A major increase in the number of cases of coccidioidomycosis (Valley fever), a fungal respiratory disease of humans, has occurred in southwestern United States during the last five years. Our goals are to develop rapid and relatively simple methods for diagnosis of this disease. Current methods are inadequate because they are primarily dependent on patient antibody detection (antibody production may be delayed or attenuated in immunocompromised patients), and because multicomponent antigens currently used for antibody detection are variable between batches and display some cross-reactivity with heterologous sera (false positive reactions). We have proposed the development of three approaches to diagnosis of coccidioidomycosis and have designed experiments to test specificity and sensitivity of the molecular probes to be used. We will employ a recombinant, complement fixation antigen (CF-Ag) in standard immunoassays for specific antibody detection, monoclonal antibody to the CF-Ag and Coccidioides-specific antigen (CS-Ag) for detection of antigenemia, and a PCR method developed in the P.I.'s laboratory for C. immitis DNA detection in patient fluids. Multiple control and positive patient fluids (>400 sera, CSF, urine, BALs, and sputum) submitted by 8 physician-consultants will be used in open and blind studies for testing the efficacy of our diagnostic methods.
This proposal outlines methods for detection of (a) pathogen-specific antibody, (b) antigenemia and (c) DNA in patient sera and other body fluid using (1) bacterial-produced recombinant antigen, (2) monoclonal antibodies and (3) a polymerase chain reaction (PCR) primer-pair derived from a pathogen-specific gene, respectively. Each reagent (molecular probe) can be produced in unlimited amounts, can be stored without loss of activity for unlimited time, can be standardized (quality-controlled), and can be incorporated into individual test kits for clinical application using established testing methods in clinical laboratories.
