Prospective, multi-site study of patient outcomes after implementation of the TREWS machine learning-based early warning system for sepsis.

Early recognition and treatment of sepsis are linked to improved patient outcomes. Machine learning-based early warning systems may reduce the time to recognition, but few systems have undergone clinical evaluation. In this prospective, multi-site cohort study, we examined the association between patient outcomes and provider interaction with a deployed sepsis alert system called the Targeted Real-time Early Warning System (TREWS). During the study, 590,736 patients were monitored by TREWS across five hospitals. We focused our analysis on 6,877 patients with sepsis who were identified by the alert before initiation of antibiotic therapy. Adjusting for patient presentation and severity, patients in this group whose alert was confirmed by a provider within 3 h of the alert had a reduced in-hospital mortality rate (3.3%, confidence interval (CI) 1.7, 5.1%, adjusted absolute reduction, and 18.7%, CI 9.4, 27.0%, adjusted relative reduction), organ failure and length of stay compared with patients whose alert was not confirmed by a provider within 3 h. Improvements in mortality rate (4.5%, CI 0.8, 8.3%, adjusted absolute reduction) and organ failure were larger among those patients who were additionally flagged as high risk. Our findings indicate that early warning systems have the potential to identify sepsis patients early and improve patient outcomes and that sepsis patients who would benefit the most from early treatment can be identified and prioritized at the time of the alert.

Factors driving provider adoption of the TREWS machine learning-based early warning system and its effects on sepsis treatment timing.

Machine learning-based clinical decision support tools for sepsis create opportunities to identify at-risk patients and initiate treatments at early time points, which is critical for improving sepsis outcomes. In view of the increasing use of such systems, better understanding of how they are adopted and used by healthcare providers is needed. Here, we analyzed provider interactions with a sepsis early detection tool (Targeted Real-time Early Warning System), which was deployed at five hospitals over a 2-year period. Among 9,805 retrospectively identified sepsis cases, the early detection tool achieved high sensitivity (82% of sepsis cases were identified) and a high rate of adoption: 89% of all alerts by the system were evaluated by a physician or advanced practice provider and 38% of evaluated alerts were confirmed by a provider. Adjusting for patient presentation and severity, patients with sepsis whose alert was confirmed by a provider within 3 h had a 1.85-h (95% CI 1.66-2.00) reduction in median time to first antibiotic order compared to patients with sepsis whose alert was either dismissed, confirmed more than 3 h after the alert or never addressed in the system. Finally, we found that emergency department providers and providers who had previous interactions with an alert were more likely to interact with alerts, as well as to confirm alerts on retrospectively identified patients with sepsis. Beyond efforts to improve the performance of early warning systems, efforts to improve adoption are essential to their clinical impact and should focus on understanding providers' knowledge of, experience with and attitudes toward such systems.

How do nematodes transfer phosphorylcholine to carbohydrates?

An unusual aspect of the biology of nematodes is the attachment of phosphorylcholine (PC) to carbohydrate. The attachment appears to play an important role in nematode development and, in some parasitic species, in immunomodulation. This article considers the nature of the biosynthetic pathway of nematode PC-containing glycoconjugates and, in particular, the identity of the final component in the pathway - the enzyme that transfers PC to carbohydrate (the 'PC transferase'). We offer the opinion that the PC transferase could be a member of the fukutin family (fukutin refers to the mutated gene product that causes Fukuyama congenital muscular dystrophy), a group of enzymes with apparent phosphoryl-ligand transferase activity that are found in organisms ranging from bacteria to humans.

Gene inactivation confirms the identity of enzymes involved in nematode phosphorylcholine-N-glycan synthesis.

An unusual feature of nematodes is the covalent attachment of immunomodulatory phosphorylcholine (PC) moieties to N-type glycans. Our previous work on the filarial nematode glycoprotein ES-62 has enabled us to predict the identity of enzymes necessary for PC-N-glycan biosynthesis. Here, we addressed these predictions using gene knockout technology applied to C. elegans and present two pieces of confirmatory data. Employing a triple null mutant worm lacking all three genes that encode active UDP-N-acetyl-D-glucosamine: alpha-3-D-mannoside beta1, 2-N-acetylglucosaminyltransferase I (GnT I) we have confirmed our earlier prediction that a crucial step in the generation of the substrate for PC transfer is addition of terminal GlcNAc to the alpha1-3-linked mannose residue of the glycan by GnT I. Second, by silencing genes responsible for expressing enzymes of the Kennedy pathway of phosphatidylcholine biosynthesis by RNA interference (RNAi), we have confirmed our belief for a role for diacylglycerol: choline phosphotransferase (CPT) in PC-N-glycan biosynthesis.

Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62.

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.

In vivo exposure of murine dendritic cell and macrophage bone marrow progenitors to the phosphorylcholine-containing filarial nematode glycoprotein ES-62 polarizes their differentiation to an anti-inflammatory phenotype.

We have previously shown in an in vitro study that the filarial nematode phosphorylcholine (PC)-containing glycoprotein ES-62 promotes a murine dendritic cell (DC) phenotype that induces T helper type 2 (Th2) responses. We now show that, in addition to directly priming Th2 responses, ES-62 can act to dampen down the pro-inflammatory DC responses elicited by lipopolysaccharide. Furthermore, we also demonstrate that murine DCs and macrophages derived ex vivo from bone marrow cells exposed in vivo to ES-62 by release from osmotic pumps are hyporesponsive to subsequent stimulation with lipopolysaccharide. These effects can be largely mimicked by exposure to the PC moiety of ES-62 conjugated to an irrelevant protein. The data we provide are, as far as we aware, the first to show that a defined pathogen product can modulate the developmental pathway of bone marrow cells of the immune system in vivo. Such a finding could have important implications for the use of pathogen products or their derivatives for immunotherapy.

Hyporesponsiveness of murine B lymphocytes exposed to the filarial nematode secreted product ES-62 in vivo.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by filarial nematodes, parasites of vertebrates including humans. We have previously demonstrated that pre-exposure to this molecule in vitro interferes with subsequent B-cell receptor (BCR)-dependent activation of murine splenic B lymphocytes. To investigate the significance of this during filarial nematode infection, we now employ mice exposed to ES-62, at concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans, via release from implanted osmotic pumps. Using this approach, we reveal that splenic and lymph node mononuclear cells, and also purified splenic B cells recovered from these mice have reduced ability ex vivo to proliferate in response to BCR ligation. The effect on BCR-induced proliferation was further investigated with respect to elucidating the mechanism of action of the parasite product and was shown to be associated with impaired signal transduction affecting the ErkMAPkinase pathway. Also, it was found that ES-62 did not act by promoting apoptosis or by priming for apoptosis following subsequent stimulation, but rather, appeared to render cells hyporesponsive to stimulation. ES-62 is thus shown for the first time to be a potent modulator of B lymphocyte function in vivo at a concentration relevant to natural filarial nematode infection. This finding considerably strengthens the idea that ES-62 plays a role in evasion of the immune response during parasitism.

Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates.

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20-30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [3H]serine and [3H]S-adenosyl methionine (SAM). Incubation of worms with [3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [14C]choline.