Project Grant R21AI180713

A NOVEL JANUS BEAD BLOOD ASSAY FOR POINT-OF-CARE ASSESSMENT OF HCV VIRAL LOAD AND ASSOCIATED LIVER DAMAGE - PROJECT ABSTRACT GLOBALLY, NEARLY 300,000 PEOPLE DIE FROM HEPATITIS C VIRUS (HCV) RELATED LIVER DISEASES, MOST OF THEM IN DEVELOPING NATIONS WITH POOR RESOURCES. UNTIL THE RECENT COVID PANDEMIC, HEPATITIS C VIRUS IS ALSO ONE OF THE LEADING CAUSE OF DEATH IN THE US BY INFECTION. WHO AIMS TO ELIMINATE HCV DEATHS BY 2030. THIS AMBITIOUS GOAL CAN ONLY BE ACHIEVED IF HCV INFECTED PATIENTS WITH SEVERE LIVER DISEASE, INCLUDING LIVER CANCER, CAN BE DIAGNOSED AND TREATED RAPIDLY. THE CURRENT GOLD STANDARD, FOR HCV DIAGNOSTICS FOR BOTH DEVELOPING AND DEVELOPED COUNTRIES, IS A POINT-OF-CARE (POC) HOST ANTIBODY TEST, BASED ON SALIVA SAMPLE OR FINGER PRICK BLOOD SAMPLE, FOLLOWED BY A LAB-BOUND REVERSE-TRANSCRIPTION PCR ESTIMATE OF THE VIRAL RNA (LOAD). THE FIRST TEST DOES NOT INDICATE ACTIVE INFECTION THAT SHOULD BE TREATED. THIS IS DETERMINED BY THE SECOND VIRAL LOAD TEST, WHICH ALSO DETERMINES THE SEVERITY OF THE INFECTION AND THE SELECTION OF PROPER TREATMENT OR MORE INVASIVE DIAGNOSIS. THE KEY OBSTACLE TO SUCCESSFULLY DIAGNOSING AND TREATING HCV INFECTED PATIENTS WITH LIVER DISEASES IN THE DEVELOPING WORLD IS THE SECOND PCR TEST FOR VIRAL LOAD. THERE ARE VERY FEW LABORATORIES FOR SUCH TESTS AND MAIL DELIVERY OF BLOOD SAMPLES IS NOT FEASIBLE IN A COUNTRY WITH LOW RESOURCE. MANY PATIENTS THAT TESTED POSITIVE BY THE POC ANTIBODY TEST DO NOT OR CANNOT TRAVEL TO THE LABORATORIES FOR THE PCR AND ANTIGEN TESTS. THIS PROPOSAL AIMS TO INTEGRATE TWO TECHNOLOGIES WITH A SMART-PHONE IMAGING DEVICE TO PROVIDE A 30-MINUTE ONE- STEP POC ASSAY WITH UNTREATED BLOOD THAT CAN QUANTIFY VIRAL LOAD AND DETERMINE THE SPECIFIC LIVER DISEASE. IT IS BASED ON A JANUS MICROPARTICLE ASSAY THAT HAS THE SENSITIVITY OF THE PCR TEST BUT REQUIRES MUCH LESS PERSONNEL ATTENTION AND IS MUCH MORE RAPID. ITS RAPIDITY (30 MINUTES) IS PARTLY BECAUSE SAMPLE PREP IS UNNECESSARY. AS ITS SIGNAL CAN ONLY BE PROVIDED BY THE VIRUS, RATHER THAN ITS RNA (OR PROTEIN), ONLY A RAPID ULTRAFILTRATION STEP IS REQUIRED TO ENRICH THE VIRUS. THE PIS HAVE DEVELOPED SUCH AN ULTRAFILTRATION TECHNOLOGY FOR EXTRACELLULAR VESICLES (EVS). SINCE THE HCV VIRUS IS THE SAME SIZE AS THE EVS, THIS ULTRAFILTRATION TECHNOLOGY WILL BE DEVELOPED FOR VIRUS AND INTEGRATED WITH THE JANUS PARTICLE ASSAY TO PROVIDE A ONE-STEP POC VIRAL LOAD QUANTIFICATION PLATFORM. THE DIRECT VIRUS ASSAY REDUCES THE SAMPLE VOLUME SO THAT CAPILLARY DRAWN BLOOD SAMPLE IS ADEQUATE. THE ONLY INSTRUMENTATION NEEDED FOR THE POC PLATFORM IS A PORTABLE SMART-PHONE BASED IMAGING SYSTEM. UPON COMPLETION OF THIS R21 PROJECT, A BUSINESS PARTNER AOPIA WILL DESIGN INTEGRATED PROTOTYPES THAT CAN PROCESS MULTIPLE SAMPLES FOR A MAJOR CLINICAL TRIAL AFTER THE FUNDING PERIOD.