Chapter 45: Medical Mycology

Mycology is the study of fungi, which are eukaryotic organisms that evolved in tandem with the animal kingdom. However, unlike animals, most fungi are nonmotile and possess a rigid cell wall. Unlike plants, fungi are nonphotosynthetic. Approximately 80,000 species of fungi have been described, but only about 400 are medically important, and less than 50 are responsible for more than 90% of the fungal infections of humans and other animals. Rather, most species of fungi are beneficial to humankind. They reside in nature and are essential in breaking down and recycling organic matter. Some fungi greatly enhance our quality of life by contributing to the production of food and spirits, including cheese, bread, and beer. Other fungi have served medicine by providing useful bioactive secondary metabolites such as antibiotics (eg, penicillin) and immunosuppressive drugs (eg, cyclosporine). Fungi have also been exploited by geneticists and molecular biologists as model systems for the investigation of a variety of eukaryotic processes, including cellular growth and development. Overall, fungi exert their greatest economic impact as phytopathogens; the agricultural industry sustains huge crop losses every year as a result of fungal diseases of rice, corn, grains, and other plants.

Like all eukaryotes, each fungal cell has at least one nucleus with a nuclear membrane, endoplasmic reticulum, mitochondria, and secretory apparatus. Most fungi are obligate or facultative aerobes. They are chemotrophic, secreting enzymes that degrade a wide variety of organic substrates into soluble nutrients which are then passively absorbed or taken into the cell by active transport.

The term mycoses refers to infections that are caused by fungi. Most pathogenic fungi are exogenous, their natural habitats being water, soil, and organic debris. The mycoses with the highest incidence—candidiasis and dermatophytosis—are caused by fungi that are frequent components of the normal human microbiota and highly adapted to survival on the human host. For convenience, mycoses may be classified as superficial, cutaneous, subcutaneous, or systemic, invading the internal organs (Table 45-1). The systemic mycoses may be caused by endemic fungi, which are usually primary pathogens and geographically restricted, or by ubiquitous, often secondary opportunistic pathogens. Grouping mycoses in these categories reflects their most common portal of entry and initial site of involvement. However, there is considerable overlap, since systemic mycoses often exhibit subcutaneous manifestations and vice versa. Most patients who develop opportunistic infections have serious underlying diseases and compromised host defenses. But primary systemic mycoses also occur in such patients, and the opportunists often infect immunocompetent individuals.

During a fungal infection, most patients develop significant cellular and humoral immune responses to fungal antigens. Much of the continuing increase in opportunistic mycoses can be attributed to medical advances that have significantly prolonged the survival of patients with cancer, AIDS, and hematopoietic stem cell or solid organ transplants. As these clinical data suggest, Th1 and Th17 immune responses are critical host defense mechanisms for natural protection against life-threatening mycoses. Pathogenic fungi do not produce potent toxins, and the features of fungal pathogenicity are complex and polygenic. In addition, experimental infections have shown that populations of a pathogenic species vary in their virulence, and genetic studies have identified numerous genes that contribute to fungal pathogenicity.

Most mycoses are difficult to treat. Because fungi are eukaryotes, they share numerous homologous genes, gene products, and pathways with their human hosts. Consequently, there are few unique targets for chemotherapy. However, there is growing interest in the search for potential therapeutic targets and new antifungal drugs are becoming available.

Fungi have two basic growth forms, as molds and yeasts. Growth in the mold (or mould) form occurs by the production of multicellular branching cylindrical tubules called hyphae that vary in diameter from 2 to 10 μm. Hyphae are extended by apical elongation due to the production of new cell wall growth at the hyphal tips. The mass of intertwined hyphae that accumulates during active growth is a mycelium. Some hyphae are divided into cells by cross-walls or septa, which typically form at regular intervals during hyphal growth. However, members of the Order Mucorales produce hyphae that are rarely septated. Vegetative or substrate hyphae penetrate the supporting medium, anchor the colony, and absorb nutrients. In contrast, aerial hyphae project above the surface of the mycelium and usually bear the reproductive structures of the mold. When a mold is isolated from a clinical specimen, its growth rate, macroscopic appearance, and microscopic morphology are usually sufficient to determine its genus and species. The most helpful phenotypic features are the ontogeny and morphology of the asexual reproductive spores, or conidia (Figures 45-2, 45-3, 45-4, 45-5, 45-6, 45-7, and 45-8).

Yeasts are single cells, usually spherical to ellipsoid in shape and varying in diameter from 3 to 15 μm. Most yeasts reproduce by budding, which is initiated by a lateral or terminal protrusion of new cell wall growth that enlarges during mitosis. One or more replicated nuclei enter the nascent bud, which subsequently forms a septum and separates from the parent cell. Some species produce buds that characteristically fail to detach and become elongated; this continuation of the budding process produces chains of elongated yeast cells called pseudohyphae. Yeast colonies are usually soft, opaque, 1–3 mm in size, and cream colored. The colonies and microscopic morphology of many species of yeasts appear quite similar, but they can be identified by physiologic tests and a few key morphologic differences. Some species, including several pathogens, are dimorphic and capable of growth as a yeast or mold depending on environmental conditions, such as temperature or available nutrients.

The life cycles of fungi are remarkably versatile. Depending on the fungus, the predominant nuclear chromosomal count may be haploid or diploid. Some species exist entirely by clonal growth or asexual reproduction, and barring spontaneous mutations, every cell will be a genetic clone. Many other species are capable of sexual reproduction, which may or may not require genetically different partners for mating and meiosis. Asexual as well as sexual reproduction can result in the production of spores, which enhance fungal survival. Spores are usually dormant, readily dispersed, more resistant to adverse conditions, and germinate to form vegetative cells when conditions for growth are favorable. Spores derived from asexual or sexual reproduction are termed anamorphic or teleomorphic, respectively. Like vegetative cells, asexual spores are mitotic progeny (ie, mitospores). The medical fungi produce two major types of asexual spores, conidia, which are produced by most pathogenic fungi, and, in the Order Mucorales, sporangiospores (see below and Glossary). Informative features of spores include their ontogeny (some molds produce complex conidiogenic structures) as well as their morphology (size, shape, texture, color, and unicellularity or multicellularity). In some fungi, vegetative cells may transform into conidia (eg, arthroconidia, chlamydospores). In others, conidia are produced by a conidiogenous cell, such as a phialide, which itself may be attached to a specialized hypha called a conidiophore. Sporangiospores result from mitotic replication and spore production within a sac-like structure called a sporangium, which is supported by a sporangiophore.

Certain fungal properties are essential but not necessarily sufficient for pathogenicity, such as the ability to proliferate in the mammalian host. Many virulence factors have evolved to enable pathogenic fungi to withstand or circumvent the defenses and stressful environment of the host. Some of these virulence determinants include morphological transformations, genetic “switching” of metabolic processes in response to the host environment, the production of surface adhesins that bind to host cell membranes, the secretion of enzymes that attack host substrates (eg, catalase, aspartyl proteinases, phospholipases), cell wall components that resist phagocytosis (eg, α-(1,3)-glucan, melanin, the capsule of Cryptococcus), and the formation of biofilms. Specific examples are provided in this chapter’s descriptions of several mycoses.

Fungi have an essential rigid cell wall that determines their shape and protects them from osmotic and other environmental stresses. Cell walls are composed largely of carbohydrate layers—long chains of polysaccharides—as well as glycoproteins and lipids. Some sugar polymers are found in the cell walls of many fungi, such as chitin (an unbranched polymer of β-1,4-linked N-acetylglucosamine); glucans, which are glucose polymers (eg, α-1,3-glucan, β-1,3-glucan, and β-1,6-glucan); and mannans, polymers of mannose (eg, α-1,6-mannose). These components are cross-linked to from a multilayered cell wall matrix. In addition, other polysaccharides may be unique to specific fungal species and therefore useful for identification. During infection, fungal cell walls exert important pathobiologic properties. The surface components of the cell wall mediate attachment of the fungus to host cells. Specific fungal cell wall moieties bind to pattern recognition receptors on host cell membranes, such as certain Toll-like receptors, to stimulate innate immune responses. Cell wall glucans and other polysaccharides may activate the complement cascade and provoke an inflammatory reaction. Most of these polysaccharides are poorly degraded by the host and can be detected with special histologic stains. Cell walls also release immunodominant antigens that may elicit cellular immune responses and diagnostic antibodies. In addition, some yeasts and molds are described as dematiaceous because their cell walls contain melanin, which imparts a brown or black pigment to the fungal colony. Several studies have shown that melanin protects these fungi from host defenses.

Taxonomy

Fungi were initially classified into phyla based largely on their modes of sexual reproduction and phenotypic data. These methods have been supplanted by molecular systematics, which more accurately reflect phylogenetic relationships. There is some ambiguity about the divergence of fungi and animals and their extant ancestors. The lower fungi were assigned to the Phylum Zygomycota, but this phylum was shown to be polyphyletic and has been replaced by the Phylum Glomerulomycota, four subphyla and two zoopathogenic Orders, the Mucorales and the Entomophthorales. However, the two largest phyla, Ascomycota and Basidiomycota, are well supported by phylogenetic analyses. All three of these phyla contain yeasts, molds, and dimorphic species. The Phylum Ascomycota (or ascomycetes) includes more than 60% of the known fungi and about 85% of the human pathogens. Most of the other pathogenic fungi are members of the Phylum Basidiomycota (basidiomycetes) or the Order Mucorales. These medically relevant taxa are distinguished by their modes of reproduction. Sexual reproduction typically occurs when mating-compatible strains of a species are stimulated by pheromones to undergo plasmogamy, karyogamy (nuclear fusion), and meiosis, resulting in the exchange of genetic information and the formation of haploid sexual spores.

For molds within the Order Mucorales, the vegetative hyphae have few septations, the product of sexual reproduction between mating-compatible isolates is a zygospore, and asexual reproduction occurs via sporangia, which are borne on aerial sporangiophores (see Glossary). Examples include species of Rhizopus, Lichtheimia, and Cunninghamella. Among the ascomycetes, sexual reproduction usually requires the fusion of mating-compatible strains and involves the formation of a sac or ascus in which karyogamy and meiosis occur, producing haploid ascospores. They reproduce asexually with the production of conidia. Ascomycetous molds have septate hyphae. Most pathogenic yeasts (Saccharomyces, Candida) and molds (Coccidioides, Blastomyces, Trichophyton) are ascomycetes. The basidiomycetes include mushrooms as well as pathogenic species of Cryptococcus. Sexual reproduction results in dikaryotic hyphae and four progeny basidiospores that are supported by a club-shaped basidium. In the diagnostic laboratory, multiple approaches are deployed to identify clinical isolates, including molecular and phenotypic features (eg, signature DNA sequences, morphology of reproductive structures, physiologic properties). Clinical isolates almost always represent infection by a single clone and reproduce asexually in the laboratory. Consequently, many pathogens were initially classified according to their asexual reproductive structures or anamorphic states, and with the subsequent discovery of a sexual cycle, such taxa acquired a teleomorphic name. During the evolution to become successful pathogens, some fungi have apparently lost the ability to reproduce sexually.

The vast majority of fungi have evolved to reside in various environmental niches where they grow readily on vicinal organic substrates and are protected from deleterious conditions. Although these exogenous fungi are unable to penetrate the intact surfaces of healthy hosts, they may be acquired accidentally by traumatic exposure to resident fungi in soil, water, air, or vegetation. Once fungal cells have breached the cutaneous or mucosal surfaces, such as the skin, or the respiratory, urinary, or gastrointestinal tract, they are repelled by innate host defenses. Potentially pathogenic fungi must be able to grow at 37°C, acquire essential nutrients from the host, and evade the immune responses. The few hundred environmental fungi with these attributes represent only a tiny percentage of global species. Unfortunately, a few highly prevalent fungi with these abilities are able to cause opportunistic, invasive infections in patients with compromised host defenses (eg, aspergillosis, cryptococcosis). Overall, most mycoses are caused by noninvasive molds that have adapted to grow on the skin, hair, or nails, and by endogenous species of Candida, which are members of the human mycobiome. However, regardless of their source, with the exception of dermatophytes, pathogenic fungi are not contagious, and transmission among humans or animals is extremely rare. This chapter describes the most prevalent mycoses, but new pathogens are reported every year. There is also brief coverage of two different mechanisms whereby fungi may cause human disease—ingestion of fungal toxins or exposure to fungal cell wall components that elicit IgE-mediated allergic responses.

In general, the most definitive methods to establish the diagnosis of a fungal infection are culture of the pathogen, microscopic examination, detection of species-specific fungal DNA, and serology. Since these methods vary in their availability, specificity, sensitivity, methods, and turnaround time, it is prudent to utilize several diagnostic strategies.

A. Specimens

Clinical specimens collected for microscopy and culture are determined by the site(s) of infection and the condition of the patient. All specimens should be obtained using aseptic technique, especially with specimens from normally sterile sites, such as blood, tissue biopsies, cerebrospinal fluid, and so forth. Specimens from nonsterile body sites include skin and subcutaneous lesions, nasopharyngeal or genital swabs, bronchoalveolar lavage fluid, urine, and wounds. To minimize bacterial growth, specimens should be transported to the diagnostic laboratory within two hours. Whenever a fungal infection is suspected, alert the diagnostic laboratory because special stains and culture media have been developed for the detection of pathogenic fungi.

B. Microscopic Examination

One or two drops of an aqueous or serous specimen, such as sputum, urine, spinal fluid, or aspirate, can be placed on a glass slide in a drop of 10–20% potassium hydroxide (KOH), and after adding a coverslip, the slide is examined under the microscope with the low- and high-power (450×) objectives. KOH dissolves any tissue cells, and the resistant, highly refractory fungal cell walls become more visible. This procedure can also be used to examine skin scrapings or minced tissue samples. The sensitivity of the KOH solution is improved by the addition of calcofluor white, which is a nonspecific fungal cell wall stain that is visible with a fluorescent microscope. The detection of fungi in pus, viscous exudates, and minced tissue can also be examined with KOH preps by gently heating the slide to dissolve excess tissue debris and inflammatory cells. Fungi can also be observed in blood smears, CSF, and other preps treated with Gram or Wright stain.

In formalin-fixed biopsy specimens, fungi can be detected with the routine hematoxylin and eosin (H&E) histopathological stain. However, specialized fungal cell wall stains are more sensitive. The two most common stains are Gomori-methenamine silver (GMS) and periodic acid-Schiff (PAS), which stain fungal walls black or red, respectively. Other specialized stains, such as capsule stains for Cryptococcus, are described in subsequent sections.

Although the sensitivity of microscopic examinations varies with the mycosis and the extent of disease, this examination can be performed very quickly and is often definitive. In the host, most fungi grow as yeasts, hyphae, or a combination of yeasts and pseudohyphae. Table 45-2 lists the spectrum of in vivo fungal structures. In many cases, the pathogens that are present only as yeasts are sufficiently distinctive in size and shape to establish an immediate diagnosis. In other cases, based on fungal appearance (eg, nonseptate hyphae or brownish cell walls) and the specimen site (eg, superficial or systemic), the list of possible fungal agents is considerably narrowed.

C. Culture

In most cases, the culture is more sensitive than the direct examination, and a portion of the material collected for microscopy should be cultured. The traditional mycological medium, Sabouraud’s dextrose agar (SDA), contains glucose and modified peptone (pH 7.0), supports the growth of fungi, and restricts the growth of bacteria. The morphologic characteristics of fungi used for identification have been described from growth on SDA. However, other media, such as inhibitory mold agar (IMA), enhance the recovery of fungi from clinical specimens. To culture medical fungi from nonsterile specimens, antibacterial antibiotics (eg, gentamicin, chloramphenicol) and cycloheximide are added to the media to inhibit bacteria and saprobic molds, respectively. After cultures are obtained, Potato Dextrose Agar stimulates the production of conidia.

