Geographic Distribution of Human Blastomycosis Cases in Milwaukee, Wisconsin, USA: Association with Urban Watersheds

Most studies of endemic blastomycosis and outbreaks have involved rural areas. Case homesites in rural Northern Wisconsin have been associated with waterways and sand soils. ARC-GIS was used to geocode addresses and to observe geographic features of homesites from 45 State-mandated reports of human blastomycosis in urban Milwaukee County, Southeastern Wisconsin 2000–2004. Each case property was directly observed, and houses and duplexes (N = 38) were compared with 151 same-street control homesites. Categorical data was analyzed using a chi-square or Fisher’s exact test; continuous variables by Kruskal–Wallis test. One case cluster was seen on Milwaukee’s North side where the estimated annual incidence was 2.8/100,000 compared to 0.96/100,000 for the entire county. Cases were less common in the most urbanized watersheds (0.49/100,000/yr) versus Lake Michigan shores (0.85) versus remaining three open watersheds (1.4) [P<0.01]. Case homesites averaged 1067 m to waterways and none were on sand soils. (Comparison is made to a Northern Wisconsin community where case homesites averaged 354 m to waterways, 24/25 were on sand soils and annual incidence was 74/100,000.) No unique features of case homesites were identified in Milwaukee County. In this urban area of Wisconsin, relatively low incidence rates may be explained, in part, by lower density of inland waterways and lack of sand soils, however, blastomycosis cases appear to be associated with open watersheds.     

Secondary Intracerebral Blastomycosis with Giant Yeast Forms

Secondary central nervous system (CNS) blastomycosis is an unusual manifestation of blastomycosis. We report a case of recurrent intracerebral blastomycosis that presented histopathologically with giant yeast-like cells and multinucleation that mimicked Coccidioides immitis. The yeast forms of Blastomyces dermatitidis usually range in size from 8 to 20 μm in diameter. Large or giant yeast forms (20–40 μm) are rare. The four cases previously reported in the literature involving giant yeast cell forms of B. dermatitidis are reviewed here. Intracerebral blastomycosis should be suspected in patients with signs and symptoms of CNS lesions and histories of primary blastomycosis, or treatment with corticosteroids, or comprised immune systems. The diagnosis should be confirmed by culture which presents typical biphasic microbiologic features.     

Blastomyces dermatitidis Lysate Antigens: Antibody Detection in Serial Serum Specimens from Dogs with Blastomycosis

Yeast phase lysate antigens prepared from different isolates of Blastomyces dermatitidis (T-58, dog-Tennessee; T-27, polar bear-Tennessee; ERC-2, dog-Wisconsin; ER-3, woodpile-Wisconsin) were compared with respect to the detection of antibodies (indirect enzyme-linked immunosorbent assay-ELISA, peroxidase system) in 126 serial serum specimens (pre-treatment, 30 and 60 days post-treatment with itraconazole) from 42 dogs with diagnosed blastomycosis. Mean absorbance values observed with the four lysate antigens at the three treatment intervals ranged from the most reactive to the least reactive as follows: T-58 (0.270, 0.210, 0.136); T-27 (0.209, 0.156, 0.096); ER-3 (0.189, 0.144, 0.089) and ERC-2 (0.158, 0.129, 0.080). Even though variations in reactivity were evidenced, the lysates prepared from isolates from various geographical regions and sources were all efficacious as antigens for the immunodiagnosis of canine blastomycosis.     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Blastomycosis in Africa

The known African cases of blastomycosis to 1987 are presented, including thirteen previously undescribed cases. This brings to 81 the total number of cases known to have occurred in Africa. The question of whether the disease in Africa is the same in all respects as that in North America is addressed; the age and sex distributions of patients are similar. Minor differences in the clinical features relate particularly to the type of skin lesion, the more frequent bone involvement and the less frequent central nervous system involvement in the African patient. Little is known about the epidemiology of blastomycosis in Africa; one noteworthy feature is the apparent absence of the disease in dogs. Isolates of Blastomyces dermatitidis from the two continents, although closely related, differ in some respects.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

An outbreak of blastomycosis in Eastern Tennessee

Most cases of blastomycosis are sporadic and only nine outbreaks representing a total of 112 cases have previously been reported. Less than half of these have been culture proven cases. Outbreaks have previously occurred in North Carolina, Minnesota, Illinois, Wisconsin and Virginia. We report three culturally confirmed cases of blastomycosis from Elizabethton, Tennessee, who had onset of illness within a one-week span of time. The patients presented with fever, chest pain, weight loss, poor appetite and myalgia. Each initially had a dry cough which became productive of purulent sputum as the illness progressed. Mild hemoptysis occurred during each patient's course. Serologic testing by immunodiffusion and enzyme immunoassay were positive and testing by complement fixation was negative in each case. The diagnosis was made by histopathology on transbronchial biopsy or transthoracic needle aspiration material. Each patient improved on ketoconazole therapy.     

