MEDICAL MYCOLOGY Fungi
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- Abraham Perkins
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1 MEDICAL MYCOLOGY Fungi In contrast to the bacteria and viruses, the fungi are eukaryotes (like animals and plants). This is medically relevant in that it impacts how fungal infections are attacked by the immune system and how they are treated pharmacologically. Fungi are scavengers. Environmentally, they play a vital role in the decomposition of dead biological materials and the maintenance of the carbon cycle. Fungi also have industrial uses, chiefly in the production of wine, beer, cheese and antibiotics (e.g., penicillin). These useful characteristics can be contrasted with the more negative roles that fungi play as pathogens of plants and animals. The fungi that infect humans fall into four main categories; superficial (hair,skin and nails only), subcutaneous, true systemic (pathogenic) and opportunistic. In the following sections we will examine the classification and biological characteristics of fungi, followed by profiles of superficial, subcutaneous, opportunistic and systemic fungi. Finally, we will examine the treatment of fungal infections. Fungal Biology and Classification Table 1 Animals Plants Fungi Bacteria Free-living Attached Attached & Free-living Free-living No Cell Wall (chitin exoskeletons) Cell Wall (cellulose) Cell Wall (chitin) Cell Wall (NAG-NAM) Heterotrophic Autotrophic Heterotrophic Both Requires O 2 Requires CO 2 & O 2 Requires O 2 Varies 80S = 60S & 40S Ribosomes 80S = 60S & 40S Ribosomes 80S = 60S & 40S Ribosomes 70S = 50S & 30S Ribosomes Nulceus Nucleus Nucleus No nucleus Table 1 illustrates similarities and differences between the fungi and other major life form groups. As you can see, the fungi share many characteristics of animals and plants. Importantly, fungal ribosomes are eukaryotic and similar to human and plant ribosomes (while different from those of bacteria). This difference means that antimicrobial agents which target the bacterial ribosomal subunits will be ineffective against fungi. Similarly, while fungi possess a cell wall (important for shape, rigidity, strength, and osmotic protection), the fungal cell wall is composed of chitin rather than peptidoglycan as in bacteria. As such, antimicrobial agents that target bacterial cell walls are ineffective against fungi. However, the presence of a fungal cell wall and unique sterol molecules (e.g., ergosterol) in the fungal 173
2 cell membrane (plasma membrane) are differences between fungi and animals that can be targeted by antifungal therapies. Many fungi can exist in either one or both of two possible forms, a hyphal or a yeast form. In the environment, the majority of fungi form a mycelium (mass of branching hyphae), secrete digestive enzymes, and absorb food by decomposing organic material. This can happen during fungal infections in people as well. The yeast form is more common in liquid environments, such as the ocean, beer, or blood. Generally, we classify the medically important fungi as having mold (hyphal), yeast (small, single cells) or dimorphic (when both, usually hyphal in the environment and yeast in humans) forms. Fungi are capable of either sexual or asexual reproduction. The details of fungal reproduction are largely beyond the scope of this guide, however there are some important points to consider. Many fungi that live in the hyphal form in the soil, and reproduce asexually or sexually through the production of spores. As with bacterial species, fungal spores are small and hardy forms that can be widely disseminated before they begin growing in a hospitable environment (e.g., human lung). Fungal reproduction varies by phylum; as information about reproduction is relevant to the clinical course of an individual fungus it will be included in the section for that fungus. Yeasts are single celled organisms, reproduce by budding (blastoconidia), may form pseudohyphae (elongated forms that look like hyphae but have recurring bud-like constrictions and less rigid cell walls than hyphae) and form bacterial like colonies on culture plates. Molds have multicellular hyphae and spores (conidia). Molds can be morphologically divided into those with septate and non-septate hyphae. Hyphal septations divide the (generally) non-septate mucormycetes from the other septate molds. A mycelium