Pulmonary Blastomycosis Following Eculizumab Therapy in a Lung Transplant Recipient.

Lung transplant recipients are at risk of developing many kinds of lung infection, such as community-acquired, nosocomial, opportunistic, and endemic. Here, we report a young lung transplant recipient who developed blastomycosis, which had most likely occurred following eculizumab treatment for atypical hemolytic uremia syndrome. We hypothesize that the agent interfering with C5 would influence the immune response against Blastomyces species. Although eculizumab has opened a new era for treatment of atypical hemolytic uremia syndrome and has led to the understanding that complementmediated pathology is needed, the risk of potentially fatal infections by blocking the complement pathway has not been fully elucidated. Careful follow-up and frequent tests to look for infections are needed after using this monoclonal antibody.

Blastomyces dermatitidis Antibody and Antigen Detection: Comparison of Four Lysate Antigens and Antibodies Prepared from Human Isolates from a Blastomycosis Outbreak.

Blastomycosis is a systemic fungal disease of humans and other animals produced by the thermally dimorphic fungal organism, Blastomyces dermatitidis. Recent studies have focused on the utilization of antibody and antigen detection in the development of immunoassays for the diagnosis of blastomycosis. This study was designed to evaluate four B. dermatitidis yeast lysate antigenic preparations from human isolates (591, 592, 597, 598) from an outbreak of blastomycosis in Eagle River, Wisconsin. The indirect enzyme-linked immunosorbent assay (ELISA) was used to compare these four antigens for their ability to detect antibodies in 28 serum specimens from immunized rabbits and in 18 sera from dogs with blastomycosis. This study also compared antibodies prepared from each of the four B. dermatitidis lysate antigens for their ability to detect antigen using the competitive enzyme-linked immunosorbent assay in 18 urine specimens from the same dogs as above with blastomycosis. All four reagents proved to be immunoreactive and were able to detect antibody in the rabbit and dog sera and antigen in each of the urine specimens with only slight variations in the mean absorbance values evidenced. Antibody detection, mean absorbance values with the four lysates, ranged from 1.522 (592 antigen) to 2.047 (597 antigen) in the rabbit sera and from 1.504 (591 antigen) to 1.878 (597 antigen) in the dog sera. Antigen detection, sensitivity values obtained with the antibodies prepared from the four lysates, ranged from 89% (598 serum) to 100% (591 and 592 serum specimens).

Investigation of Genetic Susceptibility to Blastomycosis Reveals Interleukin-6 as a Potential Susceptibility Locus.

Genetic differences are hypothesized to underlie ethnic disparities in incidence rates of the endemic systemic mycoses, including blastomycosis. Individuals of Hmong ancestry display elevated risk for this serious fungal infection. Here, we interrogated the genomes of Wisconsin (WI) Hmong blastomycosis patients using homozygosity mapping to uncover regions of the genome that are likely shared among the greater Hmong population and filtered for variants with high potential to affect disease susceptibility. This approach uncovered 113 candidate susceptibility variants, and among the most promising are those in genes involved in the interleukin-17 (IL-17) response. In particular, we identified 25 linked variants near the gene encoding IL-6 (IL6). We validated differences in cytokine production between Hmong and European volunteers and formally demonstrated a critical role for IL-6 in the development of adaptive immunity to Blastomyces dermatitidis Our findings suggest that the dysregulation of IL-17 responses underlies a recently reported and poorly understood ethnic health disparity.IMPORTANCE Blastomycosis is a potentially life-threatening infection caused by the fungus Blastomyces dermatitidis As with related fungal diseases, blastomycosis is noted to affect some populations more than others. These patterns of illness are often not related to predisposing conditions or exposure risks; thus, genetic differences are thought to underlie these health disparities. People of Hmong ancestry in Wisconsin are at elevated risk of blastomycosis compared to the general population. We studied the genetic codes of Hmong blastomycosis patients and identified candidate sites in their genomes that may explain their susceptibility to this infection. We further studied one particular region of the genome that is involved with the immune processes that fight B. dermatitidis Our work revealed population differences in the response to fungi. A better understanding of the genetic underpinnings of susceptibility to infectious diseases has broader implications for community health, especially in the paradigm of personalized medicine.

