Chapter 43: Pathogenesis and Diagnosis of Fungal Infections

Fungal infections are most often acquired from the external environment. One common mechanism of infection is by the inhalation of infectious conidia generated from environmental molds. Some of these molds are ubiquitous, whereas others are restricted to specific endemic areas and geographic regions whose climate favors their growth. Many fungi produce disease only after they are accidentally injected past the skin/mucosal barrier, especially in immunocompromised patients. Other pathogenic fungi have more sophisticated means of tissue penetration and invasion. In the case of systemic candidiasis, infection can result from systemic invasion by a fungal species that is typically an endogenous member of the resident microbiota, such as Candida albicans (Figure 43–1).

PATHOGENESIS

Compared with bacterial, viral, and parasitic disease, less is known about the pathogenic mechanisms and virulence factors involved in fungal infections. Analogies to bacterial diseases come the closest because of similarities in microbial adherence to mucosal surfaces, invasion into deeper tissue layers, production of extracellular compounds, and interaction with phagocytes (Figure 43–2). In general, the principles discussed in Chapter 22 also apply to fungal infections. Most fungi are opportunists, causing serious disease only in individuals with impaired host defense systems. Only a few fungi are able to cause disease in previously healthy persons.

Adherence

Several fungal species, particularly the yeasts, are able to colonize the mucosal surfaces of the gastrointestinal and female genital tracts. It has been shown experimentally that the ability to adhere to buccal or vaginal epithelial cells is associated with colonization and virulence. Within the genus Candida, the species that best adhere to epithelial cells are those most frequently isolated from clinical infections. Adherence usually requires a surface adhesin on the fungus and a receptor on the epithelial cell. In the case of C albicans, mannoprotein components extending from the cell wall have been implicated as specific adhesins, interacting with host fibronectin and other components of the extracellular matrix. Other fungal/host binding mediators have been identified, and this process can help to explain why certain tissues are targeted by specific fungal pathogens. For example, the neuropathogen Cryptococcus neoformans displays a unique interaction with proteins on the endothelium of the brain microvasculature, perhaps explaining how this species specifically invades the central nervous system.

Invasion

Passing an initial surface barrier—skin, mucous membrane, or respiratory epithelium—is an important step for most successful pathogens. Some fungi are introduced through mechanical breaks. For example, Sporothrix schenckii infection typically follows a thorn prick trauma to the skin. Fungi that initially infect the lung must produce conidia small enough to be inhaled past the upper airway defenses. For example, arthroconidia of Coccidioides immitis (2-6 μm) can remain suspended in air for a considerable period of time and can reach the terminal bronchioles to initiate pulmonary coccidioidomycosis.

Triggered by temperature and possibly other cues, dimorphic fungi from the environment undergo a metabolic shift similar to the heat shock response, completely changing their morphology to a more invasive form. Invasion directly across mucosal barriers by the endogenous yeast C albicans is similarly associated with a morphologic change, the formation of hyphae. For this species, the ability to transition between yeast-like and hyphal forms allows it to effectively penetrate tissue, form adherent biofilms, and disseminate to distant sites. Extracellular enzymes (eg, proteases, elastases) are associated with the advancing edge of the hyphal form of Candida species, as well as with the invasive forms of many of the dimorphic and other pathogenic fungi.

Injury

There are many mechanisms of tissue injury during fungal infection. Although many fungi produce secondary metabolites and mycotoxins in the environment, most of these extracellular toxins do not appear to be directly related to pathogenesis in human infections. Cell surface components contributing to host damage, analogous to the endotoxin of gram-negative bacteria, are lacking in most fungi. Moreover, only the most immunocompromised patients appear to have extensive injury due to direct fungal destruction of the surrounding tissue, such as neutropenic patients with invasive mold infections. In contrast, the injury experienced by the host during most fungal infections seems to be due primarily to the immune response against the infecting microorganism. As the immune system attempts to clear the fungal pathogens, there is some degree of collateral damage to the host.

IMMUNITY

Innate Immunity

Healthy persons have effective innate immunity to most fungal infections, especially the opportunistic molds. This resistance is mediated by the professional phagocytes (neutrophils, macrophages, and dendritic cells), the complement system, and pattern recognition receptors. Important receptors recognizing fungal elements include a lectin-like structure on phagocytes (dectin-1) that binds glucan, and toll-like receptors (TLR2, TLR4). In most instances, neutrophils and alveolar macrophages are able to kill the conidia of fungi if they reach the tissues.

