Cryptococcus: From Human Pathogen to Model Yeast

This chapter focuses on the genetic diversity of Cryptococcus species as assessed by sequence analysis of DNA. In 1935, a comprehensive study was conducted with numerous yeasts, which included 22 strains of pathogenic and nonpathogenic Cryptococci isolated from humans. Prior to the discovery of the genus Filobasidiella, the teleomorph of Cryptococcus neoformans and C. gattii, yeast taxonomists had speculated that C. neoformans belonged to the Basidiomycota for the following reasons. First, it is the only basidiomycetous genus that contains human pathogens that produce life-threatening infection of the central nervous system in immunocompromised as well as immunocompetent patients. Second, Filobasidiella is the only genus in the phylum Basidiomycota that produces long chains of basidiospores on the apex of the holobasidia by repetitive basipetal budding. Third, the genus contains two groups of species, one with and one without ontogenetic yeast stages. Due to taxonomic confusion about the status of the type species of the genus Cryptococcus, as well as the widely recognized importance of C. neoformans and C. gattii as human pathogens, the genus was neotypified by C. neoformans. This revision implies that if one uses a modern phylogenetic genus concept, only those anamorphic species that belong to the Filobasidiella clade should be classified as Cryptococcus species, e.g., C. neoformans, C. gattii, and C. amylolentus. Among the Tremellales, species classified as Cryptococcus occur intermingled with species of diverse genera such as Bullera, Tremella, Papiliotrema, Auriculibuller, Trimorphomyces, Kwoniella, Fibulobasidium, and Bulleribasidium.

This chapter considers over a century of clinical experience to examine the encapsulated yeast Cryptococcus, to identify the highlights of its maturation in identifying, understanding, diagnosing, and managing this major emerging fungal pathogen. In the areas of pathogenesis, diagnosis, and treatment of cryptococcosis, there are myriad events to report. A study conducted in 1998 demonstrated that cryptococcus could demonstrate morphological flexibility in that under certain environmental conditions the yeasts produce hyphae and spores without a mating reaction; in other words, some cryptococcal strains were found to have the ability to haploid fruit, and thus a new part of the life cycle of cryptococcus was identified. Another study introduced the rigorous methodology of comparative, randomized trials into antifungal therapy and particularly cryptococcosis. This study validated the efficacy of combination antifungal therapy for cryptococcosis, and represented a major starting point not just for cryptococcosis but also for other invasive fungal infections. This study initially provided much of the infrastructure for understanding the management of cryptococcal meningitis. The first large outbreak of cryptococcosis was associated with cows and mastitis in the 1950s, probably related to direct inoculation with contaminated fomites. The history of cryptococcosis has been dynamic and continues to evolve. From its humble beginnings as single case reports to its model system status in molecular studies for fungal pathogenesis, it has become a sophisticated complex organism for study and management.

Until the late 1940s, little was known about cryptococcal capsule composition or structure. Classically, the capsule is described as composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins, based on the original fractionation of shed polysaccharide material. Cell wall polymers are involved in capsule association with the cell, although they are not considered part of the capsule itself. Polysaccharide synthesis starts with the generation of precursor molecules, the most common being the activated sugars discovered by Leloir. A number of proteins involved in the synthesis of these precursors have been identified and studied in Cryptococcus neoformans. This work has been facilitated by the fact that many of these enzymes are highly conserved in terms of sequence, as would be expected from the participation of activated sugar precursors in multiple synthetic pathways across biology. In support of a lumenal location for capsule synthesis, a conditional mutant generated in both serotypes A and D that is defective in vesicle targeting to the plasma membrane accumulates post-Golgi vesicles containing GXM. The conclusion from this study is that GXM is made within the classical secretory pathway, consistent with the requirement for nucleotide sugar transporters to achieve normal capsule synthesis.

This chapter focuses on chemical, physical, antigenic, architectural, and dynamical properties of the polysaccharide capsule. It examines how changes in these parameters can influence the interaction with the host and the virulence of the yeast. The capsule is composed mainly of polysaccharide. The chemical composition of the capsule is responsible for several physical characteristics. The chapter reviews the antigenic properties of the capsule and the structural properties that could have important consequences during infection. Many different monoclonal antibodies (MAbs) that specifically recognize the Cryptococcus neoformans capsule have been obtained. The study of these antibodies has contributed to the identification of multiple structural features of the capsule. Further, the chapter discusses other important processes involved in the physical organization of the capsule, such as the polysaccharide transport mechanisms and anchoring of the polysaccharide fibers to the cell wall. Anchoring of capsular components to the cell wall is crucial for capsule assembly. However, additional interpolysaccharide interactions are expected to occur at the capsular microenvironment. The current literature indicates that at least four types of polysaccharide-polysaccharide interaction occur at the cryptococcal capsule-cell wall interface, including glucuronoxylomannan (GXM)-GXM, GXM-galactoxylomannan (GalXM), GXM-glucans, and GXM-chitin.

There is an extensive body of evidence from several laboratories that establishes a role for melanization in virulence, providing a fascinating example of how a microorganism can utilize a ubiquitous pigment to undermine host defense mechanisms. Melanin-deficient mutant strains generated by UV-irradiation are avirulent in murine models of cryptococcal infection. The majority of Cryptococcus neoformans melanin is produced by Lac1, although a second laccase enzyme, Lac2, adjacent to LAC1 in the genome exists. Various microscopy techniques were used to develop the melanin model, including atomic force and scanning electron microscopy to examine the surface structure of melanin, transmission electron microscopy (TEM) to study cross sections of melanin, and nuclear magnetic resonance (NMR) cryoporometry to investigate the porosity of melanin. In the environment, melanin is thought to protect C. neoformans from various stresses including enzymatic degradation, radiation (UV, solar, gamma), and heavy metals (silver nitrate) while providing thermotolerance and structural integrity to withstand osmotic challenges. Melanin-binding drugs may be useful as therapeutics against C. neoformans infection. In fact, administration of melanin-binding monoclonal antibodies (MAbs) prolongs the survival of mice in a lethal intravenous infection model. In this study, the fungal burdens of collected brains and lungs of infected mice were taken 7 days after infection, and mice administered MAbs to melanin had significantly lower fungal burdens than control mice administered irrelevant immunoglobulin. Early studies that linked melanin to C. neoformans virulence used classical genetic techniques to generate mutants through nonspecific mutagenesis to identify yeast cells deficient in melanin production.

The fungal cell wall is an essential organelle, vital for maintaining cell integrity against various chemical, physical, and biological stressors. This chapter covers work on the cell wall of Cryptococcus, drawing on studies from fungi such as Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Histoplasma capsulatum, and Aspergillus fumigatus, for introduction and comparison. Certain cell wall components can be up- or downregulated in response to various external conditions. The fungal cell wall contains several different components, and much is understood regarding their structures and mechanisms of synthesis. Certain proteins, such as Gas1p and Bgl2p, have been proposed to function in cell wall organization, but definitive studies are still needed. Future investigations in this area promise to dramatically increase our understanding of cell wall assembly and dynamics. Our understanding of the evolution of fungal cell wall proteins will advance with fungal genome sequencing. Studies of Cryptococcus neoformans have revealed that its cell wall is very different from and more complex than other well-characterized fungal cell walls. Mutants of Cryptococcus lacking β-1, 6-glucan, and therefore having decreased retention of cell wall proteins, appear to have shifts in chitosan localization. The fungal cell wall remains the most attractive target for the next generation of antifungal drugs.

