Infants in the neonatal ICU (NICU) have a high incidence of bacterial sepsis, predominantly caused by Gram-negative enteric pathogens and staphylococcal species, with antibiotic resistant infections increasing. Symptoms of sepsis in newborns are non-specific, leading to an overuse of antibiotics in this population. Current methods for pathogen detection and antibiotic susceptibility testing are time and labor intensive and often inadequate. Rapid, real-time diagnostics, with the capability to detect, identify and characterize pathogens with respect to antibiotic sensitivities are critically needed for improved patient care, patient outcomes and antibiotic stewardship. This application proposes development of NeoDIRECT (Detection and Identification of Resistance Equals Correct Treatment), a rapid real-time PCR-based diagnostic assay for the detection and identification of the primary pathogens of sepsis in the NICU. My Specific Aims are: 1) to complete development of NeoDIRECT to include nested-multiplex PCR (nmPCR) to detect and identify the most common pathogens of neonatal sepsis and their genetic determinants of antibiotic resistance, 2) to adapt NeoDIRECT for direct-from-specimen testing using whole blood as a model and 3) perform a clinical study to evaluate the potential impact of NeoDIRECT on vancomycin use in the NICU and a survey to evaluate physician attitudes. Development of NeoDIRECT will be of significant benefit to infants in the NICU and will provide a model for pathogen detection in the FilmArray, an innovative real-time PCR platform developed at Idaho Technology, Inc., with myriad future applications for hospitalized children and adults. In addition to my research aims, my Specific Career Development Aims include: 1) to expand my existing skills in the development of PCR-based diagnostics, 2) to expand my knowledge about hospital- acquired infection and antibiotic-resistant organisms, 3) to learn about the validation, implementation, and FDA approval of clinical assays, 4) to develop skills in clinical and health services research that will enable me to validate novel diagnostic tests in clinical settings and use them to conduct hypothesis-driven patient- oriented research,5) to develop research management skills to lead future projects and 6) to develop leadership skills through leadership training. Optimal development of new diagnostic technologies will involve an interdisciplinary approach including academia, industry, basic- and clinician- scientists. As a highly- trained physician and scientist-collaborating with biotechnology at the outset of my career-I am uniquely positioned to make significant contributions to this field. RELEVANCE (See instructions): My overarching career goal is to improve the healthcare of children and adults through improved diagnostic testing for infectious agents and antimicrobial resistance. Development of NeoDIRECT will be of significant benefit to infants in the NICU and will provide a model for pathogen detection in the FilmArray, an innovative real-time PCR platform developed at Idaho Technology, Inc., with myriad future applications for hospitalized children and adults