58: Overview of Fungal Identification Methods and Strategies

Fungi seen in the clinical laboratory generally can be categorized into two groups based on the appearance of the colonies formed. The yeasts are unicellular organisms (Chapter 62) and produce moist, creamy, opaque, or pasty colonies on media, whereas the filamentous fungi or molds produce multicellular structures (Chapters 59 and 60) and demonstrate fluffy, cottony, woolly, or powdery colonies. Several systemic fungal pathogens that 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 in the environment or when cultured on routine artificial mycology agar at 25°C to 30°C and are yeastlike in tissue or when cultured on enriched artificial medium at 35°C to 37°C.

The medically important dimorphic fungi are Histoplasma sp., Blastomyces spp., Coccidioides spp., Cokeromyces recurvatus, Emergomyces spp., and Paracoccidioides spp. Additionally, some of the medically important yeasts, particularly the Candida species, may produce yeast forms, pseudohyphae, and/or true hyphae (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. They are heterotrophic (saprophytic) and require preformed organic carbon for nutrition. 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:

• Chitin in the cell wall

• Ergosterol in the cell membrane

• Reproduction by means of spores, produced asexually or sexually

• Lack of chlorophyll

• Lack of susceptibility to antibacterial antibiotics

Significant changes have resulted in the organization of the groups, taxonomy, and nomenclature within the fungi due to the introduction of molecular methods. Traditionally, the fungi have been categorized based on phenotypic traits, which vary based on temperature, atmospheric conditions, nutrient availability, and humidity, to name a few. Because of this variation, phenotypic classification is unclear, complex, and variable. Molecular analysis of these organisms, which includes extensive DNA sequencing, is now considered the gold standard to determine taxonomic designation and classification. In addition, fungal taxonomy has been complicated by fungi names that describe the sexual (teleomorph) or asexual (anamorph) stage. This system has become obsolete. The International Botanical Congress adopted a one-fungus, one-name policy, published in the International Code of Nomenclature, Article 59.

The implementation of DNA sequencing for the classification and taxonomy of fungi is not without challenges. As with other microorganisms, fungi have the ability to exchange genetic material and demonstrate evidence of recombination among populations, making speciation difficult. Currently, no standard exists that provides for a definition of the amount of genetic diversity that is allowable, or that limits the species level in taxonomy of fungi. Some clinically relevant fungi have been identified that include molecular siblings. A molecular sibling is an organism that cannot be distinguished based on phenotypic, metabolic, and clinical presentation but is recognized as a different species based on molecular analysis. Molecular siblings that do not differ in traditional characteristics, including disease presentation, are often considered a species complex. The use of the term “complex” does not hold any taxonomic relevance; it provides a system to limit organism identification within the clinical laboratory. Clinical laboratories may choose to report species complexes as preliminary identifications until further studies can be performed on the isolate, including antifungal susceptibility profiles when appropriate.

Historically, the fungi were categorized into three well-established phyla: Zygomycota, Ascomycota, and Basidiomycota. The previous phylum, Zygomycota, contained a very diverse group of organisms. Zygomycota has now been replaced with the subphyla Mucoromycotina and Entomophthoromycotina. The subphyla Mucoromycotina includes the order Mucorales and the genera Lichtheimia, Mucor, Rhizomucor, and Rhizopus; the Entomophthoromycotina, order Entomophthorales, includes the genera Basidiobolus and Conidiobolus. These two subphyla include organisms that produce aseptate or sparsely septate hyphae and exhibit asexual reproduction by sporangiospores. Some species may reproduce sexually and form zygospores in culture.

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 produce true septate hyphae. They may exhibit a sexual form (teleomorph) but also exist in an asexual form (anamorph) (Figs. 58.1 to 58.3). Fungi that have different asexual forms of the same fungus are called synanamorphs. An example of a clinically important fungus that belongs to the phylum Ascomycota is Histoplasma capsulatum. Numerous opportunistic fungi such as Aspergillus, the atypical fungi, Pneumocystis, and yeast such as the Saccharomyces, Saprochaete, and Candida, also belong to the Ascomycota.

The phylum Basidiomycota includes fungi that reproduce sexually through the formation of basidiospores on a specialized structure called the basidia. Medically relevant basidiomycetous yeasts include the genera Cryptococcus, Malassezia, Pseudozyma, Rhodotorula, Sporobolomyces, and Trichosporon. The filamentous Basidiomycetes are uncommon causes of human respiratory and systemic disease in immunocompetent and immunosuppressed patients and are poorly characterized. In addition, several organisms that are pathogenic to humans and have been classified as fungal or parafungal organisms that phenotypically resemble protozoans or yeast and remain unculturable are included in Chapter 61.

Clinical Classification of the Fungi

The complexity associated with the taxonomic classifications of fungi makes the identification of clinically relevant organisms to the species level inherently difficult. For clinicians, dividing the fungi into four categories of mycoses according to the type of infection in combination with macroscopic and microscopic characteristics is used to provide a systematic approach to identification. The clinical categories of fungi are separated as follows:

• Superficial (cutaneous) mycoses

• Subcutaneous mycoses

• Systemic mycoses

• Opportunistic mycoses

The superficial, or cutaneous, mycoses are fungal infections that involve hair, skin, or nails without direct invasion of 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 (Chapter 60).

