Transthoracic echocardiographic assessment of IVC diameter variability to determine fluid responsiveness in children with septic shock: a pilot study

OBJECTIVE
Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-α (mediating anti-inflammatory effects) and glucocorticoid receptor-β (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T-cells. We hypothesized that 1) changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and 2) glucocorticoid receptor-α expression is controlled by microRNA-mediated gene silencing.


DESIGN
Laboratory-based study.


SETTING
University research laboratory.


SUBJECTS AND PATIENTS
Healthy volunteers, sepsis patients.


METHODS
First, T-cells from healthy volunteers (native and CD3/CD28-stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor-isoforms by quantitative polymerase chain reaction. Additionally, effects of gene silencing of glucocorticoid receptor-β by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslated-region reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α expression were analyzed in Jurkat T-cells and in T-cells from healthy volunteers (quantitative polymerase chain reaction and Western blotting). Finally, expression of glucocorticoid receptor-α, glucocorticoid receptor-β, and miR-124 was tested in T-cells of sepsis patients (n=24).


MEASUREMENTS AND MAIN RESULTS
Stimulation of T-cells induced a significant upregulation of glucocorticoid receptor-α (not glucocorticoid receptor-β) thereby possibly rendering T-cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-β doubled the inhibitory effects of glucocorticoids on interleukin-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced three-fold upregulation of microRNA-124 was found. T-cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-β expression was two-fold upregulated (p<.01) and exhibited a remarkable interindividual variability.


CONCLUSIONS
Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-α thereby limiting anti-inflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-β levels.


P3
Thalidomide modulates macrophage-mediated inflammatory innate immune response during Klebsiella pneumoniae B5055 infection in BALB/c mice V Kumar * , S Chhibber Panjab University, Chandigarh, India Critical Care 2012, 16(Suppl 3):P3 Background: Lung innate immune response plays an important role in the clearance of pathogens (that is, bacteria, virus or fungi) from lungs. However, profound activation of innate immune cells (alveolar macrophages or neutrophils) can lead to development of acute lung inflammation or injury observed during pneumonia, acute respiratory distress syndrome (ARDS), or sepsis by producing various proinflammatory cytokines (that is, IL-1α, TNFα) and molecules (that is, hydrogen peroxide (H 2 O 2 )). Thalidomide is a drug, which has its own place in the history of medicine and has been used in various types of cancers and other chronic inflammatory disorders (erythema nodosum leprosum) but its mode of action as an immunomodulatory drug in acute infections (that is, pneumonia and sepsis) is not clear. Thus, the present study was designed to investigate its effect on pulmonary innate immune response during acute lung infection in BALB/c mice. Methods: Animals were divided into four groups (n = 20 for each group). Acute lung inflammation was induced by intranasal instillation of Klebsiella pneumoniae B5055 into mice without any anesthesia and treated with thalidomide (30 mg/kg/day/p.o.) or normal saline orally using a treatment schedule shown to modulate proinflammatory innate immune response. Various proinflammatory (IL-1α, TNFα) as well as anti-inflammatory (that is, IL-10) cytokines were estimated by ELISA. Pulmonary macrophagemediated phagocytosis and bactericidal assays were performed. Alveolar macrophages were evaluated in terms of macrophage spreading ability assay. H 2 O 2 production by macrophages was assessed according to the method based on the process of horseradish peroxidase-dependent oxidation of phenol red assay. Neutrophil infiltration to the lungs was determined by histopathological analysis. Results: Thalidomide treatment modulated proinflammatory function of alveolar macrophages by significantly decreasing their phagocytic potential in terms of phagocytic uptake and intracellular killing, spreading and H 2 O 2 release. Besides that, thalidomide treatment also significantly decreased neutrophil infiltration into the lung alveoli. Remarkably, the levels of proinflammatory cytokines (IL-1α and TNFα) were found to be decreased significantly in the thalidomide-treated group but the levels of IL-10 were found to be significantly elevated. Conclusion: Thalidomide proved a promising immunomodulatory agent with a potential to modulate aggravated innate immune response observed during acute lung inflammation associated with pneumonia or sepsis caused by Gram-negative bacterial infection. Background: The literature data demonstrate that the initial stage of abdominal sepsis (AS) is characterized with typical immune shifts as white blood cell (WBC) activation, decrease of T-lymphocyte and B-lymphocyte function followed by dysimmunoglobulinemia. As AS progresses immunity deficiency acquires severe combined character. Impact of immunity deficiency on a course is not described. Methods: We investigated 33 patients (male:female = 25:8) with abdominal sepsis (total peritonitis following appendicitis, perforated duodenal ulcer, pancreonecrosis). Anti-endotoxin (AE) antibodies (anti-LPS-IgA, anti-LPS-IgM, anti-LPS-IgG) were determined by original modification of hard-phase immunoenzyme analysis. Escherichia coli K30 LPS was used as antigen for AE antibody detection. All data were compared with healthy donors (99 patients). As a method of immunity correction we selected Sandoglobulin (Novartis, Switzerland), introduced as 3 ml once intravenously. Results: According to results of immunity parameter investigation on the day of admission we shared all patients into two groups: with high initial immunity (n = 6, no evidence from normal parameters) and low initial immunity (n = 27, evident decrease of AE immunity parameters). All patients needed comprehensive medication support. The patients with high immunity did not need any immunotherapy. But the patients with low initial immunity required immune therapy to avoid severe course of peritonitis and unfavorable outcomes. Single introduction of Sandoglobulin on the fifth day after surgery was accompanied with increase of immunity indices (Table 1). Simultaneously with an increase of anti-LPS-immunoglobulin titer one noticed sharp positive clinical changes (improvement of patients' self-feeling, body temperature decrease, decrease/normalization of WBC number, neutrophil shift). We have the following data about routine laboratory parameters in abdominal sepsis patients with Sandoglobulin introduction: WBC, ×10 9 /l, 16.5 ± 0.9 before introducing and 9.8 ± 1.2 in 2 days after introducing; stab neutrophils, %, 18.3 ± 1.6 and 10.8 ± 1.4 appropriately; juvenile neutrophils, %, 4.1 ± 0.8 and 0.3 ± 0.1 appropriately (all changes are evidently proved, P < 0.001). Successful surgical treatment and immunotherapy of abdominal sepsis are accompanied by activation of AE immunity, all-class anti-LPS-immunoglobulin concentration growth that blocks mechanisms of further inflammation progress and result in rapid patients' recovery. Conclusion: Peritonitis patients with initial low immunity need passive nonspecific immunotherapy that stimulates organism protective functions and promotes rapid recovery. Data presented as mean (SD). MMP-9, matrix metalloproteinase-9; Ang-2, angiopiotein-2; vWF, von Willebrand factor. P value by Mann-Witney test. ALI/ARDS patients were significantly different compared with healthy controls. a P < 0.001.

Table 1(abstract P4) Dynamics of AE antibodies in abdominal sepsis patients with Sandoglobulin introduction
Patients with low immunity level (n = 27) Before introduction (optic units) 2 days after introduction (optic units)

P5
Procalcitonin level as a marker of severe sepsis and septic shock patients who required polymyxin-B immobilized fiber with direct hemoperfusion T Ikeda * , K Ikeda, S Suda Tokyo Medical University Hachioji Medical Center, Tokyo, Japan Critical Care 2012, 16(Suppl 3):P5 Background: When septic patients progress to endotoxin shock, the mortality rate becomes high and is within 30 to 80% in those with multiple organ failure. Methods: We evaluated the levels of procalcitonin (PCT) and markers of sepsis (IL-6, IL-8, IL-1ra and PAI-1) in 159 patients who had severe sepsis and septic shock. Before starting the polymyxin-B immobilized fiber with direct hemoperfusion (PMX-DHP) treatment, patients were divided into three groups according to their PCT levels (L group: 0.5 < PCT ≦ 2.0 ng/ml, M group: 2.0 < PCT ≦ 10.0 g/ml, H group: PCT > 10.0 ng/ml).
Results: Sixty-two percent of the patients showed high PCT levels (>10.0 ng/ml). The APACHE II score and Sequential Organ Failure Assessment score tended to be high in the H group, but there was no significant difference among the groups. The survival rate declined with high PCT levels. The levels of inflammatory (IL-6 and IL-8) and antiinflammatory (IL-1ra) cytokines tended to be high in the H group, but there was no statistically significant difference among the groups. On the other hand, the PAI-1 level was significantly increased in the H group (498 ± 499 ng/ml) compared with the L group (157 ± 103 ng/ml) and M group (311 ± 319 ng/ml). Conclusion: Sixty-two per cent of patients who required PMX-DHP treatment had high PCT levels. PCT might be a mediator of sepsis and sepsis markers. PCT can potentially affect sepsis-related markers. Background: Sepsis is a common cause of morbidity and mortality in critically ill patients. Microbiological culture is the gold standard for diagnosis of sepsis but unfortunately culture results are positive in only 30 to 50% patients. Sepsis is also difficult to distinguish from systemic inflammatory response syndrome (SIRS) because of similar clinical presentations. Procalcitonin (PCT) and IL-6 are known biochemical markers for diagnosis and prognosis of sepsis. The aim of this study was to evaluate the diagnostic role of PCT and IL-6 in differentiating sepsis (both culture positive and culture negative) from SIRS. Methods: The study comprised three patient groups, age >18 years: group 1 (n = 41), proven infection; group 2 (n = 29), clinically suspected infection but negative culture; group 3 (n = 29), patients with SIRS. Blood was collected at the time of admission for microbiological culture and estimation of PCT (TRACE, Kryptor) and IL-6 (CLIA, Access). Results: The median PCT level was significantly higher (P < 0.001) in both groups 1 and 2 as compared with group 3, whereas the median IL-6 level was significantly high (P < 0.001) only in group 1 as compared with group 3. Receiver operating characteristic (ROC) curve analysis between groups 1 and 3 for prediction of systemic infection demonstrates that both PCT and IL-6 have a significant area under the curve (AUC) of 0.923 (P < 0.001) and 0.824 (P < 0.001) respectively. The best cutoff point of PCT was at 1.48 ng/ml with 85% sensitivity, 83% specificity, 88% positive predictive value (PPV) and 80% negative predictive value (NPV). However, the best cutoff point for IL-6 was very high at 219 pg/ml, with 78% sensitivity, 76% specificity, 82% PPV and 71% NPV. Similarly, ROC curve analysis between groups 2 and 3 demonstrates that PCT has a significant AUC of 0.848 (P = 0.001), whereas the AUC of IL-6 is 0.555, which is not significant (P = 0.47). The best cutoff point for PCT was at 1.45 ng/ml with 83% sensitivity, 79% specificity, 80% PPV and 82% NPV, whereas the best cutoff point of IL-6 was 98.25 pg/ml, with only 59% sensitivity, 55% specificity, 57% PPV and 57% NPV.
Conclusion: In differentiating SIRS from sepsis, IL-6 does not have a diagnostic role in culture negative sepsis patients, whereas PCT showed a better accuracy in differentiating SIRS from both proven and suspected sepsis. Hence, inclusion of PCT in the initial diagnostic strategy may aid in early and appropriate therapeutic intervention in culture-negative sepsis patients.

P7
Evaluation of a soluble CD14 subtype in patients with surgical sepsis Y Fukui Kochi Health Sciences Center, Kochi, Japan Critical Care 2012, 16(Suppl 3):P7 Background: Surgical sepsis remains a cause of poor outcome. In order to treat surgical sepsis appropriately, it is important to reach diagnosis of sepsis early during the course and start antibiotic treatment. The gold standard of sepsis diagnosis is blood culture but there are issues with its sensitivity along with contamination. Soluble CD14 subtype (sCD14st) is an N-terminal fragment of CD14, and signal messenger of lipopolysaccharide (LPS). sCD14st is reported to increase in sepsis patients. In this study, we evaluated the specificity, clinical effectiveness of soluble sCD14st in patients with surgical sepsis. Methods: The study population was 18 operated patients in Kochi Health Sciences Center between April 2010 and March 2011. We investigated sCD14st using the compact automated enzyme immunoanalyzer PATHFAST (Mitsubishi Chemical Medience Co., Japan) and other biomarkers (procalcitonin (PCT), CRP, IL-6) at surgery (day 0) followed by 1, 3, 5, 7 days. APACHE II and Sequential Organ Failure Assessment (SOFA) were used to assess the sepsis severity. Sepsis was diagnosed according to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) criteria.
Results: There were nine patients with acute abdomen, esophageal cancer in five patients, cerebral bleeding in two patients, and a patient with Fournier's gangrene and trauma, respectively. The area under the curves (AUC) of receiver operating characteristic (ROC) curve analysis for septic infection were 0.92 for sCD14st, 0.82 for PCT, 074 for IL-6, and 0.82 for CRP. Two esophageal cancer patients who indicated over 800pg/ml in sCD14st had complicated nosocomial pneumonia. sCD14st also positively correlated with SOFA sore (r = 0.41) and APACHE II score (r = 0.28).
Conclusion: sCD14st was a useful biomarker for the diagnosis of surgical sepsis and reflected clinical severity after operation.
infections [2,3]. However, pre-analytical and analytical issues inherent to mHLA-DR measurement by flow cytometry limit the use of this marker in large multicentric clinical studies and on a routine basis. We investigated whether the whole blood mRNA expression of genes related to major histocompatibility class II (MHC class II) antigens could correlate with mHLA-DR protein expression measured by flow cytometry and predict mortality in septic shock patients. Methods: Ninety-three septic shock patients were included. PAXgene® tubes were collected at days 3 to 4 after the onset of shock. The mRNA expression of five MHC class II-related genes (that is, CD74, HLA-DRA, HLA-DMB, HLA-DMA and CIITA) was measured by qRT-PCR. In parallel, ethylene diamine tetraacetic acid-anticoagulated blood samples were collected to measure mHLA-DR expression by flow cytometry. Results: A significant correlation (r > 0.8) was found between the mRNA expression of MHC class II related genes. As observed for mHLA-DR, the mRNA levels were significantly decreased in nonsurvivors as compared with survivors. The best predictive value was obtained for the invariant chain (CD74) mRNA level (P = 0.003). This was confirmed by the receiver operating characteristic curve analysis (AUC = 0.7, 95% CI = 0.574 to 0.822; P = 0.003). Importantly, this remained significant after multivariate analysis including usual confounders (severity scores) and mHLA-DR values (P = 0.016). Survival curves showed that the decreased CD74 mRNA level was associated with increased mortality after septic shock ( Figure 1). Conclusion: Decreased invariant chain mRNA expression significantly predicts 28-day mortality in septic shock patients. After validation in a larger multicentric study, this biomarker could become a robust predictor of mortality in septic patients. This is all the more because the availability in routine laboratories of molecular biology platforms will enable standardized and routine use of such biomarkers. Background: In 2008, Kaiser Permanente Northern California (KPNC), which provides care to 3.3 million members in 21 hospitals, implemented an initiative to improve sepsis care, a critical step to reduce hospital mortality. The goals of the program were threefold: improve identification of sepsis patients, appropriately stratify risk, and reliably provide treatment, focusing on spread and sustainability across all medical centers. Methods: In spring 2008, all hospitals reviewed the last 50 deaths and sepsis was identified as a significant improvement opportunity. In May 2008, two hospitals began rapid cycle pilot testing, resulting in the development of a playbook containing treatment algorithms, standardized order sets and flow charts, and chart abstraction tools. These tools, along with expectations for implementation, were shared with leaders and champions from all 21 hospitals at the November 2008 Sepsis Summit. The summit closed with a young mother sharing the story of how her life was saved as a result of the work at the pilot hospital. Subsequently, all hospitals convened multidisciplinary sepsis teams and began training and tool adoption, focusing immediately on improving sepsis identification. Regional mentors and medical center improvement advisors supported team-building and rapid implementation; timely and actionable data allowed ongoing identification of improvement opportunities. Identification and performance monitoring were supported by the development of a web-based tool that pulled information directly from the electronic medical record.  Figure 1 and Table 1.

Conclusion:
The KPNC program is unique in its rapid rate of improvement in sepsis measures, adoption of a single standard of care across an entire 21-hospital system, sustainability well beyond the rapid adoption period, and the quantification of mortality risk beyond the shock population to the intermediate sepsis population. These results demonstrate that a strong performance improvement engine can drive large-scale, sustained improvements in care within a short duration.

P13
Dynamics of lymphocyte subpopulations during Legionnaires' disease CPC de Jager 1* , EFA Gemen 1 , J de Jongh-Leuvenink 1 , IBB Walsh 1 , RJF Laheij 1 , T van der Poll 2 , PC Wever 1 1 Jeroen Bosch Hospital, ś-Hertogenbosch, the Netherlands; 2 Academic Medical Center, Center of Infection and Immunity Amsterdam and Center of Experimental and Molecular Medicine, Amsterdam, the Netherlands Critical Care 2012, 16(Suppl 3):P13 Background: Absolute lymphocytopenia (lymphocyte count <1.0 × 10 9 /l) is recognized as an important hallmark of the immune response to severe infection and observed in patients with Legionnaires' disease (LD). Furthermore, LD is characterized by accumulation of activated T cells in the lungs. To explore the immune response in patients with LD, we studied the dynamics of peripheral blood lymphocyte subpopulations in the acute and subacute phase of the disease. Methods: EDTA-anticoagulated blood was obtained from eight LD patients on the day the diagnosis was made (acute phase) through detection of Legionella pneumophila serogroup 1 antigen in urine. A second blood Figure 1(abstract P11) Kaplan-Meier analysis of septic shock patients' 28-day survival after stratification on CD74 mRNA expression. The threshold was chosen based on the Youden index calculated on the receiver operating characteristic curve. There is a significant difference between the two curves (log-rank test, P < 0.001; hazard ratio = 7.065, 95% CI = 2.56 to 19.48).
Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 Figure 1(abstract P12) Sepsis EGDT mortality rate compared with overall bundle compliance. Kaiser Permanente Northern California Hospitals. As bundle performance passed 30%, a sharp decline in mortality was observed for septic shock patients who qualify for EGDT. sample was obtained in the subacute phase. Multiparametric flow cytometry was used to calculate absolute lymphocyte counts and B-cell, T-cell, NK-cell, CD4 + and CD8 + T-cell counts. Expression of activation markers was analyzed on CD4 + and CD8 + T cells. C-reactive protein (CRP) levels were used to monitor treatment response.
Conclusion: The acute phase of LD is characterized by absolute lymphocytopenia which recovers in the subacute phase with an increase in absolute T-cell count and emergence of activated and memory-type CD4 + and CD8 + T cells. This confirms a role for T-cell activation in the immune response to LD.