is an intertwined mass of hyphae; a vegetative mycelium acts as a root whereas an aerial mycelium bears reproductive structures. Asexual conidia arise directly from hyphae or on a stalk-like structure, the conidiophore. Microconidia and macroconidia indicate size and complexity. Chlamydoconidia or arthroconidia develop within hyphae. The most common sexual spore is an ascospore. Mold on a culture plate Non-septate hyphae (mucormycetes) Septate hyphae The dimorphic fungi of clinical interest are the thermally dimorphic pathogenic fungi that exist in the hyphal form when cool (e.g., in the environment) and as yeast when warm (e.g., at 37ºC in the human). 174
3 Superficial Fungal Infections (Dermatomycoses) Several genera of fungi cause dermatomycoses (superficial skin infections which can occur anywhere on the body), including ringworm and athletes foot. The latin name for this infection, tinea is followed by the location [e.g., corporis = body, capitis = trunk, barbae = beard, unguium = nails (a.k.a. onychomycosis) and so on]. The most common sites of infection are the hair, skin or nails. Dermatophytes are widely distributed in the environment, and the infection is generally transmitted by contact with soil, animals or person-to-person contact. Classification: The dermatophyte label applies to three genera of fungi (Epidemophyton, Microsporum, Trichophyton) that commonly cause indolent skin, hair, and nail infections in people (and animals). These fungi invade and destroy keratin and are filamentous. They are transmitted person-to-person or by contaminated objects or animal-to-human. Epidermophyton species Microsporum species Trichophyton species Epidermophyton: Epidermophyton floccosum is the only pathogenic species in this genus. Microsporum: There are several pathogenic species within this genus, including Microsporum canis. Trichophyton: Trichophyton rubrum is the most common cause of ringworm. There are several pathogenic species within this genus. Clinical Features: Patients typically present with itchy, red, raised, scaly patches on the affected skin that may blister and ooze. The patches often have a sharply defined edge. They are often more erythematous (red) around the perimeter with normal skin tone in the center, creating the appearance of a ring. The skin may also appear unusually dark or light (hyper or hypopigmented). Bald patches appear when the scalp and beard are infected. Infected nails become discolored, thick, and often crumble. Tinea pedis Ringworm 175
4 Diagnosis: The diagnosis is primarily based on the appearance of the skin. Diagnostic tests include the KOH preparation (skin scrapings are dissolved in KOH and examined under a microscope; KOH dissolves keratin material found in skin so the fungi can be visualized), and fungal culture (not commonly used because cultures are expensive and time-consuming, but used if diagnosis is uncertain or if the infection is unmanageable or recurrent; the typical Wood s Lamp KOH Preparation culture medium is Sabouraud's dextrose agar). A Wood s lamp (UV light) can be used to visualize a few fungi, that fluoresce (shown). The dermatophytes possess septate hyaline hyphae that can help distinguish them from the more invasive non-septate hyphae of the mucormycetes. Pathogenesis: All three dermatophytes produce virulence factors that allow them to invade the skin, hair, and nails; including keratinases, elastases, and proteinases. Infection is restricted to the nonliving layers of the epidermis since they lack the ability to penetrate viable tissues of an immunocompetent host; however, they may cause invasive infections in immunocompromised hosts. A ring shape often forms because the center clears as the rash spreads (shown). Epidemiology: Dermatophyte infections are the most common fungal infections worldwide. Tropical regions have a higher incidence of ringworm since the climate is hot, humid, and wet. (Humid or moist skin provides a favorable environment for the establishment of these fungal infections). Tinea pedis is the most common type in the United States and in the rest of the world. Tinea capitis is one of the most common infections in children. Tinea unguium is a common problem in adults. Trichophyton species infect the hair, skin and nails. Microsporum species infect the hair and skin. E. floccosum infects the skin and nails. When in doubt, remember that