Quantifying neutrophil phagocytes by droplet digital cell PCR for diagnosis of bacteremia

Despite extraordinary efforts to develop rapid molecular diagnostics for bacteremia, accurate and timely detection remains a challenge. While most molecular technologies are designed to detect bacteria circulating in the bloodstream, few technologies that detect bacteria engulfed by circulating neutrophils have been developed. Here, we describe a single-cell digital PCR assay to quantify circulating neutrophils harboring phagocytosed bacteria. We encapsulated a single neutrophil within a single droplet of a PCR reaction mix, and then performed in situ PCR to detect intra-neutrophil bacterial DNA. In this way, we successfully quantified neutrophils containing Staphylococcus aureus DNA, as well as human genes, at single-cell resolution. This technology, termed droplet digital cell PCR, enables rapid and accurate identification of bacteria directly from blood, thereby improving diagnosis and management of bacteremia. Despite efforts to develop rapid molecular diagnostics for bacteremia, accurate and timely detection remains a challenge. Here, a single-cell digital PCR assay quantifies circulating neutrophils harboring phagocytosed bacteria, enabling rapid and accurate identification of bacteria directly from blood.