Prospective and Operational Validation of the Tropical Fever Panel on a Point-of-Care Platform

Background: Force readiness requires tools and strategies to address infectious disease threats. Our team has developed a novel multiplex assay to detect biological agents that are important to the Warfighter and are known to pose health risks (Dengue serotype 1-4, Chikungunya, and Zika viruses, malaria, leptospirosis, and plague) with a limit of detection of ~100 copies of pathogen RNA/DNA. The panel is run on the Rapid Analysis Mobile Platform (RAMP) handheld device using a finger stick of blood that provides results in less than 70 minutes. The test is fully automated, with RNA and DNA extraction and purification followed by reverse transcriptase-polymerase chain reaction amplification of pathogen nucleic acids. This assay system eliminates the need for sample processing in the laboratory, thus allowing rapid diagnosis in the field. Rationale: Infectious diseases of military relevance are indistinguishable based on clinical presentation alone. Deployable diagnostics for priority pathogens are needed for early detection, appropriate clinical management, and interruption of transmission. Objective: Our objective is to deliver a multiplex assay capable of accurately detecting military-relevant pathogens that can be run on a handheld platform in approximately 1 hour, and to rigorously evaluate the performance characteristics of the assay system. Specific Aims: (1) Retrospective assay validation of the Tropical Fever Panel using archived samples and user feedback. (2) Prospective assay validation of acute febrile illness subjects in a highly endemic area. (3) Clinical site testing in febrile and severely ill subjects at the point of care. Study Design: We propose retrospective and prospective observational studies to evaluate the performance of the Tropical Fever Panel and RAMP for the detection of Dengue serotype 1-4, Chikungunya, Zika, malaria, leptospirosis, and plague. Archived samples collected from febrile patients with known Dengue, Zika, Chikungunya, P. vivax, P. falciparum, or Leptospira infection will be used to validate the sensitivity/specificity of the platform in Cambodia and Peru (approximately 100 samples each). We will integrate the assay into ongoing active febrile illness health clinic-based surveillance in Iquitos, Peru for prospective evaluation of the assay system when compared to gold-standard assays for each pathogen on the panel. We will also evaluate the performance of the assay system in a hospital setting at the point of care in febrile and severely ill patients in Cambodia and Peru. Subjects presenting to ACESO clinical sites will be screened for eligibility, and eligible patients will be consented. We will prospectively enroll approximately 100 febrile adult patients (>/=18 years) total, and 100 severely ill subjects per year, at both sites. The assay will be run at the clinical site in real time, and results will be compared to gold-standard assays. The primary outcome of interest is the performance of the Tropical Fever Panel run on the RAMP, which will be evaluated in terms of sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio. Impact: The lack of rapid, accurate diagnostics at the point of care has been recognized as an urgent need for Force health protection. Early detection of infection could dramatically improve clinical management, provide early outbreak detection, enhance surveillance capabilities, and improve operational readiness. Relevance: A diagnostic assay that can be performed in far-forward, deployed field conditions directly addresses the topic area for the development of highly sensitive diagnostic systems for use at the point of need. Use of a rapid diagnostic would decrease the time from presentation to treatment and would be a force multiplier, increasing combat effectiveness by returning Service members to duty earlier and reducing morbidity and mortality. Less