Successful Treatment of Cutaneous Blastomycosis in a Renal Transplant Patient With BK Virus Infection: A Case Report.

This is the first case report from Turkey to describe a renal transplant complicated by subcutaneous blastomycosis and BK infection. The cutaneous lesions were successfully treated with amphotericin B and fluconazole. The presence of BK infection led to graft failure. Infections with uncommonly seen organisms should be kept in mind due to the impaired T-cell immunity in transplantation.

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

Lung Epithelial Cells Coordinate Innate Lymphocytes and Immunity against Pulmonary Fungal Infection.

Lung epithelial cells (LECs) are strategically positioned in the airway mucosa to provide barrier defense. LECs also express pattern recognition receptors and a myriad of immune genes, but their role in immunity is often concealed by the activities of "professional" immune cells, particularly in the context of fungal infection. Here, we demonstrate that NF-κB signaling in LECs is essential for immunity against the pulmonary fungal pathogen Blastomyces dermatitidis. LECs orchestrate innate antifungal immunity by augmenting the numbers of interleukin-17A (IL-17A)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing innate lymphocytes, specifically "natural" Th17 (nTh17) cells. Innate lymphocyte-derived IL-17A and GM-CSF in turn enable phagocyte-driven fungal killing. LECs regulate the numbers of nTh17 cells via the production of chemokines such as CCL20, a process dependent on IL-1α-IL-1 receptor (IL-1R) signaling on LECs. Therefore, LECs orchestrate IL-17A- and GM-CSF-mediated immunity in an IL-1R-dependent manner and represent an essential component of innate immunity to pulmonary fungal pathogens.

Antifungal Tc17 cells are durable and stable, persisting as long-lasting vaccine memory without plasticity towards IFNγ cells.

Our understanding of persistence and plasticity of IL-17A+ memory T cells is clouded by conflicting results in models analyzing T helper 17 cells. We studied memory IL-17A+ CD8+ T-cell (Tc17) homeostasis, persistence and plasticity during fungal vaccine immunity. We report that vaccine-induced memory Tc17 cells persist with high fidelity to the type 17 phenotype. Tc17 cells persisted durably for a year as functional IL-17A+ memory cells without converting to IFNγ+ (Tc1) cells, although they produced multiple type I cytokines in the absence of residual vaccine antigen. Memory Tc17 cells were canonical CD8+ T cells with phenotypic features distinct from Tc1 cells, and were Ror(γ)thi, TCF-1hi, T-betlo and EOMESlo. In investigating the bases of Tc17 persistence, we observed that memory Tc17 cells had much higher levels of basal homeostatic proliferation than did Tc1 cells. Conversely, memory Tc17 cells displayed lower levels of anti-apoptotic molecules Bcl-2 and Bcl-xL than Tc1 cells, yet were resistant to apoptosis. Tc1 cells required Bcl-2 for their survival, but Bcl-2 was dispensable for the maintenance of Tc17 cells. Tc17 and Tc1 cells displayed different requirements for HIF-1α during effector differentiation and sustenance and memory persistence. Thus, antifungal vaccination induces durable and stable memory Tc17 cells with distinct requirements for long-term persistence that distinguish them from memory Tc1 cells.

Probable pulmonary Blastomyomycocis in an immunocompetent person.

The current case report presents a 29-year old man who worked as a hotel guard in a city in China. The patient had fever and dry cough before and after hospitalization. Chest X-ray and CT revealed nodular and patchy lesions in both lower lungs. There were no abnormal findings in the examination of sputum and bronchoalveoular lavage fluid (BALF). He was diagnosed with pulmonary blastomycosis by histological examination of lung biopsy and treated with fluconazole. Follow-up chest CT examination demonstrated that the patient was recovered through appropriate antifungal treatment.

