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Cryptococcosis ( Cryptococcus neoformans and Cryptococcus gattii )
Cryptococcus neoformans and Cryptococcus gattii are encapsulated, heterobasidiomycetous fungi that have progressed from being rare human pathogens, with just over 300 cases of cryptococcosis reported in the literature before 1955, to becoming a common worldwide opportunistic pathogen as immunocompromised human populations have dramatically increased over the past 2 decades. Cryptococcosis crosses the entire spectrum of patient populations, from the apparently immunocompetent host without an underlying disease to those severely immunocompromised from infection with the human immunodeficiency virus (HIV), an organ transplantation, or a malignancy and its treatment. Furthermore, it has a wide range of clinical presentations, which can vary from asymptomatic colonization of the respiratory airways to dissemination of infection into any part of the human body. Cryptococcus enters the host primarily through the lungs but has a special predilection for invading the central nervous system (CNS) of the susceptible host. Pulmonary infections are common and may have multiple clinical presentations and management issues. On the other hand, cryptococcal meningitis represents the primary life-threatening infection for this fungal pathogen and has required the most clinical attention.
History
The first identification of Cryptococcus from an environmental source was made by Sanfelice in 1894, from peach juice in Italy. Within a year, Busse and Buschke independently reported the first human case of cryptococcosis in a young woman who developed a chronic ulcer over the skin above her tibia, with yeasts identified in the tissue and later at autopsy. This yeast was also found to have spread to multiple organs in her body. By 1914, Versé described a human case of cryptococcal meningitis, and in 1916 Stoddard and Cutler gave a complete description of the CNS pathology for this infection, including in their report that the yeast forms had surrounding areas of clearing within the tissue. This finding was the first description of the signature structure for this yeast, the polysaccharide capsule. During the early years of clinical cryptococcosis, the names of this yeast were several and included Saccharomyces neoformans, Cryptococcus hominis, and Torula histolytica . In 1950 Benham attempted to categorize these poorly defined yeasts based on morphology, fermentation, and serologic studies. She named one yeast C. hominis and its disease “cryptococcosis.” The name was later changed to C. neoformans based on temporal priority because Sanfelice had first used the species name of neoformans. However, despite Benham's proposal, it took another 25 years before “cryptococcosis” became the primary nomenclature for this infection rather than “torulosis.” In 1976 Kwon-Chung discovered and characterized the sexual stage of Cryptococcus, and the teleomorphs were named Filobasidiella neoformans and Filobasidiella bacillispora. It was proposed in 2002 that there be two varieties— C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A)—and another species, Cryptococcus gattii (serotypes B and C). In 2005 the genome sequence of C. neoformans was released, and now hundreds of strains, including strains of C. gattii, have been sequenced for comparisons as we have entered the genome age for fungal pathogen discoveries. With whole-genome sequence information of over a thousand strains, the Cryptococcus species complex has now been further divided into genotypes and species, and taxonomic issues are dynamic.
Mycology
Life Cycle and Genetics
The life cycles of both C. neoformans and C. gattii involve two distinct forms: asexual and sexual. The asexual stage exists as encapsulated yeast cells that reproduce by simple, narrow-based budding. The haploid (occasionally diploid in nature), unicellular yeasts are the primary forms recovered from environmental sources and human infections. The asexual or yeast forms represent the primary structures seen in host tissue and recovered from cultures during clinical disease. However, this fungus has a more complex life cycle, with a bipolar mating system that can be observed under certain in vitro conditions and even on plants. For example, yeasts exist in one of two mating types, “alpha” or “a.” When two strains of opposite mating types are physically placed together on specific, nutrient-poor media such as V-8 juice agar, the cells undergo conjugation, producing filaments with true clamp connections. At the ends of these filaments, basidia form, and within these basidia, meiosis occurs and chains of basidiospores are produced. The 1- to 2-micron basidiospores, with their size and shape, have been hypothesized to be the infectious propagules. They may deposit in the lung, where the spores rapidly convert to yeasts. However, the sexual stage at present remains a laboratory observation, and the sexual structures, such as basidiospores, have yet to be identified in nature, but clearly, sexual recombination appears to be occurring in nature. Studies have made great progress in precisely understanding the molecular signaling networks that control the sexual cycle, and in some cases genes in these mating pathways have been linked to both morphogenesis and virulence of the yeast.