P14
Receptor for advanced glycation endproducts controls deleterious lung inflammation in severe Pseudomonas aeruginosa pneumonia in immunosuppressed mice C Ravinet-Trillou 1* , F Soubrier 2 , A Caron 2 , S Llopart 2 , M Fontanie 1 , F Lantreibecq 2 , PE Rouby 1 , C Menet 1 , L Leotard 1 , C Serradeil-Le Gal 1 1 Sanofi, TSU ID, Toulouse, France; 2 Sanofi, SCP Biologics, Toulouse, France Critical Care 2012, 16(Suppl 3):P14 Background: The receptor for advanced glycation endproducts (RAGE/ AGER) plays a key role in infections by sustaining tissue-damaging inflammation. This multiligand/multisignal pattern recognition receptor, overexpressed in lung and on innate immune cells, was shown to contribute to the pathogenesis of pneumococcal pneumonia and thus may be an attractive therapeutic target to treat severe pneumonia, one of the most common causes of death in western countries. Here, we characterized a new anti-human RAGE antibody (SAR) and its in vivo effect in a model of severe Gram-negative bacterial pneumonia in immunosuppressed mice. Methods: The SAR antibody was previously selected for binding affinity for the human sRAGE (Biacore) and for RAGE ligand binding inhibition (S100B, HMGB1, S100A6) by ELISA. A similar IC 50 was found for SAR and anti-RAGE XT-M4 antibody (Pfizer/Wyeth) with HMGB1. Since SAR did not cross-react with mouse RAGE, transgenic C57Bl6 mice expressing human RAGE were engineered for in vivo testing. Mice, receiving at day 4 and day 1 cyclophosphamide for immunosuppression, were challenged with intranasal 10 7 or 10 8 colony forming units (CFU) Pseudomonas aeruginosa (PA01 strain) and treated 4 hours after with ciprofloxacin alone or in the presence of the selected RAGE antibody: survival, CFU and bronchoalveolar lavage (BAL) cytokines were measured. The anti-RAGE XT-M4 antibody was used as a reference. Results: In human RAGE KO/KI humanized mice, hRAGE expression was validated at the mRNA (RT PCR) and protein (IHC) levels. In the pneumonia model, higher loads of P. aeruginosa was used in transgenic versus wild-type mice to achieve more than 80% of mortality 24 hours after infection (10 8 CFU). XT-M4 antibody was injected at 15 mg/kg intravenously 4 hours after infection (alone or with ciprofloxacin, 0.1 and 0.3 mg/kg, intraperitoneal given 4 hours and 24 hours after infection). The association of XT-M4 and ciprofloxacin significantly increased survival compared with the antibiotic alone (ciprofloxacin 0.1/XT-M4 +54%, P < 0.066, ciprofloxacin 0.3/XT-M4 +78%, P < 0.01). The SAR antibody (7.5 and 15 mg/kg) also induced significant improvement in delaying mortality in transgenic mice when given in combination with ciprofloxacin versus antibiotic alone. Moreover, 30 mg/kg SAR with ciprofloxacin (10 mg/kg) reduced (but not significantly) BAL cytokine release (TNFα, KC, IL-12) 18 hours after infection. Conclusion: A survival benefit and partial control of inflammation was observed with anti-RAGE antibodies in combination with antimicrobial agent in a model of Gram-negative (P. aeruginosa) pneumonia in immunosuppressed mice. Thus, together with existing literature reports, these data are in favor of the contribution of RAGE to the pathogenesis of severe bacterial pneumonia.

P15
Low-tidal volume ventilation as compared with conventional tidal volume ventilation in patients of sepsis: a randomized controlled trial A Waheed 1* , S Bhat 2 , V Nabi 1 , S Gurcu 1  Background: In a surgical ICU, low-tidal volume ventilation was compared with conventional tidal volume ventilation. Low-tidal volume ventilation is preferentially used to provide mechanical ventilation in support of patients with acute lung injury, acute respiratory distress syndrome, and inhalation injury. However, few prospective studies have compared low-tidal volume ventilation with normal tidal volume ventilation in sepsis. The purpose of this study was to prospectively compare the two ventilator modalities in an ICU setting in patients of sepsis. Methods: A single-center, prospective, randomized controlled trial, comparing conventional tidal volume (12 ml/kg) ventilation with low-tidal volume ventilation (6 ml/kg) in patients with sepsis. In an ICU at a tertiary care teaching hospital, adult patients ≥18 years of age with documented sepsis requiring prolonged (>24 hours) mechanical ventilation were admitted to the ICU. Subjects were randomly assigned to receive mechanical ventilation either by conventional tidal volume ventilation (n = 40) or a low-tidal volume ventilation-based strategy (n = 40). The first primary outcome was death before a patient was shifted out of the ICU or breathing without assistance. The second primary outcome was the number of days without ventilator use from day 1 to day 28. Results: At baseline, both the traditional ventilation group and the low-tidal volume ventilation group had similar demographics to include median age (interquartile range) (35 years (18 to 65) vs. 33 years (18 to 65), P = non significant), weight (64 (50 to 82) versus 62 (53 to 70)). The primary outcome was ventilator-free days in the first 28 days after randomization. Our study revealed no significant difference between the low-tidal volume ventilation and the traditional strategy ventilation groups in mean (± SD) ventilator-free days (12 ± 9 vs. 11 ± 8, P = non significant) respectively), although the number of days without ventilator use during the first 28 days after randomization was greater in the low-tidal volume group. Mortality was lower in the group treated with lower tidal volumes than in the group treated with traditional tidal volumes (27.0% vs. 30.8%, P = non significant); again the difference was not statistically significant. Conclusion: Our study revealed that in patients with sepsis, mechanical ventilation with a lower tidal volume may be beneficial in decreasing mortality and increasing the number of ventilator-free days although the difference was found to be statistically insignificant. Background: Rivers' randomized clinical trial showed that early goaldirected therapy improves patient outcome in severe sepsis and septic shock [1]. The findings were confirmed by multicenter international prospective observational studies [2,3]. In our institution, we implemented a Quality Improvement Project based on daily auditing of patients admitted to a medical intensive care unit (MICU) for severe sepsis/septic shock followed by weekly feedback on compliance of the Institute for Healthcare Improvement (IHI) resuscitation bundle, and Sepsis Response Team (SRT) activation [4]. Once the Quality Improvement Project was completed, the daily audit and weekly feedback were stopped. This study aims to assess the impact of this change on the process of care and hospital mortality. Methods: The study was conducted in a 24-bed adult MICU of an academic medical center. Only the first admission of each patient was included. Patients who did not authorize their medical records to be reviewed for research were excluded. During the first period of the study, 2009, daily auditing, weekly feedback to healthcare providers and SRT were implemented. During the second study period, 2010, the daily auditing and weekly feedback were stopped and the SRT activation was continued. Baseline data collected during the two periods of the study included gender, age, and resuscitation preference in case of cardiac arrest. Process of care was measured using the seven elements of the IHI resuscitation bundle. Patient outcome was measured by hospital mortality. Statistical comparisons between the two groups were made using Student's t and chi-square tests. Two-tailed P < 0.05 was considered statistically significant. Results: A total of 777 patients were included in the study: 378 had auditing and feedback and 399 did not. The mean (SD) age of the feedback group was 65. 4 [1]. We aimed to assess the ability of the RIFLE criteria to predict mortality in critically ill patients admitted to a medical ICU.
Methods: A retrospective cohort study in an eight-bed medical ICU of a tertiary care hospital over a period of 16 months. Data regarding patient demographics and ICU course including need for organ support and length of stay were recorded. We classified each patient according to their RIFLE class using admission creatinine values (no AKI: <1.5 × baseline, Risk: ≥1.5 × baseline, Injury: ≥2 × baseline or Failure: ≥3 × baseline), as previously described [1]. Qualitative data were analyzed using the chisquared or Fisher exact test as appropriate and quantitative data were analyzed using Student's t test. Inter-group and intra-group comparison for quantitative data was done by one-way ANOVA. The primary outcome measure was the ICU mortality, which was compared in five groups of patients: no AKI, risk (R), injury (I), failure (F), and loss or end-stage (L/E   (Table 1). Conclusion: There is a high incidence of candiduria in ICU patients, especially among those with indwelling catheters and those on antibiotic therapy. Moreover, in our cohort, an increased proportion of patients with candiduria had noncandida infection, emphasizing the need to have localized regional surveillance studies to identify the locally prevalent candida species and devise antifungal therapy protocols.

P20
Erythropoietin enhances the effects of transplanted mesenchymal stem cells in an experimental model of endotoxemia     Background: Statins target several mechanisms involved in the pathophysiology of sepsis, leading to their consideration as an adjuvant therapy. Ubiquinone is an important mitochondrial antioxidant and constituent of the electron transport chain. Ubiquinone production is inhibited by statins, whereas sepsis itself also affects mitochondrial activity. The impact of statins on mitochondrial function in sepsis has not been previously explored.
Methods: Sepsis was induced in instrumented, awake, male Wistar rats by i.p. injection of faecal slurry. Fluid resuscitation was provided by continuous intravenous infusion. Simvastatin 20 mg/kg bd was administrated by gavage commencing either 3 days pre-sepsis (pre-treatment) or from 6 hours postsepsis (post-treatment). A control group received only vehicle but no active drug (vehicle). Survival was assessed at 72 hours (16 per group). In a second set of experiments, rats were sacrificed at 24 hours post-sepsis (seven per group) for measurement of: plasma biochemistry and simvastatin acid; heart and muscle ubiquinone levels by HPLC; and oxygen consumption on permeabilized soleus muscle fibers in a Clark electrode chamber. A fourth group of naive animals was also used as healthy controls. Statistics were performed using the Wilcoxon test and repeated-measures ANOVA and post hoc Bonferroni test.
Results: Survival at 72 hours ( Figure 1) was 43.7%, 25% and 12.5% for pre-treatment, vehicle, and post-treatment groups, respectively (P < 0.05). At 24 hours post sepsis, ubiquinone was significantly decreased only in hearts taken from statin pre-treated animals (706 ± 222 vs. 1,217 ± 269 pmol/mg protein for vehicle hearts). Plasma simvastatin acid levels were significantly increased in statin pre-treated animals (41 ± 3 ng/ml) compared with naïve animals receiving the same dose (1 ± 0.4 ng/ml). Compared with vehicle-treated animals, statin pre-treatment resulted in significant decreases in urea and creatinine, and a trend towards improvement for liver enzymes and creatine kinase. Plasma lipid levels were not significantly affected by statins given either before or after the onset of sepsis. Mean values of muscle oxygen consumption were 11.9 ± 5.3, 14.4 ± 2.9, 12.1 ± 1.5 and 5.9 ± 2.4 pmol/ml/second/mg for naïve, sepsis + post-treatment, sepsis + pre-treatment and sepsis + vehicle groups, respectively. The significant reduction in muscle oxygen consumption seen in the sepsis + vehicle group was prevented in both groups receiving simvastatin.
Conclusion: This study confirms the beneficial effect of statins when given before the onset of sepsis, and this appear to be independent of its lipidlowering property. This beneficial effect is likely to be multifactorial but could be attributed in part to a protective effect on mitochondrial respiration.

P25
Abstract withdrawn Background: Several low-molecular-weight phenolic acids are present in the blood of septic patients at high levels [1,2]. The microbial origin of the most of phenylcarboxylic acids in the human body was shown previously [3], but the pathophysiological role of phenolic acids is not clear. It was shown that microbial phenolic acids produce either the antioxidant or the pro-oxidant action on mitochondria depending on the chemical structure [4]. In this work the influence of phenolic acids on reactive oxygen species (ROS) production in mitochondria and neutrophils was investigated. Methods: Mitochondria were isolated from the liver of Wistar rats. The difference of electric potentials on the inner mitochondrial membrane (Δψ M ) was determined from the distribution of the lipophilic cation of tetraphenylphosphonium whose concentration in external medium [TPP + ] out was registered using a TPP + -selective electrode. The rate of oxygen consumption by mitochondria was determined polarographically. The production of ROS by mitochondria was determined by measuring the chemiluminescence of the Cypridina luciferin. The generation of superoxide anion was induced by 25 μM menadione. The production of ROS by neutrophils was examined by the method of luminol-dependent chemiluminescence. All reagents used were from Sigma (USA). Statistical processing of the data was carried out using the program MS Excel 2003. The differences were considered significant at P ≤ 0.05. Results: ROS production by mitochondria was higher in the presence of cinnamic acid, benzoic acid, 3-phenylpropionic acid and phenylacetic acid than in the presence of menadione alone. The most effective activators of ROS production in mitochondria were those phenolic acids whose effect is mediated via the interaction with thiol (-SH) groups. ROS production by mitochondria was lower in the presence of phenyllactate, p-hydroxyphenylacetate or p-hydroxyphenyllactate compared with the control. All phenolic acids inhibited ROS formation in neutrophils to different degrees. The inhibition was related to the scavenging of the superoxide anion. Conclusion: Microbial metabolites from both the normal gut microbiota and infection sources enter the circulation and can enhance or reduce the inflammatory response. In healthy people phenyllactate and phydroxyphenyllactate, which decrease ROS production in both in mitochondria and neutrophils, can have the protecting effect on organs and tissues. They can play a role of natural antioxidants. But in septic patients deficit phenolic acids producing the pro-oxidant effects on mitochondria (for example, PPA and PAA) and high levels of other phenols can led to mitochondrial dysfunction and multiorgan failure.
Background: Sepsis and its common complication septic shock, are generally induced by the action of lipopolysaccharide (LPS) and is characterized by peripheral arteriolar vasodilatation which results in hypotension and inadequate tissue perfusion. Nitric oxide (NO) is a free radical gas, produced by the immune system in response to an immunological stimulus and is related to the pathogenesis of sepsis due to its vasodilator and cytotoxic actions. One significant finding in clinics is that man and woman respond differently to sepsis, with better prognosis related to women [1].
Objective: This study was designed to investigate the role of estrogen on immune response in an experimental model of septic shock. Methods: In the first set of experiments male and female (ovariectomized and sham surgery) rats were injected intraperitoneally (IP) for three consecutive days with estradiol cypionate (ECP), 40 μg/kg or vehicle. In the third day, after ECP injection, rats receive IP injection of 10 mg/kg of bacterial LPS or saline solution. Plasma was collected 4 and 6 hours after LPS. In the second set of experiments macrophage culture was performed from peritoneal wash of male and female rats. Culture was stimulated with β-estradiol (10 -9 M) and LPS 1 μg/ml and medium collected 12 and 24 hours after stimulation for NO measure.

Results:
In vivo experiments showed that administration of LPS increased NO plasma concentration in males and females. ECP pre-treatment decreased NO concentration in sham females in the two studied periods (4 and 6 hours); conversely, increased nitrate levels in ovariectomized and had no effect in males. Results of in vitro experiments showed that macrophages from males produced more NO than the ones from female, either in basal conditions or after LPS stimulation; however, treatment of the culture with β-estradiol, significantly increased the NO production in macrophages from male but had no effect in female rats.