Blastomyces dermatitidis serine protease dipeptidyl peptidase IVA (DppIVA) cleaves ELR+ CXC chemokines altering their effects on neutrophils.

Blastomycosis elicits a pyogranulomatous inflammatory response that involves a prominent recruitment of neutrophils to the site of infection. Although neutrophils are efficiently recruited to the site of infection, this event is paradoxically coupled with the host's inability to control infection by Blastomyces dermatitidis, the causative agent. The mechanisms underlying this characteristic pyogranulomatous response and inability of neutrophils to kill the yeast are poorly understood. We recently reported that the fungal protease dipeptidyl peptidase IVA (DppIVA) promotes B. dermatitidis virulence by cleaving a dipeptide from the N-terminus of C-C chemokines and granulocyte/macrophage-colony stimulating factor, thereby inactivating them. Herein, we present evidence that DppIVA can also truncate the N-terminus of members of the ELR+ CXC chemokine family, which are known to modulate neutrophil function. We show that the DppIVA cleaved form of human (h) CXCL-2, for example, hCXCL-2 (3-73), is a more potent neutrophil chemoattractant than its intact counterpart, but hCXCL-2 (3-73) is conversely impaired in its ability to prime the reactive oxygen species response of neutrophils. Thus, DppIVA action on ELR+ CXC chemokines may promote the pyogranulomatous response that is typical of blastomycosis, while also explaining the inability of neutrophils to control infection.

A 45-Year-Old Woman With 3 Weeks of Cough and Night Sweats.

A 45-year-old woman who received a renal transplant 7 years prior presented with a 3-week history of low-grade fever, night sweats, and a dry cough with scant sputum production. Additionally, she reported generalized weakness and increased fatigability. She denied hemoptysis or weight loss, and there had been no change in medication or foreign travel. She had no history of latent tuberculosis or sick contacts. She had recently relocated to Baton Rouge, Louisiana. She was sexually active with her boyfriend who worked as a prison guard. She also reported that she was briefly incarcerated 7 years ago shortly after her renal transplantation. Her immunosuppression consisted of tacrolimus, mycophenolate, and prednisone.

Mannose Receptor Is Required for Optimal Induction of Vaccine-Induced T-Helper Type 17 Cells and Resistance to Blastomyces dermatitidis Infection.

We investigated how innate sensing by the mannose receptor (MR) influences the development of antifungal immunity. We demonstrate that MR senses mannan on the surface of attenuated Blastomyces dermatitidis vaccine yeast and that MR(-/-) mice demonstrate impaired vaccine immunity against lethal experimental blastomycosis, compared with wild-type control mice. Using naive Blastomyces-specific transgenic CD4(+) T cells, we found that MR regulates differentiation of naive T cells into T-helper type 17 (Th17) effector cells, which are essential in vaccine immunity against systemic dimorphic fungi. Thus, MR regulates differentiation of Th17 cells and is required to induce vaccine immunity against lethal pulmonary blastomycosis.

The C-Type Lectin Receptor MCL Mediates Vaccine-Induced Immunity against Infection with Blastomyces dermatitidis.

C-type lectin receptors (CLRs) are essential in shaping the immune response to fungal pathogens. Vaccine-induced resistance requires Dectin-2 to promote differentiation of antifungal Th1 and Th17 cells. Since Dectin-2 and MCL heterodimerize and both CLRs use FcRγ as the signaling adaptor, we investigated the role of MCL in vaccine immunity to the fungal pathogen Blastomyces dermatitidis. MCL(-/-) mice showed impaired vaccine resistance against B. dermatitidis infection compared to that of wild-type animals. The lack of resistance correlated with the reduced recruitment of Th17 cells to the lung upon recall following experimental challenge and impaired interleukin-17 (IL-17) production by vaccine antigen-stimulated splenocytes in vitro. Soluble MCL fusion protein recognized and bound a water-soluble ligand from the cell wall of vaccine yeast, but the addition of soluble Dectin-2 fusion protein did not augment ligand recognition by MCL. Taken together, our data indicate that MCL regulates the development of vaccine-induced Th17 cells and protective immunity against lethal experimental infection with B. dermatitidis.