Some of the agents of systemic fungal infections include the genera Blastomyces, Coccidioides, Histoplasma, Cokeromyces, Talaromyces, Sporothrix, Emergomyces, and Paracoccidioides. Infections caused by most of these organisms involve the lungs but also may become widely disseminated and involve any organ system.

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 prolonged use of 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 by clinical relevance. Table 58.1 presents an example of a classification of infections and their etiologic agents that is useful to clinicians.

The identification of organisms in clinical samples requires examination of the macroscopic and microscopic characteristics of the fungal culture. Although the macroscopic characteristics may be suggestive of particular genera of fungi, the microscopic characteristics tend to provide a more reliable method for identification. This requires a microscopic examination of the isolate for the appearance of hyphal elements, including the presence, absence, and number of septa. If the hyphae appear to be broad and predominantly nonseptate (i.e., cells are not separated by a septum or wall), Mucoromycetes, Basidiobolomycoses, or Entomophthoromycoses should be considered. If the hyphae are septate, they must be examined further for the presence or absence of pigmentation. If a dark pigment is present in the hyphae, the organism is considered to be melanized (dematiaceous), and the conidia are then examined for their morphologic features and their arrangement on the hyphae. If the hyphae are nonpigmented, they are considered to be hyaline. The fungi are then examined for the type and the arrangement of the conidia produced. The molds are identified by recognition of their characteristic microscopic features.

Table 58.1

General Clinical Classification of Pathogenic Fungi
Cutaneous	Subcutaneous	Opportunistic	Systemic
Superficial mycoses Tinea Piedra Candidosis Dermatophytosis	Chromoblastomycosis Sporotrichosis Mycetoma (eumycotic) Phaeohyphomycosis	Aspergillosis Candidosis Cryptococcosis Geotrichosis Mucormycosis Fusariosis Trichosporonosis Entomophthoromycosis Others ^a	Aspergillosis Blastomycosis Candidosis Coccidioidomycosis Adiaspiromycosis Emmonsiosis Histoplasmosis Cryptococcosis Geotrichosis Paracoccidioidomycosis Penicilliosis Pneumocystosis Sporotrichosis Pseudoallescheriosis/scedosporiosis Mucormycosis Entomophthoromycosis Fusariosis Trichosporonosis

It is important that the clinical laboratory evaluate the isolation of a fungus from a clinical sample, because it may be regarded as a contaminant acquired during collection, transportation, processing, or incubation. It is important to consider that all contaminating fungi may be considered pathogenic in the appropriate clinical setting. Each isolate should be carefully evaluated on a case-by-case basis.

Pathogenesis and Spectrum of Disease

Fungal infection is caused by either primary pathogens or opportunistic pathogens. Infections caused by primary pathogens can occur in immunocompetent hosts, are not always as virulent, and may lead to subclinical disease. Opportunistic pathogens primarily infect immunocompromised hosts. Opportunistic pathogens include almost any fungus present in the environment. An increase in the identification of opportunistic fungal infections in humans is in large part a result of the immunocompromised nature of the host but also the increasing use of antifungals and improved diagnostic methods. In addition, organism-specific factors, called virulence factors, make invading tissues and causing disease easier. Some virulence factors include:

• The organism’s size (with inhalation, the organism must be small enough to reach the alveoli)

• The organism’s ability to grow at 37°C at a neutral pH

• Conversion of the dimorphic fungi from the mycelial (mold) form into the corresponding yeast or spherule form in the host

• Toxin production

Most of the fungi exist in environmental niches as saprophytic organisms (Table 58.2). Perhaps the fungi that cause disease in humans have developed various mechanisms that allow them to establish disease in the human host. Table 58.3 describes some of the known or speculative virulence factors of the fungi known to be pathogenic for humans.

Laboratory Diagnosis

Collection, Transport, and Culturing of Clinical Specimens

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. If a fungal clinical infection is suspected, the sample should always be cultured on fungal media. If the specimen quantity is insufficient for microscopy and culture, the culture should be performed. Many infections have a primary focus in the lungs; respiratory tract secretions are usually 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. Specimens should be processed within 2 hours of receipt. Most fungal specimens can be maintained at room temperature. If processing of intravascular catheter tips, other medical devices (stents, surgical implants, replacement joints, etc.), lower respiratory tract or urine specimens will be delayed, the samples can be refrigerated for a short time. Dermatologic specimens (skin, hair, nails) are very sensitive to cold temperatures. 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 discussed later in this chapter. Additional information on specimen collection is included in Table 5.1.

