Chapter 22 Fungi of relevance to dentistry

The study of fungi is called mycology. Fungi are eukaryotic microorganisms, as opposed to bacteria together with Archaea that are prokaryotic (Chapter 2). By far the most important fungus of relevance in dentistry is a yeast belonging to the genus Candida. It is an oral commensal in about half of the general population. In this chapter, the general characteristics of some medically important fungi will be given, but the emphasis will be on fungal infections of the oral cavity – the oral mycoses, especially those caused by the Candida species.

Morphology

Fungi exhibit two basic structural forms: the yeast form (Fig. 22.1) and the mould form. While some fungi are capable of existing as both forms (dimorphic) at different times, others exist in one form only. This morphological switching depends on factors such as the environment and nutrient supply. Generally, dimorphic fungi exist as moulds in the natural environment (and in laboratory culture) and as yeasts in tissue:

Fig. 22.1 The yeast (blastospore) and hyphal forms of Candida albicans.

• Yeasts are unicellular with spherical or ovoid bodies; all yeasts are similar morphologically on light microscopic examination.

• Moulds are multicellular with a variety of specialized structures that perform specific functions. The size and nature of these structures vary with different genera. Hyphae (singular: hypha or hyphum) are thread-like tubes containing the fungal cytoplasm and its organelles. They can be considered as the structural units of the mould. The hyphae are divided into unit cells by cross walls called septa. The septa have pores that allow the movement of cytoplasm, and even organelles, between cells. The term mycelium is given to the mass of hyphae that forms the mould colony.

Reproduction

Both asexual and sexual modes of reproduction are seen in fungi. It is believed that the sexual forms of fungi are not found in clinical material.

Classification

Taxonomy of fungi is a complex subject not dealt with here. Most of the medically important fungi are classified as fungi imperfecti as their sexual forms have not been identified. Fungi of medical importance are classified into:

• yeasts

• filamentous fungi

• dimorphic fungi.

The following methods are used in classification of fungi:

1. Yeasts are identified by biochemical reactions based on the fermentation and assimilation of carbohydrates, utilization of enzyme substrates and other metabolic activities.

2. Moulds are identified by their colour, texture and colonial and microscopic morphology. The specialized asexual reproductive structures of moulds are useful in differentiating various species of moulds.

Cultural requirements

Medically important fungi require different cultural and growth conditions in comparison to bacteria:

1. The vast majority of medically important fungi grow aerobically.

2. Sabouraud dextrose agar (SAB) and variations of it, such as SAB plus antibacterial agents, and potato dextrose agar (PDA) are commonly used for laboratory culture of pathogenic fungi.

These mycological media differ from conventional bacteriological media in having a high carbohydrate content (SAB usually contains 3% dextrose or sucrose) and an acidic pH (approximately 4.0). Both these conditions are inhibitory to most bacteria. The SAB medium may also be supplemented with antibiotics to suppress bacterial growth.

Pathogenicity

In general, medically important fungi do not possess the virulent attributes of bacteria such as exotoxins and endotoxins (an exception is the exotoxin, aflatoxin, produced by Aspergillus species); hence, they cause slowly progressive chronic infections rather than the acute disease commonly seen in bacterial or viral diseases. However, they may cause life-threatening acute infections in immunocompromised patients (e.g. those with acquired immune deficiency syndrome (AIDS)). The oral fungal pathogen Candida possesses a number of virulent attributes, including:

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• the ability to adhere to host tissues and prostheses (e.g. dentures) and form biofilms

• the potential to switch (e.g. rough to smooth colony formation) and modify the surface antigens

• the ability to form hyphae that helps in tissue invasion

• extracellular phospholipase and proteinase, and haemolysin production, which break down physical defence barriers of the host.

Human mycoses

Human infections caused by fungi can be divided into:

• superficial mycoses

• subcutaneous mycoses

• systemic mycoses.

Superficial mycoses

Superficial mycoses involve the mucosal surfaces and keratin-containing structures of the body (skin, nails and hair). These infections, relatively common in western countries, are in general cosmetic problems and are not life-threatening. Superficial mycoses include:

• yeast infections of mucosae, which lead to thrush and similar manifestations (see Chapter 35)

• dermatophyte infection of skin, hair, etc., leading to ringworm or similar diseases.

Subcutaneous mycoses

Subcutaneous mycoses involve the subcutaneous tissue and rarely disseminate. They are the result of traumatic implantation of environmental fungi leading to chronic progressive disease, tissue destruction and sinus formation. Examples include sporotrichosis and mycetoma (Madura foot), which are common in the tropics and rare in the West.