In the 1980s, an interesting epidemiologic observation was made and confirmed by others that in most areas of the world, more than 95% of environmental and clinical C. neoformans isolates appear to contain only the alpha mating locus. The reason for this genetic bias remains unclear, but two factors may be important. First, it has been discovered that under certain environmental conditions C. neoformans undergoes haploid fruiting. Haploid fruiting occurs when haploid yeast strains under specific conditions produce hyphae and basidiospores without mating and the exchange of genetic information through meiosis. It is possible that this haploid fruiting with sporulation allows wider dissemination of the fungus in the environment and thus more environmental exposure, leading to more clinical disease. Second, it has now been shown that sexual reproduction can occur between partners with the same mating type, and this allows recombination and improved fitness of progeny. The alpha mating strains are much more likely to produce haploid fruiting structures or perform same-sex matings than the “a” mating strains. An alternative explanation for the alpha mating locus bias is that the approximately 100-kilobase locus or its adjacent genomic areas contain virulence genes that make these mating-type strains more fit in the environment (or in the host). Initial studies with congenic strains of C. neoformans var. neoformans differing primarily in the mating locus did suggest that the alpha mating strain was more virulent in mice. On the other hand, the alpha and “a” mating loci have been identified in C. neoformans var. grubii, and experiments with two congenic strains in this variety differing only in the mating locus appear to be identical in virulence. It is still uncertain how much the mating loci types contribute to the virulence of this yeast, and the alpha mating–type bias is not yet precisely explained. However, as witnessed in the Vancouver Island C. gattii outbreak, sexual recombination in nature between strains can play an important role in the evolution of pathologic fitness for strains. Furthermore, in certain areas within Botswana, environmental and clinical isolates do have similar numbers of both mating types, so that simple exposure may be cause for the mating-type bias.
Taxonomy
The genus Cryptococcus comprises 19 species, loosely characterized as a variety of encapsulated yeasts. There continues to be occasional reports of human infections with several of these non- neoformans and non- gattii species, such as Cryptococcus albidus and Cryptococcus laurentii. However, such clinical reports are uncommon, and the disease is occasionally poorly documented with these rare cryptococcal species. Therefore any human infection with a cryptococcal species other than C. neoformans or C. gattii needs rigorous histopathology and cultural proof of invasive disease.
For several decades, C. neoformans strains had been grouped into two varieties that included five serotypes based on their capsular structure. C. neoformans var. neoformans and C. neoformans var. grubii included strains with serotypes A, D, and AD, and C. gattii contained strains with serotypes B and C. The serotype classification (A–D) describes antigenic differences in the structure of the polysaccharide capsule; these differences are detected by antibodies from rabbit sera or by specific monoclonal antibodies.
The stable taxonomic classification of these varieties and serotypes has now evolved through new genomic analyses, and several new changes have been proposed. With the use of specific DNA typing methods and other physiologic factors, it has been proposed that the serotype A strains be classified into a separate variety, C. neoformans var. grubii. Serotype D isolates are to remain in the variety neoformans. The varietal status of serotype AD hybrid strains has not been proposed. It has also become clear that most of the serotype AD strains represent stable diploid strains, possibly occurring as incomplete genetic crosses between varieties neoformans and grubii. However, genetic mapping of A and D strains suggests that these varieties biologically diverged from each other more than 18 million years ago. Thus with whole-genome sequencing of many cryptococcal strains, C. neoformans var. grubii (serotype A) can be divided into five genotypes (VNI, VNII, VNBI, VNBII, and VNIII), with a separate variety, C. neoformans var. neoformans (serotype D), as genotype VNIV. C gattii (serotypes B and C) has been proposed to be divided into five cryptic species: Cryptococcus gattii , Cryptococcus bacillisporus , Cryptococcus deuterogattii, Cryptococcus tetragattii, and Cryptococcus decagattii (genotypes VGI–VGV).
As rapid advances in the understanding of genetic population diversity are made among the Cryptococcus complex during the genome era, taxonomic relationships and nomenclature will remain in some flux. It has been suggested that the group be designated “ Cryptococcus complex species” until further widespread genetic and phenotypic studies are completed and analyzed. However, at present, for clinicians the standard serotype classification used for half a century and the split into two varieties, var. neoformans and var. grubii, and two species, C. neoformans and C. gattii, still remain useful nomenclature for describing the clinical strain differences in epidemiology, pathogenesis, and clinical features. As further research continues, the taxonomic name of a strain may become less important to the clinician than the identification of its specific genetic structure. In this chapter, the terms “var. grubii ” and “var. neoformans ” will continue to be appended to the designation of serotype A and serotype D, respectively, even though serotyping sera are no longer available, because so much of the literature is based on serotype.