Fungal species that cause human infections have developed strategies to avoid immune recognition and to thwart various aspects of immune-mediated clearance. The polysaccharide capsule of C neoformans shields immunogenic epitopes on the cell surface from being sensed by pattern recognition receptors and complement proteins. Moreover, capsule material secreted by the cryptococcal cell specifically inhibits the function of many immune cells. Similarly, Candida albicans is able to bind complement components in a way that interferes with phagocytosis. As the thermally dimorphic fungi convert to the pathogenic yeast-like state, they too become more resistant to killing by phagocytes than fungal opportunists because of changes in surface structures subject to pattern recognition.

In addition to preventing immune recognition, many fungal pathogens are also able to survive once sensed and engulfed by immune cells. The yeast-to-hyphal transition by C albicans favors its escape from phagocytic immune cells. As the hyphae of the thermally dimorphic fungus C immitis convert to the spherule (tissue) phase, they also become resistant to phagocytic killing because of their size and surface characteristics. Some fungi produce substances such as melanin, which interfere with oxidative killing by phagocytes. The yeast forms of Histoplasma capsulatum and C neoformans are adapted to live and multiply within macrophages by interfering with lysosomal killing mechanisms.

Adaptive Immune Response

A recurrent theme with fungal infections is the importance of an intact immune response in preventing infection and progression of disease. Most fungi are incapable of producing even a mild infection in immunocompetent individuals. The small number of species that are able to cause clinically apparent infection are usually cleared from the host, most often through a combination of the innate activity of neutrophils and through the development of an adaptive, T_H1-mediated immune response. Progressive, debilitating, or life-threatening fungal infections are commonly associated with depressed or absent cellular immune responses.

Humoral Immunity

Antifungal antibodies can be detected at some time during the course of almost all fungal infections, but the appearance of antibodies does not necessarily correlate with resistance. In coccidioidomycosis, for example, high titers of C immitis-specific antibodies are associated with dissemination and a worsening clinical course; antibody titers decrease as the infection is cured. In contrast, antibodies directed against the C neoformans capsule may actually contribute to the cell-mediated clearance of this encapsulated yeast from the site of infection. Antibody may also play a role in control of C albicans infections by enhancing fungus–phagocyte interactions.

Cellular Immunity

Considerable clinical and experimental evidence points toward the importance of cellular immunity in the resolution of fungal infections. Most patients with invasive mycoses have neutropenia, defects in neutrophil function, or depressed T_H1 immune responses. These can result from factors such as steroid treatment, leukemia/lymphoma, transplantation, and AIDS.

A basic schema for fungal-host interactions is illustrated in Figure 43–2. When hyphae or yeast cells of the fungus reach deep tissue sites, they are either killed by neutrophils or resist destruction by one of the antiphagocytic mechanisms described earlier. Surviving cells continue to grow slowly within the host in their tissue-adapted fungal forms (spherules for C immitis, hyphae for A fumigatus, intracellular yeasts for C neoformans and H capsulatum). The growth of these invasive forms may be slowed or killed by phagocytes such as neutrophils and macrophages. In healthy persons, the extent of infection is minimal, and any symptoms are caused by the inflammatory response. Fungal persistence and spread is most common in people with defective immunity.

Everything awaits the specific adaptive immune response to the invader. In fungal infections, antigen presenting cells such as dendritic cells and macrophages help to activate adaptive immune response, including antifungal antibody production and T_H1-mediated immunity. Defects that disturb this cycle lead to progressive disease. To the extent that they are known, the specifics of these reactions are discussed in the following chapters.

LABORATORY DIAGNOSIS

Direct Examination

Fungi can often be identified by directly observing their distinctive morphologic features on direct microscopic examination of infected pus, fluids, or tissues. The simplest method is to mix a clinical specimen, such as skin scrapings, with a 10% solution of potassium hydroxide (KOH) on a microscope slide under a coverslip. The strong alkali digests the tissue elements (epithelial cells, leukocytes, debris), but not the rigid cell walls of either yeasts or molds. After digestion of the material, the fungi can be observed under the light microscope with or without staining (Figure 43–3). Direct examinations can be aided by the use of calcofluor white, a dye that binds to polysaccharides in cellulose and chitin. Under ultraviolet light, calcofluor white fluoresces, enhancing detection of fungi in fluids or tissue sections. A few yeasts including C albicans can be visualized using the Gram stain (gram positive).

