First Reported Case of Cryptococcus gattii in the Southeastern USA: Implications for Travel-Associated Acquisition of an Emerging Pathogen

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient's travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina

A Diverse Population of Cryptococcus gattii Molecular Type VGIII in Southern Californian HIV/AIDS Patients

Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5′ VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts

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

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

Crosses conducted for the study and resulting progeny.

<p>Crosses conducted for the study and resulting progeny.</p

Molecular characterisation of progeny from outgroup cross CBS10090 x NIH312.

<p><b>A</b>) Self-fertility of NIH312 x CBS10090 progeny. Green coloration indicates self-fertility and red coloration indicates no signs of self-fertility after four weeks. <b>B</b>) MLST analysis of NIH312 x CBS10090 progeny was conducted at eight unlinked loci, and scored as VGIII parental (blue), VGII parental (yellow), or both VGII and VGIII (green). The mitochondrial inheritance is also indicated (uniparental). The ploidy determination is listed based on FACS analysis. Progeny 9 is indicated as 1–2N due to an unclear FACS plot, although the molecular and self-fertility assays indicate it is diploid. ^†CBS10090 mitochondrial type by MLST, but subsequent sequencing indicated that it actually carries a recombinant genome (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003771#pgen-1003771-g010" target="_blank">Figure 10</a>).</p

Molecular characterisation of the progeny from outgroup crosses of R265 x B4546.

<p><i>ATP6</i> primers were used to differentiate VGII and VGIII molecular types and allow for the documentation of uniparental mitochondrial inheritance in <i>C. gattii</i>. <b>A</b>) mtDNA PCR products for 12 strains (six VGII and six VGIII) where a shorter amplicon is produced from VGII isolates compared to VGIII isolates. <b>B</b>) Uniparental inheritance of the mitochondrial genome in 18 R265 x B4656 progeny. These progeny showed the same length as their VGIII <i>MAT</i><b>a</b> parent (isolate B4546) and not the α parental strain (isolate R265). <b>C</b>) MLST analysis of B4546 x R265 progeny was conducted at eight unlinked loci, and scored as VGIII parental (blue, B = B4546), VGII parental (yellow, R = R265), or both VGII and VGIII (green). The mitochondrial inheritance is also indicated (uniparental). The ploidy determination is listed based on FACS analysis. <b>D</b>) Self-fertility of R265 x B4546 progeny. Green coloration indicates self-fertility and red coloration indicates no signs of self-fertility after four weeks. <b>E</b>) MLST analysis of B4546 x R265 haploid progeny was conducted at eight unlinked loci, and scored as VGIII parental (blue, B = B4546) or VGII parental (yellow, R = R265). The mitochondrial inheritance is also indicated (uniparental). The ploidy determination is listed based on FACS analysis. None of the haploid progeny showed self-fertility after four weeks (indicated by red coloration).</p

Experimental design of ingroup and outgroup crosses.

<p><b>A</b>) Schematic illustration of the crosses between a low or highly virulent α parent and a high or low virulence <b>a</b> parent respectively. <b>B</b>) Summary of the strains used for mating, their ability to proliferate intracellularly within macrophages (IPR), and the number of progeny isolated from each cross. ^†Subsequent whole genome sequence analysis revealed that the progeny and restored ‘haploid’ strains are aneuploid strains. Progeny showed regions of triploidy and restored strains regions of diploidy (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003771#pgen.1003771.s001" target="_blank">Figure S1</a>).</p