Chapter 46: The Opportunistic Fungi: Candida, Aspergillus, the zygomycetes, and Pneumocystis

The “opportunistic fungi” are usually found as members of the resident human microbiota or as saprophytes in the environment. With the breakdown of host defenses, they can cause infections ranging from skin/mucous membrane involvement to life-threatening, systemic disease. The most common opportunistic infections are caused by two species: the yeast Candida albicans, a common inhabitant of the gastrointestinal and genital microbiota; and the mold Aspergillus fumigatus which is widespread in the environment. Pneumocystis, a frequent cause of pneumonia in AIDS patients, is an unusual fungus that used to be considered a parasite on morphologic grounds. However, it too is a frequent colonizer of the human respiratory tract. The diseases caused by these opportunistic fungi are summarized in Table 46–1.

Candida species grow as 4 to 6 μm, budding, round or oval yeast-like cells (Figure 46–1) under most conditions and at most temperatures. Under certain growth conditions, including those encountered during infection, certain pathogenic Candida species can also form hyphae. Of the over 150 Candida species, fewer than 10 cause human infections. Particular attention is given to the differentiation of C albicans from other species, because it is by far the most common cause of disease. For serious infections, identifying Candida isolates to the species level is important for prognostic and treatment decisions (Figure 46–1).

Most Candida species grow rapidly on Sabouraud’s agar and on enriched bacteriologic media such as blood agar. Smooth, white, 2 to 4 mm colonies resembling those of staphylococci are produced on blood agar after overnight incubation. Aeration of cultures favors their isolation. The primary identification procedure involves presumptive differentiation of C albicans from the other Candida species with the germ tube test. Germ tube–negative strains may be further identified biochemically or reported as “yeast not C albicans,” depending on their apparent clinical significance.

Candida albicans

MYCOLOGY

Candida albicans grows in multiple morphologic forms, most often as a budding yeast. Candida albicans is also able to form hyphae triggered by changes in conditions such as temperature, pH, and available nutrients. When observed in their initial stages of germination from the yeast cell, these nascent hyphae resemble sprouts and are called “germ tubes” (Figure 46–2A). Other elongated forms with restrictions at regular intervals are called pseudohyphae because they lack the parallel walls and septation of true hyphae. There is evidence that these three forms have distinct stimuli and genetic regulation, making C albicans a polymorphic fungus. Unless otherwise specified, the term hyphae is used here to encompass both the true hyphal and pseudohyphal forms. Being able to recognize these different fungal morphologies can help clinicians and laboratory personnel to rapidly distinguish C albicans from other, similar yeast species in clinical specimens.

The C albicans cell wall is made up of a mixture of the polysaccharides mannan, glucan, and chitin alone or in complexes with protein. A fibrillar outer layer extending to the surface contains several glycoproteins and complexes of mannan with protein called mannoproteins. The exact composition of the cell wall and surface components varies under different growth conditions.

CANDIDIASIS

EPIDEMIOLOGY

Candida albicans is present in the microbiota of 30% to 50% of healthy persons, especially common in the oropharyngeal, gastrointestinal, and female genital tracts. Infections are endogenous, with most symptoms arising from infections with one’s own resident species. However, transmission and new acquisition of Candida colonization can occur by direct mucosal contact with others (eg, through sexual intercourse). Similarly, in nosocomial C albicans infections, the strains involved are usually derived from the patient’s own flora rather than from cross-infection. Invasive procedures and indwelling devices may provide the portal of entry, and the number of Candida present on the skin and mucosal surfaces may be enhanced by the use of antibacterial agents.

PATHOGENESIS

Because C albicans is regularly present on mucosal surfaces; disease implies a change in the organism, the host, or both. The ability of this microorganism to change between the yeast and hyphal forms is strongly associated with its pathogenic potential, and these different morphological forms are likely required for different phases of candidiasis. In histologic preparations, hyphae are seen during Candida invasion, either superficially into the mucosa or within deep tissues. However, dissemination in the bloodstream is likely enhanced during the yeast growth phase. Therefore, it is the plasticity between the two forms, rather than one morphology or the other, that results in the ability of C albicans to so effectively colonize and infect the host. The yeast-hyphal switch can be controlled in vitro by the manipulation of a wide variety of environmental conditions (serum, pH, temperature, amino acids). Various sensors and signaling pathways for morphogenesis have been described including one in which C albicans induces its own morphological change by directly altering the local pH.