The anamorph (yeast or asexual stage) dominates clinical discussion of this encapsulated yeast. On the other hand, the teleomorph, with its more complex structure and its genome sequences, places this fungus within the basidiomycete family, and its teleomorph genus name is Filobasidiella. Thus the teleomorph of serotypes A and D strains is called Filobasidiella neoformans and the teleomorph of serotypes B and C strains is designated Filobasidiella bacillispora; however, these teleomorphic names are not used in clinical practice.
Identification
On most routine laboratory agar media, colonies of C. neoformans and C. gattii appear within 48 to 72 hours after plating a specimen. Some selective fungal media containing cycloheximide inhibit the growth of this yeast and thus should not be used. For blood cultures, the lysis-centrifugation method works well for isolating Cryptococcus but is no longer necessary because automated blood culture methods can reliably detect cryptococcemia, which is commonly observed in severely immunosuppressed patients such as patients with acquired immunodeficiency syndrome (AIDS) and disseminated disease. In some populations of the world with high rates of HIV infection, cryptococcemia is a common finding in patients during the workup of fever with blood cultures. However, cryptococcemia rarely produces symptoms of hypotension or septic shock.
On agar plates, the yeast colony grows as a white-to-cream–colored, opaque colony several millimeters in diameter. The colonies typically become mucoid with prolonged incubation, reflecting increased polysaccharide capsule formation. Colonies occasionally develop sectors that differ in pigmentation or exhibit morphologic changes (e.g., smooth or wrinkled). In fact, C. neoformans has been shown to possess the ability to produce a morphologic switching colony phenotype, which explains the variety of colony shapes in some strains and emphasizes the plasticity of the cryptococcal genome. The optimal environmental growth temperature for the majority of C. neoformans strains is between 30° and 35°C, with a maximum tolerated temperature for most strains at 40°C. Serotype A strains tend to tolerate higher environmental temperatures better than serotype D and serotype B/C strains. C. neoformans and C. gattii strains generally grow well at 37°C, with doubling generation times of 3 to 6 hours, and this high-temperature growth characteristic is a primary virulence phenotype that separates them from other cryptococcal species that generally do not either grow or survive well at mammalian body temperatures and thus are rarely found to be human pathogens.
In the clinical laboratory, C. neoformans and C. gattii can be readily differentiated from other yeasts on the basis of their morphology and biochemical tests. The specific identification can be confirmed by a battery of biochemical tests available commercially in kits. Recently the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry system has been effective in identifying and distinguishing both C. neoformans and C. gattii yeasts. However, there are three direct tests that predict that a yeast may be C. neoformans or C. gattii. First, placing the yeast into an India ink preparation for microscopy may reveal the encapsulation of the yeast. The capsule is generally better seen in direct clinical specimens from the host and may not be as apparent in wet mounts made from in vitro cultures. This finding occurs because capsule production is induced by certain environmental cues, such as elevated carbon dioxide concentrations, serum, urea, or limited iron conditions. In fact, the mammalian host environment provides an ideal environment for capsule production.
Second, a rapid urease test is positive for most Cryptococcus species. Cryptococcus species, unlike Candida species, possess urease, an enzyme that hydrolyzes urea to ammonia and increases the ambient pH. A positive urease test can be detected in the laboratory within minutes. Several nonpathogenic yeasts can produce abundant urease, and Trichosporon species may be weakly urease positive.
Third, C. neoformans is one of the few yeast species that possesses prominent activity of laccase, an enzyme that allows the conversion of diphenolic compounds into melanin. Detection of this unique biologic characteristic is possible with media containing niger seed (birdseed), caffeic acid, or dopamine. Yeast colonies that turn brown to black on these special agars are identified as melanin positive. In a clinical specimen, such a yeast colony will likely be either C. neoformans or C. gattii. However, other cryptococcal species in the general environment also possess laccase, but these selective agar assays are still particularly helpful when attempting to identify pigmented cryptococcal colonies from environmental samples contaminated by other fungi or bacteria species, or both.