This chapter reviews the discovery of both opposite-sex and unisexual reproduction and illustrates how these pathways are molecularly controlled and the central cell biology questions that remain to be addressed. Fungal sexual reproduction is genetically regulated by the mating-type locus (MAT), a specialized region of the genome that is idiomorphic or allelic between different sexes. Similar to the model fission yeast Schizosaccharomyces pombe, mating and meiosis of Cryptococcus neoformans occur sequentially in response to limitation or specific nutrient cues. Other environmental cues such as light and temperature also affect mating of C. neoformans. Those factors that have been connected to the sexual reproduction of C. neoformans are summarized in the chapter. Furthermore, several Cryptococcus species can be isolated from trees and fermenting fruits, suggesting that there might be potential plant-fungus interactions that contribute to sexual reproduction of C. neoformans and Cryptococcus gattii in nature. Apart from nutritional cues, other environmental signals also influence sexual reproduction. However, the predominance of the α mating type (>99%) in the Cryptococcus population represents a paradox as to how sexual reproduction might occur in this essentially unisexual population. The discovery of same-sex mating in C. neoformans resulted in a paradigm shift in considering how genetic diversity is generated in a unisexual population and the evolutionary role of a unisexual reproductive mode. Moreover, the transitions that occur in the fungal kingdom between heterothallic outbreeding and homothallic inbreeding modes promise to reveal general features by which sexual reproduction enhances fitness and enables evolutionary success throughout biology.

Cryptococcus neoformans and Cryptococcus gattii comprise the pathogenic Cryptococcus species complex. This chapter focuses on the population structure and ecology of C. neoformans and C. gattii. It summarizes the current knowledge of the population structure of C. neoformans and C. gattii and discusses the associations of the genetically isolated subpopulations with their ecological niches. The most reliable methods developed to genotype individuals use markers that detect polymorphisms among microsatellites and similar repetitive DNA elements, restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphisms (AFLP), and direct DNA sequencing, such as multilocus sequence typing (MLST). Most studies of the population genetics of the pathogenic species of Cryptococcus use reference strains to recognize the major clades or subpopulations. These are designated as VNI through VNIV and VNB for C. neoformans and VGI through VGIV for C. gattii. A table shows the current designation of the nine major subpopulations of the C. neoformans/C. gattii complex and their relationships to the conventional serotypes. This method of serotyping served for years to separate C. neoformans (serotypes A, D, and AD) from C. gattii (serotypes B and C). Analyses of the population structure presented in the chapter are concerned with (i) genetic diversity, (ii) mode of reproduction (clonality versus recombination), and (iii) population subdivision. The chapter also talks about the advances in the understanding of the intertwining effects of ecology and genetics on the lifestyle of C. neoformans and C. gattii.

This chapter describes the genomic resources available for Cryptococcus neoformans and C. gattii and discusses selected examples of the application of the resources to address questions relevant to virulence. It appears that in C. neoformans, natural selection is gradually lengthening short introns and shortening longer introns toward a modal size, which presumably provides an increased level of evolutionary fitness. The current picture, however, may be refined in light of future sequencing of related genomes. Although several studies employed subtractive hybridization methods and differential display to identify genes with specific patterns of regulation, the chapter focuses on experiments with microarrays and serial analysis of gene expression (SAGE) methods. The influence of temperature on gene expression with a shotgun genomic DNA microarray containing 6,274 elements is described in the chapter. The shotgun microarray was used in additional transcriptome profiling experiments to identify targets of Mga2, and these included genes for fatty acid biosynthesis. This study serves to focus attention on fatty acid and sterol metabolism as important aspects of the response to temperature and other stresses. The genome sequences have enabled a series of transcriptome analyses with oligonucleotide microarrays and serial analysis of gene expression. The studies on genome sequences are becoming standard in the analysis of complex traits and the impact of mutations and drug treatments. There is a clear need for a central, curated database for the cryptococcal genome sequences and related resources such as mutant phenotypes, transcriptome and proteome data, protein interaction data, literature, and community information.

Infection with Cryptococcus species is often considered an AIDS-defining illness, the outbreak of Cryptococcus gattii on Vancouver Island underscores the broad potential of the Cryptococcus neoformans species complex to evade host immune defenses and cause disease. There are several simple developments that need to be made to maximize the use of C. neoformans genomes. One development is the publication of the genomes of the serotype A and C. gattii strains and a finalized nomenclature for each gene in each strain sequenced, preferably as similar to other designations as possible. Examples of larger-scale screens include a series on melanin, morphology, suppressor screens of light sensitivity of mating , and growth under hypoxic conditions. In the first, "alternative" hosts have been used, with the most success obtained thus far in the nematode Caenorhabditis elegans. Importantly, both genes were found to be required for maximal virulence in murine inhalation models. Protein-protein interactions are important in a number of regulatory events involved in cryptococcal virulence and have been detected in C. neoformans by using a variety of methods. Single gene interactions have been detected by coimmunoprecipitation studies of the nuclear heat shock transcription factor, Hsf1, with a coactivating heat shock homolog, Ssa1, during laccase induction. A major experimental advantage of C. neoformans, uncommon among the human pathogenic fungi, is the established complete sexual cycle that enables classical Mendelian crosses and genetics.

The Cryptococcus mating-type locus (MAT) locus has emerged as an exemplary model to elucidate general principles governing the assembly and function of gene clusters. Moreover, comparative genomic studies of the evolutionary trajectory of MAT in the pathogenic Cryptococcus species complex mirror evolutionary steps that have been hypothesized to have given rise to the more complex sex chromosomes in mammals, insects, fish, and plants. These then represent examples of convergent evolution to specialized genomic regions governing sexual reproduction throughout the eukaryotic tree of life. Although the molecular nature of the homothallic sexual cycle of Filobasidiella depauperata remains to be elucidated, possible models for the homothallic life cycle of F. depauperata are discussed. In summary, comparative genomic analysis of the structure of MAT loci from Cryptococcus-related species has important implications for the hypothesized evolutionary model. The MAT genes present in these species have implications for the gene content of the ancestral homeodomain and P/R loci in the model. The P/R locus boundaries of C. heveanensis have expanded to include not only the pheromone and pheromone receptor genes but also at least one of the pheromone response pathway genes, but not all of the C. neoformans and C. gattii MAT locus gene homologs, which illuminates the serial expansions that created evolutionary strata in the MAT locus genes of the pathogenic Cryptococcus species.