Systemic mycoses (deep mycoses)

By far the most serious, and often fatal, systemic mycoses involve the internal organ systems of the body. The organisms are generally acquired through the respiratory tract and spread haematogenously. In the developed world, they are increasingly seen in compromised patients with impaired defence systems when the organisms behave as opportunistic pathogens. In the developing world, systemic mycoses (e.g. histoplasmosis, blastomycosis and coccidioidomycosis) occur in otherwise healthy individuals.

Opportunistic fungal infections

When fungi (such as Candida albicans) that are generally innocuous for healthy humans cause disease in compromised patient groups, they are called opportunistic pathogens. Such opportunistic mycoses are increasingly common owing to a global rise in compromised individuals such as human immunodeficiency virus (HIV)-infected patients, organ transplant recipients on immunosuppressive therapy and cancer patients on cytotoxic and radiation therapy.

Yeasts

Yeasts are unicellular, oval or spherical organisms, 2–5 µm in diameter, and stain positively by the Gram method (Fig. 22.2). They are commonly seen to have lateral projections or buds called daughter cells. These gradually enlarge in size until they split off from the parent or mother cell to produce the next generation. Most yeasts develop pseudohyphae (chains of elongated budding cells devoid of septa or cross walls) but only a few form true hyphae (septate hyphae). Yeasts of the genus Candida, the most important fungal pathogen in the oral cavity, also form pseudohyphae. It is a common yeast that lives in the oral cavity of about half of the population and is also a resident commensal of the gut. It can cause either superficial or systemic candidiasis (synonym: candidosis). The superficial disease affects:

Fig. 22.2 A Gram-stained film of a smear from the fitting surface of the denture of a patient with Candida-associated denture stomatitis showing the blastospore and hyphal forms of the organism.

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• the mucosa – mucosal candidiasis

• the skin – cutaneous candidiasis

• both the skin and the mucosa – mucocutaneous candidiasis.

The infection is usually endogenous in origin. Several species in the genus Candida are found in humans, including C. albicans, C. glabrata, C. krusei and C. tropicalis (Fig. 22.3), but C. albicans is responsible for the vast majority of infections (>90%). Candida dubliniensis is a newly recognized species of Candida very similar to C. albicans. First isolated from the oral cavity of HIV-infected patients, C. dubliniensis is now known to be a relatively common oral inhabitant in both health and disease.

Fig. 22.3 Candida albicans and Candida tropicalis growing side by side on a special medium (Pagano–Levin agar), which elicits differential colour reactions. Mixed oral candidal infections are not uncommon.

Candida albicans

Habitat and transmission

C. albicans is indigenous to the oral cavity, gastrointestinal tract, female genital tract and sometimes the skin; hence, infection is usually endogenous, although cross infection may occur, e.g. from mother to baby, and among infant siblings.

Characteristics

C. albicans typically grows as spherical to oval budding yeast cells 3–5 × 5–10 µm in size. These yeast-phase cells are also called blastospores (Fig. 22.4), but should not be confused with bacterial spores. Pseudohyphae (elongated filamentous cells joined end to end) are seen, especially at lower incubation temperatures and on nutritionally poor media.

Fig. 22.4 A transmission electron micrograph of a blastospore of Candida albicans and a budding daughter cell.

Culture and identification

Cultures grow on Sabouraud medium as creamy-white colonies, flat or hemispherical in shape with a beer-like aroma. C. albicans and C. dubliniensis may be differentiated from other Candida species by their ability to produce germ tubes and chlamydospores:

• When yeast cells are incubated for 3 h at 37°C in serum, C. albicans and C. dubliniensis form germ tubes (incipient hyphae), whereas other Candida species do not (see Fig. 6.15).

• Both C. albicans and C. dubliniensis form round, thick-walled, resting structures called chlamydospores when incubated at 22–25°C with decreased oxygen on a nutritionally poor medium (e.g. cornmeal agar).

However, definitive identification of the species is made on the basis of carbohydrate assimilation (aerobic metabolism) and fermentation (anaerobic metabolism) reactions and other biochemical tests; polymerase chain reaction (PCR) diagnostics should soon replace these methodologies in diagnostic laboratories, especially due to their rapidity.

Pathogenicity

Candida species rarely cause disease in the absence of predisposing factors, a vast number of which have been identified, both for superficial and systemic candidiasis (Table 22.1).

