Detection of Coccidioides Antigenemia following Dissociation of Immune Complexes

Abstract

Having reported that pretreatment of serum samples with EDTA at 100°C improved the sensitivity for the detection of Histoplasma antigenemia, we have evaluated this method for the detection of Coccidioides antigenemia. Urine and serum samples from patients with coccidioidomycosis were tested using the MVista Coccidioides enzyme immunoassay, and serum samples with and without EDTA-heat treatment were tested. Antigenemia was detected in 28.6% of patients whose samples were not EDTA-heat treated and in 73.1% of those whose samples were treated. Antigenuria was detected in 50% of patients. Specificity of 100% was obtained in healthy subjects, but cross-reactions were seen in 22.2% of patients with histoplasmosis or blastomycosis. EDTA-heat treatment improves the sensitivity for the detection of Coccidioides antigenemia.Antigen detection could be useful for diagnosis of coccidioidomycosis. We reported detection of antigenuria in 70.8% of cases using the MVista Coccidioides antigen enzyme immunoassay (EIA), which detects Coccidioides galactomannan (2). When combined with serology, antigen or seropositivity was detected in 88% of cases of moderate to severe coccidioidomycosis, equaling the rate of isolation of the organism in cultures (2).Others have reported the detection of antigenemia in patients with coccidioidomycosis (4,5,8). In our earlier studies, antigenemia was not detected in the few cases that were tested (L. J. Wheat, unpublished observations). More recently we noted that pretreatment of serum at 100°C in the presence of EDTA, by dissociating immune complexes and destroying the freed antibody, markedly improved the detection of antigenemia in histoplasmosis (7). Among AIDS patients with histoplasmosis and undetectable antigenemia, antigen was detected in 95% of samples following EDTA-heat treatment. Herein we describe the effect of EDTA-heat treatment on the detection of antigenemia in the Coccidioides antigen EIA. MATERIALS AND METHODSEIA. The MVista Coccidioides antigen EIA was performed as previously reported (2), using microplates coated with anti-Coccidioides antibodies. Following incubation of the test specimen in the precoated microplate, antigen that had attached to the capture antibody was detected with biotinylated rabbit antiCoccidioides detector antibody. Results greater than or equal to the 0.07 ng/ml urinary galactomannan calibrator were considered positive. A single urine or serum specimen was tested for each patient.Clinical specimens. Urine and serum specimens were available from 16 patients diagnosed with coccidioidomycosis who were entered into approved clinical studies at three institutions (El Rio Special Immunology, Tucson, AZ; Brook Army Medical Center, San Antonio, TX; Naval Medical Center, San Diego, CA) and 12 additional patients diagnosed with coccidioidomycosis whose sera were tested at MiraVista Diagnostics, for whom clinical information was incomplete; a study with 3 of these 12 patients was previously reported (2). The criteria for diagnosis included a compatible clinical illn...

Antigen Detection in Canine Blastomycosis: Comparison of Different Antibody-Antigen Combinations in Two Competitive ELISAs

“…In another study on Coccidioides antigen detection, antigenuria was detected in 50% of patients. Specificity of 100% was obtained in healthy subjects, but cross-reactions were seen in 22.2% of patients with histoplasmosis or blastomycosis [14].…”

“…In recent years other investigators have been approaching the problem of immunodiagnosis and have produced encouraging results with antigen detection assays as second-generation assays have been developed [7,[14][15][16][17][18]. Our laboratory has been focusing on the use of the competitive enzyme-linked immunosorbent assay (ELISA) in B. dermatitidis antigen detection studies in urine specimens from dogs with diagnosed blastomycosis [11].…”

Evaluation of Coccidioides Antigen Detection in Dogs with Coccidioidomycosis

“…A possible explanation for the difference in sensitivity between human patients and dogs is that the organism burden may have been higher in the human patients (2,3), as most of the human cases had severe coccidioidomycosis and were immunocompromised. Information was not collected about the severity of coccidioidomycosis, the presence of immunocompromising conditions, species or strain characteristics of the infecting strain of Coccidioides spp., or prior antifungal therapy, precluding assessment of the role of these factors in antigen production in dogs.…”

“…Second, specificity is uncertain as only 75 controls were from areas where coccidioidomycosis is endemic. Specificity was 99% in urine and serum from healthy humans from areas of endemicity, however (2,3). Third, clinical information was not available for the control dogs with histoplasmosis.…”

“…The Coccidioides quantitative antigen assay was performed as previously reported (2), using microplates coated (VWR, Batavia, IL) with anti-Coccidioides antibodies selected for maximum sensitivity and specificity for detection of the galactomannan antigen. Serum specimens were first treated by adding 200 l of 4% EDTA (Midwest Scientific, St. Louis, MO) at pH 4.6 to 600 l of serum, vortexing the mixture, and placing it in a heat block (Fisher Scientific, Pittsburgh, PA) at 104°C for 6 min, after which the samples were centrifuged and the supernatant was collected (3). Following incubation of the test specimen in the precoated microplate, antigen that had attached to the capture antibody was quantified with biotinylated rabbit anti-Coccidioides detector antibody.…”