This chapter addresses the roles of heterotrimeric and monomeric G proteins in the growth and development of Cryptococcus neoformans. The central motifs of the cAMP signal transduction pathway are highly conserved in eukaryotes, allowing diverse cell types to respond to different environmental stresses. However, the role of this pathway in microbial virulence was not predicted in studies involving nonpathogenic model organisms. Additionally, fungal pathogens of plants, such as the maize pathogen Ustilago maydis and the rice blast fungus Magnaporthe grisea, use cAMP pathways to regulate morphogenesis and virulence. Alternatively, overproduction of intracellular cyclic AMP (cAMP) may also impair virulence through mechanisms such as stress tolerance modulation. Studies of RGS proteins in Cryptococcus have revealed that the regulation of G-protein signaling is more intricate and potentially more complicated than in S. cerevisiae. The C. albicans NRG1 ortholog represses the transcription of several genes required for the yeast-hyphal transition, and this gene is required for Candida virulence. Several of the capsule and melanin genes whose transcription is controlled by cAMP do not appear to be targets of Nrg1. Additionally, the altered capsule phenotype of the nrg1 mutant strain is less severe than the acapsular phenotype of strains with defective cAMP signaling. Lastly, melanin is not significantly altered in the nrg1 mutant. The cAMP signal transduction pathway plays a central role in the stress response and virulence of microbial pathogens. Animal modeling of cryptococcal infection with MATα and MATa strains suggest that MATα strains are intrinsically more virulent.

This chapter discusses the effect of mating type, pheromone signaling, spore production, and ploidy on cryptococcal virulence. Cryptococcus may have the most unusual mating-type locus (MAT) of all the human-pathogenic fungi. The majority of basidiomycetous fungi have tetrapolar mating systems with two unlinked pheromone and sex-determining loci in which strains must differ at both loci for mating to succeed. In Cryptococcus, two traditional mating loci appear to have fused to generate one very large mating-type (MAT) locus that contains not only the sex-determining homeodomain transcription factors, pheromones, and pheromone receptors, but also genes from many other functional categories including several essential genes. The role of mating type in pathogenicity of C. neoformans var. grubii has been further characterized by examining the colonization of various organs by the KN99α/ α congenic strains in animal models. When infected individually, both mating types accumulate equivalently in all organs examined at early, intermediate, and late stages of the infection. While sexual recombination disrupts accumulation of deleterious mutations by reducing linkage, it can also disrupt the accumulation of beneficial mutations.

The chapter describes the abilities of Cryptococcus neoformans to sense and adapt to a subset of environmental conditions it encounters. The mechanisms for sensing extracellular signals such as stress, light, nitrogen, carbon dioxide, and oxygen are emerging. There are several future paths toward understanding the light responses of C. neoformans. First, the functions of the opsin and phytochrome in light sensing, if any, are currently unknown. Second, the conformational effects of light on the white collar complex are also unknown in C. neoformans or in detail from any fungal system since the proteins are notoriously difficult to purify in abundance for structural and other studies. Third, the genes that are regulated by light remain to be fully elucidated, although this is being addressed through transcript profiling microarray and genetic screens, particularly since these genes should control C. neoformans ability to proliferate in the wild and cause disease in humans. The current model of the CO2-sensing system in C. neoformans suggests that under limiting concentrations this molecule diffuses into the cell and is subsequently hydrated to HCO3 - and fixed inside the cell by the CA Can2p. Analysis of deletion mutants identified two genes in addition to SRE1 and SCP1 that were required for full growth under hypoxic conditions. It is of interest to note that mutation of SRE1 and SCP1 leads to an increased sensitivity to ROS, which led Ingavale et al. to propose that there is a link in oxygen sensing between hypoxia, mitochondrial function, and ROS generation.

This chapter describes what we have learned so far as well as many outstanding questions regarding the virulence of Cryptococcus gattii. It documents the available evidence for convergence and/or divergence of virulence mechanisms in the closely related pathogen, C. neoformans. The chapter summarizes some key features that distinguish C. gattii from C. neoformans. These distinguishing features reveal major differences in shape, antigenicity, and assimilation abilities that might underpin differences in the virulence mechanisms of C. gattii and C. neoformans. The availability of gene manipulation systems has accelerated characterization of C. gattii gene functions and evaluations of their relevance in virulence. A systematic study of host-pathogen interactions is critically dependent upon the availability of genetic tools for manipulation of the pathogens and tractable animal models to investigate the host factors critical in the infectious processes. The chapter also discusses primary and accessory determinants of C. gattii virulence. The primary determinants are capsule, melanin, thermal tolerance, psuperoxide dismutase, trehalose, and phenotype switchinghospholipases. Accessory determinants include mating locus, transcription factor STE12α, protein kinase A (PKA), comparative genomics, extracellular secretory products, ecological fitness, and antifungal susceptibility profiles. A better understanding of C. gattii virulence mechanisms would lead to more effective therapeutic options and the possible development of a C. gattii vaccine strategy, especially for areas of high incidence and for veterinary use.

This chapter describes the epidemiology of antifungal drug resistance as identified by in vitro susceptibility testing and studies of the mechanisms of resistance in Cryptococcus neoformans and C. gattii. Taking into consideration the MIC distribution profiles for the various antifungal agents, the pharmacology of the antifungal drugs, and studies of resistance mechanisms and clinical outcomes, it is reasonable to adapt the breakpoints developed by the Clinical and Laboratory Standards Institute (CLSI) for Candida spp. for use in the discussion of antifungal resistance in C. neoformans and C. gattii. Resistance to flucytosine may develop from decreased uptake (loss of permease activity) or loss of enzymatic activity required for the conversion of flucytosine to 5-fluorouracil (cytosine deaminase) and 5-fluorouridylic acid (FUMP pyrophosphorylase). The echinocandin class of antifungals is relatively ineffective against Cryptococcus sp., but the mechanism of this resistance is still unknown. The epidemiology of antifungal resistance in the pathogenic cryptococci is a dynamic process that differs considerably according to the status of health care, especially that devoted to HIV and AIDS, in the region of interest. Improved understanding of the mechanisms of antifungal resistance in cryptococci will ultimately help in the battle against emerging resistance.

