Development and characterization of a fecal-induced peritonitis model of murine sepsis: results from a multi-laboratory study and iterative modification of experimental conditions.

BACKGROUND: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2. RESULTS: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups. CONCLUSIONS: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies.

Sepsis: network pathophysiology and implications for early diagnosis.

Sepsis, a medical emergency, is the overwhelming host response to infection leading to organ failure. The pathophysiology of this heterogeneous disease includes an inflammatory response that stimulates a complex interaction between endothelial and complements with associated coagulation abnormalities. Despite a more comprehensive understanding of sepsis pathophysiology, there exists a translational gap to improve sepsis diagnosis clinically. Many of the proposed biomarkers to diagnose sepsis lack sufficient specificity and sensitivity to be used in routine clinical practice. There has also been a lack of progress in diagnostic tools due to the focus on the inflammatory pathway. Inflammation and coagulation are known to be linked to the innate immune response. Early immunothrombotic changes could result in the early switch from infection to sepsis and aid in sepsis diagnosis. This review integrates both preclinical and clinical studies that highlight sepsis pathophysiology providing a framework for how the development of immunothrombosis could be used as a starting point to investigate biomarkers for early sepsis diagnosis.

Microfluidic device for single step measurement of protein C in plasma samples for sepsis prognosis.

Protein C is a vitamin K dependant protein in plasma that plays an essential role in regulating the coagulation cascade and inflammatory response. As a result of its importance in these roles, it has been suggested as a biomarker for prognosis of patients affected by sepsis. Sepsis is a dysregulated host response to an infection that is the leading cause of mortality in U.S. hospitals and results in the highest cost of hospitalization. It was found that protein C concentration in non-surviving sepsis patients is significantly lower (1.8 µg mL-1) than in survivors and healthy patients who have a protein C concentration of 3.9-5.9 µg mL-1. Current methods for diagnosing sepsis rely on expensive immunoassays or functional assays that require multiple steps for isolation and activation of protein C. We demonstrate in this paper a low cost, single step assay for detection of protein C in blood plasma. This was done by combining isoelectric gates with barium-immobilized metal affinity trapping. The electric field was optimized for use with immobilized metal affinity using COMSOL simulation. The integrated device was tested with samples containing buffered protein C, protein C in the presence of high concentration bovine serum albumin and alpha 1-proteinase inhibitor, and in blood plasma with spiked protein C. The stability of the measured values was tested by monitoring the intensity of a mixture of protein C with BSA and A1PI every minute to determine that measurement after 40 minutes was optimal. The results showed that the device could be used to distinguish a reduction in protein C from 4.46 µg mL-1 to 1.96 µg mL-1 with greater than 98% confidence in plasma making it suitable for sepsis prognosis.

Single-step measurement of cell-free DNA for sepsis prognosis using a thread-based microfluidic device.

Cell-free DNA (cfDNA) content in plasma has been studied as a biomarker for sepsis. Recent publications show that the cfDNA content in sepsis patients entering intensive care unit who were likely to survive had a total cfDNA concentration of 1.16 ± 0.13 µg/mL compared to 4.65 ± 0.48 µg/mL of non-survivors. Current methods for measuring cfDNA content in plasma were designed to amplify and measure low concentrations of specific DNA, making them unsuitable for low-cost measurement of total cfDNA content in plasma. Here, we have developed a point of care (POC) device that uses a thread silicone device as a medium to store a fluorescent dye which eliminates the need for preparatory steps, external aliquoting and dispensing of reagents, preconcentration, and external mixing while reducing the detection cost. The device was paired with a portable imaging system with an excitation filter at 472 ± 10 nm and an emission filter of 520 ± 10 nm that can be operated with just 100 mA current supply. The device was demonstrated for use in the quantification of buffered cfDNA samples in a range 1-6 µg/mL with a sensitivity of 5.72 AU/µg/mL and with cfDNA spiked in plasma with a range of 1-3 µg/mL and a sensitivity of 5.43 AU/µg/mL. The results showed that the device could be used as a low-cost, rapid, and portable POC device for differentiating between survivors and non-survivors of sepsis within 20 min.

Diagnostic Potential of Coagulation-Related Biomarkers for Sepsis in the Emergency Department: Protocol for a Pilot Observational Cohort Study.

BACKGROUND: Between 75% and 80% of patients with sepsis arrive in the hospital through the emergency department. Early diagnosis is important to alter patient prognosis, but currently, there is no reliable biomarker. The innate immune response links inflammation and coagulation. Several coagulation -related biomarkers are associated with poor prognosis in the ICU. The role of coagulation biomarkers to aid in early sepsis diagnosis has not previously been investigated. The objective of our study is to determine the individual or combined accuracy of coagulation and inflammation biomarkers with standard biochemical tests to diagnose adult septic patients presenting to the emergency department. METHODS: in the Emergency Department is a prospective, observational cohort study with a target enrolment of 250 suspected septic patients from two Canadian emergency departments. The emergency physicians will enroll patients with suspected sepsis. Blood samples will be collected at two time points (initial presentation and 4 hr following). Patients will be adjudicated into septic, infected, or not infected status in accordance with the Sepsis-3 definitions. Patient demographics, cultures, diagnosis, and biomarkers will be reported using descriptive statistics. Optimal cut off values with sensitivity and specificity for each biomarker will be determined using C-statistics to distinguish between septic and nonseptic patients. Stepwise multiple logistic regression analysis with exclusion of nonsignificant covariates from the final model will be used to establish a panel of biomarkers. CONCLUSIONS: Our protocol describes the processes and methods for a pragmatic observational biomarker study in the emergency department. This study will seek to determine the potential diagnostic importance of early coagulation abnormalities to identify additional tools for sepsis diagnosis.

National Preclinical Sepsis Platform: developing a framework for accelerating innovation in Canadian sepsis research.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies-defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis-are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical  research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.