Development of a Comprehensive Tick-Borne Pathogen and Febrile Illness Detection Panel

Development of a comprehensive tick-borne pathogen and febrile illness detection panel Background: Progress in advanced molecular detection (AMD) technology 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 a technology development project using a novel AMD strategy to rapidly monitor and discover pathogens with applications for tick surveillance and clinical testing. Traditional molecular detection approaches rely on polymerase chain reaction (PCR) to amplify pathogen DNA or RNA in a sample and detect it with a fluorometric signal. Two major limitations of this approach are that it requires primers that are very specific to a target sequence, which limits its utility for novel pathogen discovery, and it can only combine a limited number of reactions together without compromising assay performance. Hybrid capture next-generation sequencing (HCNGS) is a method of enriching the pathogen signatures in a sample without relying primarily on PCR. It does not have either of these drawbacks and additionally, can be deployed without the need for heavy lab infrastructure. While PCR is generally limited to hundreds of primers, hybrid capture can accommodate millions. This technology is commercially available for respiratory pathogen detection, but no TBD application exists to our knowledge. Hypotheses/Objective: The long-term goal of the proposed research is to develop a rapid point-of-care (POC) test covering all major TBD and non-respiratory febrile illness pathogens known to affect humans. The goal of the study proposed here is to develop and bench-validate a hybrid capture sequencing approach using Illumina sequencing chemistry. We hypothesize that a hybrid capture approach targeting both TBD pathogens with RNA genomes (arboviruses) and DNA genomes (bacteria and parasites) will have performance comparable to PCR and sufficient flexibility to detect currently known and novel pathogens. Specific Aims: We have assembled a multidisciplinary partnership between Cornell University and Columbia University to accomplish the following Specific Aims: Specific Aim 1- Panel design and pilot scale production: A curated list of genes from all major known TBD pathogens will be completed. This will include all of the targets of our currently validated nanoscale PCR array of 17 of the major pathogens of concern in North America. In addition to pathogen targets, we will also incorporate a novel strategy for host blood meal remnant identification for vector surveillance. Specific Aim 2 - Analytic validation using spiked controls: Bench evaluation of assay performance for pathogen detection will be performed using pools of synthetic mock pathogen communities serially diluted and spiked into relevant matrices (tick, skin biopsy, and blood). Published test evaluation benchmarks for clinical molecular microbiology will be used to assess performance. 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. Study Design: 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 and mammals at our field site in order to assess accuracy for domestic pathogens circulating at the field site, comparing with established gold-standard assays. In order to systematically assess our method for ability to detect novel pathogens, we will spike negative field samples with in vitro transcribed RNA from synthetic viral genes with different levels of variation from reference sequences in order to determine the genetic variation tolerance. Impact: The vast majority of available TBD diagnostic testing cannot differentiate strains or even species. Molecular detection approaches such as the strategy proposed here are ideal for sentinel pathogen detection and subtyping in vectors and clinical samples. Because of how they are designed, hybrid capture probes will hybridize to a specific known sequence and then pull down the surrounding sequence. This will help facilitate detection of novel variants and potentially novel species in a host independent manner. This has been demonstrated by applying HCNGS to synthetic controls derived from viral species that emerged after panel design. The immediate impact of our work will be to report on pathogens present in an area of high tick burden and human-tick interactions. The long-range outcomes of the study will be in development of a POC approach that would enhance rapid TBD testing and discovery capabilities in military facilities worldwide. Less