Histopathologic examination of tissue biopsy specimens is widely used to diagnose fungal infections and shows the relation of the organism to tissue elements and responses (blood vessels, phagocytes, granulomatous reactions). Most fungi can be seen in sections stained with the basic hematoxylin and eosin (H&E) method used in histology laboratories (Figure 43–4). Specialized staining procedures such as silver impregnation methods are frequently used because they stain almost all fungi strongly, but only a few tissue components (Figure 43–5). The pathologist should be alerted to the possibility of fungal infection when tissues are submitted, because fungal-specific stains are used routinely.

Culture

Fungi can be grown by methods similar to those used to isolate bacteria. The growth of many fungal species occurs readily on enriched bacteriologic media commonly used in clinical laboratories (eg, blood agar and chocolate agar). Many fungal cultures, however, require days to weeks of incubation for initial growth; bacteria present in the specimen grow more rapidly and may interfere with isolation of a slow-growing fungus. Therefore, the culture procedures of diagnostic mycology are designed to favor the growth of fungi over bacteria and to allow incubation to continue for a sufficient time to isolate slower growing strains.

The most commonly used medium for cultivating fungi is Sabouraud’s agar, which contains only glucose and peptones as nutrients. Its pH is 5.6, which is optimal for growth of dermatophytes and satisfactory for growth of other fungi. Most bacteria fail to grow, or grow poorly, on Sabouraud’s agar. A wide variety of other media are in use, many of which use either Sabouraud’s or brain-heart infusion as their base.

Blood agar or other types of enriched bacteriologic media are used when pure cultures would be expected, such as subculturing from blood cultures bottles in which yeast species are observed. These media can be made more fungal-selective by the addition of antibacterial antibiotics such as chloramphenicol and gentamicin. Cycloheximide, an antimicrobial that inhibits some saprophytic fungi, is sometimes added to Sabouraud’s agar to prevent overgrowth of contaminating molds from the environment, particularly for skin cultures. Media containing these selective agents cannot be relied on exclusively because they can interfere with growth of some pathogenic fungi or because the “contaminant” may be producing an opportunistic infection. For example, cycloheximide inhibits C neoformans, and chloramphenicol may inhibit the yeast forms of some dimorphic fungi. In contrast to most pathogenic bacteria, many fungi grow best at 25°C to 30°C, and temperatures in this range are used for primary isolation. Paired cultures incubated at 30°C and 35°C may be used to demonstrate dimorphism.

Once a fungus is isolated, identification procedures depend on whether the growing fungus is a yeast or a mold. Yeasts are identified by biochemical tests analogous to those used for bacteria, including some that are identical (eg, urease production). The observation of specific fungal structures such as pseudohyphae is also diagnostically useful among the yeasts.

Molds are most often identified by the morphology of their conidia and conidiophores. Other features such as the size, texture, and color of the colonies help characterize molds, but without demonstrating conidiation they are not sufficient for identification. The ease and speed with which various fungi produce conidia vary greatly. Minimal nutrition, moisture, good aeration, and ambient temperature favor development of conidia.

Microscopic fungal morphology is usually demonstrated by methods that allow in situ microscopic observation of the fragile asexual conidia and their shape and arrangement. Morphology may also be examined in fragments of growth teased free of a mold and examined in preparations containing a dye called lactophenol cotton blue. The dye stains the hyphae, conidia, and spores. Conidium production may not occur for days or weeks after the initial growth of the mold. It is similar to waiting for flowers to bloom, and it can be frustrating when the result has immediate clinical application.

It is desirable, but not always possible, to demonstrate the yeast and mold phases with dimorphic fungi. In some cases, this result can be achieved with parallel cultures at 30°C and 35°C. The tissue form of C immitis is not readily produced in vitro. Demonstration of dimorphism has become less important with the development of specific DNA probes for the major systemic pathogens. These probes are rapid and can be applied directly to the mycelial growth of the readily grown mold phases of these fungi.

Antigen and Antibody Detection

Serum antibodies directed against a variety of fungal antigens can be detected in patients infected with those agents. These tests are rarely useful for diagnosing acute infections, except for C immitis in which antibody levels often correlate with extent of infection. Immunoassays to detect fungal antigens have been pursued for some time. The major targets are mannans, mannoproteins, glucan, chitin, or some other structure unique to the fungal pathogen(s). Two of these tests are extremely sensitive and specific for systemic infection: (1) the C neoformans capsular polysaccharide antigen test (cryptococcal antigen) and (2) the H capsulatum surface antigen test (Histoplasma antigen). Serum antigen tests for Aspergillus species (galactomannan) and other fungal pathogens (β-D-glucan) are less sensitive for diagnosing infection but can be useful in some cases.