Epidemiology

Fungal infections are an increasing threat to individuals. The number of nosocomial and community-acquired infections has increased dramatically. The major factors responsible for the increase in the number of fungal infections are alterations in the host, particularly the growing number of immunocompromised people. Whether caused by immunosuppressive agents or serious underlying diseases, these alterations may lead to infection by organisms normally nonpathogenic or part of the patient’s normal microbiota (i.e., normal flora). These infections may occur in patients with debilitating diseases, such as progressive infection with the human immunodeficiency virus (HIV) or diabetes mellitus, or in patients with impaired immunologic function resulting from corticosteroid or antimetabolite chemotherapy. Other common predisposing factors include complex surgical procedures and antibacterial therapy. More than 200,000 valid species of fungi exist, but only 100 to 150 species are generally recognized as causes of human disease, and approximately 25 species cause most human disease. Most of these organisms normally live a saprophytic existence (living on dead or decayed organic matter) in nature.

Fungal infections generally are not communicable in the usual sense, through person-to-person transmission. Humans become accidental hosts for fungi by inhaling spores or through the introduction of fungal elements into tissue by trauma. Except for disease caused by the dimorphic fungi, humans are relatively resistant to infections caused by fungi. Classic infections are now appearing in new forms in patients, and the old “harmless” saprophytic molds are now being implicated in serious diseases. This ability of normally saprophytic fungi to cause disease in the immunocompromised patient means that laboratories now must be able to identify and report a wide array of fungi.

The primary pathogens appear to have well-defined geographic locations. An example of this is the dimorphic fungi Coccidioides immitis. C. immitis is usually found only in the United States in the desert Southwest, northern Mexico, and Central America. Opportunistic pathogens such as Candida and Aspergillus spp. are found all over the world.

General Features of the Fungi

Fungi seen in the clinical laboratory generally can be categorized into two groups based on the appearance of the colonies formed. The yeasts produce moist, creamy, opaque or pasty colonies on media, whereas the filamentous fungi or molds (see Chapters 60 and 61) produce fluffy, cottony, woolly, or powdery colonies. Several systemic fungal pathogens exhibit either a yeast (or yeastlike) phase, and filamentous forms are referred to as dimorphic. When dimorphism is temperature dependent, the fungi are designated as thermally dimorphic. In general, these fungi produce a mold form at 25° to 30°C and a yeast form at 35° to 37°C under certain circumstances.

The medically important dimorphic fungi are Histoplasma capsulatum, Blastomyces dermatitidis, C. immitis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Penicillium marneffei (see Chapter 60). C. immitis is not thermally dimorphic. Additionally, some of the medically important yeasts, particularly the Candida species, may produce yeasts forms, pseudohyphae, and/or true hyphae (see Chapter 62). Fungi that have more than one independent form or spore stage in their life cycle are called polymorphic fungi. The polymorphic features of this group of organisms are not temperature dependent.

Taxonomy of the Fungi

Fungi are composed of a vast array of organisms that are unique compared with plants and animals. Included among these are the mushrooms, rusts and smuts, molds and mildews, and yeasts. Despite their great variation in morphologic features, most fungi share the following characteristics:

Traditionally, the fungi have been categorized into four well-established phyla: Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota. The previous phylum, Zygomycota, has contained a very diverse group of organisms. Until further distinction is resolved, the organisms have been divided into the phylum, Glomeromycota and subphylum, Mucoromycotina, and Entomophthoracortina. This diverse group of fungi includes organisms that produce sparsely septate hyphae and exhibit asexual reproduction by sporangiospores and sexual reproduction by the production of zygospores. Some of the clinically important genera in this phylum are Rhizopus, Mucor, Rhizomucor, Absidia, and Cunninghamella.

The Ascomycota include many fungi that reproduce asexually by the formation of conidia (asexual spores) and sexually by the production of ascospores. The filamentous ascomycetes are ubiquitous in nature, and all produce true septate hyphae. All exhibit a sexual form (teleomorph) but also exist in an asexual form (anamorph). Fungi that have different asexual forms of the same fungus are called synanomorphs. In general, the anamorphic form correlates well with the teleomorphic classification. However, different anamorphic forms may have the same teleomorphic form. For example, Pseudallescheria boydii (Figure 59-1), in addition to having the Scedosporium apiospermum anamorph (Figure 59-2), may exhibit a Graphium anamorph (Figure 59-3). The latter anamorph may be seen with several other fungi.

An example of another clinically important fungi that belong to the phylum Ascomycota is H. capsulatum, which has a teleomorph designated as Ajellomyces. Some species of Aspergillus have a teleomorph, Eurotium.

Numerous yeast species also belong to the Ascomycota; these include Saccharomyces spp. and some species of Candida.