Lower Respiratory Tract Secretions

Respiratory tract secretions (sputum, induced sputum, bronchial washings, bronchoalveolar lavage, and tracheal aspirations) are perhaps the most common specimens collected for fungal culture. Viscous lower respiratory tract specimens should be pretreated with a mucolytic agent and concentrated by centrifugation. The sediment should then be plated to media without antibiotic and a media containing antibacterial antibiotics to prevent overgrowth by contaminants and ensure optimal recover of fungi. The antifungal agent cycloheximide prevents overgrowth by rapidly growing molds and should be included in at least one of the culture media. As much specimen as possible (0.5 mL) should be used to inoculate each medium. Two fungal organisms commonly isolated from cystic fibrosis (CF) patients include Candida spp. and Scedosporium spp. Therefore, it is recommended that selective and differential chromogenic medium for the isolation of Candida spp. and Scedosporium-selective medium containing antifungal agents dicloran and benomyl should be added to respiratory cultures of CF patients to improve the isolation of these organisms. Specimens may be stored at room temperature if processing is completed within 2 hours; if processing is delayed, specimens should be refrigerated at 4°C.

Table 58.2

Summary of Common Pathogens
Organism	Natural Habitat	Infectious Form	Mode of Transmission	Common Sites of Infection	Clinical Form
Aspergillus spp.	Ubiquitous, plants	Conidia	Inhalation	Lungs, eyes, skin, nails	Hyphae
Blastomyces spp.	Unknown, possibly soil/wood in moist areas	Probably conidia	Usually inhalation	Lungs, skin, long bones	Yeast
Candida spp.	Human microbiota	Yeast, pseudohyphae, and true hyphae	Direct invasion/dissemination	GI and GU tracts, nails, viscera, blood	Yeast, pseudohyphae, and true hyphae
Coccidioides spp.	Soil of many arid regions	Arthroconidia	Inhalation	Lungs, skin, meninges	Spherules, endospores
Cryptococcus spp.	Bird feces, soil	Yeast ^a	Inhalation	Lungs, skin, meninges	Yeast
Histoplasma capsulatum	Bat and bird feces	Conidia	Inhalation	Lungs, bone marrow, blood	Yeast
Paracoccidioides spp.	Possibly soil, plants	Conidia	Inhalation/trauma	Lungs, skin, mucous membranes	Yeast
Sporothrix spp.	Soil, plants	Conidia/hyphae	Trauma/rarely inhalation	Skin and lymphatics, lungs, meninges	Yeast
Dermatophytes	Human disease, animals, soil	Conidia/hyphae	Contact	Skin, hair, or nails	Hyphae

Sterile Body Fluids Including Cerebrospinal Fluid

Most sterile body fluids are generally collected in heparin blood tubes to prevent clotting. Lysis centrifugation tubes may also be used for the collection of sterile body fluids. Cerebrospinal fluid lumbar puncture tubes collected for culture should be concentrated by centrifugation and the concentrated sediment used to inoculate the culture medium. Cultures should be examined daily. It is recommended that ≥2 mL of specimen should be centrifuged and up to 0.5 mL of sample be inoculated onto each type of fungal culture medium. If less than 1 mL of specimen is submitted for culture, following centrifugation, 1-drop aliquots of the sediment should be placed on several areas on the agar surface. Many laboratories utilize screw-cap tubes containing slants or culture vials to avoid contamination of fungal cultures. Media used for the recovery of fungi from sterile fluids should contain no antibacterial or antifungal agents. Cryptococcus spp., a pathogenic encapsulated yeast associated with meningitis, is inhibited by the antifungal agent cycloheximide.

Once submitted to the laboratory, sterile body fluid specimens should be processed promptly. If prompt processing is not possible, samples should be kept at room temperature. Sterile body fluid specimens should never be refrigerated.

Blood and Bone Marrow

Disseminated fungal infections are a major cause of morbidity and mortality in hospitalized patients, and blood cultures provide an accurate method for determining the cause in many instances. Currently several automated blood culture systems that utilize fungal medium modifications for the isolation of fungi, including the BACTEC (Becton Dickinson, Sparks, MD), BacT/ALERT 3D (bioMérieux, Durham, NC), and VersaTREK (Thermo Scientific, Oakwood Village, OH), are adequate systems for the recovery of yeasts, except Malassezia spp.

Laboratories that frequently recover dimorphic fungi and molds from blood or bone marrow are encouraged to use the lysis-centrifugation system, the Isolator. A heparinized syringe or pediatric Isolator tube is recommended for the collection of bone marrow samples. The Isolator has been proven optimal for the recovery of H. capsulatum and other filamentous fungi. Special automated fungal media such as the BACTEC MYCO/F lytic medium or BacT/ALERT media may also be used for the isolation of filamentous molds from blood samples. With this system, red blood cells and white blood cells, which may contain the microorganisms, are lysed, and centrifugation concentrates the organisms before culturing. The concentrate is inoculated onto the surface of appropriate culture media, and most fungi are detected within the first 4 days of incubation. However, occasional isolates of H. capsulatum may require approximately 10 to 14 days for recovery. In addition to the lysis-centrifugation system, special automated fungal media such as the BACTEC MYCO/F lytic medium or BacT/ALERT media may also be used for the isolation of filamentous molds to improve the recovery from blood. Bone marrow samples should not be placed in automated blood culture media. The optimal temperature for fungal blood cultures is 30°C, and the suggested incubation time is 21 days.

