Improved Detection of
            <i>Histoplasma</i>
            Antigenemia following Dissociation of Immune Complexes

Swartzentruber, S.; LeMonte, Ann M.; Witt, John R.; Fuller, D.; Davis, Thomas E.; Hage, Chadi A.; Connolly, Patricia; Durkin, Michelle; Wheat, L. Joseph

doi:10.1128/cvi.00409-08

Cited by 57 publications

(45 citation statements)

References 7 publications

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“…More cases were tested in the current report than the previous report: 28 and 11, respectively. However, antigenemia was detected less frequently in patients with blastomycosis than in those with histoplasmosis: 90% in patients with disseminated histoplasmosis (11) and 80% in those with acute pulmonary histoplasmosis (12).…”

Section: Discussionmentioning

confidence: 93%

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Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

Connolly¹,

Hage

Bariola

et al. 2012

Clin Vaccine Immunol

104

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The second-generation MVista Blastomyces antigen enzyme immunoassay was not quantitative; therefore, specimens obtained previously were tested in the same assay as new specimens to assess the change in antigen levels. Furthermore, the sensitivity in serum had not been fully evaluated. The purpose of this study was to evaluate a quantitative Blastomyces antigen assay and detection of antigen in serum. Calibrators containing known concentrations of Blastomyces galactomannan were used to quantify antigen in urine and serum from patients with proven blastomycosis and from controls. Paired current and previously obtained urine specimens were tested to determine if quantification eliminated the need for concurrent testing to assess change in antigen. Pretreatment of serum with EDTA at 104°C was evaluated to determine if dissociation of immune complexes improved detection of antigenemia. Antigenuria was detected in 89.9% of patients with culture-or histopathology-proven blastomycosis. Specificity was 99.0% in patients with nonfungal infections and healthy subjects, but cross-reactions occurred in 95.6% of patients with histoplasmosis. Change in antigen level categorized as increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in ng/ml determined from different assays. Pretreatment increased the sensitivity of detection of antigenemia from 35.7% to 57.1%. Quantification eliminated the need for concurrent testing of current and previously obtained specimens for assessment of changes in antigen concentration. Pretreatment increased the sensitivity for detection of antigenemia. Differentiation of histoplasmosis and blastomycosis is not possible by antigen detection.A ntigen detection is a useful method for diagnosis of blastomycosis. The sensitivity has been reported to be over 90% and specificity has been reported to be 100% in healthy subjects, but cross-reactions occur in most patients with histoplasmosis (6). In a recent report of 59 patients with proven blastomycosis (4), culture was positive in 86%, histopathology in 81%, antigen in 74%, cytopathology in 38%, and immunodiffusion for antibody in 32% (4). Antigen detection was considered to be a "reliable method to make an accurate and rapid diagnosis of blastomycosis, particularly when a large burden of disease is present, and when cytological analysis is performed at less-experienced centers" (4). Blastomyces antigen detection was also reported to be useful for diagnosis of blastomycosis in solid organ transplant patients (7).Because of interassay variability, specimens obtained earlier have been tested simultaneously with current specimens to assess the change in antigen levels. In a Histoplasma antigen assay, quantification of galactomannan antigen eliminated the need to test the previously obtained specimen with the current specimen to determine change in antigen level (5). In a recent study, quantification and improved detection of antigen in serum following EDTA treatment at 104°C in the t...

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Section: Discussionmentioning

confidence: 93%

“…The pretreatment procedure has been described for detection of Histoplasma antigen (11). A total of 200 l of 4% EDTA was added to 600 l of serum, vortex mixed, and placed in a heat block (Fisher Scientific) at 104°C for 6 min.…”

Section: Patientsmentioning

confidence: 99%

Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

Connolly¹,

Hage

Bariola

et al. 2012

Clin Vaccine Immunol

104

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“…A total of 200 l of 4% EDTA at pH 4.6 was added to 600 l of sera, and the mixture was vortexed and then placed in a heat block (Fisher Scientific) at 100°C for 6 minutes, followed by centrifugation and collection of the supernatant (7).…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

Detection of Coccidioides Antigenemia following Dissociation of Immune Complexes

Durkin¹,

Estok²,

Hospenthal

et al. 2009

Clin Vaccine Immunol

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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...

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“…One of the principal aspects that can increase the sensitivity and specificity values of PCM diagnostic tools is the treatment of the samples, such as for the elimination of immune complexes, before they are tested (22). Pretreatment has most improved the detection of antigens (antigenemia); however, some studies have also observed its importance for detecting antibodies (23).…”

mentioning

confidence: 99%

Effects of Pretreating Serum Samples on the Performance of a Latex Agglutination Test for Serodiagnosis of Paracoccidioidomycosis

Silveira-Gomes

Silva

2011

Clin. Vaccine Immunol.

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b Paracoccidioidomycosis (PCM) is a fungal disease caused by Paracoccidioides brasiliensis, and Brazil is one of the principal countries where it is endemic. Diagnosis is based on the observation of budding P. brasiliensis yeast in clinical specimens from patients; however, the sensitivity of the visualization of fungi is low, indicating that serological tests are used for early diagnosis. The double-immunodiffusion test (ID) is the "gold standard" test for serology in PCM, although the execution of this test requires the availability of laboratorial infrastructure. We report the improved performance of a latex agglutination test (LAT) by pretreating 30 serum samples from PCM patients and 71 controls (histoplasmosis and aspergillosis patients, patients with bacterial infections, and normal human sera) with a dilution buffer incubated at 37°C for 30 min. The sensitivity and specificity of the LAT test in the nonpretreated samples were 73% and 79%, respectively. However, when samples were pretreated, the sensitivity and specificity of the test increased to 90%. In this study, we did not observe cross-reactivity with histoplasmosis patient sera, but some reactions to sera from patients with aspergillosis and bacterial infections were noted. Normal human sera were not reactive in our tests. These results indicate the need for the elimination of heterologous reactions so that we can adequately use this method for screening cases of PCM.

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Improved Detection of Histoplasma Antigenemia following Dissociation of Immune Complexes

Cited by 57 publications

References 7 publications

Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

Blastomyces dermatitidis Antigen Detection by Quantitative Enzyme Immunoassay

Detection of Coccidioides Antigenemia following Dissociation of Immune Complexes

Effects of Pretreating Serum Samples on the Performance of a Latex Agglutination Test for Serodiagnosis of Paracoccidioidomycosis

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