Exome Sequencing of a Blastomycosis Case-Control Cohort From Manitoba and Northwestern Ontario, Canada.

BACKGROUND: Blastomycosis is a pulmonary disease caused by Blastomyces spp., a group of pathogenic dimorphic fungi endemic to a number of geographic regions, specifically Manitoba and northwestern Ontario, Canada. Immunosuppression is a major risk factor affecting disease susceptibility, yet host immunity is not well understood. Genetic immunodeficiencies can also influence disease, with variants in IL6, GATA2 and VDBP shown to influence susceptibility. Additional genetic factors in disease susceptibility and severity remain undetected. Our study seeks to identify potential genetic risk factors in a blastomycosis case-control cohort from Manitoba and northwestern Ontario, Canada. METHODS: Exomes from 18 blastomycosis cases and 9 controls were sequenced, variants were identified and filtered for accuracy and quality. We performed candidate gene prioritisation and variant aggregation to identify genetic associations and explored the full exome dataset. RESULTS: Ninety-nine genetic variants in 42 candidate genes were identified in the exome dataset. No variants associated with susceptibility were identified in a single-variant analysis although two non-synonymous variants in TYK2 were enriched among cases suggesting a possible role in susceptibility. Gene-based association analysis found variants in TLR1 enriched in controls (p = 0.024) suggesting a possible protective effect. Gene cluster analysis identified genetic variants in genes of chromatin remodelling, proteasome and intraflagellar transport significantly enriched in cases (false discovery rates < 14%). CONCLUSIONS: The findings in this study show novel associations with blastomycosis susceptibility. A better understanding of host immunity and genetic predisposition to Blastomyces infection can help to inform clinical practice for improved outcomes.

Genomic Epidemiology of Large Blastomycosis Outbreak, Ontario, Canada, 2021.

Using phylogenomic analysis, we provide genomic epidemiology analysis of a large blastomycosis outbreak in Ontario, Canada, caused by Blastomyces gilchristii. The outbreak occurred in a locale where blastomycosis is rarely diagnosed, signaling a possible shift in geographically associated incidence patterns. Results elucidated fungal population genetic structure, enhancing understanding of the outbreak.

Clinical Presentation of Blastomycosis is Associated With Infecting Species, Not Host Genotype.

Objective: To determine if host genetics may be a risk factor for severe blastomycosis.Design: A cohort of patients who had contracted blastomycosis underwent targeted SNP (single nucleotide polymorphism) genotyping. The genetics of these patients were compared to a set of age and gender-matched controls and between patients with severe versus mild to moderate blastomycosis.Setting: The Marshfield Clinic Health System in central and northern WisconsinParticipants: Patients with a diagnosis of blastomycosis prior to 2017 were contacted for enrollment in this study. A phone hotline was also set up to allow interested participants from outside the Marshfield Clinic Health System to request enrollment.Methods: SNP frequency was assessed for significant differences between the patient cohort and controls and between patients with severe versus mild to moderate blastomycosis. We also tested the effect of Blastomyces species identified in clinical isolates on disease symptoms and severity.Results: No significant differences were found in SNP frequency between cases and controls or between those with severe or mild to moderate blastomycosis. We did detect significant differences in symptom frequency and disease severity by Blastomyces species.Conclusions: Our study did not identify any genetic risk factors for blastomycosis. Instead, the species of Blastomyces causing the infection had a significant effect on disease severity.

Detection of Blastomyces gilchristii via metagenomic sequencing in outbreak-associated soils.

Cases of blastomycosis, a serious fungal disease globally rare but endemic to North America, can appear both sporadically and in outbreaks. Tracing these outbreaks to their environment has traditionally used culturing and polymerase chain reaction. Here, we present our method for metagenomic detection of Blastomyces in a 2015 outbreak soil sample from central Wisconsin. By sequencing this sample to multiple depths, we simulated the minimum required depth to detect Blastomyces in this outbreak. Our methods and recommendations can be used to identify the sources of blastomycosis during outbreaks and to learn about the ecology of Blastomyces.

Direct Tissue PCR and Genotyping for Species Identification in a Case of Laryngeal Blastomycosis.

