Cryptococcus neoformans
Authors: Arturo Casadevall1, John R. Perfect2Category: Fungi and Fungal Pathogenesis
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The dramatic increase in the incidence of Cryptococcus neoformans infections calls for an up-to-date look at its biology and pathogenesis. This volume provides a comprehensive review of the ecology, epidemiology, pathogenic mechanisms, host immunity, clinical features and diagnostic conditions, and principles of treatment and prevention of this important fungal pathogen.
The only recent monograph on the subject, Cryptococcus neoformans goes beyond textbook chapters and journal articles to provide the most comprehensive, current information on basic science and clinical information relating to this fungus. The authors, investigators who have direct experience with the subject matter, provide objective data as well as interpretations and opinions.
Cryptococcus neoformans, a model organism for the understanding of disseminated mycoses, has become a major concern facing infectious disease physicians and immunocompromised patients. Now this valuable resource provides both experienced investigators and those new to the field of medical mycology an opportunity to review the vast database of knowledge on this major worldwide pathogen.
Electronic only, 541 pages, illustrations, index.
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Chapter 1 : Introduction to the Pathogen
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This chapter begins with the history of Cryptococcus neoformans. By the end of the 19th century three seminal observations had been made regarding C. neoformans. First, the organism had been recovered from lesions in humans and animals, establishing its potential to cause disease. Second, the organism had been recovered from the environment, establishing that it was free-living. Finally, the organism was propagated in the laboratory and shown to cause disease in laboratory animals. Many of the early clinical reports of cryptococcosis included animal experiments with the organism isolated from patients. Examination of C. neoformans by light microscopy reveals yeast-like cells that reproduce by budding and are of variable size. C. neoformans has been extensively studied by electron microscopy, which shows that C. neoformans cells have morphological features typical of eukaryotic cells. There are many reports in the literature of C. neoformans cells with germ tube-like structure, hyphal forms, pseudohyphae, and sporulation. For an extensive discussion of the effect of various environmental conditions on the survival the effect of temperature, pH, sunlight, and moisture are briefly discussed in the chapter.
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Chapter 2 : Taxonomy
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Members of the genus Cryptococcus generally are considered to be yeasts that are nonfermentative, assimilate inositol, and produce urease. Besides the pathogenic Cryptococcus neoformans, at least 38 other Cryptococcus species have been found in a wide variety of environmental locations. Humans are frequently exposed to these species, which can be found in aqueous environments, soil, human skin such as toe webs, and bird guano, but for all practical purposes C. neoformans should be considered the only Cryptococcus species to consistently cause cryptococcosis. Despite the differences in morphology, biochemistry, ecology, and pathogenicity, F. neoformans and filobasidiella depauperata are phylogenetically related and belong in the same genus based upon DNA homology. Although taxonomic relationships between Cryptococcus and Tremella have been based on fatty acid compositions and other phenotypic characteristics, molecular phylogeny studies provide the most objective evidence for genus and species relationships. The molecular phylogeny of Filobasidiella in relationship to other closely related fungi has continued to be elucidated. As more genetic loci, such as the conserved actin gene, are sequenced and compared, the phylogenetic relationships on a molecular level can be interpreted further with regard to classical methods of taxonomy.
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Chapter 3 : Ecology of Cryptococcus neoformans
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In the past half-century, considerable progress has been made in understanding the ecology of Cryptococcus neoformans and the epidemiology of cryptococcal infections. Among the most significant observations were the division of C. neoformans strains into the varieties neoformans and gattii on the basis of phenotypic, biochemical, and genetic differences; the association of C. neoformans var. neoformans with avian excreta; and the association of C. neoformans var. gattii with eucalyptus trees. This chapter describes the distinctions between the two C. neoformans varieties and reviews the available information on the ecology of this fungal pathogen. C. neoformans strains have been divided into two varieties on the basis of phenotypic, serological, genetic, biochemical, and epidemiological criteria. Sexual reproduction of C. neoformans results in the production of haploid basidiospores. The question of which mode of reproduction is employed by C. neoformans in the environment may be important for a complete understanding of its ecology and pathogenesis. Recently, other C. neoformans characteristics have been suggested to contribute to virulence, including mannitol production and the elaboration of several enzymes that have the potential to degrade tissue, such as proteases and phospholipases.
