The frequency of neonatal sepsis has remained constant at between 1 and 5 per 1,000 live births in the United States for several years, resulting in approximately 8,000 neonates with early-onset sepsis annually. Due to the devastating consequences of the failure to diagnose and treat a neonate with sepsis, many infants undergo an evaluation for sepsis and the unnecessary administration of empiric antibiotics. Up to 600,000 neonates annually undergo an evaluation for sepsis during their birth hospitalization, and the diagnosis of """"""""rule out sepsis"""""""" is the single most frequent discharge diagnosis from neonatal intensive care units. Traditionally, the diagnosis of suspected sepsis has been based upon clinical judgment, bacterial cultures, and adjunctive laboratory tests. Unfortunately, these adjunctive laboratory tests are insensitive, non-specific, and require 48 to 72 hours for the evaluation of bacterial cultures. Therefore, there is an urgent need for rapid and reliable tests for the diagnosis of neonatal sepsis. We hypothesize that there are neonatal serum proteins that are differentially expressed as a result of sepsis that could serve as diagnostic markers. Our proposed studies will employ state-of-the-art proteomics approaches to identify novel biomarkers of neonatal sepsis that can be used to develop a rapid point-of-service diagnostic test. To address our hypothesis, we propose the following specific aim:
Specific aim 1. Identification of proteins that are differentially expressed in the cord blood of infants with sepsis. Cord blood from cases with confirmed neonatal sepsis and gestational age and birth weight-matched controls will be analyzed by (a) surface-enhanced laser desorption-ionization/time-of-flight (SELDI-TOF) mass spectrometry, (b) two-dimensional gel electrophoresis, and (c) isotope-coded affinity tagging (ICAT) to detect differentially expressed proteins. Candidate biomarkers will be subsequently identified by tandem mass spectrometry, and microsequence data will be used for antibody generation. Characterized antibodies will then be employed to verify differential expression by western immunoblotting. The proposed studies will provide the background data and reagents that will enable the subsequent design and implementation of a commercial diagnostic assay that will fulfill a critical need in neonatal medicine. ? ? ?
