Current methods for the diagnosis of sepsis have insufficient precision, causing regular misdiagnoses. Microbiological tests can help diagnose sepsis but are usually too slow to have an impact on timely clinical-decision making. Neutrophils have high sensitivity to infections, yet measurements of neutrophil surface markers, genomic changes, and phenotype alterations have had only a marginal effect on sepsis diagnosis. Here, we report a microfluidic assay that measures the spontaneous motility of neutrophils in the context of plasma, in one droplet of blood. We measured the performance of the assay in two independent cohorts of critically ill patients suspected of sepsis. In the first cohort, we developed a machine-learning-based scoring system (sepsis score) that segregated patients with sepsis from those without sepsis. In the second cohort, we validated the sepsis score in a double-blinded, prospective case-control study. For the 42 patients across the two cohorts, the assay identified sepsis patients with 97% sensitivity and 98% specificity. The neutrophil assay could potentially be used to accurately diagnose and monitor sepsis in larger populations of at-risk patients.

Polyhydroxyalkanoate (PHA) inclusions are polymeric storage inclusions formed in some bacterial species when carbon levels are high but levels of another essential nutrient, such as nitrogen, are low. Though much is known about PHA synthesis, little is known about inclusion structure. In this study, atomic force microscopy (AFM) was employed to elucidate the structure of PHA inclusions at the nanoscale level, including the characterization of different layers of structure. AFM data suggest that underneath the inclusion envelope, there is a 2-to 4-nm-thick network layer that resides on top of a harder layer that is likely to be a crystalline lamellar polymer. The network is comprised of ϳ20-nm-wide linear segments and junctions that are typically formed by the joining of three to four of the linear segments. In some cases, ϳ50-nm globular structures that are raised ϳ1 to 2 nm above the network are present at the junctions. These globular structures always have a central pore that is ϳ15 nm in diameter. To determine if the major surface protein of PHA inclusions, PhaP, is involved in the structure of this network, inclusions from Cupriavidus necator H16 ⌬phaP were examined. No network structure was detected. Instead, apparently random globular structures were found on the surfaces of the inclusions. When PhaP levels were reconstituted in this strain by the addition of phaP on a plasmid, the network was also reconstituted, albeit in a slightly different arrangement from that of the wild-type network. We conclude that PhaP participates in the formation of the inclusion network.

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