Comparison of Antibody Detection with Yeast Lysate Antigens Prepared from Blastomyces dermatitidis Dog Isolates from Wisconsin and Tennessee

Blastomyces dermatitidis, the causative agent of blastomycosis, a potentially lethal dimorphic fungal disease of humans and animals has been difficult to diagnose in the clinical laboratory. We are attempting to develop and improve immunodiagnostic assays by producing novel yeast lysate reagents for the detection of antibodies in blastomycosis. The objective of this study was to use lysate antigens prepared from four B. dermatitidis antigens isolated from dogs infected with blastomycosis from two different endemic areas (Wisconsin and Tennessee) testing for the detection of antibodies in serum specimens from immunized rabbits and infected dogs using the indirect ELISA. In the dog sera, absorbance values ranged from 0.774 to 1.350, while the rabbit sera values ranged from 0.533 to 1.191. Antigen T-58 appeared to lack any geographical specificity in antibody detection, which could prove useful in future immunodiagnostic detection of blastomycosis infections.

<i>Blastomyces dermatitidis</i>: Chitinase Homology Model, <i>in Silico</i>Docking, and Inhibition Assay

Blastomyces dermatitidis is a thermally dimorphic fungus that causes the disease blastomycosis. Currently there are a limited number of effective treatments, many of which have harsh side effects. Chitin, a component of the fungal cell wall is often broken down and recycled for cell wall remodeling and growth. Chitinase is the digestive enzyme capable of chitin hydrolysis. By inhibiting the chitinase we predicted that cells wouldn’t be able to divide and multiply normally, thereby leading to possible anti-fungal treatments. For this study we modeled the structure of B. dermatitidis chitinase, using homology modeling. By predicting a three-dimensional structure we were able to do additional analyses of the active site of the chitinase and predict the binding of a possible small molecule, acetazolamide, in silico. This binding allowed us to predict that this molecule might be capable of inhibiting the chitinase of B. dermatitidis. This inhibition was tested in vivo. No difference in the growth curves of the test and control organisms was observed, however there was a difference within the cell walls of the yeast cells. The cell walls appeared thicker with additional differences in cell wall orderly growth. These changes are consistent with changes that may occur as B. dermatitidis chitinases are inhibited.

Detection of Antibody in Dogs with Blastomycosis Using <i>Blastomyces dermatitidis</i>Yeast Phase Lysate Antigens

The objective of our study was to compare two B. dermatitidis yeast phase lysate antigens [ERC-2, dog Wisconsin;85, soil Georgia, ATCC 56,920] for detecting antibody in 38 serum specimens [pre-treatment, 30-day, and 60-day post treatment] from dogs with diagnosed blastomycosis. The mean absorbance values obtained with the two antigens (N = 38) were ERC-2 = 2.359 and 85 = 2.189. The mean absorbance values when the sera were divided into the three treatment groups were as follows pre-treatment: Isolate ERC-2 had an absorbance value of 2.418;Isolate 85 had an absorbance value of 2.688, 30-day post treatment: ERC-2 had an absorbance value of 2.452;85 had an absorbance value of 2.303 and 60-day post treatment: ERC-2 had an absorbance value of 2.150;85 had an absorbance value of 2.073 with the mean absorbance values of all treatment groups were ERC-2: 2.229 and 85: 2.141. This study indicates the potential for further evaluations of the two lysate antigens with regard to antibody detection in dog sera with the ERC-2 reagent slightly more reactive than the 85 lysate antigen.

Blastomyces dermatitidis: Stability studies on different yeast lysate antigens

In Trial 1, 19 lots of Blastomyces dermatitidis (T-58;Tennessee dog isolate) were assayed to determine the stability of the reagents following storage. The reactivity of the antigens, produced from 1989 to 2012 and stored at 4°C, was determined by comparing antibody detection (enzyme-linked immunosorbent assay;ELISA) in 12 serum specimens from immunized rabbits. All of the 19 reagents produced during this 23-year period exhibited a high degree of stability and were able to detect antibody in the sera. Mean absorbance values ranged from 0.798 (1989) to 0.827 (2012) and a mean value for all 19 antigens of 0.728. In a related evaluation, Trial 2, B. dermatitidis lysate antigens prepared from 8 isolates (dog, human, soil) at two different time periods were assayed as above to determine reactivity. The time of storage between the first and second reagents varied from 4 to 17 years. The results indicated that all 16 of the lysate antigens detected antibody in the 15 rabbit serum specimens with mean absorbance values ranging from 0.346 to 0.682, but variations in reactivity were observed depending on the lysate and the serum specimen assayed. This comparative study provided evidence that the antigenic reagents do exhibit some lot-to-lot variation in reactivity, but they did not lose any appreciable potency during prolonged storage.

Comparison of Two Enzyme Immunoassays and Four Lysate Antigens for the Detection of Antibody in Canine Blastomycosis

Blastomycosis, the systemic fungal disease of humans and animals caused by Blastomyces dermatitidis and the cryptic species Blastomyces gilchristii, is often misdiagnosed as a bacterial or viral pulmonary disease. Therefore, the development of improved immunodiagnostic assays for this disease has been the primary focus of research in our laboratory. The present study was designed to evaluate four Blastomyces yeast-phase lysate antigenic preparations (human, 597, Eagle River, WI;dog, ERC-2, WI;Human, B5927, Mountain Iron, MN;soil, 85, Georgia, ATCC 56920) for their ability to detect antibody in 48 serum specimens from dogs with diagnosed blastomycosis using an indirect ELISA (STD) compared to a biotin-streptavidin ELISA (B-SA). All four lysate antigens were able to detect antibodies in the specimens with mean absorbance values ranging from 0.930 (B5927) to 1.142 (ERC-2) with the STD ELSA and from 1.395 (B5927) to 1.775 (85) with the B-SA ELISA. The results indicated that both ELISA methods could be utilized for antibody detection, but the B-SA ELISA exhibited greater sensitivity than the STD ELISA with all four of the lysates.

Disseminated Blastomycosis during Pregnancy at 11 Weeks Gestation

Blastomyces dermatitidis is a dimorphic fungus that typically causes disease in immunocompromised hosts, but can affect immunocompetent patients as well. Here we present a case of disseminated blastomycosis in a pregnant patient at 11 weeks gestation who was successfully treated with systemic antifungal therapy. Data regarding treatment of blastomycosis in pregnancy is limited, and choice of antifungal agent remains challenging due to uncertainties regarding fetal toxicity.

Blastomycosis in China： a case report and literature review

Blastomycosis is a fungal disease that is endemic in parts of North America. It is very rare in China and also commonly misdiagnosed, often as cancer or other infectious diseases. The clinical profile of a case of disseminated blastomycosis with pulmonary changes and skin ulcers was described. He had been misdiagnosed with tuberculosis, after adequate therapy with a lipid formulation of amphotericin B, followed by itraconazole, the lung and skin lesions improved. Then the five cases reported in China and literatures were reviewed. The aim of this report was to improve the knowledge regarding blastomycosis for physicians in China to avoid delaying adequate therapy.