The phylum Basidiomycota includes fungi that reproduce sexually through the formation of basidiospores on a specialized structure called the basidia. The basidiomycetes are generally plant pathogens or environmental organisms that rarely cause disease in humans. This group includes smuts, rusts, mushrooms, and Cryptococcus neoformans complex. The teleomorphic form of C. neoformans is Filobasidiella neoformans.

The phylum Deuteromycota includes fungi that lack a sexual reproductive cycle and are characterized by their asexual reproductive structures, primarily conidia. The organisms in this group may have sexual forms that have not yet been described. The medically important fungus, Blastomyces dermatitidis, is a dimorphic fungus that has not been assigned to a phylum and is therefore placed in the order, Incertae sedis until the taxonomic placement is resolved.

Clinical Classification of the Fungi

The botanic taxonomic schema for grouping the fungi has little value in a clinical microbiology laboratory. Table 59-1 is a simplified taxonomic schema illustrating the major groups of fungi.

For clinicians, dividing the fungi into four categories of mycoses, according to the type of infection, is much more useful. The fungi are categorized as follows:

The superficial, or cutaneous, mycoses are fungal infections that involve the hair, skin, or nails without direct invasion of the deeper tissue. The fungi in this category include the dermatophytes (agents of ringworm, athlete’s foot) and agents of infections such as tinea, tinea nigra, and piedra. All of these infect keratinized tissues.

Some fungi cause infections that are confined to the subcutaneous tissue without dissemination to distant sites. Examples of subcutaneous infections include chromoblastomycosis, mycetoma, and phaeohyphomycotic cysts (see Chapter 61).

As traditionally defined, agents of systemic fungal infections include the genera Blastomyces, Coccidioides, Histoplasma, and Paracoccidioides. Infections caused by these organisms primarily involve the lungs but also may become widely disseminated and involve any organ system. P. marneffei, a geographically limited cause of systemic mycosis in a select patient population, may also be considered a part of this group.

Any of the fungi could be considered an opportunistic pathogen in the appropriate clinical setting. The list of uncommon fungi found to cause disease in humans expands every year. Fungi previously thought to be nonpathogenic may be the cause of infections. The infections these organisms cause occur primarily in patients with some type of compromise of the immune system. This may occur secondary to an underlying disease process, such as diabetes mellitus, or it may be caused by an immunosuppressive agent. Although any fungus potentially can cause disease in these patients, the most commonly encountered genera in this group are Aspergillus, Candida, and Cryptococcus, among others. All of these organisms may cause disseminated (systemic) disease. Some of the dematiaceous fungi may cause deeply invasive phaeohyphomycoses (i.e., produce brown-pigmented structures) in this patient population.

Classification by type of infection allows the clinician to attempt to categorize organisms in a logical fashion into groups having clinical relevance. Table 59-2 presents an example of a clinical classification of infections and their etiologic agents that is useful to clinicians.

Practical Working Schema

To assist individuals working in clinical microbiology laboratories with the identification of clinically important fungi, Koneman and Roberts¹ have suggested a practical working scheme designed to do the following:

Table 59-3 presents these features. However, the table includes only organisms commonly seen in the clinical laboratory. With practice, most laboratorians should be able to recognize these on a day-to-day basis. For other, less commonly encountered fungi, the microbiologist must use a variety of texts that have photomicrographs, which can aid identification.

Pathogeneis and Spectrum of Disease

Fungal infection is caused by either primary pathogens or opportunistic pathogens. Infections caused by primary pathogens usually occur in immunocompetent hosts, are not always as virulent, and may lead to subclinical disease. Opportunistic pathogens infect immunocompromised hosts. Opportunistic pathogens include almost any fungus present in the environment. An increase in the number of opportunistic fungal infections in humans is due in large part to the immunocompromised nature of the host. However, certain factors, called virulence factors, make invading tissues and causing disease easier for these organisms. Some virulence factors have been known for years:

Most of the fungi exist in environmental niches as saprophytic organisms (Table 59-4). Perhaps the fungi that cause disease in humans have developed various mechanisms that allow them to establish disease in the human host. Table 59-5 describes the known or speculative virulence factors of the fungi known to be pathogenic for humans.

The diagnosis of fungal infections depends entirely on the selection and collection of an appropriate clinical specimen for microscopic analysis and culture. Many fungal infections are similar clinically to mycobacterial infections, and often the same specimen is cultured for both fungi and mycobacteria. Many infections have a primary focus in the lungs; respiratory tract secretions are almost always included among the specimens selected for culture. It should be emphasized that dissemination to distant body sites may occur, and fungi may be recovered from nonrespiratory sites.