Efficacy of brain heart infusion-egg albumen agar,yeast extract phosphate agar and peptone glucose agar media for isolation of Blastomyces dermatitidis from sputum

The efficacy of brain heart infusion (BHI)-egg albumen agar, yeast extract phosphate agar and several modified peptone glucose agar media was evaluated for isolation of Blastomyces dermatitidis from sputum concomitantly seeded with the yeast form of the pathogen and Candida albicans. Based upon high per cent culture positivity of sputum, improved recovery (CFU/ml) of the seeded inoculum, faster growth rate of B. dermatitidis and low level of contamination, BHI-egg albumen agar, followed by yeast extract phosphate agar are recommended as the media of choice for the isolation of B. dermatitidis from contaminated clinical specimens.     

Clinical Characteristics and Outcomes in Patients with Pulmonary Blastomycosis

Background  Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. Objectives  To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. Methods  A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. Results  The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1–3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. Conclusion  Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on the molecular ecology of Blastomyces dermatitidis

The microecology of Blastomyces dermatitidis, the dimorphic etiologic agentof the potentially fatal systemic fungal infection, blastomycosis, is not well defined.Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability toutilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20° and 37° centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cluster of pulmonary blastomycosis in a rural community: Evidence for multiple high-risk environmental foci following a sustained period of diminished precipitation

Much of our understanding of the epidemiologic features of infection with Blastomyces dermatitidis has come from cluster and outbreak investigations which have established the association of human disease with recreational pursuits and the presence of infectious microfoci in areas of moist soil with high organic content. This report describes the clustering of eight cases of pulmonary blastomycosis without an apparent common source exposure which occurred during a 90 day period in a 96 square mile area (population 4,450) within Oconto County, Wisconsin. We conclude that multiple high-risk environmental foci may have existed following a sustained five-year period of diminished precipitation in the cluster area. A case-control study which included family and community controls concluded that multiple earth-disturbing activities engaged in by case-patients was statistically associated with illness. Lymphocyte-proliferation assays of whole blood samples detected previously unrecognized infection withB. dermatitidis among five of 32 family controls. This revised version was published online in June 2006 with corrections to the Cover Date.     

An Imported Case of Blastomyces Dermatitidis Infection in Mexico

Blastomycosis is an acute or chronic primary infection of the respiratory system, endemic in North America (United States of America and Canada), Africa and Asia. We report a case in Mexico, in a three years old child who had been born in California and lived in Chicago, U.S.A. The patient presented pulmonary symptoms prior to development of a skin ulcer. Blastomyces dermatitidis was identified by mycological and molecular procedures. The patient was successfully treated with amphotericin B, oral ketoconazole and itraconazole.     

Detection of IgG and IgM in Sera from Canines with Blastomycosis using Eight Blastomyces dermatitidis Yeast Phase Lysate Antigens

The objective of this study was to compare the efficacy of eight Blastomyces dermatitidis yeast phase lysate antigens (T-58: dog, Tennessee; T-27: polar bear, Tennessee; ERC-2: dog, Wisconsin; B5894: human, Minnesota; SOIL: soil, Canada; B5896: human, Minnesota; 48089: human, Zaire; 48938: bat, India) in the detection of the immunoglobulins IgG and IgM in serum specimens from canines with blastomycosis. An indirect enzyme-linked immunosorbent assay (ELISA, peroxidase system) was used to analyze sera collected during four different intervals post-infection. The yeast lysate antigen 48938 was a reactive antigen for the detection of both IgG (mean absorbance value range: 1.198–2.934) and IgM (mean absorbance value range: 0.505–0.845). For the same sera, antigen T-27 was also effective in the detection of IgG (mean absorbance value range: 0.904–3.356) and antigen 48089 was useful for the detection of IgM (mean absorbance value range: 0.377–0.554). The yeast lysate antigen B5894 proved to be a poor antigen for the detection of both IgG and IgM (mean absorbance value ranges: 0.310–0.744 for IgG, 0.025–0.069 for IgM). Inherent variations in yeast lysate antigens such as these may be utilized to develop improved immunoassay procedures for the specific detection of IgG or IgM in cases of blastomycosis.     

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. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytologic findings in 43 cases of blastomycosis diagnosed ante-mortem in naturally-infected dogs

A retrospective study of the cytology of blastomycosis was undertaken. Sixty-one samples from 43 naturally infected dogs diagnosedante-mortem by means of cytology were reviewed. Skin and lymph nodes rendered the highest number of positive samples with 17 out of 18 and 14 out of 17 respectively. Transtracheal washes contained the tissue forms ofBlastomyces dermatitidis in 3 out of 5 samples. Pyogranulomatous inflammation was diagnosed in 38 of the 61 samples, and of these, 25 cases contained multinucleated giant cells. Epithelioid cells were found in all 38 cases. Purulent inflammation was seen in 15 out of 61 samples. Three cases had a minimal or virtual lack of inflammation. The cytologic findings are described and photographically illustrated.     