Candida albicans hyphae have the capacity to form strong attachments to human epithelial cells. One mediator of this binding is a surface hyphal wall protein (Hwp1), which is found only on the surface of germ tubes and hyphae. Other surface mannoproteins that have similarities to vertebrate integrins may also mediate binding to components of the extracellular matrix (ECM), such as fibronectin, collagen, and laminin. Hyphae also secrete proteinases and phospholipases that are able to digest epithelial cells and probably facilitate invasion (Figures 46–3 and 46–4A and B). One family of hyphal enzymes, the secreted aspartic proteinases (Saps), is able to digest keratin and collagen, which would facilitate deep tissue invasion. The pattern of Sap production may be tissue-specific with individual isolates producing distinct sets of Saps depending whether they are invading gastrointestinal or vaginal epithelium.

One of the most important pathogenic features of C albicans is its ability to form biofilms. These complex structures include yeast and hyphal forms of the fungus along with host-derived proteins. Once formed, the biofilm strongly adheres to components of the ECM as well as to plastics. Neither host immune cells nor antifungal agents are able to penetrate Candida biofilms well, making this structure an important source of microbial persistence during infection. In a very practical sense, fungal biofilms that develop on prosthetic surfaces (eg, intravenous catheters, prosthetic joints, prosthetic heart valves) are almost impossible to sterilize without device removal.

Candida albicans has various mechanisms which facilitate evasion of innate immune mechanisms. These include the masking of surface structures from Toll-like receptors (TLRs) and the accelerated degradation of surface complement C3b. The latter can be accomplished by binding of serum factor H or by secretion of its own protease. Hyphae also have surface proteins that resemble the complement receptors (CR2, CR3) on phagocytes. This seems likely to confuse the phagocyte’s ability to recognize C3b bound to the candidal surface. Enhanced production of these receptors under various conditions, such as elevated glucose concentration, is associated with resistance to phagocytosis by neutrophils. If phagocytosed, hyphal growth interferes with lysosomal fusion and leads to the death of macrophages.

Many factors predispose to both local and invasive Candida infections. Antibacterial therapy reduces microbial competition on mucosal surfaces and increases the relative abundance of C albicans within the microbiota. Alterations in innate immunity (eg, leukopenia or corticosteroid therapy) or adaptive immunity (eg, AIDS) are important contributing factors to systemic and mucosal candidiasis. Additionally, anatomic disruptions of the skin and mucosa may enhance the invasion process by exposing Candida binding sites in the ECM and by allowing direct access to deeper tissues. Biofilm formation on the plastics used in medical devices also contributes to fungal persistence in this host. Diabetes mellitus predisposes to C albicans infection, possibly due to greater production of surface mannoproteins in the presence of high glucose concentrations.

IMMUNITY

Both humoral immunity and cell-mediated immunity (CMI) are involved in defense against Candida infections. Neutrophils are the primary first-line defense, and defects in neutrophil number or function are among the most common immune correlates of serious C albicans infections. Yeast forms of C albicans are readily phagocytosed and killed by these innate immune cells, especially when opsonized by antibody and complement. In the absence of specific antibody, the process is less efficient, but a naturally occurring antimannan IgG is able to activate the classical complement pathway and facilitate the alternate pathway. Hyphal forms may be too large to be ingested by polymorphonuclear neutrophils (PMNs), but these immune cells can still kill the fungi by attaching to the hyphae and discharging metabolites generated by the oxidative burst.

Many immunodeficiency syndromes involving T-lymphocyte dysfunction result in severe mucocutaneous candidiasis, emphasizing the importance of this arm of the immune system in defense against Candida infections. For example, patients with AIDS develop frequent episodes of oral and esophageal candidiasis, suggesting that protection against even superficial infections involves CD4-mediated immune responses. As with other fungi, cytokine activation of macrophages and other immune effector cells enhances their ability to kill C albicans. Therefore, a favorable outcome of infection appears to require the proper balance between T_H1- and T_H2-mediated cytokine responses, and this balance is lost in the absence of intact CD4 cell function. Typically, the cytokines associated with T_H1-mediated immunity (interleukin-2 [IL-2], IL-12, interferon-γ, tumor necrosis factor-α) are correlated with enhanced resistance against Candida infection. In contrast, T_H2 responses (IL-4, IL-6, and IL-10) are associated with chronic disease. In animal studies, the fungus appears to play an active role in regulating this host response. For example, Candida cell wall mannan has been shown to play an immunoregulatory function by downregulating protective CMI responses.

