Rapid ID and AST Directly from Whole Blood Using Single Molecule Detection

Project Summary Standard identification (ID) and antimicrobial susceptibility testing (AST) of bloodstream pathogens are conducted using slow methods that require blood culture. Diagnostic results are available three or more days after the diagnostic process is started. The lack of rapid diagnostic results forces physicians to treat patients with broad-spectrum antibiotics which are not effective against a growing number of resistant organisms and can induce the development of more antimicrobial resistance. Here, we propose the development of a rapid diagnostic platform that, if successful, will enable effective and targeted therapy of bloodstream infection patients within hours. The new platform will directly analyze whole blood samples using a simple and robust single molecule detection approach called Single MOlecule Tethering or SMOLT. The platform will be rapid (ID in one hour and AST in 4- 6 hours), easy-to-use, and cost-effective. Our preliminary results show that a SMOLT multiplex assay can detect bacteria in blood with limits of detection (LOD) between 1-10 CFU/mL with a turn-around-time of about an hour. The same technique can be used for AST. Preliminary results show that a brief incubation with an antibiotic follow by SMOLT detection can be used to determine if a strain is susceptible, intermediate or resistance in high agreement with the category assigned be a standard reference method. In aim 1, we propose the development of a SMOLT multiplex ID assay capable of detecting and identifying the rRNA of six highly relevant pathogens directly in whole blood. The goal of aim 2 is to develop a SMOLT phenotypic AST assay for combinations of the six ID species and 14 relevant drugs. Our third aim is to develop a prototype instrument for the SMOLT ID and AST assays that is easy-to-use with minimum hands-on-time. The last aim is to evaluate the ID and AST assays using blood samples seeded with a combination of fresh and stock clinical isolates as well as type strains.