Rationale: Progress in molecular diagnostics since the start of the COVID-19 pandemic has opened up immense possibilities for the next stage of diagnostic capabilities for tick-borne diseases (TBDs) and unexplained fevers. We propose to develop a technology for tick surveillance and clinical testing using a novel strategy to rapidly monitor and discover pathogens. Traditional molecular detection approaches need to be very specific to a known pathogen sequence, which limits our ability to discover variant or novel pathogens. A new technology called hybrid capture sequencing does not have this drawback and can be deployed without the need for heavy lab infrastructure. This technology is now commercially available for respiratory pathogen detection, but no TBD application exists to our knowledge.

Scientific objective: The long-term goal of the proposed research is to develop a rapid point-of-care test covering all major TBD and febrile illness pathogens known to affect humans. The goal of the study proposed here is to develop and bench-validate a hybrid capture sequencing method for this application and assess its ability to detect variants and unknown pathogens.

Aims: We have assembled a multidisciplinary partnership between Cornell University and Columbia University to accomplish the following aims:

Specific Aim 1 - Panel design and pilot scale production: A curated list of genes from all major known TBD pathogens will be completed. In addition to pathogen targets, we will also incorporate a novel strategy for host blood meal remnant identification for tick surveillance.

Specific Aim 2 - Lab bench validation: Evaluation of assay performance for pathogen detection will be performed using recommended benchmarks for clinical microbiology laboratory-developed tests.

Specific Aim 3 - Field sample and mock novel virus evaluation: Targeted collections of ticks and animals (as surrogate for human skin biopsies and blood) will be performed on Staten Island, New York City, where Columbia University has established a long-term field site. This site is ideal to capture a diverse pathogen set given large populations of people, different tick species, and urban forests with extensive contact between them.

The proposed method is in early stage development; thus, laboratory and computationally-based development will be the initial focus. In addition to testing banked specimens, we will prospectively collect adult ticks, biopsies, and blood samples from medium-sized mammals at our field site, with the goal of obtaining positive and negative samples for a subset of domestic pathogens with established gold-standard assays for determination of sensitivity and specificity. In order to systematically assess our method for ability to detect novel pathogens, we will also generate mock novel viral genes with different levels of variation from reference sequences that will be spiked into negative field samples.

Impact: This Idea Development Award proposal pertains to two of the Fiscal Year 2021 Tick-Borne Disease Research Program Focus Areas:

Diagnosis: If successful, our strategy will greatly improve resolution in TBD clinical diagnosis by allowing the rapid identification of different variations of known pathogens as well as giving early warning of new threats. Because of how they are designed, the probes we will use have the ability to enrich fragments of unknown sequences that could be pathogenic. Our technology also enables comprehensive coverage of all known TBD pathogens that military Service Members may be exposed to across the globe. The platform used is small and portable and is currently being used for pathogen discovery in low-resource settings.

Prevention: This project will develop an accessible method that, once established in the laboratory, can be immediately used for vector surveillance and vector ecology studies. It will also provide current information on pathogens present in an area of high tick burden and human-tick interactions. Having the ability to profile not only the pathogens and their subtle variations but also the bloodmeal remnants from humans and animal reservoirs will be a monumental advancement in our ability to study Lyme and TBD ecology and the effectiveness of prevention and control interventions.