Histopathologically, C. neoformans and C. gattii have several characteristic features. In most clinical cases, Cryptococcus in tissue exhibits a prominent capsule. Microscopically, most clinical isolates appear as spherical, narrow-based, budding, encapsulated yeast cells in both tissue and culture. Short hyphal or pseudohyphal structures may exist in vivo, or under certain stress conditions in vitro, but these structures are rarely observed unless certain in vitro nutrient conditions for mating or haploid fruiting are met. The yeast cells vary in size from 5 to 10 microns in diameter, and they exhibit single or multiple buds. Because the buds are readily detached from their parental cells, the majority of yeast cells in both tissue and culture lack buds. In tissue, Cryptococcus has the ability to produce large (titan) cells of 50 to 100 µm, which possess features such as aneuploidy and large antiphagocytic capsules that promote their survival. Finally, the size of the capsule under direct observation dynamically varies with the individual strain and its immediate environment.
There are three methods for identifying the four serotypes. First, commercial antibodies had been used to distinguish differences in the capsular structures but presently are not available. Second, there are known differences between the biochemical pathways of the serotypes. Most serotypes B and C ( C. gattii ) isolates assimilate glycine as a sole carbon source, whereas serotypes A and D ( C. neoformans ) isolates generally do not. An agar containing l -canavanine, glycine, and bromothymol blue (CGB) uses a color change to separate serotypes A and D from B and C. Third, analysis of DNA base composition is extremely accurate. Comparison of sequenced genomes of these serotypes shows an approximately 6% to 8% overall difference in nucleotide sequences between serotype A and D strains and an even greater difference between these strains and serotype B and C strains. A variety of molecular methods, including random amplified polymorphic DNA (RAPD), karyotypes, polymerase chain reaction (PCR) fingerprinting, multilocus sequencing typing (MLST), and direct sequencing of strains, can be used to readily identify an isolate as belonging to a certain serotype or clade. Furthermore, strains are classified into specific genotypes by PCR fingerprint patterns and sequence. There are presently multiple distinct genotypes with VNI, VNII, VNBI, VNBII, VNBIII, and VNIV for C. neoformans and VGI to VGV for C. gattii, and within these genotypes there are subgroup genotypes such as VGIIa to VGIIc, identified by MLST, which possess epidemiologic and maybe pathobiologic relevance. Along with genome sequencing, MALDI-TOF mass spectrometry has also become a precise and facile method for distinguishing cryptococcal strains in the laboratory.
Ecology
C. neoformans and C. gattii are saprobes in nature. C. neoformans was first described in fruits, but after years of investigation it is clear that it also has an environmental niche or habitat associated with certain trees and rotting wood. A second consistent finding is that C. neoformans has frequently been isolated from soil contaminated by guano from birds. On the other hand, C. gattii clearly has an environmental association with trees, from eucalyptus to a variety of coniferous trees, but not bird guano.
Cryptococcus neoformans Serotypes A, D, and AD ( grubii var. or neoformans var.)
In the 1950s, Emmons first isolated C. neoformans from soil and from the droppings and nests of pigeons. Since the original reports, the fungus has been found in soil samples from around the world. However, the soils most enriched in C. neoformans are those that are frequented by birds, especially pigeons, turkeys, and chickens. Guano from other birds, such as canaries and parrots, has also yielded the yeast. Despite this consistent ecologic observation, the precise link between the yeast's natural habitat and the birds is still not certain. Occasionally, birds develop disease that involves C. neoformans, but this is relatively unusual. The resistance of birds to disease may result from their very high body temperature, which is not conducive to growth of C. neoformans, and possibly even their innate protective immunity. However, the yeasts may transiently colonize the gastrointestinal tract of the birds. The most common environmental niche for this species remains rotting vegetation or wood of certain trees, such as coniferous trees and particularly mopane trees. The birds may simply represent vectors, spreading the fungus from these vegetations into the soils and dusts of human traffic.