For culturing blood, several commercial broth media have been developed for bacteria and/or fungi. Most yeast species in blood can be detected and subcultured from these media within three days. However, molds may require several weeks of incubation to become positive, and hence, special procedures must be used to optimize their recovery. Yeasts grow better at 37°C, and molds at 30°C. When a dimorphic fungus is suspected, multiple media and incubation temperatures are recommended. The ideal method for likely cases of fungemia is a commercial lysis-centrifugation (Isolator^®) tube to which blood is directly added. The tube contains an anticoagulant and detergent to lyse the blood cells, releasing any phagocytized fungal cells, and the tube is then centrifuged to pellet any fungi. After decanting the supernatant fluid, the pellet is suspended, streaked on agar plates of mycologic media, and incubated. Positive cultures of most yeasts or molds can be identified by morphological and physiological phenotypes. Several commercial microculture systems for yeasts are able to generate substrate assimilation profiles, and these profiles can be compared with large databases to identify most pathogenic species of yeasts. As described later, specialized media are available to assist in the rapid identification of Candida species (eg, CHROMagar^®).

D. Serology

The following sections of this chapter will explain how the detection of specific antibodies or antigens in serum or CSF can provide useful diagnostic and/or prognostic information. In immunocompetent patients, positive antibody tests may confirm the diagnosis, and negative tests may exclude fungal disease. However, the interpretation of each serologic test depends upon its sensitivity, specificity, and predictive value in the patient population being tested.

E. Molecular Methods

An increasing number of clinical laboratories have implemented methods based on the detection of fungal nucleic acids, proteins, or antigens to identify pathogenic fungi in clinical specimens or after their recovery in culture. Multiple approaches have been published, and in-house as well as commercial systems are available, but none have been widely adopted. In the next decade one or more of these methods may become routine, especially if they can be automated, provide rapid throughput, and detect multiple microbes. In addition, some platforms have the potential for point-of-care usage. Most DNA-based methods use PCR to amplify fungus-specific sequences of ribosomal DNA or other conserved genes. By comparing the DNA sequences of a clinical isolate with databases of thousands of fungal DNA sequences, the genus or species of an unknown fungus can be identified. This approach has been used to identify cultures of hundreds of fungal taxa. In addition, a variety of reports have used PCR to detect fungal DNA (mostly Candida or Aspergillus) in blood and other specimens.

Automated commercial instruments for the DNA-based identification of pathogenic fungi have been developed by several companies, including Luminex Molecular Diagnostics^®, Gen-Probe^®, LightCycler^® SeptiFast, and MicroSeq^®. For detection, they typically use oligonucleotide probes that emit a signal amplified by fluorescence, chemical reagents, or enzyme immunoassay. For detecting fungal cells in slide preparations, a PNA-FISH^® (peptide-nucleic acid fluorescent in situ hybridization) test kit with species-specific probes can be used.

DNA-based fingerprinting methods, such as multilocus sequence typing (MLST), have identified phylogenetic subpopulations of many pathogenic fungi, including species of Candida, Cryptococcus, Aspergillus, and Coccidioides. Some of these molecular subgroups are clinically relevant as they are associated with differences in geographic distribution, susceptibility to antifungal drugs, clinical manifestations, or virulence. Some of these subgroups may represent cryptic species that cannot be differentiated by phenotypic methods.

Another molecular method that is gaining currency because of its application to pathogenic bacteria as well as fungi involves the extraction of microbial proteins, which are then submitted to mass spectroscopy. Pathogens are identified by comparing their protein spectral patterns with those in databases of previously tested species. With the ease of sample preparation and the commercial availability of automated instruments, this method—matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS)—is becoming more accessible. In several studies, MALDI-TOF-MS has proven to be more accurate and faster than conventional culture methods. Most of the current data are based on the identification of species of Candida.

Similar to the rapid test for endotoxin, the clotting cascade of the horseshoe crab (Limulus) hemolymph is also triggered by fungal cell wall β-(1,3)-d-glucan. This reaction (Fungicell^®) has been exploited to quantify its concentration in blood and spinal fluid. β-(1,3)-d-glucan levels of ≥80 pg/mL are positive and associated with invasive candidiasis, aspergillosis, dimorphic pathogens, and other mycoses.

Malassezia infections

Pityriasis versicolor is a highly prevalent, chronic superficial infection of the stratum corneum caused by species of the lipophilic yeast, Malassezia. These yeasts can be isolated from normal skin and scalp and are considered part of the cutaneous mycobiota. Thus, infections are likely caused by endogenous strains. There are 14 currently recognized species of Malassezia, but the vast majority of cases of pityriasis versicolor are caused by Malassezia globosa, Malassezia furfur, or Malassezia sympodialis. The infection is characterized by discrete, serpentine, hyper-, or hypopigmented maculae that develop on the skin, usually on the chest, upper back, arms, or abdomen. These patches of discolored skin may enlarge and coalesce, but scaling, inflammation, and irritation are minimal. Indeed, this common affliction is largely a cosmetic problem. Pityriasis versicolor affects all ages, and the annual incidence is reportedly 5–8%. Predisposing conditions include the immune status of the patient, genetic factors, and elevated temperature and humidity.

Most species of Malassezia require lipid in the medium for growth. The diagnosis is confirmed by direct KOH microscopic examination of scrapings of infected skin. Short unbranched, nonpigmented hyphae and spherical cells are observed. The lesions also fluoresce under Wood’s lamp. Pityriasis versicolor is treated with daily applications of selenium sulfide. Topical or oral azoles are also effective. The goal of treatment is not to eradicate Malassezia from the skin but to reduce the cutaneous population to commensal levels.

The species of Malassezia noted above, as well as Malassezia restricta, have been implicated as causes or contributors to seborrheic dermatitis (ie, dandruff). This etiology is supported by the observation that many cases are alleviated by treatment with ketoconazole. Rarely, Malassezia may cause an opportunistic fungemia in patients—usually infants—receiving total parenteral nutrition (TPN), as a result of contamination of the lipid emulsion. In most cases, the fungemia is transient and eliminated by replacing the TPN and intravenous catheter. In addition, another less common manifestation of Malassezia is folliculitis.

Tinea Nigra

Tinea nigra (or tinea nigra palmaris) is a superficial chronic and asymptomatic infection of the stratum corneum caused by the dematiaceous fungus Hortaea (Exophiala) werneckii. This condition is more prevalent in warm coastal regions and among young women. The lesions appear as a dark (brown to black) discoloration, often on the palm. Microscopic examination of skin scrapings from the periphery of the lesion will reveal branched, septate hyphae and budding yeast cells with melanized cell walls. Tinea nigra will respond to treatment with keratolytic solutions, salicylic acid, or azole antifungal drugs.

Piedra

Black piedra is a nodular infection of the hair shaft caused by Piedraia hortae (Figure 45-9B). White piedra, due to infection with Trichosporon species, presents as larger, softer, yellowish nodules on the hairs (see Figure 45-9A). Hair of the axilla, genitalia, beard, and scalp hair may be infected. Treatment for both types consists of removal of the infected hair and application of a topical antifungal agent. Piedra is endemic in tropical countries.

Dermatophytosis

Cutaneous mycoses are caused by fungi that infect only the keratinized tissue (skin, hair, and nails). The most important of these are the dermatophytes, a group of about 40 related fungi that belong to three genera: Microsporum, Trichophyton, and Epidermophyton. Dermatophytes are restricted to the nonviable skin because most are unable to grow at 37°C or in the presence of serum. Dermatophytoses are among the most prevalent infections in the world. Although they can be persistent and troublesome, they are rarely debilitating or life-threatening—yet billions of dollars are expended annually in their treatment. Being superficial, dermatophyte (ringworm) infections have been recognized since antiquity. In skin they are diagnosed by the presence of hyaline, septate, branching hyphae, or chains of arthroconidia. In culture, the many species are closely related and often difficult to identify. They are speciated on the basis of subtle differences in the appearance of the colonies and microscopic morphology as well as a few vitamin requirements. Despite their similarities in morphology, nutritional requirements, surface antigens, and other features, many species have particular keratinases, elastases, and other enzymes that enable them to be quite host-specific. The identification of closely related and outbreak strains has been greatly aided by DNA sequence analysis. The several dermatophytic species that are capable of sexual reproduction produce ascospores and belong to the teleomorphic genus Arthroderma.

Dermatophytes are acquired by contact with contaminated soil or with infected animals or humans. The species are classified as geophilic, zoophilic, or anthropophilic depending on whether their usual habitat is soil, animals, or humans. Several dermatophytes that normally reside in soil or are associated with particular animal species are also able to cause human infections. In general, as a species evolves from habitation in soil to a specific animal or human host, it loses the ability to produce asexual conidia and to reproduce sexually. Anthropophilic species cause the greatest number of human infections. They elicit relatively mild and chronic infections, produce few conidia in culture, and may be difficult to eradicate. Conversely, geophilic and zoophilic dermatophytes, being less adapted to human hosts, produce more acute inflammatory infections that tend to resolve more quickly.

Some anthropophilic species are geographically restricted, but others, such as Epidermophyton floccosum, Trichophyton mentagrophytes var interdigitale, Trichophyton rubrum, and Trichophyton tonsurans, are globally distributed. The most common geophilic species causing human infections is Microsporum gypseum. Cosmopolitan zoophilic species (and their natural hosts) include Microsporum canis (dogs and cats), Microsporum gallinae (fowl), Microsporum nanum (pigs), Trichophyton equinum (horses), and Trichophyton verrucosum (cattle).

Morphology and Identification

The more common dermatophytes are identified by their colonial appearance and microscopic morphology after growth for 2 weeks at 25°C on SDA. Trichophyton species, which may infect hair, skin, or nails, develop cylindric, smooth-walled macroconidia and characteristic microconidia (Figure 45-10A). Depending on the variety, colonies of T mentagrophytes may be cottony to granular; both types display abundant grape-like clusters of spherical microconidia on terminal branches. Coiled or spiral hyphae are commonly found in primary isolates. The typical colony of T rubrum has a white, cottony surface and a deep red, nondiffusible pigment when viewed from the reverse side of the colony. The microconidia are small and piriform (pear-shaped). T tonsurans produces a flat, powdery to velvety colony on the obverse surface that becomes reddish brown on reverse; the microconidia are mostly elongate (see Figure 45-10A).

Microsporum species tend to produce distinctive multicellular macroconidia with echinulate walls (see Figure 45-10B). Both types of conidia are borne singly in these genera. M canis forms a colony with a white cottony surface and a deep yellow color on reverse; the thick-walled, 8- to 15-celled macroconidia frequently have curved or hooked tips. M gypseum produces a tan, powdery colony and abundant thin-walled, four- to six-celled macroconidia. Microsporum species infect only hair and skin.

E floccosum, which is the only pathogen in this genus, produces only macroconidia, which are smooth-walled, clavate, two- to four-celled, and formed in small clusters (see Figure 45-10C). The colonies are usually flat and velvety with a tan to olive-green tinge. E floccosum infects the skin and nails but not the hair.

In addition to gross and microscopic morphology, a few nutritional or other tests, such as growth at 37°C or a test for in vitro hair perforation, are useful in differentiating certain species. Atypical isolates can usually be identified by species-specific polymerase chain reaction (PCR) tests.

Epidemiology and Immunity

Dermatophyte infections begin in the skin after trauma and contact. There is evidence that host susceptibility may be enhanced by moisture, warmth, specific skin chemistry, composition of sebum and perspiration, youth, heavy exposure, and genetic predisposition. The incidence is higher in hot, humid climates and under crowded living conditions. Shoes provide warmth and moisture, a setting for infections of the feet. The source of infection is soil or an infected animal in the case of geophilic and zoophilic dermatophytes, respectively. Anthropophilic species may be transmitted by direct contact or through fomites, such as contaminated towels, clothing, shared shower stalls, and similar examples. Unlike other fungal infections, dermatophytes are contagious and frequently transmitted by exposure to shed skin scales, nails, or hair containing hyphae or conidia. These fungal elements can remain viable for long periods on fomites.

Trichophytin is a crude antigen preparation that can be used to detect immediate- or delayed-type hypersensitivity to dermatophytic antigens. Many patients who develop chronic, noninflammatory dermatophyte infections have poor cell-mediated immune responses to dermatophyte antigen. These patients often are atopic and have immediate-type hypersensitivity and elevated IgE antibody levels. In the normal host, immunity to dermatophytosis varies in duration and degree depending on the host, the site, and the species of fungus causing the infection.

Clinical Findings

Dermatophyte infections were mistakenly described as ringworm or tinea because of the raised circular lesions, and tradition has maintained this terminology. The clinical forms are based on the site of involvement. A single species is able to cause more than one type of clinical infection. Conversely, a single clinical form, such as tinea corporis, may be caused by more than one dermatophyte species. Table 45-3 lists the more prevalent etiologies of common clinical forms. Very rarely, immunocompromised patients may develop systemic infection by a dermatophyte.

A. Tinea Pedis (Athlete’s Foot)

Tinea pedis is the most prevalent of all dermatophytoses. It usually occurs as a chronic infection of the toe webs. Other varieties are the vesicular, ulcerative, and moccasin types, with hyperkeratosis of the sole. Initially, there is itching between the toes and the development of small vesicles that rupture and discharge a thin fluid. The skin of the toe webs becomes macerated and peels, whereupon cracks appear that are prone to develop secondary bacterial infection. When the fungal infection becomes chronic, peeling and cracking of the skin are the principal manifestations, accompanied by pain and pruritus.

B. Tinea Unguium (Onychomycosis)

Nail infection may follow prolonged tinea pedis. With hyphal invasion, the nails become yellow, brittle, thickened, and crumbly. One or more nails of the feet or hands may be involved.

C. Tinea Corporis, Tinea Cruris, and Tinea Manus

Dermatophytosis of the glabrous skin commonly gives rise to the annular lesions of ringworm, with a clearing, scaly center surrounded by a red advancing border that may be dry or vesicular. The dermatophyte grows only within dead, keratinized tissue, but fungal metabolites, enzymes, and antigens diffuse through the viable layers of the epidermis to cause erythema, vesicle formation, and pruritus. Infections with geophilic and zoophilic dermatophytes produce more irritants and are more inflammatory than anthropophilic species. As hyphae age, they often form chains of arthroconidia. The lesions expand centrifugally and active hyphal growth is at the periphery, which is the most likely region from which to obtain material for diagnosis. Penetration into the newly forming stratum corneum of the thicker plantar and palmar surfaces accounts for the persistent infections at those sites.

When the infection occurs in the groin area, it is called tinea cruris, or jock itch. Most such infections involve males and present as dry, itchy lesions that often start on the scrotum and spread to the groin. Tinea manus refers to ringworm of the hands or fingers. Dry scaly lesions may involve one or both hands, single fingers, or two or more fingers.

D. Tinea Capitis and Tinea Barbae

Tinea capitis is dermatophytosis or ringworm of the scalp and hair. The infection begins with hyphal invasion of the skin of the scalp, with subsequent spread down the keratinized wall of the hair follicle. Infection of the hair takes place just above the hair root. The hyphae grow downward on the nonliving portion of the hair and at the same rate as the hair grows upward. The infection produces dull gray, circular patches of alopecia, scaling, and itching. As the hair grows out of the follicle, the hyphae of Microsporum species produce a chain of spores that form a sheath around the hair shaft (ectothrix). These spores impart a greenish to silvery fluorescence when the hairs are examined under Wood’s light (365 nm). In contrast, T tonsurans, the chief cause of “black dot” tinea capitis, produces spores within the hair shaft (endothrix). These hairs do not fluoresce; they are weakened and typically break easily at the follicular opening. In prepubescent children, epidemic tinea capitis is usually self-limiting.

Zoophilic species may induce a severe combined inflammatory and hypersensitivity reaction called a kerion. Another manifestation of tinea capitis is favus, an acute inflammatory infection of the hair follicle caused by Trichophyton schoenleinii, which leads to the formation of scutula (crusts) around the follicle. In favic hairs, the hyphae do not form spores but can be found within the hair shaft. Tinea barbae involves the bearded region. Especially when a zoophilic dermatophyte is involved, a highly inflammatory reaction may be elicited that closely resembles pyogenic infection.

E. Trichophytid Reaction

In the course of dermatophytosis, the patient may become hypersensitive to constituents or products of the fungus and develop allergic manifestations—called dermatophytids (usually vesicles)—elsewhere on the body, most often on the hands. The trichophytin skin test is markedly positive in such persons.

