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.     

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.     

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.     

Molecular type distribution and fluconazole susceptibility of clinical Cryptococcus gattii isolates from South African laboratory-based surveillance, 2005–2013

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC₅₀ and MIC₉₀ values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC₅₀ value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93–55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa’s most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.     

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.     

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.     

Deubiquitinase Ubp5 Is Required for the Growth and Pathogenicity of Cryptococcus gattii

Cryptococcus gattii is a resurgent fungal pathogen that primarily infects immunocompetent hosts. Thus, it poses an increasingly significant impact on global public health; however, the mechanisms underlying its pathogenesis remain largely unknown. We conducted a detailed characterization of the deubiquitinase Ubp5 in the biology and virulence of C. gattii using the hypervirulent strain R265, and defined its properties as either distinctive or shared with C. neoformans. Deletion of the C. gattii Ubp5 protein by site-directed disruption resulted in a severe growth defect under both normal and stressful conditions (such as high temperature, high salt, cell wall damaging agents, and antifungal agents), similar to the effects observed in C. neoformans. However, unlike C. neoformans, the C. gattii ubp5Δ mutant displayed a slight enhancement of capsule and melanin production, indicating the evolutionary convergence and divergence of Ubp5 between these two sibling species. Attenuated virulence of the Cg-ubp5Δ mutant was not solely due to its reduced thermotolerance at 37°C, as shown in both worm and mouse survival assays. In addition, the assessment of fungal burden in mammalian organs further indicated that Ubp5 was required for C. gattii pulmonary survival and, consequently, extrapulmonary dissemination. Taken together, our work highlights the importance of deubiquitinase Ubp5 in the virulence composite of both pathogenic cryptococcal species, and it facilitates a better understanding of C. gattii virulence mechanisms.     

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.     

Multilocus Sequence Typing and Virulence Profiles in Uropathogenic Escherichia coli Isolated from Cats in the United States

The population structure, virulence, and antimicrobial resistance of uropathogenic E. coli (UPEC) from cats are rarely characterized. The aim of this study was to compare and characterize the UPEC isolated from cats in four geographic regions of USA in terms of their multilocus sequence typing (MLST), virulence profiles, clinical signs, antimicrobial resistance and phylogenetic grouping. The results showed that a total of 74 E. coli isolates were typed to 40 sequence types with 10 being novel. The most frequent phylogenetic group was B2 (n = 57). The most frequent sequence types were ST73 (n = 12) and ST83 (n = 6), ST73 was represented by four multidrug resistant (MDR) and eight non-multidrug resistant (SDR) isolates, and ST83 were significantly more likely to exhibit no drug resistant (NDR) isolates carrying the highest number of virulence genes. Additionally, MDR isolates were more diverse, and followed by SDR and NDR isolates in regards to the distribution of the STs. afa/draBC was the most prevalent among the 29 virulence-associated genes. Linking virulence profile and antimicrobial resistance, the majority of virulence-associated genes tested were more prevalent in NDR isolates, and followed by SDR and MDR isolates. Twenty (50%) MLST types in this study have previously been associated with human isolates, suggesting that these STs are potentially zoonotic. Our data enhanced the understanding of E. coli population structure and virulence association from cats. The diverse and various combinations of virulence-associated genes implied that the infection control may be challenging.     

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.     

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.     

Multiple phylogenetically-diverse,differentially-virulent Burkholderia pseudomallei isolated from a single soil sample collected in Thailand

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796–1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1–2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei.     

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.     

Retrospective Study of the Epidemiology and Clinical Manifestations of Cryptococcus gattii Infections in Colombia from 1997–2011

Background

Cryptococcosis due to Cryptococcus gattii is endemic in various parts of the world, affecting mostly immunocompetent patients. A national surveillance study of cryptococcosis, including demographical, clinical and microbiological data, has been ongoing since 1997 in Colombia, to provide insights into the epidemiology of this mycosis.

Methodology/Principal Findings

From 1,209 surveys analyzed between 1997–2011, 45 cases caused by C. gattii were reported (prevalence 3.7%; annual incidence 0.07 cases/million inhabitants/year). Norte de Santander had the highest incidence (0.81 cases/million/year), representing 33.3% of all cases. The male: female ratio was 3.3∶1. Mean age at diagnosis was 41±16 years. No specific risk factors were identified in 91.1% of patients. HIV infection was reported in 6.7% of patients, autoimmune disease and steroids use in 2.2%. Clinical features included headache (80.5%), nausea/vomiting (56.1%) and neurological derangements (48.8%). Chest radiographs were taken in 21 (46.7%) cases, with abnormal findings in 7 (33.3%). Cranial CT scans were obtained in 15 (33.3%) cases, with abnormalities detected in 10 (66.7%). Treatment was well documented in 30 cases, with most receiving amphotericin B. Direct sample examination was positive in 97.7% cases. Antigen detection was positive for all CSF specimens and for 75% of serum samples. C. gattii was recovered from CSF (93.3%) and respiratory specimens (6.6%). Serotype was determined in 42 isolates; 36 isolates were serotype B (85.7%), while 6 were C (14.3%). The breakdowns of molecular types were VGII (55.6%), VGIII (31.1%) and VGI (13.3%). Among 44 strains, 16 MLST sequence types (ST) were identified, 11 of them newly reported.

Conclusions/Significance

The results of this passive surveillance study demonstrate that cryptococcosis caused by C. gattii has a low prevalence in Colombia, with the exception of Norte de Santander. The predominance of molecular type VGII is of concern considering its association with high virulence and the potential to evolve into outbreaks.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

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.     

Emergence and Pathogenicity of Highly Virulent Cryptococcus gattii Genotypes in the Northwest United States

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.     

Cryptococcus gattii Infections and Virulence

Cryptococcus gattii is an emerging fungal pathogen in many areas of the world. Since the start of an outbreak in North America in 1999, scientific interest in C. gattii has increased greatly. Much discussion has centered around the comparative virulence of C. gattii infections, both in relation to the related and more common C. neoformans infections and among the different molecular types and subtypes of C. gattii. We review current data and present outstanding questions that remain to be answered regarding C. gattii virulence.