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.
Public Health Relevance Statement: NARRATIVE Current point-of-care rapid strep throat immunoassays lack the sensitivity needed for accurate diagnosis of infection. As a result, physicians frequently prescribe unnecessary antibiotics as a precaution, which contributes to the rise in antimicro- bial resistance and negatively impacts gut microbiome, particularly in children. The ultrasensitive rapid strep throat immu- noassay proposed here utilizes capillary-driven microfluidics to achieve improved sensitivity by automating all steps of a laboratory-quality immunoassay while maintaining the simplicity of a single-step assay procedure consisting of inserting the swab into the device and reading the result.
Project Terms: Antibiotic Agents; Antibiotic Drugs; Miscellaneous Antibiotic; Antibiotics; Antibodies; immunogen; Antigens; Bacteria; Biological Assay; Assay; Bioassay; Biologic Assays; Buffers; Blood capillaries; capillary; Child; 0-11 years old; Child Youth; Children (0-21); kids; youngster; Colorado; Communicable Diseases; Infectious Disease Pathway; Infectious Diseases; Infectious Disorder; Disease; Disorder; Enzyme-Linked Immunosorbent Assay; ELISA; enzyme linked immunoassay; Feedback; Future; Patient Care; Patient Care Delivery; Goals; Health Personnel; Health Care Providers; Healthcare Providers; Healthcare worker; health care personnel; health care worker; health provider; health workforce; healthcare personnel; medical personnel; treatment provider; Housing; Immunoassay; Infection; instrumentation; Kinetics; Laboratories; Manuals; Marketing; Methods; United States National Institutes of Health; NIH; National Institutes of Health; Paper; Legal patent; Patents; Patients; Pharyngitis; Pharyngeal structure; Pharynx; Throat; Physicians; Polyesters; Reading; Reagent; Running; Sensitivity and Specificity; Signal Transduction; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Streptococcus pyogenes; S pyogenes; S. pyogenes; Streptococcus Group A; Technology; Testing; Time; Treatment Protocols; Treatment Regimen; Treatment Schedule; Universities; Work; Generations; Device Designs; Film; Telemedicine; Label; Pump; improved; Procedures; Clinical; Penetration; Series; Evaluation; Training; Individual; Sore Throat; Funding; Exposure to; Antibiotic Therapy; Antibiotic Treatment; bacterial disease treatment; bacterial infectious disease treatment; Diagnostic; Molds; Filamentous Fungi; Diagnostic Procedure; Diagnostic Method; Diagnostic Technique; Clinic; Protocols documentation; Protocol; Reaction; System; Test Result; Performance; hydrophilicity; Speed; Devices; Human Resources; Manpower; personnel; Reporting; Modeling; Sampling; µfluidic; Microfluidics; Swab; Dose; Microfluidic Microchips; Microfluidic Device; Microfluidic Lab-On-A-Chip; microfluidic chip; Antimicrobial Resistance; Antimicrobial resistant; Resistance to antimicrobial; anti-microbial resistance; anti-microbial resistant; resistance to anti-microbial; resistant to anti-microbial; resistant to antimicrobial; Symptoms; Detection; Molecular; Development; developmental; point of care; cost; designing; design; nano particle; nano-sized particle; nanosized particle; nanoparticle; Outcome; stem; prototype; point-of-care diagnostics; phase II study; phase 2 study; operations; operation; screenings; screening; 3-D print; 3-D printer; 3D printer; 3D printing; three dimensional printing; 3D Print; accurate diagnosis; GI microbiome; digestive tract microbiome; enteric microbiome; gastrointestinal microbiome; gut-associated microbiome; intestinal biome; intestinal microbiome; gut microbiome; physical symptom; acute symptom; Injections; preservation; Infrastructure; 2019 novel corona virus; 2019 novel coronavirus; COVID-19 virus; COVID19 virus; CoV-2; CoV2; SARS corona virus 2; SARS-CO-V2; SARS-COVID-2; SARS-CoV-2; SARS-CoV2; SARS-associated corona virus 2; SARS-associated coronavirus 2; SARS-coronavirus-2; SARS-related corona virus 2; SARS-related coronavirus 2; SARSCoV2; Severe Acute Respiratory Coronavirus 2; Severe Acute Respiratory Distress Syndrome CoV 2; Severe Acute Respiratory Distress Syndrome Corona Virus 2; Severe Acute Respiratory Distress Syndrome Coronavirus 2; Severe Acute Respiratory Syndrome CoV 2; Severe Acute Respiratory Syndrome-associated coronavirus 2; Severe Acute Respiratory Syndrome-related coronavirus 2; Severe acute respiratory syndrome associated corona virus 2; Severe acute respiratory syndrome coronavirus 2; Severe acute respiratory syndrome related corona virus 2; Wuhan coronavirus; coronavirus disease 2019 virus; coronavirus disease-19 virus; hCoV19; nCoV2; 2019-nCoV; RadX.; Radx-; Rapid Acceleration of Diagnostics; RADx; home-based test; home test; antigen based detection; detect antigen; antigen detection; COVID-19 antigen; SARS-CoV2 antigen; coronavirus disease 2019 antigen; severe acute respiratory syndrome coronavirus 2 antigen; SARS-CoV-2 antigen; Rapid diagnostics; rapid assay; rapid tests; rapid test; detection tests; detection test; detection limit; COVID19 outbreak; SARS-CoV-2 outbreak; Severe acute respiratory syndrome coronavirus 2 outbreak; coronavirus disease 2019 outbreak; coronavirus disease-19 outbreak; outbreak of SARS-CoV-2; COVID-19 outbreak; homes; Home; point of care testing; rural health clinic; rural clinic; manufacture
