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.

Blastomyces helicus, a New Dimorphic Fungus Causing Fatal Pulmonary and Systemic Disease in Humans and Animals in Western Canada and the United States.

Background: Blastomyces helicus (formerly Emmonsia helica) is a dimorphic fungus first isolated from a man with fungal encephalitis in Alberta, Canada. The geographic range, epidemiology, and clinical features of disease are unknown. Methods: We reviewed human and veterinary isolates of B. helicus identified among Blastomyces and Emmonsia isolates at the University of Alberta Microfungus Collection and Herbarium, University of Texas Health San Antonio's Fungus Testing Laboratory, and Associated Regional and University Pathologists Laboratories. Isolates were selected based on low Blastomyces dermatitidis DNA probe values and/or atypical morphology. Species identification was confirmed for most isolates by DNA sequence analysis of the internal transcribed spacer with or without D1/D2 ribosomal RNA regions. Epidemiological and clinical data were analyzed. Results: We identified isolates from 10 human and 5 veterinary cases of B. helicus infection; all were referred from western regions of Canada and the United States. Isolates remained sterile in culture, producing neither conidia nor sexual spores in the mycelial phase, but often producing coiled hyphae. Isolates were most frequently cultured from blood and bronchoalveolar lavage in humans and lungs in animals. Most infected persons were immunocompromised. Histopathological findings included pleomorphic, small or variably sized yeast-like cells, with single or multiple budding, sometimes proliferating to form short, branching, hyphal-like elements. Disease carried a high case-fatality rate. Conclusions: Blastomyces helicus causes fatal pulmonary and systemic disease in humans and companion animals. It differs from B. dermatitidis in morphological presentation in culture and in histopathology, by primarily affecting immunocompromised persons, and in a geographic range that includes western regions of North America.

Phylogeographic Analysis of Blastomyces dermatitidis and Blastomyces gilchristii Reveals an Association with North American Freshwater Drainage Basins.

Blastomyces dermatitidis and Blastomyces gilchristii are dimorphic fungal pathogens that cause serious pulmonary and systemic infections in humans. Although their natural habitat is in the environment, little is known about their specific ecologic niche(s). Here, we analyzed 25 microsatellite loci from 169 strains collected from various regions throughout their known endemic range in North America, representing the largest and most geographically diverse collection of isolates studied to date. Genetic analysis of multilocus microsatellite data divided the strains into four populations of B. dermatitidis and four populations of B. gilchristii. B. dermatitidis isolates were recovered from areas throughout North America, while the B. gilchristii strains were restricted to Canada and some northern US states. Furthermore, the populations of both species were associated with major freshwater drainage basins. The four B. dermatitidis populations were partitioned among (1) the Nelson River drainage basin, (2) the St. Lawrence River and northeast Atlantic Ocean Seaboard drainage basins, (3) the Mississippi River System drainage basin, and (4) the Gulf of Mexico Seaboard and southeast Atlantic Ocean Seaboard drainage basins. A similar partitioning of the B. gilchristii populations was observed among the more northerly drainage basins only. These associations suggest that the ecologic niche where the sexual reproduction, growth, and dispersal of B. dermatitidis and B. gilchristii occur is intimately linked to freshwater systems. For most populations, sexual reproduction was rare enough to produce significant linkage disequilibrium among loci but frequent enough that mating-type idiomorphic ratios were not skewed from 1:1. Furthermore, the evolutionary divergence of B. dermatitidis and B. gilchristii was estimated at 1.9 MYA during the Pleistocene epoch. We suggest that repeated glaciations during the Pleistocene period and resulting biotic refugia may have provided the impetus for speciation as theorized for other species associated with temperate freshwater systems.

Blastomyces gilchristii as Cause of Fatal Acute Respiratory Distress Syndrome.

Since the 2013 description of Blastomyces gilchristii, research describing the virulence or clinical outcome of B. gilchristii infection has been lacking. We report molecular evidence of B. gilchristii as an etiologic agent of fatal acute respiratory distress syndrome. B. gilchristii infection was confirmed by PCR and sequence analysis.