Table 58.3

Virulence Factors of Medically Important Fungi
Fungal Pathogen	Putative Virulence Factor
Aspergillus spp.	Elastase-serine protease Proteases Toxins (gliotoxin, fumagillin, helvolic acid) Elastase-metalloprotease Aspartic acid proteinase Aflatoxin Catalase Lysine biosynthesis p-aminobenzoic acid synthesis
Blastomyces spp.	Cell wall alpha-1,3-glucan BAD-1 an adhesion and immune modulator
Coccidioides spp.	Extracellular proteinases
Cryptococcus spp.	Capsule Phenoloxidase melanin synthesis Varietal differences
Dematiaceous fungi	Phenoloxidase melanin synthesis
Histoplasma capsulatum	Cell wall alpha-1,3-glucan Intracellular growth Thermotolerance CBP, binds calcium
Paracoccidioides spp.	Estrogen-binding proteins Cell wall components Beta-glucan Alpha-1,3-glucan
Sporothrix spp.	Thermotolerance Extracellular enzymes

Culture Media and Incubation Requirements

A number of fungal culture media are satisfactory for use in the clinical microbiology laboratory (Table 58.4). Most are adequate for the recovery of fungi. For optimal recovery, a battery of media should be used; the following are recommended:

• Media with and without cycloheximide to prevent the overgrowth of slow-growing fungi by more rapidly growing species. It is important to note that cycloheximide may also be inhibitory to some fungi.

• Media with and without an antibacterial agent (media with an antibacterial agent are used for specimens likely to contain contaminating bacteria; they are not necessary for specimens from sterile sites).

• Inhibitory agar controls bacterial contamination more effectively than Sabouraud dextrose agar.

• Chloramphenicol is an inhibitory agent for the growth of contaminating bacteria; however, it is important to note that it is also inhibitory to Nocardia and other aerobic actinomycetes.

• Growth of dimorphic fungi is enhanced on enriched media such as BHI containing antibiotics and 5% to 10% sheep blood. This enhances growth but inhibits sporulation. Once isolated, the fungi should be immediately subcultured to blood-free enriched media for identification.

• Birdseed agar may be used for the cultivation of Cryptococcus spp. from CSF, pleural fluid, bone marrow, tissue, and lower respiratory specimens.

• Specific chromogenic agar may be used to identify some species of yeast.

• Additional specialized media may be required for additional fungal isolates that have unique nutritional or incubation requirements.

Agar plates (petri dishes), rectangle agar vials, or screw-capped agar tubes are satisfactory for the recovery of fungi; however, plates or vials are preferred, because they provide better aeration of cultures and a large surface area for better isolation of colonies. Agar plates provide greater ease of handling by laboratory professionals when making microscopic preparations for examination. Agar tends to dehydrate during the extended incubation period required for fungal recovery, but this problem can be minimized by using agar plates or vials containing at least 40 mL of agar and placing them in a humidified incubator. Agar plates should be opened and examined in a certified biologic safety cabinet (BSC). Many laboratories discourage the use of agar plates because of safety considerations; however, the advantages outweigh the disadvantages.

Compared with agar plates, screw-capped culture tubes are more easily stored, require less space for incubation, and are easily handled. In addition, they have a lower dehydration rate, and most laboratory workers believe cultures are less hazardous to handle when in tubes. However, disadvantages, such as relatively poor isolation of colonies, a reduced surface area for culturing, and a tendency to promote anaerobiosis, discourage routine use in most clinical microbiology laboratories. If culture tubes are used, the tube should be as large as possible to provide an adequate surface area for isolation. After inoculation, tubes should be placed in a horizontal position for at least 1 to 2 hours to allow the specimen to absorb to the agar surface and prevent settling at the bottom of the tube.

Cultures should be incubated at room temperature, or preferably at 30°C, for 21 to 30 days before they are reported as negative. A relative humidity in the range of 40% to 50% can be achieved by placing an open pan of water in the incubator. Cultures should be examined at least three times weekly during incubation.

As previously mentioned some clinical specimens are contaminated with bacteria or rapidly growing fungi or both, requiring the use of antifungal and antibacterial agents. The addition of 0.5 μg/mL of cycloheximide and 16 μg/mL of chloramphenicol to media traditionally has been advocated to inhibit the growth of contaminating molds and bacteria, respectively. However, better results have been achieved using a combination of 5 μg/mL of gentamicin and 16 μg/mL of chloramphenicol as antibacterial agents. Ciprofloxacin at a concentration of 5 μg/mL may be used.

Cycloheximide may be added to any of the media that contain or lack antibacterial antibiotics. However, if cycloheximide is included in the battery of culture media, a medium lacking this ingredient should also be included. Pathogenic fungi, such as Cryptococcus spp., Candida krusei and other Candida spp., Trichosporon spp., P. boydii, and Aspergillus spp., are partially or completely inhibited by cycloheximide.

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 cases by direct examination (particularly with 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 cause of disease in patients with fungal infection and guide the selection of appropriate media to support growth.

