The Yeasts

1. Explain the etiology and epidemiology of Candida, Cryptococcus, Trichosporon, and Malassezia spp.

2. Describe the macroscopic phenotypes and microscopic structures used to identify yeasts.

3. Differentiate yeast species based on microscopic, macroscopic, and biochemical test results.

4. Compare and contrast the methods used to identify yeasts, including staining, antigen testing, and biochemical and growth characteristics (i.e., morphology).

5. Diagram an algorithm for identifying the yeasts discussed in this chapter.

Genera and Species to be Considered

General Characteristics

Yeasts are eukaryotic, unicellular organisms that are round to oval and range in size from 2 to 60 µm. The microscopic morphologic features have limited usefulness in helping to differentiate or identify these organisms. The microscopic morphology on cornmeal agar is most useful when considered in conjunction with the biophysical profile (i.e., a combination of the biochemical and physical characteristics used in the identification of a microorganism) obtained using a commercial system. Differentiation of yeasts in direct microscopic and histopathologic examination of clinical specimens is often impossible, but sometimes particular characteristics are seen that suggest the identification or are pathognomonic (i.e., unique) for a particular organism. Important morphologic characteristics that are useful in differentiating yeasts include the size of the yeasts, the presence or absence of a capsule, and broad-based or narrow-necked budding. For example, variability in size with evidence of a capsule and narrow-necked budding are features that can be helpful for distinguishing Cryptococcus spp. from Candida spp. The medically important yeasts and yeastlike organisms belong to different taxonomic groups, including the Ascomycota, Basidiomycota, and Deuteromycota.

In general, the yeasts reproduce asexually by blastoconidia formation (budding) (Figure 63-1) and sexually by the production of ascospores or basidiospores. The process of budding begins with a weakening and subsequent outpouching of the yeast cell wall. This process continues until the bud, or daughter cell, is completely formed. The cytoplasm of the bud is contiguous with the cytoplasm for the original cell. Finally, a cell wall septum is created between mother and daughter yeast cells. The daughter cell often eventually detaches from the mother cell, and a residual defect occurs at the budding site (i.e., a bud scar).

Figure 63-1 Blastoconidia (budding cells *[arrow]*) characteristic of the yeasts.

With certain environmental stimuli, yeast can produce different morphologies. An outpouching of the cell wall that becomes tubular and does not have a constriction at its base is called a germ tube; it represents the initial stage of true hyphae formation (Figure 63-2). Alternatively, if buds elongate, fail to dissociate, and form subsequent buds, pseudohyphae are formed; to some, these resemble links of sausage (Figure 63-3). Pseudohyphae have cell wall constrictions rather than true intracellular septations delineating the fungal cell borders.

Figure 63-2 Germ tube test for *C. albicans* showing yeast cells with germ tubes.

The number of fungal infections caused by yeasts and yeastlike fungi has increased significantly during recent years. Most of these infections have been caused by various Candida spp. However, other yeasts also cause significant disease, particularly in immunocompromised hosts, as yeasts are the cause of many opportunistic infections. In addition to causing disease in immunocompromised patients, infections also are common in postsurgical patients, trauma patients, and patients with long-term indwelling venous catheters. Some of these yeasts are resistant to commonly used antifungal agents, which emphasizes the need for prompt, appropriate identification and, in some cases, antifungal susceptibility testing.

The extent to which the laboratory should identify all yeast species is the subject of debate. Each laboratory director must decide how much time, effort, and expense is to be spent on the identification of yeasts in the laboratory.

The development of commercially available yeast identification systems has provided laboratories of all sizes with accurate, standardized methods. However, these methods should be used in conjunction with cornmeal agar morphology to prevent misidentifications. Some commercial systems have extensive computer databases that include biochemical profiles of a large number of yeasts. Variations in the reactions of carbohydrates and other substrates utilized are considered in the identification of yeasts provided by these systems.

Commercially available systems are recommended for all laboratories. They may be used in conjunction with some less expensive and rapid screening tests that provide presumptive identification of C. neoformans and definitive identification of Candida albicans.

More recent diagnostic tools that have been introduced for quicker characterization of yeasts include CHROMagar Candida (BD Diagnostics, Sparks, Maryland) and C. albicans PNA FISH (AdvanDX, Woburn, Massachusetts). Because some laboratories might prefer to use conventional systems, the information presented in this section discusses rapid screening methods for presumptive identification of yeasts, commercially available systems, and a conventional schema for identifying commonly encountered yeast species.

Epidemiology

Cryptococcus Neoformans

Genus Cryptococcus

Cryptocococcosis is an acute, subacute, or chronic fungal infection that has several manifestations. Crytococcus neoformans var. grubii, C. neoformans var. neofromans, and C. gatti are considered the major human pathogens. Differences in the infections by Cryptococcus neoformans appear to be dependent on the host immune status and not the variant. C. neoformans infections can present initially as a chronic or subacute pulmonary infection. C. neoformans eventually makes its way to the central nervous system, where the yeast can cause cryptococcal meningitis. Disseminated disease with meningitis is commonly seen in immunocompromised patients. Patients with a moderately compromised immune system or who are early in the disease process of cryptococcal fungemia may present without concomitant meningitis. Disseminated cryptococcosis and cryptococcal meningitis became well recognized in patients with acquired immunodeficiency syndrome (AIDS), and they remain an important cause of morbidity and mortality in these patients in resource-poor countries that do not have access to highly active antiretroviral therapy.

