Point-of-Care Differential Diagnosis of Hemorrhagic Fevers

Introduction: The United States military, with significant deployments in regions like Africa and Asia, faces substantial risks from infectious pathogens such as malaria, chikungunya, and dengue virus (DENV). Of particular concern are viral hemorrhagic fevers (VHFs) such as Ebola (EBOV), Lassa (LASV), and Marburg (MARV) viruses, which pose global threats. These diseases present with similar initial symptoms—fever, malaise, vomiting, and diarrhea—making them difficult to distinguish through syndromic surveillance alone​​. To address this diagnostic challenge, the FeverPhone, a mobile-enabled multiplexed point-of-care diagnostic platform, was developed under NIH funding. This platform initially targeted agents causing acute febrile illnesses, including DENV, chikungunya, malaria, and typhoid. The current project aims to extend this platform to include differential detection of VHFs like EBOV, LASV, and MARV​​.

The joint effort between the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) and Cornell University seeks to enhance biomarker-guided clinical management for severe infectious diseases in austere settings. The project will co-develop a tri-plex EBOV/LASV/MARV platform that targets antigens and IgM/IgG antibodies to each virus, differentiated using a patented color encoding scheme. Clinical validation will involve USAMRIID biorepositories and non-human primates (NHPs)

Materials and

Methods: The project has two specific aims: first, to develop and validate single-virus antigen/IgM/IgG tests for EBOV, LASV, and MARV individually at Cornell and USAMRIID, and then combine them into a multiplexed system; second, to validate the triplex FeverPhone system using live viruses and animal model samples at USAMRIID​​.

For the development of single-virus tests, color-coded latex nanoparticles functionalized with specific antigens will be used. The antigen detection and antibody tests will be spatially separated on the test strip to prevent cross-reactivity. The antigen tests will use monoclonal antibodies conjugated to color-coded nanoparticles for visual labeling, while antibody tests will target IgM and IgG using recombinant proteins​​. Initial validation will occur at Cornell using contrived blood and serum samples, followed by testing with live viruses at USAMRIID's BSL-4 facilities​​.

The integration phase will combine individual tests into a tri-plex format using a color-splitting algorithm to measure the intensities of red, blue, and green nanoparticles, representing EBOV, LASV, and MARV concentrations, respectively. This phase will also involve anti-cross reactivity procedures to ensure specificity​​.

Validation will involve contrived samples and feedback from multiple users at both Cornell and USAMRIID. Approximately 200 tri-plex tests and six FeverPhone units will be produced for detailed validation studies​​.

Results, Conclusions, and Discussions: Preliminary data from the FeverPhone system demonstrated high sensitivity and specificity in differentiating DENV and chikungunya infections in human samples. For the current project, the development of single-virus tests for EBOV, LASV, and MARV has shown promising results in terms of antigen and antibody detection​​. The single-virus tests will be integrated into a tri-plex format and validated with live viruses. This system will enable rapid, point-of-care diagnosis, reducing the need for extensive laboratory infrastructure and trained personnel​.

The FeverPhone's unique color multiplexing allows for differentiation of multiple pathogens on a single lateral flow assay, enhancing diagnostic capabilities in resource-limited settings. The platform's adaptability for use in both military and civilian healthcare contexts highlights its potential impact on public health and disease management strategies​​. The validation studies aim to achieve at least 90% sensitivity and specificity for antigen and antibody detection. Feedback from multiple users will help refine the system for practical deployment. If successful, the FeverPhone 2.0 will be ready for broader field validation and regulatory clearance.

Acknowledgements (Optional): This project is a collaborative effort between USAMRIID and Cornell University, supported by NIH funding. We extend our gratitude to the teams involved in the development, testing, and validation phases of the FeverPhone project. Special thanks to the USAMRIID biorepositories and the non-human primate facilities for their crucial role in clinical validation. We also acknowledge the contributions of various researchers and technicians whose expertise and dedication have been instrumental in advancing this diagnostic platform.