Sepsis 2016 Paris

P17 Evaluation of a novel molecular host response assay to diagnose infection in patients after high-risk gastro-intestinal surgery: a pilot study P18 Validation of sepsis-3 diagnostic criteria in intensive care unit patients


Background
Sepsis affects over 26 million people worldwide each year resulting in a death every 3 to 4 seconds [1]. For every hour that antibiotics are delayed after the first episode of hypotension, there is a 7.6 % increase in the risk of mortality [2]. Thus, international sepsis guidelines recommend the administration of broad spectrum antimicrobial therapy within 1 hour of recognition [3]. In 2011, the New South Wales Clinical Excellence Commission (CEC) developed the Sepsis Kills program including the Adult Emergency Sepsis Pathway [4]. This pathway was introduced to Emergency Departments (ED) and auditing of time to first antibiotics commenced. Belmont Hospital Emergency Department has approximately 25000 presentations per year. In 2012, 163 patients were diagnosed with sepsis. Time to first antibiotics for sepsis patients peaked at 254 minutes ( Fig. 1). Discussion of these results highlighted the need to develop education strategies in order to reduce time to first administration of antibiotics.

Materials and methods
Audits of the CEC Sepsis database [5] over 4 years included a total of 769 patients. Collected data entered included: age, triage time and category, clinical observations, time and amount of intravenous fluid, time to first antibiotics, and diagnosis. The tables provided reflect median time to antibiotics. In 2012 a Clinical Nurse Specialist from the ED was designated Sepsis Lead. Over 4 years, the Sepsis Lead worked collaboratively with clinical staff to develop and implement several strategies to decrease time to first antibiotics Results A multimodal approach strategy was adopted, which included: regular audits; targeted education programs for triage nurses and nursing team leaders; the introduction of Sepsis September -a month dedicated to sepsis awareness and education; and the Sepsis Road show. These interventions were well received by Emergency staff. As a direct result time to first antibiotics was reduced to a median of 41 minutes (Fig. 2).

Conclusions
The collaborative effort between the Sepsis Lead and clinical staff has produced a significant reduction in time to first antibiotics from 254 minutes to 41 minutes at last audit. The success achieved at Belmont Hospital due to this multimodal approach strategy has the potential to be translated globally. It also serves to highlight the importance of the Emergency Nurse in early recognition and initiation of treatment for sepsis.

Background
Currently there is little data on identification, management and outcomes of patients with sepsis in developing countries. In low income countries major concerns regarding accessibility to healthcare, limitations due to costs, lack of resources and delayed presentations of patients with sepsis make implementing protocols, based on developed countries patients profile difficult. Thus in Sub-Saharan Africa there has been a widespread shift towards protocol development that is cost effective and specific for the epidemiologic and ecologic data.

Materials and methods
The aim of our study is to assess the efficacy of clinicians at a district level hospital in the Western Cape at identifying and managing sepsis. Furthermore, we will assess the outcome of patients in terms of in-hospital mortality and length of hospital stay given the above management. A retrospective study design was applied when analyzing data from the routine burden of disease audit done on a 3 monthly basis at Karl Bremer Hospital.

Results
The total sample size obtained was 70 patients. A total of 18/70 (26 %) patients had an initial triage blood pressure indicative of sepsis induced hypotension however only 1/18 (5.5 %) of these patients received an initial crystalloid fluid bolus of 30 ml/kg. The median time for antibiotic administration in septic shock was 4.65 hours. Further a positive delay in antibiotic administration (p value = 0.0039) was demonstrated. A total of 7/70 patients received no antibiotics in the first 24 hours of hospital admission. Of these 7, 3 of these patients could be classified as septic shock at presentation. All 3 patients died within 36 hours of hospital admission. The data showed 8/12 (66 %) of patients with septic shock received inappropriate amounts of fluids. The in-hospital mortality for sepsis was found to be 4/24 (17 %), for severe sepsis 11/34 (32 %) and a staggering 9/12(75 %) for septic shock. A positive association between in-hospital mortality and the following was found: Time to first dose antibiotic administration (OR = 1.07, P value = 0.027, 95%CI = 1.008-1.14). For every 1-hour delay in antibiotic administration the chance of death increased by 7 %. Source appropriate antibiotics (OR = 0.17, P value = 0.005, 95 % CI = 0.048-0.59). The chance of death amongst patients that received source appropriate antibiotics is 83 % less than those who did not. Early appropriate intravenous fluid administration (OR = 0.33, P value = 0.040,95 % CI = 0.11-0.95). Appropriate intravenous fluids was associated with a 67 % reduction in in-hospital mortality.