Conclusion:
Our results indicate that estradiol may have pro or antiinflammatory actions depending on the gender; however, estradiol in female seems to be protective, since it decreased NO plasma concentration. These results may explain in part the better outcomes of woman during sepsis. Background: Correct classification of the source of infection is important in observational and interventional studies of sepsis. The Centre for Disease Control (CDC) criteria are most commonly used for this purpose, but the robustness of these definitions in critically ill patients is not known. We determined the interobserver agreement for classifying infections in the ICU.
Methods: Data were collected as part of a prospective cohort of 1,214 critically ill patients admitted to two hospitals in the Netherlands between January 2011 and June 2011. Eight observers assessed a random sample of 168 out of 554 patients who had experienced at least one infectious episode in the ICU. Each patient was assessed by two randomly selected observers who independently scored the source of infection (by affected organ system or site), the plausibility of infection (rated as none, possible, probable, or definite), and the most likely causative pathogen. Assessments were based on a post hoc review of all available clinical, radiological and microbiological evidence. The observed diagnostic agreement for source of infection was classified as partial (that is, matching on organ system or site) or complete (that is, matching on specific diagnostic terms), for plausibility as partial (two-point scale) or complete (four-point scale), and for causative pathogens as an approximate or exact pathogen match. Interobserver agreement was expressed as percentage agreement and as a kappa statistic.
Results: A total of 206 infectious episodes were observed in 168 patients. Agreement regarding the source of infection was 89% (183/206) and 69% (142/206) for partial and complete diagnostic agreement, respectively (Table 1). This resulted in an overall kappa of 0.85 (95% CI = 0.79 to 0.90). Agreement varied from 70 to 100% within major diagnostic subgroups.
In the subgroup of 142 episodes with full diagnostic agreement on source of infection, the interobserver agreement for plausibility of infection was 83% and 65% on a two-point and four-point scale, respectively. For causative pathogen, agreement was 78% and 70% for an approximate and exact pathogen match, respectively. Conclusion: In this study, overall interobserver agreement of classifying infections using CDC criteria was excellent, whereas an exact match on all aspects of the diagnosis between independent observers was limited for some infections. Background: Sepsis is one of the leading causes of admission to the ICU. After the initial proinflammatory response a gradual change towards a more anti-inflammatory pattern can be seen. In this latter stage, immune cell function has been suggested to be downregulated leading to an immunoparalysis, lending the patient more vulnerable to deleterious secondary infections. Mitochondrial dysfunction has been suggested to play a role in this immunoparalytic state and we therefore investigated mitochondrial respiratory function in peripheral blood immune cells (PBICs) in patients with sepsis and its evolvement over time.
Methods: Twenty patients with severe sepsis or septic shock were included. PBICs were isolated from freshly drawn blood via density gradient centrifugation and analyzed three times during the first week after admission to the ICU (within 48 hours, days 3 to 4 and days 6 to 7). Mitochondrial respiration was examined with high-resolution respirometry in intact and permeabilized cells in order to evaluate both whole cell respiration as well as contribution of individual complexes. Mitochondrial DNA (mtDNA), cytochrome c (Cyt c) and citrate synthase (CS) were measured as indicators of change in cellular mitochondrial content.
Results: In intact PBICs from septic patients, with endogenous substrates, there was a gradual increase in cellular respiration that was 73% higher after 1 week compared with controls (P = 0.003). In permeabilized cells, complex I, II and IV displayed increased respiration compared with controls already at days 1 to 2 (37%, 30% and 73% respectively, P < 0.001) and continued to increase to days 6 to 7 (68%, 68% and 108% respectively). The rise in mitochondrial respiration was paralleled by higher levels of CS activity and increased mtDNA and Cyt c content in cells from septic patients (90%, 143% and 231% for the respective parameter at days 6 to 7 compared with controls, P < 0.0001). Mortality for the septic patients was 25% by day 7. There was no difference in respiratory capacity between survivors and nonsurvivors at any of the time points measured. Conclusion: In PBICs from patients with severe sepsis or septic shock there is a gradual increase in mitochondrial respiratory capacity. This increase is probably due to mitochondrial biogenesis as indicated by increases in mitochondria-specific markers. Nonsurvivors displayed the same increase in respiration as survivors arguing against mitochondrial respiratory dysfunction and defect mitochondrial biogenesis, in PBICs, as a mediator of increased mortality in the septic condition.  Background: A compromising endothelial cell (EC) monolayer affects vascular permeability and leads to fluid extravasation. Tight junctions and more particularly Zonula occludens 1 (ZO-1) play a major role in maintaining vascular barrier integrity and are regulated by cytoskeletal proteins. During sepsis, endothelial barrier disruption occurs in most organs and contributes to organ dysfunction. We and others have demonstrated that the α 1 isoform of AMP-activated protein kinase (α 1 AMPK) controls cytoskeleton organisation in various cell types including endothelial cells. Therefore, we hypothesised that α 1 AMPK preserves tight junction organisation and vascular permeability during sepsis in the coronary microcirculation. Methods: In vitro, tight junction organisation (ZO-1 staining), cytoskeleton organisation (phalloïdin staining) and vascular permeability were measured in endothelial cells in culture (HCAECs). Endothelial cells were pretreated with 1 mM AICA riboside (AICAr) before LPS challenge (50 μg/ml O55:B5).
In vivo, wild-type (α 1 AMPK +/+ ) mice were treated with LPS (O55:B5,10 mg/kg) and compared with α 1 AMPK knockout animals (α 1 AMPK -/-). ZO-1 localisation was determined on frozen heart sections. Vascular permeability was evaluated using Evans Blue dye leakage. In addition, myocardial wall oedema was assessed by magnetic resonance imaging (MRI). Results: In vitro, LPS-challenged cells displayed a significant disruption of the ZO-1 linear configuration after 24 hours and exhibited a decrease in peripheral actin filaments. Gap areas appeared in the cellular monolayer exposed to LPS, unlike untreated cells for which the monolayer remained unaltered. Consequently, LPS treatment gradually increased endothelial cells monolayer permeability in a time-dependent manner. AMPK activation by AICAr preserved the linear pattern of ZO-1 and prevented gap areas in the monolayer. More interestingly, AMPK activation reduced LPS-induced hyperpermeability. In vivo, according to the in vitro experiments, a dramatic decrease in ZO-1 staining was observed in α 1 AMPK -/hearts compared with α 1 AMPK +/+ , after LPS challenge. This resulted in an increased Evans Blue extravasation and, more importantly, in an exaggerated myocardial wall oedema observed by MRI. Finally, treating C57Bl6 mice with AICAr reduced cardiac vascular hyperpermeability in our model of sepsis. Conclusion: The AMPK signalling pathway protects the endothelial barrier during sepsis by preserving tight junction organisation. Since AMPK counteracts the molecular events involved in the LPS-induced barrier disruption in coronary microcirculation, it could consequently represent a new therapeutic molecule during sepsis. Background: Gram-positive bacterial lung infection is commonly associated with sepsis, and particularly important in older individuals. Systemic vascular dysfunction associated with sepsis is characterized by vascular permeability that can lead to tissue hypoxia. Our studies focus on angiopoietin-2 (Ang-2), whose increased plasma concentration is associated with severity of lung injury and with mortality. Ang-2 is stored in the Weibel-Palade body (WPB), an endothelial-specific secretory organelle. Here, we examine the release of Ang-2 from primary human pulmonary microvascular endothelial cells (HPMECs) in vitro stimulated with Gram-positive bacterial cell wall components. We then used our model of pulmonary infection to investigate the use of an inhibitor of WPB exocytosis to prevent systemic inflammation and hemodynamic instability. Methods: HPMECs were treated with lipoteichoic acid (LTA; 50 to 100 μg/ml) and peptidoglycan (PGN; 167 to 333 μg/ml) in the presence or absence of TAT-NSF700 fusion peptide (10 μM) to inhibit WPB exocytosis. Ang-2 in culture medium was measured by ELISA, and WPB exocytosis assessed using immunofluorescent (IF) staining of Ang-2. HPMEC monolayer permeability was measured using FITC-dextran. Male C57/Bl6 mice, 8 to 10 weeks old (n = 6/group), were pretreated with TAT-NSF700 (0.5 mg/kg) or saline intraperitoneally. Thirty minutes later, LTA (150 μg) and PGN (500 μg) or saline alone were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to and 6 hours post instillation. The mice were then euthanized by exsanguination under anesthesia and bronchoalveolar lavage (BAL) was performed. BAL fluid total and differential counts were evaluated and protein and cytokine concentrations in plasma and BAL were assessed using commercial assays. Results: LTA-PGN increased Ang-2 levels dose dependently (up to ninefold, P = 0.003) in HPMEC culture medium within 30 minutes, which was blocked by TAT-NSF700 pretreatment. IF staining showed aggregation and localization of Ang-2 in the cytoplasm suggesting WPB exocytosis within 30 minutes. LTA-PGN also induced HPMEC permeability. LTA-PGN induced both local and systemic inflammation resulting in decreased heart and breath rates and oxygen saturation (94% vs. 97%, P = 0.038) at 6 hours. These physiological changes were prevented in mice pretreated with TAT-NSF700.
Conclusion: LTA-PGN induced rapid Ang-2 secretion via WPB exocytosis and this release is associated with significant changes in permeability.
In vivo, LTA-PGN induces significant changes in heart and breath rates and oxygen saturation that was prevented by inhibition of WPB exocytosis. Thus, we have defined WPB, a storage organelle for multiple proinflammatory mediators, as a potential target to control overwhelming inflammation in Gram-positive sepsis and improve tissue oxygenation and hemodynamics. . Median time to decolonization was 6.0 (IQR 5.6 to 6.4) and 9.0 days (IQR 8.1 to 10.9) in patients receiving and not receiving NAB, respectively (log-rank test, P = 0.03) ( Figure 1). Adjustment for age, sex and time from ICU admission to acquirement of Candida did not alter the results.
Conclusion: Inhaled amphotericin B treatment in mechanically ventilated patients with acquired Candida colonization of the lower respiratory tract significantly increases the rate of decolonization. Background: In sepsis, the sympathetic nerve activity (SNA) is differentially increased to individual organs [1]. It has been suggested that inhibition of central sympathetic outflow with clonidine would improve outcome in sepsis [2] but the cardiovascular and renal effects of clonidine in sepsis are unknown. Accordingly, we sought to assess the effect of the central α 2agonist clonidine on renal function in an ovine model of severe sepsis. Methods: Animals had renal and cardiac flow probes implanted to continuously measure the cardiac index (CI) and renal blood flow (RBF). Mean arterial pressure (MAP) was continuously monitored and hourly Figure 1(abstract P32) Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 urine collection was performed. After the first 24 hours of sepsis every animal was randomly and blindly allocated to receive vehicle or clonidine 0.25 μg/kg/hour for 8 hours. Animals were followed for further 40 hours during recovery and, at least 2 weeks later, crossed over to the other arm of the study. Two-way repeated-measures ANOVA was performed and P < 0.05 was considered significant. Data are mean ± standard error of the average of 4 hours preceding each time point. Results: Eight animals per group were studied. Two animals per group died during the first experiment, leaving six animals for crossover. Values presented refer to all eight animals. Animals in both groups had similar baseline values and developed a similar hyperdynamic state with 50% increase in CI, 15 mmHg drop in MAP and threefold increase in arterial lactate. Clonidine ( Figure 1) reduced the HR by 23(7) bpm (P = 0.013) but did not affect MAP, RBF and lactate levels. Clonidine increased urine output from 23(4) to 71(23) ml/hour (P = 0.004), but did not improve creatinine clearance (from 44(6) to 42(4) ml/minute). Conclusion: In experimental hyperdynamic sepsis, treatment with clonidine appears safe, but it does not improve the glomerular filtration rate. Background: The pathophysiology of septic acute kidney injury (AKI) is poorly understood. Renal medullary hypoxia has been proposed as a cause of AKI, but the changes in intrarenal oxygenation in hyperdynamic sepsis are unknown. Accordingly, we sought to determine the changes in regional renal perfusion and tissue oxygenation in the cortex and in the medulla in an ovine model of severe sepsis. Methods: Mean arterial pressure (MAP), cardiac index (CI) and renal blood flow (RBF) were continuously monitored in conscious sheep. Fibre optic probes (Oxford Optronix) were used to measure tissue perfusion and oxygen partial pressure at 30-second intervals. Arterial and renal venous blood samples were collected for oximetry. After 24 hour of baseline data collection, sepsis was induced with live Escherichia coli infusion for 24 hours. Gentamycin was given to terminate sepsis. The animals were followed for a further 24 hours of recovery. Histology was performed to confirm the position of the catheter tips and to assess tissue viability around the probes. Data are mean (± standard error) of the average of the periods. One-way repeated-measures ANOVA was used and P < 0.05 considered significant. Results: Eight animals were studied. All animals developed a hyperdynamic state with a doubling in heart rate and CI, and a 50% increase in RBF. MAP decreased by 15 mmHg, with a fourfold increase in arterial lactate. Urine output halved, serum creatinine doubled and creatinine clearance decreased by one-third. Two animals died 28 hours after induction of sepsis. Baseline cortical and medullar pO 2 were 29.6 (± 4.3) and 29.1 (± 4.1) mmHg, respectively. During sepsis (see Figure 1), cortical perfusion and oxygenation did not change significantly. In contrast, medullary perfusion showed an early trend towards reduction, while medullary tissue pO 2 had decreased by 53 ± 11% at 24 hours of sepsis (P < 0.05). Calculated total renal oxygen consumption did not change significantly (from 33 to 32 ml O 2 /minute). Conclusion: This is the first study to measure intrarenal perfusion and oxygenation in hyperdynamic sepsis. In a conscious large animal model of septic AKI, we showed decreased medullary oxygenation, possibly due to mismatched local perfusion and oxygen consumption. Background: Selective decontamination of the digestive tract (SDD) was shown to reduce acquisition of resistant bacteria and mortality in ICUs. A significant reduction of bacteremia, candidemia, ventilator-associated pneumonia, respiratory tract and rectal colonization have been found in multicenter, randomized studies. SDD was proven to be clinically safe and cost-effective in multiple clinical studies but has not been accepted by the critical care community as a standard of care. Reluctance to use SDD despite proof to the contrary is mostly explained by its perceived potential to raise bacterial resistance to parenteral and enteral antibiotics or rebound infection after their cessation. We studied the effect of a simplified SDD protocol on Gram-negative colonization of the respiratory tract and bloodstream infection.

