G. M. Scalarone

Preparation and ELISA evaluation of Blastomyces dermatitidis yeast phase lysate antigens

Enzyme-linked immunosorbent assay (ELISA) comparative studies are described in which 10 Blastomyces dermatitidis antigenic preparations from human, dog, and soil strains were used for the detection of rabbit serum antibodies directed against B. dermatitidis killed yeast cells of human and dog origin as well as detection of antibodies in human serum samples. Reactive reagents were produced from all 10 strains, but two antigens (K-Le and T-043324) demonstrated exceptional sensitivity with moderate cross-reactivity with histoplasmosis specimens. Additionally, these reagents were able to detect anti-B. dermatitidis antibodies produced in rabbits in response to human (H-100) or dog (T-042976) strains in a comparable manner. These data suggest that a single antigenic reagent might be suitable for ELISA detection of B. dermatitidis antibodies in different species with blastomycosis.

Blastomyces Dermatitidis 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.

Turbidometric characterization of the postantifungal effect: comparative studies with amphotericin B, 5-fluorocytosine and miconazole on Candida albicans.

Summary. The phenomenon of persistent suppression of Candida albicans yeast cell growth after short drug exposures (postantifungal effect; PAFE) was determined by performing comparative studies using different concentrations of 5‐fluorocytosine (5‐FC), amphotericin B (AMPH) and miconazole (MCZ). An in vitro turbidometric method was used to measure cell growth and to quantitate the PAFE after removal of the drug by dilution following exposure to C. albicans yeast cells for 0.5 h, 1 h or 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the antifungal agent. A PAFE was demonstrated with each agent and generally the length of the PAFE was dependent upon the concentration of the drug and the time of exposure. An exposure time of 0.5 h resulted in PAFEs ranging from 0.6 to 16.7 h with 5‐FC, 0 to 16.5 h with AMPH and 0.1 to 14.1 h with MCZ. In most instances exposure of the cells to each drug for 1 or 2 h resulted in slightly longer PAFEs, respectively. Longer PAFEs were induced with lower concentrations of 5‐FC as compared to AMPH and MCZ.

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.

Sensitivity and specificity of an isoelectric focusing fraction of Blastomyces dermatitidis yeast lysate antigen for the detection of canine blastomycosis

Summary. Blastomyces dermatitidis yeast lysate antigen (T‐58, dog isolate) fractions prepared using the Rotofor® preparative isoelectric focusing (IEF) cell (Bio‐Rad) were compared with B. dermatitidis yeast lysate and filtrate reagents with respect to the detection of antibodies in sera from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis. A horseradish peroxidase enzyme immunoassay with Turbo TMB substrate was used in the study. One particular IEF fraction (pH 4.3) was optimal in the assay, and it exhibited greater sensitivity (100%) and specificity (93%) than the lysate or filtrate preparations. The highest degree of cross‐reactivity was encountered with the histoplasmosis and coccidioidomycosis specimens and considerably less with the cryptococcosis and aspergillosis sera. Studies are in progress to purify further the optimal IEF fraction.

Detection of antibodies and delayed hypersensitivity with Rotofor® preparative IEF fractions of Blastomyces dermatitidis yeast phase lysate antigen

Blastomyces dermatitidis (dog isolate T-58) yeast phase lysate antigen was concentrated and separated by Rotofor preparative isoelectric focusing cell (Bio-Rad). The pH values of the fractions were determined and equilibrated to pH 7.2 and then analysed by enzyme-linked immunosorbent assay using horseradish peroxidase enzyme system against serum specimens from dogs with blastomycosis, histoplasmosis, aspergillosis, and coccidioidomycosis. The results showed a peak absorbance at pH 3.89-4.31 (fractions 4 and 5) with the blastomycosis serum specimens. This was a single sharp peak while the rest of the fractions were lower. In contrast the sera from dogs with histoplasmosis showed a peak absorbance at pH 5.54-5.97 (fractions 9 and 10), while the other mycoses showed patterns that did not resemble the blastomycosis or histoplasmosis specimens. Serum specimens from dogs with blastomycosis being treated with itraconazole were also assayed (pre-treatment and 1, 2, 3, and 12 months post-treatment sera). The characteristic peak for blastomycosis was observed and a decrease in the peak was seen as the treatment progressed. Fractions 3-12 were also used to detect delayed dermal hypersensitivity in hyperimmunized hairless guinea-pigs. Fraction 5 (pH 4.31) elicited the optimal response in B. dermatitidis-immunized animals, while no cross-reactivity was observed in guinea-pigs sensitized with Histoplasma capsulatum killed cells.