Conclusion
The outcomes of the study concluded that there is room for improvement by our clinicians with regards to appropriate identification and management of patients with sepsis. Flawed management has inevitably shown to have an impact on in-hospital mortality. Simple, cost effective measures that can implemented regardless of resources include early appropriate antibiotics and early aggressive fluid therapy; with the potential for an inordinate impact on mortality.  PC pulmonary complications, ARF acute respiratory failure, PSI pneumonia severity index, r correlation coefficient, P Pvalue at two sided significance level  Fig. 3. The standard mortality ratios of observed mortality to predicted mortality were between 0.81 and 1.1. The SSS presented good discrimination with an area under the receiver operating characteristic curve (AUC) of 0.892. The performances of each severity scoring system are shown in Table 2.
The discriminative performance of the SSS was not statistically significant from the APACHE II (P = 0.07), SAPS II (P = 0.06) or SAPS 3 (P = 0.11). However, the APACHE IV score showed the best discrimination with an AUC of 0.948 and the overall performance by a Brier score of 0.096. The AUC of the APACHE IV score was statistically greater than the SSS (P < 0.0001), APACHE II (P < 0.0001), APACHE III (P = 0.0002), SAPS II (P < 0.0001) and SAPS 3 (P < 0.0001) (Fig. 4). The calibration of all scores was poor (Hosmer-Lemeshow goodness-of-fit H test <0.05). The calibration curves for the SSS and APACHE scores are presented in Fig. 5.

Conclusions
The SSS provided good discrimination as well as the APACHE II, SAPS II and SAPS 3 scores. However, the APACHE IV score had the best discrimination and overall performance in our severe sepsis and septic shock patients. The SSS needs to be adapted and modified with new parameters to improve the performance.   Background Sepsis in a public health problem and septic patients suffer high mortality [1,2]. Few data are available about physicians' knowledge and capacity to manage sepsis outside the Intensive Care Unit (ICU) [3]. The physicians' knowledge about sepsis could explain discrepancies in management between Emergency Department (ED) and ward septic patients, who suffer a poorer quality of care and a poorer outcome [4]. We aimed to assess knowledge and ability to handle sepsis management through a questionnaire.

Materials and methods
Observational, prospective, monocentric study during 6 months (May to December 2015). A 26 questions sheet was distributed to all physicians. Questionnaires were anonymous, included age, experience and specialty. Questions involved definitions, epidemiology, outcome, diagnosis and management of severe sepsis/septic shock. Eight questions out of 25 were core questions, and a 26th evaluated physicians' self-appreciation of ability. Evaluation included levels of good, wrong answers (WA) or "no knowledge" (NK) with specialties and selfestimated ability. Physicians were compared as fellows or senior, and specialties between "referent" (ED or Anesthesiologists/Intensivists vs others The induction of inflammatory responses by lipopolysaccharides (LPSs, endotoxins) is achieved by the coordinate and sequential action of four principal LPS-binding proteins: LPS-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and Myeloid differentiation protein -MD-2 [1]. CD14, the cell surface receptor of monocytes and neutrophils, is required for LPS activation of phospholipases, protein tyrosine kinases, protein kinases A and C as well as MAP kinases [2]. TLR4 independently of CD14 cannot mobilize all of the adapter proteins that it requires for full signaling activity [3]. CD14 functional multiplicity provides the reason for their implication in target therapy of inflammatory response to LPS by development of blocking monoclonal antibodies (mAbs) specific to human CD14 [4] or TLR4 [5]. In the present study we have examined CD14 involvement in endotoxin priming of human  polymorphonuclear leukocytes (PML) for respiratory burst triggered by N-formyl-methionyl-leucyl-phenylalanine (fMLP).

Materials and methods
Human PML were isolated from heparinized blood of healthy volunteers by standard procedure and incubated with or without anti-CD14 mAbs UCHM-1 or isotype-matched IgG2a for 30 min before priming by LPS. Control cells and cells that had been preexposed to mAbs or IgG2a were placed into chemiluminometer chambers containing solution for luminol-enhanced chemiluminescence (CL) measurement supplemented with autologous serum (2 %) and 0.01 mM CaCl2. PML priming was achieved by addition of 100 ng/ml S-or Re-LPS from Escherichia coli O55:B5 or JM103 followed by continuous gentle shaking for 30 min at 37°C. CL had been triggered by adding of 1 μM fMLP and the light emission was recorded continuously for 20 min. Statistical significance was determined by applying the Wilcoxon's signed-rank date analysis. Differences were considered to be significant when p < 0.05.