Signal transduction cascades are utilized by all organisms to convey signals perceived at the cell surface to effectors within the cell. These enzymatic signaling cascades are important in the pathogenesis of many infections, including cryptococcosis. This chapter summarizes the significance and functional interactions involved in the cell wall integrity, phospholipase, and calcineurin signaling pathways for the establishment of Cryptococcus neoformans virulence. The fungal Plc enzymes referred to in this review preferentially hydrolyze phosphatidylinositol (PI)-based substrates within the cryptococcal cell and affect multiple cellular functions, including the secretion of (phospholipase B ) Plb1. It was found that the Plb1 MW could be as high as 125 kDa due to extensive asparagine N-linked glycosylation, which is responsible for at least 30% of the MW of Plb1 and essential for its activity. It was recently demonstrated that PI-PLC1 (Plc1) regulates cryptococcal virulence, acting in part through interactions with the Pkc/Mpk1 cell wall integrity pathway. In contrast to Plcs from higher eukaryotes, Plcs from the parasite Trypanosoma brucei preferentially hydrolyze the glycosylphosphatidylinositol (GPI) anchor of variant surface glycoprotein or GPI biosynthetic intermediates, in addition to PI, but not the phosphorylated intermediates, despite their localization to the peripheral cytoplasmic face of intracellular vesicles. Metabolic labeling studies performed in S. cerevisiae implicated a Plc enzyme and a secondary-acting protease in hydrolysis of the GPI anchor of certain proteins in the plasma membrane, resulting in their subsequent localization in the cell wall. ScPlc1, the only Plc1 in S. cerevisiae, like CnPlc1, lacks a secretory signal leader peptide.

This chapter focuses on the environmental habitats of the dominant human-pathogenic species, Cryptococcus neoformans and Cryptococcus gattii, and their varieties and subgroups. The procedures for isolating C. gattii from any environmental sample are the same as those for C. neoformans. Several researchers have sampled specific sites temporally and spatially. Many have included a variety of telluric, arboreal, aquatic, and aerial niches. C. neoformans was recovered from 62 samples of decayed wood from the hollows of five pink shower trees, two fig trees, and one November shower tree. The majority of patients diagnosed with cryptococcal meningitis in Teresina resided in Piauḭ or the adjacent state of Maranhão. Strains of VNI can readily be isolated from pigeon feces, and they are able to grow and mate on media containing pigeon feces. As cited in the chapter, the rare environmental strains of VNII have included isolations from pigeon and arboreal habitats, but their numbers have been too low to define the ecological niche of this molecular type. C. gattii is rarely isolated from pigeon feces but is associated with various tree species in Australia, Asia, North America, and South America. These different environmental niches have led to the hypothesis that C. neoformans is ubiquitous in the environment due to dissemination by pigeons following migratory and trade routes and that C. gattii is restricted to tropical and subtropical regions because it is not associated with pigeons.

Several nonvertebrate hosts has been used to investigate virulence traits and therapies for several bacterial and fungal pathogens. This chapter details the use of heterologous hosts for the study of Cryptococcus neoformans. A striking finding was the correlation between known mammalian virulence factors such as the capsule, melanin, and phospholipase and their necessity for survival in amoebae. Many of the assays used when working with macrophages can be modified for use with Acanthamoeba castellanii, including killing assays, trypan blue staining, and phagocytosis assays. Although many virulence traits have shown some concordance in their requirement for both mammalian and protozoan virulence, there are some discrepancies. Two assays have been developed using Caenorhabditis elegans as a host to study C. neoformans. The first assay, termed the ‘’killing assay’’, was initially utilized to identify C. neoformans mutants reduced in virulence. Another assay has been developed based on the observation of smaller nematode brood size when infected with pathogenic C. neoformans. Mutants of Pka1 or Gpa1 (the α subunit) are reduced in virulence in C. elegans, similar to the observed virulence in mice studies. Acapsular mutants also displayed attenuated virulence in Dictyostelium discoideum and Galleria mellonella infection assays, although they remain virulent in D. melanogaster infection assays. Since the Cryptococcus genus includes a large set of organisms, all of which are also presumably under ameboid predator selection in soils and are able to survive in their environments, the avirulence of most species represents the absence of a complete trait set for animal hosts.

This chapter reviews the epidemiology, diagnosis, clinical manifestations, treatment, and prognosis of cryptococcosis in Africa and the ecology and population genetics of African isolates of Cryptococcus. In the pre-highly active antiretroviral therapy (HAART) era in sub-Saharan Africa, cryptococcosis was often a sentinel opportunistic infection among HIV-infected adults, heralding the diagnosis of AIDS in more than 88% of cryptococcal cases. The initial diagnosis of cryptococcal meningitis in HAART naive, HIV-infected patients is relatively straightforward and involves microscopy and culture of clinical specimens, as well as serology. Maintenance therapy with fluconazole is usually prescribed for those patients who survive their initial episode of disease. The diagnosis of cryptococcal disease in HAART-treated patients with paradoxical immune reconstitution inflammatory syndrome (IRIS) is slightly more difficult. The formidable challenges of cryptococcosis in sub-Saharan Africa involve significant problems with diagnosis, management, and prevention. In sub-Saharan Africa, cases of cryptococcosis due to C. neoformans far exceed the number of infections caused by C. gattii. Sub-Saharan Africa is the global epicenter of cryptococcosis and the HIV/AIDS pandemic. It is possible to elucidate the evolutionary relationships among African strains of Cryptococcus and their global ancestors.

This chapter includes sections on aspects of the clinical epidemiology and features of cryptococcal infection particular to Asia and management practices and experience from Asia, especially where this differs in some respect from other regions. In Thailand, prior to the AIDS epidemic, Cryptococcus gattii was the most common cause of cryptococcosis and accounted for over half of all isolates that were recovered from human patients. The duration from onset of symptoms to diagnosis of cryptococcal meningitis in immunocompetent patients was longer than their predisposed counterparts. In immunocompetent patients, the cerebrospinal fluid (CSF) white cell count tended to be higher, and seizures, hydrocephalus, and shunt procedures were more frequent. Immunocompromised patients were more frequently found to have high fever and parenchymal lesions in cranial magnetic resonance imaging. A recent Cochrane review identified five randomized, controlled trials of antifungal therapy for primary prevention of cryptococcal disease in a total of 1,316 HIV-infected patients who mostly had CD4 cell counts less than 150 cells/μl. A study from Thailand showed that the use of temporary external lumbar drainage can be a reasonably safe and effective management strategy for intractable elevated CSF pressure not responding to serial lumbar puncture in HIV-infected patients with cryptococcal meningitis, even in relatively resource-limited settings.

The current knowledge of sexual reproduction of Cryptococcus gattii from a population genetics perspective is reviewed in this chapter. In genetic studies, cryptic species are usually detected as exclusive groups of organisms occupying strongly supported branches on phylogenetic trees that have been derived from different, independent loci. Assessing the relative numbers of α and a cells is therefore an important aspect of determining if populations are likely to have undergone sexual recombination. The initial studies of mating type in C. gattii found 84% of clinical isolates to be of the α mating type. Mating type was assessed by coculture with tester strains, and the majority of strains (~90%) were fertile, producing basidia and basidiospores. Amplified fragment length polymorphisms (AFLPs), which are highly discriminatory molecular markers, were therefore selected to establish multilocus genotypes. The population genetics of clinical collections is generally more complicated than the study of environmental populations as humans travel and may acquire an infection far from where they eventually present with clinical disease and an isolate is obtained. The pattern of pairwise compatibility among global VGII isolates presents a striking contrast to that seen in VGI. All VGII populations studied to date are heavily biased for one or the other mating type, and the most probable scenario is that mating occurs between isolates of the same sex.