Table 58.4

Fungal Culture Media: Indications for Use
Media	Indications for Use	Media Composition	Mode of Action
Primary Recovery Media
Brain-heart infusion agar	Primary recovery of saprobic and pathogenic fungi	Brain-heart infusion, enzymatic digest of animal tissue, enzymatic digest of casein, dextrose, sodium chloride	The agar provides a rich medium for bacteria, yeast, and pathogenic fungi.
Brain-heart infusion agar (fungal formulation) with antibiotics	Primary recovery of pathogenic fungi exclusive of dermatophytes	Brain-heart infusion, enzymatic digest of animal tissue, enzymatic digest of casein, dextrose, sodium chloride, 10% sheep blood, antibiotics (chloramphenicol, cycloheximide, and gentamicin)	The agar provides a rich medium for yeast and pathogenic fungi, including systemic dimorphic fungi.
Chromogenic agar	Isolation and presumptive identification of yeast and filamentous fungi	Chromopeptone Glucose Chromogen mix Chloramphenicol	Chromogen mix contains substrates that react with enzymes produced by different organisms that result in the production of characteristic color changes.
Dermatophyte test medium	Primary recovery of dermatophytes; recommended as screening medium	Soy, peptone, dextrose, cycloheximide, gentamicin, chloramphenicol, phenol red	Dermatophytes produce alkaline metabolites, which raise the pH and change the medium from red to yellow.
Inhibitory mold agar	Primary recovery of pathogenic, cycloheximide sensitive fungi exclusive of dermatophytes	Chloramphenicol, casein, dextrose, starch, sodium phosphate, magnesium sulfate, sodium chloride, manganese sulfate	Examine plates for growth. Chloramphenicol inhibits bacterial growth.
Potato flake agar	Primary recovery of saprobic and pathogenic fungi and the stimulation of conidia formation.	Potato flakes, glucose, cycloheximide, chloramphenicol, bromthymol blue	Growth is enhanced by a pH alkaline reaction of fungus. Chloramphenicol and antibiotics inhibit the growth of bacteria and nonpathogenic fungi.
Mycobiotic or mycosel agar	Primary recovery of dermatophytes but may also be used for the recovery of other pathogenic fungi.	Pancreatic digest of soybean meal and dextrose with cycloheximide, chloramphenicol.	Inhibits bacteria and saprophytic fungi
Sabouraud dextrose with brain-heart infusion (SABHI) agar	Primary recovery of saprobic and pathogenic fungi	Sabouraud dextrose, brain-heart infusion agar. With chloramphenicol, cycloheximide, penicillin, and/or streptomycin. 10% sheep blood may be added.	Isolates and enhances growth of all fungi including the yeast phase of dimorphic fungi.
Yeast-extract phosphate agar with ammonia	Primary recovery of pathogenic fungi exclusive of dermatophytes	Yeast extract, dipotassium phosphate, chloramphenicol. 1 drop of ammonium hydroxide is applied to the agar surface prior to inoculation and allowed to diffuse into the medium.	Enhances the recovery and sporulation of Blastomyces and Histoplasma capsulatum from contaminated specimens
Differential Test Media
Acetate Ascospore agar	Detection of ascospores in ascosporogenous yeasts (e.g., Saccharomyces spp.)	Potassium acetate, yeast extract, dextrose	Potassium acetate is necessary, and yeast extract increases the sporulation of yeasts.
Table Continued

Media	Indications for Use	Media Composition	Mode of Action
Christensen’s urea agar	Identification of Cryptococcus, Trichosporon, and Rhodotorula spp. Separation of Trichophyton mentagrophytes from Trichophyton rubrum	2% urea, phenol red	Produces urease and a change in the pH.
Cornmeal agar with Tween 80 and trypan blue	Differentiation of Candida spp. by chlamydospore production	Cornmeal, Tween 80, cornmeal infusion, and trypan blue	Addition of Tween 80 enhances the production of chlamydospores, pseudohyphal and arthrospore formation. The addition of trypan blue provides a contrasting background for observing the morphologic features of yeasts.
Czapek’s agar	Differential identification of Aspergillus spp.	Sodium nitrate, sucrose, yeast extract	Produces characteristic features of yeast and fungus of any organism that can use sodium nitrate.
Niger seed agar (birdseed agar)	Identification of Cryptococcus spp., particularly Cryptococcus neoformans and Cryptococcus gattii	Guizotia abyssinica seeds or niger seeds, dextrose, creatinine, chloramphenicol	C. neoformans and C. gattii produce the enzyme phenol oxidase, resulting in a brown pigment through metabolism of caffeic acid. Creatinine enhances the melanization of some strains of C. neoformans.
Potato dextrose agar	Demonstration of conidia formation and the pigment production by Trichophyton rubrum; preparation of microslide cultures and sporulation of dermatophytes	Potato infusion, dextrose, tartaric acid Note: Some laboratories use potato flake agar, because it may be more stable.	Carbohydrate and potato infusion promotes the growth of yeasts and molds, and the low pH (tartaric acid) partially inhibits bacterial growth.
Trichophyton agars 1–7	Identification of Trichophyton spp.	Dextrose, monopotassium phosphate, magnesium sulfate, amino acids 1. Casamino acids; vitamin free 2. Casamino acids plus inositol 3. Casamino acids plus inositol and thiamine 4. Casamino acids plus thiamine 5. Casamino acids plus niacin 6. Ammonium nitrate 7. Ammonium nitrate plus histidine	Trichophyton spp. may be differentiated by growth in the presence of different amino acids.
Yeast fermentation broth	Identification of yeasts by determining fermentation	Yeast extract, peptone, bromcresol purple, and a specific carbohydrate (e.g., dextrose, maltose, sucrose)	Most yeasts produce acid, which is indicated by a change in the solution from purple to yellow as a positive fermenter.
Yeast nitrogen-base agar	Identification of yeasts by determining carbohydrate assimilation	Ammonium sulfate, carbon source (e.g., glucose, sucrose, raffinose)	Assimilation of carbon by yeast cells produces a positive result.