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 Antigen Detection for Diagnosis and Management of Blastomycosis.

Blastomyces spp. antigen testing was evaluated over a 10-year period in an area where blastomycosis is endemic. Antigen testing was less sensitive than previously reported, but serial urine testing was useful in monitoring disease resolution or progression. Culture and cytopathology remain the gold standard for diagnosis and exclusion of this infection.

A 66-year-old woman with fever, cough, and a tongue lesion.

A 66-year-old woman presented with acute onset of fever, chills, and productive cough associated with right-sided chest pain. During a recent hospitalization for dyspnea, she had been diagnosed with Coombs-positive autoimmune hemolytic anemia and had been taking a tapering dose of prednisone starting approximately 6 weeks prior to admission. In the interim, her dyspnea had resolved on treatment with steroids. At the time of presentation, her prednisone dose was 40 mg. Additional medical history included VTE, for which the patient was receiving anticoagulation therapy, and steroid-induced diabetes mellitus. Many years earlier, she had been treated for TB in her home country. The patient had immigrated to Queens, New York, from a Nepalese village 8 years prior. While still in Nepal, she had worked on a farm and had been in close proximity to cows. In Queens, she lived with her family in a house with a small garden but had no pets. Recent travel included a visit to Nepal 9 months ago and a trip to Syracuse, New York, one month prior to presentation. She was a never smoker and did not consume alcohol.

Blastomyces dermatitidis in a renal transplant recipient.

Fungal pathogens can be the source of serious and sometimes fatal infections following organ transplantation. To the best of our knowledge, we present the first case of cutaneous blastomycosis in a renal allograft recipient in India, a country outside the known endemic regions. This case, with the very rare and unexpected diagnosis of blastomycosis, not only reflects the tremendous diversity of infections in transplant recipients but also emphasizes the utility of serological methods even in the immunosuppressed host.

Serum and urine Blastomyces antigen concentrations as markers of clinical remission in dogs treated for systemic blastomycosis.

BACKGROUND: Serum and urine Blastomyces antigen concentrations can be used to diagnose blastomycosis in dogs. OBJECTIVES: Blastomyces antigen concentrations correlate with clinical remission in dogs during antifungal treatment, and detect disease relapse after treatment discontinuation. ANIMALS: 21 dogs with newly diagnosed blastomycosis monitored until clinical remission (Treatment Phase), and 27 dogs monitored over 1 year from the time of antifungal discontinuation or until clinical relapse (After Treatment Phase). METHODS: Prospective study. Dogs were monitored monthly during treatment and every 3 months after treatment discontinuation, with a complete history, physical exam, chest radiographs, and ocular exam. Urine and serum Blastomyces antigen concentrations were measured at each visit using a quantitative enzyme immunoassay. RESULTS: At enrollment in the Treatment Phase, Blastomyces antigen was positive in all 21 urine samples (100% sensitivity; 95% CI 85-100%), and in 18 of 20 serum samples (90% sensitivity; 95% CI 70-97%). At 2-4 months of treatment, urine antigen was more sensitive for clinically detectable disease (82%; CI 60-94%) than serum antigen (18%; CI 6-41%). The sensitivity of the urine test for clinical relapse was 71% (CI 36-92%), with close to 100% specificity (CI 84-100%) during after treatment surveillance in this population. CONCLUSIONS: Urine Blastomyces antigen testing has high sensitivity for active disease at the time of diagnosis and during treatment, and moderate sensitivity but high specificity for clinical relapse. Urine testing should be useful at the time of diagnosis, when treatment discontinuation is being considered, and anytime there is poor clinical response or suspicion of relapse.