This chapter outlines issues that were quickly explored, starting with the realization in 2001 that Vancouver Island was a hot spot for a pathogen not previously described as endemic and not restricted to tropical and subtropical climates. The picture of the outbreak that has developed has implications for global travel, climate change, land use patterns, and environmental colonization. Importantly, cryptococcosis caused by Cryptococcus gattii now serves as an excellent illustration of the impact of pathogen spread into a clement ecological niche, in this case, one that happened to be in a major population center of western Canada and the Pacific Northwest. Symptoms and clinicopathological changes in animals on Vancouver Island were consistent with disease reported elsewhere. The most common primary system involved was respiratory, followed by the central nervous system (CNS), in both cats and dogs. Animal cryptococcosis due to C. gattii is a nonregulated disease in Canada. Molecular typing of C. gattii environmental isolates from Vancouver Island using PCR fingerprinting and/or restriction fragment length polymorphism methodologies revealed that the majority of isolates belonged to the VGII molecular type and a small number belonged to the VGI molecular type. A number of solid culture media, biochemical tests, and stains can confirm the diagnosis of Cryptococcus to the genus level. Ecological niche modeling was employed to identify geographical areas in British Columbia with suitable environmental conditions to support the permanent colonization of C. gattii in the environment.

A large number of molecular typing techniques have been applied over the years to discriminate between individual isolates that had been indistinguishable using conventional techniques and to obtain further insights into the epidemiology and population structure of this species complex. This chapter aims to summarize the diverse typing techniques applied to the Cryptococcus neoformans/C. gattii species complex, to correlate the obtained results, and to describe the global distribution of the major genotypes. The enzymes malate dehydrogenase, alcohol dehydrogenase, phosphoglyceromutase, and glutamate dehydrogenase could separate C. gattii from C. neoformans. Electrophoretic karyotyping was for the first time applied to the C. neoformans/C. gattii species complex to study the genetic diversity between seven cryptococcal strains representing all four serotypes. PCR fingerprinting using the primer (GACA)4 was applied to 110 cryptococcal isolates obtained mainly from Germany and Africa as well as additional globally collected reference strains. The restriction fragment length polymorphism (RFLP) patterns result from the presence of a restriction enzyme cleavage site at one place in the genome in one individual and the absence of that specific site in another individual. Intergenic spacer (IGS) sequence analysis is a powerful tool to delineate the two varieties of C. neoformans and separate the four major molecular types of C. gattii.

A study on the identification of hybrids identified six distinct clusters based on the genotypes of a large number of Cryptococcus neoformans and Cryptococcus gattii global isolates using amplified fragment length polymorphism (AFLP). A combination of genotype and ploidy analyses are able to discriminate AD hybrids from haploid serotype A or D isolates. Serological reactions and a set of genotyping techniques have been used to determine the serotype of Cryptococcus strains. Serotype A strains presented a rim pattern characterized by a sharp increase in the optical gradient at the capsular edge followed directly by an immediate decrease. The estimation of the time of hybridization showed that recombination occurred very recently, indicating that hybridization is important during Cryptococcus evolution. Gene genealogical analysis supported the hypothesis of multiple origins of hybrid strains, suggesting that hybridization events occurred in different times probably coinciding with the continuing dispersion of C. neoformans in the environment that allows the contact between genetic divergent populations. In conclusion, hybridization, in addition to a low rate of sexual reproduction, represents an important mechanism for Cryptococcus to introduce genetic variants in a population with a prevalent clonal expansion.

This chapter focuses on the dynamic interaction of Cryptococcus neoformans with alveolar macrophages (AMs) and how the consequential actions of both the host and fungal pathogen can affect the outcome of C. neoformans exposure. In macrophages, glucuronoxylomannan (GXM) binding to Toll-like receptors 2 (TLR2) and TLR4, in conjunction with CD14, triggers NF κB activation and its translocation to the nucleus. Recognition and binding of C. neoformans by phagocytes occurs with greater avidity when cells are opsonized. The alveolar spaces of the lung are coated with an epithelial lining fluid containing components essential to the induction of the host immune response, including complement and immunoglobulins (Ig). Administration of mAbs against GXM can increase C. neoformans internalization and prolong survival in murine models of cryptococcosis via complement pathway-independent and/or -dependent mechanisms. In neutrophils, GXM is rapidly degraded, but in rat macrophages, GXM can persist for months. Thus, the role of GXM in the regulation of the host immune response through macrophages is clearly more complex than that occurring through neutrophils. C. neoformans has developed mechanisms to survive within the phagolysosome. The enlargement of the polysaccharide capsule following phagocytosis provides resistance to microbicidal agents within the lumen of the phagolysosome, such as reactive oxygen species and antimicrobial peptides. The majority of research attempting to reveal novel C. neoformans virulence attributes mostly produces new mechanisms by which the three archetypal virulence factors modulate pathogenesis.

This chapter reviews current knowledge regarding the role of dendritic cells (DCs) and T cells in the generation of protective immunity against Cryptococcus neoformans infections. DCs function as sentinels in the innate immune system. They are the most effective antigenpresenting cells (APCs) for inducing cell-mediated immune responses and are uniquely capable of activating naive T cells. DCs phagocytose pathogens, endocytose foreign antigens, process and present antigens to T cells, and are key mediators in the initiation of adaptive immune responses. DCs are uniquely capable of decoding fungal-associated information and translating it into different adaptive Th-type immune responses. The protective immune responses correlated with accumulation of myeloid DCs in the draining lymph nodes, while nonprotective responses were associated with accumulation of lymphoid DCs. Peripheral blood mononuclear cells from HIV-infected donors have profoundly impaired proliferative and cytokine responses to cryptococcal antigens. Immunization of mice with heat-killed C. neoformans conferred protection against challenge in wild-type mice but did not induce protection in nude mice (lacking T cells), demonstrating the importance of T cells in protection in the central nervous system (CNS). The lungs and brain are the most common sites of infection for C. neoformans and C. gattii. In pulmonary, systemic, and CNS infections, Th1-type cytokines are required for a protective cell-mediated immune response. The necessity of T cells for host defenses against cryptococcosis has prompted research into identifying immunoreactive cryptococcal antigens that could serve as vaccine candidates and as diagnostic reagents to measure T-cell responses in infected or at-risk patients.

Research initiated in the late 1980s has been the driving force behind a major expansion of one's knowledge about Ab immunity (AI) to cryptococcal disease (cryptococcosis) and a sea change in the understanding of mechanisms of AI to Cryptococcus neoformans and other microbes for which AI is not thought to be the predominant protective host defense mechanism. This chapter highlights this information, placing emphasis on how new knowledge about AI to C. neoformans has advanced one's understanding of pathogenesis, virulence, and mechanisms of Ab action. Levels of IgG to glucuronoxylomannan (GXM) were higher in human immunodeficiency virus (HIV)-infected than HIV-uninfected individuals in studies using a direct GXM capture enzyme-linked immunosorbent assay (ELISA), but lower in studies using different ELISA methods. The major effect of monoclonal Ab (MAb) to GXM on effector cell function is achieved by enhancing expression of costimulatory molecules, cytokine receptors, and cytokines that polarize the cellular response toward a Th1-like response. A long-recognized mechanism that is central to AI to cryptococcal disease is Ab-mediated clearance of GXM. The remarkable expansion of knowledge about AI to cryptococcal disease since the late 1980s has already translated into potential therapies to ameliorate human cryptococcal disease. The study of AI to C. neoformans has provided precedents that have informed and influenced one's views on Ab immunity in general and the efficacy of Abs against other microbes.

