Diagnoses of opportunistic fungal infections constitute an increasing clinical problem. Conventional diagnostic tests are time consuming and lack specificity and sensitivity for accurate and timely prognoses. The lack of precise and prompt diagnoses has undermined the health of patients and contributed to high mortality rates in immunocompromised individuals undergoing aggressive medical treatments. This condition has led to an increase in the emergence of new antifungal resistant strains and the need for rapid non-culture based methods for the detection of pathogens at species and strain levels. In cooperation with a unique team of researchers from government (USDA, Peoria, Ill), academia (University of Miami), two clinical laboratories (John Hopkins Medical Institute and Allogen Laboratories of the Cleveland Clinic) and industry (Luminex Corp, Austin, TX) we propose to develop a sensitive, fast and reliable high throughput DNA based method aimed at detection of such important fungal pathogens as species of Aspergillus, Candida, Cryptococcus and Trichosporon. The detection method will combine the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of LabMap, a new flow cytometric technique by Luminex Corp. The multiplexed assay analysis will consist of a combination of fluorescent microspheres covalently bound to species-specific fungal oligonucleotide probes, with the capability to simultaneously test as many as 100 different species-specific probes per tube/well at rates 0.47 min/well probes per minute. In the preliminary stages of this research, the system will be developed with extensive in-house databases and culture collections from USDA and UM and from clinical isolates from JHMI to produce a detection technique for cultured organisms. Validation of the assay with archived clinical specimens will be in conjunction with the Allogen Lab, which will lead to a detection system directly from clinical specimens. The fungal detection system will be expanded to include all known yeasts, including medical, foodborne and industrial species as well as other filamentous pathogens. We foresee that this multiplex testing fungal platform can be expanded to other microorganisms with important applications in areas of public health, bioterrorism, food safety and the environment. Resulting data will be disseminated via GenBank, publications in leading scientific journals and as an inexpensive commercial detection system.
