Research

Tropical disease control and elimination efforts benefit from the sensitivity and specificity of nucleic acid amplification tests (NAAT).  However, commonly employed NAAT technologies require expensive equipment, such as real-time PCR instrumentation, not readily adaptable for use in many resource-limited settings including many harboring neglected tropical disease (NTD) infections.  Such settings stand to benefit from the cost-savings and practicality of field-friendly, point-of-collection-based approaches, but assays capitalizing on such technologies for NTD diagnosis remain uncommon.  As a means of filling this diagnostic void, my research group will exploit our previously identified DNA targets to design point-of-collection-based assays.  Reasoning that optimal DNA targets will provide maximal sensitivity and specificity regardless of NAAT technology used, researchers in my lab will utilize CRISPR-Cas to create diagnostic assays for soil-transmitted helminth (STH) pathogens of human importance.  CRISPR diagnostics for the detection of DNA targets make use of the Cas12 enzyme, which, upon recognition of a sequence-specific target molecule facilitates the non-specific cleavage of dsDNA.  Exploiting this property, it becomes possible to detect a target of interest through the introduction of a fluorescently-labeled probe into the master mix.  While intact, this probe has a quenched chemistry.  However, upon cleavage by Cas12 (following recognition of the assay-specific target) fluorescence occurs due to the physical separation of fluorophore and quencher molecules.  In this manner, fluorescence increases with increased probe cleavage, allowing for detection of target and a positive test result.

While qPCR-based diagnostics can efficiently detect pathogens in vector insects, they require prior knowledge of which pathogens may be present. Next-generation sequencing (NGS)-based diagnostics overcome this challenge by detecting all pathogens in a sample. To determine whether NGS could be used as an alternative to qPCR to identify neglected tropical diseases in vectors, the Pilotte Lab is comparing the two methods using field collected mosquitoes from pathogen-endemic regions. 

Working with Quinnipiac Professor of Biology Dr. Dennis Richardson, Pilotte lab members will utilize established bioinformatics approaches to develop a species-specific real-time PCR assay for the detection of Haemonchus contortus.  Currently, Dr. Richardson is utilizing microscopy-based approaches to diagnose the presence of H. contortus in stool samples obtained from New England dairy goat farmers.  However, current microscopy-based approaches are not capable of differentiating closely related strongyle eggs at the species level.  This shortcoming leaves the dairy goat community unable to ascribe infections to a specific pathogenic agent, resulting in uncertainty regarding the rate of infection and the burden of infection, as well as an inability to accurately gauge infection-associated morbidity.  For these reasons, the development of a sensitive and species-specific assay, capable of accurately diagnosing H. contortus infections would provide the dairy goat community with a valuable tool.

Following the development of an H. contortus-specific assay, Pilotte lab members will work with Dr. Richardson to comparatively analyze samples obtained from New England dairy goat farmers using both the developed real-time PCR assay and established microscopy techniques.  This analysis will also serve as the driving factor in the generation of additional research questions, as it is anticipated that many strongyl-positive, H. contortus-negative samples will be identified, providing avenues for the exploration of additional infectious agents.

Using a bioinformatics-based pipeline for qPCR assay design, previously developed by members of the Pilotte Lab, and in collaboration with a leading veterinarian pharmaceutical company, the Pilotte lab is working to advance veterinary pathogen detection. 

In collaboration with Dr. Scott Davies (Biological Sciences), we aim to better understand the parasite diversity within the Song Sparrow.