T. rubrum is the most common cause of ringworm. Subcutaneous Fungal Infections The subcutaneous fungal infections clinically significant for this guide include sporotrichosis and mycetoma. Sporotrichosis (also known as Rose Gardener s Disease) is caused by the fungus Sporothrix schenckii that produces nodular and ulcerative lesions that follow the lymphatics. Mycetoma, in contrast, is a symptomatic description of a large tumor-like subcutaneous lesion that can be caused by a number of etiologic agents including bacteria and fungi. Actinomycotic mycetoma is caused by aerobic Actinomycetes, which, as you remember, are filamentous bacteria that were originally mistaken for fungi. Eumycotic mycetoma is of fungal origin, the causative agent being one of several genera of filamentous fungi. For our purposes, the principle etiologic agents of eumycotic and actinomycotic mycetoma are Pseudallescheria boydii and Nocardia brasiliensis, respectively. 176
5 Sporotrichosis History: A sporotrichosis history will usually include a patient with exposure to the outdoors and a cutaneous lesion. Pathogenesis: (Subcutaneous infection). Sporothrix schenckii is introduced into the skin and subcutaneous tissue by trauma (such as pricking a finger on a rose bush). In cases of lymphocutaneous infection, lymphatics that drain the primary lesion transport the organism to regional lymph nodes. The tissue response is usually mixed, with suppuration and granuloma formation in the dermis and subcutaneous tissues. The lesions spread along routes of lymphatic drainage. Disseminated infection occurs as a result of hematogenous spread (typically in immunocompromised hosts). Lymphocutaneous Sporotrichosis Epidemiology: Sporotrichosis is found worldwide, most commonly in warm temperate climates, especially Mexico, Central and South America. Cases have been reported in almost every state. The mold form is commonly found in soil, on decaying vegetation, and on or in rose bushes, wood splinters, thorns, sphagnum moss, straw, potting soil and other woody plants. Infection is associated with gardening and any activity that enhances traumatic implantation of the organism into uncovered skin. (Aerosolization of the organism may result in pulmonary infection.) Diagnosis: The organism has a mold form in the environment (where it forms conidia in clusters or flowerettes) but has a yeast form in human tissue. S. schenkii has round to oval to elongated (cigar-shaped) yeast cells, 2-6 µm in diameter. Diagnosis is by direct observation or by serology, fungal stains, and fungal culture. S. schenkii yeast form S. schenkii conidia in flowerettes 177
6 Mycetoma (Actinomycotic and Eumycotic) Mycetoma is a symptomatic description of a bacterial or fungal infection that causes large, tumor-like swollen subcutaneous lesions that form draining sinus tracts and sulfur granules. Multiple species of fungi and aerobic actinomycetes (bacteria) are associated with mycetoma. Bacterial mycetoma is caused by Gram-positive aerobic branching Mycetoma bacteria; some may be partially acid-fast (e.g., Nocardia spp.). Fungal mycetoma is caused by hyaline or dematiaceous organisms such as Pseudallescheria boydii (most common) or Scedosporium apiospermum. Histories: outdoor activities and certain occupations, including walking barefoot or working in endemic areas that predispose one to mycetoma. Pathogenesis: Infection is acquired by traumatic implantation of organisms into the skin and subcutaneous tissue. The organisms form grains (granules). Some may produce extensive lytic lesions in bone. Diagnosis: Direct examination is helpful in confirming a diagnosis of mycetoma and determining whether it is bacterial or fungal. Grains of aerobic actinomycetes range Sulfur granule in size from 0.05 to 0.6 mm and are composed of delicate branching filaments. If the cause is fungal, a diagnosis may be possible based on direct microscopic examination. (P. boydii produces ascospores while S. apiospermum produces single oval to pear-shaped conidia at the tips of short or long conidiophores and may produce ascospores.) Fungal culture provides the most definitive proof of etiology Systemic Fungal Infections Systemic fungal infections are caused by truly pathogenic fungi, and can occur in immunocompetent hosts. We will explore four systemic fungal diseases: histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis. In studying the systemic fungi, it is particularly useful to pay attention to their geographic distribution, as they vary greatly in where they are found. Similarly, taking a complete and detailed travel history from a patient with a suspected fungal infection may provide clues as to the etiology. It is also important to note that all of the pathogenic (systemic) fungi are dimorphic, and that it is the mold form found in the environment that is usually infectious through the respiratory tract. In the human host, the systemic fungi exist as yeasts. 178