Results
LPS is known to be engaged by CD14 and then transferred to TLR4 [2,6,7]. To assess CD14 involvement in endotoxin priming of human PML for fMLP-triggered respiratory burst we used the full anti-CD14 mAbs containing Fc-regions. We have therefore examined the effect of anti-CD14 mAbs as well as IgG2a on fMLP-triggered respiratory burst of control LPS-unprimed PML. As described in Fig. 6a incubation of PML with anti-CD14 mAbs decreased the level of fMLPtriggered CL caused by reactive oxygen species (ROS) production (p < 0.05). The same pattern was recorded when isotype-matched IgG2a had been employed (p < 0.05) (Fig. 6a). It is not yet known whether this inhibitory effect of the IgG2a's Fc-regions on fMLPtriggered ROS generation will be still maintained during PML priming by endotoxins. When PML had been exposed to anti-CD14 mAbs as well as to IgG2a and then primed by Re-LPS near the same suppression of fMLP-triggered CL was detected (p < 0.05) (Fig. 6b) (Fig. 6b). Since "quenching" effects of Fc-regions of full anti-CD14 mAbs as well as isotype-matched IgG2a on fMLP-triggered respiratory burst have been almost identical it is not possible at the moment to make decision what is the real impact of F(ab')2regions of used anti-CD14 mAbs in PML priming by LPS. Some indication on the impact of F(ab')2-regions in this process may came from the comparison of anti-CD14 mAbs effect on PML priming by different LPS-glycoforms, namely S-or Re-LPS. As follows from Fig. 7a and b the most pronounced inhibitory effect of anti-CD14 mAbs on PML priming by S-LPS from E. coli has been achieved (by 40 %). Especially role of CD14 in delivery of S-LPS to TLR4 is well described [8].
Conclusions LPS is known to be engaged by CD14 and then transferred to TLR4 [2,6,7]. To assess CD14 involvement in endotoxin priming of human PML for fMLP-triggered respiratory burst we used the full anti-CD14 mAbs containing Fc-regions. We have therefore examined the effect of anti-CD14 mAbs as well as IgG2a on fMLP-triggered respiratory burst of control LPS-unprimed PML. As described in Fig. 6a incubation of PML with anti-CD14 mAbs decreased the level of fMLPtriggered CL caused by reactive oxygen species (ROS) production (p < 0.05). The same pattern was recorded when isotype-matched IgG2a had been employed (p < 0.05) (Fig. 6a). It is not yet known whether this inhibitory effect of the IgG2a's Fc-regions on fMLP-triggered ROS generation will be still maintained during PML priming by endotoxins. When PML had been exposed to anti-CD14 mAbs as well as to IgG2a and then primed by Re-LPS near the same suppression of fMLP-triggered CL was detected (p < 0.05) (Fig. 6b) (Fig. 6b). Since "quenching" effects of Fc-regions of full anti-CD14 mAbs as well as isotype-matched IgG2a on fMLP-triggered respiratory burst have been almost identical it is not possible at the moment to make decision what is the real impact of F(ab')2-regions of used anti-CD14 mAbs in PML priming by LPS. Some indication on the impact of F(ab')2-regions in this process may came from the comparison of anti-CD14 mAbs effect on PML priming by different LPS-glycoforms, namely S-or Re-LPS. As follows from Fig. 7a and b the most pronounced inhibitory effect of anti-CD14 mAbs on PML priming by S-LPS from E. coli has been achieved (by 40 %). Especially role of CD14 in delivery of S-LPS to TLR4 is well described [8].

Background
Bacterial pneumonia is a life threatening condition causing acute lung injury (ALI) leading to development of sepsis depending on the invasiveness of the bacteria and host immune status. This present study was designed to find out the protective immunomodulatory action of macrolide antibiotic (i.e. clarithromycin) in mouse model of pneumonia induced by K. pneumoniae B5055.

Materials and Methods
Pneumonia was induced by intranasal instillation of Klebsiella pneumoniae B5055 (10 4 cfu/ml) by holding the mice in upright position without any anaesthesia. Experimental animals were divided in two groups (i.e. Group A; control (oral saline), Group B; clarithromycin (30 mg/kg/day/os). At designated period of time (1, 2, 3, 5 and 7 days) mice were euthanized by cervical dislocation and lungs were harvested and homogenized in 1 ml of sterile normal saline under sterile conditions. The lung homogenate was used for measuring bacterial load, malondialdehyde (MDA), nitric oxide (NO) and myeloperoxidase (MPO) and pro-inflammatory (i.e. IL-1α, IL-6, TNF-α) antiinflammatory (i.e. IL-10) cytokine levels in lungs. Histopathological examination of the lungs was also carried out along with lung alveolar macrophage function (i.e. macrophage spreading and phagocytosis) assays.

Results
Clarithromycin treatment led to the significant (p < 0.05) reduction in pulmonary neutrophil infiltration along with significant (p < 0.05) decrease in other markers of inflammation (TNF-α, IL-α, IL-6, MDA, NO and MPO) associated with acute tissue injury or inflammation. The increase in bacterial clearance in lungs of mice treated with clarithromycin was associated with significant (p < 0.05) increase in macrophage spreading as well as their phagocytic potential (i.e. phagocytic uptake and intracellular killing) of alveolar macrophages.

Conclusions
Clarithromycin has a great potential as an immunomodulatory antibiotic during K. pneumoniae B5055 induced pneumonia associated acute lung inflammation or injury (ALI).

P9
Histone levels determined by an in-house immunoglobulin Y assay are associated to clinical outcomes in sepsis Jerico R. Background Experimental models have shown that histones are involved in the progression of sepsis and may be candidate prognostic biomarkers. There are limited prospective cohort studies of suspected septic patients aimed at determining the risk associated with increased level of histones to their progression to severe sepsis. To address this, we (1) developed an IgYbased immunoassay that can detect and quantify blood histone levels; (2) tested whether there is a difference in histone levels among septic and febrile patients that present in the emergency room; and (3) tested whether there is an association between histone levels in serum and the progression of patient to developing hypotension (sBP < 90 mmHg), azotemia, (creatinine > 2.0 mg/dL (176.8 μmol/L)) or thrombocytopenia (platelet count < 100,000/μL) using the developed IgY-based immunoassay.