Proper collection of specimens and rapid transport to the clinical laboratory are crucial to the recovery of fungi. Specimens often contain not only the etiologic agent, but also contaminating bacteria or fungi that rapidly overgrow some of the slower-growing pathogenic fungi. The viability of fungi decreases over time. If processing will be delayed, specimens can be refrigerated for a short time, except for dermatologic specimens (skin, hair, nails), blood, and cerebrospinal fluid (CSF). Yeasts (e.g., Candida spp.) commonly are recovered on routine bacteriology media and fungal culture media. A few specific comments concerning specimen collection and culturing are included in this chapter.

Direct Microscopic Examination

Direct microscopic examination of clinical specimens has been used for many years; however, its usefulness should be reemphasized. Because the mission of a clinical microbiology laboratory is to provide a rapid and accurate diagnosis, the mycology laboratory can provide this service in many instances by direct examination (particularly the Gram stain) of the clinical specimen submitted for culture. Microbiologists are encouraged to become familiar with the diagnostic features of fungi commonly encountered in clinical specimens and to recognize them when stained by various dyes. This important procedure often can provide the first microbiologic proof of the etiology of disease in patients with fungal infection. This is the most rapid method currently available.

Tables 59-7 and 59-8 present the methods available for direct microscopic detection of fungi in clinical specimens and a summary of the characteristic microscopic features of each. Figure 59-4 presents photomicrographs of some of the fungi commonly seen in clinical specimens.

TABLE 59-7

Summary of Methods Available for Direct Microscopic Detection of Fungi in Clinical Specimens

Method	Use	Time Required	Advantages	Disadvantages
Acid-fast stain and partial acid-fast stain	Detection of mycobacteria and Nocardia spp., respectively	12 min	Detects Nocardia spp.* and some isolates of Blastomyces dermatitidis	Tissue homogenates are difficult to observe because of background staining.
Auramine-rhodamine stain	Detection of mycobacteria and Nocardia spp., respectively	10 min	Excellent screening tool; sensitive and affordable	Not as specific for acid-fast organisms as Ziehl-Neelsen stain
Calcofluor white stain	Detection of fungi	1 min	Can be mixed with KOH: detects fungi rapidly because of bright fluorescence	Requires use of a fluorescence microscope; background fluorescence prominent, but fungi exhibit more intense fluorescence; vaginal secretions are difficult to interpret.
Gram stain	Detection of bacteria	3 min	Commonly performed on most clinical specimens submitted for bacteriology; detects most fungi.	Some fungi stain well, but others (e.g., Cryptococcus spp.) show only stippling and stain weakly in some instances; some isolates of Nocardia spp. fail to stain or stain weakly.
India ink stain	Detection of Cryptococcus neoformans in CSF	1 min	Diagnostic of meningitis when positive in CSF	Positive in fewer than 50% of cases of meningitis; not sensitive in non–HIV-infected patients
Lactophenol cotton blue wet mount	Most widely used method of staining and observing fungi	1 min	Lactic acid preserves structures; slides can be made permanent.	Mechanical treatment dislodges fungal structures.
Potassium hydroxide	Clearing of specimen to make fungi more readily visible	5 min; if clearing is not complete, an additional 5-10 min is necessary	Rapid detection of fungal elements	Requires experience, because background artifacts are often confusing; clearing of some specimens may require an extended time.
Masson-Fontana stain	Examination of melanin pigment in fungal cell walls	1 hr, 10 min	Aids differentiation of melanin and hemosiderin pigments	Difficult to interpret when only rare granular staining is present
Methenamine silver stain	Detection of fungi in histologic section	1 hr	Best stain for detecting fungal elements	Requires a specialized staining method that is not usually readily available to microbiology laboratories
Papanicolaou stain	Examination of secretions for malignant cells	30 min	Cytotechnologist can detect fungal elements.	Fungal elements stain pink to blue.
Periodic acid-Schiff (PAS) stain	Detection of fungi	20 min; 5 min additional if counterstain is used	Stains fungal elements well; hyphae of molds and yeasts can be readily distinguished.	Nocardia spp. do not stain well.
Saline wet mount	Examination of fungal elements	1 min	Quickly performed and cost-effective	Specimen must be fresh; not all elements are visible with this preparation.
Wright’s stain	Examination of bone marrow or peripheral blood smears	7 min	Detects Histoplasma capsulatum and C. neoformans	Most often used to detect H. capsulatum and C. neoformans complex in disseminated disease.

CSF, Cerebrospinal fluid; HIV, human immunodeficiency virus; KOH, potassium hydroxide.

^*Partially acid-fast bacterium.

From Versalovic J: Manual of clinical microbiology, ed 10, Washington, DC, 2011, ASM Press.