7 Histoplasmosis Organism: Histoplasma capsulatum Epidemiology: Histoplasmosis is found in the Mississippi, Ohio, Tennessee, and Missouri river valleys. States with highest prevalence are Missouri, Kentucky, Arkansas, Tennessee, Ohio, Illinois and Indiana. It can also be found around the world. It is found in areas where soil is contaminated with bird excrement, and also in association Geographic distribution of histoplasmosis with bat guano (e.g., in caves and old trees where bat roosting sites exist). History: Patients with histoplasmosis often have a history cleaning of old chicken houses, spreading chicken manure fertilizer, excavating of sites for construction, cleaning old bird roosting sites, cutting trees infested with bats, spelunking or demolishing contaminated building surfaces. Pathogenesis: Microconidia or hyphal fragments are inhaled and travel to the alveoli where they are phagocytosed by macrophages and convert to yeast forms. Lymphatics spread the organism to the mediastinal lymph nodes and to the systemic circulation where the rmononuclear phagocyte system is infected. Granuloma formation is characteristic. Calcification of primary and residual lesions is common. Disseminated disease may occur in immunocompromised persons. Yeast form of H. capsulatum Diagnosis: Microscopy is useful for detection of H. capsulatum. It may be seen on peripheral blood smear or impression smears of an oropharyngeal lesion. Small yeast cells, 2-5µm in diameter, may be seen within mononuclear cells in bone marrow or other tissues. Other tests include fungal culture, serology, PCR, and Histoplasma urine antigen detection. Blastomycosis Organism: Blastomyces dermatitidis Epidemiology: Blastomycosis is found in North America, primarily in the Ohio, Missouri and Mississippi river valleys, and especially in Kentucky, Missouri, Wisconsin, Iowa, Arkansas, Louisiana, Tennessee, Illinois and Wisconsin. (It is also found in Manitoba, Ontario, Alberta and Quebec and has been reported in Africa, South America, India, Eastern Europe, Israel, Saudi Arabia and Mexico.) Outbreaks have occurred in Minnesota, Iowa, Wisconsin and Illinois. Its ecology has not been well defined, but it is thought to be associated with soil, wood and/or areas near water. Yeast forms of B. dermatitidis with arrow pointing to a broad-based bud 179
8 History: Infected patients may have a history of activity associated with aerosolization of the organism from soil or wood. Pathogenesis: It is acquired by inhalation of infectious particles. Disseminated infection occurs as a result of hematogenous spread. Blastomycosis often has dermatologic manifestations, hence the name dermatitidis. Suppuration (abscess formation) is most often seen; granuloma formation may also occur. Diagnosis: Fungal culture and microscopic fungal examination of lower respiratory tract, bone, skin and/or mucous membranes is recommended. Direct microscopic exam may provide the earliest diagnosis; a KOH preparation is helpful. The pathologic finding is broad-based budding yeast cells, 8-15 μm in diameter. Other assays are serology, PCR, and Blastomyces urine antigen detection. Coccidioidomycosis (Valley Fever) Organisms: Coccidioides immitis (found in California); Coccidioides posadasii Epidemiology: This organism is found in the Southwestern region of North America (e.g., Arizona, California), as well as Central and South America, in the soil of areas that typify the Lower Sonoran Life Zone where semi-arid conditions exist. In these areas, temperatures reach 100 F during the summer and fall to near freezing in the winter. The soil is alkaline and has a high salt content supporting growth of short stubby vegetation such as the creosote bush, mesquite trees, cacti and yucca plants. Burrowing rodents found in these areas have been associated with distribution of the