Methods:
During 2011, all adult ICU patients on mechanical ventilation for more than 3 days were included. Enteral medication of neomycin and polymixin-E was given four times daily until gastric tube removal or ICU discharge. Blood, sputum cultures and rectal screening were taken on admission and biweekly until 3 days after ICU discharge. The emergence of multidrug-resistant (MDR) bacteria was investigated and compared with 2010. The in-hospital length of stay and 28-day mortality were compared. Results: Out of 506 patients, 277 (74% of eligible) received SDD during 2011, compared with none of 458 patients in 2010. There was a 40% reduction of positive blood cultures (P = 0.01) and a 27.3% reduction of positive sputum cultures with MDR bacteria (P = 0.04). There was a shift from MDR towards sensitive bacteria. This was mostly prominent in the Acinetobacter, Klebsiella and Pseudomonas species. There was no concomitant elevation of other pathogenes as MRSA, VRE, Clostridium or Candida. There was an 18.4% relative reduction in 28-day mortality and a 2-day reduction in median hospital length of stay (P = 0.006). Conclusion: The simplified SDD protocol reduced ICU acquisition of Gramnegative resistant infections without a rise in Gram-positives or fungi. The bacterial epidemiology of the unit changed shifting to sensitive bacteria, and the 28-day mortality and hospital length of stay have improved. This study supports the safety and efficacy of this SDD protocol and justifies its further investigation in more ICUs. Background: Acupuncture could increase vagal activity. We hypothesize that acupuncture could activate the cholinergic anti-inflammatory Figure 1(abstract P35) Haemodynamic and regional perfusion/oxygenation changes. Data are mean (standard error). Rectangle: 24-hour sepsis period.
Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 pathway, inhibit systemic inflammation, and improve the clinical prognosis in severe multiple trauma patients. Methods: The design was a prospective, randomized intervention trial. The setting was the Trauma Center, Department of Emergency Medicine, Southwest Hospital, Chongqing, P.R. China. Consecutive multiple trauma patients with an Injury Severity Score (ISS) of more than 16 points meeting the criteria between January 2007 and December 2008 were included. The intervention acupuncture was applied to the ST-36 and PC-6 acupoint twice a day until day 7 after injury or sham acupuncture was applied to non-acupoints. One hundred and fifty severe multiple traumatic patients were randomly divided into three groups: acupuncture group (group A; n = 50), sham acupuncture group (group B; n = 50), and control group (group C; n = 50).
Results: Acupuncture at ST-36 and PC-6 acupoints significantly increased the normalized unit of the high-frequency component (HFnu; P < 0.01) and decreased the ratio of low-frequency and high-frequency component (LF/HF; P < 0.05), which indicated an increased vagal activity and a tilt of the sympathovagal balance. Serum TNFα and IL-6 concentrations significantly decreased after stimulation at ST-36 and PC-6 acupoints (P < 0.01 and P < 0.05, respectively), but remained unchanged following stimulation at the non-acupoint area. During the whole study period, much lower concentrations of TNFα and IL-6 were detected in severe traumatic patients with acupuncture intervention in comparison with those in sham acupuncture and control patients (P < 0.05). Most importantly, we observed that the incidences of SIRS, and some clinical complications, such as ARDS, sepsis or MOF, and mortality in the acupuncture group were much lower in comparison with the other two groups.
Conclusion: Acupuncture has beneficial effects on modulation of inflammatory response and will improve the clinical outcomes in severe multiple traumatic patients. We suggest that larger cohort studies or multiple center studies are needed to validate whether acupuncture is another effective stimulation pattern to activate the cholinergic antiinflammatory pathway. The detailed mechanisms are worth further investigation. Background: Advances in modern medicine and patient management have seen increased use of prosthetic materials, leading to biofilm formation, and the constituent organisms are different in their growth rates and susceptibility patterns and cause many complications. Candida species are increasingly seen to be important nosocomial pathogens and are major causes of morbidity and mortality in immunocompromised patients of intensive care and postoperative wards. Hence our aims were to isolate and identify specific Candida species associated with the luminal biofilm in endotracheal tubes/i.v. catheters in ICU patients and to study the pathogenicity of Candida species by testing for virulence mechanisms including biofilm formation and antifungal susceptibility. Methods: One hundred and twenty-five medical/postoperative patients admitted to the ICU for more than 48 hours were studied. Tracheal tubes at the time of extubation and intravenous/other catheters removed from patients and blood samples and other relevant samples from potentially infected sites were collected and processed. Direct microscopy done by KOH mount and Gram staining and cultures were done on blood agar, brain heart infusion agar, and Sabouraud's Dextose agar with antibiotics and were incubated at 25 and 37°C. Antifungal susceptibility and virulence testing of Candida species was done by phospholipase, proteinase estimation and adherence assay.
Results: The patients ranged from 13 to 90 years with a male predominance seen in most ages. A total of 86.29% were admitted for less than 1 week followed by 9.68% for 1 to 2 weeks. The most common organ system involved was the GIT followed by the CNS, respiratory and multiple organ systems. In total, 356 samples including indwelling devices, blood and urine samples were processed. A total of 253 samples were sterile while fungal isolations were obtained in 103 samples with 51.4% from indwelling devices and 39.8% from urine samples. Candida tropicalis (40%) and Candida albicans (39%) were the commonest followed by Candida glabrata (11%), Candida parapsilosis (4%) and Candida krusei, Candida kefyr and Candida sphaerica (each 2%). Candida biofilm formation was elicited in 53 indwelling devices. A total of 58.25% Candida species were resistant to fluconazole. Phospholipase and proteinase activities were seen in 73.8% and 55.3% Candida isolates with different species showing a wide range of activities, while 71 Candida isolates showed (4+) adherence activity. Conclusion: Candida species continue to be important fungal pathogens in the formation of biofilms in ICU patients and knowing drug susceptibility and virulence characteristics will help in reducing associated complications, device control strategies and consequently reducing morbidity, mortality and treatment costs. Background: Differentiating the systemic inflammatory response syndrome (SIRS) from sepsis is very important to clinicians. Procalcitonin (PCT) has been studied extensively as a marker of sepsis; however, its clinical utility remains uncertain [1]. Alternative approaches involving analysis of circulating biomarkers using gene expression (GE) show promise [2]. The primary objective of this study was to compare the diagnostic performance of a GE biomarker set, SeptiCyte® Triage, with PCT in a mixed patient population. Methods: The dataset was derived from two clinical trials conducted across four tertiary care settings in Australia between 2008 and 2011 (ACTRN12610000465055). Critical care patients (n = 87) were enrolled if they fulfilled the 1992 Consensus Statement [3] for sepsis, severe sepsis or septic shock. Postsurgical patients were included as an infection-negative systemic inflammatory cohort (n = 31), with 71 healthy controls (HC) for comparison. Blood samples were collected within 24 hours of the surgical procedure or upon admission to ICU for sepsis patients. PCT was measured using a commercially available assay kit (Brahms PCT) and GE assessed using Affymetrix GeneChip Human Exon 1.0 ST arrays, where a set of biomarkers were identified a priori. A Support Vector Machine algorithm was used to calibrate the molecular biomarkers with respect to specific clinical groups. Bootstrap and permutation tests were performed on the difference in area under the receiver operating characteristic curves (AUC ROC) for PCT and crossvalidated posterior probabilities. Results: The SeptiCyte® Triage molecular biomarker set was significantly better than PCT at differentiating non-infectious systemic inflammation from all sepsis groups with an AUC ROC of 99.1% versus 90.8% (95% CI of difference = 4%, 16%), respectively. The differentiating ability of PCT and the SeptiCyte® Triage biomarker set is demonstrated in Figure 1. The 99.1% AUC ROC of the biomarker set could be maintained when using a set of less than 10 genes, important for transferring the biomarkers to a rapid assay format. As a quality step, a classifier was developed that differentiated HC from sepsis with an AUC ROC of 100%. Conclusion: In comparison with the diagnostic performance of PCT, a limited set (<10) of molecular biomarkers potentially has a significantly better sensitivity and specificity profile for the detection of sepsis within a mixed systemic inflammatory patient group. Background: Leukocyte immunophenotyping could improve sepsis diagnostics [1,2]. Our hypothesis was that monocytic and neutrophilic CD11b and CD64 antigen fluorescence intensities differ between severe sepsis, non-inflammatory ICU patients and nonseptic inflammation (offpump coronary artery bypass (OPCABG)). Methods: Monocytic and neutrophilic CD11b and CD64 expressions were analyzed from 27 patients with severe sepsis, seven OPCABG patients and from eight ICU patients who did not fulfill any SIRS criteria. Blood samples were collected within 48 hours from the beginning of severe sepsis or in non-SIRS patients from ICU admission and two consecutive days (D0, D1, D2). From surgical patients, the first samples were taken on the day of surgery before the skin incision and two consecutive days (D0, D1, D2). In addition 10 healthy individuals served as controls. Samples were collected, processed and analyzed using flow cytometry as previously described [3]. Results: The maximum fluorescence intensities of monocytic and neutrophilic CD11b and CD64 were highest in septic patients compared with the other groups (P < 0.05) ( Figure 1). In severe sepsis, fluorescence intensities decreased over time (P < 0.05). In OPCABG the fluorescence intensities of other antigens increased from D0 to D1 except neutrophilic CD11b (P < 0.05). The intensities of other antigens except neutrophilic CD64 were lower in the healthy than in all the other groups (P < 0.05). Neutrophilic CD64, as well as other antigens, were lower in healthy controls compared with severe sepsis at all time points (P < 0.05). Conclusion: Based on this study, monocytic CD11b and neutrophilic CD64 could be helpful in distinguishing severe sepsis from nonseptic inflammation and healthy controls.     Background: Diagnostic and prognostic markers are of outmost importance in the critical patient referred to the ICU. The aim of the present study was the evaluation of procalcitonin (PCT), IL-10 and soluble CD25 (sCD25) as potential markers in the diagnosis and prognosis of cohorts of critical patients evaluated following a prospective design in a tertiary-center university hospital. Methods: We studied 52 consecutive SIRS patients with suspected sepsis that were firstly stratified based on culture results (culture-positive and culture-negative), then subsequently divided into survivors and nonsurvivors. Venous blood samples were obtained from each patient within 6 hours of hospital/ICU admission (T-0) and at the end of first week of hospital/ICU stay (T-1). Serum samples were collected and used for simultaneous determination of IL-10 and sCD25, using a cytokine biochip array on the Evidence Investigator analyser (Randox Laboratories Ltd, Crumlin, UK), while PCT was assayed by an enzyme-linked fluorescent assay (VIDAS BRAHMS PCT; bioMerieux, France). Statistically significant differences between groups were established by the Mann-Whitney U test. The receiver-operating characteristic (ROC) curve was used to evaluate the diagnostic accuracy (defined by the area under the ROC curve (AUROCC)) of the analyzed cytokines and to determine the sensitivity and specificity at selected cutoff values. The statistical analyses were performed using SPSS 14.0 software (SPSS, Chicago, IL, USA).
Results: PCT, IL-10 and sCD25 were significantly (P < 0.05) increased in infectious versus non-infectious SIRS patients at hospital admission (T-0) and after 1 week of hospital stay (T-1). Diagnostic accuracy of PCT, IL-10 and sCD25 was evaluated by AUROCC and exhibited a significance index of 0.0001, 0.0021 and 0.0095 respectively at T-0, while at T-1 the P values were 0.0011, 0.0016 and 0.0201 respectively. Regarding prognostic markers, PCT showed a prognostic significance only at T-1 (P = 0.04). However when IL-10 and sCD25 values from survivors were compared with those from nonsurvivors, significant differences were found for the former and the latter marker with P = 0.0014 and P = 0.014 respectively at T-0, as well as with P = 0.0002 and P = 0.014 respectively at T-1.
Conclusion: In our study, well-known PCT and IL-10 markers and novel sCD25 significantly contributed to diagnosis and prognosis of SIRS and septic patients. Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice [1]. But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis.
Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% ( Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% ( Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 ( Figure 1C,D) while poly(I:C)-induced Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells ( Figure 1E). Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation.
In vivo experiments will clarify its impact on systemic inflammation. Background: Sepsis, a common cause of morbidity and mortality in critically ill patients, is associated with systemic inflammatory response syndrome due to upregulation of cyclooxygenase-2 and increase in the levels of PGE 2 . It is also associated with increase in the levels of proinflammatory cytokines like TNFα and IL-1β. Calotropis procera is a plant that grows in the wild producing latex. The aqueous and methanolic extracts of dried latex of this plant (AqDL and MeDL) and proteins isolated from the fresh latex (LP) have shown anti-inflammatory and anti-arthritic properties. AqDL and MeDL are orally effective, LP is effective parenterally. The current study was designed to evaluate the efficacy of these extracts against yeast-induced pyrexia and the levels of TNFα and PGE 2 in the hypothalamus of rats.
Methods: Pyrexia was induced in rats by subcutaneous injection of yeast in the nape of the neck and the rectal temperature was measured at 0 hours (basal temperature), 3 hours and 6 hours. Rats were divided into groups (n = 6) and were treated with AqDL and MeDL given orally and LP given intravenously at 6 hours. Group I: NC (normal control); Group II: YC (yeast control); Group III: AqDL (200 mg/kg); Group IV: AqDL (400 mg/kg); Group V: MeDL (100 mg/kg); Group VI: MeDL (250 mg/kg); Group VII: LP (5 mg/kg); Group VIII: LP (25 mg/kg); Group IX: paracetamol (PCM 100 mg/kg). Rectal temperature was measured hourly until 9 hours. The levels of TNFα and PGE 2 were measured in the excised hypothalamus region of the brain using ELISA kits.
Results: Subcutaneous injection of yeast produced a marked increase in rectal temperature of rats with a maximum effect at 6 hours (101.17°C). Like paracetamol, treatment of rats with AqDL and MeDL produced a significant decrease in body temperature from 101.17°C at 6 hours to 97.9°C and 98.2°C at 9 hours respectively at higher doses and their effect was dose dependent while LP was found to be ineffective. Background: Natural killer (NK) and natural killer T (NKT) cells play a key role in bacterial infection and sepsis since they contribute to the bridging of innate and acquired immune responses. We have previously shown that in vivo depletion of these cell populations in a murine pneumococcal pneumonia sepsis model affected mortality.
Twenty-four hours prior to bacterial inoculation, NK cell depletion was achieved by intravenous (i.v.) administration of anti-asialoGM1 rabbit polyclonal antibody in one group (NK DEPL ), or anti-CD1d monoclonal antibody, clone 1B1 was given for NKT cell depletion in a second group (NKT DEPL ). The control group received equal volume of isotype antibody control i.v. (C) and a fourth group received sham intratracheal installation of normal saline (S). All animals were euthanized 48 hours post infection. Serum and tissue samples were analyzed for bacterial colony counts, cytokine levels, splenocyte apoptosis rates and cell population analysis by flow cytometry. In parallel, specific miRNA expression analyses in splenocytes and lung histologic examination were also performed.
Comparisons of numeric data between groups were made using the oneway ANOVA test for multiple groups.
Results: We found that upon NK cell depletion there was a significant increase in the spleen NKT (CD3 + /CD1d + ) cell population compared with NKT DEPL , C and S (P = 0.014, P = 0.021 and P = 0.033, respectively). Interestingly, upon NKT cell depletion, spleen NK (CD3 -/NK1.1 + ) cells increased significantly compared with NK DEPL , C and S (P < 0.0001, P < 0.0001 and P = 0.001, respectively). NKT depletion led to decreased lymphocyte apoptosis compared with C (P = 0.035), higher bacterial load in the lung compared with C and NK DEPL (P = 0.014 and P = 0.022 respectively) and in the liver compared with C (P = 0.012). In addition, serum levels of IFNγ were significantly increased and splenocytes from NKT depleted animals, incubated ex vivo in the presence or absence of IL-2, produced more IFNγ in comparison with all other groups. Furthermore, splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in the NKT depleted animals compared with all other groups. Conclusion: For the first time we have shown that NKT cell depletion resulted in an increase in spleen NK (CD3 -/NK1.1 + ) cells and a higher IFNγ production, which were associated with specific changes in splenocyte miRNA expression. Background: Undergoing systemic inflammation, the innate immune system releases excessive proinflammatory mediators, which finally can lead to organ failure. Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), form the interface between bacterial and viral toxins and innate immunity. During sepsis, patients with diagnosed adrenal gland insufficiency are at high risk of developing a multiorgan dysfunction syndrome, which dramatically increases the risk of mortality. To date, little is known about the mechanisms leading to adrenal dysfunction under septic conditions. Here, we investigated the sepsisrelated activation of the PRRs, cell inflammation, and apoptosis within adrenal glands. Methods: Two sepsis models were performed: the polymicrobial sepsis model (caecal ligation and puncture (CLP)) and the LTA-induced intoxication model. All experiments received institutional approval by the Regierungspräsidium Darmstadt. CLP was performed as previously described [1], wherein one-third of the caecum was ligated and punctured with a 20-gauge needle. For LTA-induced systemic inflammation, TLR2 knockout (TLR2 -/-) and WT mice were injected intraperitoneally with pure LTA (pLTA; 1 mg/kg) or PBS for 2 hours. To detect potential direct adrenal dysfunction, mice were additionally injected with adrenocorticotropic hormone (ACTH; 100 μg/kg) 1 hour after pLTA or PBS. Adrenals and plasma samples were taken. Gene expressions in the adrenals (rt-PCR), cytokine release (multiplex assay), and the apoptosis rate (TUNEL assay) within the adrenals were determined. Results: In both models, adrenals showed increased mRNA expression of TLR2 and TLR4, various NLRs, cytokines as well as inflammasome components, NADPH oxidase subunits, and nitric oxide synthases (data not shown). In WT mice, ACTH alone had no effect on inflammation, while pLTA or pLTA/ACTH administration showed increased levels of the cytokines IL-1β, IL-6, and TNFα. TLR2 -/mice indicated no response as expected (Figure 1, left). Interestingly, surviving CLP mice showed no inflammatory adrenal response, whereas nonsurvivors had elevated cytokine levels (Figure 1, right). Additionally, we identified a marked increase in apoptosis of both chromaffin and steroid-producing cells in adrenal glands obtained from mice with sepsis as compared with their controls (Figure 2). Conclusion: Taken together, sepsis-induced activation of the PRRs may contribute to adrenal impairment by enhancing tissue inflammation, oxidative stress and culminate in cellular apoptosis, while mortality seems to be associated with adrenal inflammation.   Background: Triggering receptor expressed on myeloid cells-1 (TREM-1) is expressed on innate immune cells and plays a crucial role during the onset of sepsis by amplifying the host immune response. TREM-like transcript-1 (TLT-1) belongs to the TREM family and is selectively expressed on activated platelets. We recently showed that TLT-1 and a TLT-1-derived peptide (LR12) exhibit anti-inflammatory properties by dampening TREM-1 signalling and thus behave as naturally occurring TREM-1 inhibitors [1]. We also, however, demonstrated that the same peptide modulates in vivo the inflammatory cascade triggered by infection, thus inhibiting hyper-responsiveness, organ damage and death during sepsis in mice. As mouse models of septic shock are far from recapitulating the human physiology, we investigated the effects of LR12 during peritonitis in adult mini-pigs. Here we show that sepsis-induced cardiovascular dysfunction and organ failure was prevented by LR12 administration. The objective was to determine the effects of a TLT-1 derived peptide (LR12) administration during septic shock in pigs (13 adult male mini-pigs).
Methods: Two hours after induction of a fecal peritonitis, anesthetized and mechanically ventilated mini-pigs were randomized to receive LR12 (n = 6) or its vehicle alone (normal saline, n = 5). Two animals were operated and instrumented without the induction of peritonitis and served as controls (sham). Resuscitation was achieved using hydroxyethyl starch (up to 20 ml/kg) and norepinephrine infusion (up to 10 μg/kg/ minute). Results: Hemodynamic parameters were continuously recorded. Gas exchange, acid-base status, organ function, and cytokines were measured at regular intervals until 24 hours after the onset of peritonitis when animals were sacrificed under anesthesia. Peritonitis induced profound hypotension, myocardial dysfunction, lactic acidosis, coagulation abnormalities, and multiple organ failure. These disorders were largely attenuated by LR12. In particular, cardiovascular failure was prevented as attested by better mean arterial pressure, cardiac index, cardiac power index, and SvO 2 , despite lower norepinephrine requirements ( Figure 1). Finally, 24-hour mortality rates were respectively 60% and 0% for control and LR12 groups.
Conclusion: LR12, a TLT-1 derived peptide, exhibits salutary properties during septic shock in adult mini-pigs.   Background: Triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor of the immunoglobulin superfamily expressed on the surface of neutrophils and monocytes/macrophages. It plays an important role during sepsis by amplifying the inflammatory response. Modulation of TREM-1 through the administration of a short synthetic peptide (LR12) increases survival during experimental sepsis. This study aimed to explore the mechanisms by which LR12 prevented sepsis-induced cardiovascular dysfunction. Methods: We studied the effect of TREM-1 modulation by a synthetic peptide LR12 (3 mg/kg) on MAP and blood lactate during experimental sepsis (CLP). Aortic and mesenteric arterial vessels of these animals were collected to study the vasoreactivity to phenylephrine (Phe) and acetylcholine (Ach) ex vivo (Myograph). Alternatively, vasoreactivity was studied in the vessels of healthy animals (with and without endothelial lawyer) stimulated with LPS or with a specific agonist of TREM-1 (αTREM-1, 10 μg/ml), with or without LR12 (20 mg/ml). The effect of LR12 on arterial vessels was also studied through western blotting (eNOS, iNOS, Akt, COX-1, COX-2) and qRT-PCR. Mouse lung microvascular endothelial cells (LUMECs; CD146 + ) were analyzed by flow cytometry, qRT-PCR, and ELISA to decipher the effect of LR12 on LPS-induced endothelial activation.
Results: CLP induced MAP decrease and lactate elevation were prevented by the administration of LR12. Arterial vessels from septic animals treated with LR12 showed better response to Phe and Ach compared with controls. The reactivity of aortic and mesenteric vessels (contraction and relaxation) stimulated in vitro with LPS or by αTREM-1 was altered: this phenomenon was reversed by LR12. LR12 restored the phosphorylation of Akt and eNOS while it reduced the activation of inducible pathways (iNOS, COX-2). FACS analysis showed that TREM-1 is constitutively expressed by LUMECs (CD146 + /VEGFR2 + ). In vivo, the expression of Trem-1 was increased in septic animals and was inducible in vitro upon stimulation with LPS. Trem-1, Tnf-a and Il-6 expression was upregulated by LPS; once again LR12 attenuated this upregulation. Finally, the production of several cytokines by LPS-stimulated LUMECs was decreased by LR12.
Conclusion: Here we show that TREM-1 is expressed on endothelial cells from the aorta, mesenteric artery, and microvascular endothelial cells, and that TREM-1 might be directly involved in endothelial dysfunction during experimental sepsis. Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression [1]. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation [2]. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure [3,4]. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade [5].   Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators [1]. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production [2] and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections [3]. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2 [4]. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased. Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS. Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis. Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.  Background: Cholecystokinin (CCK) was firstly described as a gastrointestinal hormone, but immune cells express their receptors, suggesting a possible involvement of this peptide in pathophysiological processes. Our aims were to evaluate the role of CCK on resistance against Gram-positive Staphylococcus aureus-induced sepsis, as well as cell influx to infectious focus. Furthermore, since nitric oxide, TNFα and IL-10 play a key role in the innate immune system controlling bacterial infection, we also evaluated the synthesis of these inflammatory mediators during this sepsis model. Methods: Male Wistar rats (180 to 200 g) received an intraperitoneal injection of proglumide (P) (nonselective CCK receptor antagonist; 30 or 50 mg/kg) 30 minutes before bacterial S. aureus inoculum (0.5 to 1 × 10 10 CFU/animal). At 4 and 24 hours after sepsis induction, blood and peritoneal lavage fluid (PLF) were collected for microbiological analysis, cytokines and nitrate quantifications and also differential cell counting. Nitrate was detected by chemiluminescence, while TNFα and IL-10 were determined by ELISA sandwich kits.
Results: The pretreatment with P at higher dose (50 mg/kg) increased bacteremia in comparison with the saline-injected group (2,052 ± 810.7 vs. 154.3 ± 47.0 CFU/ml, P < 0.01) at 4 hours after sepsis induction. At the same time point, the bacterial counting in PLF increased in a dosedependent manner in the P-treated rats (P < 0.05). On the other hand, only the higher P dose elevated significantly the CFU/ml in the PLF at 24 hours (97.75 ± 12.77 × 10 4 vs. 35 ± 10.05 × 10 4 CFU/ml, P < 0.05). The plasma TNFα and nitrate concentrations were not changed by treatment or time after sepsis induction. However, the administration of CCK receptors antagonist reduced the TNFα production in comparison with the control group in PLF, at both time points. The plasma IL-10 concentration increased at 4 hours in P-treated rats, while at 24 hours it was reduced (85.83 ± 48.0 vs. 1,698 ± 265.6 pg/ml, P < 0.001). In PLF, the rats pretreated with P reduced the IL-10 measurements (P < 0.05) when compared with the control group. In agreement, the macrophage influx to peritoneal infectious focus was compromised by treatment with a high P dose at 24 hours after S. aureus-induced sepsis (  Background: Sepsis is a common condition in newborns, which has significant morbidity and mortality worldwide. In recent years, multiple factors have led to an increase in its incidence, such as the use of invasive diagnostic procedures, broad-spectrum antimicrobial therapy and an increase of immunocompromised patients. There have also been changes in the profile of the agents causing sepsis. The aims of this study were the determination of the annual incidence of neonatal sepsis (early-onset and late-onset), analyzing their clinical course, significant microorganisms isolated and the profile of antimicrobial resistance. Conclusion: K. pneumoniae was the organism responsible for more episodes of sepsis in neonates. As in other studies in neonatology, this study highlights the prevalence of nonalbicans species in candidemia due to the frequency of C. parapsilosis. Late-onset sepsis has increased by Gram-negative bacilli in the last year, probably due to the occurrence of a nosocomial outbreak of ESBL-producing E. cloacae. However, the mortality rate from sepsis has remained stable. When clinically evaluated, cases of sepsis can be detected as a predominance of enterobacteria against S. epidermidis, highlighting the importance of careful analysis of clinical outcomes.