CANDIDIASIS: CLINICAL ASPECTS

MANIFESTATIONS

Superficial invasion of the mucous membranes by C albicans produces a white, cheesy plaque that is loosely adherent to the mucosal surface. Oral lesions, called thrush, occur on the tongue, palate, and other mucosal surfaces as ragged white patches (Figure 46–5). Scraping the fungal plaque with a tongue blade will reveal varying degrees of underlying mucosal invasion and inflammation, helping to differentiate this infectious process from other causes of superficial oral films. A similar infection in the vagina, vaginal candidiasis, produces a thick, curd-like discharge and itching of the vulva. Although many women have at least one episode of vaginal candidiasis in a lifetime, a small proportion suffers chronic, recurrent infections. No general or specific immune defect has yet been linked to this syndrome.

Superficial C albicans infections also occur in skinfolds and other areas in which wet, macerated skin surfaces are opposed. For example, one type of diaper rash is caused by C albicans (Figure 46–6A). Other infections of the skinfolds and appendages occur in association with recurrent immersion in water (eg, dishwashers). The initial lesions are erythematous papules or confluent areas of erythema, tenderness, and skin fissures. Infection usually remains confined to the chronically irritated area with adjacent “satellite” lesions.

Rarely, chronic and relapsing Candida infections occur in patients with a specific defect in T_H1 immune defenses. This condition, known as chronic mucocutaneous candidiasis (CMC), manifests with recurrent severe skin and mucosal lesions. With time, patients with CMC experience considerable skin disfigurement. Although lesions may become extensive, they usually do not result in fungal dissemination.

In contrast, most people live their entire lives in constant contact with C albicans but without developing symptomatic infections. This observation largely reflects the ability of normal hosts to effectively control the growth of this fungus. However, Candida colonization without associated symptomatic inflammation may also represent a clinical example of immunologic tolerance—to be able to be exposed to a microorganism without developing an excessive immune response. Therefore, it is possible that a subset of patients with frequent episodes of symptomatic candidiasis (eg, recurrent vulvovaginal candidiasis) actually has difficulty suppressing an overexuberant immune reaction to resident fungi, as opposed to trouble controlling microbial growth.

Inflammatory patches similar to those in thrush may also develop in the esophagus and upper GI tract. These lesions occur most frequently in immunocompromised patients and are characterized by painful swallowing or substernal chest pain. Extensive ulcerations, deformity, and occasionally perforation of the esophagus may ensue.

In addition to infection of mucosal surfaces, Candida infection often involves the urinary tract. Ascending infections may produce cystitis, pyelonephritis, renal abscesses, or expanding fungus ball lesions in the renal pelvis. Patients with urinary catheters, kidney transplants, or other types of chronic urinary devices are at particular risk for these infections.

Disseminated infections are particularly serious forms of candidiasis. The fungus often gains access to the bloodstream through skin lesions (eg, burns, intravascular catheters), disruption of the GI tract (eg, intestinal perforations, abdominal surgery), or prosthetic devices colonized with Candida biofilms. Once in the bloodstream, Candida species can infect many organs, including the kidneys, brain, and heart valves; however, symptoms are generally not sufficiently characteristic to suggest C albicans over the bacterial pathogens. Importantly, disseminated candidiasis frequently involves the eye. Candida endophthalmitis has the characteristic funduscopic appearance of a white cotton ball expanding on the retina or floating free in the vitreous humor. Endophthalmitis and infections of other eye structures can lead to blindness, and ocular complications must be considered in every case of disseminated candidiasis.

DIAGNOSIS

Exudate or epithelial scrapings examined by KOH preparations (Figure 46–6B) demonstrate abundant budding yeast cells; if associated hyphae are present, the infection is almost certainly caused by C albicans. C albicans is readily isolated in culture from clinical specimens including blood. Cultures from specimens, such as sputum, run the risk of contamination from yeasts present in the normal flora.

Deep organ involvement is difficult to prove without a direct aspirate or biopsy. However, Candida species often grow in routine blood cultures, and every episode of candidemia must be carefully evaluated for evidence of dissemination of involvement of prosthetic devices.