Glucuronoxylomannan (GXM) is the most prominent of the capsular constituents and is the primary target for anticapsular antibody and the most likely mediator of capsule-complement interactions. The goal of this chapter is to describe (i) variability in the interactions of GXM antibodies with the capsular matrix, (ii) the mechanisms for interaction of the capsule with proteins of the complement system, (iii) the nexus of the anticapsular antibody and complement system in capsule interactions, and (iv) the consequences of antibody and complement binding for modification of the capsular matrix. Differential interference contrast (DIC) systems detect optical gradients, preserve its sign, and convert the gradients into visible intensity gradients. Binding of antibody to the capsule edge to produce the annular rim could be due to localization of epitopes only at the capsule perimeter; monoclonal antibodies (MAbs) producing puffy patterns may bind to epitopes located throughout the capsule. Evidence of the importance of the complement system is provided in several reports of the course of cryptococcosis in normal versus complement deficient mice. Activation of the alternative pathway leads to conversion of C3 to C3b, which in turn, binds to the cryptococcal cell. Incubation of acapsular cryptococci in adsorbed serum leads to deposition of C3 onto the cell wall, but C3 binding occurs via the slow kinetics characteristic of the alternative pathway. In contrast, encapsulated cryptococci are unable to adsorb the antibodies from normal serum that mediate the rapid, early deposition of C3 onto acapsular cryptococci.

This chapter portrays the current understanding of the mechanisms involved in direct killing of Cryptococcus by innate cytotoxic lymphocytes. Binding results in natural killer (NK)- cell reorganization of the actin and microtubular cytoskeleton that is important for formation of the immunological synapse (NKIS). Syngeneic, nylon wool nonadherent, splenic cells (NK cells) were adoptively transferred into the depleted mice. The mice were injected intravenously with Cryptococcus in the presence or absence of anti-asialo GM1, which depletes NK cells. The response of NK cells to Cryptococcus has been extended to human studies. Human NK cells can be obtained from the blood, where they are present as a small percentage of the total lymphocyte population. Perforin is the effector molecule required for NK-cell killing of Cryptococcus. Via exocytosis of lytic granules, NK cells secrete effector molecules that are involved in the killing of tumor targets. NK cells can receive signals from other innate cells in response to Cryptococcus as well as produce cytokines that stimulate other effector cells and shape adaptive immune responses. Human primary NK cells also secrete gamma interferon (IFN-γ) that correlates with increased killing of Cryptococcus in vitro. Cytokines and chemokines are important mediators of immune cell function. In addition, similar to the observations of NK cells, NKT cells enhanced host defense against Cryptococcus in the presence of interleukin-12 (IL-12) and IFN-γ. Thus, similar to NK cells, cytokines are important in eliciting NKT cell responses to Cryptococcus and in direct killing of the organism.

This chapter reviews the evidence of disease after acute infection and reactivation of latent infection, considering the newer epidemiological and serological studies and the older anatomical evidence. Cryptococcal meningitis was diagnosed in an HIV-infected patient who helped dismantle an aviary, and another patient had pigeons nesting in the ceiling above his desk. Cryptococcal infection occurs in children as demonstrated by the existence of antibodies to Cryptococcus neoformans in children. Primary pulmonary cryptococcosis may be asymptomatic or confused with viral infections frequently observed during childhood. The high incidence of disease reported in the Vancouver Island outbreak may not be the norm, even though it appears from epidemiological studies that C. neoformans has a greater potential for reactivation of infection leading to disease than C. gattii. The overwhelming majority of individuals with persistent/latent infection would remain asymptomatic as long as they retained an intact immune system. In murine inhalational models acute pulmonary infection with C. neoformans is rapidly followed by the appearance of cryptococci both free and within macrophages in the lymphoid sinuses of hilar (draining) lymph nodes. The best animal model available for the study of latency and reactivation is experimental rat pulmonary cryptococcosis.

This chapter discusses the ability of the pathogenic cryptococci to proliferate intracellularly and examines the different strategies cryptococci use to exit host cells. The intracellular location provides a dual benefit to Cryptococcus, both in avoiding extracellular host immune mechanisms, such as complement, and in reducing exposure to antifungal agents. The establishment of different in vitro systems that utilize cell lines and the application of techniques such as live cell imaging have contributed to rapid advances in one's understanding of the molecular mechanisms influencing Cryptococcus's ability to proliferate intracellularly. Intracellular parasitism is associated with a continuous struggle between the pathogen and its host cell. Within host cells, Cryptococcus encounters a harsh environment of reactive oxygen and nitrogen species; oxygen, nutrient, and metal ion deprivation; and low pH and high temperatures. Therefore, the yeast expresses multiple virulence factors including a capsule, melanin, and a variety of secreted enzymes that can modify the host’s defense mechanisms to achieve intracellular replication. The melanized Cryptococcus neoformans strain 145 is more resistant to cell death caused by lymphocytes than the less melanized strain 52. Resistance to cryptococcal infection is associated with a Th1 response and the consequent phagocyte activation, whereas Th2-polarized host responses lead to inhibition of phagocyte activity and enhanced susceptibility to C. neoformans. Whole-genome microarray analysis has identified a large number of candidate genes that may influence proliferative capacity and revealed an unexpected role for mitochondrial genes in regulating cryptococcal hypervirulence in the Vancouver Island strains.

Clinical and experimental studies clearly substantiate the role of acquired cell-mediated immunity in the establishment of long-term protection against pulmonary cryptococcosis. However, the innate immune system is also centrally involved in both immediate antifungal immune responses as well as promoting adaptive immunity. Surfactant is secreted by type II cells in the alveolar space of the lung, and it is composed of a complex mixture of phospholipids and proteins, including surfactant proteins A and D (SP-A, SP-D), which play a role in innate host defense. Complement plays an important role during innate immune responses to Cryptococcus neoformans cells in extrapulmonary sites as a consequence of its ability to bind to encapsulated yeast cells and function as an opsonin. Toll-like receptors (TLRs) have been implicated in airway inflammation, and recent evidence suggests the involvement of certain TLRs in mediating a protective C. neoformans response by promoting a Th1-type T-cell adaptive immune response. C. neoformans enters the pulmonary system in both a spore and yeast form and has the ability to remain dormant for many years within the phagosomal compartment before infection is activated. The immune response to this organism is flexible and involves both cellular and humoral factors. However, environmental, genetic, or cryptococcal factors can exert pressures on this flexible program of host defense, leading to chronic and/or progressive infections, including disseminated disease.