TABLE 59-8

Summary of Characteristic Features of Fungi Seen in Direct Examination of Clinical Specimens

Morphologic Form Found in Specimens	Organism	Size Range (diameter, mm)	Characteristic Features
Yeastlike	Histoplasma capsulatum	2-5	Small; oval to round budding cells; often found clustered in histiocytes; difficult to detect when present in small numbers
	Sporothrix schenckii	2-6	Small; oval to round to cigar shaped; single or multiple buds present; uncommonly seen in clinical specimens
	Cryptococcus neoformans complex	2-15	Cells exhibit great variation in size; usually spherical but may be football shaped; buds single or multiple and “pinched off”; capsule may or may not be evident; occasionally, pseudohyphal forms with or without a capsule may be seen in exudates of cerebrospinal fluid
	Malassezia furfur (in fungemia)	1.5-4.5	Small; bottle-shaped cells, buds separated from parent cell by a septum; emerge from a small collar
	Blastomyces dermatitidis	8-15	Cells are usually large, double refractile when present; buds usually single; however, several may remain attached to parent cells; buds connected by a broad base
	Paracoccidioides brasiliensis	5-60	Cells are usually large and are surrounded by smaller buds around the periphery (“mariner’s wheel appearance”); smaller cells may be present (2-5 µm) and resemble H. capsulatum; buds have “pinched-off” appearance
Spherules	Coccidioides immitis	10-200	Spherules vary in size; some may contain endospores, others may be empty; adjacent spherules may resemble B. dermatitidis; endospores may resemble H. capsulatum but show no evidence of budding; spherules may produce multiple germ tubes if a direct preparation is kept in a moist chamber ≥24 hr
	Rhinosporidium seeberi	6-300	Large, thick-walled sporangia containing sporangiospores are present; mature sporangia are larger than spherules of C. immitis; hyphae may be found in cavitary lesions
Yeast and pseudohyphae or hyphae	Candida spp. except C. glabrata	5-10 (pseudohyphae)	Cells usually exhibit single budding; pseudohyphae, when present, are constricted at the ends and remain attached like links of sausage; hyphae, when present, are septate
	M. furfur (in tinea versicolor)	3-8 (yeast) 2.5-4 (hyphae)	Short, curved hyphal elements are usually present, along with round yeast cells that retain their spherical shape in compacted clusters
Pauciseptate hyphae	Mucorales: Mucor, Rhizopus, and other genera	10-30	Hyphae are large, ribbonlike, often fractured or twisted; occasional septa may be present; smaller hyphae are confused with those of Aspergillus spp., particularly A. flavus
Hyaline septate hyphae	Dermatophytes, skin and nails	3-15	Hyaline, septate hyphae are commonly seen; chains of arthroconidia may be present
	Hair	3-15	Arthroconidia on periphery of hair shaft producing a sheath indicate ectothrix infection; arthroconidia formed by fragmentation of hyphae in the hair shaft indicate endothrix infection
		3-15	Long hyphal filaments or channels in the hair shaft indicate favus hair infection
	Aspergillus spp.	3-12	Hyphae are septate and exhibit dichotomous, 45-degree branching; larger hyphae, often disturbed, may resemble those of zygomycetes
	Geotrichum spp.	4-12	Hyphae and rectangular arthroconidia are present and sometimes rounded; irregular forms may be present
	Trichosporon spp.	2-4 by 8	Hyphae and rectangular arthroconidia are present and sometimes rounded; occasionally, blastoconidia may be present
Dematiaceous septate hyphae	Bipolaris spp., Cladosporium spp., Curvularia spp., Drechslera spp., Exophiala spp., Exserohilum spp., Hortaea werneckii, Phialophora spp.	2-6	Dematiaceous polymorphous hyphae are seen; budding cells with single septa and chains of swollen rounded cells are often present; occasionally, aggregates may be present in infection caused by Phialophora and Exophiala spp.
	Wangiella dermatitidis	1.5-5	Usually large numbers of frequently branched hyphae are present, along with budding cells
Sclerotic bodies	Cladosporium carrionii Fonsecaea compacta Fonsecaea pedrosoi Phialophora verrucosa Rhinocladiella aquaspersa	5-20	Brown, round to pleomorphic, thick-walled cells with transverse septations; commonly, cells contain two fission planes that form a tetrad of cells (sclerotic bodies)
Granules	Acremonium  A. falciforme  A. kiliense  A. recifei	200-300	White, soft granules without a cementlike matrix
	Aspergillus  A. nidulans	500-1000	Black, hard grains with a cementlike matrix at periphery
	Curvularia  C. geniculata  C. lunata	65-160	White, soft granule without a cementlike matrix
	Exophiala  E. jeanselmei	200-300	Black, soft granules, vacuolated, without a cementlike matrix, made of dark hyphae and swollen cells
	Fusarium  F. moniliforme	200-500	White, soft granules without a cementlike matrix
	F. solani	300-600
	Leptosphaeria  L. senegalensis	400-600	Black, hard granules with cementlike matrix present
	L. tompkinsii	500-1000	Periphery composed of polygonal swollen cells and center of a hyphal network
	Madurella  M. grisea	350-500	Black, soft granules without a cementlike matrix, periphery composed of polygonal swollen cells and center of a hyphal network
	M. mycetomatis	200-900	Black to brown, hard granules, two types: (1) rust brown, compact, filled with cementlike matrix; (2) deep brown, filled with numerous vesicles, 6-14 µm in diameter, cementlike matrix in periphery, central area of light-colored hyphae
	Neotestudina  N. rosatii	300-600	White, soft granules with cementlike matrix at periphery
	Pseudallescheria  P. boydii	200-300	White, soft granules composed of hyphae and swollen cells at periphery in cementlike matrix
	Pyrenochaeta  P. romeroi	300-600	Black, soft granules composed of polygonal swollen cells at periphery; center is network of hyphae; no cementlike matrix present