organism. These organisms disappear from the soil during the wet months but reappears during the dry summer and fall months. Winds disseminate arthroconidia that are inhaled by humans. Geographic distribution of coccidioidomycosis History: Infected patients may have a history of exposure to archaeological digs, dust storms, or construction or agricultural activities. Sometimes only a walk around a golf course or another area is enough to acquire an infection. Pathogenesis: Hyphae segment into rectangular arthroconidia. The often barrel-shaped arthroconidia alternate with empty disjunctor cells that fracture easily, releasing the arthroconidia. These are easily aerosolized; infection occurs via inhalation. In the body, arthroconidia transform into rounded spherules that are antiphagocytic. The spherule form contains endospores that resemble yeasts but do not exhibit budding. Diagnosis: Direct fungal microscopic examination of clinical specimens is useful. The microscopic finding is rounded spherules filled with non-budding endospores. Importantly, these organisms are extremely infectious in the mold form, and therefore hazardous to work with in the laboratory. The full battery of diagnostic tests includes fungal culture, serology, and PCR. 180
9 Paracoccidioidomycosis (South American Blastomycosis) Organism: Paracoccidioides brasiliensis Epidemiology: This infection is usually found in South America (e.g., Brazil). Diagnosis: On microscopy, the key finding is the ship s wheel morphology of buds around a single larger cell. Fungal culture may be helpful. Penicilliosis septa Organism: Penicillium marneffei Epidemiology: This infection is usually found in immunocompromised persons (eg. HIV-positive) who live in or have traveled to Southeast Asia. Diagnosis: On microscopy, the key finding after growth at C is a yeast-like cell that reproduces by fission with a central septa dividing the cell. Opportunistic Fungal Infections Opportunistic infections are caused by saprophytic (i.e., eat dead or decaying matter) organisms that are either normal flora or found in nature. Under normal circumstances the immune system prevents overgrowth of these organisms. An immunocompromised host may be susceptible to infections by opportunistic fungi. Factors that can contribute to the development of invasive opportunistic fungal infection include immunosuppression (e.g., cyclosporine, steroids, or neutropenia), damage to skin/mucosa (e.g., as a result of surgery or devices), malignancy, age extremes, alteration of microbial flora, chronic illness, renal/liver failure, and intensive care unit stays. There is a wide range of fungal organisms capable of causing opportunistic infections. The opportunistic infections we will focus on include zygomycosis, aspergillosis, candidiasis, cryptococcosis, and others (e.g., pneumocystosis, trichosporonosis, fusariosis, penicilliosis). In reality, any fungi found in nature may be opportunistic mycoses. 181
10 Mucormycosis (Zygomycosis) Organisms: The mucormycetes (zygomycetes) include members of the genus Rhizopus, Mucor, Lichthemia (Absidia) and others. Epidemiology: Mucormycetes are found worldwide and are ubiquitous in nature; they are found in or on fruits, bread, decomposing plants, grain, or soil (e.g., bread molds). History: Activities that provide an opportunity for inhalation of spores or traumatic implantation of an organism into tissue pose a risk for zygomycosis. Contaminated wound dressings have been associated with infection. Hyphae of a mucormycete Pathogenesis: Infection occurs in patients with diabetes or neutropenia, usually beginning in either the sinuses or lungs. Spores are deposited on the palate or nasal mucosa, spread through the sinuses to the orbital space to the brain. Pulmonary and disseminated infections may also occur. Mucormycetes are angioinvasive; vascular invasion and a propensity for spread through tissue planes are characteristic. Embolization, thrombosis, local ischemia and necrosis may occur. Diagnosis: Direct fungal examination and/or culture of respiratory tract, central nervous system, nose and sinus, and/or skin and mucous membrane specimens is recommended. Under the microscope, one sees fragments of large, ribbon-like, sparsely septate branching hyphae (the angle of branching is ~90, contrast this with Aspergillus, which is septate and branches at ~45 ). (The common mucormycetes may be separated into genera by their microscopic morphologic features: Mucor sp., no rhizoids; Rhizopus sp., rhizoids at the point where they connect to the hyphae; Lichthemia sp., rhizoids arising between areas where sporangiophores attach to hyphae.) Colonies are wooly and rapidly growing; they fill the culture dish overnight. These organisms invade vessels within the same time frame. In addition to direct exam and fungal culture, in situ hybridization probes can be used for identification of organisms in biopsy tissue. Hyphae of a mucormycete 182
