Blastomyces Induced Otomastoiditis With Local Soft Tissue Invasion: A Case Report.

The aim of this report is to document a very rare case of Blastomycosis dermatitidis mastoiditis with extension into the retromastoid soft tissue and surrounding muscle. Blastomycosis dermatitidis is a dimorphic fungus of endemic areas which classically infiltrates the lungs; however, dissemination presenting as otomastoiditis is exceedingly rare. The patient was an immunocompetent 27-year-old male with no significant preexisting health conditions. He had significant work exposure to dust and soil and was referred to our department for evaluation of otalgia with headaches, hearing loss, and intermittent facial paralysis. Initially, the extent of the infection was unknown. Based on extensive disease on magnetic resonance imaging, the patient was scheduled for urgent tympanoplasty and mastoidectomy. Postoperative treatment with itraconazole resolved any further manifestations and halted further soft tissue invasion. It is important to consider uncommon fungal infections in the workup of persistent otalgia, especially when presenting with facial paralysis and a history of environmental exposure to soil and dust. This type of infection should be considered regardless of immunodeficiency status. Early detection may prevent hearing loss and local invasion into surrounding structures.

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.

Safety Evaluation of an Alpha-Emitter Bismuth-213 Labeled Antibody to (1→3)-ß-Glucan in Healthy Dogs as a Prelude for a Trial in Companion Dogs with Invasive Fungal Infections.

Background: With the limited options available for therapy to treat invasive fungal infections (IFI), radioimmunotherapy (RIT) can potentially offer an effective alternative treatment. Microorganism-specific monoclonal antibodies have shown promising results in the experimental treatment of fungal, bacterial, and viral infections, including our recent and encouraging results from treating mice infected with Blastomyces dermatitidis with 213Bi-labeled antibody 400-2 to (1→3)-ß-glucan. In this work, we performed a safety study of 213Bi-400-2 antibody in healthy dogs as a prelude for a clinical trial in companion dogs with acquired invasive fungal infections and later on in human patients with IFI. Methods: Three female beagle dogs (≈6.1 kg body weight) were treated intravenously with 155.3, 142.5, or 133.2 MBq of 213Bi-400-2 given as three subfractions over an 8 h period. RBC, WBC, platelet, and blood serum biochemistry parameters were measured periodically for 6 months post injection. Results: No significant acute or long-term side effects were observed after RIT injections; only a few parameters were mildly and transiently outside reference change value limits, and a transient atypical morphology was observed in the circulating lymphocyte population of two dogs. Conclusions: These results demonstrate the safety of systemic 213Bi-400-2 administration in dogs and provide encouragement to pursue evaluation of RIT of IFI in companion dogs.

Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia.

Fungal infections have skyrocketed in immune-compromised patients lacking CD4+ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A+ CD8+ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4+ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ+ CD8+ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8+ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.

Blastomyces Virulence Adhesin-1 Protein Binding to Glycosaminoglycans Is Enhanced by Protein Disulfide Isomerase.

UNLABELLED: Blastomyces adhesin-1 (BAD-1) protein mediates the virulence of the yeast Blastomyces dermatitidis, in part by binding host lung tissue, the extracellular matrix, and cellular receptors via glycosaminoglycans (GAGs), such as heparan sulfate. The tandem repeats that make up over 90% of BAD-1 appear in their native state to be tightly folded into an inactive conformation, but recent work has shown that they become activated and adhesive upon reduction of a disulfide linkage. Here, atomic force microscopy (AFM) of a single BAD-1 molecule interacting with immobilized heparin revealed that binding is enhanced upon treatment with protein disulfide isomerase and dithiothreitol (PDI/DTT). PDI/DTT treatment of BAD-1 induced a plateau effect in atomic force signatures that was consistent with sequential rupture of tandem binding domains. Inhibition of PDI in murine macrophages blunted BAD-1 binding to heparin in vitro. Based on AFM, we found that a short Cardin-Weintraub sequence paired with a WxxWxxW sequence in the first, degenerate repeat at the N terminus of BAD-1 was sufficient to initiate heparin binding. Removal of half of the 41 BAD-1 tandem repeats led to weaker adhesion, illustrating their role in enhanced binding. Mass spectroscopy of the tandem repeat revealed that the PDI-induced interaction with heparin is characterized by ruptured disulfide bonds and that cysteine thiols remain reduced. Further binding studies showed direct involvement of thiols in heparin ligation. Thus, we propose that the N-terminal domain of BAD-1 governs the initial association with host GAGs and that proximity to GAG-associated host PDI catalyzes activation of additional binding motifs conserved within the tandem repeats, leading to enhanced avidity and availability of reduced thiols. IMPORTANCE: Pathogenic fungi and other microbes must adhere to host tissue to initiate infection. Surface adhesins promote this event and may be required for disease pathogenesis. We studied a fungal adhesin essential for virulence (BAD-1; Blastomyces adhesin-1) and found that host products induce its structural reconfiguration and foster its optimal binding to tissue structures.

