PROJECT SUMMARY Otitis media (middle ear infection) is a highly prevalent disease, especially in young children. It has been documented that more than 75% of children will have at least one episode of otitis media (OM) by age 3, and many children will have recurrent or chronic OM. In most cases, diagnosis of an ear infection is performed based on the appearance of the tympanic membrane and presence of fluid via an otoscope. In the absence of a method to identify bacterial infections, broad-spectrum antibiotics are prescribed without definitive knowledge of whether i) an active infection present, and ii) whether it is a bacterial infection (versus a viral infection). Previous clinical data also suggests that antibiotic therapy is only effective for one-third of OM patients, with two thirds of cases likely caused by antibiotic resistant bacteria, or more likely, viral pathogens, resulting in a misdirected expense of more than $700 million, annually. Therefore, there is a clear and urgent need for a point-of-care diagnostic tool to determine the presence of an active infection and whether an infection is bacterial in origin. Further, if we can distinguish the bacterial species present, unnecessary administration of broad-spectrum antibiotics can be eliminated, thus improving the efficacy of treatment and management of this highly prevalent disease. This proposal seeks to fulfill this unmet need by developing integrated Raman Spectroscopy-Optical Coherence Tomography (RSOCT) for the real-time detection and bacterial differentiation of pathological microorganisms in the middle ear. The scientific premise of the proposed research is that OCT can be used for image-guided placement of the RS probing beam, as well as visualization of any biofilm affixed to the tympanic membrane which would be indicative of a bacterial infection. This imaging system will be coupled with Raman spectroscopy for a more direct assessment of any biofilm and/or effusion by determining the presence of OM-causing bacteria and speciation via its biochemical fingerprint. The overall objective of the project will be accomplished by technological innovation and integration, followed by a series of systematic in vitro, ex vivo, and in vivo human studies with rigorous data analysis methods outlined in the specific aims. This research will have a profound impact on how we diagnose and care for these common ear infections. Most importantly, this project will provide critical data (identifying specific bacterial species) to monitor the development of antibiotic resistance, to reduce the overuse of antibiotics, and ultimately, to efficiently improve the health of patients.