Traditionally, the potassium hydroxide preparation has been the recommended method for direct microscopic examination of specimens. However, the calcofluor white stain now is believed to be superior (see Procedure 59-1 on the Evolve site). Slides prepared by this method may be observed using fluorescent or bright-field microscopy, as is used for the potassium hydroxide preparation; the former is optimal, because fungal cells fluoresce.

Procedure 59-1   Calcofluor White–Potassium Hydroxide Preparation

Method

1. Place a drop of calcofluor white (CW) reagent and a drop of 10% potassium hydroxide (KOH) glycerin in the center of a microscope slide.

2. Add a portion of the clinical specimen to the CW-KOH solution and apply a coverslip.

3. If necessary, dissociate particles by applying gentle pressure to the coverslip with a pencil eraser. Allow to stand for 5 minutes. If particles do not dissociate, repeat this step.

4. Examine the slide for fungal elements using a fluorescent microscope with a 400 to 500 nm exciter filter and a 500 to 520 nm barrier filter. Slides are scanned at ×10 magnification for fluorescent fungal elements. The presence and nature of fungal elements are discerned using ×40 magnification.

Reagents

KOH reagent: (10 g KOH, 10 mL glycerin, 80 mL distilled water)

CW reagent: (0.05 g CW, 0.02 g Evans blue, 50 mL distilled water)

Calcofluor white is an industrial textile brightener that nonspecifically binds to chitin and other elements in the fungal cell wall. Calcofluor white fluorescence occurs maximally at an excitation wavelength of 440 nm. Under these conditions, fungal elements fluoresce blue-white. Because CW is a nonspecific stain, an appreciation for fungal element morphology on direct examination is crucial for adequate specimen interpretation. The presence of KOH in the solution dissolves human cellular elements and debris, which allows for easier visualization of fungal elements.

Limitations

Careful interpretation is crucial, because nonspecific fluorescence may be observed with the presence of cotton fibers or certain tissues from patients with tumors.

General Considerations for the Identification of Yeasts

Most often yeasts are identified through the use of a combination of tests (Figure 59-5). Identification factors and techniques include:

Several key characteristics can be seen macroscopically. Colonies have a wide variety of colors, shapes, and textures. Chromogenic agar can be used to differentiate yeast presumptively. Wet preps and lactophenol cotton blue stain can aid microscopic identification by improving the visualization of spore structure. Sexual and asexual characteristics are very important. Often a genus can be determined by the microscopic and macroscopic characteristics alone. India ink stain is useful when Cryptococcus organisms are suspected. Because carbon and nitrogen source differences are the key to differentiating yeasts, many automated and semiautomated commercial systems have been designed with assimilation and fermentation tests. Supplemental testing takes advantage of restriction of a limited set of characteristics to further aid identification.

General Considerations for the Identification of Molds

Filamentous fungi are also identified by a combination of tests (Figure 59-6). Molds are identified using a combination of the following:

In most cases the microscopic morphologic features provide the most definitive means of identification. Determination of the growth rate can be most helpful when a mold culture is examined. However, this may have limited value, because the growth rate of certain fungi varies, depending on the amount of inoculum present in a clinical specimen. Slow-growers form mature colonies in 11 to 21 days, and intermediate-growers form mature colonies in 6 to 10 days. Rapid-growers form mature colonies in 5 days or less.

The growth of C. immitis is often rapid and is hazardous to microbiologists. In general, the growth rate for the dimorphic fungi, B. dermatitidis, H. capsulatum, and P. brasiliensis, is slow; 1 to 4 weeks usually are required before colonies become visible. In some instances, cultures of B. dermatitidis and H. capsulatum may be detected within 3 to 5 days. This is a somewhat uncommon circumstance, encountered only when large numbers of the organism are present in the specimen. Colonies of Mucorales may appear within 24 hours, whereas the other hyaline and dematiaceous (melanized) fungi often exhibit growth in 1 to 5 days. The growth rate of an organism, therefore, is important, but it must be used in combination with other features before a definitive identification can be made.