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Chapter 4 : Biochemistry
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This chapter surveys the available information on Cryptococcus neoformans biochemistry. The C. neoformans capsule is important for virulence and, as a result, the capsular polysaccharide has been studied extensively. Glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoprotein (MP), the three major components of the C. neoformans capsular exopolysaccharides, can each elicit antibody responses, but only the MP component elicits cell-mediated immunity as measured by delayed-type hypersensitivity reaction. The immune response to C. neoformans polysaccharide antigens is discussed. The detection and analysis of capsular polysaccharides in tissue remain dependent upon serological assays. Many assays have been described for the measurement of cryptococcal capsular polysaccharides based on the use of antibody reagents. Electron microscopy of the process of cell wall digestion with snail gut enzymes shows that protoplast-spheroplast formation is a two-stage process. First, the enzymes induce a hole in the equatorial region of the cell wall, through which the protoplast-spheroplast emerges from a cell wall “ ghost”. Second, continued digestion of the cell wall leads to the disappearance of these structures. The biochemistry of melanin and the assembly of melanin on the cell wall remain poorly understood. Melanogenesis is interesting because of its association with virulence and because it is a potential target for antifungal drug design. Mouse passage of environmental C. neoformans isolates produced isolates with higher amphotericin B and fluconazole MICs, suggesting that sterols and antifungal drug resistance could be altered by mammalian infection without a need for exposure to antifungal drugs.
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Chapter 5 : Molecular Biology
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The understanding of the epidemiology and pathogenic features for Cryptococcus neoformans infections can now be aided by the use of molecular biological techniques. In the last three decades, model systems for eukaryotic molecular biology have developed around certain fungi, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, and Aspergillus nidulans. In the late 1980s, studies for the present foundation of molecular biology in C. neoformans began. The early development of molecular biological techniques for studying a pathogen generally involves questions of epidemiology in relationship to pathogenicity. In fact, little work has yet been performed on the genetics and molecular biology of mitochondrial DNA in C. neoformans. The use of polymerase chain reaction (PCR) technology combined with repetitive primers (arbitrary primed PCR) has allowed investigators to identify the DNA fingerprints of different fungal strains rapidly, specifically, and with a minimal number of DNA purifications. Recent work in molecular phylogeny of C. neoformans has focused on other genes and their ancestral lineages. Investigations into C. neoformans gene structures and the understanding of gene regulation in this yeast are in their infancy.
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Chapter 6 : Virulence Factors
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A common term in the description of pathogens is virulence factor. A fungal infection is generally an accidental encounter in the life cycle of the fungus. It is likely that there are many subtle virulence factors in fungi, such as the simple ability to survive and grow within the host, that are important to their ability to be pathogens. The focus on Cryptococcus neoformans and its melanin production has two potential benefits. First, it may help to understand the regulation of virulence of this infection with a well-defined phenotype that is easy to measure. Second, with further understanding of the molecular biology and biochemistry of melanin, it could become a unique and selective target for antifungal drugs against C. neoformans and other dematiaceous fungi. Identification and characterization of extracellular proteins in C. neoformans are important because proteinases, esterases, and lipases have been associated with virulence in other pathogens, and these proteins may also elicit strong immune responses from the host. The potential importance of urease in the virulence composite could involve its ability to change local pH in the host, such as when the yeast resides in a phagolysosome. Production of the phospholipases appears to be extracellular, and at least one enzymatic component appears to be phospholipase B.
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Chapter 7 : Physical Defenses and Nonspecific Immunity
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The low frequency of symptomatic cryptococcal infections, despite the presumably high frequency of exposure, suggests that physical barriers and nonspecific immunity provide adequate defenses to protect the host against infection. This chapter describes physical barriers such as skin, nasal passages, and eye, and humoral and cellular nonspecific defense mechanisms such as polymorphonuclear cells (PMNs), natural killer (NK) cells and macrophages that are believed to provide the first line of defense against cryptococcal infection. Antibody-mediated phagocytosis occurs through Fc receptors, which are constitutively expressed in macrophages, but it should be emphasized that opsonic capsule-binding antibody is not consistently present during human and animal infections. In human macrophages, complement-mediated binding is an energy-dependent process that requires three C3 complement receptors (CR1, CR3, and CR4) and actin but does not necessarily lead to phagocytosis. In summary, there is considerable evidence that macrophages from patients with AIDS are less effective against Cryptococcus neoformans than are those from HIV-seronegative individuals. The relative importance of oxidative and nonoxidative antimicrobial mechanisms of host effector cells in inhibiting and killing C. neoformans is uncertain. In vivo, it is likely that the oxidative and nonoxidative antimicrobial mechanisms cooperate to produce the antifungal effects described for host effector cells.
