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.