Chapter 60 1. Describe where mucorales (zygomycetes) are found, how they are transmitted to humans, and the diseases they cause. 2. Describe the characteristic colony morphology of the mucorales (zygomycetes). 3. Outline the tests needed to diagnose a Trichophyton species. 4. List the key features that distinguish Trichophyton rubrum and Trichophyton mentagrophytes. 5. Compare and contrast the ways Microsporum audouinii and Microsporum canis are spread and the populations at risk. 6. Define ectothrix and endothrix. 7. Explain why diagnosing an opportunistic fungal infection in an immunocompromised patient is difficult. 8. Compare and contrast Aspergillus and Penicillium spp., both macroscopically and microscopically. 9. Discuss the dimorphic molds in relation to their endemic areas, disease states, and associated diagnostic methods for identification. The mucorales (zygomycetes) characteristically produce large, ribbonlike hyphae that are irregular in diameter and contain occasional septa. Because the septa may not be apparent in some preparations, this group sometimes has been characterized as aseptate. The specific identification of these organisms is confirmed by observing the characteristic saclike fruiting structures (sporangia), which produce internally spherical, yellow or brown spores (sporangiospores) (Figure 60-1). Each sporangium is formed at the tip of a supporting structure (sporangiophore). During maturation, the sporangium becomes fractured and sporangiospores are released into the environment. Sporangiophores are usually connected to one another by occasionally septate hyphae called stolons, which attach at contact points where rootlike structures (rhizoids) may appear and anchor the organism to the agar surface. Identification of the mucorales (Mucor, Rhizopus, Lichtheimia, and Absidia spp.) is partly based on the presence or absence of rhizoids and the position of the rhizoids in relation to the sporangiophores. See General Considerations for the Laboratory Diagnosis of Fungal Infections in Chapter 59. 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. 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. Rhizopus spp. have unbranched sporangiophores with rhizoids that appear opposite the point where the stolon arises, at the base of the sporangiophore (see Figure 60-1). In contrast, Mucor spp. are characterized by sporangiophores that are singularly produced or branched and have a round sporangium at the tip filled with sporangiospores. They do not have rhizoids or stolons, which distinguishes this genus from the other genera of the mucorales (Figure 60-4). Lichtheimia spp. and Absidia spp. are characterized by the presence of rhizoids that originate between sporangiophores (Figure 60-5). The sporangia of Lichtheimia spp. are pyriform and have a funnel-shaped area (apophysis) at the junction of the sporangium and the sporangiophore. Usually a septum is formed in the sporangiophore just below the sporangium. Other genera of Glomeromycota that are encountered much less frequently in the clinical laboratory are Rhizomucor, Saksenaea, Cunninghamella, Apophysomyces, Conidiobolus, and Basidiobolus spp. See General Considerations for the Laboratory Diagnosis of Fungal Infections in Chapter 59. 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. 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. TABLE 60-1 Characteristics of Dermatophytes Commonly Recovered in the Clinical Laboratory *These organisms are not commonly seen in the United States. 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. 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. 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 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. 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. 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. 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. 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. Several Aspergillus spp. are among the most frequently encountered fungi in the clinical laboratory (Table 60-2); any is potentially pathogenic in the immunocompromised host, but some species are more frequently associated with disease than others. The aspergilli are widespread in the environment, where they colonize grain, leaves, soil, and living plants. Conidia of the aspergilli are easily dispersed into the environment, and humans become infected by inhaling them. Assessing the significance of Aspergillus organisms in a clinical specimen may be difficult. They are found frequently in cultures of respiratory secretions, skin scrapings, and other specimens. TABLE 60-2 Species of Aspergillus Recovered from Clinical Specimens During a 10-Year Period at the Mayo Clinic CNS, Central nervous system. *Numerator, Number of cultures; denominator, number of patients.
Hyaline Molds, Mucorales (Zygomycetes), Dermatophytes, and Opportunistic and Systemic Mycoses
The Mucorales
General Characteristics
Laboratory Diagnosis
Specimen Collection and Transport
Direct Detection Methods
Stains.
Cultivation.
