Abstract
Early and accurate diagnosis of coccidioidomycosis, also known as Valley fever, is critical for appropriate disease treatment and management. Current serodiagnosis is based on the detection of patient serum antibodies that react with tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides. IgM is the first class of antibodies produced by hosts in response to coccidioidal insults. The highly glycosylated β-glucosidase 2 (BGL2) is a major active component of the TP antigen that stimulates IgM antibody responses during early Coccidioides infection. The predominant IgM epitope on BGL2 is a unique 3-O-methyl-mannose moiety that is not produced by commonly used protein expression systems. We genetically engineered and expressed a recombinant BGL2 (rBGL2ur), derived from Coccidioides, in non-pathogenic Uncinocarpus reesii, a fungus phylogenetically related to the Coccidioides pathogen. The rBGL2ur protein was purified from the culture medium of transformed U. reesii by nickel affinity chromatography, and the presence of 3-O-methyl mannose was demonstrated by gas chromatography. Seroreactivity of the purified rBGL2ur protein was tested by enzyme-linked immunosorbent assays using sera from 90 patients with coccidioidomycosis and 134 control individuals. The sensitivity and specificity of the assay with rBGL2ur were 78.8% and 87.3%, respectively. These results were comparable to those obtained using a proprietary MiraVista Diagnostic (MVD) IgM (63.3% sensitivity; 96.3% specificity), but significantly better than the ID-TP assay using non-concentrated patient sera (33.3% sensitivity; 100% specificity). Expression of rBGL2ur in U. reesii retains its antigenicity for coccidioidomycosis serodiagnosis and greatly reduces biosafety concerns for antigen production, as Coccidioides spp. are biological safety level 3 agents.
Highlights
Coccidioidomycosis is a fungal infection caused by Coccidioides immitis and Coccidioides posadasii in endemic areas including the southwest region of United States, Mexico, and South America
Some reports have shown that coccidioidomycosis is responsible for 15%-29% of patients presenting with community acquired pneumonia (CAP) in endemic regions, whereas less than 5% of CAP patients are tested for coccidioidomycosis [8, 9]
The pCE-tube precipitin (TP) plasmid contained a hygromycin-B-phosphotransferase encoding gene (HPH) flanked by a promoter element of the gpdA gene and a terminator region of the trpC gene of Aspergillus nidulans. This HPH gene cassette is commonly used for creating Coccidioides transformants that are resistant to hygromycin B at up to 100 μg/ml
Summary
Coccidioidomycosis is a fungal infection caused by Coccidioides immitis and Coccidioides posadasii in endemic areas including the southwest region of United States, Mexico, and South America. The minimum number of spores needed to cause symptomatic disease in human is not known, intranasal inoculation with approximately 10 viable spores in BALB/c mice is sufficient to cause disseminated disease and death in two to three weeks post-challenge [2] Because of their low infectious dose and capacity as airborne pathogens, Coccidioides species are listed as risk group 3 agents, and require biosafety level 3 (BSL3) containment. Diagnosis of coccidioidomycosis relies on clinical presentation of the disease, radiographic findings, delayed type hypersensitivity (skin) test results, culture and histopathological examination of tissue biopsy, and serodiagnosis [3,4,5,6,7]. Detection of specific antibody responses to Coccidioides antigens is usually required to establish diagnosis of coccidioidomycosis
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