The colonial morphologic features may have limited value for identifying molds because of natural variation among isolates and colonies grown on different culture media. Although common organisms recovered repeatedly in the laboratory may be more easily recognized, colonial morphology is an unreliable criterion that should be used only to supplement the microscopic morphologic features of the organism.

The color of the colony can be important. The examiner must be sure to notice both the front and reverse sides of the culture. The colony topography describes the various elevations of the colony on the agar plate. Topography can be described as verrucose (furrowed or convoluted), umbonate (slightly raised in the center), and rugose (furrows radiate out from the center).

The colony’s texture should also be noted. Various textures can be seen, such as cottony (loose, high aerial mycelium), velvety (low aerial mycelium resembling a velvet cloth), glabrous (smooth surface with no aerial mycelium), granular (dense, powdery, resembling sugar granules), and wooly (high aerial mycelium that appears slightly matted down).

Incubation conditions and culture media must also be considered. For example, H. capsulatum, which appears as a white-to-tan fluffy mold on brain-heart infusion agar, may have a yeastlike appearance when grown on the same medium containing blood enrichment.

In general, the microscopic morphologic features of the molds are stable and show minimal variation. Definitive identification is based on the characteristic shape, method of reproduction, and arrangement of spores; however, the size of the hyphae also provides helpful information. The large, ribbonlike, pauciseptate hyphae of the mucorales are easily recognized; small hyphae, approximately 2 µm in diameter, may suggest the presence of one of the dimorphic fungi or a dermatophyte.

The fungi may be prepared for microscopic observation using several techniques. The procedure traditionally used by most laboratories is the cellophane (Scotch) tape preparation (see Procedure 59-2 on the Evolve site; Figure 59-7). It can be done easily and quickly and often is sufficient to make the identification for most fungi. However, some laboratories prefer the wet mount (see Procedure 59-3 on the Evolve site; Figure 59-8) or tease mount (see Procedure 59-4 on the Evolve site). A microslide culture method (see Procedure 59-5 on the Evolve site; Figure 59-9) may be used when greater detail of the morphologic features is required.

Procedure 59-2   Cellophane (Scotch) Tape Preparation

Method

1. Touch the adhesive side of a small length of transparent tape to the surface of the colony.

2. Add a drop of lactophenol cotton or aniline blue to the surface of a microscope slide; then, adhere the length of tape to the surface the slide (Figure 59-7).

3. Observe microscopically for the characteristic shape and arrangement of the spores.

The transparent adhesive tape preparation allows the laboratorian to observe the organism microscopically approximately the way the fungi sporulates in culture. The relationship of the spores, spore-producing structures (e.g., conidiophores), and the body of the fungus are usually intact, and microscopic identification of an organism can be made easily. If the tape is not pressed firmly enough to the surface of the colony, the sample may consist only of conidia and may not be adequate for identification. When spores are not observed, a wet mount should be made. In some cases, the macroconidia of Histoplasma capsulatum have been seen in wet mount preparations when the adhesive tape preparation revealed only hyphal fragments. In other instances, cultures have sporulated and revealed only the presence of conidia when the adhesive tape preparation was observed. In this type of situation, a second adhesive tape preparation should be made from the periphery of the colony where sporulation is not as prominent.

Some laboratories prefer to use the microslide culture (see Procedure 59-5) for microscopic identification of an organism. This method might appear to be the most suitable, because it allows the examiner to observe microscopically the fungus growing directly underneath the coverslip. Microscopic features should be easily discerned, structures should be intact, and many representative areas of growth are available for observation.

Procedure 59-3   Wet Mount

Method

1. With a wire bent at a 90-degree angle, cut out a small portion of an isolated colony. The portion should be removed from a point intermediate between the center and the periphery and should contain a small amount of the supporting agar.

2. Add a drop of lactophenol cotton or aniline blue to a slide and then place the portion on the slide (Figure 59-8).

3. Place a coverslip in position and apply gentle pressure with a pencil eraser or other suitable object to disperse the growth and the agar. Examine the slide microscopically.

Limitations

The major disadvantage of the wet mount is that the characteristic arrangement of spores is disrupted when pressure is applied to the coverslip. However, this method is suitable in many instances, because characteristic spores are often seen but their arrangement cannot be determined. In some instances, the wet mount is not adequate for making a definitive identification.

Procedure 59-5   Microslide Culture

Method

1. Cut a small block of a suitable agar medium that has been previously poured into a culture dish to a depth of approximately 2 mm. The block may be cut using a sterile scalpel blade or with a sterile test tube that has no lip (which produces a round block).

2. Place a sterile microscope slide on the surface of a culture dish containing sterile 2% agar. Alternatively, place a round piece of filter paper or paper towel in a sterile culture dish, add two applicator sticks, and position the microscope slide on top.