This chapter reviews the current state of knowledge regarding how Cryptococcus neoformans invades into the central nervous system (CNS). An understanding of the unique characteristics of the blood-brain barrier (BBB) is essential to comprehend the potential mechanisms by which blood-borne microbes, including C. neoformans, transverse it. Brain microvascular endothelial cells (BMECs), which line the capillaries supplying blood to the brain, have unique tight junctions that contribute to the barrier function of the BBB. A major function of the BBB is maintenance of the neural microenvironment by regulating the passage of molecules into and out of the brain. The role of monocytes in CNS invasion by C. neoformans relies first on clinical observations. When occlusion occurs in blood vessels supplying the CNS, invasion into damaged tissue can occur. Additionally, at early time points, invasion of the CNS by C. neoformans did not occur from the surface of the brain via the invasion of the blood vessels supplying the leptomeninges (pia mater and arachnoid). The other cryptococcal virulence factor that has been linked to brain invasion is urease, which is produced by nearly all clinical isolates of C. neoformans. Urease expression contributes to the CNS invasion by enhancing yeast sequestration within cerebral microcapillary beds during hematogenous spread. Meningoencephalitis is the most common and serious clinical manifestation of cryptococcosis. Remarkable progress has been made toward elucidating the means by which C. neoformans gains access into the CNS.

This chapter presents salient examples in the use and performance of animal models of cryptococcal infection, particularly murine and rabbit models, and demonstrate how these models in conjunction with the application of emerging technologies have advanced our knowledge about this disease. In the current era of increasingly more stringent regulations on the use of laboratory animals, it is no longer acceptable to most Institutional Animal Care and Use Committees for the investigator to allow the experimental animals to die of infection. Mice are the most frequently used species for cryptococcal models and can be infected by one of several routes. Intraperitoneal infection using 105 to 107 yeast cells is technically easy to master, and many mice can be infected quickly. Human cryptococcosis is acquired via inhalation, and thus, pulmonary models of infection are better mimics of the natural disease progression. The use of rabbits in models of fungal infection has been much more limited than the use of rodents. Prevention of infection is even more desirable, and vaccine studies in animal models have been done in the field of cryptococcosis.

This chapter focuses on naturally occurring disease in animals by concentrating on an approach based on the concept of comparative pathology and one medicine/one health. It should be emphasized at the outset that members of the Cryptococcus neoformans species complex, comprising the two species C. neoformans and C. gattii, are environmental organisms. Some animal species are inherently more resistant to cryptococcosis. The reasons for this are still not clear, although this is a well-accepted concept for other primary pathogens, such as Mycobacterium tuberculosis. Resistance may occur at the level of phagocytic cell function. Intact innate immunity, such as an effective cough reflex and good functionality of the mucociliary escalator, and timely development of acquired cell mediated and antibody-mediated immunity all favor elimination of the infective agent, either prior to development of tissue invasion or after limited tissue invasion. Veterinary clinicians interested in fungal disease are to be commended for having been important players in this quest for new knowledge. There can be no doubt that studying disease in companion animals, production animals, and wildlife can play a critical role in unraveling the environmental associations of the C. neoformans species complex.

Cryptococcosis is one of the most significant opportunistic fungal infections in solid organ transplant (SOT) recipients. Disease presentation and principles of management of cryptococcosis in other hosts are relevant in transplant recipients. This chapter summarizes the current knowledge and topical developments in the epidemiologic characteristics, clinical manifestations, diagnosis, and management of cryptococcosis in transplant recipients. Cryptococcosis is the third most commonly occurring invasive fungal disease in SOT recipients. Most cryptococcal disease in SOT recipients is due to Cryptococcus neoformans var. grubii (serotype A), which has no particular geographic predilection. Calcineurin inhibitors are currently the mainstay of immunosuppression in SOT recipients. Cutaneous cryptococcosis can present as papular, nodular, or ulcerative lesions; cellulitis; or necrotizing fasciitis. While cutaneous lesions largely represent hematogenous dissemination, skin has also been identified as a portal of entry of Cryptococcus and a potential source of subsequent disseminated disease in SOT recipients. Rapid reduction of immunosuppressive therapy in conjunction with initiation of antifungal therapy in SOT recipients may lead to the development of immune reconstitution syndrome (IRS), the clinical manifestations of which mimic worsening disease due to cryptococcosis. Cryptococcosis occurs rarely in hematopoietic stem cell transplant (HSCT) recipients. Lipid formulations of amphotericin B are recommended in lieu of amphotericin B deoxycholate owing to the risk of nephrotoxicity when concurrently administered with a calcineurin inhibitor and owing to the significant number of transplant patients with underlying renal dysfunction at the time of diagnosis. Reduction in immunosuppressive therapy should be considered in transplant recipients diagnosed with cryptococcosis.

Infection with HIV is the most common predisposing condition for developing cryptococcosis. This chapter focuses on aspects that are specific to the coinfection of HIV and Cryptococcus neoformans. These include clinical presentation, treatment regimens, alterations of the host immune response, and concerns about drug toxicities and interactions. Soon after the HIV pandemic was recognized in the United States and Europe, it became clear that cryptococcosis was an important opportunistic pathogen in patients with AIDS. C. neoformans infection in AIDS patients can either be the result of a newly acquired primary infection or alternatively constitute a reactivation of latent C. neoformans infection. Patients with AIDS who respond to highly active antiretroviral therapy (HAART) show a greatly reduced incidence of opportunistic infections such as cryptococcosis. Experimental evidence suggests that C. neoformans coinfection can affect HIV replication. Another factor that may affect HIV-infected patients’ ability to mount a successful antifungal host response is the common abuse of methamphetamine. This drug exposure facilitates intracellular replication and inhibits intracellular killing of C. neoformans and thus affects pathogenesis in these patients. The most common manifestation of cryptococcosis in patients with HIV infection is meningoencephalitis, and central nervous system (CNS) involvement is found in the vast majority of patients with AIDS in whom cryptococcal infection is diagnosed. The treatment of AIDS-associated cryptococcal meningitis is usually divided into three stages: induction, consolidation, and maintenance.

This chapter briefly describes the history of treatment for CNS cryptococcosis and discusses novel approaches to therapy with traditional agents, the newer antifungal agents, experimental agents, and the potential role of immunotherapy. The availability of fluconazole, itraconazole, the lipid formulations of amphotericin B, and the expanded spectrum triazoles provided additional options in the antifungal armamentarium. There are several newer antifungal agents that could offer significant advantages over more traditional agents. Specifically, three new triazoles, voriconazole, posaconazole, and isavuconazole, demonstrate excellent in vitro activity against most strains of clinically relevant Cryptococcus species. Passive immunotherapy has been advocated as a potentially important adjunct to conventional antifungal therapy in the management of invasive mycoses. The hypothetical value of cryptococcal antigen removal from the infected host relates to its function as a virulence factor: cryptococcal antigen is known to inhibit leukocyte migration, promote cerebral edema, promote cytokine dysregulation, induce T-lymphocyte secretion of immunosuppressive molecules, and induce the shedding of L-selectin. There are good data supporting the role of adjuvant corticosteroids in the early management of bacterial meningitis, and this has become a standard approach to patients with proven or suspected disease. Tacrolimus demonstrates synergistic activity with several antifungals including bafilomycin A (an experimental agent), caspofungin, and fluconazole. In the developed world, cryptococcosis in transplantation represents a large and inadequately studied complication among transplant recipients.