Phylogenetic analysis reveals a cryptic species Blastomyces gilchristii, sp. nov. within the human pathogenic fungus Blastomyces dermatitidis.

BACKGROUND: Analysis of the population genetic structure of microbial species is of fundamental importance to many scientific disciplines because it can identify cryptic species, reveal reproductive mode, and elucidate processes that contribute to pathogen evolution. Here, we examined the population genetic structure and geographic differentiation of the sexual, dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. METHODOLOGY/PRINCIPAL FINDINGS: Criteria for Genealogical Concordance Phylogenetic Species Recognition (GCPSR) applied to seven nuclear loci (arf6, chs2, drk1, fads, pyrF, tub1, and its-2) from 78 clinical and environmental isolates identified two previously unrecognized phylogenetic species. Four of seven single gene phylogenies examined (chs2, drk1, pyrF, and its-2) supported the separation of Phylogenetic Species 1 (PS1) and Phylogenetic Species 2 (PS2) which were also well differentiated in the concatenated chs2-drk1-fads-pyrF-tub1-arf6-its2 genealogy with all isolates falling into one of two evolutionarily independent lineages. Phylogenetic species were genetically distinct with interspecific divergence 4-fold greater than intraspecific divergence and a high Fst value (0.772, P<0.001) indicative of restricted gene flow between PS1 and PS2. Whereas panmixia expected of a single freely recombining population was not observed, recombination was detected when PS1 and PS2 were assessed separately, suggesting reproductive isolation. Random mating among PS1 isolates, which were distributed across North America, was only detected after partitioning isolates into six geographic regions. The PS2 population, found predominantly in the hyper-endemic regions of northwestern Ontario, Wisconsin, and Minnesota, contained a substantial clonal component with random mating detected only among unique genotypes in the population. CONCLUSIONS/SIGNIFICANCE: These analyses provide evidence for a genetically divergent clade within Blastomyces dermatitidis, which we use to describe a novel species, Blastomyces gilchristii sp. nov. In addition, we discuss the value of population genetic and phylogenetic analyses as a foundation for disease surveillance, understanding pathogen evolution, and discerning phenotypic differences between phylogenetic species.

Variation in clinical phenotype of human infection among genetic groups of Blastomyces dermatitidis.

BACKGROUND: Blastomyces dermatitidis, the etiologic agent of blastomycosis, has 2 genetic groups and shows varied clinical presentation, ranging from silent infections to fulminant respiratory disease and dissemination. The objective of this study was to determine whether clinical phenotype and outcomes vary based on the infecting organism's genetic group. METHODS: We used microsatellites to genotype 227 clinical isolates of B. dermatitidis from Wisconsin patients. For each isolate, corresponding clinical disease characteristics and patient demographic information were abstracted from electronic health records and Wisconsin Division of Health reportable disease forms and questionnaires. RESULTS: In univariate analysis, group 1 isolates were more likely to be associated with pulmonary-only infections (P < .0001) and constitutional symptoms such as fever (P < .0001). In contrast, group 2 isolates were more likely to be associated with disseminated disease (P < .0001), older patient age (P < .0001), and comorbidities (P = .0019). In multivariate analysis, disease onset to diagnosis of >1 month (P < .0001), older age at diagnosis (P < .0001), and current smoking status (P = .0001) remained predictors for group 2 infections. CONCLUSIONS: This study identified previously unknown associations between clinical phenotype of human infection and genetic groups of B. dermatitidis and provides a framework for further investigations of the genetic basis for virulence in B. dermatitidis.

Population genetic structure of clinical and environmental isolates of Blastomyces dermatitidis, based on 27 polymorphic microsatellite markers.