TREATMENT

Candida albicans is usually susceptible to amphotericin B, nystatin, flucytosine, the echinocandins, and the azoles. Superficial infections are generally treated with topical nystatin or azole preparations. Measures to decrease moisture and chronic trauma are important adjuncts in treating Candida skin infections. All C albicans infections may also require addressing predisposing conditions. For example, removal of an infected catheter, control of diabetes, or an increase in peripheral leukocyte counts can be important aspects of the complete treatment of infection. Systemic therapy with amphotericin B, echinocandins, or azoles is required for disseminated or deep tissue infections. The choice of treatment is often guided by speciation and antifungal susceptibility testing. Fluconazole has been effective treatment for chronic mucocutaneous candidiasis and recurrent mucosal infections, although antifungal resistance can develop with the prolonged use of this agent.

Other Candida Species

Candida species other than C albicans can produce very similar infections to those described earlier, especially disseminated and urinary tract infections. However, these nonalbicans Candida species are isolated almost exclusively from patients with nosocomial infections. Antibiotic use, wounds, and prosthetic devices also predispose hospitalized patients to infections with diverse Candida species. Some species, such as C glabrata and C krusei, display increased levels of resistance to the azole antifungals. Therefore, selection for colonization by these species may occur in patients previously treated with azoles. Other species, such as C tropicalis and C parapsilosis, are also isolated mostly from hospitalized patients. Importantly, nosocomial transmission of Candida species may occur with poor adherence to proper handwashing and other infection control practices. Little is known of the pathogenesis of these species with the exception of Candida tropicalis. Both experimental and clinical evidence indicate that C tropicalis has virulence at least equal to that of C albicans. Candida tropicalis produces extracellular proteinases similar to those of C albicans, which may enhance its invasiveness.

MYCOLOGY

Aspergillus species are rapidly growing molds that are frequently isolated from the environment. Since we are all exposed continuously to this group of fungi, it is not surprising that they are common causes of infections in patients with severe immune compromising conditions.

In culture and during infection, Aspergillus species grow as branching septate hyphae. With prolonged culture in the laboratory, fruiting bodies develop as sites of spore (conidia) formation. Individual species are defined on the basis of differences in the structure of the conidiophore and the arrangement of the conidia. (Figure 46–7A to C). The most common infections in humans are caused by A fumigatus, but other species, such as A flavus, A niger, and A terreus may infrequently cause human disease. For all of these species, fluffy colonies appear in 1 to 2 days; by 5 days, they may cover an entire plate with hyphal growth.

ASPERGILLOSIS

EPIDEMIOLOGY

Aspergillus species are widely distributed in nature and throughout the world. They seem to adapt to a wide range of environmental conditions, and the heat-resistant conidia provide a good mechanism for dispersal. Like bacterial spores, the fungal conidia survive well in the environment, with inhalation as the mode of human infection. Outbreaks of pulmonary aspergillosis in hospitals have been traced to fungal spores transmitted through air ducts, emphasizing the potential for environmental acquisition of infection. Similarly, building construction and remodeling have also been associated with increased frequency of Aspergillus infections. Hospital wards with highly immunocompromised patients often use specialized air filtration devices to limit patient exposure to these environmental fungi.

PATHOGENESIS

Once inhaled, Aspergillus conidia are small enough to readily reach the terminal airways and alveoli. However, the resident innate immune cells are able to suppress the germination of these cells into growing hyphae. Therefore, lung infections due to Aspergillus species are exceeding rare in people with normal immune systems. Host factors favoring Aspergillus growth include anatomic abnormalities of the lungs (eg, bronchiectasis, severe emphysema) and immunosuppression (neutropenia, lung transplantation).

The factors that aid the fungus in the initial stages of infection are incompletely defined. However, surface glucans and galactomannans likely favor adherence to host proteins such as fibrinogen and laminin, as well as masking the fungal cell from immune recognition. The more virulent species produce extracellular elastase, proteinases, and phospholipases, perhaps assisting invasion of host tissue. Like many filamentous fungi, Aspergillus species produce an array of “secondary metabolites,” compounds that are not required for basic metabolism of the fungal cell but that may assist its survival in a competitive environment. These include substances such as gliotoxin, a molecule that inhibits steps in the oxidative killing mechanisms of phagocytes, and aflatoxin, a potent carcinogen. The ability of Aspergillus to form biofilms has been implicated in infections developing in medical devices and implants.