Materials and methods
An indirect ELISA was developed for detecting human histones that used polyclonal immunoglobulin Y (IgY) extracted from eggs laid by calf histone-challenged chickens as a primary antibody. Chickens were inoculated with calf histones and the IgY from eggs were extracted by means of a delipidation, salt precipitation and affinity chromatography.
To determine its clinical utility as a prognostic kit, a prospective cohort study of 4 febrile patients and 21 septic patients seeking consult at the emergency room were enrolled based on the inclusion criteria. Higher total histone levels measured by immunoglobulin Y was then determined if it was related to the development of hypotension, azotemia or thrombocytopenia.

Results
The limit of detection of this IgY-based immunoassay was determined to be 2036 pg/mL. Comparison of histone levels between 4 febrile patients, and 21 septic patients within 3 days of admission in the hospital showed that septic patients have significantly higher (p = 0.0314) levels of histones as compared to febrile patients (Fig. 8).
In this cohort, those that developed hypotension, azotemia or thrombocytopenia during their hospital stay have higher levels of histones upon admission (p = 0.0257) compared to those that did not (Fig. 9). Receiver operator curve analysis showed that at 2101 pg/uL (Fig. 10 Background Acute lung injury (ALI) is an important cause of morbidity and mortality in critically ill patients. Although the condition has many etiologies, pneumonia and sepsis are leading triggers. Age is a major determinant of clinical outcome, and there are age-dependent alterations in the responses to pulmonary challenge that can trigger kidney inflammation (a precursor of chronic kidney disease) and higher mortality rates.
Staphylococcus aureus infection is a frequent cause of pneumonia, especially in elderly patients. To develop a better understanding of the pathophysiology, and assist in defining future targets, we developed a lung model of staphylococcal pneumonia in rodents. Using this model we have examined inflammatory responses, hemodynamics, and cardiac proteasome activation, neutrophil trafficking in pulmonary inflammation, and age-related changes [1][2][3]. Here we examined renal inflammation following pulmonary challenge.

Materials and methods
C57Bl6 Mice (8-10wks, n = 6-8/group) were challenged intratracheally with staphylococcal cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN): (0.2 μg + 0.66 μg); (30 mg + 100 mg); (150 mg + 500 mg); or saline alone. Pulse oximetry was performed on awake animals before challenge and immediately prior to euthanasia. At 6, 24 or 72 hrs post challenge, animals were euthanized, bronchoalveolar lavage (BAL) performed, and tissues harvested. Concentrations of plasma and BAL cytokines were assessed by ELISA. The left kidney was fixed for histological analysis, and the right kidney was assessed for cytokine mRNA by qPCR, cytokine protein using ELISA, and activity of specific proteasome subunits by ProCISE [4].

Results
BAL from LTA/PGN challenged mice showed significant increases in neutrophils, total protein, IL-6, KC, MCP-1, TNFa and MIF as in previous studies. Additionally, while there was no LTA or PGN detected in the plasma, LTA/PGN challenged mice had increased plasma concentrations of IL-6, IL-1b, KC and TNFa. In renal tissue, there were time and concentration dependent changes in IL1b, IL-6, TNFa, IP10, and MCP-1 and an increase in proteasome b5 subunit activity.

Conclusions
Even in these young animals pulmonary challenge induced significant inflammatory changes within the lung, blood and kidney. Notably, in the kidney there was increased activity of proteasome b5, a regulator of IκB/NFκB activation and signaling pathways. Our recent study uncovered a time dependent, NF-kB switch that regulates transition from endothelial barrier injury to repair [5]. If we can define changes in the injury-to-repair phase in the kidney, we may reveal specific targets for therapeutic manipulation in a clinically relevant time frame to improve outcome from ALI associated renal involvement, particularly in the older individual.

Background
Accumulating evidence suggests that the intestinal barrier function is impaired during systemic inflammation as in sepsis. Animal models of sepsis revealed several ileal pathological changes. These include epithelial apoptosis, disruption of tight junctions and increased intestinal permeability [1,2]. The impaired gut barrier function may increase the risk of bacterial translocation from the gut lumen to the bloodstream, aggravating systemic inflammation. The molecular mechanism associated with this phenotype remains largely unknown. Cathelicidin represents one of the most important classes of antimicrobial peptides in mammals. In addition to bactericidal property, this peptide inhibits endotoxininduced pyroptosis of leukocytes, suppresses the release of inflammatory mediators and protects endothelial cells from apoptosis [3][4][5]. In this study, we aimed to investigate the role of murine cathelicidin-related antimicrobial peptide (mCRAM), a rodent antimicrobial peptide analogous to human LL-37, in maintaining gut barrier function in sepsis. Materials and methods 129X1/SvJ mice that were wild type (cnlp+/+) or deficient for cathelicidin (cnlp−/−) were used. Polymicrobial sepsis was induced by cecal-ligation and puncture (CLP) [6]. Animals were divided into four groups: cathelicidin wild type and knockout mice receiving either CLP or sham surgery. At 24 h, blood and ileal tissues were harvested for total bacterial DNA quantification, immunoblotting and histological staining. The survival rates and septic severity were recorded every 12 hours until seven days after the surgery. Sepsis morbidity was evaluated by Murine Sepsis Severity (MSS) score [7]. At 20 h after CLP or sham surgery, the animals were orally fed with 4 kDa fluorescein-dextran. Three hours later, serum was harvested and fluorescence was measured.

