Ticks transmit many pathogenic viruses and bacteria and in the United States are responsible for more human disease than any other arthropod group. Alarmingly, the severity and incidence of disease transmission has been steadily increasing. For instance Lyme disease (LD) caused by Borrelia burgdorferi sensu lato genospecies, first identified in the 1970s in a small number of cases in Connecticut, is now the most commonly reported arthropod-borne disease in the US. Early detection leading to antibiotic treatment is usually curative, but if left undiagnosed, LD frequently develops into disseminated disease that can affect neurologic, cardiac and muscoskeletal systems. Unfortunately, early diagnosis is often confounded by non-specific symptoms and lack of risk awareness. Since increased infected tick density is correlates well with human disease incidence, B. burgdorferi surveillance can help estimate disease endemicity and risk of spread, which in turn can be used to raise awareness, heighten prevention and engender aggressive monitoring of early clinical signs and molecular markers of LD. However, current molecular surveillance techniques, mostly based on polymerase chain reaction (PCR), require technical expertise, complex instruments, and extensive infrastructure typically only available in centralized laboratories. Innovative molecular diagnostic tools are critically needed to improve vector surveillance across states in geographically remote or resource-limited areas. To fill this critical need, we propose to develop a low cost point-of-care (POC) nucleic acid diagnostic that will identify Ixodes scapularis and B. burgdorferi sensu stricto, the principal vector and pathogen, respectively, responsible for LD. Our one-pot assay designed for field-use will generate clear `Yes/No' answers with minimal user intervention or data analysis. Our POC surveillance platform will rely on several molecular innovations: adapting a chemically-modified, high amplification efficiency version of loop-mediated isothermal amplification, coupling isothermal amplification with nucleic acid logic processing to improve diagnostic surety, developing wavelength-shifting TAO probes to color code specific amplicons, optimizing assays and probes to generate fluorescence amplitude that can be readily imaged with smartphones, and proofing diagnostic performance with field-caught ticks supplied by our partner, Dr. Maria Esteve-Gassent at Texas A&M University. R21 funding will propel our research to the point where we can apply for Early Translational Research Awards and begin to partner for larger scale manufacturing and field trials. Moreover, development of this simple-but-robust approach to fast and accurate pathogen detection can be readily diversified for surveillance of a wider array of vectors and pathogens. We envision that by reducing cost and increasing end user accessibility, our POC diagnostic platform will facilitate greater geographic and demographic penetration of surveillance while enabling rapid reporting of epidemiological data via cloud networks.
Rapid methods to accurately detect Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease, directly in Ixodes scapularis tick vectors would significantly facilitate in-field surveillance of pathogen prevalence as well as identification of potential patients. We propose to develop a multiplex amplification assay for `Yes/No' identification of both I. scapularis and B. burgdorferi from a single tube one-pot assay, and to adapt this assay to a portable sample-to-smartphone device that will accept crushed ticks and transduce only a few copies of tick and bacterial nucleic acids into color-coded fluorescent signals that can be read via unmodified smartphones within 60 min. Our device would be used at point-of-care to streamline the time required for epidemiological monitoring and identification of infected ticks, allowing timely and appropriate management, and will also pave the way to developing point-of-care assays and devices for a much wider variety of pathogens.