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Is urinary kidney injury molecule-1 a good marker for acute kidney injury in septic shock? Methods: This clinical prospective study -approved by the local ethics committee -was performed to assess urinary levels of KIM-1 by ELISA technique in 38 patients with septic shock (Table 1). They were prospectively enrolled within 24 hours of onset of signs of infection, if they met the criteria for septic shock as defined by the members of the ACCP/SCCM Consensus Conference Committee. KIM-1 levels were quantified on admission (baseline, day 0) and on days 4, 7 and 10 of the ICU stay. The patients were classified by AKIN and RIFLE criteria. Data were analyzed with regard to the prognostic value of survival, need for renal replacement therapy (RRT) and correlation with renal function parameters (plasmatic urea and creatinine, creatinine elimination and clearance, free water clearance, fractional sodium excretion) or hepatic laboratory findings (albumin, total bilirubin, ASAT, ALAT, γ-GT). Data are given as mean ± SEM. Results: The urinary KIM-1 concentration on admission showed no significant difference with respect to survival of patients. However, the urinary KIM-1 concentration determined on days 4, 7 and 10 was higher in patients surviving septic shock (P < 0.001 on days 4 and 7). Elimination displayed lower levels in deceased patients (P < 0.05 on days 0 and 4), whereas urinary output was higher in survivors during the whole ICU stay (P < 0.05 on day 7). Urinary KIM-1 concentration did not differ between AKIN and RIFLE classification groups. Urinary KIM-1 elimination per 24 hours on days 0, 4 and 7 was higher in stage 1 than in stage 2 or 3 of the AKIN classification, respectively (both P < 0.05). If patients were classified by RIFLE criteria, urinary KIM-1 elimination was also higher in the risk group as compared with the injury or failure group without reaching significance. However, the need for RRT was reflected by a higher urinary KIM-1 concentration after admission (P < 0.05 on days 4 and 10), a lower KIM-1 elimination and urinary output during the whole ICU stay (KIM-1 elimination: P < 0.05 on day 0; urinary output: P < 0.001 on days 0 and 4, P < 0.05 on day 7; Figure 1A,B,C). Neither urinary KIM-1 concentration or elimination correlated with any renal and hepatic function parameter. Conclusion: Urinary KIM-1 can be used as a prognostic factor for survival and need for RRT in septic shock patients.

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Necrotizing Background: Erysipelas in some patients can be complicated with SIRS and sepsis, even with septic shock that may be explained by the hyperactivity of the complement system. The complement system in erysipelas has not been explored thoroughly. Previously a reduction of the general hemolytic activity of different complement components was reported. The study of the complement system is absolutely required to determine the functional state of the immune system and inflammation process in erysipelas. Methods: Plasma samples from 38 patients with different clinical erysipelas forms (two primary erysipelas, 12 recurrent erysipelas) were obtained in 2010. The erythematous form was diagnosed in 10 patients with primary infection and in four patients with recurrent infection, the erythematous-hemorrhagic form in eight and seven patients, and the bullous-hemorrhagic form in seven and two patients accordingly. Blood samples were taken on day 1 (admission to the hospital) and on days 7 to 10 (on discharge from the hospital).   Background: The Acute Physiology and Chronic Health Evaluation (APACHE II) score is widely used in the ICU and has been well validated against the other critical illness scoring systems in predicting ICU mortality. The aim of this audit was to determine survival to hospital discharge for patients admitted to our ICU during a period of 6 months. Methods: We performed a prospective audit of 80 consecutive patients admitted to our 16-bed respiratory intensive care unit (RICU) in a university teaching hospital. Patients were excluded if the observation period was less than 24 hours and age less than 12 years. An audit form was completed by a fellow intensivist who followed up the patients until they were discharged from the RICU. Patient data were stratified according to the outcome (S, survivors; E, expired) and compared for different age groups (12 to 29 years, 30 to 60 years, >60 years), gender (males vs. females) and diagnostic disease groups. Student's t test, chi-square test/ Fisher's exact test and analysis of variance were used for comparison of data as appropriate and P < 0.05 was considered significant. Results: The mean APACHE II score, mean age and duration of ICU stay were 12.4 ± 10.8, 42.1 ± 20.6 years and 12.8 ± 12.2 days respectively. The patients' number of days of ICU stay was independent of patients' outcome (S or E) (P = 0.476). Similarly the gender distribution did not affect the mean APACHE II score on admission (P = 0.273) and duration of ICU stay (P = 0.166). The survival rate among the eight different diagnostic groups was similar (P = 0.064). A higher APACHE II score and a higher age was associated with increased mortality (P < 0.001 and P = 0.001 respectively). Conclusion: Our RICU had a mortality of 32% (26/80 patients) and patients who died belonged to the higher age group and had high mean APACHE II score. Although our audit is small and may not represent the cohort adequately, our mortality rate was similar to that obtained from a large cohort [1]. The higher mortality rate was probably because our centre is a tertiary-care referral hospital and many referred cases reached us late. Introduction: Central venous catheterization (CVC) is a frequently performed procedure in ICUs for both monitoring and definitive central venous access. Although the apical approach is the most preferred technique in our practice, a modified landmark guided technique that uses only the carotid artery pulsation as a landmark (paracarotid approach) to locate the puncture site for internal jugular venous (IJV) catheterization attained a high success rate with few complications. The aim of the study was to compare two approaches used for IJV cannulation: the apical approach and the paracarotid approach. The primary endpoint was the rate of success. The secondary endpoints were the related adverse events and the difficulty factors (number of attempts). Methods: A prospective, randomized clinical trial in a tertiary-care university teaching hospital. After obtaining approval from the Hospital Ethics Committee, 50 adult patients admitted to our ICU with an absolute indication for CVC were randomized to undergo one of the two techniques. We compared the demographics, success rates, difficulty factors and adverse events.
Conclusion: This study shows that the paracarotid approach of IJV cannulation is a better technique in providing a higher first-attempt success rate and has fewer complications.

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Citrate anticoagulation protocol to treat septic shock patients with liver dysfunction in CPFA extracorporeal therapy Background: Regional citrate anticoagulation has emerged in critically ill patients to safely treat patients with risk of bleeding. However, liver dysfunction may lead to citrate accumulation and change patients' acidbase status. In 10 years, 100 septic shock patients were treated with CPFA, an extracorporeal therapy that combines unselective plasma adsorption Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 resin (MediaSorb) with continuous hemofiltration. Some patients suffered from liver dysfunction (total bilirubin ≥2 mg/dl) and we treated them with our citrate protocol. The aim of this study is to evaluated the safety of citrate-like anticoagulation on patients with liver dysfunction. Methods: Four consecutive mechanically ventilated patients (three male, one female) with septic shock and liver dysfunction were treated. Prescribed CPFA parameters were: Qb 150 ml/minute, plasma flow rate (Qp) 30 ml/minute, predilution solution (Na + 136 mmol/l, citrate 10 mmol/l, citric acid 2 mmol/l) infused to keep inlet citratemia at 3 mmol/l, postdilution solution (Na + 139 mmol/l, K + 1.5 mmol/l, Ca 2+ 2 mmol/l, Mg 2+ 0.75 mmol/l, HCO 3 -35 mmol/l, glucose 5.55 mmol/l) and postdilution CaCl 2 at a rate restoring the plasma Ca 2+ to 1.1 mmol/l ( Table 1) and adjusted according to patients' need. We evaluated five clinical parameters pre and post CPFA: pH, bicarbonate, lactate, Ca 2+ /iCa 2+ , total bilirubin. Results: Twenty-two treatments were performed accounting for 163 hours, mean duration 10.2 ± 2.1 hours, mean plasma volume of 16.4 ± 5.4 l, Qb 150 ml/minute, Qp 28.4 ± 1.8 ml/minute, and a treated plasma dose/kg body weight of 0.8 ± 0.4 l/kg. Mean CaCl 2 10% infusion of 4.5 ± 1.3ml/hour, with citratemia, evaluated as total Ca 2+ /iCa 2+ ratio (Figure 1), was always <2.5 (mean 1.8 ± 0.2). For pH (mean pre 7.45 ± 0.06 vs. mean post 7.39 ± 0.07), bicarbonate (24.6 ± 3.5 vs. 24.8 ± 4.7), and lactate (2.9 ± 1.5 vs. 2.1 ± 1.6) there are no statistically significant differences between pre and post treatment. Instead we observed a decrease of bilirubin ( Figure 2). Mean SOFA and SAPS II pre CPFA were 14 ± 3 and 54 ± 17, respectively. During treatment, the acid-base patients' status were kept under control with no significant electrolyte correction (Mg 2+ , K + , Thamesol 3.6%, NaHCO 3 8.4%). Conclusion: In these four patients treated with CPFA and citrate in liver dysfunction, we have observed the absence of alteration: pH, bicarbonate, lactate and Ca 2+ /iCa 2+ . We also observed a decrease of bilirubin. Background: As mortality of patients with severe sepsis and septic shock is still inappropriately high [1], innovative therapeutic approaches are urgently needed. In the presence of hypovolemia, fluid therapy is typically initiated to compensate for intravascular volume deficits [2]. However, administration of fluids may not necessarily correct a disturbed blood flow on the microcirculatory level [3]. Microcirculatory failure during sepsis is, at least in part, caused by pathological nitric oxide (NO) levels [4,5]. To achieve an optimal NO availability, approaches including NO donors or inhibitors may be useful. The aim of the present study was to assess the ability of our test substance S-nitrosothiol-HES (S-NO-HES) to act as NO donor and exert a pharmacological activity. Methods: The investigated test substance S-NO-HES is a novel molecule consisting of NO coupled to a thiolated derivative of hydroxyethyl starch (HES). The ability of S-NO-HES to release NO was demonstrated. Furthermore, the effect of S-NO-HES on myocardial function was studied in isolated Langendorff-perfused hearts from guinea pigs and compared with that of the reference substance sodium nitroprusside. The thiolated HES derivative (SH-HES) served as negative control. In addition, isolated aortic rings from rats were pre-contracted by phenylephrine. After defined incubation periods with reference, test, or control items, the NO-induced relaxation was determined. S-nitrosoglutathione served as reference compound, HES and in one experiment also SH-HES as control substances.   At the end of the 180-minute experiment, papaverine was applied in order to completely relax the aortic rings and to define the 100% relaxation level.
Results: S-NO-HES significantly increased the heart rate of Langendorffperfused guinea pig hearts and additionally reduced both the QT interval and QTc-B values. In addition, S-NO-HES exerted a significant vasodilatory effect on phenylephrine pre-contracted rat aortic rings that was dose dependent. The effect was not only observed under light, which is known to trigger NO release from S-nitroso compounds, but also under exclusion of light and therefore more physiological conditions. Conclusion: We demonstrated for the first time that the S-NO-HES molecule released NO and exhibited corresponding pharmacological properties. In future experiments, the effectiveness of S-NO-HES to substitute NO deficiency under septic conditions has to be studied. Background: Bacterial sepsis is a very severe infection, potentially lethal, that requires an etiologic diagnosis and fairly complex treatment, quickly set. Plex-ID is the newest method in etiologic diagnoses of bacterial infections. It can detect bacterial DNA found in various pathological products: blood, CSF, joint fluid, pleural liquid. Methods: We conducted a 6-month study from January to June 2012 on children admitted to our pediatric ICU of the National Institute of Infectious Diseases for severe forms of bacterial sepsis. In these children we monitored the following parameters: sex, age and origin (nosocomial or community-acquired infection). Positive diagnosis of sepsis was established on clinical and laboratory criteria (hemocultures, CSF cultures). As well as classical cultures, we found causal agents through the newest modern technique -Plex-ID. We watched for correlation of data obtained by classical methods versus molecular methods and the clinical and biological evolution of patients under treatment Results: In the 6 months of study, 21 children met the clinical and biological criteria for bacterial sepsis. Seventy-six percent of patients came from other hospitals, the reason for which we consider that the etiology is most probably nosocomial pathogens. Sex distribution was approximately equal boys and girls, and in terms of distribution by age group prevailed in children aged 0 to 3 years (80%). We obtained 15 positive results (71%) by molecular methods that were correlated with conventional methods of diagnosis. The seat in the distribution of case etiology was as follows: Niessiera meningitides (four cases), Streptococcus pneumoniae (two cases), Staphylococcus spp. (four cases), Klebsiella pneumoniae (one case), Acinetobacter baumannii (one case), Pseudomonas aeruginosa (one case), Propionibacterium acnes (one case), and Haemophilus influenzae (one case). Conclusion: Bacterial sepsis in children is a serious condition resulting in four deaths (19%) with septic shock and multiple organ failure in our study. The patients require rapid etiologic diagnosis and establishment of appropriate emergency treatment. Plex-ID is an effective and rapid diagnosis method, identifying casual agent in 71% of cases. The major advantage of this new method is the possibility to establish the etiological diagnosis in the early hours of patient admission. Also the speed and specificity of the modern diagnosis methods increase the survival index of the hospitalized patients with severe bacterial sepsis.

