Overview of Fungal Identification Methods and Strategies

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Overview of Fungal Identification Methods and Strategies

Objectives

1. Define the terms mycology, saprophytic, dermatophyte, and polymorphic, dimorphic, and thermally dimorphic fungi.

2. Define and differentiate superficial, cutaneous, subcutaneous, and systemic mycoses, including the tissues involved.

3. Differentiate the colonial morphology of yeasts and filamentous fungi (molds).

4. Define and differentiate anamorph, teleomorph, and synanamorph.

5. Describe three ways in which fungi reproduce.

6. List the media that should be used for optimal recovery of fungi, including their incubation requirements.

7. List the common antibacterial agents used in fungal media.

8. Explain and differentiate the characteristic colonial morphology of fungi, including topography (rugose, umbonate, verrucose), texture (cottony, velvety, glabrous, granular, wooly) and surface described (front, reverse).

9. Describe and differentiate the sexual and asexual reproduction of the Ascomycota.

10. Define and differentiate rapid, intermediate, and slow growth rates with regard to fungal reproduction and cultivation.

11. Describe the proper method of specimen collection for fungal cultures, including collection site, acceptability, processing, transport, and storage.

12. Give the advantages and disadvantages of using screw-capped culture tubes, compared with agar plates, in the laboratory.

13. Describe the chemical principle and methodologies used to identify fungi, including calcofluor white–potassium hydroxide preparations, hair perforation, cellophane (Scotch) tape preparations, saline/wet mounts, lactophenol cotton blue, potassium hydroxide, Gram stain, India ink, modified acid-fast stain, periodic acid-Schiff stain (PAS), Wright’s stain, Papanicolaou stain, Grocott’s methenamine silver (GMS), hematoxylin and eosin (H&E) stain, Masson-Fontana stain, tease mount and microslide culture.

Mycology is a specialized discipline in the field of biology concerned with the study of fungi, including their taxonomy, environmental impact, and genetic and biochemical properties. Historically, the fungi were regarded as relatively insignificant causes of infection. However, in the early to mid-twentieth century, these microorganisms began to be recognized as important causes of disease, particularly because of changes in patient profiles, and this trend continues today. Because of the endemic systemic mycoses, which may cause disease in healthy hosts, a number of fungal species normally found in the environment have been recognized as important causes of human disease, particularly in the immunocompromised host. The modern clinical laboratory, therefore, must provide methods for isolating and identifying the common causes of mycologic disease. Susceptibility testing of these isolates is often necessary.

Some clinical microbiology laboratories have kept pace with changing times and have developed more extensive mycologic testing methods. However, the economic constraints of the current health care environment have prevented other laboratories from offering these services. In such cases, diagnostic clinical mycology is performed by reference laboratories, which have varying degrees of experience. The lack of experience in clinical mycology has been influenced by a shortage of trained individuals, lack of quality educational programs, and inability of clinical laboratories to support the cost of sending personnel to training courses. Commonly, individuals with experience who retire or leave their position are replaced by someone with considerably less experience. Training and continuing education programs are needed to assist in the development of such individuals, if quality laboratory services are to be offered. A real concern is that the changing health care environment and implementation of cost containment measures, without continuing education, will prevent future generations from being well trained in diagnostic clinical mycology.

This chapter is designed to assist technologists and microbiologists with the basics of diagnostic clinical mycology, in the hope that the information will allow some laboratories to offer clinical mycology services.

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.

