Whole genome sequence analysis of Cryptococcus gattii from the Pacific Northwest reveals unexpected diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.     

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.     

Galleria mellonella Model Identifies Highly Virulent Strains among All Major Molecular Types of Cryptococcus gattii

Cryptococcosis is mainly caused by Cryptococcus neoformans. However, the number of cases due to C. gattii is increasing, affecting mainly immunocompetent hosts. C. gattii is divided into four major molecular types, VGI to VGIV, which differ in their host range, epidemiology, antifungal susceptibility and geographic distribution. Besides studies on the Vancouver Island outbreak strains, which showed that the subtype VGIIa is highly virulent compared to the subtype VGIIb, little is known about the virulence of the other major molecular types. To elucidate the virulence potential of the major molecular types of C. gattii, Galleria mellonella larvae were inoculated with ten globally selected strains per molecular type. Survival rates were recorded and known virulence factors were studied. One VGII, one VGIII and one VGIV strain were more virulent (p <0.05) than the highly virulent Vancouver Island outbreak strain VGIIa (CDCR265), 11 (four VGI, two VGII, four VGIII and one VGIV) had similar virulence (p >0.05), 21 (five VGI, five VGII, four VGIII and seven VGIV) were less virulent (p <0.05) while one strain of each molecular type were avirulent. Cell and capsule size of all strains increased markedly during larvae infection (p <0.001). No differences in growth rate at 37°C were observed. Melanin synthesis was directly related with the level of virulence: more virulent strains produced more melanin than less virulent strains (p <0.05). The results indicate that all C. gattii major molecular types exhibit a range of virulence, with some strains having the potential to be more virulent. The study highlights the necessity to further investigate the genetic background of more and less virulent strains in order to recognize critical features, other than the known virulence factors (capsule, melanin and growth at mammalian body temperature), that maybe crucial for the development and progression of cryptococcosis.     

Cryptococcus gattii VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal

Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MAT a isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.     

Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia

Background

Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.

Methodology/Principal Findings

A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MAT a VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MAT a populations. Our results also support the hypothesis of a “smouldering” outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.

Conclusions/Significance

The detection of sexual recombination in MATα-MAT a population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.     

Genetic Diversity of the Cryptococcus Species Complex Suggests that Cryptococcus gattii Deserves to Have Varieties

The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV). To investigate the phylogenetic relationships among these haploid genotypes, we selected 73 strains from 2000 globally collected isolates investigated in our previous typing studies, representing each of these genotypes and carried out multigene sequence analyses using four genetically unlinked nuclear loci, ACT1, IDE, PLB1 and URA5. The separate or combined sequence analyses of all four loci revealed seven clades with significant support for each molecular type. However, three strains of each species revealed some incongruence between the original molecular type and the sequence-based type obtained here. The topology of the individual gene trees was identical for each clade of C. neoformans but incongruent for the clades of C. gattii indicating recent recombination events within C. gattii. There was strong evidence of recombination in the global VGII population. Both parsimony and likelihood analyses supported three major clades of C. neoformans (VNI/VNB, VNII and VNIV) and four major clades of C. gattii (VGI, VGII, VGIII and VGIV). The sequence variation between VGI, VGIII and VGIV was similar to that between VNI/VNB and VNII. MATa was for the first time identified for VGIV. The VNIV and VGII clades are basal to the C. neoformans or the C. gattii clade, respectively. Divergence times among the seven haploid monophyletic lineages in the Cryptococcus species complex were estimated by applying the hypothesis of the molecular clock. The genetic variation found among all of these haploid monophyletic lineages indicates that they warrant varietal status.     

Molecular characterization of Cryptococcus gattii genotype AFLP6/VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire,Dutch Caribbean

BackgroundThe basidiomycetous yeast Cryptococcus gattii is an emerging and primary pathogen. There is a lack of information about its environmental spread outside outbreak regions in Mediterranean Europe, North and South America. Environmental sampling for C. gattii and molecular characterization of the obtained isolates will provide an insight into the global spread of the various genotypes.MethodsWoody debris of native divi-divi (Caesalpinia coriaria) trees were sampled across Bonaire, Dutch Caribbean. Colonies suspected for Cryptococcus species were subjected to standard mycology investigations and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Isolates identified as C. gattii were subjected to amplified fragment length polymorphism genotyping, mating-type analysis and multi-locus sequence typing.ResultsTen colonies of C. gattii were cultured from different trunk hollows of the same divi-divi tree. Molecular characterization showed that all isolates were genotype AFLP6/VGII and mating-type α. Multi-locus sequence typing revealed that all isolates were genetically indistinguishable from each other.ConclusionsC. gattii is present in the environment of Bonaire, which suggests that this yeast is likely to be present in the environment of other Caribbean islands.     

Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest

Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.     

Molecular epidemiology and microbiological characteristics of Cryptococcus gattii VGII isolates from China

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from China exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China.     

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported.     

Population Genetic Analysis Reveals a High Genetic Diversity in the Brazilian Cryptococcus gattii VGII Population and Shifts the Global Origin from the Amazon Rainforest to the Semi-arid Desert in the Northeast of Brazil

Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence.     

MLST and Whole-Genome-Based Population Analysis of Cryptococcus gattii VGIII Links Clinical,Veterinary and Environmental Strains,and Reveals Divergent Serotype Specific Sub-populations and Distant Ancestors

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome.     

Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil

BackgroundThe differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients.AimsThe aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008.MethodsIt was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments.ResultsPatients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58 μg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type.ConclusionsThis study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil.     

Promiscuous mitochondria in Cryptococcus gattii

Cryptococcus gattii is a primary pathogenic basidiomycetous yeast comprising four genotypic groups. Here we present data on two mitochondrial loci (MtLrRNA and ATP6 ). Two of the genotypic groups, namely amplified fragment length polymorphism (AFLP)5/VGIII and AFLP6/VGII, formed monophyletic lineages. The AFLP4/VGI genotypic group, however, possessed five different mitochondrial genotypes that did not form a monophyletic lineage. The majority of these isolates contained mitochondrial genomes that are partially identical to those found in isolates belonging to AFLP6/VGII, which is causing the ongoing and expanding Vancouver Island outbreak. Two out of four AFLP7/VGIV isolates contained an AFLP4/VGI allele of MtLrRNA. These observations are best explained by assuming a process of mitochondrial recombination. If this is true, mitochondrial recombination seems possible between cells belonging to different genotypic groups of C. gattii , especially between AFLP6/VGII or AFLP7/VGIV and AFLP4/VGI. We also have to assume that mitochondria, most likely, were transferred from cells belonging to AFLP6/VGII to AFLP4/VGI. As such a process of mitochondrial recombination is only possible after cell–cell conjugation, this may also allow the further exchange of genetic material, for example nuclear or plasmid in nature, between different genotypes of C. gattii . This may be relevant as it may provide a possible mechanism contributing to the modulation of virulence attributes of isolates, such as has been observed in the ongoing Vancouver Island outbreak of C. gattii .     

Detection of Cryptococcus gattii in Selected Urban Parks of the Willamette Valley, Oregon

Human and animal infections of the fungus Cryptococcus gattii have been recognized in Oregon since 2006. Transmission is primarily via airborne environmental spores and now thought to be locally acquired due to infection in non-migratory animals and humans with no travel history. Previous published efforts to detect C. gattii from tree swabs and soil samples in Oregon have been unsuccessful. This study was conducted to determine the presence of C. gattii in selected urban parks of Oregon cities within the Willamette Valley where both human and animal cases of C. gattii have been diagnosed. Urban parks were sampled due to spatial and temporal overlap of humans, companion animals and wildlife. Two of 64 parks had positive samples for C. gattii. One park had a positive tree and the other park, 60 miles away, had positive bark mulch samples from a walkway. Genotypic subtypes identified included C. gattii VGIIa and VGIIc, both considered highly virulent in murine host models.     

Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas

Cryptococcus gattii has recently emerged as a pathogen of humans and animals in the temperate climate of Vancouver Island, British Columbia (B.C.). The majority (approximately 95%) of the isolates from the island belong to the VGII molecular type, and the remainder belong to the VGI molecular type. The goals of this study were to compare patterns of molecular variation among C. gattii isolates from B.C. with those from different areas of the world and to investigate the population structure using a comparative gene genealogy approach. Our results indicate that the C. gattii population in B.C. comprises at least two divergent lineages, corresponding to previously identified VGI and VGII molecular types. The genealogical analysis of strains suggested a predominantly clonal population structure among B.C. isolates, while there was evidence for sexual recombination between different molecular types on a global scale. We found no geographic pattern of strain relationships, and nucleotide sequence comparisons revealed that genotypes among isolates from B.C. were also present among isolates from other areas of the world, indicating extensive strain dispersal. The nucleotide sequence diversity among isolates from B.C. was similar to that among isolates from other areas of the world.     

Microbiological,Epidemiological, and Clinical Characteristics and Outcomes of Patients with Cryptococcosis in Taiwan, 1997–2010

Background

Among members of Cryptococcus neoformans- Cryptococcus gattii species complex, C. neoformans is distributed worldwide whereas C. gattii is considered to be more prevalent in the subtropics and tropics including Taiwan. This nationwide study was undertaken to determine the distribution of genotypes, clinical characteristics and outcomes of 219 patients with proven cryptococcosis at 20 hospitals representative of all geographic areas in Taiwan during 1997–2010.

