-growing organisms, requiring 7 to 21 days for visible growth to appear at 25° to 30°C. However, exceptions to this rule occur with some frequency. Occasionally cultures of B. dermatitidis and H. capsulatum are recovered in as short a time as 2 to 5 days when many organisms are present in the clinical specimen. In contrast, when a small number of colonies of B. dermatitidis and H. capsulatum are present, sometimes 21 to 30 days of incubation are required before they are detected. C. immitis is consistently recovered within 3 to 5 days of incubation, but when many organisms are present, colonies may be detected within 48 hours. Cultures of P. brasiliensis are commonly recovered within 5 to 25 days, with a usual incubation period of 10 to 15 days. The growth rate, if slow, might lead the laboratorian to suspect the presence of a dimorphic fungus; however, considerable variation in the time for recovery exists. The exceptions to this slow growth are C. immitis and P. marneffei, which may be recovered within 3 to 5 days.

Textbooks present descriptions of the dimorphic fungi that the reader assumes are typical for each particular organism. As is true in other areas of microbiology, variation in the colonial morphologic features also occurs, depending on the strain and the type of medium used. The laboratorian must be aware of this variation and must not rely heavily on colonial morphologic features to identify members of this group of fungi.

The pigmentation of colonies is sometimes helpful but also varies widely; colonies of B. dermatitidis and H. capsulatum are described as being fluffy white, with a change in color to tan or buff with age. Some isolates initially appear darkly pigmented, with colors ranging from gray or dark brown to red. On media containing blood enrichment, these organisms may appear heaped, wrinkled, glabrous, neutral in color, and yeastlike; often tufts of aerial hyphae project from the top of the colony. Some colonies may appear pink to red, possibly because of the adsorption of hemoglobin from the blood in the medium. C. immitis is described as fluffy white with scattered areas of hyphae adherent to the agar surface, giving an overall “cobweb” appearance to the colony. However, numerous morphologic forms have been reported, including textures ranging from wooly to powdery and pigmentation ranging from pink-lavender or yellow to brown or buff.

The definitive traditional identification method for dimorphic fungus includes observing both the mold and tissue or parasitic forms of the organism. In general 25° to 30°C is the optimal temperature for recovery and identification of the dimorphic fungi from clinical specimens. Temperature (35° to 37°C), certain nutritional factors, and stimulation of growth in tissue independent of temperature are among the factors necessary to initiate the transformation of the mold form to the tissue form. Previously, B. dermatitidis and H. capsulatum were identified definitively by the in vitro conversion of a mold form to the corresponding yeast form through in vitro conversion on a blood-enriched medium incubated at 35° to 37°C; definitive identification of C. immitis involved conversion to the spherule form by animal inoculation. Except for C. immitis, the conversion of dimorphic molds to the yeast form can be accomplished with some difficulty (see Procedure 60-3 on the Evolve site). Some laboratories use the exoantigen test (see Procedure 60-4 on the Evolve site) to identify the dimorphic pathogens. However, this test requires extended incubation before cultures may be identified.

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 60-39); 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.

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 (see Figure 60-28). Some strains may produce yeast cells as small as 2 to 5 µm, called microforms. These small forms may resemble C. neoformans var. neoformans or H. capsulatum. Although these microforms 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 right-angle 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 (see Figure 60-24). 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 laboratory-acquired 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:

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 60-40; see Figure 60-23). 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.

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 60-41). The macroconidia are produced either on short or long conidiospores. Some isolates produce round to pyriform, smooth microconidia (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.

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 mushrooms, 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 60-42). Most often cultures reveal only fine septate hyphae and numerous chlamydoconidia.

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 (see Figure 60-35). If in vitro conversion to the yeast form is unsuccessful, the exoantigen test (see Procedure 60-4 on the Evolve site) 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 (see Figure 60-36).

Sporothrix schenckii.

Microscopically, hyphae are delicate (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 60-43).

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 60-44). 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.

Table 60-3 presents a summary of the colonial and microscopic morphologic features of the dimorphic fungi in addition to other organisms previously discussed.