Diagnostic Laboratory Tests

A. Specimens and Microscopic Examination

Specimens consist of scrapings from both the skin and the nails plus hairs plucked from involved areas. In KOH preps of skin or nails, regardless of the infecting species, branching hyphae or chains of arthroconidia (arthrospores) are seen (Figure 45-11). In hairs, most Microsporum species form dense sheaths of spores around the hair (ectothrix). The ectothrix spores of Microsporum-infected hairs fluoresce under Wood’s light in a darkened room. T tonsurans and Trichophyton violaceum are noted for producing arthroconidia inside the hair shaft (endothrix).

B. Culture

The identification of dermatophytic species requires cultures. Specimens are inoculated onto IMA or SDA slants containing cycloheximide and chloramphenicol to suppress mold and bacterial growth, incubated for 1–3 weeks at room temperature, and further examined in slide cultures if necessary. Species are identified on the basis of colonial morphology (growth rate, surface texture, and any pigmentation), microscopic morphology (macroconidia, microconidia), and, in some cases, nutritional requirements.

Treatment

Therapy consists of thorough removal of infected and dead epithelial structures and application of a topical antifungal drug. To prevent reinfection the area should be kept dry, and sources of infection, such as an infected pet or shared bathing facilities, should be avoided.

Scalp (tinea capitis) infections are treated for several weeks with oral administration of griseofulvin or terbinafine. Frequent shampoos and miconazole cream or other topical antifungal agents may be effective if used for weeks. Alternatively, ketoconazole and itraconazole are quite effective.

For tinea corporis, tinea pedis, and related infections, the most effective drugs are itraconazole and terbinafine. However, a number of topical preparations may be used, such as miconazole nitrate, tolnaftate, and clotrimazole. If applied for at least 2–4 weeks, the cure rates are usually 70–100%. Treatment should be continued for 1–2 weeks after clearing of the lesions. For troublesome cases, a short course of oral griseofulvin can be administered.

Nail infections (tinea unguium) are the most difficult to treat, often requiring months of oral itraconazole or terbinafine as well as surgical removal of the nail. Relapses are common. A new topical imidazole, luliconazole, has been formulated to penetrate the nail plate and demonstrated potent effectiveness against dermatophytes and onychomycosis.

• Superficial and cutaneous mycoses are among the most common of all communicable diseases.

• Most superficial and cutaneous fungal infections are caused by species of Malassezia, dermatophytes, or Candida (discussed later).

• The growth of dermatophytes is inhibited by serum and body temperature, and these fungi rarely become invasive.

• Geophilic and zoophilic dermatophytes usually cause acute, inflammatory lesions that respond to topical treatment within weeks and rarely recur.

• Anthropophilic dermatophytes usually cause relatively mild, chronic lesions that may require months or years of treatment and frequently recur.

The fungi that cause subcutaneous mycoses normally reside in soil or on vegetation. They enter the skin or subcutaneous tissue by traumatic inoculation with contaminated material. For example, a superficial cut or abrasion may introduce an environmental mold with the ability to infect the exposed dermis. In general, the lesions become granulomatous and expand slowly from the area of implantation. Extension via the lymphatics draining the lesion is slow except in sporotrichosis. These mycoses are usually confined to the subcutaneous tissues, but in rare cases they become systemic and produce life-threatening disease.

Sporothrix schenckii is a thermally dimorphic fungus that lives on vegetation. It is associated with a variety of plants—grasses, trees, sphagnum moss, rose bushes, and other horticultural plants. At ambient temperatures, it grows as a mold, producing branching, septate hyphae and conidia, and in tissue or in vitro at 35–37°C as a small budding yeast. Following traumatic introduction into the skin, S schenckii causes sporotrichosis, a chronic granulomatous infection. The initial episode is typically followed by secondary spread with involvement of the draining lymphatics and lymph nodes.

Morphology and Identification

S schenckii grows well on routine agar media, and at room temperature the young colonies are blackish and shiny, becoming wrinkled and fuzzy with age. Strains vary in pigmentation from shades of black and gray to whitish. The organism produces branching, septate hyphae and distinctive small (3–5 μm) conidia, delicately clustered at the ends of tapering conidiophores. Isolates may also form larger conidia directly from the hyphae. S schenckii is thermally dimorphic, and at 35°C on a rich medium it converts to growth as small, often multiply budding yeast cells that are variable in shape but often fusiform (about 1–3 × 3–10 μm), as shown in Figure 45-12.

Antigenic Structure

Heat-killed saline suspensions of cultures or carbohydrate fractions (sporotrichin) will elicit positive delayed skin tests in infected humans or animals. A variety of serologic tests have been developed, and most patients, as well as some normal individuals, have specific or cross-reactive antibodies.

Pathogenesis and Clinical Findings

The conidia or hyphal fragments of S schenckii are introduced into the skin by trauma. Patients frequently recall a history of trauma associated with outdoor activities and plants. The initial lesion is usually located on the extremities but can be found anywhere (children often present with facial lesions). About 75% of cases are lymphocutaneous; that is, the initial lesion develops as a granulomatous nodule that may progress to form a necrotic or ulcerative lesion. Meanwhile, the draining lymphatics become thickened and cord-like. Multiple subcutaneous nodules and abscesses occur along the lymphatics.

Fixed sporotrichosis is a single nonlymphangitic nodule that is limited and less progressive. The fixed lesion is more common in endemic areas such as Mexico, where there is a high level of exposure and immunity in the population. Immunity limits the local spread of the infection.

There is usually little systemic illness associated with these lesions, but dissemination may occur, especially in debilitated patients. Rarely, primary pulmonary sporotrichosis results from inhalation of the conidia. This manifestation mimics chronic cavitary tuberculosis and tends to occur in patients with impaired cell-mediated immunity.

Diagnostic Laboratory Tests

A. Specimens and Microscopic Examination

Specimens include biopsy material or exudate from granulomatous or ulcerative lesions. Although specimens can be examined directly with KOH or calcofluor white stain, the yeasts are rarely found. Even though they are sparse in tissue, the sensitivity of histopathologic sections is enhanced with routine fungal cell wall stains, such as Gomori methenamine silver, which stains the cell walls black, or the periodic acid-Schiff stain, which imparts a red color to the cell walls. Alternatively, they can be identified by fluorescent antibody staining. The yeasts are 3–5 μm in diameter and spherical to elongated.

Another structure termed an asteroid body is often seen in tissue, particularly in endemic areas such as Mexico, South Africa, and Japan. In hematoxylin and eosin-stained tissue, the asteroid body consists of a central basophilic yeast cell surrounded by radiating extensions of eosinophilic material, which are depositions of antigen–antibody complexes and complement.

B. Culture

The most reliable method of diagnosis is culture. Specimens are streaked on IMA or SDA containing antibacterial antibiotics and incubated at 25–30°C. The identification is confirmed by growth at 35°C and conversion to the yeast form.

C. Serology

High titers of agglutinating antibodies to yeast cell suspensions or antigen-coated latex particles are often detected in sera of infected patients. However, these tests are generally not useful because elevated titers do not develop early in the course of disease and uninfected or previously exposed patients may give false-positive results.

Treatment

In some cases, the infection is self-limited. Although the oral administration of saturated solution of potassium iodide in milk is quite effective, it is difficult for many patients to tolerate. The treatment of choice is oral itraconazole or another azole. For systemic disease, amphotericin B is given.

Epidemiology and Control

S schenckii occurs worldwide in close association with plants. For example, cases have been linked to contact with sphagnum moss, rose thorns, decaying wood, pine straw, prairie grass, and other vegetation. About 75% of cases occur in males, either because of increased exposure or because of an X-linked difference in susceptibility. The incidence is higher among agricultural workers, and sporotrichosis is considered an occupational risk for forest rangers, horticulturists, and workers in similar occupations. Prevention includes measures to minimize accidental inoculation and the use of fungicides, where appropriate, to treat wood. Animals are also susceptible to sporotrichosis.

Chromoblastomycosis (chromomycosis) is a subcutaneous mycotic infection that is usually caused by traumatic inoculation of any of the recognized fungal agents, which reside in soil and vegetation. All are dematiaceous fungi, having melanized cell walls: Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Rhinocladiella aquaspersa, and Cladophialophora carrionii. The infection is chronic and characterized by the slow development of progressive granulomatous lesions that in time induce hyperplasia of the epidermal tissue.

Morphology and Identification

The dematiaceous fungi are similar in their pigmentation, antigenic structure, morphology, and physiologic properties. The colonies are compact, deep brown to black, and develop a velvety, often wrinkled surface. The agents of chromoblastomycosis are identified by their modes of conidiation. In tissue they appear the same, producing spherical brown cells (4–12 μm in diameter) termed muriform or sclerotic bodies that divide by transverse septation. Septation in different planes with delayed separation may give rise to a cluster of four to eight cells (Figure 45-13). Cells within superficial crusts or exudates may germinate into septate, branching hyphae.

Cultures of these dematiaceous molds can be distinguished as follows:

A. P verrucosa

The conidia are produced from flask-shaped phialides with cup-shaped collarettes. Mature, spherical to oval conidia are extruded from the phialide and usually accumulate around it (Figure 45-14A).

B. F pedrosoi

The Fonsecaea genus is polymorphic. Isolates may exhibit (1) phialides; (2) chains of blastoconidia, similar to Cladosporium species; or (3) sympodial, rhinocladiella-type conidiation. Most strains of F pedrosoi form short branching chains of blastoconidia as well as sympodial conidia (see Figure 45-14B).

C. F compacta

The blastoconidia produced by F compacta are almost spherical, with a broad base connecting the conidia. These structures are smaller and more compact than those of F pedrosoi.

D. R aquaspersa

This species produces lateral or terminal conidia from a lengthening conidiogenous cell—a sympodial process. The conidia are elliptical to clavate.

E. Cladophialophora (Cladosporium) carrionii

Species of Cladophialophora and Cladosporium produce branching chains of conidia by distal (acropetalous) budding. The terminal conidium of a chain gives rise to the next conidium by a budding process. Species are identified based on differences in the length of the chains and the shape and size of the conidia. C carrionii produces elongated conidiophores with long, branching chains of oval conidia.

Pathogenesis and Clinical Findings

The fungi are introduced into the skin by trauma, often of the exposed legs or feet. Over months to years, the primary lesion becomes verrucous and wart-like with extension along the draining lymphatics. Cauliflower-like nodules with crusting abscesses eventually cover the area. Small ulcerations or “black dots” of hemopurulent material are present on the warty surface. Rarely, elephantiasis may result from secondary infection, obstruction, and fibrosis of lymph channels. Dissemination to other parts of the body is very rare, though satellite lesions can occur due either to local lymphatic spread or to autoinoculation. Histologically, the lesions are granulomatous and the dark sclerotic bodies may be seen within leukocytes or giant cells.

Diagnostic Laboratory Tests

Specimens of scrapings or biopsies from lesions are examined microscopically in KOH for dark, spherical cells. Detection of the sclerotic bodies is diagnostic of chromoblastomycosis regardless of the etiologic agent. Tissue sections reveal granulomas and extensive hyperplasia of the dermal tissue. Specimens should be cultured on IMA or SDA with antibiotics. The dematiaceous species is identified by its characteristic conidial structures, as described above. There are many similar saprophytic dematiaceous molds, but they differ from the pathogenic species in being unable to grow at 37°C and being able to digest gelatin.

Treatment

Surgical excision with wide margins is the therapy of choice for small lesions. Chemotherapy with flucytosine or itraconazole may be efficacious for larger lesions. The application of local heat is also beneficial. Relapse is common.

Epidemiology

Chromoblastomycosis occurs mainly in the tropics. The fungi are saprophytic in nature, probably occurring on vegetation and in soil. The disease occurs chiefly on the legs of barefoot agrarian workers following traumatic introduction of the fungus. Chromoblastomycosis is not communicable. Wearing shoes and protecting the legs probably would prevent infection.

Phaeohyphomycosis is a term applied to infections characterized by the presence of darkly pigmented septate hyphae in tissue. Both cutaneous and systemic infections have been described. The clinical forms vary from solitary encapsulated cysts in the subcutaneous tissue to sinusitis to brain abscesses. Over 100 species of dematiaceous molds have been associated with various types of phaeohyphomycotic infections. They are all exogenous molds that normally exist in nature. Some of the more common causes of subcutaneous phaeohyphomycosis are Exophiala jeanselmei, Phialophora richardsiae, Bipolaris spicifera, and Wangiella dermatitidis. These species and others (eg, Exserohilum rostratum, Alternaria species, and Curvularia species) may be implicated also in systemic phaeohyphomycosis. The incidence of phaeohyphomycosis and the range of pathogens have been increasing in recent years in both immunocompetent and compromised patients.

In tissue, the hyphae are large (5–10 μm in diameter), often distorted and may be accompanied by yeast cells, but these structures can be differentiated from other fungi by the melanin in their cell walls (Figure 45-15). Specimens are cultured on routine fungal media to identify the etiologic agent. In general, itraconazole or flucytosine is the drug of choice for subcutaneous phaeohyphomycosis. Brain abscesses are usually fatal, but when recognized, they are managed with amphotericin B and surgery. The leading cause of cerebral phaeohyphomycosis is Cladophialophora bantiana.

Mycetoma is a chronic subcutaneous infection induced by traumatic inoculation with any of several saprophytic species of fungi or actinomycetous bacteria that are normally found in soil. The clinical features defining mycetoma are local swelling of the infected tissue and interconnecting, often draining, sinuses or fistulae that contain granules, which are microcolonies of the agent embedded in tissue material. An actinomycetoma is a mycetoma caused by an actinomycete; a eumycetoma (maduromycosis, Madura foot) is a mycetoma caused by a fungus. The natural history and clinical features of both types of mycetoma are similar, but actinomycetomas may be more invasive, spreading from the subcutaneous tissue to the underlying muscle. Of course, the therapy is different. Mycetoma occurs worldwide but more often among impoverished people who reside in tropical areas and wear less protective clothing. Mycetomas occur only sporadically outside the tropics but are particularly prevalent in India, Africa, and Latin America. Actinomycetomas are discussed in Chapter 12.

Morphology and Identification

The fungal agents of mycetoma include, among others, Pseudallescheria boydii (anamorph, Scedosporium apiospermum), Madurella mycetomatis, Madurella grisea, E jeanselmei, and Acremonium falciforme. In the United States, the prevalent species is P boydii, which is self-fertile (homothallic) and has the ability to produce ascospores in culture. E jeanselmei and the Madurella species are dematiaceous molds. These molds are identified primarily by their mode of conidiation. P boydii may also cause pseudallescheriasis, which is a systemic infection of compromised patients.

In tissue, the mycetoma granules may range up to 2 mm in size. The color of the granule may provide information about the agent. For example, the granules of mycetoma caused by P boydii and A falciforme are white; those of M grisea and E jeanselmei are black; and M mycetomatis produces a dark red to black granule. These granules are hard and contain intertwined, septate hyphae (3–5 μm in width). The hyphae are typically distorted and enlarged at the periphery of the granule.

Pathogenesis and Clinical Findings

Mycetoma develops after traumatic inoculation with soil contaminated with one of the agents. Subcutaneous tissues of the feet, lower extremities, hands, and exposed areas are most often involved. Regardless of the agent, the pathology is characterized by suppuration and abscesses, granulomata, and draining sinuses containing the granules. This process may spread to contiguous muscle and bone. Untreated lesions persist for years and extend deeper and peripherally, causing deformation and loss of function.

Very rarely, P boydii may disseminate in an immunocompromised host or produces infection of a foreign body (eg, a cardiac pacemaker).

Diagnostic Laboratory Tests

Granules can be dissected out from the pus or biopsy material for examination and culture on appropriate media. The granule color, texture, and size and the presence of hyaline or pigmented hyphae (or bacteria) are helpful in determining the causative agent. Draining mycetomas are often superinfected with staphylococci and streptococci.

Treatment

The management of eumycetoma is difficult, involving surgical debridement or excision and chemotherapy. P boydii is treated with topical nystatin or miconazole. Itraconazole, ketoconazole, and even amphotericin B can be recommended for Madurella infections and flucytosine for E jeanselmei. Chemotherapeutic agents must be given for long periods to adequately penetrate these lesions.

Epidemiology and Control

The organisms producing mycetoma occur in soil and on vegetation. Barefoot farm laborers are therefore commonly exposed. Properly cleaning wounds and wearing shoes are reasonable control measures.