TABLE 59-1

Phylogenetic Position of Medically Significant Fungi

Phylum/Class Order Genus/Species
Phylum Glomeromycota    
 Subphylum
 Mucormycotina
Mucorales Absidia
Cunninghamella
Mucor
Rhizopus
Syncephalastrum
 Subphylum
 Entomophthoromycotina
Entomophthorales Basidiobolus
Conidiobolus
Phylum Ascomycota    
 Dothideomycetes Capnodiales Cladosporium
Piedraia hortae
Dothideales
Pleosporales
Aureobasidium
Alternaria
Bipolaris
Curvularia
Drechslera
Exserohilum
Helminthosporium
Stemphylium
Ulocladium
Epicoccum
Phoma
 Saccharomycetes Saccharomycetales Endomyces (Geotrichum sp.)*
Kluyveromyces (Candida pseudotropicalis)*
Candida
Geotrichum
 Sordariomycetes Hypocreales Acremonium
Gliocladium
Fusarium
Scopulariopsis
Sepedonium
Trichoderma
Microascales Pseudallescheria boydii
Scedosporium prolificans
Sporothrix
Sordariales Madurella
Trichosphaeriales Nigrospora
 Eurotiomycetes Chaetothyriales Cladophialophora
Exophiala
Fonsecaea
Phialophora
Rhinocladiella
Eurotiales Emericella (Aspergillus nidulans)*
Aspergillus
Paecilomyces
Penicillium
Onygenales Ajellomyces (Histoplasma capsulatum, Blastomyces dermatitidis)*
Arthroderma (Trichophyton sp. and Microsporum sp.)*
Chrysosporium
Coccidioides
Epidermophyton
Histoplasma
Microsporum
Paracoccidioides
Trichophyton
Phylum Basidiomycota    
 Tremellomycetes Filobasidiales Filobasidium (Cryptococcus neoformans)*
Not assigned Incertae sidis Blastomyces

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*When the sexual form is known.

Most commonly encountered as causes of infection.

Modified from the Catalogue of Life. November 20, 2012. www.catalogueoflife.org/col/browse/classification and Hibbet DS, Binder M, Bischoff JF, et al. A higher-level of classification of the fungi, Mycological Research, 509-547, 2007.

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.

TABLE 59-2

General Clinical Classification of Pathogenic Fungi

Cutaneous Subcutaneous Opportunistic Systemic

 

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*Virtually any fungus may cause disease in a profoundly immunocompromised host.

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.

image

*Rudimentary hyphae may be present.

From Koneman EW, Roberts GD: Practical laboratory mycology, ed 3, Baltimore, 1985, Williams & Wilkins.

Use of the identification scheme just described requires examination of the fungal culture for 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), zygomycetes 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 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 (see Table 59-3). Murray2 has developed an expanded morphologic classification of medically important fungi based on general microscopic features and colonial morphology. The color pigmentation of colonies is presented as a useful diagnostic feature (Box 59-1).

Box 59-1

Phenotypic Classification of Medically Important Fungi

Thermally Monomorphic Molds

1. White, cream, or light gray surface; nonpigmented reverse

2. White, cream, beige, or light gray surface; yellow, orange, or reddish reverse (genera)

3. White cream, beige, or light gray surface; red to purple reverse (genera)

4. White, cream, beige, or light gray surface; brown reverse (genera)

5. White, cream, beige, or light gray surface; black reverse (genera)

6. Tan to brown surface

7. Yellow to orange surface (genera)

8. Pink to violet surface (genera)

9. Green surface; light reverse (genera)

10. Dark gray or black surface; light reverse (genera)

11. Green, dark gray, or black surface; dark reverse

From Murray PR: ASM pocket guide to clinical microbiology, vol 3, Washington, DC, 2004, ASM Press.

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.

TABLE 59-4

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 dermatitidis Unknown(?), soil/wood Probably conidia Usually inhalation Lungs, skin, long bones Yeast
Candida spp. Human flora Yeast, pseudohyphae, and true hyphae Direct invasion/dissemination GI and GU tracts, nails, viscera, blood Yeast, pseudohyphae, and true hyphae
Coccidioides immitis Soil of many arid regions Arthroconidia Inhalation Lungs, skin, meninges Spherules, endospores
Cryptococcus neoformans complex Bird feces, soil Yeast* Inhalation Lungs, skin, meninges Yeast
Histoplasma capsulatum Bat and bird feces Conidia Inhalation Lungs, bone marrow, blood Yeast
Paracoccidioides brasiliensis (?)Soil, plants Conidia Inhalation/trauma Lungs, skin, mucous membranes Yeast
Sporothrix schenckii 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

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GI, Gastrointestinal; GU, genitourinary.

*Possibly the conidia of the teleomorphic stage (Filobasidiella neoformans).

Blastomyces dermatitidis
BAD-1 Coccidioides immitis Cryptococcus neoformans complex Dematiaceous fungi Histoplasma capsulatum Paracoccidioides brasiliensis Sporothrix schenckii

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Laboratory Diagnosis

Collection, Transport, and Culturing of Clinical Specimens

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