Patients with disseminated infection may have painless papular skin lesions that may ulcerate. Other, less common manifestations of cryptococcosis include endocarditis, hepatitis, renal infection, and pleural effusion. Interestingly, a review of patient records at the Mayo Clinic revealed that more than 100 immunocompetent patients with C. neoformans colonization of the respiratory tract did not develop subsequent infection. Follow-up on these patients was as long as 6 years, and none in this group were considered to be immunocompromised. This makes the clinical significance of C. neoformans somewhat difficult to assess. However, given the severity of disease it can cause, its presence in clinical specimens should be considered significant. In many instances, the clinical symptoms are suppressed by corticosteroid therapy, which is a risk factor for disease, and culture or serologic evidence (detection of cryptococcal antigens) provides the earliest proof of infection. Cryptococcal infection is strongly associated with such debilitating diseases as leukemia and lymphoma and the immunosuppressive therapy that may be required for these and other underlying diseases. In some cases the presence of C. neoformans in clinical specimens precedes the symptoms of an underlying disease.

C. neoformans can exhibit a very characteristic polysaccharide capsule. The capsule collapses and protects the yeast from desiccation under drying conditions. The capsule of C. neoformans is thought to help the organism survive through the pigeon gut before it is excreted. The reduction in the yeast’s cell size caused by capsular collapse places the organism in the ideal size range for alveolar deposition in the human host. In addition, a virulent property of the polysaccharide capsule is that it contains compounds that phagocytes do not recognize. The so-called acapsular strains of C. neoformans, which actually just have a very reduced capsule, are more easily phagocytosed. In some instances, C. neoformans elicits minimal tissue response in infected individuals, particularly severely immunocompromised patients.

Phenoloxidase, an enzyme found in C. neoformans, is responsible for melanin production. Some have speculated that melanin might act as a virulence factor by making the organism resistant to leukocyte attack. Evidence also has been presented that increased melanin production can decrease immune system functions, such as lymphocyte proliferation and tumor necrosis factor production. Whether phenoloxidase is truly a virulence factor has yet to be determined (Li SS, et al, 2010. (Cryptococcus. 2010 Proc Am Thorac Soc Vol 7 pp 186-196, 2010). An interesting question is whether the interactions of substances in the brain that are known to react with phenoloxidase may play a role in the affinity of C. neoformans and certain neurotropic dematiaceous fungi for invading the central nervous system (Li et al, 2010).

Cryptococcus Gattii

C. gattii was thought to be a variant of C. neoformans until genetic studies proved it to be a distinct species. Since the 1990s this organism has emerged in the Pacific Northwest as a pathogen in immunocompetent hosts—a major difference from C. neoformans, which causes disease in primarily immunodeficient hosts. Some speculate that C. gattii is able to modulate the host’s immune system by reducing the inflammatory response or evading the immune system completely.

Trichosporon spp.

Trichosporonosis is caused by a variety of Trichosporon spp., which have undergone changes in nomenclature based on DNA sequence comparisons. The yeastlike fungus causes disease almost exclusively in immunocompromised patients, particularly those with leukemia. Disseminated trichosporonosis is the most common clinical manifestation. Skin lesions accompanied by fungemia are frequently seen. Endocarditis, endophthalmitis, and brain abscess have been reported. Trichosporon organisms occasionally are recovered from respiratory tract secretions, skin, the oropharynx, and the stool of patients with no evidence of infection and may represent transient fungal colonization of those individuals.

White piedra, an uncommon fungal infection of immunocompetent patients, is found in both tropical and temperate regions of the world. It is characterized by the development of soft, yellow or pale brown aggregations around hair shafts in the axillary, facial, genital, and scalp regions of the body. The Trichosporon spp. that cause this disease frequently invade the cortex of the hair, causing damage.

Malassezia spp.

Malassezia furfur causes tinea versicolor, a skin infection characterized by superficial, brownish, scaly areas on light-skinned individuals and lighter areas on dark-skinned people. The lesions occur on the smooth surfaces of the body, namely, the trunk, arms, shoulders, and face. The disorder has a worldwide distribution. M. furfur is also a cause of disseminated infection in infants and young children and even in adults given lipid replacement therapy. Malassezia pachydermatis, another species, may be recovered from skin lesions. In rare cases it may cause fungemia in immunocompromised patients.

Direct microscopic examination of clinical specimens containing Candida organisms reveals budding yeast cells (blastoconidia) 2 to 4 μm in diameter and/or pseudohyphae (Figure 63-4) showing regular points of constriction, resembling links of sausage. True septate hyphae (filamentation) may also be produced by C. albicans and C. dubliniensis. The blastoconidia, hyphae, and pseudohyphae are strongly gram positive. The approximate number of such forms should be reported, because the presence of large numbers in a fresh clinical specimen may have diagnostic significance. Microscopically C. glabrata blastoconidia are notably smaller (at 1 to 4 µm) than those of other medically significant Candida spp.