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Chapter 8 : Specific Immunity and Cytokines
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Specific immune mechanisms are composed of humoral and cellular arms. Specific immunity develops in response to infection or immunization with Cryptococcus neoformans products and functions to contain and/or eradicate infection. The human antibody response to C. neoformans consists of antibodies to the polysaccharide capsule and to protein antigens. The antibody response to the capsular polysaccharide antigen has been extensively studied. The conjugate vaccine strategy is based on the premise that high-titer antibody responses in patients at risk for C. neoformans infection can enhance the efficacy of nonspecific (e.g., macrophage, neutrophil, natural killer cell) and cellular immunity to prevent clinical infection. In summary, the role of humoral immunity in defense against C. neoformans is complex and incompletely understood. There is convincing evidence that administration of glucuronoxylomannan (GXM)-binding monoclonal antibodies (MAbs) can protect mice. The study of cytokines in mice is aided by the availability of many murine reagents and several mouse strains deficient in specific cytokines. Many cytokines have now been shown to have an important role in host defense against cryptococcal infection.
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Chapter 9 : Tissue Responses and Special Topics in Immunity
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This chapter explores the host response to infection, with emphasis on integrating clinical observations with those from animal studies and in vitro observations. The chapter describes the host response to infection for lung, brain, and skin, since these are the organs for which most information is available. The inflammatory response to Cryptococcus neoformans infection has four unusual features. First, the inflammatory response can range in intensity from florid, intense granulomatous inflammation to the virtual absence of inflammatory cells. The variables responsible for differences in the magnitude and intensity of the inflammatory response include the immune status of the host and poorly defined characteristics of C. neoformans strains. Second, C. neoformans can be found in tissue as both an intracellular pathogen and an extracellular pathogen. Third, the type of inflammatory response is a function, in part, of the organ tissue where the infection occurs. Fourth, there is considerable variation in the type of effector cells found in inflammatory responses. A study compared the inflammatory response to pulmonary infection in CBA/J mice using two strains of C. neoformans, and strongly suggested that some of the differences observed in the human inflammatory response may be the result of unique C. neoformans strain differences in host inflammatory cell recruitment. In summary, the literature strongly supports the view that impairment of cell-mediated immunity is associated with enhanced susceptibility to C. neoformans infection.
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Chapter 10 : Animal Models and Veterinary Aspects of Cryptococcosis
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Cryptococcus neoformans has been reported to cause infection in a wide variety of mammalian hosts. The clinical history of both natural and experimental animal cryptococcosis began over 100 years ago with the initial description of the pathogenic yeast. It was at this time in the late 19th century that experimental animal models of cryptococcosis were first used. Several pathological findings in this study were of further interest. Mice are extremely attractive for the study of cryptococcosis for two very important reasons. First, they provide both a very uniform set of immune responses and a wide variety of these responses in a series of inbred strains. The second reason that mice are attractive for the study of cryptococcosis is the ability to immunologically alter them, either genetically or through immunological reagents. Mouse models have also been used to dissect the importance of specific cell-mediated immunity in cryptococcosis. The rabbit is an interesting model for the study of cryptococcosis and has both positive and negative features for the study of cryptococcal pathogenesis. Although much of the discussion on animal model systems has emphasized the four major models, C. neoformans has also been inoculated into other animals.
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Chapter 11 : Epidemiology
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The epidemiology of cryptococcal infection is an important undertaking because it provides clues as to the pathogenesis of infections and permits an assessment of infection risk in a given population. Knowledge of how and where Cryptococcus neoformans infects the human host is crucial for understanding the epidemiology and pathogenesis of infection. In a study the mean time that patients were on immunosuppressive therapy before the onset of cryptococcal infection was 48 months, and mortality was 30%. Among neoplastic disorders, disseminated cryptococcal infection has been historically associated with lymphoproliferative malignancies. Cryptococcosis is a relatively common infection among renal transplant patients despite the fact that renal transplantation has the lowest incidence of fungal infection among solid organ transplantation procedures. Insights on the epidemiology and pathogenesis of infection have resulted from comparative studies of cryptococcosis in various geographic regions. Researchers have interpreted the genetic diversity of C. neoformans strains as consistent with the theory of adaptive polymorphism, which predicts that the genetic diversity of a species is proportional to the diversity of ecological sites occupied.
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Chapter 12 : Diagnosis and Laboratory Techniques
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The India ink examination is a particularly useful and rapid diagnostic test for cryptococcal meningitis. Although Cryptococcus neoformans can become fragile, collapse, or be crescentic when dried or fixed, the encapsulated cells are rapidly distinguished in a colored colloidal medium such as India ink when mixed with fluids such as cerebrospinal fluid (CSF). There are two clinical situations in which cultures of the CSF for identification of viable C. neoformans may be difficult. First, in some cases of chronic cryptococcal meningitis, the lumbar fluid is found to be culture negative. Second, some patients who receive an antifungal treatment course will have positive India ink examinations at the end of therapy despite clinical improvement. Serological tests for the diagnosis of cryptococcosis are one of the true success stories for rapid and accurate diagnosis of fungal infections in the last 30 to 40 years. The popularity of the diagnostic test for cryptococcal polysaccharide antigen is readily apparent when it is noted that there are at least five commercial latex agglutination kits for cryptococcal polysaccharide. DNA and RNA isolation, karyotyping, and DNA transformation techniques are discussed because they are more C. neoformans-specific; most other molecular techniques for working with C. neoformans are standard molecular biology procedures.
