Specific Aim 3: Biomarker development for early differentiation of sepsis and CAP pathogens Currently, initial antimicrobial treatment of sepsis and CAP is empiric. Common etiologic agents in sepsis are gram-positive bacteria (Staphylococcus spp. and Streptococcus spp.), gram-negative bacteria (e.g., Escherichia coli, Klebsiella spp., and Enterobacter spp.), and fungi (Candida spp.). Common etiologic agents in CAP are Streptococcus pneumoniae, Legionella pneumophila, Mycoplasma spp., and viruses. The ability to distinguish these pathogens at time of presentation of sepsis or CAP would potentially allow more targeted rather than broad-spectrum initial therapy. Earlier administration of appropriate antimicrobials would lower patient management cost associated with ineffective therapy and lessen likelihood of antibiotic resistance. We propose to identify host biomarkers for early differentiation of up to 4 common etiologic agents in sepsis and CAP. Our preliminary studies have established proof-of-concept for differentiation between classes of pathogens in sepsis based on specific differences in soluble host proteins in a blood sample. Based on our preliminary studies, it is anticipated that approximately 25% of patients in the Specific Aim 1 clinical study will have a positive blood culture. At least 33 of these patients are anticipated to have S. aureus bacteremia and 20 gram negative bacteremia. Specific Aim 3 proposes to compare plasma samples from patients with S. aureus and gram negative bacteremia in order to identify host biomarkers for early differentiation of specific class agent in sepsis. As in specific aims 1 and 2, bivariable and multivariable analyses of biomarkers is proposed to develop a biomarker panel for early differentiation of staphylococcal and gram-negative sepsis. Similar analysis is proposed to differentiate CAP pathogens. However, given the absence of a high-sensitivity, gold-standard method for determination of causal pathogen in CAP, Specific Aim 3 proposes the more conservative goal of differentiating pneumococcal CAP from atypical CAP based on quantitative differences in host blood biomarkers. The pneumococcal CAP group will be selected from the clinical studydataset based on rigorous criteria: S. pneumoniae from blood or sputum culture or detection of pneumococcal antigen in urine, clinical evidence of CAP and typical (lobar consolidation) chest radiograph. The nonpneumococcal CAP group will be determined by negative pneumococcal cultures and urine antigen, clinical evidence of CAP, and an atypical chest radiograph. It is anticipated that at least 20 patients (15% of the 133 with CAP) will have confirmed pneumococcal CAP and 40 patients (30%) atypical, non-pneumococcal CAP. Biomarkers for differentiation of i. S. aureus bacteremia from gram-negative bacteremia, and ii. pneumococcal CAP from atypical CAP, will be identified by mass spectrometry of patient EDTA plasma samples. Subject to availability of multiplexed immunoassays, some of these biomarkers will be replicated by immunoassay in the same samples. It should be noted that given budget-imposed reduction in patient enrollment of one third from that originally proposed, we are uncertain that sufficient patients will be enrolled for all Specific Aim 3 analyses to be meaningful. We propose to evaluate the group sizes of enrolled patients by specific class agent in order to select two specific comparisons between sepsis and CAP pathogens that are of sufficient size to permit meaningful analysis.