Results
Knocking out cnlp gene has significantly increased septic severity and reduced 7-day survival of 129X1/SvJ mice. Real time PCR targeting bacterial 16S rDNA revealed that total bacterial DNA load increased more than threefold (Fig. 11). Without cathelicidin, the expression of mucus has also reduced considerably, as revealed by Alcian blue periodic acid Schiff reaction and RT-PCR targeting MUC1, MUC2, MUC3 and MUC4 genes (Fig. 12). Immunoblotting revealed cleavage of caspase 3 and PARP involving in apoptosis (Fig. 13). Consistently, confocal microscopy using TUNEL staining identified greater number of puncta per villus. Deletion of cnlp gene increased ileal permeability to 4kD Fluorescein-labelled dextran, accompanied with reduced expression of tight junction proteins claudin-1 and occludin (Fig. 14).

Conclusions
This study demonstrated the critical role of cathelicidin in maintaining gut barrier integrity in polymicrobial sepsis. This may partially explain the association between lower LL-37 and higher mortality in septic patients [8].   To date, there is no standard diagnostic test to identify septic patients, although early identification is necessary to improve their outcome [1]. We sought to identify novel biomarkers by studying the enzymatic activity of the proline-specific peptidases dipeptidyl peptidase 4 (DPP4), prolyl carboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) in the plasma of 32 controls and 40 patients with septic shock on days 1, 3, 5 and 7.

Materials and methods
Enzymatic activity measurements were done using in-house developed specific assays [2][3][4]. On the same days, inflammatory (e.g. TNF-α, IL-6) , hemodynamic (e.g. mean arterial pressure, pulse) and metabolic parameters (e.g. bilirubin, lactate) were measured as well. The data was used to generate receiver operating characteristic (ROC) curves and to identify associations. A survival analysis (up to 90 days) was also performed.

Results
Significant differences between the control group and the patients with septic shock could be found for all four enzymes. PREP and PRCP showed a higher enzymatic activity on day 1 compared to the controls (p ≤ 0.05 and p ≤ 0.01, respectively The systemic inflammation represents a primary injury to the intestinal epithelium structure. In acute phase, the massive production of inflammatory mediators (cytokines and nitric oxide) contributes to the intestinal permeability, bacterial translocation and tight junction rearrangement. During the past decades, the clinical administration of glucocorticoids has been widely discussed and the Surviving Sepsis Campaign's recommendation is the low-dose treatment for those  The protein level of occludin and claudin-1 was determined by immunoblotting. Error bars denote standard error of the mean patients refractory to vasopressor therapy. Our aim was to investigate the possible role of dexamethasone on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction. Furthermore, we evaluated its ability to modulate the inflammatory response and also the expression of proteins of the intestinal tight junctions.

Materials and methods
To conduct these experiments, male rats had their jugular vein cannulated for endotoxin administration, one day before the experiment. Rats were pre-treated with dexamethasone (synthetic glucocorticoid; 0.1 or 1 mg/kg, intraperitoneal) before LPS administration (1.5 mg/kg, intravenous). At 6 h after endotoxemia induction, the intestinal permeability was evaluated by injecting FITC-dextran 4 kDa in the ileum, mesenteric lymph nodes were collected for microbiological analysis and also cytokines were quantified in the plasma and intestinal mucosa by ELISA technique. Additionally, the integrity of the tight junctions was determined by transmission electron microscopy, histological analysis, the expression of tight junction proteins (occludin, claudin-1, claudin-2, junctional adhesion molecule-A) as well as their localization.

Results
Our results demonstrated that dexamethasone administration reduces the LPS-induced permeability in the ileum and prevented the bacterial translocation to the mesenteric lymph nodes. The plasma and mucosa concentrations of TNF-α, IL-1β, IL-6, IL-10 and IFN-γ were significantly reduced in dexamethasone-treated rats. Furthermore, treatment with dexamethasone reverted the LPS-induced epithelial barrier dysfunction, increasing the expression of occludin and claudin-1, reducing the claudin-2 cleavage and also the localization of these proteins at the apical portion. Moreover, the histological damages and the morphology of the tight junctions were preserved by the dexamethasone administration, therefore reducing their opening induced by endotoxemia.

Conclusions
Together these results suggest a protective role for dexamethasone preventing the intestinal barrier dysfunction induced by systemic inflammation, possibly modulating the inflammatory response. Furthermore, these experimental findings reinforce the Surviving Sepsis Campaign's recommendation for the administration of low glucocorticoid doses in septic shock patients.

