Research Project
9253915
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 10 days. In contrast, bacterial meningitis has a high mortality rate that approaches 100% 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 `empiric' 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 manufacturer'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 2-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 1 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 2 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.