Tables 58.5 and 58.6 present the methods available for direct microscopic detection of fungi in clinical specimens and a summary of the characteristic microscopic features of each. Fig. 58.4 presents photomicrographs of some of the fungi commonly seen in clinical specimens.

Traditionally the potassium hydroxide preparation has been the recommended method for direct microscopic examination of specimens in dermatological samples. Calcofluor white (CW) (Evolve Procedure 58.1) and lactophenol blue (LPCB) bind specifically to polysaccharides, chitin, and cellulose present in fungal cell walls. In addition, LPCB contains lactic acid that aids in the preservation of fungal structures and phenol that acts as a killing agent. Slides prepared using these methods may be observed using fluorescent (CW) or bright-field microscopy (LPCB).

Procedure 58.1

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 cover slip.

3. If necessary, dissociate particles by applying gentle pressure to the cover slip 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 400–500 nm exciter filter and a 500–520 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.

Serologic Testing

Molecular diagnostics may eventually replace the use of serologic testing for the diagnosis of fungal infections. These methods currently lack standardization for performance when taken directly from patient specimens. No commercially available procedures exist for serologic testing of most fungi. However, serology testing is a useful tool for the diagnosis of invasive fungal infections with select organisms, such as Cryptococcus, Coccidioides, Blastomyces, Histoplasma, and Aspergillus spp.

Antibody testing has proven useful but not for immunocompromised patients, who are incapable of producing a measurable humoral response. Acute and convalescent titers need to be monitored during treatment of the fungal infection.

Immunodiffusion testing is a simple, cost-effective procedure. Although it is 100% specific, it is relatively insensitive and is not used as a screening tool. This test also requires 2 to 3 weeks to exhibit a positive result.

Enzyme immunoassays for both antibody and antigen have been used. These tests are also commonly negative in immunocompromised patients, especially early in the infection.

Point-of-care (POC) testing using lateral flow assay-based devices have the potential to improve the diagnosis of fungal infections, especially in developing countries with limited laboratory resources. POC devices generally are inexpensive and portable while providing rapid and reproducible results that are highly sensitive and specific. Undoubtedly, the development of these devices will be useful in monitoring and detecting fungal antibodies.

(1,3)-β-D-Glucan Detection

(1,3)-β-D-Glucan, a polysaccharide present in the cell wall of some fungi, is found in the blood of patients that have invasive fungal infections. Although detection of the polysaccharide has demonstrated success in the diagnosis and monitoring of fungal meningitis, variation in assay sensitivity and specificity, along with the need for what are considered significant levels in the blood of a patient with fungal infections, indicates further studies are necessary.

Molecular Methods

Phenotypic and biochemical identification methods are extremely time-consuming for the identification of fungal pathogens. One of the most critical risk factors associated with mortality from systemic mycoses is the time to diagnosis, making molecular detection methods ideal in the clinical laboratory. In addition to diagnosis, drug resistance among invasive Candida and Cryptococcus spp. has increased, requiring the development of new assays to detect drug resistance. Ideally, a molecular direct hybridization assay or amplification assay panel of primers specific for the detection of fungi in clinical specimens would include the most common organisms known to cause disease in immunocompromised patients (including the dimorphic fungi and Pneumocystis spp.). The current literature contains references to all of the major human fungal pathogens, describing species and strain-specific primers and probes, yet no commercial methods are available to the clinical laboratory. Sequence-based molecular identification of fungal isolates is a useful diagnostic tool and has resulted in the development of commercial diagnostic assays such as the MicroSEQ D2 rDNA Fungal Sequencing Kit (Thermo Fisher, Grand Island, NY). Currently, DNA sequencing technology, including whole genome sequencing, remains confined to research and reference laboratories due to the large capital investment and expertise required for implementation.

Currently, a few FDA-cleared molecular diagnostic assays are available, which include amplification and hybridization techniques. These assays primarily focus on the identification of Candida, Cryptococcus, Aspergillus spp., and the systemic dimorphic fungi.

Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a biophysical method that significantly reduces the time required to specifically identify fungal organisms. The major disadvantage of this technique is the need to have pure cultures of the clinical isolates for sample preparation, adding days and weeks to the process. The protein profiles for fungi also vary significantly based on environmental and culture conditions, making standardization of the analytical process extremely difficult in fungal identification. The level of fungal organisms, such as yeast in the bloodstream and other clinical fluid samples is generally too low for direct detection by MALDI-TOF MS. In addition, proteins and hemoglobin tend to interfere with the spectra analytics making identification problematic. Further clinical studies are needed prior to implementation of direct identification using MALDI-TOF MS for systemic fungal infections. An overview of the technique is discussed in more detail in Chapter 7.