IMMUNITY

Aspergillus infection is extremely rare in healthy persons. Macrophages, particularly pulmonary alveolar macrophages, are the first line of defense against inhaled Aspergillus conidia, phagocytosing and killing them by nonoxidative mechanisms. For the conidia that survive and germinate, PMNs become the primary defense. They are able to attach to the growing hyphae, generate an oxidative burst, and secrete reactive oxygen intermediates. Patients with AIDS rarely develop pulmonary or disseminated aspergillosis, suggesting that adaptive, T-cell mediated immunity is less important than innate immune mechanisms in controlling and preventing this infection. Antibodies to Aspergillus fungal elements are formed during infection, but their protective value is unknown.

ASPERGILLOSIS: CLINICAL ASPECTS

MANIFESTATIONS

Aspergillus infections typically present in one of four major ways, with the clinical findings completely dependent on the immune status of the host. These clinical presentations include: (1) Aspergillus pneumonia, (2) disseminated aspergillosis, (3) allergic respiratory disease, and (4) aspergilloma (fungus ball).

Aspergillus pneumonia. As the main site of fungal cell entry into the body, the lung is the primary organ involved in most cases of aspergillosis. Although innate immune responses in the lung are usually able to clear Aspergillus spores as they are inhaled, anatomic and immune defects can allow this fungus to grow and establish infection. Patients with severe forms of emphysema and bronchiectasis have regions of anatomic abnormalities within their lungs that offer protected sites for fungal germination and growth. Resident macrophages and neutrophils in the lung are less effective at controlling fungal growth in these regions of scarring within the airways and lung parenchyma. Therefore, patients with emphysema or cystic fibrosis can develop microinvasive lung infections due to Aspergillus species. In the presence of an intact immune system, the infection is unable to deeply penetrate into the surrounding lung tissue or to disseminate. However, this smoldering infection can result in symptoms of chronic bronchopneumonia with flares of cough and worsening respiratory function.

In contrast, patients with severe defects of immunity, such as solid-organ transplantation or neutropenia, can develop a progressive and immediately life-threatening pneumonia due to Aspergillus species. This rapidly progressive infection does not require prior lung anatomic abnormalities, and it emphasizes the importance of the innate immune system in normally preventing fungal colonization and growth. During invasive pulmonary aspergillosis, the fungal hyphae penetrate intact lung tissue, leading to local necrosis (Figure 46–7C). Symptoms include fever, cough, hemoptysis, and respiratory failure. Rapidly progressive lung infiltrates are often observed radiographically. Untreated, this infection quickly leads to the death of the patient. Even in the presence of antifungal therapy, cure of this infection often requires restoration of immune function (eg, recovery from neutropenia).

Disseminated aspergillosis. Once a primary Aspergillus infection is established in an immunocompromised patient, the fungus can disseminate through the bloodstream to affect any organ system in the body. The most dreaded site of spread is the central nervous system. The symptoms of disseminated infection will depend on the organs affected. However, changes in mental status, or other specific organ dysfunction, should always prompt a thorough investigation in immunosuppressed patients who are at risk for this devastating complication. When feasible, surgical debridement of infected tissue may favor patient survival. However, disseminated aspergillosis carries a high mortality rate. Therefore, much effort has gone into developing rapid diagnostic and preventive strategies in neutropenic patients, since delays in the treatment of invasive aspergillosis are associated with worse survival.

Allergic respiratory disease. As ubiquitous components of the air that we breathe, fungal spores are typically captured by the mucus present in our respiratory tract. In patients with allergic tendencies, the accumulation of excessive mucus in the airways may provide a growth substrate for inhaled fungi. After germinating, these fungal elements are not able to invade the underlying respiratory tissue, nor are they able to disseminate from this initial growth site. However, as potent allergens, the fungi may induce a cycle of progressive inflammation creating enhanced mucinous substrate for fungal growth. The growth of environmental Aspergillus species in the sinuses and larger airways can cause chronic allergic symptoms in susceptible patients.

Of note, children with severe forms of asthma are especially prone to develop a condition known as allergic bronchopulmonary aspergillosis (ABPA), characterized by eosinophilia, symptomatic asthma flares, cough, and respiratory distress. Anti-Aspergillus antibodies can also be detected in the bloodstream. During flares of ABPA, fleeting infiltrates may be seen on chest radiographs. These infiltrates do not reflect invasive fungal disease, but rather the extensive inflammation due to the allergic reaction to fungal antigens. This condition is treated with antiallergy therapy (eg, inhaled/systemic corticosteroids, antihistamines) rather than antifungal therapy. Allergic sinus disease due to fungi is thought to be an especially common cause of chronic sinusitis.