Table 22.1 Factors predisposing to oral candidiasis

Chronic local irritants

Ill-fitting appliances

Inadequate care of appliances

Disturbed oral ecology or marked changes in the oral microbial flora by antibiotics, corticosteroids, xerostomia

Dietary factors

Immunological and endocrine disorders (e.g. diabetes mellitus)

Malignant and chronic diseases

Severe blood dyscrasias

Radiation to the head and neck

Abnormal nutrition

Age (e.g. very young or very old)

Hospitalization

Oral epithelial dysplasia

Heavy smoking

Superficial candidiasis

1. Mucosal infection: the characteristic mucosal lesion of Candida is thrush. This is classically a white pseudomembrane on buccal mucosa and vagina, which can be easily removed by wiping. Other oral manifestations include erythematous and hyperplastic variants (see Chapter 35). Candidal vulvovaginitis is common in women using oral contraceptives and is accompanied by a thick, yeasty-smelling discharge, vaginal itching and discomfort.

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2. Skin infection is seen particularly on surfaces that are warm and moist. Candidal intertrigo consists of vesicular pustules that enlarge, rupture and cause fissures, and is especially seen in the obese.

3. Nappy rash in children may be caused by C. albicans derived from the lower gastrointestinal tract. Scaly macules or vesicles, associated with intense burning and pruritus, are common in nappy rash.

4. Candidal paronychia is a localized inflammation around and under the nails, caused by Candida when the hands are frequently immersed in water (e.g. in dishwashers and laundry workers).

Mucocutaneous candidiasis

Mucocutaneous candidiasis involves both the skin and the oral and/or vaginal mucosae. This rare disease is due to heritable or acquired defects in the host immune system or metabolism. Chronic mucocutaneous candidiasis is a rare condition associated with T cell deficiency (see Chapter 35).

Systemic or deep candidiasis

This may involve the lower respiratory tract and urinary tract, with resultant candidaemia (Candida in blood); localization in endocardium, meninges, bone, kidney and eye is common. Untreated disseminated disease is fatal. Susceptible settings include organ transplantation, heart surgery, prosthetic implantation and long-term steroid or immunosuppressive therapy. Rarely, a superficial infection is the cause of disseminated disease.

Diagnosis

1. Demonstration of yeasts in Gram-stained smear, followed by culture of specimen on Sabouraud agar.

2. Serology or novel PCR-based molecular methods or blood culture (in suspected candidaemia) are helpful in the diagnosis of disseminated candidiasis.

3. Histopathological examination of a biopsy of the lesion; demonstration of tissue invasion by fungal hyphae helps establish a causal relationship.

Treatment

Candida infections can be treated by three groups of agents: the polyenes, the azoles and the DNA analogues (see Chapter 7). The agents used depend upon the type and severity of infection. Superficial infections can be treated topically with a polyene (nystatin or amphotericin) or an imidazole (miconazole, clotrimazole). Polyenes are also very effective for oral candidal infections. Systemic infections and disseminated candidiasis require intravenous amphotericin, either alone or in combination with flucytosine. The triazole agent fluconazole, effective for both superficial and systemic mycoses, is the drug of choice in treating Candida infections in HIV disease. (However, C. krusei is resistant to fluconazole). The use of newer agents such as echinocandins and terbinafine in dentistry are as yet ill defined.

Prevention

Candidiasis is almost always endogenous in origin; therefore, prevention entails correction of predisposing factors. Those who are compromised may require long-term prophylactic antifungal treatment, either continuously or intermittently.

Cryptococcus

Cryptococcus neoformans is a pathogenic yeast belonging to the Cryptococcus genus. It causes cryptococcosis, especially cryptococcal meningitis.

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Habitat and transmission

This yeast is a ubiquitous saprophyte commonly isolated from soil enriched by pigeon droppings. Infection is initiated by inhalation of airborne yeast cells.

Characteristics

C. neoformans is a budding yeast with a thick capsule, 5–15 µm in diameter.

Culture and identification

Identification is by sputum and spinal fluid culture on Sabouraud agar. Latex agglutination is used to detect the polysaccharide antigen in urine, blood or spinal fluid. Indian ink preparations of spinal fluid are used in the demonstration of the encapsulated yeast (Fig. 22.5).

Fig. 22.5 Indian ink preparation of Cryptococcus neoformans showing capsules of yeasts, which appear as translucent haloes.

(Courtesy of Centres for Disease Control, Atlanta, USA and Dr. Leanor Haley.)

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Pathogenicity

The thick polysaccharide capsule of the yeast is highly resistant to host immune defences. Life-threatening infections caused by C. neoformans have been steadily increasing over the past few decades due to the onset of AIDS, and the expanded use of immunosuppressive drugs. Cryptococci cause an influenza-like syndrome or pneumonia. Subsequent fungaemia causes infection of the meninges. Reduced cell-mediated immunity exacerbates the infection; immunocompetent people may occasionally develop cryptococcal meningitis. Rarely cryptococcal oral ulceration can be seen in compromised patients such as those with HIV infection.