Cryptococcus gattii Serotypes B and C
Unlike C. neoformans, C . gattii has never been cultured from bird guano. Furthermore, there appears to be a certain geographic limitation to the occurrence of infections with this variety. With this knowledge base, investigators were initially able to culture C. gattii from vegetation around and associated with the river red gum trees ( Eucalyptus camaldulensis ) and forest red gum trees ( Eucalyptus tereticornis ) in Australia. Because these trees were exported to other areas of the world where C. gattii is also observed, it was reasoned that Eucalyptus species may be a vector for infection. It was suggested that the poorly encapsulated yeasts or basidiospores might be released in relationship to the flowering of these trees, but this has not yet been proved. However, despite the association of these trees with C. gattii, the outbreak of cryptococcosis on Vancouver Island, British Columbia, revealed that other trees such as firs, maples, and oaks may also be an ecologic niche for specific strains of C. gattii. It is proposed that these new geographic locations and specific environmental niches for C. gattii could be due to recent climate changes.
Another ecologic factor that may be important to the human pathogenicity of this fungus is its association with other organisms. For example, it has been found in soil associated with a variety of bacteria, amebas, mites, worms, and sow bugs. The stress of this biotic area with its abundant predatory scavengers may have selected for a yeast species that can survive such harsh conditions. In fact, studies have shown that C. neoformans can survive within amebas, which in some respects may provide an environment similar to that in a human macrophage. Furthermore, nonpathogenic cryptococci can act as food for the nematode Caenorhabditis elegans, but C. neoformans can actually kill the worm, and several invertebrate models (worms, grubs, and amebae) have been used in studies of cryptococcal pathogenesis and treatment experiments.
Epidemiology
C. neoformans is not generally considered to be a routine constituent of the human microbiota. There are clinical reports of Cryptococcus being isolated from nonsterile body sites on patients with no signs or symptoms of cryptococcosis. It can also be detected as a commensal in cats and dogs. Furthermore, endobronchial colonization is more frequently observed in humans in the presence of an underlying chronic pulmonary disease. When C. neoformans is isolated from nonsterile clinical specimens, the clinician must examine the patient for evidence of disease and analyze risk factors for the potential development of disease before planning further management strategies. Several methods have been used to study the existence of prior infection with C. neoformans without evidence for disease. Research has shown that patients with cryptococcosis have delayed hypersensitivity to cryptococcal antigens, and the prevalence of positive skin test reactions in pigeon fanciers and laboratory workers engaged in research activities with this yeast has been reported to be high. Unfortunately, there is no established skin test for routine clinical use in patients with cryptococcosis today, and this reduces our ability to assess the magnitude of this infection. However, most adults possess specific antibodies to C. neoformans antigens; for instance, in New York City, most children acquire antibodies to cryptococcal antigens before the age of 10 years. These observations suggest that there are frequent asymptomatic infections. These studies examining serologic evidence of cryptococcal exposure in children do emphasize that in some respects there are certain areas of high exposure to this fungus and other geographic sites with much lower exposure. Although exposure to this yeast is likely limited in certain areas of the world, cryptococcal disease has been reported worldwide.
The vast majority of patients with symptomatic disseminated cryptococcosis have a clearly identified underlying immunocompromised condition ( Table 262.1 ). The most common underlying conditions worldwide include AIDS, prolonged treatment with corticosteroids, organ transplantation, advanced malignancy, diabetes, and sarcoidosis. In fact, occurrence of cryptococcosis may identify an underlying idiopathic CD4 lymphocytopenia or the development of certain autoantibodies, or be associated with the use of the specific immune-modifying monoclonal antibodies, such as alemtuzumab, infliximab, etanercept, and adalimumab. Finally, it has been estimated that approximately 20% of patients who have cryptococcosis without HIV infection have no apparent underlying disease or risk factor. The genetic susceptibility to cryptococcal disease has been clearly determined in murine models but needs more studies in humans. However, recent studies on associations with polymorphisms in immunoglobulin genes and mannose-binding protein gene might be the start of understanding the group of patients with cryptococcal disease without an apparent immunosuppressive event, or how these associations might add to the risk of an immunosuppressed individual. Furthermore, recent studies have suggested a link between autoantibodies to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and risk for cryptococcosis.
TABLE 262.1
Conditions Known to Be or Possibly Associated with Predisposition to Cryptococcus neoformans Infections
Modified from Casadevall A, Perfect JR. Cryptococcus neoformans . Washington, DC: ASM Press; 1998:410.