Otolaryngologic manifestations of infection with Blastomyces species are extremely rare and restricted geographically to recognized endemic regions. Here, we describe a case of laryngeal blastomycosis that presented as slowly progressive dysphonia. While a preliminary diagnosis was made using routine histopathology, a species identification of Blastomyces dermatitidis was made using polymerase chain reaction amplification and rapid genotyping without the need for fungal culture. All symptoms resolved following 1 month of antifungal therapy. Rapid molecular differentiation of B dermatitidis from Blastomyces gilchristii provides important insights into pathogenesis given recent recognition of differences in clinical spectra.

Molecular Epidemiology of Blastomyces gilchristii Clusters, Minnesota, USA.

We characterized 2 clusters of blastomycosis cases in Minnesota, USA, using whole-genome sequencing and single-nucleotide polymorphism analyses. Blastomyces gilchristii was confirmed as the cause of infection. Genomic analyses corresponded with epidemiologic findings for cases of B. gilchristii infections, demonstrating the utility of genomic methods for future blastomycosis outbreak investigations.

Variation in Host Resistance to Blastomyces dermatitidis: Potential Use of Genetic Reference Panels and Advances in Immunophenotyping of Diverse Mouse Strains.

Host genetic determinants that underpin variation in susceptibility to systemic fungal infection are poorly understood. Genes responsible for complex traits can be identified by correlating variation in phenotype with allele in founder strains of wild mice with known genetic variation, assembled in genetic reference panels. In this work, we describe wide natural variation in both primary and acquired resistance to experimental pulmonary blastomycosis in eight founder strains, including 129, A/J, BL/6, CAST, NOD, NZO, PWK, and WSB of the Collaborative Cross collection, and the inbred DBA strain. These differences in susceptibility across strains were accompanied by sharp differences in the accumulation and function of immune cells in the lungs. Immune perturbations were mapped by identifying reagents that phenotypically mark immune cell populations in the distinct strains of mice. In particular, we uncovered marked differences between BL/6 and DBA/2 mouse strains in the development of acquired resistance. Our findings highlight the potential value in using genetic reference panels of mice, and particularly the BXD (recombinant inbred strains of mice from a cross of C57BL/6J and DBA/2J mice) collection harboring a cross between resistant BL/6 and susceptible DBA/2 mice, for unveiling genes linked with host resistance to fungal infection. IMPORTANCE Host genetic variation significantly impacts vulnerability to infectious diseases. While host variation in susceptibility to fungal infection with dimorphic fungi has long been recognized, genes that underpin this variation are poorly understood. We used a collection of seven mouse strains that represent nearly 90% of the genetic variation in mice to identify genetic variability among the strains in resistance to pulmonary infection with the dimorphic fungus Blastomyces dermatitidis. We analyzed differences between the strains in innate resistance by infecting naive mice and in acquired resistance by infecting vaccinated mice. We identified extreme variations in both innate and acquired resistance among the strains. In particular, we found sharp differences between C57BL/6 and DBA/2 strains in the ability to acquire vaccine-induced resistance. We also identified commercial reagents that allowed the phenotyping of immune cells from this strain collection of mice. Because there are additional mice harboring a genetic cross of the C57BL/6 and DBA/2 strains (BXD collection), such mice will permit future investigations to identify the genes that underlie differences in the ability to acquire resistance to infection.

Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi.

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

Blastomycosis in 64 Wisconsin Children: Unanticipated Infection Risk and Severity in Urban Residents.

BACKGROUND: Blastomycosis, an endemic mycosis of immunocompetent individuals, is typically seen after exposure to waterways within rural wooded regions. It is not considered a disease of urban environments. Infection can be solely pneumonic or disseminate to skin, bone or central nervous system. Unknown factors influence disease acquisition and severity in children. METHODS: We analyzed acquisition risks and disease characteristics of blastomycosis in children seen at a tertiary care center from 1998 to 2018 to identify potential exposure sources, measure disease severity and assess the effect of race upon disease severity. RESULTS: Of 64 infected children, mean age was 12.9 years, with median time to diagnosis 38.5 days. About 72% were male, 38% resided in urban counties and 50% had typical environmental exposure. Isolated pulmonary infection occurred in 33 (52%). The remainder had evidence of dissemination to skin (N = 13), bone (N = 16; 7 clinically silent) and cranium (N = 7; 3 clinically silent). Infection was moderate/severe in 19 (30%). Two children (3%) died. About 79% of children with moderate/severe disease (P = 0.008) and 71% of urban children (P = 0.007) lacked typical environmental exposure. Comparing children from urban counties to other residences, 63% versus 5% were black (P < 0.001) and 71% versus 35% developed extrapulmonary dissemination (P = 0.006). Moderate/severe disease was seen in 7/17 (42%) black children but only 12/47 (26%) children of other races (P = 0.23). CONCLUSIONS: Blastomycosis, can be endemic in urban children in the absence of typical exposure history, have frequent, sometimes clinically silent, extrapulmonary dissemination and possibly produces more severe disease in black children.