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Insulin exerts anti-inflammatory effects through reduction of IKK/IB/ NF-B pathway activation in septic rats Background: During the onset of sepsis, the inflammatory system becomes hyperactive, leading to an overproduction of proinflammatory mediators, which contribute to septic shock, multiple organ failure, and death. Animal studies indicate that insulin may have direct antiinflammatory effects, independent of its effect on hyperglycemia. However, the mechanism by which insulin reduces inflammation in the absence of hyperglycemia is unknown. The aim of the present study was to investigate of the molecular mechanisms of action and response of insulin administration in relation to its anti-inflammatory effect in septic animals. We analyzed the modulation of IKK/IB/NF-B pathways and insulin signaling pathway (IR, IRS-1 and Akt) and the expression of NF-B p65 DNA binding in liver, muscle and adipose tissue in septic animals. Methods: For the experiment, diffuse sepsis was induced by cecal ligation and puncture surgery in male adult Wistar rats. The septic animals were randomly divided into four different groups. Three of them were submitted to insulin injection in the portal vein and were stimulated for 1, 3 and 5 minutes, and the other group (control) was not stimulated (saline). After the stimulation time, insulin sensitive tissues were extracted and the expression and phosphorylation of the proteins were analyzed through western blot. NF-B p65 activation was determined in nuclear extracts from muscle, liver and adipose tissue from septic rats by ELISA.
Results: In a time-course experiment we observed an increase in insulininduced phosphorylation of IR-β, IRS1 tyrosine phosphorylation, and Akt serine phosphorylation that reached a peak at 3 minutes post injection in muscle, liver and adipose tissue in septic animals. In parallel, there was a reduction in the phosphorylation of IKK in the time 1 and 3 minutes after stimulation by insulin returned to basal levels at 5 minutes. There was also a reduction in the phosphorylation of IB in the time 3 and 5 minutes after insulin injection. The DNA binding of NF-B p65 in the cell nucleus showed a reduction in expression levels of NF-B at all times investigated, in accordance with the reduction of IKK and IB phosphorylation after insulin injection in liver, muscle and adipose tissue of septic animals. Conclusion: In summary, we report evidence indicating that the acute infusion of insulin activated the PI3K-Akt pathway and reduced IKK/IB/ NF-B indicate an important molecular mechanism to the anti-inflammatory effect of the hormone in liver, muscle and adipose tissue of septic animals. This helps explain a correlation between activation of insulin signaling and its effect on the inflammatory pathway.
petechial exanthema. From the child's medical history we would like to mention: untreated perianal fissure and excised warts on terminal phalanges of his third finger of the left hand, big toe and the fifth toe of his left leg as the overgrowth and necrosis at the site of excision. Shortly after admission the patient presents an episode of acute respiratory failure followed by apnea and he is tracheal intubated and mechanically ventilated.
Results: During admission the general condition remains serious, the patient remains sedated and respiratory assisted with petechial exanthema, with hypotension, oligo-anuria, anal fissure, and necrotic lesions in the distal finger phalanges. Biological: severe pancytopenia (WBC = 700/mmc, Hb = 8.2 g/dl, platelets = 34.000/mmc), coagulation disorders, nitrogen retention syndrome, hypoalbuminemia, hepatic cytolysis syndrome, procalcitonin and inflammatory tests intensely positive. On smears made from the skin lesions, Gram-negative bacilli were visualized. The cultures of skin lesions were suggestive for P. aeruginosa, confirmed after 48 hours by blood culture identification and PCR determination (Plex-ID). We established comprehensive treatment: antibiotic (meropenem, ciprofloxacin, linezolid), antifungal, inotropic drugs (dobutamine, norepinephrine), ENP (rebalance acid-base and electrolyte), human immunoglobulin, blood and blood products transfusions and Neupogen. The evolution was favorable to the resumption of diuresis after 24 hours, increasing white blood cells and the lesions diminished.
Conclusion: Positive diagnosis of severe sepsis was based on clinical and laboratory findings. We have established the likely starting point of the patient's severe condition to be the multiple skin lesions and untreated anal fissure. Although the patient was immunocompetent, he developed a severe form of sepsis with septic shock caused by P. aeruginosa hardly responsive to treatment. We believe that the patient's favorable development was due to this germ's increased sensitivity to antibiotics, most probably community acquired. Background: Polymicrobial infection is associated with systemic inflammatory response, which is involved in the pathogenesis and development of acute kidney injury (AKI) [1,2]. In this study, we examined the effect of sesamol against AKI in cecal-ligation-and-puncture (CLP)treated rats. Methods: Rats were given two subcutaneous doses of sesamol (10 mg/kg) 0 and 6 hours after CLP. Serum and kidney tissue were sampled 12 hours after CLP. Renal function and proinflammatory mediators, such as IL-1β, IL-6, and nitrite production were detected. Systemic oxidative stress was assessed by determining nitric oxide, superoxide anion, and xanthine oxidase activities. In addition, inducible nitric oxide synthase (iNOS) expression was also assessed in leukocytes from rats with AKI.
Results: The levels of serum BUN, CRE, IL-1β, IL-6, nitrite, iNOS expression, superoxide anion, and xanthine oxidase activity were significantly higher in rats after CLP. Sesamol significantly inhibited all parameters in CLP-treated rats.
Conclusion: Sesamol attenuated AKI by inhibiting neutrophil-initiated systemic inflammation and oxidative stress in CLP-treated rats. Background: Neurotransmitters derived from the autonomic nervous system can regulate inflammatory cytokine secretion. This has been extensively studied in the parasympathetic nervous system, where acetylcholine regulates the secretion of TNF from splenic macrophages during LPS-induced inflammation. However, the role of noradrenergic neurons during mouse models of infection has not been characterized. The goal of these experiments was to study the influence of noradrenergic neurons on the immune response during Gram-negative septic peritonitis in mice. Methods: Peripheral noradrenergic nerves were ablated using 6hydroxydopamine (6-OHDA), a commonly employed method for studying noradrenergic neurons. Four days later, septic peritonitis was induced by i.p. injection of 150 CFU Klebsiella pneumoniae. Survival, serum and peritoneal bacterial loads, inflammatory cytokine production and leukocyte recruitment were studied at multiple time points after infection. To assess the importance of the NE containing splenic nerve, survival experiments on splenectomized mice with or without 6-OHDA treatment were performed. Results: Ablation of noradrenergic nerves improved survival following K. pneumoniae septic peritonitis ( Figure 1A). Mice in which noradrenergic nerves had been ablated showed a more robust immune response 4 hours after infection with higher systemic IL-6, higher intraperitoneal MCP-1 and a fourfold increase in monocyte recruitment into the peritoneum. Bacterial loads were lower 24 and 48 hours after infection in mice in which noradrenergic nerves had been ablated ( Figure 1B). Four hours after infection, 6-OHDA-treated mice recruited more inflammatory monocytes to the infected peritoneum ( Figure 1D, CTRL vs. 6-OHDA). Splenectomy prior to noradrenergic nerve ablation abrogated the beneficial effects of 6-OHDA during infection ( Figure 1C) and reduced the recruitment of monocytes to the infected peritoneum ( Figure 1D, 6 Background: Staphylococcus aureus is a major cause of bloodstream infections (BSI), and is associated with a higher morbidity and mortality compared with other BSI pathogens. Innate pattern recognition receptors like mannose-binding lectin (MBL) of the complement system and NOD2 (nucleotide-binding oligomerization domain-containing protein 2), an intracellular sensor for a variety of pathogens, have been shown to be crucially involved in the immune response against S. aureus in knockout animal models [1,2], but human data are lacking. Low MBL levels and NOD2 mutations can be found in up to 30% and 10% in the general population, respectively [3,4]. This study aimed to investigate whether MBL deficiency and NOD2 mutations predispose to and influence the severity of S. aureus BSI. Methods: A matched case-control study was undertaken involving 70 patients with S. aureus BSI and 70 age-matched and sex-matched hospitalized controls recruited prospectively at two major tertiary hospitals. Participant blood samples were analyzed for MBL levels by mannan-binding ELISA and for four MBL2 and three NOD2 polymorphisms by real-time PCR. Clinical and microbiological data were reviewed. MBL deficiency was defined as functional MBL level ≤0.1 μg/ml. Univariate and multivariate conditional logistic regression was used to investigate the risk of BSI in matched controls and cases. Results: S. aureus BSI were nosocomially acquired (60%) and intravenous catheter associated (50%) in the majority of cases with an in-hospital mortality of 10%. After adjusting for diabetes, immunosuppression, chronic kidney disease and long-term intravenous catheters, MBL deficiency was found less frequently in cases than controls (8.6% vs. 20%, OR = 0.38, P = 0.07) as were low-producing MBL genotypes (11% vs. 23%, OR = 0.37, P = 0.05), whereas NOD2 polymorphisms were similarly distributed (14% vs. 10%, P = 0.4). In line with MBL2 genotypic results, MBL levels were significantly higher in cases than in controls (adjusted OR = 1.35 per 1 μg/ml increase, P = 0.002; Figure 1). Cases with NOD2 polymorphisms had less severe disease manifestations as shown by a lower SOFA score (mean 2.1 vs. 4.4, P = 0.04) and a reduced rate of multiorgan dysfunction and death (40% vs. 60%, P = 0.06), whereas MBL deficiency had no influence on the severity of S. aureus BSI. Conclusion: Neither MBL deficiency nor NOD2 polymorphisms were associated with an increased risk of S. aureus BSI. In fact, contrary to experimental data, MBL deficiency seemed to confer protection in acquiring S. aureus BSI, and NOD2 mutations were less frequently associated with multiorgan dysfunction in this matched case-control study. Background: Plasminogen activator inhibitor type 1 (PAI-1) inhibits fibrinolysis and its plasma increases correlate with exacerbated sepsis mortality. However, its exact role in abdominal sepsis outcomes Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 is unclear. We aimed to test the effects of fibrinolysis restoration upon survival during acute polymicrobial sepsis using a custom-developed anti-PAI-1 monoclonal antibody. We specifically studied whether the effects of the treatment vary between all-inclusive populations and homogeneous cohorts stratified based on the risk of death. Methods: Mice underwent cecal ligation and puncture (CLP) and received a single intravenous injection of either anti-PAI-1 (10 mg/kg; MA-MP6H6) or control antibody (MA-Control). Three approaches were used: approach I, co-treatment (CLP and MA-MP6H6 administered at 0 hours) without stratification; approach II, post treatment (MA-MP6H6 at 18 hours post CLP) without stratification; and approach III, post treatment (MA-MP6H6 at 30 hours) with prospective stratification. Then 20 μl blood (facial vein) was sampled daily from all mice until day 5 and survival was followed for 28 days. In approach I, a group of mice was sampled at 6 hours and sacrificed at 24 hours to test the MA-MP6H6 efficacy. In approach III, mice were stratified into either predicted to survive (P-SUR) or die (P-DIE) based on circulating IL-6 measured at 24 hours post CLP. Results: MA-MP6H6 co-treatment blocked 60 to 100% of active plasma PAI-1 and restored fibrinolysis (fibrin plate assay) in septic animals at 24 hours post application. Without stratification, MA-MP6H6 co-treatment and 18-hour (also 30-hour) post treatment neither conferred benefit nor was detrimental. Post treatment with MA-MP6H6 after prospective IL-6 stratification at 14 ng/ml cutoff (48% sensitivity for P-DIE and 100% specificity for P-SUR) showed 15% exacerbation of mortality (P > 0.05). Cutoff readjustment to 3.3 ng/ml maximized P-DIE cohort homogeneity (82% sensitivity; 100% specificity) revealing harmful MA-MP6H6 effects: in P-SUR, no MA-Control-treated mice ever died, but 30% of MA-MP6H6treated did (P < 0.05). In P-DIE, all MA-MP6H6-treated mice died within 72 hours, while MA-Control mice lived up to 8 days (P < 0.05). In cotreatment, MA-MP6H6 increased circulating IL-6 and CXCL-1/KC by threefold and twofold at 24 hours (MCP-1, MIP-1α, IL-1β, IL-10 and TNFα unaffected), but had no effect in post treatment (24 to 96 hours). Regardless of the treatment approach, MA-MP6H6 did not significantly alter hematological and/or organ function parameters. Conclusion: Restoration of fibrinolysis by neutralization of PAI-1 in acute sepsis lacks benefit and may prove fatal. Remarkably, the detrimental effect of MA-MP6H6 became apparent only after a maximally homogeneous separation of high/low risk-of-death cohorts. A prospective separation of septic patients into various well-defined, homogenous cohorts is critical in revealing negative side effects of tested anti-sepsis therapeutics. Acknowledgements: Supported by WWTF Grant LS07-065. Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 technique for differential diagnostics of heparin-induced thrombocytopenia is using 4T scales [1]. Methods: An observational retrospective cohort study of 106 patients in the period from 2010 to 2011. The aim of the study was to determine the effect of heparin on the severity of clinical signs of heparin-induced thrombocytopenia in patients with severe sepsis. Inclusion criteria were the presence of severe sepsis in a patient (Surviving Sepsis Campaign 2008), the need for extracorporeal blood purification, and thrombocytopenia. The mean age of all the patients was 54 ± 15.91, mean SOFA score was 7.4 ± 0.9 (2 to 13). Reduced platelet counts were observed in 23 patients. Patients at high risk for heparin-induced thrombocytopenia type II (4 to 5 points for the 4T scale) in the study were not included. Ten of the patients (9.4%) conducted a session intermittent high-volume hemofiltration (group IHVH) within 4 hours, c volume replacement of 100 ml/kg/hour. Eleven patients (10.4%) did not receive extracorporeal blood purification (group Stand), because of uncontrolled bleeding. All patients received therapy according to Surviving Sepsis Campaign 2008. The standard dose of heparin was 8 to 15 units/kg/hour. Results: In group IHVH the increase in platelet count at the end of the second day treatment was 105.75 ± 52.8 × 10 9 /l ( Figure 1). This level is significantly different from the number of platelets in group Stand to the same phase of the study (10.7 ± 4.0 × 10 9 /l). During treatment in the groups studied there were no thromboses. The 28-day period mortality in group IHVH was 20% (two patients), and in group Stand was 9% (one patient). Conclusion: The use of heparin, including the extracorporeal blood purification, can be safe with heparin-induced thrombocytopenia type I in patients with severe sepsis. Background: Recent improvements in the treatment of severe Gramnegative sepsis have not resulted in a substantial decrease in mortality. LPS, originating from Gram-negative bacteria, is a major mediator in the development of septic shock [1]. Decreasing the load of LPS in these patients may be beneficial for these patients. The Alteco LPS Adsorber® selectively binds the lipid A moiety of LPS. Positive effects of the adsorber have been reported [2][3][4].
Methods: Nineteen patients with Gram-negative sepsis were treated with the Alteco LPS Adsorber®, in addition to standard therapy according to SSC Guidelines 2008. All patients needed inotropic support and mechanical lung ventilation. The mean APACHE II score at the start of treatment was 25.9 ± 1.8. The time to initiating the perfusion varied between 1.7 and 8.6 hours after the diagnosis of septic shock. The treatment lasted 120 minutes and was repeated after 24 hours. The SOFA score, oxygenation index (PaO 2 /FiO 2 ) and the dose of dopamine was noted before and 48 hours after the treatment.
Results: At baseline, the severity of MODS was SOFA score 9.2 ± 2.8. At 48 hours the mean score SOFA was 4.3 ± 2.7, at the expense of an increase in the index of oxygenation from 128.6 ± 36.2 to 253.5 ± 44.8; and a decrease in doses of inotropic support (dopamine) from 17.1 ± 1.8 mkg/kg/minute to 4.2 ± 1.8 mkg/kg/minute. We found an inverse correlation between the time to initiate treatment with the adsorbtion and the decrease in SOFA score (R = -0.69; P < 0.1) (Figure 1). The degree  Figure 2. The 28-day mortality was 26.3% in this patient group. There were no adverse events related to use of the adsorber. Conclusion: Since LPS is a major mediator in Gram-negative sepsis, there is a rationale for rapid removal of LPS from patients with sepsis. Early initiation of LPS adsorption is most effective for reducing the signs of MODS. Our experiences with the use of Alteco LPS Adsorber® are promising as shown by our data above and are in support of previous findings [2][3][4]. The results are limited by the lack of controls; however, we believe that further randomized controlled clinical studies using this therapy are warranted, more so in view of the fact that no specific treatment against septic shock is available. Background: The Surviving Sepsis 6-hour bundle [1] was created to promote effective identification and management of patients with severe sepsis and septic shock. Efficient and timely implementation of the 6-hour bundle including early intravenous antibiotics has been shown to improve patient outcome [2]. We aimed to determine the efficiency of severe sepsis identification, implementation of the 6-hour bundle and overall management of ward-based patients. Methods: During a 4-month period, ward-based patients with severe sepsis were identified by nurse practitioners and the critical care outreach team at a large teaching hospital in North West England. Analysis of patient medical records was performed to assess the efficiency and quality of care received compared with the gold standard sepsis 6-hour bundle. Promptness of doctor attendance and patient 30-day outcome were also analysed. Results: Twenty-eight patients with severe sepsis were identified and analysed. Suspicion of sepsis was documented in medical records for all patients; however, the 6-hour sepsis bundle was completed in only one case. Appropriate i.v. antibiotics were prescribed to all patients. Median duration from the documented onset of severe sepsis (time 0) until review by a doctor was 90 minutes (0 to 19 hours). The median Modified Early Warning Score (MEWS), determined from patients' vital signs, at documented onset of severe sepsis was 3.5 (1 to 9) with a subsequent increase of median MEWS to 4 by the time of doctors' first attendance. The 30-day patient outcome showed 13 patients discharged home and seven patients deceased. Comparison of deceased patients with patients who were discharged alive (see Table 1) demonstrated an increased median duration from time 0 until doctor attendance in the deceased group. However, interestingly, timely antibiotics (given within 1 hour of patient assessment by a doctor) were administered with greater frequency in the deceased patient group. Conclusion: Despite the widespread recognition of patients with severe sepsis, underperformance of the 6-hour bundle remains a major factor in The sensitivity and negative predictive value of the neutrophil CD64 assay was consistently ≥90% in identifying infected and non-infected infants. In fact, during the 3-year analysis period only three infants were identified with positive blood cultures that had a normal CD64 index. Two of these infants had blood cultures that were positive for Staphylococcus epidermidis and one had a positive blood culture that was obtained from a colonized central line. Although absolute CD64 levels did not correlate with severity of illness in our population (as determined by the need for ventilation (P = 0.87) or inotropic support (P = 0.90)), relative decreasing CD64 levels did correlate well with the resolution of infection within a given infant. When compared with infants with an elevated CD64 level, a normal CD64level decreased unnecessary antibiotic exposure by 3.9 days. By comparison, in infants evaluated with the traditional methods that did not include a CD64 level, a normal CBC only decreased antibiotic exposure by 1 day when compared with infants with an abnormal CBC. Conclusion: In summary, the clinical care that we provide our infants has improved with the use of neutrophil CD64 levels in our infectious evaluations.