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GM-CSF, Granulocyte-macrophage colony-stimulating factor; HIV, human immunodeficiency virus; IgE, immunoglobulin E; IgM, immunoglobulin M.
a Immunosuppressive therapy may account for the predisposition.
b Diabetes mellitus has historically been considered a risk factor for cryptococcal infection. However, diabetes is a common disease, and it is unclear whether this condition is truly a specific risk factor for cryptococcosis.
The best estimates for rates of cryptococcosis in the United States in the pre-AIDS era predicted an overall incidence of 0.8 case per 1 million persons per year. In 1992, during the peak of the AIDS epidemic in the United States, the rate reached almost 5 cases of cryptococcosis per 100,000 persons per year in several large cities. In the mid-1990s, before highly active antiretroviral therapy (ART) but with widespread use of fluconazole for oral candidiasis, the rate was reduced, and it stabilized in the cities at approximately 1 case per 100,000 persons per year. With the widespread use of ART in developed countries by the beginning of the 21st century, the incidence of cryptococcosis has declined further and appears to have reached a stable number of new infections. In the AIDS population in developed countries, it now generally represents an infection that identifies a disadvantaged patient or an untreated and undiagnosed HIV infection. Thus cryptococcosis in patients with AIDS identifies a group as having or wanting less access to medical care, or both.
In less medically resourced countries with major ongoing epidemics of HIV, such as in sub-Saharan Africa, cryptococcosis reached high prevalence. Some reports estimate that 15% to 45% of individuals with advanced HIV infection succumb to cryptococcosis. In a population-based surveillance study for cryptococcosis in an antiretroviral-naïve South African population, the overall incidence in HIV-infected patients was 95 cases per 100,000 patients, and those with AIDS had a rate of 14 cases per 1000 patients. A recent sobering study in Botswana with generally excellent access to ART showed an initial drop in cases of cryptococcosis, but over the last 4 to 5 years the incidence has stabilized at a still very high level. A general report from the US Centers for Disease Control and Prevention emphasized that at the peak of the AIDS epidemic, there were annually almost a million new cases of disseminated cryptococcosis with more than 600,000 deaths per year, and in sub-Saharan Africa mortality is higher than with infections caused by tuberculosis in similar areas. In many African medical centers, cryptococcosis represents the most common cause of culture-proven meningitis, even surpassing Neisseria meningitidis and Streptococcus pneumoniae meningitis. In fact, the risk of cryptococcosis appears higher for African-born individuals even when they move to industrialized nations. Increasing cases of cryptococcosis have consistently followed the pattern of HIV infections, and in countries such as Thailand, blood cultures done before widely available ART frequently contained this yeast. However, with increased availability of ART in sub-Saharan Africa and Asia, the magnitude of these observations has changed. An updated assessment of cryptococcosis during widespread ART now predicts over 200,000 cases per year with over 100,000 deaths per year from cryptococcosis. However, the incidence of cryptococcosis will not be dramatically reduced further secondary to undiagnosed HIV infections, noncompliance with ART, or antiretroviral drug resistance.
The varieties or species of Cryptococcus that are identified as causing disease differ by geographic location and by whether the patient has a concomitant HIV infection. Before the AIDS epidemic, Kwon-Chung and Bennett found that at least 80% of clinical isolates worldwide were C. neoformans serotype A (var. grubii ). Cryptococcus gattii serotype B was almost exclusively found in tropical and subtropical areas, such as southern California, Hawaii, Brazil, Australia, Southeast Asia, and central Africa. Serotype C was rare in all localities but seemed to follow the same geographic distribution as serotype B. However, with outbreaks in Vancouver and the Pacific Northwest in the United States and even isolated reports of C. gattii infections east of the Mississippi River and in Europe, the epidemiology is clearly changing with this species. C. neoformans serotype D was predominantly isolated from Europe, especially Denmark, Germany, Italy, France, and Switzerland, and some strains of this variety were found in the United States. In AIDS patients, the vast majority of isolates are serotype A, although serotype D has constituted a significant percentage of isolates in several areas of France. A small, measurable portion of cases in AIDS patients have been reported to be caused by C. gattii and specifically by VGIII and VGIV genotypes. On the other hand, the numbers of C . gattii infections remain small even in areas where this species was commonly observed to cause disease in the pre-AIDS era, and clinical presentations and outcomes remain similar to C. neoformans infections in a comparable risk group.
