Otitis media (OM) is the most common cause of acquired pediatric hearing loss and may delay language and cognitive development. It is the leading diagnosis in the U.S. for children and is the primary reason for prescribing antibiotics and performing surgery. The etiology and pathogenesis of OM are poorly understood. This has hampered the development of rational therapeutic approaches. The primary goal of this proposal is to gain a better understanding of the role of bacteria and viruses in OM. Elucidation of the pathogenic cascade in OM has been impeded by the inherent limitations of conventional cultural methods for these microbes. These obstacles can be overcome by use of the polymerase chain reaction (PCR) which has revolutionized the study of infectious disease. Chronic OM effusions are being obtained from a well- characterized pediatric population during myringotomy and tube placement. These specimens are then subjected to comparative analysis for microbial infection using PCR-based assays and culture methods. Using this molecular method, Hemophilus influenzae and Streptococcus pneumonia DNA have been detected in the majority of culturally-sterile middle-ear effusions of children. To determine if these findings are indicative of low-grade active infections the molecular environment of the middle-ear will be characterized. The fate of nucleic acids. and DNase activity, will be ascertained using a well-characterized animal model (Chinchilla laniger). A multiplex-PCR-based assay will be developed to detect and differentiate upper respiratory tract viruses in pediatric middle-ear effusions. The results of this analysis will be compared with culture. The chinchilla model will be joined with one of the most recent advances in molecular medicine to gain a fundamental understanding of viral pathogenesis in OM. In situ PCR is the molecular equivalent of immunohistochemistry an can demonstrate the location of amplified nucleic acids in histologic sections. PCR and in situ PCR will be used in the chinchilla model to determine if OM is a consequence of direct viral invasion of the middle- ear mucoperiosteum following nasopharyngeal infection, or rather a sequelae of viral-induced Eustachian tube (ET) dysfunction. In future studies this molecular diagnostic system will be used in children to establish whether acute OM is the result of a direct viral infection of the middle ear, or rather the result of virally-induced ET dysfunction with concomitant bacterial infection.