Blastomyces dermatitidis, a thermally dimorphic fungus, is the etiologic agent of North American blastomycosis. Clinical presentation is varied, ranging from silent infections to fulminant respiratory disease and dissemination to skin and other sites. Exploration of the population genetic structure of B. dermatitidis would improve our knowledge regarding variation in virulence phenotypes, geographic distribution, and difference in host specificity. The objective of this study was to develop and test a panel of microsatellite markers to delineate the population genetic structure within a group of clinical and environmental isolates of B. dermatitidis. We developed 27 microsatellite markers and genotyped B. dermatitidis isolates from various hosts and environmental sources (n=112). Assembly of a neighbor-joining tree of allele-sharing distance revealed two genetically distinct groups, separated by a deep node. Bayesian admixture analysis showed that two populations were statistically supported. Principal coordinate analysis also reinforced support for two genetic groups, with the primary axis explaining 61.41% of the genetic variability. Group 1 isolates average 1.8 alleles/locus, whereas group 2 isolates are highly polymorphic, averaging 8.2 alleles/locus. In this data set, alleles at three loci are unshared between the two groups and appear diagnostic. The mating type of individual isolates was determined by PCR. Both mating type-specific genes, the HMG and α-box domains, were represented in each of the genetic groups, with slightly more isolates having the HMG allele. One interpretation of this study is that the species currently designated B. dermatitidis includes a cryptic subspecies or perhaps a separate species.

Identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species by repetitive-sequence-based PCR.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.

Attempted isolation of Blastomyces dermatitidis from native shrews in northern Wisconsin, USA.

The precise ecological niche of Blastomyces dermatitidis is unknown. The related dimorphic fungus, Paracoccidioides brasiliensis, has been isolated from South American ground-dwelling insectivorous armadillos. We attempted to isolate Blastomyces from shrews, North American ground-dwelling insectivores that have been shown to harbor Histoplasma capsulatum in endemic areas. Forty-seven masked shrews (Sorex cinereus) and 13 northern short-tailed shrews (Blarina brevicauda) were collected in endemic areas of northern Wisconsin and Michigan using pitfall traps. Specimens were collected between 1998 and summer 2002, stored frozen, then necropsied. Cultures of nasopharynx, lungs, liver, spleen and large and small bowel were placed on yeast extract phosphate agar with one or two drops of ammonium hydroxide. Cultures for Blastomyces were negative from all 60 shrews and two deer mice (Peromyscus maniculatus) and three southern red-backed voles (Clethrionomys gapperi), which were trapped inadvertently. Histological examination of 36 of these specimens revealed no Blastomyces yeast forms. Northern Wisconsin shrews do not appear to be carriers of B. dermatitidis.

Coccidioidomycosis and blastomycosis: advances in molecular diagnosis.

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.

Serological differences in two Blastomyces dermatitidis isolates from different geographical regions of North America.

Yeast phase lysate antigens were prepared from two isolates (T-58 and ERC-2) from different geographic locations. Tennessee and Wisconsin. These lysate were evaluated with respect to their ability to detect antibody in dogs infected with blastomycosis and rabbits immunized with the lysates by an enzyme linked immunosorbent assay (ELISA). Both the dog sera and rabbit sera assays demonstrated that there were serological differences in these two isolates, which implied that there was antigenic variance in geographical populations of B. dermatitidis. These results correlated with a previous molecular study that indicated that there are genetic differences in different geographical populations of the organism.

Small subunit ribosomal DNA sequence shows Paracoccidioides brasiliensis closely related to Blastomyces dermatitidis.