Background
Shakoory and colleagues performed a post hoc analysis of a randomized clinical trial of anakinra, an interleukin-1 receptor antagonist [1], and reported that severe sepsis patients with features of macrophage activation syndrome (MAS), defined by the combined presence of hepatobiliary dysfunction and disseminated intravascular coagulation, represented 5.6 % of the severe sepsis population. Additionally, anakinra, in comparison with placebo, reduced 28-day mortality (34.6 % vs 64.7 %; p < 0.05) in a subset of patients with features of MAS [1]. Based on these findings, we hypothesized that the Protocolized Care for Early Septic Shock (ProCESS) trial [2] would contain a similar proportion of septic patients with features of MAS and a similar mortality rate. We further hypothesized that MAS patients would have elevated circulating biomarkers related to macrophage activation, including ferritin, interleukin-18 (IL-18), interleukin-1β (IL-1β), and interferonγ (IFN-γ).

Materials and methods
We selected all patients with features of MAS and age-matched controls without features of MAS from the ProCESS trial. Concentrations of circulating ferritin, IL-18, IL-1β, and IFN-γ were measured in plasma samples collected on day 1, and mortality was assessed at hospital discharge and at 90 days. The MAS patients were further stratified according to their circulating ferritin levels, and a threshold of 1200 ng/ml was established based on the upper limit of the 95 % confidence interval for the mean ferritin concentration in MAS survivors.

Conclusions
Post hoc analysis of ProCESS patients illustrated that features of MAS are relatively uncommon but lethal in severe sepsis. Septic patients with features of MAS and ferritin levels greater than 1200 ng/ml had the highest mortality and increased IL-18, IL-1β, and IFN-γ concentrations compared to non-MAS septic patients, making this an attractive subset to target in future anti-inflammatory agent trials.

Conflicts of interest
The authors declare that they do not have a conflict of interest related to the subject matter. Background Patients after esophagectomy are prone to develop sepsis due to pneumonia, anastomotic leakage, or wound infection. A new diagnostic test (SeptiCyte LAB) that measures the expression of four blood gene biomarkers has diagnostic utility in distinguishing sepsis from SIRS [1]. However, test performance in the immediate postoperative setting has not been investigated.

Materials and methods
We studied patients undergoing esophagectomy in two Dutch university hospitals from 2011 to 2013 who had developed a complication requiring ICU (re)admission within 30 days. Blood was collected postoperatively and when a complication occurred. After RNA extraction, samples were analyzed on an Applied Biosystems® 7500 fast Dx Real-Time PCR instrument. SeptiCyte LAB results (a score, range 0-10) were categorized into four probability bands according to the manufacturer's specification.

Results
Among 384 patients 113 (29 %) subjects had developed at least one complication and were thus eligible for study inclusion; paired samples (i.e., both a direct postoperative and follow-up sample) were available for 71 (63 %) of these. Sample preparation or processing issues resulted in exclusion of 4 pairs, leaving 67 (59 %) patients for analysis (Table 5). Five (7 %) patients had active infection during postoperative sampling, and 55 (82 %) on the day of their first complication. SeptiCyte results were highly variable immediately after surgery and at follow-up (Fig. 15)

Conclusion
SeptiCyte LAB is a novel sepsis biomarker which may have clinical utility in monitoring patients at high risk for postoperative infections, but its diagnostic value in this setting has to be further evaluated.

Conflicts of interest
The authors declare that they do not have a conflict of interest related to the subject matter.

Background
The Third International Consensus definitions for sepsis aimed to improve the discrimination between sepsis and uncomplicated infections [1]. Our goal was to assess the construct and criterion validity of the new definitions in a cohort of ICU patients with infection.

Methods
We analyzed ICU patients with suspected infection who were enrolled as part of a prospective cohort study in the Netherlands between June 2011 and April 2015 [2]. Infections were classified as either present at admission or newly-acquired in the ICU (onset >48 hours). Sepsis and septic shock were defined according to both old and updated criteria [1,3]. The apparent incidences of sepsis and septic shock according to both definitions, and associated hospital mortality rates were compared. In addition, we explored minor variations in the timing of diagnostic criteria relative to infection onset, and inclusion of lactate for the definition of septic shock. Sepsis status was added to a baseline logistic model (including age, gender, race and chronic comorbidity) to evaluate the value of the sepsis-3 criteria in predicting mortality.

Conclusion
Agreement between new and old sepsis definitions was higher in infections at admission than in ICU-acquired infections. Mortality was similar using the various sepsis definitions. Sepsis-3 had no clinical relevant additive value in predicting mortality infections at admission.    The likelihood of infection was based on post hoc physician assessment using validated definitions [2].

Introduction
Metabolites of aromatic amino acids -phenylcarboxylic acids (PhCAs) -significantly increased in critically ill patients with infection, especially with sepsis [1] and associated with mortality [2,3]. They have demonstrated good predictive ability in patients with different critical illness [3,4]. Unfortunately, accurate prediction does not save the patient's life. We need better criteria for evaluating of treatment efficiency in patients with infection. This work is our first step in the development of laboratory monitoring based on PhCA clearance.

