Ultrasensitive detection of six sepsis-associated proteins in neonatal saliva

Sepsis is a life-threatening condition that affects millions of newborns every year. Current diagnostic practices require painful, often repetitive, blood collections to isolate and culture the causative microorganism. Definitive return of results can take up to 5 days, exposing newborns to potentially unnecessary antibiotics. Developing a more rapid, non-invasive assessment of infectious status based upon host immune response provides an alternative diagnostic approach to infection screening. However, additional blood collection for multiplex quantification of inflammatory biomarkers in neonates remains prohibitive. Alternatively, saliva may serve as an informative biofluid, though detection of biologically relevant proteins in saliva remains challenging, with analyte concentrations 100 to 1000 times lower compared to blood. To address this challenge, we developed a panel of six ultra-sensitive single-molecule array (Simoa) assays for inflammatory biomarkers known to be associated with neonatal sepsis: serum amyloid A1 (SAA1), lipopolysaccharide-binding protein (LBP), chemokines CCL20, CXCL6, CXCL12; and the adipokine resistin. When these assays were tested on 40 neonatal salivary samples, we found that CXCL6 and CCL20 were significantly elevated in infected and septic neonates compared to those uninfected. The small volumes of fluid used (~10 μL saliva) and limited concentrations of these analytes in saliva (pg/mL) underscore the importance of ultra-sensitive measurements in the development of non-invasive diagnostics and reinforces the value of neonatal saliva as a viable sample matrix for monitoring infection. We hypothesize these assays could be useful in future diagnostic tests to discriminate between infected and uninfected neonates.