Table 58.5

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. ^a and some isolates of Blastomyces spp.	Tissue homogenates are difficult to observe because of background staining.
Alcian blue or mucicarmine stain.	Mucopolysaccharide stains used to visualize the capsule of Cryptococcus spp. in histological tissue sections.	30 min	Detects encapsulated yeast in tissue sections	Blastomyces dermatitidis and Rhinosporidium seeberi may also react positively with this stain.
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. Nonspecific reactions may be observed, such as cotton fibers from swabs and brain tumor biopsies, both falsely resembling branching hyphae.
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 (nigrosin) stain	Detection of Cryptococcus spp. 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. Artifacts such as erythrocytes, leukocytes, and talc particles from gloves or bubbles may mimic yeast, resulting in false positives.
Lactophenol cotton (aniline) blue wet mount	Most widely used method of staining and observing fungi	1 min	Lactic acid and glycerol preserves structures; slides can be made permanent.	Mechanical treatment dislodges fungal structures.
Potassium hydroxide	Clearing of specimen using 10%–20% KOH 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. 0.1% thimerosal (Sigma Chemical Co.) may be added to preserve the specimen.	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 h, 10 min	Aids differentiation of melanin and hemosiderin pigments.	Difficult to interpret when only rare granular staining is present.
Table Continued

Method	Use	Time Required	Advantages	Disadvantages
Methenamine silver stain	Detection of fungi in histologic section	1 h	Best stain for detecting fungal elements (black) against a pale green or yellow background.	Requires a specialized staining method that is not usually readily available to microbiology laboratories.
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. Time-consuming and has been replaced in many laboratories by calcofluor white staining procedures.
Toluidine blue O	Rapid detection of P. jiroveci from lung biopsy and BAL specimens.	1 min	Quickly performed, easy, rapid results, and cost-effective.	Trophozoites are not discernable.
Wright’s stain	Examination of bone marrow or peripheral blood smears	7 min	Detects Histoplasma capsulatum and Cryptococcus spp.	Most often used to detect H. capsulatum and Cryptococcus spp. in disseminated disease.

General Considerations for the Identification of Yeasts

Most often yeasts are identified through the use of a combination of differential test media (Fig. 58.5). Identification factors and techniques include the following:

Several key characteristics can be seen macroscopically. Colonies have a wide variety of colors, shapes, and textures. Chromogenic agar or a combination of rapid tests can be used to differentiate some pathogenic yeast presumptively. Presumptive identification is indicated when the test or combination of tests do not identify characteristics that are unique to that species. The results of these tests must be correlated with the macroscopic and microscopic morphological characteristics and the site of infection (i.e., clinical specimen). Wet preps and lactophenol cotton blue stain can aid microscopic identification by improving the visualization of the fungal reproductive structures. Sexual and asexual characteristics are very important. Often a genus can be determined by the microscopic and macroscopic characteristics. India ink stain is useful when Cryptococcus spp. 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 a limited set of characteristics to further aid identification.

The clinical microbiology laboratory has historically operated under the idea that if the identification of the isolate to the species level is incorrect, the patient will not be adversely affected once the susceptibility is completed. This is problematic, when the specific species identifications are used to determine minimum inhibitory concentrations and breakpoints for treatment for bacterial and fungal infections. Treatment failures, along with the increasing occurrence of resistance to echinocandins, fluconazole, and voriconazole, make accurate identification and susceptibility testing required. Molecular diagnostic techniques, proteomic and genomic, clearly demonstrate more accurate and reliable identification for yeasts than conventional biochemical methods. The use of these methods in the routine clinical laboratory is currently limited by the need for standardization and the expansion of the current databases.

Table 58.6

Summary of Characteristic Features of Select 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 spp.	2–6	Small; oval to round to cigar-shaped; single or multiple buds present; uncommonly seen in clinical specimens.
	Cryptococcus spp.	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 spp.	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 spp.	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 spp.	10–200	Spherules vary in size; some may contain endospores, others may be empty; adjacent spherules may resemble Blastomyces spp.; 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 h.
Spherules	Rhinosporidium seeberi (protozoan [parafungal] pathogen that is studied in mycology)	6–300	Large, thick-walled sporangia containing sporangiospores are present; mature sporangia are larger than spherules of Coccidioides; hyphae may be found in cavitary lesions.
Table Continued

Morphologic Form Found in Specimens	Organism	Size Range (diameter, mm)	Characteristic Features
Yeast and pseudohyphae or hyphae	Candida and Candida dubliniensis	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.
Yeast and pseudohyphae or hyphae	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; “spaghetti and meatballs.”
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 Aspergillus flavus.
Hyaline septate hyphae	Dermatophytes, skin and nails	3–15	Hyaline, septate hyphae are commonly seen; chains of arthroconidia may be present.
	Dermatophytes, 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. 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 Mucorales.
	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., Cladophialophora spp., Cladosporium spp., Curvularia spp., Exophiala spp., Exserohilum spp., Hortaea werneckii, Phialophora spp., and other genera.	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.
Sclerotic bodies	Cladophialophora (formerly Cladosporium) carrionii Fonsecaea spp., Phialophora verrucosa, and 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).
Table Continued