Treatment

Intravenous combination therapy with amphotericin and flucytosine, although the beneficial role of the latter has been questioned. Fluconazole, which penetrates the central nervous system, is also useful.

Filamentous and dimorphic fungi and oral disease

The foregoing text describes one major group of fungi – yeasts – which are of dental and medical relevance. The other two main groups, filamentous and dimorphic fungi, usually do not cause oral disease except in immunocompromised groups. Organisms that are noteworthy and cause oral ulceration are Penicillium marneffei, Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum and Histoplasma duboisii (Table 22.2).

Table 22.2 Dimorphic fungi that may cause oral ulceration, especially in immunocompromised patients

Fungus	Disease	Geographic distribution
Penicillium marneffei	Penicilliosis	South-East Asia
Blastomyces dermatitidis	North American blastomycosis	North America, especially Mississippi and Ohio valleys
Coccidioides immitis	Coccidioidomycosis	USA from California to Texas; South and Central America
Histoplasma capsulatum	Histoplasmosis	Eastern and central USA; occasionally other parts of the world
Histoplasma duboisii	African histoplasmosis	Equatorial Africa

Pathogenicity

In all these cases, infection is usually acquired by inhalation, and the primary lesions are seen in the lungs. In a majority, the initial lesion heals, often asymptomatically, and delayed hypersensitivity develops, with a positive skin test reaction to the appropriate antigen. Progressive disease may affect the lungs, causing cavitation, and/or disseminate widely to involve the skin, oral and other mucous membranes and internal organs. Ulceration is the most common presentation in the oral mucosa.

Diagnosis

Diagnosis may be by direct demonstration in exudate, sputum or biopsy specimens, isolation in appropriate culture media and/or serology.

Treatment

Amphotericin is the drug of choice; itraconazole is an alternative.

Key facts

• Fungi are eukaryotic microorganisms, as opposed to bacteria, which are prokaryotes.

• Fungi exhibit two basic structural forms – the yeast form and the mould form: yeasts are unicellular with spherical/ovoid bodies, while moulds are multicellular with a variety of specialized structures.

• Hyphae (singular: hypha or hyphum) are thread-like tubes containing the fungal cytoplasm and its organelles (mycelium, a mass of hyphae).

• Fungi of medical importance are classified into yeasts, filamentous fungi and dimorphic fungi.

• The vast majority of medically important fungi grow aerobically on Sabouraud dextrose agar (SAB) or its variations.

• Candida albicans possesses a number of virulent attributes, including the ability to adhere to host tissues/prostheses and form hyphae, colonial switching and the production of extracellular phospholipase and proteinases.

• Human infections caused by fungi can be broadly categorized as superficial, subcutaneous or systemic (deep) mycoses.

• When fungi (such as C. albicans) that are generally innocuous in healthy humans cause disease in compromised patients, they are called opportunistic infections.

• Candida, a common yeast that lives in the oral cavity of some 50–60% of the population, can cause either superficial (mucosal, cutaneous or mucocutaneous) or systemic candidiasis.

• Species in the Candida genus found in humans include C. albicans, C. glabrata, C. dubliniensis, C. krusei and C. tropicalis; C. albicans is responsible for the vast majority of infections (>90%).

• C. albicans is indigenous to the oral cavity, gastrointestinal tract, female genital tract and sometimes the skin; hence, the infection is usually endogenous.

• C. albicans and C. dubliniensis may be differentiated from other Candida species by their ability to produce germ tubes and chlamydospores.

• Candida species rarely cause oral disease in the absence of predisposing factors, such as intraoral environmental changes (e.g. unhygienic prostheses, xerostomia) and/or systemic factors such as diabetes and immunodeficiency.

• The three major clinical manifestations of oral candidiasis are pseudomembranous, erythematous and hyperplastic variants (see Chapter 35).

• Demonstration of yeasts in Gram-stained smear, positive culture on Sabouraud agar and subsequent confirmation by biochemical or genetic techniques constitute a mycological diagnosis of candidiasis.

• Candida infections can be treated by three main groups of agents: the polyenes, the azoles and the DNA analogues, depending on the type and severity of infection.

• The triazole agent fluconazole is effective for both superficial and systemic mycoses, and is the drug of choice in treating Candida infections in HIV disease.

• Resistance to azoles is seen in Candida species, usually following prolonged treatment, while resistance to DNA analogues and polyene group drugs is rare.

• Treatment of candidiasis entails correction of predisposing factors, with or without oral or systemic antifungals.

• Oral lesions due to fungi other than Candida are rare. These, such as cryptococcosis, histoplasmosis and penicilliosis, may be seen in HIV disease and usually respond to intravenous amphotericin therapy.

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