Methods and Findings

Of 219 isolates analyzed, C. neoformans accounted for 210 isolates (95.9%); nine isolates were C. gattii (4.1%). The predominant genotype was VNI (206 isolates). The other genotypes included VNII (4 isolates), VGI (3 isolates) and VGII (6 isolates). Antifungal minimal inhibition concentrations higher than epidemiologic cutoff values (ECVs) were found in nine VNI isolates (7 for amphotericin B). HIV infection was the most common underlying condition (54/219, 24.6%). Among HIV-negative patients, liver diseases (HBV carrier or cirrhosis) were common (30.2%) and 15.4% did not have any underlying condition. Meningoencephalitis was the most common presentation (58.9%), followed by pulmonary infection (19.6%) and “others” (predominantly cryptococcemia) (18.7%). The independent risk factors for 10-week mortality, by multivariate analysis, were cirrhosis of liver (P = 0.014) and CSF cryptococcal antigen titer ≥512 (P = 0.020). All except one of 54 HIV-infected patients were infected by VNI genotype (98.1%). Of the 13 isolates of genotypes other than VNI, 12 (92.3%) were isolated from HIV-negative patients. HIV-infected patients compared to HIV-negative patients were more likely to have meningoencephalitis and serum cryptococcal antigen ≥1∶512. Patients infected with C. gattii compared to C. neoformans were younger, more likely to have meningoencephalitis (100% vs. 57%), reside in Central Taiwan (56% vs. 31%), and higher 10-week crude mortality (44.4% vs. 22.2%).

Conclusions

Cryptococcus neoformans in Taiwan, more prevalent than C. gatii, has a predominant VNI genotype. Isolates with antifungal MIC higher than ECVs were rare.     

Characterization of Environmental Sources of the Human and Animal Pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States

Cryptococcus gattii has recently emerged as a primary pathogen of humans and wild and domesticated animals in British Columbia, particularly on Vancouver Island. C. gattii infections are typically infections of the pulmonary and/or the central nervous system, and the incidence of infection in British Columbia is currently the highest reported globally. Prior to this emergence, the environmental distribution of and the extent of colonization by C. gattii in British Columbia were unknown. We characterized the environmental sources and potential determinants of colonization in British Columbia. C. gattii was isolated from tree surfaces, soil, air, freshwater, and seawater, and no seasonal prevalence was observed. The C. gattii concentrations in air samples were significantly higher during the warm, dry summer months, although potentially infectious propagules (<3.3 μm in diameter) were present throughout the year. Positive samples were obtained from many different areas of British Columbia, and some locations were colonization “hot spots.” C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization. C. gattii soil colonization also was associated with low moisture and low organic carbon contents. Most of the C. gattii isolates recovered belonged to the VGIIa genetic subtype; however, sympatric colonization by the VGIIb strain was observed at most locations. At one sampling site, VGIIa, VGIIb, VGI, and the Cryptococcus neoformans serotype AD hybrid all were coisolated. Our findings indicate extensive colonization by C. gattii within British Columbia and highlight an expansion of the ecological niche of this pathogen.     

A Decade of Experience: Cryptococcus gattii in British Columbia

It has been over a decade since Cryptococcus gattii was first recognized as the causative organism of an outbreak of cryptococcosis on Vancouver Island, British Columbia. A number of novel observations have been associated with the study of this emergent pathogen. A novel genotype of C. gattii, VGIIa was described as the major genotype associated with clinical disease. Minor genotypes, VGIIb and VGI, are also responsible for disease in British Columbians, in both human and animal populations. The clinical major genotype VGIIa and minor genotype VGIIb are identical to C. gattii isolated from the environment of Vancouver Island. There is more heterogeneity in VGI, and a clear association with the environment is not apparent. Between 1999 and 2010, there have been 281 cases of C. gattii cryptococcosis. Risk factors for infection are reported to be age greater than 50 years, history of smoking, corticosteroid use, HIV infection, and history of cancer or chronic lung disease. The major C. gattii genotype VGIIa is as virulent in mice as the model Cryptococcus, H99 C. neoformans, although the outbreak strain produces a less protective inflammatory response in C57BL/6 mice. The minor genotype VGIIb is significantly less virulent in mouse models. Cryptococcus gattii is found associated with native trees and soil on Vancouver Island. Transiently positive isolations have been made from air and water. An ecological niche for this organism is associated within a limited biogeoclimatic zone characterized by daily average winter temperatures above freezing.