11 Aspergillosis Organisms: About 200 species of Aspergillus are known, but only a few are clinically important. Aspergillus fumigatus, causes most cases of invasive disease. Aspergillus niger is a frequent cause of aspergilloma. Aspergillus flavus commonly produces disease in leukemic patients. Aspergillus terreus is becoming a more common cause of invasive disease. Many other species have been reported to cause disease but their frequency is low. Epidemiology: Aspergillus spp. have a worldwide distribution and are found on decaying vegetation, soil, grass, compost, tobacco, grain, Hyphae of Aspergillus sp. wicker, dust, raw construction materials, ground pepper, potted plants and ventilation systems. They are common colonizing agents of normal hosts. History: Patients often have a history of exposure to dusts produced by demolition or construction activities both exterior and interior, potted plants on hospital premises, handling of compost, grain, exposure to air from contaminated duct systems, or other activities associated with aerosolization of conidia of Aspergillus sp. into the environment. Cell mediated immune deficiency or neutropenia increase the risk of aspergillosis. Pathogenesis: Respiratory tract and disseminated infection occur after inhalation of conidia by the immunocompromised host. Aspergilli may infect the sinuses, external auditory canal, nails, or cornea, or grow in the bronchial tree eliciting a hypersensitivity reaction. Aspergillus infections can manifest in a number of different ways including allergic bronchopulmonary (ABPA) [septate and dichotomously (45 ) branching hyphae within mucous plugs in the lungs] and invasive aspergillosis [septate branching hyphae in the vasculature, often accompanied by hemorrhage]. An aspergilloma is a fungus ball in a previously existing cavity. Aspergillus sp. in tissue Diagnosis: Aspergillus sp. can be found in the respiratory tract or multiple systemic sites. On microscopic examination, look for septate, dichotomously branched hyphae, 3-10 μm in diameter. It is impossible to distinguish the hyphae of Aspergillus sp. from those of several other fungi. Fungal culture is recommended. Colonies vary from green to yellow to tan to black. Most species cannot be Aspergillus fungus ball (aspergilloma) in a recognized by the colonial morphology. Common species are easily small cavitary lesion identified based on microscopic examination of colonies. Identification is made based on the morphologic features of the "fruiting heads. Serology can be used for ABPA. Aspergillus antigen (galactomannan) detection or PCR may be helpful for invasive disease. 183
12 Candidiasis Organisms: Candida albicans (most common species), Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida dubliniensis, Candida krusei, and numerous other species of Candida cause candidiasis. Epidemiology: Most species are part of the normal flora of the mouth, skin, vagina, gastrointestinal or respiratory tracts; they are found worldwide. They are also found on plants, in water and dairy products, on fruits and vegetables, etc. Candida sp. is the most common cause of opportunistic fungal infections, and are the fourth most common cause of septicemia. C. albicans is the most frequent species found in clinical specimens; recently there has been a shift such that non-albicans Candida species are increasingly encountered. History: Infection is associated with interruption of mechanical barriers or indwelling intravascular catheters, gastrointestinal surgical procedures, foreign bodies (e.g., prosthetic joints, chronic indwelling urinary catheters, cardiac valve placement, pacemakers, severe burns, cardiac or dental surgery, injection drug use). Pathogenesis: Infections are usually endogenous in origin. There are two major forms of disease, mucosal candidiasis (e.g., oropharyngeal disease, vulvo-vaginitis, cystitis) and systemic candidiasis (e.g., candidemia, endocarditis, chronic disseminated candidiasis). Breaches in the mucous membranes of the host are of a common source of candidemia in neutropenic patients. A break occurs in a mucous membrane or tissue, the organism enters the Germ-tube formation by Candida albicans systemic circulation and becomes phagocytosed by neutrophils and monocytes where intracellular myeloperoxidase kills the organism (in patients with normal host defense mechanisms). In neutropenic patients or those with immune defects in which they lack myeloperoxidase, cells remain viable and easily spread into systemic circulation. Candida species are adept biofilm formers, contributing to the pathogenesis of foreign body-related infection. Diagnosis: The presence of budding yeast cells and pseudohyphae (constricted at the ends) and/or hyphae with regular septations are diagnostic. Candida sp. can be cultured from the blood using blood cultures designed to detect bacteria. Colonies of Candida are white to buff, pasty, and grow on usual laboratory culture media within hours. C. albicans produces germ-tubes (see photo). (The definitive identification of the species of Candida is based on characteristic biochemical profiles, nucleic acid sequences or protein analysis.) 184
13 Cryptococcosis Organism: Cryptococcus neoformans (also Cryptococcus gattii) Epidemiology: C. neoformans is found in association with pigeon excreta, in old buildings, barns, pigeon roosts and nests, in demolition sites and on window ledges. It is the most common fungus to infect the central nervous system (e.g., meningitis, meningoencephalitis). C. gattii has emerged in the Pacific Nortwest in the past several years and can affect immunocompetent individuals. History: Contact with dust in buildings contaminated with pigeon excreta, demolition of buildings, cleaning of old barns, and spelunking are risk factors. At risk India ink stain of Cryptococcus patients often have compromised cell-mediated immunity. neoformans highlighting the capsules Pathogenesis: The portal of entry is the respiratory tract from which hematogenous spread may occur. C. neoformans has a marked affinity for the central nervous system (but may infect to any part of the body). The organism has an antiphagocytic capsule that is solubilized over time. Diagnosis: Cryptococcus capsular antigen can be sensitively and rapidly detected in blood and/or cerebrospinal fluid. Fungal stains and fungal cultures of clinical specimens are also helpful. C. neoformans yeast cells are spherical, vary in size from 2-15 μm, have narrow-necked buds (contrast with B. dermatitidis), and often times are encapsulated. The capsule can be visualized with India ink). Cultured organism is identified by urease production, carbohydrate utilization, and pigment production on niger seed agar. Pneumocystosis Organism: Pneumocystis jiroveci (formerly Pneumcocystis carinii) is different than the other fungi we have been discusing. It was previously classified as a protozoan parasite, is susceptible to antibacterial agents to which other fungi are not (e.g., trimethoprim-sulfamethoxazole), and lacks ergosterol in its cell membrane (which other fungi possess). Is not susceptible to amphotericin B (which targets ergosterol) and has a fragile cell wall compared to other fungi. Trimethoprim-sulfamethoxazole is commonly given to at-risk patients for prophylaxis (and can also be used for therapy). Multiple other agents can be used for treatment, P. jiroveci including pentamidine. Pathogenesis: It exclusively causes disease in patients with compromised cell-mediated immunity (e.g., human immunodeficient virus-infected patients) and is acquired by inhalation. It causes an interstitial pneumonia; the alveoli become filled with a foamy alveolar exudate containing cells, degenerated cell membranes and host proteins. Gas exchange can be seriously compromised. Disseminated disease can (rarely) occur. Diagnosis: P. jiroveci cannot be cultured in vitro. Trophozoites make up a majority of the organisms in the lung, though thick-walled cysts containing intracystic bodies may also be present. Diagnosis is made by stain of lung biopsy or bronchoalveolar lavage fluid. (The best stains to use are methenamine silver stain, calcofluor white or Giemsa stains.) PCR, performed on respiratory secretions, is also available for the diagnosis. 185