Production of IL-6, in contrast to other cytokines and chemokines, in macrophage innate immune responses: effect of serum and fungal (Blastomyces) challenge

Murine peritoneal macrophages, after adherence and establishment in culture in vitro in the presence of medium containing fetal bovine serum (FBS) for 20 h, then cultured for 20 h, produced several cytokines. If, in the second 20 h period, a fungus (heat-killed Blastomyces, HK-Bd) was introduced, a more complex pattern of cytokine (particularly TNF) and chemokine production ensued. The cytokine production, assayed by antibody array and also quantitation in supernatants, was depressed (particularly TNF) by the addition of mouse serum to these cultures, with the exception of IL-6. Macrophages could be cultured in the presence or absence of serum during the initial 20 h adherence and establishment period, enabling study of the effect of serum factors. In the absence of serum, with or without fungal stimulation, cytokine and chemokine production was more restricted, largely to TNF and IL-6. The addition of mouse serum [corrected] resulted in marked depression of TNF and enhancement of IL-6. The combination of HK-Bd and mouse serum resulted in more IL-6 production than either component alone. The enhancement of IL-6 by mouse serum was concentration-dependent and maximal at 8 h. The effects of fungus or serum on macrophage production of cytokines were similar in an outbred and an inbred mouse strain. The larger repertoire of cytokine production in the macrophages that had been cultured longer (20 h+20 h) in serum may be related to maturation of cell receptors. IL-6 production in vivo in response to fungal-serum complexes could affect pathogenesis by opposing the host defense modulation by proinflammatory cytokines or by modulating the destructive effects of inflammation on host tissues.     

Attachment of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages: Characterization of binding the elicitation of respiratory burst

We studied the mechanisms of adherence of Blastomyces dermatitidis conidia to murine bronchoalveolar macrophages and the ability of the conidia to elicit an increase in macrophage O _inf2 ^sup- production, using an avirulent fungal strain. The number of cell associated conidia was counted by visual inspection of 2 hour macrophage monolayers incubated with conidia and O _inf2 ^sup- was measured by reduction of ferricytochrome c. Adherence of conidia to bronchoalveolar macrophages was time dependent and reached a plateau after 30 min (36±5%, 51±22%, and 36±17% macrophages with adherent conidia after 15, 30, and 60 min, respectively). Both Ca⁺² and Mg⁺² were required. The carbohydrates mannose, mannan, fucose, alpha-methylmannoside, beta-glucan, galactose, N-acetylglucosamine and chitotriose (100–1000 g/ml) did not inhibit adherence of conidia to macrophages. Trypsin treatment of macrophages or conidia did not affect binding. Conidia did not stimulate bronchoalveolar macrophage production of O _inf2 ^sup- above baseline concentrations (2.0±0.9 vs 0.8±0.5 nmol O _inf2 ^sup- , p>0.05). We conclude that murine bronchoalveolar macrophage-B. dermatitidis conidia interactions occur primarily by a non-lectin-like attachment and do not result in the production of macrophage derived O _inf2 ^sup- .     

Controls on chlorophyll-a in nutrient-rich agricultural streams in Illinois, USA

Nitrogen and phosphorus are the primary nutrients that affect water quality in streams in the midwestern USA and high concentrations of these nutrients tend to increase algal biomass. However, how nutrients interact with physical controls in regulating algal biomass is not well known in agricultural streams. Eighteen streams in east-central Illinois (USA) were sampled during June and September 2003 to analyze factors possibly regulating algal biomass. Additionally, two shaded and two non-shaded sites in the Embarras River in east-central Illinois were sampled intensively from June to December 2003. Both sestonic and periphytic chlorophyll-a (chl-a) were analyzed, and periphytic chl-a was assessed on natural substrata and unglazed ceramic tiles. Although high concentrations of nutrients were found in these streams (mean total P = 0.09–0.122 mg l⁻¹ and mean NO₃-N=4.4–8.4 mg l⁻¹), concentrations of sestonic chl-a were low among all sites and both sampling periods (<18 mg m⁻³, median values of 5 and 3 in June and September, respectively). Filamentous algae were an important component of the algal communities in streams with stable substrata. Periphytic chl-a was generally not related to the concentration of N or P in the water column, and in non-shaded streams periphyton appeared at times to be light-limited due to turbid water. Turbidity was found to be an important factor controlling chl-a on ceramic tiles across the 18 sites and for the Embarras River sites; chl-a decreased exponentially in concentration (132–0 mg m⁻²) as turbidity increased from 4 to 39 NTU (r ² = 0.80). In general, the interaction between hydrology and light (turbidity) likely controlled algal biomass in these nutrient-rich, agricultural streams.     

Identification of lower fungi isolated from larvae of mangrove crab,Scylla serrata, in Indonesia

Since 1992, seed production of mangrove crab,Scylla serrata, has been attempted at the Gondol Research Station for Coastal Fisheries, Bali, Indonesia. During the production process, almost all of the larvae have died due to fungal infection. Fungi isolated from the larvae with fungal infection were classified into three species in the order Lagenidiales:Lagenidium callinectes, Haliphthoros milfordensis andHalocrusticida baliensis sp. nov. based on detailed morphological characteristics. The effects of temperature, pH and mineral content of the water on their growth were also examined.