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).

Potential clinical utility of ERC-2 yeast phase lysate antigen for antibody detection in dogs with blastomycosis.

Four Blastomyces antigens ERC-2 (B. gilchristii, dog, Wisconsin), B5929 (human, Minnesota), 597 (human, Wisconsin), and T-27 (polar bear, Tennessee) were tested against 31 serum specimens from dogs with blastomycosis and 19 from healthy dogs. All antigens detected antibody; efficacy varied. ERC-2 showed the highest ELISA mean absorbance value of 3.00 followed by T-27. Test performance varied by sample geographic origin. Further study is needed to determine if ERC-2 antigens may be clinically useful, and whether the combination of the particular fungal species as antigen source, host animal, and the species and geographic location of the patient being tested is important for optimum test characteristics.

Blastomyces dermatitidis Yeast Lysate Antigen Combinations: Antibody Detection in Dogs with Blastomycosis.

The systemic fungal infection, blastomycosis, which infects both humans and animals has presented a diagnostic challenge for clinicians for many years. The aim of this study was to evaluate the diagnostic sensitivity of Blastomyces dermatitidis yeast lysate antigens with respect to antibody detection in dogs with blastomycosis. Lysate antigens were prepared from B. dermatitidis isolates T-58 and T-66 (dogs, Tennessee) and WI-R and WI-J (dogs, Wisconsin). Based on results obtained from a preliminary comparative study, five combinations of these isolates and one individual isolate were tested against 92 serum specimens from dogs with culture-proven or histologically-confirmed blastomycosis, using the indirect enzyme-linked immunosorbent assay (ELISA). Mean absorbance values obtained from the sera ranged from 0.905 with the individual T-58 antigen to 1.760 using an antigen combination (T-58 + T-66 + WI-R). All of the 6 antigenic preparations were able to detect antibody in the serum specimens, but the antigen combinations detected antibody to a higher degree than the individual antigen. This study provides evidence that combinations of the yeast lysate reagents seem to be more efficacious for antibody detection in dog sera, but our laboratory is continuing to evaluate antigen lysate combinations for detection of antibodies in blastomycosis.

Development of a slide agglutination assay for detection of blastomycosis.

Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection.

Isoelectric focusing and ELISA evaluation of a Blastomyces dermatitidis human isolate.

Blastomyces dermatitidis is a dimorphic fungal organism and the causative agent of blastomycosis. This organism is endemic east of the Mississippi river as is the fungal organism Histoplasma capsulatum. This study was performed to determine if sensitive and specific antigens from the B. dermatitidis yeast phase lysate (human isolate 592) could be separated using isoelectric focusing (IEF) to eliminate antigens that are cross-reactive with H. capsulatum. Indirect enzyme linked immunosorbent assays were performed to test for reactivity and cross-reactivity and indicate that certain fractions (4-6) were highly reactive. Fraction 16 exhibited a high degree of cross-reactivity with H. capsulatum. This study indicates that IEF may be a useful method for the separation of B. dermatitidis proteins.

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.

Molecular approaches to the study of Coccidioides immitis.

The study of the molecular biology of Coccidioides sp. is only just beginning. As the importance of coccidioidomycosis grows as a public health problem, our need for understanding of pathogenesis, immune responses, and improved antifungal therapy also increases in proportion. Tools have now become available to study gene manipulation in this pathogen and this will allow molecular approaches to be used. Genetic experiments will also be accelerated by the availability of the whole coccidioidal genome, expected to be made public in the spring of 2003 (see http://www.tigr.org/tdb/tgi/cigi/GenInfo.html). Thus, there seems to be several reasons to expect considerable progress in the coming years.