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Chapter 13 : Human Cryptococcosis
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In the first 100 years of clinical cases of Cryptococcus neoformans, the infection was described in most organs of the human and animal host. The clinical presentations of human cryptococcosis may be varied, and because of the variety of signs and symptoms, clinical diagnosis without laboratory studies can be difficult at times in both high- and low-risk patients. Clinical manifestations by organ system and demonstrates the breadth of clinical signs attributed to C. neoformans such as central nervous system (CNS), lung and skin. The relative differences in frequency of clinical symptoms and findings are compared in patients with or without human immunodeficiency virus (HIV) infection. A new factor in liver transplants has been the use of tacrolimus and its possible effect on cryptococcosis. It is known that all the immunophilins (cyclosporine, tacrolimus, and sirolimus) have anticryptococcal activity in vitro, but the effect of these medications on human cryptococcosis in this group remains uncertain. A review of pulmonary infections in immunocompromised patients prior to the AIDS epidemic described 34 of 41 patients with pulmonary cryptococcosis who had an underlying immunocompromised condition other than HIV. The most common radiographic findings (in order of frequency of occurrence) were alveolar or interstitial infiltrates, single or multiple-coin lesions, masses, cavitary lesions, and pleural effusions. A clinical principle emphasized in this study is that immunocompromised patients with pulmonary cryptococcosis frequently have disseminated infection and always require antifungal therapy. In animals, C. neoformans strains have been identified that appear to localize/produce infection directly in skin tissue.
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Chapter 14 : Therapy of Cryptococcosis
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The hypothesis that estrogen therapy might be useful in treatment was based on the observation that in both humans and animals, males appear to be more susceptible to cryptococcosis than females. Infact, Mohr et al. performed two insightful studies on sex hormones. These studies showed that patients with cryptococcal meningitis who were given doses of the estrogenic hormone diethylstilbestrol increased their host-cell phagocytic capacities. This work, like serotherapy, was one of the first studies on immune modulation for treatment of cryptococcosis. Shadomy et al. examined 77 Cryptococcus neoformans strains against amphotericin B, hamycin, and flucytosine, using a broth dilution technique, and found that all strains were very susceptible to amphotericin B at MICs of <0.2 μg/ml. In this work, the importance of media for these antifungal tests was first recognized. With the combination of more antifungal agents and the concern about antifungal drug resistance, there was a renewed focus on antifungal drug susceptibility testing. Flucytosine has been very successfully combined with amphotericin B to take advantage of their synergistic or additive mechanisms of actions and their different but complementary pharmacokinetics. First, this combination has reduced concerns about the development of drug-resistant strains and, in fact, flucytosine resistance has not generally been reported during combination therapy. Second, the combination therapy has allowed a reduction in the amount of amphotericin B needed to treat cryptococcosis, and this helps to limit its known toxicity.
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Chapter 15 : Prevention of Human Infection
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The benefits of prevention are obvious given that cryptococcal infections require prolonged therapy, have high mortality and morbidity despite appropriate therapy, and are virtually incurable in patients with severely impaired immune function. The inadequacy of existing therapies is further exemplified by their inability to eradicate most cryptococcal infections in patients with severe immunological impairment, such as those with late-stage human immunodeficiency virus (HTV) infection. Chemoprophylaxis involves the administration of antifungal drugs to high-risk patients to reduce the probability of primary infection or recurrence of infection. The introduction of safe and effective oral azole drugs in the early 1990s made feasible the design of effective, low-toxicity regimens for preventing cryptococcal infections. The experience with Cryptococcus neoformans infections in patients with AIDS is different from that in other patient groups in that cryptococcal infection in patients with AIDS is seldom curable. It is important to consider that non-antifungal drugs and/or dietary practices may conceivably reduce the risk of cryptococcal infection in some patients. The availability of a sensitive and specific diagnostic test for C. neoformans infection in the form of the cryptococcal antigen detection assay suggests the feasibility of screening high-risk populations for early signs of cryptococcosis. Several studies have shown that screening of high-risk patients for cryptococcal antigen can identify some cases of cryptococcal infection. The conjugate vaccine is based on the premise that a vaccine that elicits a strong antibody response will protect against C. neoformans infection in susceptible hosts.
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