1. Subcutaneous mycoses may be caused by dozens of environmental molds associated with vegetation and soil.

2. These infections are usually acquired when minor cuts or scratches introduce soil or plant debris (eg, splinters, thorns) containing the pathogenic fungus. The ensuing infections are frequently chronic but rarely spread to deeper tissues.

3. S schenckii, the cause of sporotrichosis, is a dimorphic fungus that converts from hyphal growth to yeast cells within the host.

4. The diagnostic feature of chromoblastomycosis is the microscopic observation of brownish (melanized), spherical sclerotic bodies within the lesions.

5. The diagnostic feature of phaeohyphomycosis is the presence of brownish (melanized), septate hyphae within the lesions.

6. The hallmark of a mycetoma is localized swelling and the formation of fistulae that contain hard granules composed of hyphae and inflammatory tissue (eg, macrophages, fibrin).

Each of the four primary systemic mycoses—coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis—is geographically restricted to specific areas of endemicity. The fungi that cause coccidioidomycosis and histoplasmosis exist in nature in dry soil or in soil mixed with guano, respectively. The agents of blastomycosis and paracoccidioidomycosis are presumed to reside in nature, but their habitats have not been clearly defined. Each of these four mycoses is caused by a thermally dimorphic fungus, and the infections are initiated in the lungs following inhalation of the respective conidia. Most infections are asymptomatic or mild and resolve without treatment. However, a small but significant number of patients develop pulmonary disease, which may involve dissemination from the lungs to other organs. With rare exceptions, these mycoses are not transmissible among humans or other animals. Table 45-4 summarizes and contrasts some of the fundamental features of these systemic or deep mycoses.

For all of these infections, the initial host defenses are provided by the alveolar macrophages, which are usually capable of inactivating the conidia and inducing a robust immune response. This process typically leads to granulomatous inflammation and the production of both antibodies and cell-mediated immunity. The induction of Th1 cytokines (eg, interleukin-12, interferon-Γ, tumor necrosis factor α) will amplify the cellular defenses, activating macrophages, and enhancing their fungicidal capacity. In an immunocompetent host, these responses lead to resolution of the inflammatory lesions. However, residual granulomata may retain dormant organisms with the potential for subsequent reactivation, constituting a latent form of the disease. Within the endemic areas for these fungi, most infections occur in immunocompetent individuals, but persons with impaired cellular immunity, such as patients with HIV/AIDS, have a greatly increased risk of serious infection. Strains of most pathogenic fungi exhibit large variations in laboratory assays of pathogenicity. For the agents of endemic mycoses, virulence is associated with α-glucan in their cell walls, perhaps by masking pathogen-associated molecular patterns that trigger protective immune responses.

Coccidioidomycosis is caused Coccidioides posadasii or C immitis. These sibling species were recognized by genotyping and phylogenetic analyses. However, they are phenotypically indistinguishable, cause similar clinical manifestations, and are not differentiated in the clinical laboratory. Coccidioidomycosis is endemic in well-circumscribed semiarid regions of the southwestern United States, Central America, and South America. Infection is usually self-limited, and dissemination is rare but always serious, and it may be fatal. Clinical and environmental isolates of Coccidioides have revealed that the two species are not equally distributed within the endemic regions. Although there is some overlap, C immitis is largely confined to California, whereas C posadasii predominates in Arizona, Texas, and South America.

Morphology and Identification

Most infections are probably due to C posadasii. However, since the two species cannot be readily identified in the laboratory and since the clinical manifestations are the same with either C immitis or C posadasii, only the former, more familiar species name will be used in this chapter.

On most laboratory media, C immitis produces a white to tan cottony colony. The hyphae form chains of arthroconidia (arthrospores), which often develop in alternate cells of a hypha. These chains fragment into individual arthroconidia, which are readily airborne and highly resistant to adverse environmental conditions (Figure 45-16A). These small arthroconidia (3 × 6 μm) remain viable for years and are highly infectious. Following their inhalation, the arthroconidia become spherical and enlarge, forming spherules that contain endospores (see Figure 45-16B). Spherules can also be produced in the laboratory by cultivation on a complex medium.

In histologic sections of tissue, sputum, or other specimens, the spherules are diagnostic of C immitis. At maturity, the spherules have a thick, doubly refractile wall and may attain a size of 80 μm in diameter. The spherule becomes packed with endospores (2–5 μm in size). Eventually, the wall ruptures to release the endospores, which may develop into new spherules (see Figure 45-16B).

Antigenic Structure

Two clinically useful antigens are available. Coccidioidin is an antigen preparation extracted from the filtrate of a liquid mycelial culture of C immitis. Spherulin is produced from a filtrate of a broth culture of spherules. In standardized doses, both antigens elicit positive delayed skin reactions in infected persons. They have also been used in a variety of serologic tests to measure serum antibodies to C immitis.

Pathogenesis and Clinical Findings

Inhalation of arthroconidia leads to a primary infection that is asymptomatic in 60% of individuals. The only evidence of infection is the development of serum precipitins and conversion to a positive skin test within 2–4 weeks. The precipitins will decline, but the skin test often remains positive for a lifetime. The other 40% of individuals develop a self-limited influenza-like illness with fever, malaise, cough, arthralgia, and headache. This condition is called valley fever, San Joaquin Valley fever, or desert rheumatism. After 1–2 weeks, about 15% of these patients develop hypersensitivity reactions, which present as a rash, erythema nodosum, or erythema multiforme. On radiographic examination, patients typically show hilar adenopathy along with pulmonary infiltrates, pneumonia, pleural effusions, or nodules. Pulmonary residua occur in about 5%, usually in the form of a solitary nodule or thin-walled cavity (Figure 45-17).

Less than 1% of persons infected with C immitis develop secondary or disseminated coccidioidomycosis, which is often debilitating and life-threatening. The risk factors for systemic coccidioidomycosis include heredity, sex, age, and compromised cell-mediated immunity. The disease occurs more frequently in certain racial groups. In decreasing order of risk, these are Filipinos, African Americans, Native Americans, Hispanics, and Asians. There is clearly a genetic component to the immune response to C immitis. Males are more susceptible than females, with the exception of women who are pregnant, which may relate to differences in the immune response or a direct effect of sex hormones on the fungus. For example, C immitis has estrogen-binding proteins, and elevated levels of estradiol and progesterone stimulate its growth. The young and the aged are also at greater risk. Because cell-mediated immune responses are required for adequate resistance, patients with AIDS and other conditions of cellular immunosuppression are at risk for disseminated coccidioidomycosis.

Some individuals develop a chronic but progressive pulmonary disease with multiplying or enlarging nodules or cavities. Dissemination will usually occur within a year after the primary infection. The spherules and endospores are spread hematogenously or by direct extension. A number of extrapulmonary sites may be involved, but the most frequent organs are the skin, the bones and joints, and the meninges. There are distinctive clinical manifestations associated with C immitis infections in each of these and other areas of the body.

Dissemination occurs when the immune response is inadequate to contain the pulmonary foci. In most persons, a positive skin test signifies a strong cell-mediated immune response and protection against reinfection. However, if such individuals become immunocompromised by taking cytotoxic drugs or by disease (eg, AIDS), dissemination can occur many years after primary infection (reactivation disease). Coccidioidomycosis in AIDS patients often presents with a rapidly fatal diffuse reticulonodular pneumonitis. Because of the radiologic overlap between this disease and Pneumocystis pneumonia and the different therapies for these two entities, it is important to be aware of the possibility of coccidioidal pneumonia in AIDS patients. Blood cultures are often positive for C immitis.

On histologic examination, the coccidioidal lesions contain typical granulomas with giant cells and interspersed suppuration. A diagnosis can be made by finding spherules and endospores. The clinical course is often characterized by remissions and relapses.

Diagnostic Laboratory Tests

A. Specimens and Microscopic Examination

Specimens for culture include sputum, exudate from cutaneous lesions, spinal fluid, blood, urine, and tissue biopsies.

Materials should be examined fresh (after centrifuging, if necessary) for typical spherules. KOH or calcofluor white stain will facilitate finding the spherules and endospores (see Figure 45-16B). These structures are often found in histologic preparations stained with H&E, GMS, or PAS.

B. Cultures

Cultures on IMA or Brain-Heart Infusion blood agar slants can be incubated at room temperature or at 37°C. The media can be prepared with or without antibacterial antibiotics and cycloheximide to inhibit contaminating bacteria or saprophytic molds, respectively. Because the arthroconidia are highly infectious, suspicious cultures are examined only in a biosafety cabinet (see Figure 45-16A). Identification must be confirmed by detection of a C immitis-specific antigen, animal inoculation, or use of a specific DNA probe.

C. Serology

Within 2–4 weeks after infection, IgM antibodies to coccidioidin can be detected with a latex agglutination test. Specific IgG antibodies are detected by the immunodiffusion (ID) or complement fixation (CF) test. With resolution of the primary episode, these antibodies decline within a few months. In contrast, in disseminated coccidioidomycosis, the CF antibody titer continues to rise. Titers above 1:32 are indicative of dissemination, and their fall during treatment suggests improvement (Figure 45-18 and Table 45-5). However, CF titers less than 1:32 do not exclude coccidioidomycosis. Indeed, only half of the patients with coccidioidal meningitis have elevated serum antibodies, but antibody levels in the cerebrospinal fluid are usually high. In AIDS patients with coccidioidomycosis, these serologic tests are often negative.

D. Skin Test

The coccidioidin skin test reaches maximum induration (≥5 mm in diameter) between 24 and 48 hours after cutaneous injection of 0.1 mL of a standardized dilution. If patients with disseminated disease become anergic, the skin test will be negative, which implies a very poor prognosis. Cross-reactions with antigens of other fungi may occur. Spherulin is more sensitive than coccidioidin in detecting reactors. Reactions to skin tests tend to diminish in size and intensity years after primary infection in persons residing in endemic areas, but skin testing exerts a booster effect. Following recovery from primary infection, there is usually immunity to reinfection.

Treatment

In most persons, symptomatic primary infection is self-limited and requires only supportive treatment, although itraconazole may reduce the symptoms. However, patients who have severe disease require treatment with amphotericin B, which is administered intravenously. This regimen may be followed by several months of oral therapy with itraconazole. Cases of coccidioidal meningitis have been treated with oral fluconazole, which has good penetration of the central nervous system; however, long-term therapy is required, and relapses have occurred. The azoles are not more efficacious than amphotericin B, but they are easier to administer and associated with fewer and less severe side effects. The newer lipid emulsions of amphotericin B promise to deliver higher doses with less toxicity. Surgical resection of pulmonary cavities is sometimes necessary and often curative.

Epidemiology and Control

The areas of endemicity for Coccidioides are semiarid regions, resembling the Lower Sonoran Life Zone. They include the southwestern states—particularly the San Joaquin and Sacramento Valleys of California, areas around Tucson and Phoenix in Arizona, the Rio Grande Valley—and similar areas in Central and South America. Within these regions, Coccidioides can be isolated from the soil and indigenous rodents, and the level of skin test reactivity in the population indicates that many humans have been infected. The infection rate is highest during the dry months of summer and autumn, when dust is most prevalent. A high incidence of infection and disease may follow dust storms. During an epidemic of coccidioidomycosis in the San Joaquin Valley of California in 1991–1993, the rate of coccidioidomycosis increased more than tenfold. Increased precipitation in the spring months of these years was suggested as an environmental stimulus. However, since 1998, the incidence has risen another tenfold, and this increase cannot be attributed to population growth or improved diagnoses. Furthermore, recent reports have documented the spread of coccidioidomycosis to the northwestern states, including Washington, and in patients without a history of travel to the endemic areas.

The disease is not communicable from person to person, and there is no evidence that infected rodents contribute to its spread. Some measure of control can be achieved by reducing dust, paving roads and airfields, planting grass or crops, and using oil sprays.

Histoplasma capsulatum is a dimorphic soil saprophyte that causes histoplasmosis, the most prevalent pulmonary fungal infection in humans and animals. In nature, H capsulatum grows as a mold in association with soil and avian habitats, being enriched by alkaline nitrogenous substrates in guano. H capsulatum and histoplasmosis, which is initiated by inhalation of the conidia, occur worldwide. However, the incidence varies considerably, and most cases occur in the United States. H capsulatum received its name from the appearance of the yeast cells in histopathologic sections; however, it is neither a protozoan nor does it have a capsule.

Morphology and Identification

At temperatures below 37°C, primary isolates of H capsulatum often develop brown mold colonies, but the appearance varies. Many isolates grow slowly, and specimens require incubation for 4–12 weeks before colonies develop. The hyaline, septate hyphae produce microconidia (2–5 μm) and large, spherical thick-walled macroconidia with peripheral projections of cell wall material (8–16 μm) (Figure 45-19B). In tissue or in vitro on rich medium at 37°C, the hyphae and conidia convert to small, oval yeast cells (2 × 4 μm). In tissue, the yeasts are typically seen within macrophages, as H capsulatum is a facultative intracellular parasite (see Figure 45-19A). In the laboratory, with appropriate mating strains, a sexual cycle can be demonstrated, yielding Ajellomyces capsulatus, a teleomorph that produces ascospores.

Antigenic Structure

Histoplasmin is a crude but standardized mycelial broth culture filtrate antigen. After initial infection, which is asymptomatic in over 95% of individuals, a positive delayed type skin test to histoplasmin is acquired. Antibodies to both yeast and mycelial antigens can be measured serologically (Table 45-5). An uncharacterized polysaccharide antigen can be detected serologically in serum and other specimens (see Table 45-6).

Pathogenesis and Clinical Findings

After inhalation, the conidia develop into yeast cells and are engulfed by alveolar macrophages, where they are able to replicate. Within macrophages, the yeasts may disseminate to reticuloendothelial tissues such as the liver, spleen, bone marrow, and lymph nodes. The initial inflammatory reaction becomes granulomatous. In over 95% of cases, the resulting cell-mediated immune response leads to the secretion of cytokines that activate macrophages to inhibit the intracellular growth of the yeasts. Some individuals, such as immunocompetent persons who inhale a heavy inoculum, develop acute pulmonary histoplasmosis, which is a self-limited flu-like syndrome with fever, chills, myalgias, headaches, and nonproductive cough. On radiographic examination, most patients will have hilar lymphadenopathy and pulmonary infiltrates or nodules. These symptoms resolve spontaneously without therapy, and the granulomatous nodules in the lungs or other sites heal with calcification.

Chronic pulmonary histoplasmosis occurs most often in men and is usually a reactivation process, the breaking down of a dormant lesion that may have been acquired years before. This reactivation is usually precipitated by pulmonary damage such as emphysema.

Severe disseminated histoplasmosis develops in a small minority of infected individuals—particularly infants, the elderly, and the immunosuppressed, including AIDS patients. The reticuloendothelial system is especially apt to be involved, with lymphadenopathy, enlarged spleen and liver, high fever, anemia, and a high mortality rate without antifungal therapy. Mucocutaneous ulcers of the nose, mouth, tongue, and intestine can occur. In such individuals, histologic study reveals focal areas of necrosis within granulomas in many organs. The yeasts may be present in macrophages in the blood, liver, spleen, and bone marrow.

Diagnostic Laboratory Tests

A. Specimens and Microscopic Examination

Specimens for culture include sputum, urine, scrapings from superficial lesions, bone marrow aspirates, and buffy coat blood cells. Blood films, bone marrow slides, and biopsy specimens may be examined microscopically. In disseminated histoplasmosis, bone marrow cultures are often positive. The small ovoid cells may be observed within macrophages in histologic sections stained with fungal stains, such as GMS or PAS, or in Giemsa-stained smears of bone marrow or blood (see Figure 45-19A).

B. Culture

Specimens are cultured in rich media, such as glucose-cysteine blood agar at 37°C and on SDA or IMA at 25–30°C. Cultures must be incubated for a minimum of 4 weeks. The laboratory should be alerted if histoplasmosis is suspected because special blood culture methods, such as lysis centrifugation or fungal broth medium, can be used to enhance the recovery of H capsulatum. Because the mold form resembles saprobic fungi, the identification of H capsulatum must be confirmed by in vitro conversion to the yeast form, detection of a species-specific antigen, or PCR testing for specific DNA sequences.