Methods
High-risk patients with median age 60 (IR 53-74) years with acute abdomen (n = 45, male 51 %) were admitted to the ICU immediately after emergency surgery. Acute abdomen development was associated with intestinal perforation (n = 26) and/or acute intestinal obstruction (n = 19). Serum samples were collected on the first and the third days of treatment in the ICU. The total level (C) of three PhCAs (phenyllactic, phydroxyphenyllactic and p-hydroxyphenylacetic acids) was measured using previously described gas chromatography methodology [5]. Lactate concentration (C) and SOFA scores were registered. PhCA and lactate clearance was calculated as followed: [(C first -C third )/C first ]x100%. The efficiency of treatment was judged based on the ICU outcome. Statistical processing was performed using IBM SPSS Statistics 22 program.

Results
The results were divided into two sub-groups and were presented as medians with 25-75 % interquartile range depending on the outcome (Table 6)  The cholesteryl-ester transfer protein isoform exclusively expressed in the small intestine (CETPI) and present in human plasma, although discovered by our group a few years ago, it had been waiting for a functional description and a plausible role of physiological relevance [1]. Unlike the cholesteryl-ester transfer protein (CETP), CETPI does not present exon 16 and 54 bases contained in intron 15 form part of the new mRNA replacing the 24 C-terminus residues present in CETP with a sequence of 18 residues containing a high concentration of prolines and positively charged amino acids [1]. Now, the present study introduces CETPI as a new protein with the potential capability to recognize, bind and neutralize lipopolysaccharides (LPS) in  circulation during septic shock. Employing several peptides derived from the C-terminus domain of CETPI we demonstrate their property not only to interact with several LPS serotypes but also to displace LPS bound to the surface of cells [2].

Results
Peptide VSAK derived from the last 18 residues of CETPI protects against the cytotoxic effect of LPS upon macrophages, hepatocytes and microglial cells, does not show cytotoxicity by itself, and prevents against the expression of pro-inflammatory cytokines and the generation of oxidative stress [2]. For many years, the phenomenon of recognition of bacterial LPS as components of the cell membrane of Gram-negative bacteria carried out by the innate immune system was thought to involve simple pathways if compared to the acquired immune system. Nowadays this view has dramatically changed since complex pathways involving multiple receptors and protein-protein interactions have been described. Nevertheless, despite the efforts made to counteract what it is known as the inflammatory disequilibrium syndrome and overall the outcome of septic shock, current therapeutic possibilities mainly involving inflammation and metabolic control and antibiotic treatment, have not changed for many years. Therefore, since CETPI efficiently binds LPS in vitro and it is overexpressed in small intestine cell cultures and proved to be present in human plasma, the efficacy of the administration of peptide VSAK against LPS cytotoxicity was studied in vivo. Employing a septic shock model in rabbits, we demonstrate that the infusion of peptide VSAK presents the ability to protect against the deleterious effects of LPS and the property of reducing the presence of TNFα in plasma [2,3]. Conclusions CETPI presents itself as a new protein that undoubtedly will advance the possibilities to better understand and treat the dangerous effects of LPS present during the treatment of sepsis caused by Gramnegative bacteria and its common consequence, a septic shock condition.

P21
Highly multiplexed molecular pathogen ID followed by phenotypic AST from whole blood using a novel fully automated system Jonas Jarvius 1  To address the need of more rapid diagnosis of potential pathogens causing sepsis or septic shock in patients, Q-linea is developing a fully automated, high-throughput diagnostic platform, ASTrID®.The system will perform both molecular pathogen identification and phenotypic antibiotic susceptibility testing (AST), directly from whole blood, delivering ID after four hours and AST in additional six. The pathogen panel will cover 95 % of relevant pathogens including 33 unique pathogens and 10 groups, as well as 11 resistance markers. The panel of antibiotic substances will contain 30 antibiotics and bacterial growth or inhibition reported as Minimum Inhibitory Concentration values.

Materials/Methods
In an ongoing study clinical blood samples are collected from patients suspected of having sepsis attending the Infectious Disease ER, University hospital of Örebro, Sweden. In addition to the standard 4 blood culture flasks drawn per patient for routine diagnostics, an extra, 5-10 ml, blood sample were taken for the present study. Pathogen ID and AST analysis was performed in a prototype ASTrID system, starting with target identification through a proprietary nucleic acid amplification reaction. Highly specific and selective padlock probes forming circularized DNA strands [1,2] are amplified via rolling-circle amplification (RCA) [3] and subsequent circle-to-circle amplification (C2CA) [4]. The resulting RCA products are labelled with fluorescence and detected on a microarray. The proprietary growth-based AST analysis was performed on a subset of clinical isolates spiked in blood (inoculum consistent with 10 CFU/ml blood).