Aspergilloma (fungus ball). Patients with prior lung infections can develop pulmonary scarring and cavities. Historically, this was especially common in patients with old, healed tuberculosis. Fungal spores that found their way into these cavities were often able to grow into macroscopic fungal colonies. These “balls” of fungal hyphae appear radiographically as round regions of consolidation inside of old lung cavities. Unable to penetrate into the surrounding tissues, these fungus balls nonetheless are often able to induce mechanical trauma to the wall of the lung cavity. Patients with aspergillomas therefore often experience recurrent episodes of hemoptysis. If large blood vessels are present near these cavities, life-threatening bleeding can occur. Antifungal therapy may help some patients with this condition. However, cavity removal or catheter-guided embolization of involved lung vessels may also be required for recurrent hemorrhages due to aspergillomas.

DIAGNOSIS

Aspergillus can be isolated and identified in cultures of infected tissue. Its rapidly spreading mold growth, and all too frequent contamination of cultures, cause it to be regarded by microbiologists as a kind of weed. The diagnostic problem is distinguishing Aspergillus contamination and colonization from invasive disease. The diagnosis cannot be made for certain without the use of lung aspiration, biopsy, or bronchoalveolar lavage. Fungal growth from infected material demonstrates the presence of large, branching, septate hyphae (Figure 46–7B and C). Infrequently, the complete fruiting bodies are produced in vivo, creating a striking and diagnostic histologic picture (Figure 46–7A). Serologic methods have been developed to demonstrate Aspergillus antibodies. Although these tests may be helpful in suggesting allergic aspergillosis, they have little value in invasive disease because anti-Aspergillus antibody is common in healthy persons. Detection of circulating Aspergillus antigens (galactomannan, glucan) by immunoassay or nucleic acid amplification techniques shows promise, but cross-reactions with other fungi have been a problem.

TREATMENT AND PREVENTION

The mold-active azoles, especially voriconazole, are the preferred treatments for invasive aspergillosis. Caspofungin and amphotericin B are alternatives. No regimen is considered highly effective because the mortality rate of invasive disease is high. Surgical removal of localized lesion is sometimes helpful, even in the brain. Construction of rooms with filtered air has been effective in reducing exposure to environmental conidia.

Zygomycosis (mucormycosis) is the term applied to infection with any of a group of zygomycetes, the most common of which are Absidia, Rhizopus, and Mucor. These fungi are ubiquitous saprophytes in soil and are commonly found on bread and many other foodstuffs. They occasionally cause disease in persons with diabetes mellitus and in immunosuppressed patients receiving corticosteroid therapy. Diabetic ketoacidosis has a particularly strong association with zygomycosis.

Pulmonary or rhinocerebral disease is acquired by inhalation of conidia. The pulmonary form has clinical findings similar to those of other fungal pneumonias. The rhinocerebral form, however, produces a dramatic clinical syndrome in which agents of zygomycosis show striking invasive capacity. They penetrate the mucosa of the nose, paranasal sinuses, or palate, often resulting in ulcerative lesions. Once beyond the mucosa, they progress through tissue, nerves, blood vessels, fascial planes, and often the vital structures at the base of the brain. The clinical syndrome begins with sinus symptoms and headache and may rapidly progress through orbital cellulitis and hemorrhage to cranial nerve palsy, vascular thrombosis, coma, and death.

The pathologic findings in mucormycosis are distinctive: the zygomycetes involved all show ribbon-like, nonseptate (aseptate) hyphae in tissue which are so large that their branch points can be difficult to visualize (Figure 46–8). Conidia are not seen in tissue. As with Aspergillus, tissue biopsies are necessary to demonstrate the invasive hyphae, unless they can be seen on scrapings from palatal or nasal ulcers. For reasons that are obscure, cultures are sometimes negative, even those from tissue containing characteristic hyphae. Therapy involves control of underlying disease (eg, recovery from neutropenia, treatment of hyperglycemia) and high-dose antifungal therapy (lipid-associated amphotericin B, selected azoles). Surgical debridement is the most important intervention favoring survival and cure of infection.