The similarities of paracoccidioidomycosis and blastomycosis are highly suggestive of a close relation of the two etiological agents. Whereas the agent of the first disease is exclusively endemic in Latin America, the agent of the latter one is endemic in North America and Africa. In symptomatic travelers visiting both areas of endemicity, differentiation of the diseases might be impossible, even though therapy and prognosis for these two diseases differ significantly. In order to identify differences in the 18S rRNA gene (rDNA) for use as molecular diagnostic tools, we sequenced this gene from five isolates of Paracoccidioides brasiliensis and compared them to known sequences of other fungi. Neighbor-joining, maximum parsimony, and maximum likelihood analyses and, finally, the Kishino-Hasegawa test revealed that P. brasiliensis, Blastomyces dermatitidis, and Emmonsia parva are more closely related than Histoplasma capsulatum and B. dermatitidis, whose teleomorphic forms belong to one genus, Ajellomyces. In accordance with the work of other investigators who have used internal transcribed spacer and large subunit rDNA sequences, our small subunit rDNA data show that the dimorphic fungus P. brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae. There are hints in the molecular phylogenetic analysis that the family Onygenaceae might be further divided into two families. The subgroup that includes P. brasiliensis comprises all zoopathogenic species. The differences in the 18S rDNAs appear to be too small to allow species identification of the members of the family Onygenaceae pathogenic for humans by use of target sequences within this gene.

Molecular epidemiology of Blastomyces dermatitidis.

The inhalation of conidia of Blastomyces dermatitidis, a fungus found in soil, causes disease in humans and animals. We studied the genetic diversity of this pathogen by extracting DNA yeasts and analyzing them with a polymerase chain reaction (PCR)-based typing system we developed, which used restriction fragment analysis of amplicons from the regions between the rDNA repeats and allowed us to class isolates into 3 major groups. Strains were further differentiated by use of PCR fingerprinting with 3 different primers. Fifty-nine isolates collected over 35 years from 15 regions (United States, India, Africa, Canada) were analyzed. Genotypic groups A, B, and C contained 17, 23, and 19 isolates, which were divided into 5, 15, and 12 types, respectively. All 16 isolates from North America in group A were from the upper midwestern United States or Canada, whereas 0 of 20 isolates from the southeastern United States were in group A. Studies of the largest collection from 1 locale (Eagle River, WI), revealed that the soil isolates studied were not responsible for the majority of cases in this outbreak, as previously proposed, and that >1 strain was present in the environment and in patients. Overall, these results provide a tool for the epidemiological study of blastomycosis and illuminate the genetic and geographic diversity of this important pathogen.

Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens.

Emmonsia crescens, an agent of adiaspiromycosis, Blastomyces dermatitidis, the agent of blastomycosis, and Histoplasma capsulatum, the agent of histoplasmosis, are known to form meiotic (sexual) stages in the ascomycete genus Ajellomyces (Onygenaceae, Onygenales), but no sexual stage is known for E. parva, the type species of the genus Emmonsia. To evaluate relationships among members of the putative Ajellomyces clade, large-subunit ribosomal and internal transcribed spacer region DNA sequences were determined from PCR-amplified DNA fragments. Sequences were analyzed phylogenetically to evaluate the genetic variation within the genus Emmonsia and evolutionary relationships to other taxa. E. crescens and E. parva are distinct species. E. crescens isolates are placed into two groups that correlate with their continents of origin. Considerable variation occurred among isolates previously classified as E. parva. Most isolates are placed into two closely related groups, but the remaining isolates, including some from human sources, are phylogenetically distinct and represent undescribed species. Strains of B. dermatitidis are a sister species of E. parva. Paracoccidioides brasiliensis and Histoplasma capsulatum are ancestral to most Emmonsia isolates, and P. brasiliensis, which has no known teleomorph, falls within the Ajellomyces clade.

[Etiology of the first African case of systemic blastomycosis]. / A propos de l'étiologie du premier cas africain de blastomycose systémique.

Several authors have suggested that african and north american forms of Blastomyces dermatitidis appeared separate taxa. Morphologic and pathogenic differences not only prevail, but also the failing to mate between them. African forms lose their mycelium-yeast conversion capability in old cultures. So I propose to create the complementary appellation Blastomyces dermatitidis tunisiens nov.subsp. from the african forms, in memory of the first case of systemic blastomycosis in this continent, discovered in Tunisia.

Molecular taxonomy and epidemiology of Blastomyces and Histoplasma species.