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Pancreatic stone protein as a novel marker for neonatal sepsis L Schlapbach 1* , R Graf 2 , A Woerner 3  Background: Early-onset sepsis (EOS) represents one of the main causes for ICU admission in newborns and therefore imposes a considerable burden on the healthcare system. Performance of traditional infection markers to diagnose EOS is poor and therefore insufficient to guide the decision to start or stop antibiotic treatment. While procalcitonin (PCT) has become an established sepsis marker in adults and children, sensitivity and specificity of PCT are only moderate in EOS due to the physiologic PCT increase during the first days of life. Pancreatic stone protein (PSP) is a promising sepsis marker in adults. This study investigated whether determining PSP improves diagnosis of EOS in comparison with other infection markers.
Methods: A prospective multicentre study including 137 infants >34 weeks gestational age admitted with suspected EOS. PSP, PCT, soluble human triggering receptor expressed on myeloid cells-1 (sTREM-1), macrophage migration inhibitory factor (MIF) and C-reactive protein (CRP) were measured at admission. Receiver-operating characteristics (ROC) curve analysis and multivariate logistic regression were performed. Bioscores were constructed using two, three and four sepsis markers.
Results: PSP was significantly higher in infected compared with uninfected infants (median 11.3 vs. 7.5 ng/ml, P = 0.001). The ROC area under the curve resulted at 0.69 (95% CI = 0.59 to 0.80, P < 0.001) for PSP, at 0.77 (95% CI = 0.66 to 0.87, P < 0.001) for PCT, at 0.66 (95% CI = 0.55 to 0.77, P = 0.006) for CRP, at 0.62 (0.51 to 0.73, P = 0.055) for sTREM-1 and at 0.54 (0.41 to 0.67, P = 0.54) for MIF. In multivariate models, increased PSP levels showed the strongest association of all markers with EOS and PSP >9 ng/ml independently of PCT predicted EOS (odds ratio = 26.4, 95% CI = 4.0 to 172.5, P < 0.001). Combining both markers significantly increased the ability to diagnose EOS (P < 0.001). The bioscore based on PSP and PCT performed best with an AUC of 0.83 (95% CI = 0.74 to 0.93, P < 0.001) and was superior Introduction: High morbidity and mortality rates are associated with sepsis in newborns, as well as low recovery rates, extended recovery times, and delayed culture times of blood culture. To surmount these parameters, new molecular techniques are required by the clinical laboratory. The LightCycler Septifast® technique, which identifies up to 25 microorganisms known to cause over 90% of admissions to ICUs due to infection, has proven its usefulness in adult patients in several countries. The objective was to standardize the Septifast® Roche technique for diagnostic use in newborns with suspected sepsis. Methods: Eighty-six newborn samples with suspected sepsis (according to the NOSEP-1 scale) were included. We analyzed two blood samples per patient, the first one collected in an EDTA-anticoagulated tube (0.5 to 1.0 ml), and a second sample of 1 cm 3 in a hemoculture tube for automated hemoculture procedure using BacT/ALERT®3D (Biomerieux). Briefly, the whole sample was lysed with MagNA Lyser (Roche), and nucleic acid purification was performed with Septifast prep M grade (Roche). DNA from Gram-negative, Gram-positive, and fungi was detected using multiplex LightCycler 2.0 real-time methodology with LightCycler SeptiFast M grade kit (Roche). Finally, results analysis was performed with Roche Septifast identification software (SIS). Results: Of the 86 samples analyzed, 31 (36.04%) were positive in at least one of the identification procedures. Thirteen samples (15.11%) were rendered positive by hemoculture, and 27 (31.39%) by Septifast® protocol. Fifty-seven samples (66.20%) were negative by both analytic procedures, and a total of 31 pathogens (15 Gram-negative, 11 Gram-positive and five fungi species) were identified by Septifast® (four of them in combination) versus nine species recovered from hemocultures. The response time ranged from 6 to 24 hours by Septifast® procedure, compared with 4 to 7 days by hemoculture detection. Conclusion: The use of the multiplex real-time PCR Septifast® technique allows one to detect a wider number of pathogenic species and a faster response time than hemoculture, with a small quantity of blood sample (1 ml). Background: Endotoxic shock during infection by Gram-negative bacteria could frequently lead to mortality. Bacterial endotoxins (lipopolysaccharides (LPS)) play a major role in pathogenesis of Gram-negative sepsis. The infection after injury, burn or surgery could lead to accumulation of released endotoxins in the bloodstream. LPS are able to interact with specific receptors on the surface of target host cells (monocytes/macrophages, neutrophils, endothelial cells, and so forth). After interaction with LPS, phagocytes change their morphological and functional properties, namely receptor expression, synthesis of proinflammatory cytokines and phagocytic activity. In the LPS signaling pathway, Toll-like receptor 4 (TLR4, CD284) is the main cell surface molecule inducing activation of NF-B-dependent genes. Recent reports suggest that TLRs are involved in phagocytosis as the assistance receptors of phagocytes helping them to recognize invading bacteria or their molecular patterns [1,2]. Intensity of phagocytosis is potentiated by prior neutrophil exposure to endotoxins [3]. TLRs ligation by endotoxin molecules induces adaptor protein MyD88-dependent signaling involving IL-1 receptor-associated kinase (IRAK) and p38 mitogen-activated protein kinase leading finally to upregulation of phagocytosis-inducing genes. The integrated collaboration between TLR4 and phagocytosis has been shown early [4]. As shown during phagocytosis, TLR4 was only integrated in this functional property of the cells but not involved as the primary receptor [5]. It is well known that appropriate cell response to LPS is Figure 1(abstract P80) Influence of antibodies against TLR4, CD14 and CD11b on neutrophil phagocytic activity in whole blood.

Control cells without any incentives (C), cells activated by S-LPS (C1) and cells activated by Re-LPS (C2).
Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 provided by engagement of CD14, TLR4 and CD11b receptors. Thus, we have studied the functional role of these receptors in phagocytosis of fluorescein-labeled Escherichia coli by human neutrophils in whole blood activated by S-form or Re-form endotoxins from E. coli. Methods: S-LPS from E. coli O55:B5 (Sigma) and Re-LPS from E. coli JM103 were obtained in our laboratory according to standard procedure. Fluorescein-labeled bioparticles E. coli K12 (Molecular Probes), mouse antihuman TLR4 mAbs HTA125 (IgG2a isotype; Serotec), anti-human CD14 clone UCHM-1 mAbs (IgG2a isotype; Sigma), and anti-human CD11b mAbs clone 44 (IgG1 isotype; Sigma). LPS were dissolved in pyrogen-free water to make 10 μg/ml stock solution, and sonicated vigorously for 20 minutes to increase LPS solubility. Experiments were performed in whole blood from nine donors (male, female, age 20 to 27). Blood was collected in sterile conditions into Monovette heparin-treated tubes 10 U/ml (Sarsted, Germany). Each tube contained 90 μl whole blood and 50 μl suspension of FITC-labeled bacteria (2 × 10 7 cells/ml; 1:10 leukocytes: bacteria ratio) was gently mixed and incubated for 30 minutes at 37°C. Then erythrocytes were lysed by hypotonic buffer for 5 minutes at room temperature. Samples were centrifuged (5 minutes, 200 × g, 21°C) and washed twice by cooled PBS containing 0.02% EDTA. The pellet was resuspended in 400 μl PBS-EDTA solution. To assess the influence of cell activation on phagocytosis the 100 ng/ml of S-LPS or Re-LPS were added before FITC-labeled bacteria and incubated during 30 minutes at 37°C. To determine the role of CD14, TLR4 and CD11b receptors in phagocytic activity, corresponding mAbs (1 μg, 30 minutes) were added to whole blood before cell activation by LPS and induction of phagocytosis. The phagocytic activity was monitored using the EPICS XL-MCL flow cytometer (Beckman Coulter). Results given as median fluorescence intensity. Phagocytic activity of cells without LPS activation or mAb treatment was established as 100%. Experiments were done two times using duplicate determinations in each experiment. The mean ± standard error was calculated using standard calculations available in the OriginPro spreadsheet. Statistically significant differences were determined by Student's t test.
Results: Phagocytic activity of leukocytes was increased by pre-exposure of whole blood to S-LPS or Re-LPS (Figure 1). Pretreatment of whole blood by anti-CD14 or anti-TLR4 mAbs decreased to some extent the phagocytic activity of leukocytes activated by endotoxins independently of their glycoform (Figure 1). Significant inhibition of phagocytosis using anti-CD11b mAbs was achieved (Figure 1). Abs against CD11b more pronounced decreased S-LPS activated phagocytosis by 30% but were less effective in the case of Re-LPS (by 15%). It is known that CD11b receptors play an important role during phagocytosis of opsonized bacteria. Conclusion: LPS activate phagocytosis of Gram-negative bacteria independently of their glycoforms (Figure 1). The effect of HTA125 mAbs may be explained by their interaction with TLR4 leading to inhibition of LPS-induced signaling into the nucleus and blocking synthesis and surface expression additional receptors such as class A macrophage scavenger receptor (SR-A) [6] and CD11b [7] involved in phagocytosis. Activation of integrin-dependent signaling pathways can also be blocked by these HTA125 mAbs [8]. Anti-CD11b decreased most pronounced LPSactivated phagocytosis. Taking these results into consideration one can speculate that the Fc-chain of anti-CD11b (IgG 1 ) mAbs through interaction with CD32A may mediate downregulation of TLR4 responses. Background: Delayed neutrophil apoptosis is often detected in inflammatory pathologies, including sepsis [1]. CD24 is a small heavily glycosylated cell-surface protein, linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor in a wide variety of cells [2]. Cross-ligation of CD24 triggers apoptosis in a human B-cell subset during the early activation stage [3]. Since CD24 is also expressed on neutrophils, we aimed to study its expression in sepsis and its putative role in apoptosis.
Methods: Blood samples were either collected from healthy donors or from sepsis patients at the onset of sepsis and the two following days and surface CD24 expression on neutrophils was analyzed by flow cytometry. Peripheral blood neutrophils were purified from sepsis patients and healthy individuals by positive selection. Whole blood or neutrophils were challenged with lipopoplysaccharide (LPS), TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF), heat-killed Staphylococcus aureus or heat-killed Escherichia coli and CD24 expression assessed by flow cytometry. Neutrophils were cross-linked with anti-human CD24 and assessed for apoptosis after 24 hours of culture. In some experiments, neutrophils were preincubated with caspase inhibitor (z-VAD-fmk) before crosslinking. Results: Surface expression of CD24 assessed by flow cytometry was significantly altered in neutrophils from sepsis patients compared with healthy controls. Activation of neutrophils with LPS or heat-killed bacteria in whole blood triggers a strong upregulation of CD24 at surface level despite no enhanced expression being observed when the activation was carried out in purified neutrophils. In contrast, TNF and GM-CSG upregulated CD24 in whole blood and in purified neutrophils. CD24 cross-ligation induces caspase-independent apoptosis in human neutrophils. Ex vivo death responses after CD24 ligation in neutrophils from sepsis patients are currently under study. Conclusion: This is the first report studying the role of CD24 in sepsis patients, linking homeostasis and apoptosis. various intestinal and extraintestinal diseases. It is a known fact that the digestive tract may serve as a portal of entry into the bloodstream. Therefore, the presence of highly resistant E. coli strains in the gut presents a threat to the patients with predispositions such as chronic illnesses and poor immune status. Methods: This study was undertaken with the aim to determine the resistance pattern among the E. coli strains isolated from the gut of chronically ill patients across wide clinical settings. The study was conducted over a period of 1 year from 1 January 2011 to 31 December 2011. Stool samples from patients admitted for more than 14 days in wards of all major clinical specialities were collected after proper counselling and informed consent. E. coli were identified on the basis of cultural characteristics and biochemical reactions. Strains isolated from pediatric patients were subjected to serotyping by the slide agglutination test with specific antisera (Denka Seiken Co., Ltd, Tokyo, Japan) to identify the enterovirulent strains. All E. coli strains were subjected to antimicrobial susceptibility testing and ESBL identification by the disk diffusion methods in accordance with the CLSI guidelines. Results: Two hundred and fifty-four patients were included in the study with the following distribution: 71 from a pediatrics ward, 62 from a general medicine ward, 54 from a general surgery ward, 42 from a gynaecology ward, 16 from an orthopaedics ward and nine from an ENT ward. E. coli was isolated from 112 samples. Out of 34 E. coli strains isolated from paediatric patients, 14 were determined to be enterovirulent E. coli by serotyping. Antimicrobial susceptibility testing of all E. coli strains showed 100% resistance to nalidixic acid and a high degree of resistance to ampicillin (87.5%), doxycyline (83.0%), cotrimoxazole (75.9%), ciprofloxacin (73.2%) and third-generation cephalosporins (71.4%). ESBL production was detected in 71 strains (63.4%). However, no resistance was found for carbapenems and tigecycline. Conclusion: A large population of chronically ill patients who were tested was found to be carrying highly resistant E. coli in their guts. These usually commensal strains may serve as a source of bloodstream infection especially in cases of immunosuppression. Whether these strains were acquired in the hospital or from the community needs to be studied further. Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.

