Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from multiplexed RT-PCR/PCR-based detection assays

Rapid design and fielding of four diagnostic technologies in Sierra Leone, Thailand, Peru, and Australia: Successes and challenges faced introducing these biosensors

Febrile illnesses are among the most common reasons for visits to hospitals and clinics worldwide. Since fevers can arise from a wide range of diseases, identifying the causative pathogen is essential not only for effective personal treatment but also for early detection of outbreaks. The Defense Threat Reduction Agency (DTRA) tasked a coalition of commercial, academic, and government researchers with moving diagnostic technology concepts from ideation to field use as rapidly as possible using scientifically sound evaluations. DTRA's 24 Month Challenge program examined >30 technologies before fielding four technologies on four continents. >10,000 in field test results were recorded. Here we discuss our tiered evaluation system to assess candidate technologies developed by commercial partners and the process of field testing those technologies at various front-line clinics in Sierra Leone, Thailand, Peru, and Australia. We discuss successes and challenges for introducing two multiplexed lateral flow immunoassay (LFI) tests that detect malaria, dengue fever, melioidosis, and the plague. Additionally we discuss the use of a LFI reader that assisted the interpretation of the assay, communicated results to a data cloud, and greatly facilitated reach-back support. Lastly, we discuss the concurrent field testing of a multiplexed PCR assay on the FilmArray platform, which had an assay pouch specially designed for the 24 Month Challenge. Either standard-of-care or gold-standard testing were run alongside our fielded technologies to benchmark their performance