Cryptococcosis has a measurable rate of infection in two other major risk groups: cancer patients and recipients of solid-organ transplants. Since the 1950s, it has been known that patients with lymphoproliferative disorders and certain hematologic malignancies, such as chronic lymphocytic leukemia, were at higher risk than the general population for cryptococcosis. A retrospective analysis of case reports from a single large cancer center from 1989 to 1999 reported that the incidence of cryptococcosis was 18 cases per 100,000 admissions, and the occurrence is predicted to increase with further frequent use of cell-mediated immune inhibitors, such as alemtuzumab, ibrutinib, and fludarabine, in the management of certain malignancies. On the other hand, checkpoint inhibitors to programmed cell death protein-1 for cancer treatment may actually be preventive with their actions against cryptococcosis. Because of their profound and prolonged immunosuppression, organ transplant recipients have also been a prime target for this infection. In one cohort, cryptococcosis occurred in 2.8% of all solid-organ transplant recipients. Kidney and liver transplant recipients appear to have the highest risk for cryptococcosis. In contrast, in bone marrow transplant recipients, who have a high incidence of fungal infections, cryptococcosis is not common. In rare circumstances, the transplanted organ (e.g., cornea, kidney, lung) has been shown to carry the cryptococcal infection into a susceptible recipient. This clinical scenario represents one of the few instances of person-to-person transmission of this infection.
Sarcoidosis, with or without corticosteroid therapy, predisposes to cryptococcosis and can be a diagnostic dilemma. The lung, skin, bone, and CNS lesions of the two diseases overlap clinically and by histopathology. Despite uncertain pathophysiology, diabetes as an underlying disease or cofactor is frequently mentioned in those patients without HIV or transplant recipient risk factors in most reviews.
There has always been a small but consistently higher rate of cryptococcosis in males than in females, and at times there are subtle differences in immunologic responses between sexes, suggesting hormonal influences on cryptococcal disease. Cryptococcosis can occur before puberty, but even in children with several known risk factors the incidence is uncommon, and in an area of high HIV prevalence only 2% of cryptococcosis cases were in children. Interestingly, there have been several reports of cryptococcosis in children with a hyperimmunoglobulin M syndrome. In adults, idiopathic CD4 + T-cell lymphocytopenia may be identified by the development of disseminated cryptococcosis, and paradoxically this underlying condition with cryptococcosis actually may have a good prognosis for treatment outcome. With much less precision, it has been suggested that smoking and outdoor activities may increase the risk of cryptococcosis, and that recent intravenous drug abuse might provide risk even without HIV infection.
There is general agreement that most cryptococcal infections are acquired primarily by inhalation of infectious propagules, but there are occasional cases of direct traumatic inoculation through contaminated environmental projectiles or laboratory/clinical accidents such as needlesticks. However, neither the environmental source of infection nor the infectious form of C. neoformans has been precisely established in most cases of cryptococcosis. It is hypothesized that either dehydrated, poorly encapsulated yeast cells or basidiospores (<5 µm) are needed as infectious propagules for alveolar deposition in the lungs. Studies at sites with contaminated soils or trees have found that the surrounding air contains the correct size of propagules for airway infection. Molecular typing methods have confirmed that clinical isolates can be indistinguishable from environmental isolates. Although associations between infection and environmental exposure have been reported for many of the classic dimorphic fungi, this association is rare for C. neoformans. However, the outbreak of C. gattii infections on Vancouver Island and the Pacific Northwest has convincingly linked human and animal infections to common environmental exposures. There has not been a consistent seasonal association for the occurrence of cryptococcosis worldwide, although one study did observe more cases in the fall and winter. These uncertain observations likely reflect the influence of the host-reactivation pathophysiology of this disease in many cases, or differences in environmental humidity for aerosol production. Recent fundamental niche mediation models have begun to trace and map the best environmental conditions for certain cryptococcal species’ survival.
Human-to-human transmission of cryptococcosis has not been reported except in cases of contaminated transplant tissue. Many species of animals, including dogs and cats, can develop cryptococcosis, but there is infrequent evidence of zoonotic transmission between them and humans. In one case, C. neoformans isolated from the cage of a pet cockatoo was molecularly linked with the strain that caused infection in a transplant recipient who was exposed to the cage. Also, several cryptococcal cases have been linked to intense bird exposures, and even a possible nosocomial outbreak of cryptococcosis in an intensive care unit has been reported.