14 Therapy for Fungal Infections Polyene antifungal agents include amphotericin B and nystatin. The polyenes bind to sterols in the fungal cell membrane and increase permeability by punching holes in the membrane. Amphotericin B is active against most pathogenic fungi, including yeasts and filamentous fungi. It is administered intravenously. Nephrotoxicity is a dose limiting adverse effect. Infusion related side-effects include chills, fever, tachypnea, high and low blood pressure. Other dose dependent effects include renal toxicity, anemia, and potassium wasting. Lipid formulations of amphotericin B such as Ambisome (a liposomal compound) are most commonly used in clinical practice. The lipid formulations are less toxic than the older deoxycholate formulation but significantly more expensive. Azole antifungal agents are cytochrome p450 inhibitors that block the conversion of lanosterol to ergosterol, interfering with fungal cell membrane synthesis. They include ketoconazole, fluconazole, itraconazole, voriconazole and posaconazole. [Ketoconazole is an oral fungistatic azole active against Candida sp., H. capsulatum, B. dermatitidis, and C. immitis, but not against Aspergillus sp. and the mucormycete. It requires acid ph for absorption from the gut. It is metabolized in the liver. It has several drug interactions (e.g., rifampin) and can cause hepatitis, nausea, and sterol synthesis inhibition (i.e., decreased androgens). It is now rarely used as systemic antifungal therapy because other azoles have fewer toxicities.] Fluconazole is active against Candida sp., C. neoformans, and C. immitis. C. glabrata (usually), C. krusei, Aspergillus sp., and the mucormycetes are resistant to fluconazole. It can be given orally or intravenously. It has good oral bioavailability and achieves high cerebrospinal fluid levels. It is excreted in the urine. Fluconazole is less toxic than ketoconazole, although there are still drug interactions (e.g., rifampin) and potential hepatotoxicity. Itraconazole has a similar spectrum to ketoconazole with the addition of Aspergillus sp. and some dermatophytes. It is available as both capsule (requires acid ph for absorption) and liquid (somewhat better absorption) formulations. It is metabolized by the liver. C. glabrata (usually), C. krusei, and the mucormycetes are resistant to itraconazole. Its toxicities are the same as ketoconazole. Voriconazole has the same spectrum as itraconazole with the addition of improved activity against Aspergillus sp. The mucormycetes are resistant to voriconazole. It is available as both oral and intravenous formulations. The intravenous form must be used with caution in patients with renal failure. Voriconazole is metabolized by the liver. Its toxicities and drug interactions are the same as fluconazole. Additionally, it can cause visual disturbances. There are some data that suggest that Voriconazole treatment predisposes patients to infection by mucormycetes. Posaconazole has the spectrum of voriconazole but is also active against mucormycetes. It is only available in an oral formulation. Drug absorption is variable and enhanced by coadministration with fatty foods. Posaconazole is metabolized by the liver. Its toxicities and drug interactions are similar to those of itraconazole. Echinocandins inhibit synthesis of ß (1, 3)-D-glucan, a component of the fungal cell wall. Echinocandins have relatively few side-effects, as this enzymatic pathway is not found in humans. They are active against all species of Candida and Aspergillus. Caspofungin, micafungin and anidulafungin are the currently available echinocandins; all are only formulated for intravenous administration. 186
15 Flucytosine (5-fluorocystosine) is an oral DNA synthesis inhibitor (a fluorine analog of cytosine.) It is used in combination with amphotericin B to treat some cases of cryptococcal meningitis and some candidal infections. It can cause potentially fatal bone marrow suppression, hepatic dysfunction, or enterocolitis. Terbinafine is a synthetic allylamine that interferes with ergosterol synthesis by inhibiting fungal squalene epoxidase. It is given orally and only achieves therapeutic concentrations in the skin and nails. It is used for treatment of onychomycosis and fungal dermatophyte infections. 187
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