The unappreciated intracellular lifestyle of Blastomyces dermatitidis.

Blastomyces dermatitidis, a dimorphic fungus and the causative agent of blastomycosis, is widely considered an extracellular pathogen, with little evidence for a facultative intracellular lifestyle. We infected mice with spores, that is, the infectious particle, via the pulmonary route and studied intracellular residence, transition to pathogenic yeast, and replication inside lung cells. Nearly 80% of spores were inside cells at 24 h postinfection with 10(4) spores. Most spores were located inside of alveolar macrophages, with smaller numbers in neutrophils and dendritic cells. Real-time imaging showed rapid uptake of spores into alveolar macrophages, conversion to yeast, and intracellular multiplication during in vitro coculture. The finding of multiple yeast in a macrophage was chiefly due to intracellular replication rather than multiple phagocytic events or fusion of macrophages. Depletion of alveolar macrophages curtailed infection in mice infected with spores and led to a 26-fold reduction in lung CFU by 6 d postinfection versus nondepleted mice. Phase transition of the spores to yeast was delayed in these depleted mice over a time frame that correlated with reduced lung CFU. Spores cultured in vitro converted to yeast faster in the presence of macrophages than in medium alone. Thus, although advanced B. dermatitidis infection may exhibit extracellular residence in tissue, early lung infection with infectious spores reveals its unappreciated facultative intracellular lifestyle.

Thermally Dimorphic Human Fungal Pathogens--Polyphyletic Pathogens with a Convergent Pathogenicity Trait.

Fungi are adept at changing their cell shape and developmental program in response to signals in their surroundings. Here we focus on a group of evolutionarily related fungal pathogens of humans known as the thermally dimorphic fungi. These organisms grow in a hyphal form in the environment but shift their morphology drastically within a mammalian host. Temperature is one of the main host signals that initiates their conversion to the "host" form and is sufficient in the laboratory to trigger establishment of this host-adapted developmental program. Here we discuss the major human pathogens in this group, which are Blastomyces dermatiditis, Coccidioides immitis/posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis/lutzii, Sporothrix schenckii, and Talaromyces marneffei (formerly known as Penicillium marneffei). The majority of these organisms are primary pathogens, with the ability to cause disease in healthy humans who encounter them in endemic areas.

Ependymal and periventricular magnetic resonance imaging changes in four dogs with central nervous system blastomycosis.

Rapid detection of central nervous system (CNS) involvement is important for dogs with blastomycosis, as this can affect antifungal drug selection and has been associated with an increased risk of death. Previous reports describing magnetic resonance imaging (MRI) characteristics of canine CNS blastomycosis primarily identified mass lesions. The purpose of this retrospective study was to determine whether other MRI characteristics of CNS blastomycosis may also occur. Medical records of the Purdue University Veterinary Teaching Hospital were searched and four dogs met inclusion criteria. Magnetic resonance imaging characteristics included periventricular edema, periventricular and meningeal contrast enhancement, and ventriculomegaly. Periventricular lesions most commonly involved the rostral horn of the lateral ventricles and the third ventricle. Increased meningeal contrast enhancement involved the cerebrum, thalamus, sella turcica, and brainstem. Findings indicated that, in addition to mass lesions, MRI characteristics of periventricular hyperintensity, contrast enhancement, and ventriculomegaly may also occur in dogs with CNS blastomycosis.

Identification of the mating-type (MAT) locus that controls sexual reproduction of Blastomyces dermatitidis.

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

Uncommon yeast infections in hematological patients: from diagnosis to treatment.

Invasive fungal diseases have been recognized with increasing frequency as major pathogens in patients with cancer over the past few decades, as a result of new and more aggressive anticancer treatments and supportive care, and this has been especially reported for patients suffering from hematological malignancies. In these settings, typically uncommon yeasts and filamentous fungi have recently emerged as significant human pathogens, frequently as breakthrough infections in patients receiving empirical antifungal therapy or antifungal prophylaxis and with reported high crude mortality rates. The aim of this article is to discuss certain aspects of the approach to invasive fungal diseases due to uncommon yeasts (e.g., Trichosporon spp., Blastomyces spp. and Cryptococcus spp.) in patients with hematological malignancies, focusing on epidemiology, diagnosis, treatment outcomes and the role of novel antifungal drugs (i.e., new triazoles and echinocandins).

Diagnosis of blastomycosis in surgical pathology and cytopathology: correlation with microbiologic culture.