C. Serology

CF tests for antibodies to histoplasmin or the yeast cells become positive within 2–5 weeks after infection. CF titers rise during progressive disease and then decline to very low levels when the disease is inactive. With progressive disease, the CF titers are ≥ 1:32. Because cross-reactions may occur, antibodies to other fungal antigens are routinely tested. In the ID test, precipitins to two H capsulatum-specific antigens are detected: The presence of antibodies to the h antigen often signifies active histoplasmosis, while antibodies to the m antigen may arise from repeated skin testing or past exposure (see Table 45-5).

One of the most sensitive tests is a radioassay or enzyme immunoassay for circulating polysaccharide antigen of H capsulatum (Table 45-6). Nearly all patients with disseminated histoplasmosis have a positive test for antigen in the serum or urine; the antigen level drops following successful treatment and recurs during relapse. Despite cross-reactions with other mycoses, this test for antigen is more sensitive than conventional antibody tests in AIDS patients with histoplasmosis.

E. Skin Test

The histoplasmin skin test becomes positive soon after infection and remains positive for years. It may become negative in progressive disseminated histoplasmosis. Repeated skin testing stimulates serum antibodies in sensitive individuals, interfering with the diagnostic interpretation of the serologic tests.

Immunity

Following initial infection, most persons appear to develop some degree of immunity. Immunosuppression may lead to reactivation and disseminated disease. AIDS patients may develop disseminated histoplasmosis through reactivation or new infection.

Treatment

Acute pulmonary histoplasmosis is managed with supportive therapy and rest. Itraconazole is the treatment for mild to moderate infection. In disseminated disease, systemic treatment with amphotericin B is often curative, though patients may need prolonged treatment and monitoring for relapses. Patients with AIDS typically relapse despite therapy that would be curative in other patients. Therefore, AIDS patients require maintenance therapy with itraconazole.

Epidemiology and Control

The incidence of histoplasmosis is highest in the United States, where the endemic areas include the central and eastern states and in particular the Ohio River Valley and portions of the Mississippi River Valley. Numerous outbreaks of acute histoplasmosis have resulted from exposure of many persons to large inocula of conidia. These occur when H capsulatum is disturbed in its natural habitat, that is, soil mixed with bird feces (eg, starling roosts, chicken houses) or bat guano (caves). Birds are not infected, but their excrement provides superb culture conditions for growth of the fungus. Conidia are also spread by wind and dust. The largest urban outbreak of histoplasmosis occurred in Indianapolis.

In some highly endemic areas, 80–90% of residents have a positive skin test by early adulthood. Many will have miliary calcifications in the lungs. Histoplasmosis is not communicable from person to person. Spraying formaldehyde on infected soil may destroy H capsulatum.

In Africa, in addition to the usual pathogen, there is a stable variant, H capsulatum var duboisii, which causes African histoplasmosis. This form differs from the usual disease by causing less pulmonary involvement and more skin and bone lesions with abundant giant cells that contain the yeasts, which are larger and more spherical.

B dermatitidis is a thermally dimorphic fungus that grows as a mold in culture, producing hyaline, branching septate hyphae and conidia. At 37°C or in the host, it converts to a large, singly budding yeast cell (Figure 45-20). B dermatitidis causes blastomycosis, a chronic infection with granulomatous and suppurative lesions that is initiated in the lungs, whence dissemination may occur to any organ but preferentially to the skin and bones. The disease has been called North American blastomycosis because it is endemic and most cases occur in the United States and Canada. Despite this high prevalence in North America, blastomycosis has been documented in Africa, South America, and Asia. It is endemic for humans and dogs in the eastern United States.

Morphology and Identification

When B dermatitidis is grown on SDA at room temperature, a white or brownish colony develops, with branching hyphae bearing spherical, ovoid, or piriform conidia (3–5 μm in diameter) on slender terminal or lateral conidiophores (see Figure 45-20B). Larger chlamydospores (7–18 μm) may also be produced. In tissue or culture at 37°C, B dermatitidis grows as a thick-walled, multinucleated, spherical yeast (8–15 μm) that usually produces single buds (see Figure 45-20A). The bud and the parent yeast are attached with a broad base, and the bud often enlarges to the same size as the parent yeast before they become detached. The yeast colonies are wrinkled, waxy, and soft.

Antigenic Structure

Extracts of culture filtrates of B dermatitidis contain blastomycin, probably a mixture of antigens. As a skin test reagent, blastomycin lacks specificity and sensitivity. Patients are often negative or lose their reactivity, and false-positive cross-reactions occur in people exposed to other fungi. Consequently, skin test surveys of the population to determine the level of exposure have not been conducted. The diagnostic value of blastomycin as an antigen in the CF test is also questionable because cross-reactions are common; however, many patients with widespread blastomycosis have high CF titers. In the ID test, using adsorbed reference antisera, antibodies can be detected to a specific B dermatitidis antigen, designated antigen A. More reliable is an enzyme immunoassay for antigen A (see Table 45-5). The immunodominant motif probably responsible for generating a protective cell-mediated immune response is part of a cell-surface and secreted protein, termed BAD.

Pathogenesis and Clinical Findings

Human infection is initiated in the lungs. Mild and self-limited cases have been documented, but their frequency is unknown because there is no adequate skin or serologic test with which to assess subclinical or resolved primary infections. The most common clinical presentation is a pulmonary infiltrate in association with a variety of symptoms indistinguishable from other acute lower respiratory infections (fever, malaise, night sweats, cough, and myalgias). Patients can also present with chronic pneumonia. Histologic examination reveals a distinct pyogranulomatous reaction with neutrophils and noncaseating granulomas. When dissemination occurs, skin lesions on exposed surfaces are most common. They may evolve into ulcerated verrucous granulomas with an advancing border and central scarring. The border is filled with microabscesses and has a sharp, sloping edge. Lesions of bone, the genitalia (prostate, epididymis, and testis), and the central nervous system also occur; other sites are less frequently involved. Although immunosuppressed patients, including those with AIDS, may develop blastomycosis, it is not as common in these patients as are other systemic mycoses.

Diagnostic Laboratory Tests

Specimens consist of sputum, pus, exudates, urine, and biopsies from lesions. Upon microscopic examination, wet mounts of specimens may show broadly attached buds on thick-walled yeast cells. These may also be apparent in histologic sections (see Figure 45-20A). In culture, colonies usually develop within 2 weeks on Sabouraud’s or enriched blood agar at 30°C (see Figure 45-20B). The identification is confirmed by conversion to the yeast form after cultivation on a rich medium at 37°C, by extraction and detection of the B dermatitidis-specific antigen A, or by a specific DNA probe. As indicated in Table 45-5, antibodies can be measured by the CF and ID tests. In the enzyme immunoassay (EIA), high antibody titers to antigen A are associated with progressive pulmonary or disseminated infection. Overall, serologic tests are not as useful for the diagnosis of blastomycosis as they are in the case of the other endemic mycoses.

Treatment

Severe cases of blastomycosis are treated with amphotericin B. In patients with confined lesions, a 6-month course of itraconazole is very effective.

Epidemiology

Blastomycosis is a relatively common infection of dogs (and rarely other animals) in endemic areas. Blastomycosis cannot be transmitted by animals or humans. Unlike C immitis and H capsulatum, B dermatitidis has only rarely (and not reproducibly) been isolated from the environment, so its natural habitat is unknown. However, the occurrence of several small outbreaks has linked B dermatitidis to rural river banks.

Paracoccidioides brasiliensis is the thermally dimorphic fungal agent of paracoccidioidomycosis (South American blastomycosis), which is confined to endemic regions of Central and South America.

Morphology and Identification

Cultures of the mold form of P brasiliensis grow very slowly and produce chlamydospores and conidia. The features are not distinctive. At 36°C, on rich medium, it forms large, multiply budding yeast cells (up to 30 μm). The yeasts are larger and have thinner walls than those of B dermatitidis. The buds are attached by a narrow connection (Figure 45-21).

Pathogenesis and Clinical Findings

P brasiliensis is inhaled, and initial lesions occur in the lung. After a period of dormancy that may last for decades, the pulmonary granulomas may become active, leading to chronic, progressive pulmonary disease or dissemination. Most patients are 30–60 years of age, and over 90% are men. A few patients (<10%), typically less than 30 years of age, develop an acute or subacute progressive infection with a shorter incubation time. In the usual case of chronic paracoccidioidomycosis, the yeasts spread from the lung to other organs, particularly the skin and mucocutaneous tissue, lymph nodes, spleen, liver, adrenals, and other sites. Many patients present with painful sores involving the oral mucosa. Histology usually reveals either granulomas with central caseation or microabscesses. The yeasts are frequently observed in giant cells or directly in exudate from mucocutaneous lesions.

Skin test surveys have been conducted using an antigen extract, paracoccidioidin, which may cross-react with coccidioidin or histoplasmin.

Diagnostic Laboratory Tests

In sputum, exudates, biopsies, or other material from lesions, the yeasts are often apparent on direct microscopic examination with KOH or calcofluor white. Cultures on Sabouraud’s or yeast extract agar are incubated at room temperature and confirmed by conversion to the yeast form by in vitro growth at 36°C. Serologic testing is most useful for diagnosis. Antibodies to paracoccidioidin can be measured by the CF or ID test (see Table 45-5). Healthy persons in endemic areas do not have antibodies to P brasiliensis. In patients, titers tend to correlate with the severity of disease.

Treatment

Itraconazole appears to be most effective against paracoccidioidomycosis, but ketoconazole and trimethoprim-sulfamethoxazole are also efficacious. Severe disease can be treated with amphotericin B.

Epidemiology

Paracoccidioidomycosis occurs mainly in rural areas of Latin America, particularly among farmers. The disease manifestations are much more frequently in males than in females, but infection and skin test reactivity occur equally in both sexes. Since P brasiliensis has only rarely been isolated from nature, its natural habitat has not been defined. As with the other endemic mycoses, paracoccidioidomycosis is not communicable.

1. The endemic mycoses (coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis) are characterized by distinct geographic areas of distribution and caused by dimorphic environmental molds.

2. More than 90% of the endemic mycoses are initiated by inhaling conidia of the causative fungi. Within the lungs, the conidia convert to distinctive yeast cells (H capsulatum, B dermatitidis, and P brasiliensis) or spherules (Coccidioides).

3. In their endemic areas, the rates of infection with Coccidioides H capsulatum, and P brasiliensis are very high, but approximately 90% of the infections occur in immunocompetent individuals, and the infections are asymptomatic or self-limited.

4. Individuals with compromised cell-mediated immune (Th1) defenses have a significantly greater risk of developing disseminated disease (eg, patients who are immunodeficient or immunosuppressed, HIV seropositive, congenitally predisposed, malnourished, very young, or very old).

5. For all four endemic mycoses, the incidence of disseminated disease is significantly higher in males.

6. Serologic tests for serum antibodies to the endemic fungi have diagnostic and prognostic value.

Patients with compromised host defenses are susceptible to ubiquitous fungi to which healthy people are exposed but usually resistant. In many cases, the type of fungus and the natural history of the mycotic infection are determined by the underlying predisposing condition of the host. As members of the normal mammalian microbiota, Candida and related yeasts are endogenous opportunists. Other opportunistic mycoses are caused by exogenous fungi that are globally present in soil, water, and air. The coverage here will focus on the more common pathogens and the diseases they cause—candidiasis, cryptococcosis, aspergillosis, mucormycosis, Pneumocystis pneumonia, and penicilliosis. However, as medical advances in solid organ and stem cell transplantation as well as the treatment of cancer and other debilitating diseases continue to prolong the lives of patients with impaired host defenses, the incidence and the roster of fungal species causing serious opportunistic mycoses in compromised individuals continues to increase. Every year, there are novel reports of infections caused by environmental fungi that were previously thought to be nonpathogenic. In patients with HIV/AIDS, the susceptibility and incidence of opportunistic mycoses are inversely correlated with the CD4⁺ lymphocyte count. In general, AIDS patients with CD4⁺ counts less than 200 cells/μL are highly susceptible to infection with opportunistic fungi.

Several species of the yeast genus Candida are capable of causing candidiasis. They are members of the normal flora of the skin, mucous membranes, and gastrointestinal tract. Candida species colonize the mucosal surfaces of all humans soon after birth, and the risk of endogenous infection is ever-present. Candidiasis is the most prevalent systemic mycosis, and the most common agents are Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida guilliermondii, and Candida dubliniensis. The widespread use of fluconazole has precipitated the emergence of more azole-resistant species, such as C glabrata, Candida krusei, and Candida lusitaniae. As indicated in Table 45-1, species of Candida cause both cutaneous and systemic infections, and these clinical manifestations have different mechanisms of pathogenesis. In addition, there are several other clinical forms of candidiasis.

Morphology and Identification

In culture or tissue, Candida species grow as oval, budding yeast cells (3–6 μm in size). They also form pseudohyphae when the buds continue to grow but fail to detach, producing chains of elongated cells that are pinched or constricted at the septations between cells. Unlike other species of Candida, C albicans is dimorphic; in addition to yeasts and pseudohyphae, it can also produce true hyphae (Figure 45-22). On agar media or within 24 hours at 37°C or room temperature, Candida species produce soft, cream-colored colonies with a yeasty odor. Pseudohyphae are apparent as submerged growth below the agar surface. Two simple morphologic tests distinguish C albicans, the most common pathogen, from other species of Candida: After incubation in serum for about 90 minutes at 37°C, yeast cells of C albicans will begin to form true hyphae or germ tubes (Figure 45-23), and on nutritionally deficient media C albicans produces large, spherical chlamydospores. Sugar fermentation and assimilation tests can be used to confirm the identification and speciate the more common Candida isolates, such as C tropicalis, C parapsilosis, C guilliermondii, Candida kefyr, C krusei, and C lusitaniae. C glabrata is unique among these pathogens because on routine culture media it produces only yeast cells and no pseudohyphae.

Antigenic Structure

The use of adsorbed antisera have defined two serotypes of C albicans: A (which includes C tropicalis) and B. During infection, cell wall components—such as mannans, glucans, other polysaccharides and glycoproteins, as well as enzymes—are released. These macromolecules typically elicit innate host defenses and Th1, Th17, and Th2 immune responses. For example, sera from patients with systemic candidiasis often contain detectable antibodies to candidal enolase, secretory proteases, and heat shock proteins.

Pathogenesis and Pathology

Cutaneous or mucosal candidiasis is established by an increase in the local census of Candida and damage to the skin or epithelium that permits local invasion by the yeasts and pseudohyphae. The histology of cutaneous or mucocutaneous lesions is characterized by inflammatory reactions varying from pyogenic abscesses to chronic granulomas. The lesions contain abundant budding yeast cells and pseudohyphae. The administration of broad-spectrum antibacterial antibiotics often promotes large increases in the endogenous population of Candida in the gastrointestinal tract as well as the oral and vaginal mucosa. Systemic candidiasis occurs when Candida enters the bloodstream and the innate phagocytic host defenses are inadequate to contain the growth and dissemination of the yeasts. The yeasts can enter the circulation by crossing the intestinal mucosa. Many nosocomial cases are caused by contamination of indwelling intravenous catheters with Candida. Once in the circulation, Candida can infect the kidneys, attach to prosthetic heart valves, or produce candidal infections almost anywhere (eg, arthritis, meningitis, endophthalmitis). The critical host defense against systemic candidiasis is an adequate number of functional neutrophils capable of ingesting and killing the yeast cells.

As noted above, Candida cells elaborate polysaccharides, proteins, and glycoproteins that not only stimulate host defenses but facilitate the attachment and invasion of host cells. C albicans and other Candida species produce a family of agglutinin-like sequence (ALS) surface glycoproteins, some of which are adhesins that bind host cell membrane receptors and mediate attachment to epithelial or endothelial cells. The innate host defense mechanisms include pattern recognition receptors (eg, lectins, Toll-like receptors, macrophage mannose receptor) that bind to pathogen-associated molecular patterns. A key example is the host cell lectin, dectin-1, which binds to the β-1,3-glucan of C albicans and other fungi to stimulate a robust inflammatory response. This response is characterized by the production of cytokines, especially tumor necrosis factor-α, interferon-g, and granulocyte colony-stimulating factor, which activate anti-fungal effector cells, neutrophils, and monocytes. In addition, the binding of β-glucan to dectin 1 on dendritic cells induces the Th17 lymphocytes, which secrete interleukin-17. Th17 lymphocytes differ from T and B cells. They are activated by innate, usually mucosal defense mechanisms as well as adaptive immune responses.

