Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents

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Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents Confidential PI: Shafer, David A., PhD PROJECT SUMMARY In this study, we propose developing a simple, lyophilized molecular test for detecting the causative agents of Valley fever (coccidioidomycosis), histoplasmosis, or blastomycosis, based on our proprietary internal DNA- Detection Switch (iDDS) probe technology. Timely diagnosis of these fungal infections is key for selecting effective treatments and preventing severe morbidity or death. Over 500,000 such cases are thought to occur in the US annually, although only one FDA-approved molecular test exists for diagnosing Valley fever, and no FDA- approved molecular tests exist for diagnosing histoplasmosis or blastomycosis. The symptoms of coccidioidomycosis, histoplasmosis, and blastomycosis are frequently vague and chest X-rays do not distinguish between these and other lung diseases. Thus, differential diagnosis between these diseases and other that present with similar symptoms (acute respiratory distress syndrome, lung cancer, lymphoma, pericarditis, sarcoidosis, or solitary pulmonary nodules) is key for proactive healthcare interventions. Moreover, diagnosis using culture- or serology-based methods may have a turnaround time of weeks or months, or show false-positive results, and the only related FDA-approved test (the GeneSTAT.MDx Coccidioides Assay) is only approved for use on the manufacturer’s instrument, which dissuades from use at reference labs. GeneTAG Technology (www.genetagtech.com) specializes in developing sensitive and reliable qPCR probe systems. Our error-resistant iDDS probe system employs a fluorescently labeled target specific probe and a slightly mismatched quencher-labeled antiprobe. In the absence of the intended target, the antiprobe hybridizes to the probe, quenching its fluorescence and preventing off-target detection. We have also developed dual-iDDS probe assays for increased specificity and automatic confirmation of the test results. This added layer of specificity is key for accurate diagnosis, especially with low levels of fungal infection. Redundant detection at multiple target sites greatly reduces the need for reflex/confirmatory testing. Our Specific Aims are (1) to develop a 3-tube dual-iDDS probe assay for C. immitis, C. posadasii, H. capsulatum, and B. dermatitidis, and (2) to evaluate key performance characteristics of the multiplex test for endemic fungal agents in lyophilized format. Specifically, in Aim 1, we will (i) develop locked nucleic acid (LNA)-enhanced dual-iDDS probes for diagnostic sequence-specific targets in each pathogen, (ii) convert the dual-iDDS probes to lyophilized format, and (iii) conduct cross-reactivity testing with the lyophilized assays to assess false-positive detection of other pathogens commonly found in bronchoalveolar fluid. In Aim 2, we will determine the (i) linearity, (ii) limit of detection (LoD), (iii) precision, (iv) effects of interfering substances (if any), and (v) stability of the dual-iDDS probe assays. Success in this endeavor will justify expanded testing with clinical specimens in Phase II through a BSL III- certified contract research organization.