3. Add the agar block to the surface of the sterile microscope slide.

4. With a right-angle wire, inoculate the four quadrants of the agar plug with the organism (Figure 59-9).

5. Apply a sterile coverslip to the surface of the agar plug.

6. If the filter paper applicator stick method is used, add a small amount of sterile water to the bottom of the culture dish. Replace the lid of the culture and allow it to incubate at 30°C.

7. After a suitable incubation period (and working inside a biologic safety cabinet), remove the coverslip and place it on a microscope slide containing a drop of lactophenol cotton or aniline blue. Some suggest placing the coverslip near the opening of an incinerator-burner to allow the organism to dry rapidly on the coverslip before adding it to the stain.

8. Observe microscopically for the characteristic shape and arrangement of spores.

9. If the microslide culture is unsatisfactory for microscopic identification, the remaining agar block may be used later if it is allowed to incubate further. The agar plug is then removed and discarded, and a drop of lactophenol cotton or aniline blue is placed on the area of growth and a coverslip is positioned in place. Many laboratorians like to make two cultures on the same slide so that if characteristic microscopic features are not observed on examination of the first culture, the second is available after an additional incubation period.

Limitations

Although this method is ideal for definitive identification of an organism, it is the least practical of all the methods described. It should be reserved for cases in which an identification cannot be made based on an adhesive tape preparation or a wet mount.

Caution: Do not make slide cultures of slow-growing organisms suspected to be dimorphic pathogens (e.g., Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, or Sporothrix schenckii). Microslide cultures must be observed only after a coverslip has been removed from the agar plug and not while it is in position on top of the agar plug. The latter method of observation is very dangerous, because it could cause a laboratory-acquired infection.

General Morphologic Features of the Molds

Specialized types of vegetative hyphae may be helpful for categorizing an organism into a certain group. For example, dermatophytes often produce several types of hyphae, including antler hyphae, so named because they are curved, freely branching, and have the appearance of antlers (Figure 59-10). Racquet hyphae are enlarged, club-shaped structures (Figure 59-11). In addition, certain dermatophytes produce spiral hyphae that are coiled or exhibit corkscrewlike turns in the hyphal strand (Figure 59-12). These structures are not characteristic for any certain group; however, they are found most commonly in dermatophytes.

Some species of fungi produce sexual spores in a large, saclike structure called an ascocarp (Figure 59-13). The ascocarp contains smaller sacs, called asci, each of which contains four to eight ascospores. This type of sexual reproduction is not commonly seen in the fungi recovered in the clinical microbiology laboratory; most exhibit only asexual reproduction. It is possible that all fungi have a sexual form, but for some species it has not yet been observed on artificial culture media. Conidia, which are produced by most fungi, represent the asexual reproductive cycle. The type of conidia and their morphology and arrangement are important criteria for definitively identifying an organism (Figure 59-14).

The simplest type of sporulation is the development of a spore directly from the vegetative hyphae. Arthroconidia are formed directly from the hyphae by fragmentation through the points of septation (Figure 59-15). When mature, they appear as square, rectangular, or barrel-shaped, thick-walled cells. These result from the simple fragmentation of the hyphae into spores, which are easily dislodged and disseminated into the environment. Chlamydoconidia (chlamydospores) are round, thick-walled spores formed directly from the differentiation of hyphae in which there is a concentration of protoplasm and nutrient material (Figure 59-16). These appear to be resistant resting spores produced by the rounding up and enlargement of the cells of the hyphae. Chlamydoconidia may be intercalary (within the hyphae) or terminal (on the end of the hyphae).

A variety of other types of spores occur with many species of fungi. Conidia are asexual spores produced singly or in groups by specialized hyphal strands, conidiophores. In some instances, the conidia are freed from their point of attachment by pinching off, or abstriction. Some conidiophores terminate in a swollen vesicle. From the surface of the vesicle are formed secondary small, flask-shaped phialides, which in turn give rise to long chains of conidia. This type of fruiting structure is characteristic of the aspergilli. A single, simple, slender, tubular conidiophore (phialide) that produces a cluster of conidia, held together as a gelatinous mass, is characteristic of certain fungi, including the genus Acremonium (Figure 59-17). In other instances, conidiophores form a branching structure called a penicillus, in which each branch terminates in secondary branches (metulae) and phialides, from which chains of conidia are borne (Figure 59-18). Species of Penicillium and Paecilomyces are representative of this type of sporulation. In other instances, fungi may produce conidia of two sizes: microconidia, which are small, unicellular, round, elliptical, or pyriform in shape, or macroconidia, which are large, usually multiseptate, and club or spindle shaped (Figure 59-19). Microconidia may be borne directly on the side of a hyphal strand or at the end of a conidiophore. Macroconidia are usually borne on a short to long conidiophore and may be smooth or rough walled. Microconidia and macroconidia are seen in some fungal species and are not specific, except as they are used to differentiate a limited number of genera.