Blastomycosis, a worldwide disease caused by the inhalation of Blastomyces dermatitidis spores, can be diagnosed by microbiologic culture or morphologic identification in tissue or cytologic material. A retrospective review of cases diagnosed as blastomycosis in surgical pathology and cytopathology was undertaken at a University Medical Center to assess the diagnostic value of morphologic methods and their correlation with microbiologic cultures. Surgical pathology/cytology records were reviewed for the period between January 1998 and April 2007 and 53 cases diagnosed as blastomycosis were retrieved: 38 males, 15 females; age 14 to 77 years, median 48. Twenty-nine cases (54.7%) involved lung, 14 (26.4%) soft tissue/bone, 5 (9.4%) skin, 3 (5.6%) other sites, and 2 (3.7%) involved both lung and skin. Forty-six of the 53 patients (87%) had concomitant cultures: 31 (67.4%) were positive for blastomycosis, 11 (23.9%) negative and 4 (8.7%) showed other fungal organisms. A review of microbiology laboratory results for the same period identified a total of 39 patients who were diagnosed with blastomycosis based on isolation of B. dermatitidis. These included 31 cases (79.5%) that were also diagnosed on histology/cytology specimens, 4 (10.25%) that were not submitted to surgical pathology and 4 (10.25%) cases in which pathologic examination failed to identify Blastomyces. This study shows that blastomycosis encountered in surgical/cytopathology can be reliably diagnosed by morphologic examination allowing for prompt treatment. However, microbiologic cultures still play a major role in clinical management of patients suspected of infection because 10.25% were false negative on morphology in our study.

Ecologic niche modeling of Blastomyces dermatitidis in Wisconsin.

BACKGROUND: Blastomycosis is a potentially fatal mycosis that is acquired by inhaling infectious spores of Blastomyces dermatitidis present in the environment. The ecology of this pathogen is poorly understood, in part because it has been extremely difficult to identify the niche(s) it occupies based on culture isolation of the organism from environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the ecology of blastomycosis by performing maximum entropy modeling of exposure sites from 156 cases of human and canine blastomycosis to provide a regional-scale perspective of the geographic and ecologic distribution of B. dermatitidis in Wisconsin. Based on analysis with climatic, topographic, surface reflectance and other environmental variables, we predicted that ecologic conditions favorable for maintaining the fungus in nature occur predominantly within northern counties and counties along the western shoreline of Lake Michigan. Areas of highest predicted occurrence were often in proximity to waterways, especially in northcentral Wisconsin, where incidence of infection is highest. Ecologic conditions suitable for B. dermatitidis are present in urban and rural environments, and may differ at the extremes of distribution of the species in the state. CONCLUSIONS/SIGNIFICANCE: Our results provide a framework for a more informed search for specific environmental factors modulating B. dermatitidis occurrence and transmission and will be useful for improving public health awareness of relative exposure risks.

Blastomycosis in a South Indian patient after visiting an endemic area in USA.

We describe a case of blastomycosis in a diabetic patient from South India who had visited Milwaukee, Wisconsin, an endemic area for blastomycosis in the USA. After his return to Bangalore, India, the patient developed intermittent fever of moderate to high grade, cough, loss of weight and appetite, and abscesses in the left cubital fossa and thigh regions. Systemic examination at our hospital revealed that he had dullness to percussion over the chest region and decreased breath sounds. Direct examination of Gram-stained smears of the pus from an abscess showed many broad-based budding yeast cells and culture yielded a dimorphic fungus later identified as Blastomyces dermatitidis. Histologic examination of the curettage tissue slides stained with hematoxylin and eosin, periodic acid Schiff's reagent, and Gomori's methenamine silver stain procedures showed many broad-based budding cells characteristic of B. dermatitidis. The patient was successfully treated, initially with amphotericin B, followed by oral itraconazole for a period of 6 months. Blastomycosis cases in India are reviewed and the likely source of infection in this patient is discussed.

Differential requirements of T cell subsets for CD40 costimulation in immunity to Blastomyces dermatitidis.

Cell-mediated immunity and production of type 1 cytokines are the main defenses against pathogenic fungi. Ligation of CD40 by CD40L on T cells is critical for the induction of these immune responses in vivo. We explored the role of CD40/CD40L interactions in vaccine immunity to Blastomyces dermatitidis by immunizing CD40(-/-) and CD40L(-/-) mice and analyzing their resistance to reinfection in a murine pulmonary model. In the absence of CD40 or CD40L, CD4(+) cells failed to get primed or produce type 1 cytokine and impaired the generation of CD8(+) T1 cells. The CD8(+) T cell defect was not due to regulatory T cells or impaired APC maturation or Ag presentation to T cells. If CD4(+) cells were first eliminated, vaccination of CD40(-/-) and CD40L(-/-) mice restored priming of CD8(+) cells, type 1 cytokine production, and resistance. Hence, CD4(+) and CD8(+) cells differ sharply in their requirement for CD40/CD40L interaction during the generation of antifungal immunity. Despite the plasticity of T cell subsets in vaccine immunity, in absence of CD40/CD40L interaction, CD4(+) cells may impede the priming of CD8(+) cells at the cost of host survival against a lethal infectious disease.