In addition to the family of eight ALS adhesion genes, many other virulence factors have been identified in C albicans and other species of Candida. They include 10 secreted aspartyl proteinases (SAP) that are able to degrade host cell membranes and destroy immunoglobulins. Another virulence factor is phospholipase (PLB1), which is secreted by yeasts and pseudohyphae. In addition, on a variety of biological and prosthetic surfaces, the accumulation of yeasts and pseudohyphae readily form biofilms. The fungal biofilm is protected by extracellular matrix material that resists penetration by host immune responses and antifungal drugs.

Clinical Findings

A. Cutaneous and Mucosal Candidiasis

The risk factors associated with superficial candidiasis include AIDS, pregnancy, diabetes, young or old age, birth control pills, and trauma (burns, maceration of the skin). Thrush can occur on the tongue, lips, gums, or palate. It is a patchy to confluent, whitish pseudomembranous lesion composed of epithelial cells, yeasts, and pseudohyphae, which can lead to the formation of an intractable biofilm. Thrush develops in most patients with AIDS. Other risk factors include treatment with corticosteroids or antibiotics, high levels of glucose, and cellular immunodeficiency. Yeast invasion of the vaginal mucosa leads to vulvovaginitis, characterized by irritation, pruritus, and vaginal discharge. This condition is often predisposed by conditions such as diabetes, pregnancy, or antibacterial drugs that alter the microbiota, local acidity, or secretions. Other forms of cutaneous candidiasis include invasion of the skin, which occurs when the skin is weakened by trauma, burns, or maceration. Intertriginous infection occurs in moist, warm parts of the body such as the axillae, groin, and intergluteal or inframammary folds; it is most common in obese and diabetic individuals. Before newborns establish a balanced microbiome, they are susceptible to extensive diaper rash and skin infection caused by Candida. The infected areas become red and moist and may develop vesicles. Interdigital involvement between the fingers follows repeated prolonged immersion in water; it is most common in homemakers, bartenders, cooks, and vegetable and fish handlers. Candidal invasion of the nails and around the nail plate causes onychomycosis, a painful, erythematous swelling of the nail fold resembling a pyogenic paronychia, which may eventually destroy the nail.

B. Systemic Candidiasis

Candidemia can be caused by indwelling catheters, surgery, intravenous drug abuse, aspiration, or damage to the skin or gastrointestinal tract. In most patients with normal innate immune responses and circulating neutrophils, the yeasts are eliminated and candidemia is transient. However, patients with compromised innate phagocytic defenses may develop occult lesions anywhere, especially the kidney, skin (maculonodular lesions), eye, heart, and meninges. Systemic candidiasis is most often associated with chronic administration of corticosteroids or other immunosuppressive agents; with hematologic diseases such as leukemia, lymphoma, and aplastic anemia; or with chronic granulomatous disease. Candidal endocarditis is frequently preceded by the deposition and growth of the yeasts and pseudohyphae on prosthetic heart valves or vegetations and the formation of recalcitrant biofilms. Kidney infections are usually a systemic manifestation, whereas urinary tract infections are often associated with Foley catheters, diabetes, pregnancy, and antibacterial antibiotics.

C. Chronic Mucocutaneous Candidiasis

Chronic mucocutaneous candidiasis (CMC) is a rare but distinctive clinical manifestation characterized by the formation of granulomatous candidal lesions on any or all cutaneous and/or mucosal surfaces. There are several classifications of CMC based on the age of onset, endocrinopathy, genetic predisposition, and immune status. The most common forms present in early childhood and are associated with autoimmunity and hypoparathyroidism. The patients may develop chronic, raised, and crusty highly disfiguring keratitic lesions on the skin, oral mucosa, and scalp. Many patients with chronic mucocutaneous candidiasis are unable to mount an effective Th17 response to Candida.

Diagnostic Laboratory Tests

A. Specimens and Microscopic Examination

Specimens include swabs and scrapings from superficial lesions, blood, spinal fluid, tissue biopsies, urine, exudates, and material from removed intravenous catheters.

Tissue biopsies, centrifuged spinal fluid, and other specimens may be examined in Gram-stained smears or histopathologic slides for pseudohyphae and budding cells (Figure 45-24). As with dermatophytosis, skin or nail scrapings are first placed in a drop of KOH and calcofluor white.

B. Culture

All specimens are cultured on fungal or bacteriologic media at room temperature or at 37°C. Yeast colonies are examined for the presence of pseudohyphae (see Figure 45-22). C albicans is identified by the production of germ tubes (see Figure 45-23) or chlamydospores. Other yeast isolates are speciated phenotypically by using any of several commercial kits to test for the metabolic assimilation of a battery of organic substrates. CHROMagar(^®) is a useful commercial medium for the rapid identification of several Candida species based on fungal enzymatic action on chromogenic substrates in the medium. After incubation for 1–4 days on CHROMagar, colonies of C albicans are green, C tropicalis is blue, C glabrata is dark purple, and C parapsilosis, C lusitaniae, C guilliermondii, and C krusei acquire a pinkish hue.

The interpretation of positive cultures varies with the specimen. Any positive culture from normally sterile body sites is significant. The diagnostic value of a quantitative urine culture depends on the integrity of the specimen and the yeast census. Contaminated Foley catheters may lead to “false-positive” urine cultures. Positive blood cultures may reflect systemic candidiasis or transient candidemia due to a contaminated intravenous line. Sputum cultures have no value because Candida species are part of the oral microbiota. Cultures of skin lesions are confirmatory and distinguish cutaneous candidiasis from dermatophytosis or another infection.

C. Molecular Methods

In many clinical laboratories, blood cultures for Candida are augmented by real-time PCR with species-specific primers, which are commonly designed from sequences of the multicopy ribosomal DNA genes. The specificity of DNA tests for candidemia is excellent, but the sensitivity can be compromised by a low census of yeast cells in the blood sample. A crucial problem is the method used to extract the DNA from the yeast cells, as well as eliminating inhibition of the PCR by human DNA and hemoglobin. The ideal molecular test would detect candidemia early in the course of infection before the yeasts have developed chronic infection in the kidneys and other organs, when blood cultures are usually negative.

Yeast cultures, especially non–C albicans species often take several days for definitive identification. With its ease of sample preparation and automation, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), has become a popular rapid method of identifying species of Candida, as well as other pathogenic fungi and bacteria.

D. Serology

Serum antibodies and cell-mediated immunity are demonstrable in most people as a result of lifelong exposure to Candida. In systemic candidiasis, antibody titers to various candidal antigens may be elevated, but there are no clear criteria for establishing a diagnosis serologically. The detection of circulating Candida cell wall mannan, using a latex agglutination test or an enzyme immunoassay, is much more specific, but the test lacks high sensitivity because many patients are only transiently positive or because they do not develop significant and detectable antigen titers until late in the disease. Nevertheless, a positive test can be most helpful (see Table 45-6). The biochemical test for circulating β-(1,3)-D-glucan, described earlier in this chapter, has become widely used to screen for fungemia in patients at risk who often have negative blood cultures. Although the test is not specific for Candida, most patients with an invasive fungal infection have serum β-glucan levels above 80 pg/mL. Normal levels are 10–40 pg/mL.

Immunity

The basis of resistance to candidiasis is complex and incompletely understood. Innate immune responses, especially circulating neutrophils, are crucial for resistance to systemic candidiasis. Several Candida polysaccharide antigens are recognized by host pattern recognition receptors (PRR), such as dectin-1, which binds β-(1,3)-glucan, and β-(1,2)-mannan, which binds Toll-like receptor (TLR)-4. As noted above, cell-mediated immune responses are important for controlling mucosal candidiasis. Stimulation of specific Th17 lymphocytes triggers a cascade of cytokines that activate macrophages, inflammation, and enhance phagocytic activity.

Treatment

Thrush and other mucocutaneous forms of candidiasis are usually treated with topical nystatin or oral ketoconazole or fluconazole. The clearing of cutaneous lesions is accelerated by eliminating contributing factors such as excessive moisture or antibacterial drugs. Systemic candidiasis is treated with amphotericin B, sometimes in conjunction with oral flucytosine, fluconazole, or caspofungin. Chronic mucocutaneous candidiasis responds well to oral ketoconazole and other azoles, but patients have a genetic cellular immune defect and often require lifelong treatment.

It is often difficult to establish an early diagnosis of systemic candidiasis—the clinical signs are not definitive, and blood cultures are often negative. Furthermore, there is no established prophylactic regimen for patients at risk, though the administration of an azole or a short course of low-dose amphotericin B is often indicated for febrile or debilitated patients who are immunocompromised and do not respond to antibacterial therapy (see below).

Epidemiology and Control

The most important preventive measure is to avoid disturbing the normal balance of microbiota and intact host defenses. Candidiasis is not communicable, since virtually all persons normally harbor the organism. However, molecular epidemiological studies have documented outbreaks caused by the nosocomial transmission of particular strains to susceptible patients (eg, leukemics, translplants, neonates, ICU patients). Candida species are the fourth most common blood culture isolate and the attributable mortality ranges from 30% to 40%.

Cryptococcus neoformans and Cryptococcus gattii are environmental, basidiomycetous yeasts. Unlike other pathogenic fungi, these yeast cells possess large polysaccharide capsules (Figure 45-25). C neoformans occurs worldwide in nature and is isolated readily from dry pigeon feces, as well as trees, soil, and other sites. C gattii is less common and typically associated with trees in tropical areas. Both species cause cryptococcosis, which follows inhalation of desiccated yeast cells or possibly the smaller basidiospores. From the lungs, these neurotropic yeasts typically migrate to the central nervous system where they cause meningoencephalitis (Figure 45-26). However, they also have the capacity to infect many other organs (eg, skin, eyes, prostate). C neoformans occurs in immunocompetent persons but more often in patients with HIV/AIDS, hematogenous malignancies, and other immunosuppressive conditions. Cryptococcosis due to C gattii is rarer and usually associated with apparently normal hosts. Overall, approximately one million new cases of cryptococcosis occur annually, and the mortality approaches 50%. More than 90% of these infections are caused by C neoformans. Although C gattii is less prevalent globally, for the past decade, there has been an expanding outbreak of infections with this species in the Pacific Northwest.

Morphology and Identification

In culture, Cryptococcus species produce whitish mucoid colonies within 2–3 days. Microscopically, in culture or clinical material, the spherical budding yeast cells (5–10 μm in diameter) are surrounded by a thick nonstaining capsule (see Figure 45-25). All species of Cryptococcus, including several nonpathogenic species, are encapsulated and possess urease. However, C neoformans and C gattii differ from nonpathogenic species by the abilities to grow at 37°C and the production of laccase, a phenol oxidase, which catalyzes the formation of melanin from appropriate phenolic substrates (eg, catecholamines). Both the capsule and laccase are well-characterized virulence factors. Clinical isolates are identified by demonstrating the production of laccase or a specific pattern of carbohydrate assimilations. Adsorbed antisera have defined five serotypes (A–D and AD); strains of C neoformans may possess serotype A, D, or AD, and isolates of C gattii may have serotype B or C. In addition to their capsular serotypes, the two species differ in their genotypes, ecology, some biochemical reactions, and clinical manifestations. Sexual reproduction can be demonstrated in the laboratory, and successful mating results in the production of mycelia and basidiospores; the corresponding teleomorphs of the two teleomorphic species are Filobasidiella neoformans and F. bacillispora.

Antigenic Structure

The capsular polysaccharides, regardless of serotype, have a similar structure. They are long, unbranched polymers consisting of an α-1,3-linked polymannose backbone with β-linked monomeric branches of xylose and glucuronic acid. During infection, the capsular polysaccharide, or glucuronoxylomannan (GXM), is solubilized in spinal fluid, serum, or urine and can be detected by an enzyme immunoassay or by the agglutination of latex particles coated with antibody to the polysaccharide. With proper controls, this test is diagnostic of cryptococcosis. Patient antibodies to the capsule can also be measured, but they are not used in diagnosis.

Pathogenesis

Infection is initiated by inhalation of the yeast cells, which in nature are dry, minimally encapsulated, and easily aerosolized. The primary pulmonary infection may be asymptomatic or may mimic an influenza-like respiratory infection, often resolving spontaneously. In patients who are compromised, the yeasts may multiply and disseminate to other parts of the body but preferentially to the central nervous system, causing cryptococcal meningoencephalitis (see Figure 45-26). Other common sites of dissemination include the skin, adrenals, bone, eye, and prostate gland. The inflammatory reaction is usually minimal or granulomatous.

Clinical Findings

The major clinical manifestation is chronic meningitis, which can resemble a brain tumor, brain abscess, degenerative central nervous system disease, or any mycobacterial or fungal meningitis. Cerebrospinal fluid pressure and protein may be increased and the cell count elevated, whereas the glucose is normal or low. Patients may complain of headache, neck stiffness, and disorientation. In addition, there may be lesions in skin, lungs, or other organs.

The course of cryptococcal meningitis may fluctuate over long periods, but all untreated cases are ultimately fatal. Globally, about 5–8% of patients with AIDS develop cryptococcal meningitis. The infection is not transmitted from person to person.

Diagnostic Laboratory Tests

A. Specimens, Microscopic Examination, and Culture

Specimens include cerebrospinal fluid (CSF), tissue, exudates, sputum, blood, cutaneous scrapings, and urine. Spinal fluid is centrifuged before microscopic examination and culture. For direct microscopy, specimens are often examined in wet mounts, both directly and after mixing with India ink, which delineates the capsule (see Figure 45-25).

Colonies develop within a few days on most media at room temperature or 37°C. Media with cycloheximide inhibit Cryptococcus and should be avoided. Cultures can be identified by growth at 37°C and detection of urease. Alternatively, on an appropriate diphenolic substrate, the phenol oxidase (or laccase) of C neoformans and C gattii produces melanin in the cell walls and colonies develop a brown pigment.

B. Serology

Tests for GXM, capsular antigen, can be performed on CSF, serum, and urine. The latex slide agglutination test or enzyme immunoassay (EIA) for cryptococcal antigen is positive in 90% of patients with cryptococcal meningitis. With effective treatment, the antigen titer drops—except in AIDS patients, who often maintain high antigen titers for long periods (see Table 45-6). The newest test for GXM is a lateral flow assay (LFA), in which monoclonal antibodies to GXM are prepared in an EIA format on a dipstick that can be placed in serum, CSF, or urine and produces a positive test color change within 20 minutes. This LFA test has been used extensively as a point-of-care screen for cryptococcosis in sub-Saharan Africa.

Treatment

Combination therapy of amphotericin B and flucytosine has been considered the standard treatment for cryptococcal meningitis, though the benefit from adding flucytosine remains controversial. Amphotericin B (with or without flucytosine) is curative in most non-AIDS patients. Inadequately treated AIDS patients will almost always relapse when amphotericin B is withdrawn and require suppressive therapy with fluconazole, which offers excellent penetration of the central nervous system.

HIV/AIDS patients treated with highly active antiretroviral therapy (HAART) have a lower incidence of cryptococcosis, and cases have a much better prognosis. Unfortunately, up to a third of HAART-treated AIDS patients with cryptococcal meningitis develop immune reconstitution inflammatory syndrome (IRIS), which greatly exacerbates the illness. The diagnosis, pathogenesis, and management of IRIS are problematic. In addition to causing a paradoxical relapse of cryptococcal disease, IRIS may “unmask” undiagnosed cryptococcosis. IRIS also occurs in AIDS patients with tuberculosis and other chronic infections.

Epidemiology and Ecology

Bird droppings (particularly pigeon droppings) enrich for the growth of C neoformans and serve as a reservoir of infection. The organism grows luxuriantly in pigeon excreta, but the birds are not infected. In addition to patients with AIDS or hematologic malignancies, patients being maintained on corticosteroids are highly susceptible to cryptococcosis. In sub-Saharan Africa, the epicenter of HIV/AIDS, C neoformans is the leading cause of meningitis with an estimated one million new cases and 600,000 deaths per year.

The vast majority of global cases of cryptococcosis are caused by C neoformans (serotype A). However, the normally tropical species C gattii has emerged in the Pacific Northwest, where it has been isolated from several local species of trees, soil, and water. Since 2000, human and veterinary cases have expanded from Vancouver Island to mainland British Columbia, Washington, Oregon, California, and Idaho.

