Strep-CaDI: A fast, accurate, and sensitive point-of-care test for Group A Streptococcus

SUMMARY

The current rapid antigen detection tests for group A strep lack sensitivity and result in upwards of 10 million unnecessary

antibiotic treatments provided to children each year. Current diagnostic methods utilized when a patient presents at a clinic

with a sore throat include a throat swab and rapid strep test. A positive rapid test results in antibiotic prescription, but due

to the low sensitivity of existing rapid strep immunoassays, physicians often prescribe antibiotics as a precaution while waiting for culture results after a negative rapid test. It is estimated that up to 70% of these doses are unnecessary, contributing to the rise in antimicrobial resistance. While ELISAs and molecular (PCR-based) assays with improved

sensitivity have been reported to reduce unnecessary antibiotic treatment, these tests rely on laboratory instrumentation and

significant infrastructure, making them unsuitable for point-of-care settings and rural clinics, and impossible to implement

as at-home testing protocols for telemedicine purposes. Thus, there is an urgent unmet need for an improved, ultrasensitive

rapid strep test that could reduce the unnecessary use of antibiotics, stemming the increase in antimicrobial resistance and

preserving the gut microbiome of children. Furthermore, if an improved rapid strep test could be optimized for robustness

and ease of use, as well as sensitivity, it could be performed at home enabling telemedicine protocols and reducing the need

for a potentially infected individual to expose frontline healthcare workers. The outcome of this project will be a proof-of-

principle demonstration of an ultrasensitive rapid strep test with onboard reagents that requires a simple swab insertion step

to initiate to completion with limits of detection 10-100x lower than current dipstick methods. Improved sensitivity will be achieved through use of the Capillary-Driven Immunoassay (CaDI) technology in which we are able to integrate all steps of highly sensitive, laboratory-performed ELISAs (washing, labeling, and amplification) on a simple microfluidic device

without requiring instrumentation or highly trained personnel. This first-in-its-class microfluidic point-of-care (POC) device

will be realized through the following aims. First, proof of concept for a Strep-CaDI rapid strep test will be demonstrated

for the ultrasensitive detection of Streptococcus pyogenes antigen in spiked buffer through antibody screening and assay parameter optimization. Secondly, the bacterial extraction method and corresponding CaDI parameters and device design

will be optimized for use with clinical samples spiked with whole bacteria. The final, optimized design will be compared to

current market solutions to compare assay sensitivity (LOD), time to result, and complexity (steps to result). Lastly, to

increase the likelihood of approval for at-home use and to provide a clear market advantage over current solutions, we will

prototype (through modeling, 3D printing, and testing) an assay housing capable of simplifying CaDI operation to a simple swab->insert-> read mechanism which initiates the assay to completion and isolates all reagents from the operator. Once

the required functionality is achieved, the housing and immunoassay will be tested by untrained individuals. Feedback from

these trials will be used for housing design iterations. Completion of these three aims will result in a prototype rapid strep test with the ease-of-use of a simple dipstick test and the sensitivity of a laboratory ELISA.