Accessory sperm: a biomonitor of boar sperm fertilization capacity.

The number of accessory sperm found in the zona pellucida of porcine embryos was correlated to their individual quality and to the embryo quality range found within a single sow. Our goal was to determine whether accessory sperm counts provide semen evaluation with additional, useful information. Accessory sperm count was highest when only normal embryos were found in a given sow and diminished if oocytes or degenerated embryos were present (P<0.01). Within a given sow, normal embryos had higher (P<0.05) accessory sperm counts than degenerated embryos, although not when oocytes were also present. Fertilization capacity of sperm is optimal when only normal embryos are found in a given sow; this capacity is indicated by high accessory sperm counts. A decrease in fertilization capacity is reflected in diminishing accessory sperm counts. The boar had a significant effect (P<0.01) on accessory sperm count, but not on the percentage of normal embryos; this suggests that accessory sperm may be more sensitive indicators of the fertilization capacity of sperm than the percentage of normal embryos. We conclude that accessory sperm count can be used for the detection of compensable defects in sperm and is a valid parameter for assessing sperm fertilization capacity.

Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii.

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

Delayed diagnosis of osseous blastomycosis in two patients following environmental exposure in nonendemic areas.

Blastomycosis generally results from inhalation of Blastomyces dermatitidis conidia following exposure to contaminated soil in an endemic area. Primary infections commonly involve the lungs, although secondary dissemination to other body sites may occur. We describe 2 cases of osseous blastomycosis in people living outside the endemic areas. Both patients reported exposure to soilfollowing injury to the knee from occupational activities. Mold isolated from each case was identified as B dermatitidis by micromorphologic characteristics including yeast conversion testing and by a positive AccuProbe Blastomyces dermatitidis test (GenProbe, San Diego, CA). Retrospective review of histologic slides, initially reported as negative, identified rare poorly staining, broad-based budding yeast forms in each case. Both patients were treated successfully with itraconazole with no evidence of recurrent infection after 1 year These cases illustrate the importance of considering blastomycosis in the differential diagnosis of bony lesions, even though the patient may live outside an endemic area for B dermatitidis.

A technique for creating localized subcutaneous Blastomyces granulomas in rats.

Our purpose was to develop a simple, reliable method for creating subcutaneous Blastomyces dermatitidis nodules in rats and to describe the histologic appearance of these lesions. We used B. dermatitidis isolated from a dog with blastomycosis to prepare a Blastomyces yeast suspension. Four rats were used to test initial dose concentrations of 10(5), 10(6), 10(9), and 10(10) yeast organisms. The dose was administered subcutaneously over the distal tibia in a volume of 0.1 ml. We then inoculated 35 additional rats with 10(9) or 10(10) yeast organisms. Rats were euthanized 7, 10, 14, 21, or 28 days after inoculation, and the histologic appearance of the nodules was described. A full post-mortem examination sought evidence of systemic spread of Blastomyces organisms. We successfully induced subcutaneous Blastomyces abscesses in 34 of 37 rats injected with 10(9) or 10(10) organisms. Nodules first appeared 3 to 7 days after injection and reached 2 to 15 mm in diameter by 7 to 28 days after inoculation. Histologically the lesions were characterized by a necrotic center surrounded by a layer of viable yeast and granulomatous inflammation. Live yeast organisms were recovered from all lesions. No adverse effects or systemic spread of Blastomyces organisms were observed. We conclude that subcutaneous Blastomyces abscesses can be induced safely and reliably in rats after injection of 10(9) and 10(10) organisms. Histologically, the experimentally induced lesions share both similarities to and differences with lesions of naturally occurring blastomycosis.

Molecular basis of pathogenicity in Blastomyces dermatitidis: the importance of adhesion.

An understanding of the molecular bases of pathogenicity in Blastomyces dermatitidis and related systemic dimorphic fungi has been limited until recent years. Yeast cells of B. dermatitidis display an adhesion promoting protein termed WI-1. Recent studies entailing homologous gene targeting and mutation of WI-1 have provided null mutants at this locus and demonstrated the crucial role of the WI-1 adhesin in pathogenesis of blastomycosis. Ongoing studies are pointing to a link between phase-specific expression of WI-1 and the observation that transition to yeast cells is essential for the acquisition of pathogenicity by B. dermatitidis. Recombinant attenuated yeast that lack WI-1 are serving as invaluable tools for induction of vaccine resistance and are pointing to new insights about adaptive immunity to B. dermatitidis.