Pneumocystis jirovecii is a ubiquitous colonizer of the human airway. It does not cause infections in most people, but it can cause a lethal pneumonia in immunocompromised persons, particularly those with AIDS. The more familiar name to many clinicians, Pneumocystis carinii, is now used for a Pneumocystis species found in rats. Pneumocystis jirovecii has not been grown in culture and was long considered a parasite rather than a fungus based on the morphology of forms seen in infected tissue.

MYCOLOGY

Because it has not been possible to cultivate Pneumocystis, our knowledge about its basic biology is limited. Observations rest on the study of organisms purified from infected lungs and genomic analysis of Pneumocystis DNA. The Pneumocystis “life cycle” is deduced from static images seen in infected tissues. The observed stages include delicate 5 to 8 μm cystic structures (Figure 46–9) within which elliptical subunits grow and repeat the cycle on rupture. These have been placed in three stages called trophic, precyst, and cysts. No filamentous form has been observed.

The trophic form is bounded by a cell wall and cytoplasmic membrane that enclose a nucleus and several mitochondria. As the precyst matures, the nuclei divide to form eight “spores” within the original structure to form the cyst. The spores have an eccentric nucleus, a nucleolus, and a single mitochondrion in the cytoplasm. If this is sexual reproduction, the surrounding structure would be called a spore case or ascus and the subunits would be called sporocytes.

The cell wall lacks the rigidity typical of other fungi; however, biochemical elements of typical fungal cell walls are present. These include glucan and N-acetylglucosamine, the primary subunit of chitin, as well as a major surface glycoprotein (Msg). The dominant sterol of the cytoplasmic membrane is cholesterol, rather than the ergosterol characteristic of fungi. Other biochemical analyses, however, support its fungal classification, such as the presence of elements of protein synthesis (elongation factor 3), which is unique to fungi. The designation of Pneumocystis as a fungus is most strongly supported by genome sequencing. This molecular phylogenic analysis places Pneumocystis among the ascomycetes.

PNEUMOCYSTOSIS

EPIDEMIOLOGY

Pulmonary infection with Pneumocystis occurs worldwide in humans and in a broad spectrum of animal life. Exposure is nearly ubiquitous; specific antibodies are present in most children by the age of 4. The reservoir and mode of transmission remain unknown, but laboratory rodents acquire Pneumocystis colonization with the first breaths of life. Pneumocystis is not typically observed in the respiratory tract of asymptomatic persons, but it was detected in one study by nucleic acid amplification techniques in over half the fatal victims of automobile accidents. Among HIV-infected individuals, the strains involved in second and third episodes are frequently antigenically different, suggesting frequent reacquisition of new strains.

Before the AIDS pandemic, Pneumocystis pneumonia (PCP) occurred sporadically among infants with congenital immunodeficiencies and in older children and adults as a complication of immunosuppressive therapy. Now AIDS has become the most common predisposing condition, and PCP is often the presenting manifestation of AIDS. In fact, before the development of effective chemoprophylactic regimens (see Treatment and Prevention), PCP occurred in approximately 50% of all AIDS patients at the time of initial diagnosis. Depending on the effectiveness of their HIV treatment regimen, AIDS patients may develop one or more bouts of PCP, often in conjunction with another opportunistic infection.

PATHOGENESIS

Pneumocystis is an organism of low virulence, which seldom produces disease in a host with normal T-lymphocyte function. In experimental animals, progressive infection can be initiated with starvation or corticosteroid administration, and in AIDS patients the risk of developing pneumocystosis increases dramatically once the CD4+ T-lymphocyte count has fallen below 200 cells/mm³. Concurrent viral, bacterial, fungal, and protozoan infections are found frequently in humans with PCP, suggesting that Pneumocystis may require the presence of another microbial agent for its multiplication.

Little is known about the early stages of disease. The Msg abundant on the surface of P jirovecii may act as an attachment ligand to several host proteins, including fibronectin, vitronectin, and surfactant proteins. Major surface glycoprotein undergoes antigenic variation, which could aid in its persistence in human hosts. Histologically, PCP is characterized by alveoli filled with desquamated alveolar cells, monocytes, organisms, and fluid, producing a distinctive foamy, honeycombed appearance (Figure 46–10); hyaline membranes may be present, and round cell infiltrates may be visible in the septa.

