Edmond J. Byrnes

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.

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Spread of Cryptococcus gattii into Pacific Northwest Region of the United States

This organism should be recognized as an emerging pathogen in the United States.

Molecular Evidence That the Range of the Vancouver Island Outbreak of Cryptococcus gattii Infection Has Expanded into the Pacific Northwest in the United States

Cryptococcus neoformans frequently causes fungal meningitis in immunocompromised patients, whereas the related species C. gattii is restricted to tropical and subtropical regions,where it usually infects immunocompetent individuals.An outbreak of C. gattii infection that began in 1999 on Vancouver Island has resulted in endemic C. gattii infection and caused numerous human and veterinary infections; the outbreaks range has spread to mainland British Columbia. The outbreak-related isolates have been molecular type VGIIa, the major genotype, or VGIIb, the minor genotype. Since 2006, human and veterinary cases of C. gattii infection have emerged in Washington and Oregon. Multilocus sequence typing demonstrates the spread of C. gattii VGIIa and VGIIb from Vancouver Island to the Pacific Northwest. Clinical strains recovered in Oregon represent a unique VGIIc genotype.

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals

Infectious fungi are among a broad group of microbial pathogens that has and continues to emerge concomitantly due to the global AIDS pandemic as well as an overall increase of patients with compromised immune systems. In addition, many pathogens have been emerging and re-emerging, causing disease in both individuals who have an identifiable immune defect and those who do not. The fungal pathogen Cryptococcus gattii can infect individuals with and without an identifiable immune defect, with a broad geographic range including both endemic areas and emerging outbreak regions. Infections in patients and animals can be severe and often fatal if untreated. We review the molecular epidemiology, population structure, clinical manifestations, and ecological niche of this emerging pathogen.

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 MAT a 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.

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 patients 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.

Cryptococcus gattii outbreak expands into the Northwestern United States with fatal consequences

In the past decade, the primary fungal pathogen Cryptococcus gattii has evolved and adapted to the temperate climate of the Pacific Northwest region of North America. This pathogen is now endemic and an increasingly common cause of life-threatening pulmonary and central nervous system infections that are difficult to manage and, in some cases, fatal to humans and other mammals throughout the region. A series of recent reports provide evidence that evolutionary, climatic, and anthropogenic factors may be causing the expansion of the Vancouver Island outbreak genotype into the United States, with the concomitant emergence of a unique genotype in the state of Oregon. Ongoing studies address the molecular epidemiology, roles of mating and genetic exchange, and geographic origins of this unprecedented outbreak of fungal infection of considerable public health magnitude.

The Outbreak of Cryptococcus gattii in Western North America: Epidemiology and Clinical Issues.

Over the previous decade, we observed the emergence of the fungal pathogen, Cryptococcus gattii, as a cause of disease in humans and animals in a temperate climate. This outbreak, first documented on Vancouver Island, has since expanded throughout Western North America, with non–travel-associated cases now in British Columbia, Washington, Oregon, and California. Additionally, a secondary outbreak, originating in and still restricted to Oregon, has also occurred. During the past several years, several studies detailing molecular typing, virulence, antifungal susceptibilities, epidemiology, and clinical issues have been published. These studies begin to address the complex dynamics of this novel emergence of a rare and fatal fungus, outline clinical characteristics of human cases, and also opened several new areas that should be explored in the upcoming years.