Approach to Identification
The Dermatophytes
General Characteristics
Laboratory Diagnosis
Specimen Collection and Transport
Direct Detection Methods
Stains.
Cultivation.
Dermatophyte
Colonial Morphology
Growth Rate
Microscopic Identification
Microsporum audouinii*
Downy white to salmon-pink colony; reverse tan to salmon-pink
2 weeks
Sterile hyphae; terminal chlamydoconidia, favic chandeliers, and pectinate bodies; macroconidia rarely seen (bizarre shaped if seen); microconidia rare or absent
Microsporum canis
Colony usually membranous with feathery periphery; center of colony white to buff over orange-yellow; lemon-yellow or yellow-orange apron and reverse
1 week
Thick-walled, spindle-shaped, multiseptate, rough-walled macroconidia, some with a curved tip; microconidia rarely seen
Microsporum gypseum
Cinnamon-colored, powdery colony; reverse light tan
1 week
Thick-walled, rough, elliptical, multiseptate macroconidia; microconidia few or absent
Epidermophyton floccosum
Center of colony tends to be folded and is khaki green; periphery is yellow; reverse yellowish brown with observable folds
1 week
Macroconidia: large, smooth walled, multiseptate, clavate, and borne singly or in clusters of two or three; microconidia not formed by this species
Trichophyton mentagrophytes
Different colonial types; white, granular, and fluffy varieties; occasional light yellow periphery in younger cultures; reverse buff to reddish brown
7-10 days
Many round to globose microconidia, most commonly borne in grapelike clusters or laterally along the hyphae; spiral hyphae in 30% of isolates; macroconidia are thin walled, smooth, club shaped, and multiseptate; numerous or rare, depending upon strain
Trichophyton rubrum
Colonial types vary from white downy to pink granular; rugal folds are common; reverse yellow when colony is young, but wine/ red color commonly develops with age
2 weeks
Microconidia usually teardrop-shaped, most commonly borne along sides of the hyphae; macroconidia usually absent but when present are smooth, thin walled, and pencil shaped
Trichophyton tonsurans
White, tan to yellow or rust, suedelike to powdery; wrinkled with heaped or sunken center; reverse yellow to tan to rust red
7-14 days
Microconidia are teardrop or club shaped with flat bottoms; vary in size but usually larger than other dermatophytes; macroconidia rare(balloon forms found when present)
Trichophyton schoenleinii*
Irregularly heaped, smooth, white to cream colony with radiating grooves; reverse white
2-3 weeks
Hyphae usually sterile; many antler-type hyphae seen (favic chandeliers)
Trichophyton violaceum*
Port wine to deep violet colony, may be heaped or flat with waxy-glabrous surface; pigment may be lost on subculture
2-3 weeks
Branched, tortuous. sterile hyphae; chlamydoconidia commonly aligned in chains
Trichophyton verrucosum
Glabrous to velvety white colonies; rare strains produce yellow-brown color; rugal folds with tendency to skin into agar surface
2-3 weeks
Microconidia rare, large and teardrop-shaped when seen; macroconidia extremely rare but form characteristic rat-tail types when seen; many chlamydoconidia seen in chains, particularly when colony is incubated at 37°C
Approach to Identification
Trichophyton spp.
Microsporum spp.
Epidermophyton sp.
The Opportunistic Mycoses
General Characteristics
Epidemiology and Pathogenesis
Aspergillus spp.
Organisms
CLINICAL SPECIMEN SOURCE
Respiratory Secretions
Gastrointestinal
Genitourinary
Skin, Subcutaneous Tissue
Blood, Bone, CNS, Other
A. clavatus
*97/93*
1/1
–
1/1
–
A. flavus
1298/740
10/10
11/11
177/131
2/2
A. fumigatus
3247/2656
11/9
14/14
175/137
8/8
A. glaucus
503/307
1/1
–
8/8
1/1
A. nidulans
52/48
–
–
5/3
–
A. niger
1484/1376
18/18
17/17
151/124
11/11
A. terreus
164/146
–
–
23/21
3/3
A. versicolor
1237/1202
6/6
24/22
226/224
16/16
Other Aspergillus species
3463/3418
18/14
32/32
319/314
16/16