Fungal Bioreporters to Monitor Outcomes of Blastomyces: Host-Cell Interactions.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.

Blastomycosis in Africa and the Middle East: A Comprehensive Review of Reported Cases and Reanalysis of Historical Isolates Based on Molecular Data.

BACKGROUND: Blastomycosis has been reported from countries in Africa and the Middle East, but a decades-long debate has persisted regarding whether this is the same disease known in North America and caused by Blastomyces dermatitidis and Blastomyces gilchristii. METHODS: We reviewed published cases of human and veterinary blastomycosis from Africa and the Middle East. We abstracted epidemiological and clinical features of cases, including sites of disease, diagnosis, management, outcomes, and, where available, genetic and antigenic typing of case isolates. In addition, we sequenced nucleic acids from 9 clinical isolates from Africa deposited in global collections as B. dermatitidis; for 5, we sequenced the internal transcribed spacer regions, and for the other 4 we sequenced the whole genomes. RESULTS: We identified 172 unique human patients with blastomycosis, including 159 patients from 25 African countries and 12 patients from 5 Middle Eastern countries, and also identified 7 reports of veterinary blastomycosis. In humans, cutaneous disease predominated (n = 100/137, 73%), followed by pulmonary (n = 73/129, 57%) and osteoarticular involvement (n = 61/128, 48%). Unusual direct microscopy/histopathological presentations included short hyphal fragments in tissues (n = 23/129, 18%). There were 34 genotyped case isolates that comprised 4 species: Blastomyces percursus (n = 22, 65%), from 8 countries throughout all regions; Blastomyces emzantsi (n = 9, 26%), from South Africa; B. dermatitidis (n = 1, 3%), from the Democratic Republic of Congo; and B. gilchristii (n = 2, 6%), from South Africa and Zimbabwe. CONCLUSIONS: Blastomycosis occurs throughout Africa and the Middle East and is caused predominantly by B. percursus and, at least in South Africa, B. emzantsi, resulting in distinct clinical and pathological patterns of disease.

Development of a Duplex Real-Time PCR Assay for the Differentiation of Blastomyces dermatitidis and Blastomyces gilchristii and a Retrospective Analysis of Culture and Primary Specimens from Blastomycosis Cases from New York (2005 to 2019).

Blastomycosis due to Blastomyces dermatitidis and Blastomyces gilchristii is a significant cause of respiratory mycoses in North America with occasional reported outbreaks. We developed a highly sensitive, specific, and reproducible TaqMan duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019) and primary clinical specimens from blastomycosis cases (2013 to 2019) from New York patients. We identified B. dermatitidis as the predominant pathogen in 38 cases of blastomycosis, while B. gilchristii was a minor pathogen involved in five cases; these findings expand understanding of blastomycosis in New York. The duplex real-time PCR assay could be implemented in reference and public health laboratories to further understand the ecology and epidemiology of blastomycosis due to B. dermatitidis and B. gilchristii.

Gene Editing in Dimorphic Fungi Using CRISPR/Cas9.

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.

Spiromastigoides asexualis: Phylogenetic Analysis and Evaluation as a Cause of False-Positive Blastomyces DNA Probe Test Results.

This is the first case of Spiromastigoides asexualis human infection, and it notably gave a false-positive Blastomyces DNA probe laboratory result. We further investigated other Spiromastigoides isolates as a cause of false-positive testing results, their phylogenetic relationship, and their susceptibility profiles to clinically available antifungal agents. Other S. asexualis isolates also resulted in positive Blastomyces DNA probe results, while Spiromastigoides species other than S. asexualis did not.