Results
Of the clinical samples collected, 32 samples identified as positive in normal clinical routine diagnostics were selected and are presented in this study. Pathogen identification direct from patient in ASTrID achieved sensitivity of 96 % and specificity of 100 %, compared to conventional analysis on positive blood culture flasks. When compared with broth microdilution, the ASTrID AST results showed 99 % essential agreement and 99 % categorical agreement. Overall, the results were highly correlated with traditional technologies and EUCAST guidelines.

Conclusion
With ASTrID, both pathogen identification and antibiotic susceptibility testing can be done within 10 hours from blood draw. This dramatically shortens the time to adjusted treatment. The molecular pathogen identification method allows high multiplex, high sensitivity testing. The phenotypic antibiotic susceptibility profiling method developed reports reliable data directly from patient samples, without the need to wait for positive blood cultures. The new ASTrID platform from Q-linea has the potential to become a crucial tool against the global challenge of antibiotics resistance, helping to save lives and reduce healthcare costs. Background Obligate anaerobic bacteria (OAB) are the dominant microflora of the human body. OAB are often the primary cause of the disease and can also become aggravating factors. OAB cause extensive tissue destruction and toxin production that result in multiorgan failure, thus OAB-associated pathology is usually very difficult to treat. Mixed behavior of anaerobic-aerobic microflora hampers the choice of antimicrobials, often OAB are antibiotic-resistant. To date reliable biomarkers for prognostic evaluation of septic critical patients, are still needed. A firm pathogenetic and mechanistic association between a laboratory parameter/mediator and a clinical phenotype is a necessary assumption to adopt such parameter/mediator as a clinical biomarker [1]. CARS phase of sepsis has aroused an increasing interest among clinicians. In our previous studies, we demonstrated that sCD25 and IL-10 are reliable sepsis markers [2]. Recently presepsin (soluble CD14 subtype) has been shown to increase in plasma of septic critical subjects [3]. IL-35, a well known immunoregulatory cytokine, has been reported among novel sepsis biomarkers [4]. Aim of this study was the assessment of IL-35 and presepsin as prognostic sepsis markers in critical patients.

Materials and methods
For this observational prospective study, were sequentially enrolled critical patients admitted to the Unit of Intensive Care (ICU) of the University Hospital of Catanzaro Italy. For such patients admission SOFA scores were calculated. Based on 28 days survival, subjects were stratified in survivors (n = 32) and nonsurvivors (n = 17), while a group of healthy volunteers were also included as negative controls (n = 10). Moreover, clinical and microbiological data including blood culture were recorded. Plasma and serum specimens were collected at the patient admission; samples were tested for presepsin (PATHFAST Presepsin assay), IL-35 (Bio-Ocean LLC) and procalcitonin (VIDAS B.R.A.H.M.S PCT, bioMérieux, Italy). Data were subjected to statistical analysis by ANOVA plus post-hoc PLSD Fisher's test. Selected data were processed by ROC analysis.

Results
At the admission SOFA scores were found significantly higher (p < 0.001) in nonsurvivor vs. survivor patients. Admission levels of presepsin were significantly more elevated (p < 0.05) (Fig. 17), in nonsurvivors vs. survivors at the same time. Presepsin concentrations were found significantly increased (p < 0.05), in blood culture-positive in comparison to culturenegative patients at the same time. PCT admission levels did not exhibit significantly different values between nonsurvivor patients and survivor subjects. However, culture-positive patients showed PCT levels at admission significantly increased vs. culture-negative (p < 0.05). Presepsin admission data from dead/alive individuals were subjected to ROC analysis, which revealed a very high accuracy and significant AUROCC (p < 0.0001).
Concentrations of IL-35 were found significantly (p < 0.05) higher in nonsurvivors as well as survivors, when compared with healthy subjects.

Conclusions
Presepsin may substantially improve PCT contribution to clinical decisions in the patients evaluated. IL-35 appeared a promising future sepsis biomarker, which deserves further assessment. However present data indicated presepsin as a dependable prognostic tool with an elevated clinical value.

Background
Previous studies have shown that during sepsis, brain oxidative stress and damage are induced by overproduction of reactive oxygen species (ROS). Although there are recent reports about the benefits of statins in experimental sepsis and endotoxemia in peripheral organs, little is known about its effect in the central nervous system. Our objective was to investigate the antioxidant properties of simvastatin and its possible neuroprotective effect during experimental sepsis.

Materials and methods
Male Wistar rats (250-300 g) were submitted to cecal ligation and puncture (CLP, n = 34) or remained as non-manipulated control (or naive, n = . Both groups were treated by gavage with simvastatin (20 mg/kg) or an equivalent volume of saline. The animals submitted to CLP were treated 4 days before and 48 h after surgery. One animal group was decapitated and blood and brain were collected to quantify plasma levels of cytokines and assess astrogliosis and apoptosis in the prefrontal cortex and hippocampus. Another group was perfused with PBS (0,01 M), and the same brain structures were dissected to analyze oxidative damage.

Conclusions
The results suggest a neuroprotective effect of simvastatin in structures responsible for the spatial learning and memory. Behavioral studies are needed to evaluate its impact on cognitive damage triggered by experimental sepsis encephalopathy.
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