This chapter talks about recent developments in the vaccine field and the publication of two recent comprehensive reviews that include additional information on the problem of vaccination against Cryptococcus neoformans. C. neoformans is an encapsulated pathogen, and polysaccharide-based vaccines have an excellent track record in eliciting protective immunity against bacteria with polysaccharide capsules. Human MAbs produced from immortalized B cells from glucuronoxylomannan (GXM)-TT-vaccinated individuals were protective in mice, and serum antibodies from vaccinated individuals enhanced the antifungal activity of phagocytes against C. neoformans. Peptide mimotopes of GXM selected by mouse and human MAbs have been reported. An advantage of peptide mimotopes is that they can be made easily and reproducibly and thus provide an attractive option for generating vaccines. Site-directed mutagenesis of the binding contacts of a protective MAb revealed that different amino acids were involved in binding to polysaccharide and mimotope epitopes, raising the possibility that different types of antibodies are elicited by carbohydrate and peptide antigens. Cryptococcal infection elicits strong and rapid antibody responses to fungal proteins, such as heat shock proteins (HSPs), but currently there is no evidence that these antibody responses contribute to host protection. In the late 1990s murine MAbs were made to C. neoformans melanin with the goal of using these reagents for the study of melanization in vivo and in vitro. Subsequently, it was hypothesized that if these MAbs bound to human melanin, they could be used to deliver tumoricidal radiation to melanoma cells.

The polysaccharide capsule is a unique feature of Cryptococcus neoformans that distinguishes the organism from other medically important yeasts. Various histochemical stains react with cryptococci, including mucin stains such as mucicarmine, alcian blue, and periodic acid-Schiff that stain the capsule and Fontana-Masson stain, which interacts with the cell wall. The sensitivity of cerebrospinal fluid (CSF) antigen detection is highest for diagnosing cryptococcal meningitis in HIV-infected persons and varies based upon factors such as the fungal burden in the CSF and the presence of well-encapsulated organisms. Since the serum cryptococcal antigen is positive in almost all AIDS patients with cryptococcal meningitis, there has been interest in using the test to screen high-risk HIV-infected persons, such as those with fevers, neurological symptoms, and/or low CD4 counts. At the current time, there are four commercially available assays that detect β-Glucan (BG) in serum. Over the past two decades, a myriad of molecular techniques have been explored for the detection of pathogenic fungi. Molecular fingerprinting techniques, e.g., randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis, amplified fragment length polymorphism (AFLP), PCR fingerprinting with minisatellite (M13) or microsatellite primers [e.g., (GACA)4 or (CTG)5], karyotypes, sequencing, multilocus microsatellite typing, mating-type locus, and multilocus sequence typing (MLST), are among the most popular typing techniques that have been applied to discern and characterize the genetic heterogeneity within the species of the C. neoformans species complex.

This chapter discusses the current management of cryptococcal meningoencephalitis in terms of antifungal drug therapy, the management of complications, notably raised cerebrospinal fluid (CSF) pressure, the issues of the timing and choice of antiretroviral therapy (ART) in HIV-infected patients, and the management of symptomatic relapse, including immune reconstitution syndromes. The result is that if patients anywhere in the world with access to ART survive the initial critical months of cryptococcal meningoencephalitis, they have an excellent long-term prognosis. The chapter highlights opportunities for improved intervention, including for earlier diagnosis and treatment. The emphasis is on HIV-associated infection in resource-limited settings, which constitute the majority of the global burden of cryptococcal infection. Examination of outcomes of therapy for HIV-associated cryptococcal meningitis in resource-limited settings illustrates that a high proportion of patients with abnormal mental status at presentation is associated with high mortality irrespective of the antifungal treatment available. The chapter also summarizes some of the reasons underlying the high mortality of patients with cryptococcal meningitis, including the constraints on optimal management in developing-country settings.

This chapter focuses primarily on HIV-associated cryptococcosis, due to its much greater public health burden, and discusses the challenges to public health agencies in addressing the threat of Cryptococcus gattii. The vast majority of disease caused by Cryptococcus is found in persons who are infected with HIV, especially those who develop AIDS. The large global burden of cryptococcal disease presents a number of challenges to public health, particularly in the resource-poor regions of high HIV prevalence in sub-Saharan Africa and South/Southeast Asia. Public health surveillance is an essential element in public health practice and allows factual insight into the epidemiology of a disease in order to inform decisions and take action. Cryptococcal antigenemia has been shown to be prevalent in persons without symptomatic disease. The study from Cambodia treated 10 persons with asymptomatic antigenemia with fluconazole 200 mg/day for 12 weeks. By 12 weeks after initiation of therapy, none of these had developed cryptococcal meningitis. From a public health standpoint, one of the more challenging aspects of the epidemiology of Cryptococcus is the emergence of the primary pathogen C. gattii in North America. In order to fully understand the epidemiology of this emerging infection in the United States, both human and animal public health agencies at the federal, state, and local levels are working together to form a surveillance network.

Human cryptococcosis is most commonly due to infection with Cryptococcus neoformans and C. gattii, with C. neoformans var. grubii (serotype A) accounting for the great majority of infections worldwide. Cryptococcosis is uncommon in hematopoietic stem cell transplantation; in a large multicenter study in the United States, only 2 of 306 cases were associated with hematopoietic stem cell transplantation, and 54 with solid organ transplantation (SOT). Any condition associated with prolonged or high-dose corticosteroid exposure predisposes to cryptococcosis. Other than malignancy and organ transplantation, such conditions include connective tissue disorders (e.g., systemic lupus erythematosus and vasculitides), chronic obstructive pulmonary disease, and sarcoidosis. Visual loss is one of the most serious, debilitating sequelae (if not the most serious) of central nervous system (CNS) cryptococcosis. Rapid diagnosis and treatment is the key to achieving good outcomes. Molecular tests can distinguish between C. neoformans and C. gattii but are seldom required for diagnosis of cryptococcosis due to either species. A decrease in susceptibility to azole drugs, especially fluconazole, was noted among isolates of C. neoformans var. grubii from patients with AIDS, in parallel with widespread use of fluconazole prior to the advent of highly active antiretroviral therapy (HAART). The major determinants of outcome include neurological status, intracranial pressure (ICP), the presence of cerebral mass lesions, and cryptococcal load at presentation.