Aspergillosis is a spectrum of diseases that may be caused by a number of Aspergillus species. Aspergillus species are ubiquitous saprobes in nature, and aspergillosis occurs worldwide. A fumigatus is the most common human pathogen, but many others, including Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus lentulus may cause disease. This mold produces abundant small conidia that are easily aerosolized. Following inhalation of these conidia, atopic individuals often develop severe allergic reactions to the conidial antigens. In immunocompromised patients—especially those with leukemia, stem cell transplant patients, and individuals taking corticosteroids—the conidia may germinate to produce hyphae that invade the lungs and other tissues.

Morphology and Identification

Aspergillus species grow rapidly, producing aerial hyphae that bear characteristic conidial structures: long conidiophores with terminal vesicles on which phialides produce basipetal chains of conidia (see Figure 45-6). The species are identified according to morphologic differences in these structures, including the size, shape, texture, and color of the conidia.

Pathogenesis

In the lungs, alveolar macrophages are able to engulf and destroy the conidia. However, macrophages from corticosteroid-treated animals or immunocompromised patients have a diminished ability to contain the inoculum. In the lung, conidia swell and germinate to produce hyphae that have a tendency to invade preexisting cavities (aspergilloma or fungus ball) or blood vessels.

Clinical Findings

A. Allergic Forms

In some atopic individuals, development of IgE antibodies to the surface antigens of Aspergillus conidia elicits an immediate asthmatic reaction upon subsequent exposure. In others, the conidia germinate and hyphae colonize the bronchial tree without invading the lung parenchyma. This phenomenon is characteristic of allergic bronchopulmonary aspergillosis, which is clinically defined as asthma, recurrent chest infiltrates, eosinophilia, and both type I (immediate) and type III (Arthus) skin test hypersensitivity to Aspergillus antigen. Many patients produce sputum with Aspergillus and serum precipitins. They have difficulty breathing and may develop permanent lung scarring. Normal hosts exposed to massive doses of conidia can develop extrinsic allergic alveolitis.

B. Aspergilloma and Extrapulmonary Colonization

Aspergilloma occurs when inhaled conidia enter an existing cavity, germinate, and produce abundant hyphae in the abnormal pulmonary space. Patients with previous cavitary disease (eg, tuberculosis, sarcoidosis, emphysema) are at risk. Some patients are asymptomatic; others develop cough, dyspnea, weight loss, fatigue, and hemoptysis. Cases of aspergilloma rarely become invasive. Localized, noninvasive infections (colonization) by Aspergillus species may involve the nasal sinuses, the ear canal, the cornea, or the nails.

C. Invasive Aspergillosis

Following inhalation and germination of the conidia, invasive disease develops as an acute pneumonic process with or without dissemination. Patients at risk are those with lymphocytic or myelogenous leukemia and lymphoma, stem cell transplant recipients, and especially individuals taking corticosteroids. The risk is much greater for patients receiving allogeneic (rather than autologous) hematopoietic stem cell transplants. In addition, AIDS patients with CD4 cell counts less than 50 CD4 cells/μL are predisposed to invasive aspergillosis. Symptoms include fever, cough, dyspnea, and hemoptysis. Hyphae invade the lumens and walls of blood vessels, causing thrombosis, infarction, and necrosis. From the lungs, the disease may spread to the gastrointestinal tract, kidney, liver, brain, or other organs, producing abscesses and necrotic lesions. Without rapid treatment, the prognosis for patients with invasive aspergillosis is grave. Persons with less compromising underlying disease may develop chronic necrotizing pulmonary aspergillosis, which is a milder disease.

Diagnostic Laboratory Tests

A. Specimens, Microscopic Examination, and Culture

Sputum, other respiratory tract specimens, and lung biopsy tissue provide good specimens. Blood samples are rarely positive. On direct examination of sputum with KOH or calcofluor white or in histologic sections, the hyphae of Aspergillus species are hyaline, septate, and uniform in width (about 4 μm) and branch dichotomously (Figure 45-27). Aspergillus species grow within a few days on most media at room temperature. Species are identified according to the morphology of their conidial structures (see Figure 45-6).

B. Serology

The ID test for precipitins to A fumigatus is positive in over 80% of patients with aspergilloma or allergic forms of aspergillosis, but antibody tests are not helpful in the diagnosis of invasive aspergillosis. For the latter, the serologic test for circulating cell wall galactomannan is diagnostic, although not entirely specific for aspergillosis (see Table 45-6). In addition to testing for circulating galactomannan, the detection of β-glucan is also helpful in diagnosing invasive aspergillosis as well as candidiasis.

Treatment

Aspergilloma is treated with itraconazole or amphotericin B and surgery. Invasive aspergillosis requires rapid administration of either the native or lipid formulation of amphotericin B or voriconazole, often supplemented with cytokine immunotherapy (eg, granulocyte-macrophage colony-stimulating factor or interferon Γ). Amphotericin B-resistant strains of A terreus and other species, including A flavus and A lentulus, have emerged at several leukemia treatment centers, and the new triazole, posaconazole, may be more effective for these infections. The less severe chronic necrotizing pulmonary disease may be treatable with voriconazole or itraconazole. Allergic forms of aspergillosis are treated with corticosteroids or disodium cromoglycate.

Epidemiology and Control

For persons at risk for allergic disease or invasive aspergillosis, efforts are made to avoid exposure to the conidia of Aspergillus species. Most bone marrow transplant units employ filtered air-conditioning systems, monitor airborne contaminants in patients’ rooms, reduce visiting, and institute other measures to isolate patients and minimize their risk of exposure to the conidia of Aspergillus and other molds. Some patients at risk for invasive aspergillosis are given prophylactic low-dose amphotericin B or itraconazole.

Mucormycosis (zygomycosis) is an opportunistic mycosis caused by a number of molds classified in the order Mucorales of the Phylum Glomerulomycota and Subphylum Mucoromycotina. These fungi are ubiquitous thermotolerant saprobes. The leading pathogens among this group are species of the genera Rhizopus (see Figure 45-2), Rhizomucor, Lichtheimia, Cunninghamella (see Figure 45-3), Mucor, et al. The most prevalent agent is Rhizopus oryzae. The conditions that place patients at risk include acidosis—especially that associated with diabetes mellitus—leukemias, lymphoma, corticosteroid treatment, severe burns, immunodeficiencies, and other debilitating diseases as well as dialysis with the iron chelator deferoxamine.

The major clinical form is rhinocerebral mucormycosis, which results from germination of the sporangiospores in the nasal passages and invasion of the hyphae into the blood vessels, causing thrombosis, infarction, and necrosis. The disease can progress rapidly with invasion of the sinuses, eyes, cranial bones, and brain. Blood vessels and nerves are damaged, and patients develop edema of the involved facial area, a bloody nasal exudate, and orbital cellulitis. Thoracic mucormycosis follows inhalation of the sporangiospores with invasion of the lung parenchyma and vasculature. In both locations, ischemic necrosis causes massive tissue destruction. Less frequently, this process has been associated with contaminated wound dressings and other situations.

Direct examination or culture of nasal discharge, tissue, or sputum will reveal broad hyphae (10–15 μm) with uneven thickness, irregular branching, and sparse septations (Figure 45-28). These fungi grow rapidly on laboratory media, producing abundant cottony colonies. Identification is based on the sporangial structures. Treatment consists of aggressive surgical debridement, rapid administration of amphotericin B, and control of the underlying disease. Many patients survive, but there may be residual effects such as partial facial paralysis or loss of an eye.

Pneumocystis jiroveci causes pneumonia in immunocompromised patients; dissemination is rare. For years, P jiroveci was thought to be a protozoan, but molecular biologic studies have proved that it is a fungus with a close relationship to ascomycetes. Pneumocystis species are present in the lungs of many animals (rats, mice, dogs, cats, ferrets, rabbits) but rarely cause disease unless the host is immunosuppressed. P jiroveci is the human species, and the more familiar Pneumocystis carinii is found only in rats. Until the AIDS epidemic, human disease was confined to interstitial plasma cell pneumonitis in malnourished infants and immunosuppressed patients (corticosteroid therapy, antineoplastic therapy, and transplant recipients). Prior to the introduction of effective chemoprophylactic regimens, it was a major cause of death among AIDS patients. Chemoprophylaxis has resulted in a dramatic decrease in the incidence of pneumonia, but infections are increasing in other organs, primarily the spleen, lymph nodes, and bone marrow.

P jiroveci has morphologically distinct forms: thin-walled trophozoites and cysts, which are thick-walled, spherical to elliptical (4–6 μm), and contain four to eight nuclei. Cysts can be stained with silver stain, toluidine blue, and calcofluor white. In most clinical specimens, the trophozoites and cysts are present in a tight mass that probably reflects their mode of growth in the host. P jiroveci contains a surface glycoprotein that can be detected in sera from acutely ill or normal individuals.

P jiroveci is an extracellular pathogen. Growth in the lung is limited to the surfactant layer above alveolar epithelium. In non-AIDS patients, infiltration of the alveolar spaces with plasma cells leads to interstitial plasma cell pneumonitis. Plasma cells are absent in AIDS-related Pneumocystis pneumonia. Blockage of the oxygen exchange interface results in cyanosis.

To establish the diagnosis of Pneumocystis pneumonia, specimens of bronchoalveolar lavage, lung biopsy tissue, or induced sputum are stained and examined for the presence of cysts or trophozoites. Appropriate stains include Giemsa, toluidine blue, methenamine silver, and calcofluor white. A specific monoclonal antibody is available for direct fluorescent examination of specimens. Pneumocystis cannot be cultured. While not clinically useful, serologic testing has been used to establish the prevalence of infection.

In the absence of immunosuppression, P jiroveci does not cause disease. Serologic evidence suggests that most individuals are infected in early childhood, and the organism has worldwide distribution. Cell-mediated immunity presumably plays a dominant role in resistance to disease, as AIDS patients often have significant antibody titers, and Pneumocystis pneumonia is not usually seen until the CD4 lymphocyte count drops below 400/μL. A test for antigenemia is now available (see Table 45-6).

Acute cases of Pneumocystis pneumonia are treated with trimethoprim-sulfamethoxazole or pentamidine isethionate. Prophylaxis can be achieved with daily trimethoprim-sulfamethoxazole or aerosolized pentamidine. Other drugs are also available.

No natural reservoir has been demonstrated, and the agent may be an obligate member of the normal flora. Persons at risk are provided with chemoprophylaxis. The mode of infection is unclear, and transmission by aerosols may be possible.

Only one of the numerous and ubiquitous environmental species of Penicillium is dimorphic, Penicillium marneffei, and this species has emerged as an endemic, opportunistic pathogen. P marneffei is found in several regions of Southeast Asia, including southeastern China, Thailand, Vietnam, Indonesia, Hong Kong, Taiwan, and the Manipur state of India. Within these endemic areas, P marneffei has been isolated from soil and especially soil that is associated with bamboo rats and their habitats. At ambient temperatures, the mold form grows rapidly to develop a green-yellow colony with a diffusible reddish pigment. The septate, branching hyphae produce aerial conidiophores bearing phialides and basipetal chains of conidia, similar to the structures in Figure 45-4. In tissue, the hyphal forms convert to unicellular yeast-like cells (ca. 2 × 6 μm) that divide by fission. The major risk for infection is immunodeficiency due to HIV/AIDS, tuberculosis, corticosteroid treatment, or lymphoproliferative diseases. The clinical manifestations include fungemia, skin lesions, and systemic involvement of multiple organs, especially the reticuloendothelial system. Early signs and symptoms are nonspecific and may include cough, fever, fatigue, weight loss, and lymphadenopathy. However, 70% of patients, with or without AIDS, develop cutaneous or subcutaneous papules, pustules, or rashes, which are often located on the face. From specimens of skin, blood, or tissue biopsies, the diagnosis can be established by microscopic observation of the yeast-like cells and positive cultures. The treatment usually entails a defined course of amphotericin B followed by itraconazole. Without treatment, the mortality has exceeded 90%.

Individuals with compromised host defenses are susceptible to infections by many of the thousands of saprobic molds that exist in nature and produce airborne spores. Such opportunistic mycoses occur less frequently than candidiasis, aspergillosis, and mucormycosis because the fungi are less pathogenic. Advances in medicine have resulted in growing numbers of severely compromised patients in whom normally nonpathogenic fungi may become opportunistic pathogens. Devastating systemic infections have been caused by species of Fusarium, Paecilomyces, Bipolaris, Curvularia, Alternaria, and many others. Some opportunists are geographically restricted. Another contributing factor is the increasing use of antifungal antibiotics, which has led to the selection of resistant fungal species and strains.

Perhaps the most dramatic example of an opportunistic fungal infection by a normally environmental fungus was the outbreak of central nervous system infection with Exserohilum rostratum. The outbreak began in September, 2012, and spread throughout the United States. It was caused by contaminated batches of methylprednisolone that were formulated for epidural injection. The vials of steroid were supplied by a single company in the northeast, and they were contaminated with the dematiaceous mold, E rostratum, a rare cause of phaeohyphomycosis. Nearly 13,000 patients received the injections, and depending upon their immune status and the random size of the fungal inocula, many developed chronic to acute meningitis, cerebral abscesses, and other manifestations. Over a period of months, infections were documented in more than 750 patients in 20 states. The CDC rapidly developed a PCR test to detect the fungus in culture-negative specimens. Patients were treated with amphotericin B, posaconazole, and/or isavuconazole, but 63 patients died.

1. Opportunistic mycoses are caused by globally distributed fungi that are either members of the human microbiota, such as Candida species, or environmental yeasts and molds. Among the categories of fungal infections, the incidence, severity, and mortality of systemic opportunistic mycoses are the highest.

2. Innate host defenses (eg, neutrophils, monocytes) provide crucial protection from systemic candidiasis, invasive aspergillosis, and mucormycosis. Patients at risk include those with hematologic dyscrasias (eg, leukemia, neutropenia) as well as those treated with immunosuppressive (eg, corticosteroid) or cytotoxic drugs.

3. Most patients with HIV/AIDS develop mucosal candidiasis (eg, thrush, esophagitis). Those with CD4 counts less than 100 cells/μL are at risk for cryptococcosis, Pneumocystis pneumonia, aspergillosis, penicilliosis, endemic mycoses, and other infections.

4. The diagnosis of invasive aspergillosis or candidiasis is often difficult because blood cultures are invariably negative in patients with aspergillosis, and less than 50% are positive in patients with systemic candidiasis.

5. Successful management of opportunistic mycoses involves early diagnosis, rapid administration of appropriate antifungal therapy, and control of the underlying condition or disease.

Opportunistic mycoses are increasing among immunocompromised patients, especially in patients with hematologic dyscrasias (eg, leukemia), hematopoietic stem cell recipients, and solid organ transplant patients, and others receiving cytotoxic and immunosuppressive drugs (eg, corticosteroids). For example, the incidence of systemic mycoses among patients with acute lymphocytic or myelogenous leukemia is 5–20%, and among allogeneic stem cell transplant patients, 5–10%. Many of these high-risk patients have depressed innate host defenses, such as a reduction in the number and/or functionality of circulating neutrophils and monocytes. In addition, AIDS patients are highly susceptible to a variety of systemic mycoses when their CD4 cell counts drop below 100 cells/μL.

The list of invasive opportunistic pathogens include species of Candida, Cryptococcus, Saccharomyces, and other yeasts; Aspergillus and other ascomycetous molds, such as Fusarium, Paecilomyces, and Scopulariopsis; dematiaceous molds (eg, species of Bipolaris, Phialophora, Cladosporium); and molds in the Order Mucorales (Rhizopus). Because it is usually difficult to establish a definitive diagnosis early in the course of infection, many high-risk patients are treated empirically or prophylactically with antifungal drugs. However, there is no universal consensus on the criteria for administering antifungal prophylaxis or the specific chemotherapy and regimen. Rather, most tertiary care hospitals have developed their own protocols for the administration of prophylactic antifungal chemotherapy to patients at high risk for invasive mycoses. Most hospitals will give oral fluconazole; others prescribe a short course of low-dose amphotericin B. Some of the criteria for administering antifungal prophylaxis to a patient with an underlying high-risk disease or condition are persistent fever that is unresponsive to antibacterial antibiotics, neutropenia lasting more than 7 days, the observation of new and unexplained pulmonary infiltrates on radiographic examinations, or progressive, unexplained organ failure.