The hyphae of the mucorales are sparsely septate. Sporulation takes place by progressive cleavage during maturation in the sporangium, a saclike structure produced at the tip of a long stalk (sporangiophore). Sporangiospores (spores produced in the sporangium) are produced and released by the rupture of the sporangial wall (Figure 59-20). In rare cases some isolates may produce zygospores, rough-walled spores produced by the union of two matching types of a mucorales; this is an example of sexual reproduction.

Clinical Relevance for Fungal Identification

The question of when and how far to go with the identification of fungi recovered from clinical specimens presents an interesting challenge. The current emphasis on cost containment and the ever-increasing number of opportunistic fungi causing infection in compromised patients prompts consideration of whether all fungi recovered from clinical specimens should be thoroughly identified and reported. A study by Murray et al.⁸ focused on the time and expense involved in identifying yeasts from respiratory tract specimens. Because these are the specimens most commonly submitted for fungal culture, the researchers questioned whether identifying every organism recovered was important. After evaluating the clinical usefulness of information provided through the identification of yeast recovered from respiratory tract specimens, they suggested the following:

The extent of identification of yeasts from other specimen sources is discussed in Chapter 63. The usefulness of identification and susceptibility testing of non-Cryptococcus yeast isolates was studied by Barenfenger.⁹ She found that, compared with only superficial characterization (i.e., “Yeast present, not C. neoformans), identification and susceptibility testing of Candida isolates from respiratory secretions led to unnecessary treatment and increased costs. No statistical difference was seen in the mortality of these groups.

When and how far to proceed in the identification of a mold is a difficult question to answer. Except for obvious plate contaminants, all commonly encountered molds should be identified and reported if recovered from patients at risk for invasive fungal disease. Immunocompromised patients may have serious or even fatal disease caused by fungi that were once thought to be clinically insignificant. Organisms that fail to sporulate after a reasonable time should be reported as present, but identification is not required if the dimorphic fungi have been ruled out or if the clinician believes the organism is not clinically significant. Ideally, all laboratories should identify all fungi recovered from clinical specimens; however, the limits of practicality and economic considerations play a definite role in the decision-making process. The laboratory director, in consultation with the clinicians being served, must make this decision after considering the patient population, laboratory practice, and economic impact.

As shown in Table 59-9, an increasing number of fungi may be isolated in the clinical microbiology laboratory. They are considered environmental flora, but in reality must be regarded as potential pathogens because infections with a number of these organisms have been reported. Less commonly encountered fungal pathogens that have been shown to cause human infections include but are not limited to P. boydii; Scedosporium prolificans; Bipolaris, Exserohilum, Trichosporon, and Aureobasidium spp., and others. The laboratory must identify and report all organisms recovered from clinical specimens so that their clinical significance can be determined. In many instances, the presence of environmental fungi is unimportant; however, that is not always the case. Tables 59-10 and 59-11 present the molds and yeasts implicated in causing human infection, the time required for their identification, the most likely site for their recovery, and the clinical implications of each.

Laboratory Safety

Although the handling of fungi recovered from clinical specimens poses risks, a common sense approach to the handling of these specimens protects the laboratory from contamination and workers from becoming infected.

Without exception, mold cultures and clinical specimens must be handled in a class II BSC. Some laboratory directors believe that mold cultures must be handled in an enclosed BSC equipped with gloves; however, this is not necessary if a laminar flow BSC is used. Yeast cultures may be handled on the bench top. An electric incinerator or a gas flame is suitable for decontaminating a loop used to transfer yeast cultures. Cultures of organisms suspected of being pathogens should be sealed with tape to prevent laboratory contamination and should be autoclaved as soon as the definitive identification is made. If common safety precautions are followed, few problems should occur with laboratory contamination or infection acquired by laboratory personnel.

Prevention

Preventing and controlling fungal infections continue to be a challenge to individuals, researchers, laboratorians, and hospitals. Very few formal recommendations are available to prevent exposure to community-acquired fungal infections. Good personal hygiene may be the best course for prevention. However, many strategies can be followed to prevent nosocomial infections. Hospital staff members should be aware of the pathogenesis of fungal infections. Fungi are easily spread in ventilation systems, water, and skin-to-skin contact. Hospitals should follow an infection control plan that includes periodic monitoring of air-handling systems and regular testing for environmental spores. Staff members, patients, and visitors should practice good personal hygiene to minimize exposure to potential fungal infections.

The laboratory also plays an important role in fungal prevention and control. Lack of rapid and specific testing continues to be a factor in a timely diagnosis. Early definitive diagnosis ensures that the appropriate therapy is given promptly and prevents mortality.