Pathogenicity
The encapsulated yeast C. neoformans has been studied extensively for more than 50 years. In the past 2 decades, genetic and molecular biologic research, in concert with well-established and robust animal models, has rapidly increased our understanding of its pathobiology. Progress in cryptococcal molecular biology has led to the use of karyotypes, repetitive elements, transposons, and whole-genome sequencing to identify yeast strains through a variety of analyses, including restriction fragment length polymorphism, RAPD, PCR fingerprints, and MLST. Recently, the entire sequenced genomes of many strains of C. neoformans and C. gattii have been published, and with present sequencing methods many more strains will have their entire genomes sequenced and analyzed. Several transformation systems are available for introducing DNA into this yeast, and site-specific gene disruptions and replacements are routine; a comprehensive whole-genome deletion library has even been created. Dozens of specific null mutants in a variety of pathways or for specific enzymes have been produced to examine their impact on the virulence composite of the yeast in several robust animal models, including zebrafish, in which pathogenesis can visually be observed. Furthermore, differential display PCR, complementary DNA subtraction techniques, serial analysis of gene expression, microarray analysis, and RNA sequencing have been used to document and understand C. neoformans transcriptional profiles. Proteomic and metabolomic approaches have also been used to study its pathophysiology. It is also observed that the cryptococcal genome shows plasticity and rapid changes, and variations can influence its virulence and resistance to drugs. With the use of whole-genome sequencing and following persistent or relapse isolates in the human host, the rates and location of genetic and epigenetic changes have been reported in the human host.
All these molecular tools have been employed to determine the components and mechanisms that make this yeast such an efficient and deadly pathogen. The following paragraphs describe its most prominent virulence phenotypes.
Capsule
The most distinctive feature of C. neoformans and C. gattii is a polysaccharide capsule containing an unbranched chain of α-1,3-linked mannose units substituted with xylosyl and β-glucuronyl groups. The serotype specificity appears to be determined by structural differences in the glucuronoxylomannan (GXM) related to the number of xylose residues and the degree of O -acetylation of hydroxyl groups. The capsular polysaccharide has a highly negative cell surface charge and is attached to the cell wall by α-1,3-glucan residues. However, it is easily released into the immediate growth media or tissue. Capsular thickness, which varies between isolates, is regulated by several environmental cues, including ambient partial pressure of carbon dioxide, serum, urea, and low iron concentrations, which increase capsular size in many strains. These environmental signals appear to augment the yeast's ability to produce disease and may help explain why the capsule may be small in in vitro cultures and the general environment but is much larger when observed within the mammalian host. Mutant cryptococci that are made to be hypocapsular or acapsular are dramatically less virulent than their parental strains in animal models. However, infections caused by capsule-free or poorly encapsulated strains have been rarely observed in the mammalian host.
The impact of the capsular polysaccharide on host immunity can be profound at many pathophysiologic levels. For example, it has been shown to produce the following effects on the host : (1) it acts as an antiphagocytosis barrier, (2) it depletes complement, (3) it produces antibody unresponsiveness, (4) it dysregulates cytokine secretion, (5) it interferes with antigen presentation, (6) it produces brain edema, (7) it creates selectin and tumor necrosis factor receptor loss, (8) it allows a highly negative charge around yeast cells, (9) it extrudes itself into the intracellular environment with the potential for local toxicity on cellular organelles, and (10) it enhances HIV replication. Furthermore, it confers resistance to oxidative stress, which may improve its intracellular survival. When the GXM is shed into the host environment, it affects host immunity at many levels, but fortunately, its detection in host fluids permits a very successful diagnostic test.
The biochemistry of this imposing structure remains poorly understood, but new insights continue to occur for this structure. For example, multiple genes related to capsule synthesis have been identified. Through creation of specific null mutants, it has been shown that any disturbance in efficient capsular synthesis (e.g., reduced formation, secretion, or elimination of the structure) attenuates the ability of the mutated yeast to produce disease. Furthermore, there have been new insights into the many molecular signaling pathways that control expression of the capsule. For example, one critical pathway necessary for efficient capsular production uses a G protein that signals through a cyclic adenosine monophosphate–mediated pathway. Downregulation of this pathway with a concomitant reduction in capsule size produces an attenuated virulence phenotype, but if a mutation in the pathway upregulates capsule production, the mutant yeast becomes hypervirulent.
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