With advances in comparative genomics, the prospects for newer approaches have improved for both sides of the fungus–host interaction. Sequence analyses of virulent and benign strains of pathogenic fungal species have identified many of the genes, processes, and pathways that are essential for virulence. This information has provided new strategies for combating fungal infections, such as blocking the attachment of fungal cells to host cells or inhibiting the in vivo transformation of dimorphic fungi. Studies of the mammalian genetic and immunologic responses to fungi are identifying signature cytokines and inflammatory biomarkers that characterize the innate and adaptive host responses to invading fungi.

Throughout life, the respiratory tract is exposed to airborne conidia and spores of many saprophytic fungi. These particles often possess potent surface antigens capable of stimulating and eliciting strong allergic reactions. These hypersensitivity responses do not require growth or even viability of the inducing fungus, though in some cases (allergic bronchopulmonary aspergillosis) both infection and allergy may occur simultaneously. Depending on the site of deposition of the allergen, a patient may exhibit rhinitis, bronchial asthma, alveolitis, or generalized pneumonitis. Atopic persons are more susceptible. The diagnosis and range of a patient’s hypersensitivity reactions can be determined by skin testing with fungal extracts. Management may entail avoidance of the offending allergen, treatment with corticosteroids, or attempts to desensitize patients.

Indoor air exposure to large numbers of fungal spores has led to the recognition of a condition described as “sick building syndrome” whereby moisture in construction materials, such as wood and fiberboard, allows contaminating molds to flourish. The production and contamination of the indoor air with large numbers of conidia have resulted in debilitating cases of systemic allergic or toxic reactions. Often the mold infestation is too extensive to eliminate with fungicides or filtration, and many such buildings have been demolished. The offending molds are usually noninfectious ascomycetes such as Stachybotrys, Cladosporium, Fusarium, and others.

Many fungi produce poisonous substances called mycotoxins that can cause acute or chronic intoxication and damage. The mycotoxins are secondary metabolites, and their effects are not dependent on fungal infection or viability. A variety of mycotoxins are produced by mushrooms (eg, Amanita species), and their ingestion results in a dose-related disease called mycetismus. Cooking has little effect on the potency of these toxins, which may cause severe or fatal damage to the liver and kidney. Other fungi produce mutagenic and carcinogenic compounds that can be extremely toxic for experimental animals. One of the most potent is aflatoxin, which is elaborated by A flavus and related molds and is a frequent contaminant of peanuts, corn, grains, and other foods.

A limited but increasing number of antibiotics can be used to treat mycotic infections. Most have one or more limitations, such as profound side effects, a narrow antifungal spectrum, poor penetration of certain tissues, and the selection of resistant fungi. Finding suitable fungal targets is difficult because fungi, like humans, are eukaryotes. Many of the cellular and molecular processes are similar, and there is often extensive homology among the genes and proteins.

The classes of currently available drugs include the polyenes (amphotericin B and nystatin), which bind to ergosterol in the cell membrane; flucytosine, a pyrimidine analog; the azoles and other inhibitors of ergosterol synthesis, such as the allylamines; the echinocandins, which inhibit the synthesis of cell wall β-glucan; and griseofulvin, which interferes with microtubule assembly. Currently under investigation are inhibitors of cell wall synthesis, such as nikkomycin and pradimicin, and sordarin, which inhibits elongation factor 2.

In recent years, the number of antifungal drugs has increased, and additional compounds are currently under evaluation in clinical trials. Table 45-7 provides an abridged summary of the available drugs. Many of the newer chemotherapeutics are variations of the azole class of fungistatic drugs, such as the triazoles (voriconazole and posaconazole). These drugs and the newer compounds were designed to improve the antifungal efficacy and pharmacokinetics, as well as to reduce the adverse side effects.

Amphotericin B

A. Description

The major polyene antibiotic is amphotericin B, a metabolite of streptomyces. Amphotericin B is the most effective drug for severe systemic mycoses. It has a broad spectrum, and the development of resistance is rare. The mechanism of action of the polyenes involves the formation of complexes with ergosterol in fungal cell membranes, resulting in membrane damage and leakage. Amphotericin B has greater affinity for ergosterol than cholesterol, the predominant sterol in mammalian cell membranes. Packaging of amphotericin B in liposomes and lipoidal emulsions has shown superb efficacy and excellent results in clinical studies. These formulations are currently available and may replace the conventional preparation. The lipid preparations are less toxic and permit higher concentrations of amphotericin B to be used.

B. Mechanism of Action

Amphotericin B is given intravenously as micelles with sodium deoxycholate dissolved in a dextrose solution. Though the drug is widely distributed in tissues, it penetrates poorly to the cerebrospinal fluid. Amphotericin B firmly binds to ergosterol in the cell membrane. This interaction alters the membrane fluidity and perhaps produces pores in the membrane through which ions and small molecules are lost. Unlike most other antifungals, amphotericin B is cidal. Mammalian cells lack ergosterol and are relatively resistant to these actions. Amphotericin B binds weakly to the cholesterol in mammalian membranes, and this interaction may explain its toxicity. At low levels, amphotericin B has an immunostimulatory effect.

C. Indications

Amphotericin B has a broad spectrum with demonstrated efficacy against most of the major systemic mycoses, including coccidioidomycosis, blastomycosis, histoplasmosis, sporotrichosis, cryptococcosis, aspergillosis, mucormycosis, and candidiasis. The response to amphotericin B is influenced by the dose and rate of administration, the site of the mycotic infection, the immune status of the patient, and the inherent susceptibility of the pathogen. Penetration of the joints and the central nervous system is poor, and intrathecal or intra-articular administration is recommended for some infections. Amphotericin B is used in combination with flucytosine to treat cryptococcosis. Some fungi, such as P boydii and A terreus, do not respond well to treatment with amphotericin B.

D. Side Effects

All patients have adverse reactions to amphotericin B, though these are greatly diminished with the new lipid preparations. Acute reactions that usually accompany the intravenous administration of amphotericin B include fever, chills, dyspnea, and hypotension. These effects can usually be alleviated by prior or concomitant administration of hydrocortisone or acetaminophen. Tolerance to the acute side effects develops during therapy.

Chronic side effects are usually the result of nephrotoxicity. Azotemia almost always occurs with amphotericin B therapy, and serum creatinine and ion levels must be closely monitored. Hypokalemia, anemia, renal tubular acidosis, headache, nausea, and vomiting are also frequently observed. While some of the nephrotoxicity is reversible, permanent reduction in glomerular and renal tubular function does occur. This damage can be correlated with the total dose of amphotericin B given. Toxicity is greatly diminished with the lipid formulations of amphotericin B (ie, Abelcet, Amphotec, and AmBisome).

Flucytosine

A. Description

Flucytosine (5-fluorocytosine) is a fluorinated derivative of cytosine. It is an oral antifungal compound used primarily in conjunction with amphotericin B to treat cryptococcosis or candidiasis. It is effective also against many dematiaceous fungal infections. It penetrates well into all tissues, including cerebrospinal fluid.

B. Mechanism of Action

Flucytosine is actively transported into fungal cells by a permease. It is converted by the fungal enzyme cytosine deaminase to 5-fluorouracil and incorporated into 5-fluorodexoyuridylic acid monophosphate, which interferes with the activity of thymidylate synthetase and DNA synthesis. Mammalian cells lack cytosine deaminase and are therefore protected from the toxic effects of fluorouracil. Unfortunately, resistant mutants emerge rapidly, limiting the utility of flucytosine.

C. Indications

Flucytosine is used mainly in conjunction with amphotericin B for treatment of cryptococcosis and candidiasis. In vitro, it acts synergistically with amphotericin B against these organisms, and clinical trials suggest a beneficial effect of the combination, particularly in cryptococcal meningitis. The combination has also been shown to delay or limit the emergence of flucytosine-resistant mutants. By itself, flucytosine is effective against chromoblastomycosis and other dematiaceous fungal infections.

D. Side Effects

While flucytosine itself probably has little toxicity for mammalian cells and is relatively well tolerated, its conversion to fluorouracil results in a highly toxic compound that is probably responsible for the major side effects. Prolonged administration of flucytosine results in bone marrow suppression, hair loss, and abnormal liver function. The conversion of flucytosine to fluorouracil by enteric bacteria may cause colitis. Patients with AIDS may be more susceptible to bone marrow suppression by flucytosine, and serum levels should be closely monitored.

Azoles

A. Description

The antifungal imidazoles (eg, ketoconazole) and the triazoles (fluconazole, voriconazole, and itraconazole) are oral drugs used to treat a wide range of systemic and localized fungal infections (Figure 45-29). The indications for their use are still being evaluated, but they have already supplanted amphotericin B in many less severe mycoses because they can be administered orally and are less toxic. Other imidazoles—miconazole and clotrimazole—are used topically and are discussed below.

B. Mechanism of Action

The azoles interfere with the synthesis of ergosterol. They block the cytochrome P450-dependent 14α-demethylation of lanosterol, which is a precursor of ergosterol in fungi and cholesterol in mammalian cells. However, the fungal cytochrome P450s are approximately 100–1000 times more sensitive to the azoles than mammalian systems. The various azoles are designed to improve their efficacy, availability, and pharmacokinetics and reduce their side effects. These are fungistatic drugs.

C. Indications

The indications for the use of antifungal azoles will broaden as the results of long-term studies—as well as new azoles—become available. Accepted indications for the use of antifungal azoles are listed below.

Ketoconazole is useful in the treatment of chronic mucocutaneous candidiasis, dermatophytosis, and nonmeningeal blastomycosis, coccidioidomycosis, paracoccidioidomycosis, and histoplasmosis. Of the various azoles, fluconazole offers the best penetration of the central nervous system. It is used as maintenance therapy for cryptococcal and coccidioidal meningitis. Oropharyngeal candidiasis in AIDS patients and candidemia in immunocompetent patients can also be treated with fluconazole. Itraconazole is now the agent of first choice for histoplasmosis and blastomycosis as well as for certain cases of coccidioidomycosis, paracoccidioidomycosis, and aspergillosis. It has also been shown to be effective in the treatment of chromomycosis and onychomycosis due to dermatophytes and other molds. Voriconazole, which can be given orally or intravenously, exhibits a broad spectrum of activity against many molds and yeasts, especially aspergillosis, fusariosis, pseudallescheriasis, and other less common systemic pathogens. The newest triazole is posaconazole (Figure 45-30A), which has a wide spectrum and demonstrated efficacy against fluconazole-resistant Candida species, aspergillosis, mucormycosis, and other opportunistic invasive molds. It is also well tolerated.

D. Side Effects

The adverse effects of the azoles are primarily related to their ability to inhibit mammalian cytochrome P450 enzymes. Ketoconazole is the most toxic, and therapeutic doses may inhibit the synthesis of testosterone and cortisol, which may cause a variety of reversible effects such as gynecomastia, decreased libido, impotence, menstrual irregularity, and occasionally adrenal insufficiency. Fluconazole and itraconazole at recommended therapeutic doses do not cause significant impairment of mammalian steroidogenesis. All the antifungal azoles can cause both asymptomatic elevations in liver function tests and rare cases of hepatitis. Voriconazole causes reversible visual impairment in about 30% of patients.

Since antifungal azoles interact with P450 enzymes that are also responsible for drug metabolism, some important drug interactions can occur. Increased antifungal azole concentrations can be seen when isoniazid, phenytoin, or rifampin is used. Antifungal azole therapy can also lead to higher than expected serum levels of cyclosporine, phenytoin, oral hypoglycemics, anticoagulants, digoxin, and probably many others. Serum monitoring of both drugs may be necessary to achieve a proper therapeutic range.

Echinocandins

The echinocandins are a new class of antifungal agents that perturb the synthesis of the pervasive cell wall polysaccharide β-glucan by inhibiting 1,3-β-glucan synthase and disrupting cell wall integrity. The first licensed drug, caspofungin, has shown efficacy against invasive aspergillosis and systemic candidiasis due to a wide range of Candida species (see Figure 45-30B). This intravenous agent may be especially indicated for refractory aspergillosis. Caspofungin is well tolerated.

Similar to caspofungin, two newly approved echinocandins, micafungin and anidulafungin, also inhibit the synthesis of β-glucan and have a similar spectrum of activity against species of Candida and Aspergillus, as well as several other molds. Micafungin (see Figure 45-30C) and anidulafungin were recently licensed for the treatment of esophageal candidiasis and for the antifungal prophylaxis of hematopoietic stem cell transplant patients. Both seem to have better pharmacokinetics and in vivo stability than caspofungin. Clinical studies suggest that they will be useful in the treatment of mucosal and systemic candidiasis, refractory invasive aspergillosis, and in combination with amphotericin B or some of the newer triazoles.

Griseofulvin

Griseofulvin is an orally administered antibiotic derived from a species of penicillium. It is used to treat dermatophytoses and must be given for long periods. Griseofulvin is poorly absorbed and concentrated in the stratum corneum, where it inhibits hyphal growth. It has no effect on other fungi.

After oral administration, griseofulvin is distributed throughout the body but accumulates in the keratinized tissues. Within the fungus, griseofulvin interacts with microtubules and disrupts mitotic spindle function, resulting in inhibition of growth. Only actively growing hyphae are affected. Griseofulvin is clinically useful for the treatment of dermatophyte infections of the skin, hair, and nails. Oral therapy for weeks to months is usually required. Griseofulvin is generally well tolerated. The most common side effect is headache, which usually resolves without discontinuation of the drug. Less frequently observed side effects are gastrointestinal disturbances, drowsiness, and hepatotoxicity.

Terbinafine

Terbinafine is an allylamine drug; it blocks ergosterol synthesis by inhibiting squalene epoxidase (see Figure 45-30D). Terbinafine is given orally to treat dermatophyte infections. It has proved quite effective in treating nail infections as well as other dermatophytoses. Side effects are not common but include gastrointestinal distress, headaches, skin reactions, and loss of sense of taste. For the long-term treatment of tinea unguium, terbinafine—as well as itraconazole and fluconazole—may be given intermittently, using a pulse treatment protocol.

Nystatin

Nystatin is a polyene antibiotic, structurally related to amphotericin B and having a similar mode of action. It can be used to treat local candidal infections of the mouth and vagina. Nystatin may also suppress subclinical esophageal candidiasis and gastrointestinal overgrowth of candida. No systemic absorption occurs, and there are no side effects. However, nystatin is too toxic for parenteral administration.

Clotrimazole, Miconazole, and Other Azoles

A variety of antifungal azoles too toxic for systemic use are available for topical administration. Clotrimazole and miconazole are available in several formulations. Econazole, butoconazole, tioconazole, and terconazole are also available. All these compounds seem to have comparable efficacy.

Topical azoles have a broad spectrum of activity. Tinea pedis, tinea corporis, tinea cruris, tinea versicolor, and cutaneous candidiasis respond well to local application of creams or powders. Vulvovaginal candidiasis can be treated with vaginal suppositories or creams. Clotrimazole is also available as an oral troche for treatment of oral and esophageal thrush in immunocompetent patients. For treating nail infections, which are often intractable, the new topical azole, luliconazole, has shown dramatic effectiveness.

Other Topical Antifungal Agents

Tolnaftate and naftifine are topical antifungal agents used in the treatment of many dermatophyte infections and tinea versicolor. Formulations available include creams, powders, and sprays. Undecylenic acid is available in several formulations for the treatment of tinea pedis and tinea cruris. Although it is effective and well tolerated, antifungal azoles, naftifine, and tolnaftate are more effective. Haloprogin and ciclopirox are other topical agents commonly used in dermatophyte infections.

1. Effective therapy relies on rapid identification of the fungus, administration of the appropriate drug, and management of any underlying disease or condition.

2. The fungicidal polyene, amphotericin B, has a broad spectrum, and resistance is rare. Renal toxicity and other side effects must be monitored and managed.

3. Compared to polyenes, the azoles are fungistatic and have a limited range of antifungal activity, but they manifest less severe toxicities. Voriconazole and posaconazole exhibit broader antifungal spectra than ketoconazole, itraconazole, and fluconazole.

4. The echinocandins—caspofungin, micafungin, and anidulafungin—are fungistatic and proven effective against species of Candida.