Abstract
Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.
Highlights
Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases
Strategies to improve sepsis and septic shock outcomes must include the rapid identification of infectious pathogens and their antibiotic-resistance patterns, which typically depends on culturing of relevant biological samples
By using droplet digital-PCR readout in the Proximity ligation assay (PLA) protocol, we enabled simultaneous measurement of GN- and GP-specific 16S rRNA genes, and the blaTEM gene together with IL-6 and TNF-α protein levels in the same patient sample
Summary
Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. In order to identify patients most likely to benefit from targeted interventions, new tools are needed that allow accurate characterization of the diverse pathogen and host factors that shape outcomes for patients with sepsis and septic shock. Pathogens, their toxins, and their nucleic acids can trigger the dysregulated host response of sepsis. Strategies to improve sepsis and septic shock outcomes must include the rapid identification of infectious pathogens and their antibiotic-resistance patterns, which typically depends on culturing of relevant biological samples. A major advantage of our digital amplification method is its ability to quantify very small changes in the concentration of these molecules. ddPCR has a resolution of a single-DNA molecule in samples[20], and we were able to achieve sub-femtomolar resolution for protein targets
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