Human Blastomycosis in South Africa Caused by Blastomyces percursus and Blastomyces emzantsi sp. nov., 1967 to 2014.

We reevaluated 20 cases of blastomycosis diagnosed in South Africa between 1967 and 2014, with Blastomyces dermatitidis considered to be the etiological agent, in light of newly described species and the use of more advanced technologies. In addition to histopathological and/or culture-based methods, all 20 isolates were phenotypically and genotypically characterized, including multilocus typing of five genes and whole-genome sequencing. Antifungal susceptibility testing was performed as outlined by Clinical and Laboratory Standards Institute documents M27-A3 and M38-A2. We merged laboratory and corresponding clinical case data, where available. Morphological characteristics and phylogenetic analyses of five-gene and whole-genome sequences revealed two groups, both of which were closely related to but distinct from B. dermatitidis, Blastomyces gilchristii, and Blastomyces parvus The first group (n = 12) corresponded to the recently described species Blastomyces percursus, and the other (n = 8) is described here as Blastomyces emzantsi sp. nov. Both species exhibited incomplete conversion to the yeast phase at 37°C and were heterothallic for mating types. All eight B. emzantsi isolates belonged to the α mating type. Whole-genome sequencing confirmed distinct species identities as well as the absence of a full orthologue of the BAD-1 gene. Extrapulmonary (skin or bone) disease, probably resulting from hematogenous spread from a primary lung infection, was more common than pulmonary disease alone. Voriconazole, posaconazole, itraconazole, amphotericin B, and micafungin had the most potent in vitro activity. Over the 5 decades, South African cases of blastomycosis were caused by species that are distinct from B. dermatitidis Increasing clinical awareness and access to simple rapid diagnostics may improve the diagnosis of blastomycosis in resource-limited countries.

Molecular Diagnosis of Emmonsia-Like Fungi Occurring in Wild Animals.

Using specific primers based on the ribosomal operon, positive DNA amplification was obtained from lungs of 11/215 tested small burrowing animals, both terrestrial and aquatic, and including frozen (n = 4) and formalin-fixed paraffin-embedded (n = 7) samples. The main species detected in Europe in mice, otters and river rats was Emmonsia crescens. Two strains from otters and weasels were Blastomyces parvus. Two Australian wombats revealed the presence of a hitherto unknown species of the geophilic genus Emmonsiellopsis.

Turning on virulence: Mechanisms that underpin the morphologic transition and pathogenicity of Blastomyces.

This review article focuses on the mechanisms underlying temperature adaptation and virulence of the etiologic agents of blastomycosis, Blastomyces dermatitidis, Blastomyces gilchristii, and Blastomyces percursus. In response to temperature, Blastomyces undergoes a reversible morphologic switch between hyphae and yeast known as the phase transition. The conversion to yeast for Blastomyces and related thermally dimorphic fungi is essential for virulence. In the yeast phase, Blastomyces upregulates the essential virulence factor, BAD1, which promotes attachment to host cells, impairs activation of immune cells, and blunts cytokine release. Blastomyces yeast also secrete dipeptidyl-peptidase IVA (DPPIVA), a serine protease that blunts the action of cytokines released from host immune cells. In vivo transcriptional profiling of Blastomyces yeast has uncovered genes such as PRA1 and ZRT1 involved in zinc scavenging that contribute to virulence during murine pulmonary infection. The discovery and characterization of genes important for virulence has led to advances at the bedside regarding novel diagnostics, vaccine development, and new targets for drug discovery.

Molecular detection of Blastomyces in an air sample from an outbreak associated residence.

Based on epidemiologic data during a blastomycosis outbreak, exposure within the home was suspected for two case patients that resided together. Soil and air samples were collected from the basement of their residence. Samples were tested for Blastomyces by culture and polymerase chain reaction (PCR) to compare with an available clinical isolate. An air sample from the basement of the residence was PCR positive for Blastomyces. Sequence data from the air sample and the outbreak clinical isolate were identified as different Blastomyces spp. Despite this, our findings suggest that the basement was suitable for the growth of Blastomyces and airborne organism was circulating.