Synergistic postantifungal effect of flucytosine and fluconazole on Candida albicans

The in vitro efficacy of flucytosine and fluconazole, separately and in combination, with respect to induction of a postantifungal effect (PAFE) on Candida albicans was studied. PAFE refers to the persistent suppression of fungal cell growth following a short period of exposure to an antifungal agent. A turbidometric method was used to measure cell growth and to quantitate the PAFE following exposure of C. albicans yeast cells to different concentrations of the two agents for 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the drug by dilution. Minimum (MIC) and fractional inhibitory concentration determinations were made and the data used for selecting the concentrations used in the PAFE evaluations. A synergistic interaction of the two drugs at concentrations well below their individual MICs was evidenced. Flucytosine:fluconazole ratios of 1:16-1:32 at concentrations ranging from 0.024-0.098 micrograms ml-1 and from 0.78-1.56 micrograms ml-1, with flucytosine and fluconazole, respectively, induced PAFEs which persisted for 2.5 h longer than those achieved when each of the two agents was assayed separately.

Enzyme Immunoassay Detection of Antibodies in North American Blastomycosis: Comparison of Three Blastomyces dermatitidis Antigens/Enzymimmunoassay zum Antikörpernachweis bei Nordamerikanischer Blastomykose: Vergleich dreier Blastomyces dermatitidis-Antigene

Summary: Three Blastomyces dermatitidis commercial immunodiffusion antigens, Meridian Diagnostics, Nolan/Scott Laboratories and American Microsan, were utilized in an enzyme immunoassay (ELISA) for the detection of antibodies in blastomycosis. Thirty two serum specimens from patients with blastomycosis were assayed by the indirect ELISA using the alkaline phosphatase enzyme system. Greater reactivity, based on the ELISA index value, was evidenced with the Meridian Diagnostics antigen; the other two antigens exhibited lower but comparable reactivity. Specificity was determined by performing cross‐reactivity assays on sera from patients with histoplasmosis or on specimens from individuals that More Histoplasma capsulatum skin test positive. Comparable specificity results were obtained with all three antigens.

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.

Serological differences in three Blastomyces dermatitidis strains

Summary.  Yeast phase lysate antigens, prepared from three isolates of Blastomyces dermatitidis (T‐58, Tennessee dog; 48089, Zaire human; ERC‐2, Wisconsin dog) were assayed for their ability to detect antibodies in human sera, dog sera and sera from rabbits immunized with each of the lysate antigens. The dog sera were from animals diagnosed with blastomycosis from various endemic regions in North America. T‐58 and ERC‐2 lysate antigens exhibited a high reactivity with the serum from dogs infected with blastomycosis; however, 48089 lysate showed low reactivity with the same sera. With the immunized rabbit sera, 48089 lysate was the only lysate with a high reactivity with the 48089 serum and it exhibited little reactivity with the heterologous sera. The T‐58 and ERC‐2 lysate antigens reacted minimally with the 48089 serum but reacted highly with both the T‐58 and ERC‐2 sera. The human sera were from individuals potentially exposed to B. dermatitidis while working on a prairie dog relocation project in Colorado. Remarkably, all three lysate antigens could detect antibodies in the individuals diagnosed with blastomycosis. This study indicated that there were serological differences in the 48089 Zaire lysate compared with the other lysate antigens and it may be designated serotype 2.