Discriminating viral and bacterial meningitis infections with iDDS probes

Discriminating viral and bacterial meningitis infections with iDDS probes Confidential PI Shafer David A PhD PROJECT SUMMARY Rapid and accurate molecular diagnostics assays empower physicians to make informed treatment decisions especially when a disease state may result from variable causes that are indeterminable without such testing Meningitis may be caused by infection with microorganisms such as viruses gram negative GN and gram positive GP bacteria fungi or parasites Most frequently meningitis results from infection with non polio enteroviruses EV and parechoviruses PV which typically self resolve within days In contrast bacterial meningitis has a high mortality rate that approaches if not treated Patients present with similar symptoms regardless of origin thus timely diagnosis of the causative agent is paramount for patient care Patients are commonly treated with an `empiricandapos regimen of antibiotics until a definitive diagnosis can be made Nucleic acid amplification tests NAATs reduce diagnosis turnaround times by up to several days compared to standard culturing methods allowing shortened hospital stays appropriate use of antibiotics and a reduced financial burden to patients and the health care system Currently there are only two FDA approved NAATs for diagnosing viral and or bacterial meningitis Unfortunately these tests are only approved for use on the manufacturerandapos s fully automated systems the cost of which is often prohibitive Further these assays detect the highly variable viral genomes at a single target site Collectively these limitations highlight the need for an instrument independent assay that can discriminate between viral and bacterial meningitis with error checking properties and confirmatory detection at redundant sites in the viral genomes GeneTAG Technology Inc has developed error checking DNA Detection Switch iDDS probes which employ a fluorescent labeled probe and a slightly mismatched quencher labeled antiprobe In the absence of the intended target the antiprobes hybridize to the probes quenching their fluorescence and preventing off target detection Recently we developed assays for detecting EV and PV employing two iDDS probes for each target providing simultaneous color signaling and automatic confirmation of positive or negative test results We previously developed iDDS probes for GN and GP detection Ultimately we are interested developing an FDA approved iDDS probe based meningitis assay in a Sample ReadyTM lyophilized plate format that will parallel similar tests developed by our collaborator BioGX Inc where all reagents primers and probes are included and the test would only require adding sample and molecular grade water Aim studies will focus on optimizing the dual iDDS probe EV and PV assays to perform well in multiplex format with commercial EV and PV strains and patient cerebrospinal fluids converting the multiplex test to Sample ReadyTM format and re testing the Sample ReadyTM assay with the original samples A similar strategy will be employed in Aim to prepare a Sample ReadyTM GN GP bacteria assay Delivering a high fidelity cost effective meningitis assay should positively impact meningitis diagnostics and provide a direct benefit to public health Discriminating viral and bacterial meningitis infections with iDDS probes Confidential PI Shafer David A PhD PROJECT NARRATIVE It is critical for patient care to distinguish between potentially deadly bacterial meningitis infections caused by gram negative GN or gram positive GP bacteria and less severe viral meningitis caused by enteroviruses EV or parechoviruses PV Unfortunately the only FDA approved nucleic acid amplification tests for bacterial and viral meningitis require in many cases cost prohibitive instrumentation and these assays only detect the variable viral genomes at a single target site This Phase I study seeks to develop a combined EV PV and GN GP assay based on real time PCR amplification using our more advanced error checking iDDS probe technology using double target sites for the variable EV and PV genomes to automatically confirm positive detection and to avoid false negatives This proposal will evaluate the performance characteristics of the assay with clinical samples in preparation for expanded Phase II validation studies and FDA approval Delivering a high fidelity cost effective meningitis assay with built in confirmation of test results should positively impact meningitis diagnostics and provide a direct benefit to public health