IMMUNITY

The nature of the immunodeficiencies in patients with pneumocystosis points to the primacy of T_H1 immune responses in resolution of infection with Pneumocystis. Alveolar macrophages are the first line of defense, with activated macrophages and CD4+ lymphocytes playing essential roles in the resolution of the infection. Activated macrophages release several cytotoxic factors, including O₂-derived radicals, reactive nitrogen intermediates, and cytokines (tumor necrosis factor-α, IL-2). Specific antibody responses to the Msg and other antigens appear in the course of pneumocystosis. A significant role for humoral immunity is suggested by the ability of Msg antibody to protect against experimental PCP in animals.

PNEUMOCYSTOSIS: CLINICAL ASPECTS

MANIFESTATIONS

In the immunocompromised host, the disease presents as a progressive, diffuse pneumonitis. Illness may begin after discontinuation or a decrease in the dose of corticosteroids or, in the case of acute lymphocytic leukemia, during a period of remission. These observations suggest that the immune response to the organism results in many of the symptoms accompanying the infection. In infants and patients with AIDS, symptom onset is typically insidious, and the clinical course is often 3 to 4 weeks in duration. Fever is mild or absent. In older persons and patients who have previously been on high doses of corticosteroids, the onset can be more abrupt. In both populations, the cardinal manifestations are progressive dyspnea and tachypnea; cyanosis and hypoxia eventually supervene. A nonproductive cough is present in 50% of all patients. Clinical signs of pneumonia are usually absent, despite the presence of infiltrates on X-ray. These infiltrates are alveolar in character and spread out symmetrically from the hilar regions of the lungs, eventually affecting most of the lung. Occasionally, unilateral infiltrates, coin lesions, lobar infiltrates, cavitary lesions, or spontaneous pneumothoraces are observed. Pleural effusions are uncommon. Clinical and radiographic abnormalities are generally accompanied by a decrease in arterial oxygen saturation, diffusion capacity of the lung, and vital capacity. Death occurs by progressive asphyxia.

Lesions outside the lung were rarely seen before the AIDS epidemic, but they now appear with some regularity in this patient population. The sites most often involved are lymph nodes, bone marrow, spleen, liver, eyes, thyroid, adrenal glands, gastrointestinal tract, and kidneys. The extrapulmonary clinical manifestations range from incidental autopsy findings to progressive multisystem disease.

DIAGNOSIS

Definite diagnosis of pneumocystosis depends on finding organisms of typical morphology in appropriate specimens. Because the pathologic process is alveolar rather than bronchial, the organisms are not readily seen in expectorated specimens such as sputum. The diagnostic yield is much better from specimens obtained by more invasive procedures. Of these, bronchoalveolar lavage (BAL) gives the best results with the least morbidity. Percutaneous needle aspiration of the lung, transbronchial biopsy, and open lung biopsy, though somewhat more sensitive techniques, are accompanied by more complications, including pneumothorax and hemothorax.

Pneumocystis can be demonstrated by a variety of staining procedures. The standard stain is methenamine silver (Figure 46–9), but direct fluorescent antibody (DFA) method, if available, is slightly more sensitive. Laboratories often perform a rapid stain (Wright, Giemsa, Papanicolaou) first and confirm by methenamine silver or DFA later. Methods developed for detection of Pneumocystis DNA in BAL and other specimens by polymerase chain reaction may soon be practical for clinical laboratories. The detection of fungal antigens in the blood, such as β-D glucan, supports the diagnosis.

TREATMENT AND PREVENTION

The fixed combination of trimethoprim and sulfamethoxazole (TMP-SMX) is the treatment of choice for all forms of pneumocystosis. It is administered orally or intravenously for 14 to 21 days. Patients with AIDS receive the longer course because they start with a higher organism burden, respond more slowly, and suffer relapse more often. Unfortunately, patients with AIDS have a high incidence of adverse effects to TMP-SMX, particularly the sulfonamide component. This requires the use of other antimicrobials (eg, clindamycin, primaquine, dapsone) alone or in combination with trimethoprim. Adjunctive corticosteroids in hypoxic patients with PCP results in improved survival, likely due to prevention of an excessive immune reaction to dying microbes.

Low-dose administration of TMP-SMX has been shown to significantly decrease the incidence of PCP in high-risk patients and prevents relapse in patients with AIDS. This chemoprophylaxis is indicated for patients who have CD4+ lymphocyte counts lower than 200/mm³, unexplained fever, or a previous episode of PCP. Chemoprophylaxis for PCP is also used in solid-organ transplant patients immediately after transplantation and during treatment for allograft rejection.