Cladistic analysis of partial 26S rRNA sequences was used to estimate evolutionary distances among species and varieties of the dimorphic onygenalean genera Blastomyces, Coccidioides, Emmonsia, Histoplasma and Paracoccidioides. With the exception of Coccidioides, all genera were closely related, with about 5% base differences and even less (1-2%) between Blastomyces and Emmonsia. These data were supported by a teleomorph in the same genus Ajellomyces. In a phylogenic study of a wide range of ascomycete orders and families, Coccidioides immitis was found to be closest to Aphanoascus fulvescens and Chrysosporium keratinophilum, and to have relative distances to the remaining dimorphic genera (family Onygenaceae) similar to those of the dermatophytes (family Arthrodermataceae). The sequencing data were confirmed by genomic comparisons. All dimorphic genera had a nuclear DNA base composition in the same range of 46.6-47.3% G + C. The DNA melting curves of Blastomyces and Histoplasma strains showed irregularities that were ascribed to the presence of AT-rich stretches in satellite DNA rather than in mitochondrial DNA. Derivative profiles proved to be highly reproducible within regional populations and coincided with differences in clinical behaviour of each species. Blastomyces dermatitidis generated two kinds of curves, corresponding to the geographically distinct serotypes 1 and 2. The African type (serotype 2) was characterized by a classical sigmoidal melting curve similar to that for all strains of Coccidioides, Emmonsia and Paracoccidioides. In contrast, the American type (serotype 1) contained satellite DNA (27% G + C). A rRNA base difference of 1.5% was observed between geographical types, a value slightly higher than that noted between Histoplasma capsulatum and its variety farciminosum (0.9%). All three H. capsulatum varieties presented irregularities in their DNA melting curves. The molecular data support the recognition of two of them as agents of blastomycosis and the assignment of more than one species and two varieties to the genus Emmonsia.

African strains of Blastomyces dermatitidis that do not express surface adhesin WI-1.

African strains of Blastomyces dermatitidis differ from North American strains in their growth, morphology, and clinical disease phenotype. In addition, two serotypes, designated 1 and 2, have been described. We investigated African strains of B. dermatitidis for expression of the surface protein adhesin WI-1 and found that serotype 2 strains do not express it because they lack the coding sequence in their genome. The defect will make the strains useful for gene complementation and for testing the pathogenetic role of the WI-1 adhesin.

Ajellomyces crescens sp. nov., taxonomy of Emmonsia spp., and relatedness with Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis).

Adiaspiromycosis is known primarily as a pulmonary infection of small burrowing mammals and rarely of humans, in which the tissue spore form consists of a large, globose, thick-walled, non-proliferating structure called an adiaspore. The causative agents have been placed in Emmonsia or Chrysosporium and treated as either two species or varieties. Emmonsia parva (= Chrysosporium parvum var. parvum) has been distinguished from E. crescens (= C parvum var. crescens) by differences in maximum growth temperature, size of adiaspores, host range and geographical distribution. Phenotypic similarities between Emmonsia spp. and Blastomyces dermatitidis and chance observation of Ajellomyces-type ascomatal hyphae led to the hypothesis that the teleomorph of Emmonsia spp. could occur in Ajellomyces. Isolates preliminarily identified as E. parva or E. crescens were examined by morphology and physiology and tested for compatibility in mating experiments. Ajellomyces crescens Sigler sp. nov. is described for the teleomorph of Emmonsia crescens based on compatibility among 12 of 22 strains, stellate gymnothecial ascomata composed of obtuse diamond-shaped cells, helically coiled appendages and small, globose, muriculate ascospores. The agents of adiaspiromycosis are here treated as species with adiaspore size and morphology and temperature of induction as their major defining features. The species differ also in cycloheximide tolerance and in their abilities to form a teleomorph. With evidence of a connection between Emmonsia crescens and a teleomorph in Ajellomyces, Emmonsia is favoured over Chrysosporium as the correct name for the agents of adiaspiromycosis. This finding also corroborates earlier suggestions of a close phylogenetic relationship between Emmonsia spp. and the dimorphic pathogens Blastomyces dermatitidis and Histoplasma capsulatum.