Morphologic Form Found in Specimens	Organism	Size Range (diameter, mm)	Characteristic Features
Granules	Acremonium spp.	200–300	White, soft granules without a cementlike matrix.
	Aspergillus Aspergillus nidulans	500–1000	Black, hard grains with a cementlike matrix at the periphery.
	Curvularia Curvularia geniculata Curvularia lunata	65–160	White, soft granule without a cementlike matrix.
	Exophiala Exophiala jeanselmei	200–300	Black, soft granules, vacuolated, without a cementlike matrix, made of dark hyphae and swollen cells.
	Fusarium Fusarium verticillioides (formerly F. moniliformis)	200–500	White, soft granules without a cementlike matrix.
	Fusarium solani	300–600
	Trematosphaeria grisea (formerly Madurella grisea)	350–500	Black, soft granules without a cementlike matrix; the periphery is composed of polygonal swollen cells, and the center has a hyphal network.
	Madurella 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 Neotestudina rosatii	300–600	White, soft granules with cementlike matrix at the periphery.
	Pseudallescheria Pseudallescheria boydii	200–300	White, soft granules composed of hyphae and swollen cells at the periphery in a cementlike matrix.

General Considerations for the Identification of Molds

Filamentous fungi are also identified by a combination of tests (Fig. 58.6). Molds are identified using a combination of the following:

• Growth rate

• Colonial morphologic features

• Microscopic morphologic features

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 Coccidioides is often rapid and is hazardous to microbiologists. In general, the growth rate for the dimorphic fungi, Blastomyces 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, Talaromyces marneffei, 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 melanized (dematiaceous) 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 to supplement the microscopic morphologic features of the organism.

The color of the colony and uniformity of the color can be important, along with the presence of diffusible pigments in the media. The examiner must be sure to note the color of 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), or 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 appears as a white-to-tan fluffy mold on BHI agar and 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 tape preparation (Evolve Procedure 58.2; Fig. 58.7). It can be prepared easily and quickly and often is sufficient to make the identification for most fungi. However, some laboratories prefer the wet mount (Evolve Procedure 58.3; Fig. 58.8) or tease mount (Evolve Procedure 58.4). A microslide culture method (Evolve Procedure 58.5; Fig. 58.9) may be used when greater detail of the morphologic features is required.

Fig. 58.5 Traditional identification of yeasts in clinical specimens. Manual methods, kits, and panels have been predominantly replaced with proteomic and genomic methods.

Procedure 58.2

Cellophane 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 of the slide (Fig. 58.7).
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 may be seen in wet mount preparations when the adhesive tape preparation reveals only hyphal fragments. In other instances, cultures may have sporulated and reveal only the presence of conidia when the adhesive tape preparation is 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 (Evolve Procedure 58.5) 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 cover slip. Microscopic features should be easily discerned, structures should be intact, and many representative areas of growth are available for observation.

Procedure 58.3

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 an intermediate point between the center and the periphery of the colony 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 (Fig. 58.8).
3. Place a cover slip 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 cover slip. However, this method is suitable in many situations, because characteristic spores are visible, but their arrangement cannot be determined. The wet mount is not always adequate for making a definitive identification.

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 (Fig. 58.9).
5. Apply a sterile cover slip 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 cover slip and place it on a microscope slide containing a drop of lactophenol cotton or aniline blue. Some suggest placing the cover slip near the opening of an incinerator-burner to allow the organism to dry rapidly on the cover slip 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 cover slip 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 spp., Coccidioides spp., Paracoccidioides brasiliensis, Emmonsia spp., or Sporothrix spp.). Microslide cultures must be observed only after a cover slip has been removed from the agar plug and not while it is in position on top of the agar plug. This 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 (Fig. 58.10). Racquet hyphae are enlarged, club-shaped structures (Fig. 58.11). In addition, certain dermatophytes produce spiral hyphae that are coiled or exhibit corkscrewlike turns in the hyphal strand (Fig. 58.12). These structures are not characteristic for any certain group; however, they are found most commonly in dermatophytes.

Some species of fungi (Ascomycota) produce sexual spores in a large, saclike structure called an ascocarp (Fig. 58.13). 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 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 (Fig. 58.14).

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 (Fig. 58.15). 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 (Fig. 58.16). 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. Sometimes 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 (Fig. 58.17). In other cases, 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 (Fig. 58.18). Species of Penicillium and Paecilomyces are representative of this type of sporulation. Some fungi may produce conidia of two sizes: microconidia, which are small, unicellular, round, elliptical, or pyriform (pear-shaped), or macroconidia, which are large, usually multiseptate, and club- or spindle-shaped (Fig. 58.19). 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.

Fig. 58.6 Traditional identification of filamentous fungi from clinical specimens. Some molds may be identified using proteomic and genomic methods.

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 (Fig. 58.20). In rare cases, some isolates may produce zygospores, rough-walled spores produced by the union of two mating types of a Mucorales; this is an example of sexual reproduction.

Fig. 58.12 Spiral hyphae (arrow) showing corkscrewlike turns (×430).

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. The extent of identification of yeasts from various specimen sources is discussed in Chapter 62.

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, must make this decision after considering the patient population, laboratory practice, and economic implications.