P84
Natural killer cell status and tolerance in mouse and human bacterial sepsis Background: As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as Tolllike receptors (TLRs). Natural killer (NK) cells contribute to inflammatory processes by producing proinflammatory cytokines such as IFNγ and GM-CSF [1]. Our aim was to characterize the immune status of NK cells in a murine model of sepsis and in patients with systemic inflammatory response syndrome (SIRS) and sepsis. Methods: Cecal puncture (CP) was employed as a murine model of polymicrobial sepsis. TLR expression in murine and human NK cells was studied by flow cytometry. Ex vivo IFNγ production was analyzed either by ELISA or by flow cytometry. Results: In mice, the expression of TLR2 and TLR4 in spleen NK cells is mainly intracellular, similarly to TLR9. In vitro cell responsiveness of purified NK cells to TLR2, TLR4 or TLR9 agonists, in synergy with accessory cytokines (IL-2, IL-15 and IL-18), allowed a significant production of IFNγ and GM-CSF. In contrast, NK cells, purified from spleen of mice with sepsis, showed a dramatic reduction of their capacity to produce cytokines in response to TLR agonists. Depletion of regulatory T cells (Tregs) before CP led to a complete reversion of NK cell tolerance to TLR agonists. IL-10 and TGF-β1 are two main inhibitory cytokines produced by Tregs. We showed in vivo, using IL-10 knockout mice and by inhibiting TGF-βR signaling, that the tolerization mechanism of NK cells was mostly mediated by TGF-β [2]. In humans, the expression of TLR2, TLR4 and TLR9 in peripheral blood NK cells (both CD3 -CD56 high and CD3 -CD56 dim subsets) was mainly intracellular. The ex vivo responsiveness of the blood NK cells to their agonists in synergy with accessory cytokines (IL-15 and IL-18), allowed a significant secretion of IFNγ. Similar to the murine model of sepsis, in SIRS and sepsis patients the secretion of IFNγ by NK cells was significantly decreased. Conclusion: NK cells express TLR2 and TLR4 intracellularly, as already reported for other cell types (epithelial, endothelial, and dendritic cells). Furthermore, NK cells undergo tolerance to TLR agonists during SIRS or sepsis, as already described for monocytes in these clinical settings.
Background: Enterococcus faecalis gained importance in the last years for causing tough and troublesome nosocomial infections especially in the urinary tract, sometimes even leading to sepsis. Causes of concern are increasing resistances of enterococci. Therefore, it is of utmost interest to identify resistance patterns of bacteria within a very short timeframe to select the right therapy and efficiently prevent spreading. Within our junior research group we want to develop an on-chip device to probe antibiotic susceptibility patterns of sepsis pathogens based on optical spectroscopy without the need for time-consuming bacterial cultivation. In this contribution, we present our first results with a focus on E. faecalis. Methods: E. faecalis was grown in liquid culture and characterized in the presence and absence of vancomycin by means of Raman spectroscopy. Multivariate statistical analysis was applied to extract the spectral differences due to antibiotic treatment. To enable the spectroscopic analysis directly with patient samples (such as urine) Raman spectroscopy was combined with dielectrophoresis. Bacteria from suspensions were captured and kept at well-defined positions in a nonuniform electric field for the time of Raman measurement. Currently, bacteria-spiked model urine is used. However, the investigations shall be extended to urine from patients. Results: A clear distinction between Raman spectra of E. faecalis with and without antibiotic treatment is possible even 30 minutes after incubation with vancomycin. A quadrupole electrode design is presented that allows the efficient capturing of E. faecalis and Escherichia coli, by means of negative dielectrophoresis [1] (Figure 1a). From the captured bacteria in solution on the dielectrophoretic chip, high-quality Raman spectra have been recorded within 1 second (Figure 1b). These spectra allowed a reliable differentiation of the two commonly encountered bacterial species in urinary tract infections: E. faecalis and E. coli. First steps have been undertaken to implement such dielectrophoretic capturing structures into a microfluidic device for simplified sample handling. Conclusion: Raman spectroscopy in combination with statistical analysis holds the potential for a fast evaluation of bacterial antibiotic susceptibility without the need of time-consuming cultivation. Background: Hemodynamic monitoring plays a key role in the early recognition, optimization of interventions and monitoring of therapeutic response in children with septic shock. Assessing the need for fluids followed by rapid and timely fluid resuscitation is crucial for improved outcomes. It is in this context that fluid responsiveness, defined as an increase in cardiac output in response to a fluid challenge, assumes importance. We have done this prospective clinical study to evaluate the degree of IVC diameter variability in predicting fluid responsiveness (increment in stroke index ≥15%) in children with septic shock post 20 ml/kg of crystalloid (0.9% saline) resuscitation.
Methods: A total of 166 episodes of preload responsiveness check were echocardiographically evaluated in 41 children with septic shock. In each episode, IVC diameter variability ((maximum -minimum IVC diameter)/ maximum IVC diameter), stroke index and ejection fraction were assessed at two points (before preload T0 and after preload T1). Adequate sedation was ensured before each echocardiographic assessment. Infants <1 month of completed age, any clinical evidence of increased intraabdominal pressure, children with previously diagnosed heart disease, requiring high-frequency ventilation, and for whom family did not give consent were excluded from the study. Results: Of the 166 episodes, 120 (72%) were fluid responsive and 46 (28%) were nonresponsive. One hundred and twenty-five episodes occurred on patients who were on positive pressure ventilation, whilst 41 occurred during spontaneous breathing. The decrease in the heart rate with preload was significant in the responsive as compared with the nonresponsive group (24 ± 9 vs. 4 ± 7; P = 0.001) independent of the ventilator support. IVC diameter variability at T0 correlated significantly (r = 0.39; P = 0.001) with stroke index increment following preload. The AUC of ROC for IVC diameter variability was 0.75 (0.66 to 0.85). A cutoff value of 14% variability showed 84.4% sensitivity and 65.9% specificity to positively predict fluid responsiveness in ventilated as well in spontaneously breathing children.
Conclusion: IVC diameter variability showed a significant correlation with stroke index increment after preload and can act as a useful bedside tool in predicting preload responsiveness in children with septic shock. Utility of a serial ΔIVC percentage instead of a single measurement to assess volume changes needs to be explored. Background: Rapid and timely fluid resuscitation is key to improved outcomes in septic shock. Hemodynamic monitoring is vital for preload assessment. Central venous pressure (CVP) is one such traditional tool that is currently falling out of favor as adult data have shown it to be less reliable in predicting preload responsiveness. Pediatric data are, however, limited. We conducted this study to assess the predictive utility of CVP in determining the preload responsive status defined as a stroke index increment (≥15%) after preload (20 ml/kg 0.9% saline) administration.
Methods: A total of 166 episodes of preload administrations were included. Hemodynamic variables (heart rate, mean arterial blood pressure, CVP) were measured before (T0) and after (T1) preload administration in all. Both spontaneously breathing and mechanically ventilated children were enrolled. HR decrement and CVP increment as response to preload were calculated using T1 and T0 measurements. The stroke index was measured by transthoracic echocardiography at T0 and T1. Infants <1 month of age, improper position of central venous catheter tip, clinical evidence of increased intra-abdominal pressure, previously diagnosed heart disease, need for high-frequency ventilation, and children in whom family had declined consent were excluded from the study.
Results: Of the 166 episodes, 120 (72%) were fluid responsive and 46 (28%) were nonresponsive. One hundred and twenty-five episodes occurred in patients on positive pressure ventilation and 41 during spontaneous breathing. The internal jugular vein was catheterized in 34 (142 episodes; 85%) and the femoral vein in seven patients (24 episodes; 14.5%). There was a significant decrease in heart rate independent of ventilator support in the preload responsive as compared with the nonresponsive group (24 ± 9 vs. 4 ± 7; P = 0.001). However, there was no significant difference in CVP between the fluid responsive and nonresponsive episodes (6.2 ± 1.5 vs. 6.0 ± 1.4; P = 0.71), although CVP was significantly higher in mechanical ventilation as compared with episodes on spontaneous breathing (6.5 ± 1.3 vs. 5.2 ± 1.6; P = 0.001). Baseline CVP (T0) (r = 0.01; P = 0.10) and CVP increment at T1 (r = 0.18; P = 0.14) showed a poor correlation with stroke index increment. Conclusion: CVP increment with preload showed a poor correlation with stroke index increment, suggesting that CVP measurements are not useful in predicting a preload responsiveness state in critically ill children. Decrement in heart rate was the only conventional hemodynamic variable that signified preload response but that also in retrospect, thus limiting its utility as a predictive tool. lung-protective ventilation, 22.1% (15/68). Compliance rates for the entire resuscitation and management bundles were 6.8% (11/162) and 8% (13/162) respectively. There was a trend to improving survival with bundle compliance. Compliance with the resuscitation and management bundles was associated with mortality rates of 18.2% and 19%, respectively, while mortality rates with noncompliance were 39.7% and 41.1%. Of the resuscitation bundle elements, achieving targets for fluid resuscitation and vasopressors was associated with survival, but this disappeared when adjusted for APACHE II score. Of the elements in the management bundle, achieving glucose target and low tidal volume ventilation in patients with acute lung injury as well as the APACHE II score were independently associated with survival. Conclusion: While mortality from severe sepsis is high, compliance to the resuscitation and management bundles is generally poor in ICUs in India. We also carried out daily process surveillance including compliance to hand hygiene, the ventilator bundle and the central line care bundle. We then introduced the following interventions: routine use of heat-moisture exchanger filters and closed suction systems to reduce VAP, and use of antibiotic-coated central venous catheters to reduce CLABSI. We also reinforced general hygiene practices for all ICU personnel and use of checklists. We then remeasured VAP and CLABSI rates and process compliance over the next 1 year, from March 2010 to February 2011 (period 2). Results: In period 1, VAP comprised 61% and CLABSI 35% of all devicerelated nosocomial infections in the ICU. The CLABSI and VAP rates were 33 and 58 per 1,000 device-days, respectively. VAP and CLABSI accounted for 20% and 7.7% increase in mortality, respectively. The hand hygiene compliance rate was 68% for physicians and 82% for nurses, and compliance with vascular catheter care and ventilator bundles was 65% and 70% respectively. After introduction of the interventions, in period 2, the incidence of VAP was reduced from 58/1,000 ventilator-days to 30.9/ 1,000 ventilator-days; the CLABSI rate fell to 26.7/1,000 device-days. There was no significant difference in compliance with hand hygiene or the central line and ventilator bundles compared with the previous period. Conclusion: Our rates of VAP and CLABSI were much higher than those reported for developing countries and the US NHSN. This may be partly due to the immunosuppressed nature of cancer patients with neutropenia and prior chemotherapy. Our interventions resulted in a significant reduction in the rate of VAP, and to a lesser extent for CLABSI. Process compliance did not change. It is essential to improve process compliance to further reduce the incidence of nosocomial infections in our ICU. Background: Herein we examined the hypothesis that preserving splanchnic microcirculation with lasting visceral parasympathetic vasodilatation plus a rapid hyperfluid therapy, during the early phase of severe sepsis (DL 60 ), could delay and/or reduce the acutely exacerbating inflammatory feature of sepsis and modify its progression to death. In our previous work, this sepsis determined splanchnic tissue hypoxia, gut bacterial overgrowth, and bacterial translocation (BT) and these events were inflammatory amplification causal factors. Methods: Adult Wistar-EPM rats (n = 86) were anesthetized and submitted to: sepsis (S) (2 ml Escherichia coli 10 8 CFU/ml i.v.) and/or thoracic epidural analgesia (E) (0.2 ml of 0.05% bupivacaine) and/or hyperfluid therapy (H) (30 ml/kg/20 minutes of Ringer lactate) according to the group (G) design: SG; SEG; SHG; SEHG; EG and; NG (naive). Epidural analgesia and fluid therapy were initiated 30 minutes after sepsis. At the 24-hour period, animals were monitored to: microcirculatory hemodynamics (duodenum, jejunum, ileum, liver and kidney) with Figure 1(abstract P92) SDF images of organs. Liver (1 to 4) and ileum (9 to 12). Arrows indicate altered microvessels and perivascular tissue areas.
Results: SDF findings showed that both EG and EHG preserved the microvascular and perivascular tissue architecture following sepsis induction in all organs. However, the SEH combination group showed larger microvessel diameter as compared with SEG, suggesting a better withholding of the intravascular blood volume subsequently improved tissue perfusion (Figure 1). A significant gut bacterial overgrowth was observed only in SG as compared with NG, EG, HG and EHG. The BT index to MLN was proportional to bacterial overgrowth, being higher in SG (67%), lower in SHG (33%) and SEG (17%), and absent in SEHG and EG. The mortality rate was 60% (SG), 30% (SEG) and 10% (HG). All animals with sepsis survived with E + H combination therapy. Background: The microcirculation dysfunction sequence in sepsis has not been well acknowledged to support therapy and prognosis. Herein, the abdominal organs' microcirculation and their perivascular tissue derangements captured by SDF microscopy were correlated with the whole organ histological findings in severe sepsis. Methods: Adult Wistar-EPM rats (n = 54) were distributed into: sepsis group (n = 36), animals submitted to 10 9 CFU/ml Escherichia coli inoculation through the jugular vein; and sham group (n = 18), animals with physiologic saline 0.9%. At 0, 2, 6 and 24 hours, liver, kidney and ileum microvascular and perivascular images were monitored by SDF in vivo and also by histology. Results: The liver and kidney surface SDF images showed that microcirculation and perivascular tissue alterations are a concomitant event, which were a focal event initially that turned progressively generalized in proportion to sepsis worsening. Vascular patterns were from complete absence to hyperflow and narrowed to dilated venules, showing that altered and nonaltered microvessels occur simultaneously in sepsis. The expansion of perivascular tissue fuzziness with narrowed or vanished microvessels in sepsis suggested local dysfunction in progression. Confronting these areas with histology, the enlarged perivascular areas were composed mostly of cytoplasm edema at the early sepsis phase and of varying stages of the cell necrosis process at the further periods. These processes were very similar in both the liver and kidney and occurred throughout the organ, showing that the surface findings can be extensive to deeper areas (Figure 1). In contrast, the small bowel SDF images showed that gut surface microcirculation was composed primarily of high-flow capillaries and only the vascular density was focally reduced in sepsis; besides, the perivascular tissues could not be identified, showing that the vascular and tissue architectural pattern in the gut differs substantially from solid organs, suggesting a different pathophysiology response of the intestinal microcirculation in sepsis. The gut histology showed generalized cell edema and varying necrosis phases in muscle layers of the intestine in sepsis, and such events could not be suspected by SDF. Conclusion: Organ dysfunction in sepsis is better detectable in solid organs by SDF imaging as compared with gut muscular compartment.
These results demonstrated the importance of the solid organ SDF monitoring in experimental sepsis treatment studies. Acknowledgements: Supported by FAPESP. Background: The management of the uncontrolled systemic inflammatory response in the early phase of severe sepsis is still a challenge. Herein we aimed to minimize the acute inflammation phase of sepsis by clarithromycin associated with intravascular volume restoration with an aggressive fluid therapy. Methods: Wistar rats were distributed into five groups: S, animals submitted to sepsis (DL 80 ; 3 ml Escherichia coli 10 9 CFU/ml, i.v., n = 6); SCH, animals induced to sepsis after clarithromycin (14 mg/kg, i.v., given 24 and 0 hours before sepsis) and hyperhydration (Ringer lactate 40 ml/kg, i.v., in 20 minutes after the sepsis induction) (n = 6); SC, animals induced to sepsis and treated with clarithromycin (n = 6); SH, animals induced to sepsis and treated with hyperhydration (n = 6); and Sham, animals induced to sham sepsis (n = 4). All invasive procedures were undergone after general anesthesia. The liver, kidney and ileum microcirculation were monitored by Laser Doppler and sidestream darkfield imaging at 2-hour and 26-hour periods. The mortality index was observed for 30 days. Results: All animals of the SCH and Sham groups survived while the S, SC and SH groups showed 20% survival. Hyperhydration or clarithromycin in sepsis showed a partial and transient improvement of the microcirculation of the abdominal organs, although the association of hyperhydration with clarithromycin (SCH group) showed better effects. In addition, the beneficial microhemodynamic effect under combined therapy was better evidenced in the liver and intestine as compared with the kidney. Conclusion: The association of clarithromycin and hyperhydration showed a beneficial effect in severe sepsis, possibly by modulating inflammatory response and microcirculation damage, respectively, resulting in subsequent survival; nevertheless, their individual effects were not efficient to inhibit severe sepsis mortality and microcirculation dysfunction. Acknowledgements: Supported by FAPESP.

P95
Preliminary results for the use of proteinase K to achieve release of LPS from the Alteco LPS Adsorber® after perfusion with LPS containing blood E Hansen 1* , L Pierre 2 , S Blomqvist 1, Background: The effect of Alteco LPS Adsorber® to remove LPS from the circulation is based on the incorporation of a synthetic peptide that binds to the lipid A moiety of LPS, the binding capacity in one adsorber exceeding 7.5 μg LPS. Positive clinical effects of the adsorber when used in patients with Gram-negative sepsis have been reported [1]. Measurement of LPS in human blood, however, is hampered by difficulties such as contamination and interference. The aim of this pilot study was to evaluate a method to release LPS from the LPS adsorber after perfusion with LPS containing blood. Methods: Two adsorbers (A1, A2) containing the active peptide and one dummy adsorber (D) with no peptide were used. Whole blood (500 ml) from healthy pigs was collected in a bag containing heparin, 37 μg LPS was added. A roller pump was used to recirculate the blood through the adsorber. The pump flow was set at 150 ml/minute and the duration of the perfusion was 2 hours. After the end of perfusion the adsorbers were rinsed with 500 ml Krebs solution. A solution of 20 mg proteinase K in 50 ml Tris buffer with pH 7.4 was prepared and 2.5 ml CaCl 2 was added. The adsorbers were kept at 37°C and the proteinase K solution [2] was perfused through the adsorbers at 5 ml/minute for 6 hours. Samples for LPS analysis (chromogenic LAL) [3] were drawn before the perfusion and then after 30, 240 and 360 minutes. The enzyme activity was checked using a synthetic substrate [4]. Results: The concentrations of LPS before and during perfusion with proteinase K are shown in Table 1.

Conclusion:
The LPS values found before the start of the perfusion indicate contamination of the solution. The increase in LPS seen in all adsorbers after 30 minutes is probably due to traces of blood components. The later increase in LPS after treatment with proteinase K in the active adsorbers indicates that the adsorber is effective in capturing LPS from whole blood and that proteinase K is able to dislodge LPS bound to the adsorber. Background: Major trauma is characterized by a proinflammatory response, followed by an immunosuppression, increasing the risk for ICUacquired infection. Several prognostic markers of sepsis in ICU patients have been identified and need to be assessed. Recently, Pancreatic Stone Protein/regenerating protein (PSP/reg) was shown to be increased during post-traumatic sepsis [1]. The N-terminal fragment of the C-type natriuretic peptide precursor (NT-proCNP) was assessed as a predictor of sepsis in multiple traumatized patients without brain injury [2]. The main objective of this study was to determine whether measurements of those biomarkers could help in the prediction of sepsis in severe trauma patients.
Methods: This retrospective observational study was carried over 24 months in a trauma ICU at a university hospital. Trauma patients under mechanical ventilation with an Injury Severity Score (ISS) >25 and age >18 were included. Patients dying in the first 48 hours after trauma, having pulmonary inhalation/gut perforation during trauma or after the onset of infection, were excluded. We used already described ELISA protocols for the detection of PSP/reg [1] and NT-proCNP (Biomedica, BI-20872). Both biomarkers have been measured every 2 days from day 1 to day 4 after trauma. After descriptive analysis of clinical data (using medians with interquartile ranges), we evaluated the standard fold-change (SFC) and the area under the curve (AUC) between patients being infected (sepsis group) and patients not being (nonsepsis group). Results: We analyzed 61 trauma patients: age 37 (25; 50), ISS 38 (33; 45). Among them, 24 developed sepsis in the first week after trauma (pneumonia; median delay 4 days). There were no differences between sepsis and nonsepsis groups at admission regarding demographic data. Neither PSP/reg nor NT-proCNP showed significant differences between sepsis and nonsepsis groups, whatever time point was considered (respectively days 1 to 2 and days 3 to 4; Table 1). The computed values for SFC and AUC were lower than the minimal detectable values. Conclusion: In this pilot study, neither PSP/reg nor NT-proCNP can help in the prediction of sepsis in severe trauma patients. Contrary to the results published by Bahrami and colleagues [2], we did not show significant difference between sepsis and nonsepsis groups in patients without brain injury. Our study illustrates the complexity of validating biomarkers for sepsis prediction in independent cohort of patients. Background: Time is precious for patients and clinicians facing septic events. In recent past years, many efforts have been made to develop rapid and reliable tests, especially by molecular methods, which still have several limits in determining antimicrobial susceptibility. Nowadays, MALDI-TOF mass spectrometry allows almost instantaneous bacterial identification, but antibiotic susceptibility results are usually delayed 24 to 48 hours, compared with the timing of microorganism identification. Methods: In this work, two short procedures, one for Gram-negative and one for Gram-positive bacteria, were developed. The two methods were employed to purify bacteria from positive blood cultures (Becton Dickinson) after microscopic examination, and to prepare bacterial cells for identification. Bacteria were then identified by MALDI-TOF mass spectrometry (Bruker Daltonics) with excellent scores, similar to those obtained from agar plate colonies. After identification, short-term broth cultures were inoculated with an aliquot of the recovered bacteria, with or without (growth controls) antibiotics and incubated in an HB&L (Alifax SpA) instrument, which monitored each vial for bacterial growth during the following 3 hours. Absence or reduction in growth in a vial containing a certain antibiotic was interpreted as sensibility, while resistance was assessed by a bacterial growth comparable with the control vial. Background: NGAL appears to be a biomarker of acute kidney injury. It is produced by neutrophils and renal tubular cells. In healthy patients, plasma NGAL levels have been shown to vary with white blood cell concentrations. The predictive ability of NGAL to predict AKI is attenuated by leucocytosis in patients being treated with vancomycin. The relationship between NGAL and white cell indices in the critically ill is poorly understood. The aim is to explore the relationship between different measures of NGAL and white cell count (WCC) and neutrophil count (NC) in critically ill patients at risk of AKI. Methods: Patients with systemic inflammatory response syndrome and oliguria, or a creatinine rise >25 μmol/l, or both, in a tertiary referral university hospital ICU, within 48 hours of admission, were studied. We measured kidney-specific monomeric urinary NGAL (muNGAL), plasma (pNGAL) and urinary NGAL (ucNGAL) by commercial assay, and routine haematological and biochemical parameters. Institutional ethics approval was obtained. Results: We studied 84 patients (51.2% male, median age 66.5 (52 to 74)) with a median APACHE III score of 61 (45 to76). Fifty-one patients had WCC >10 × 10 9 /l, 58 had NC >7.5 × 10 9 /l. No useful correlations were demonstrated between white cell indices and any form of NGAL measurement. A stronger correlation existed between creatinine and sNGAL than urinary indices. The strength of correlation between creatinine and all measures of NGAL was greater in those patients with high white cell indices (Table 1); the increase was significant for sNGAL in those with elevated WCC (r = 0.27 in nonleucoytotic patients, r = 0.65 in leucocytotic patients, P < 0.05). Conclusion: The stronger correlation between creatinine and pNGAL suggests systemic factors may be more important than local factors in the pathophysiology of AKI. The increased strength of relationship in those with leukocytosis suggests NGAL may be a general index of illness severity. Multicentre and longitudinal studies are required to further increase our understanding of the natural history of NGAL release and the clinical utility of these tests. Background: Acquired glucocorticoid resistance frequently complicates the therapy of sepsis. It leads to an exaggerated proinflammatory response and has been related to altered expression profiles of glucocorticoid receptor isoforms glucocorticoid receptor-α (mediating anti-inflammatory effects) and glucocorticoid receptor-β (acting as a dominant negative inhibitor). We investigated the impact of glucocorticoid receptor isoforms on glucocorticoid effects in human T cells. We hypothesized that changes of the ratio of glucocorticoid receptor isoforms impact glucocorticoid resistance and that glucocorticoid receptor-α expression is controlled by microRNA-mediated gene silencing. Methods: First, T cells from healthy volunteers (native and CD3/CD28stimulated cells with or without addition of hydrocortisone) were analyzed for the expression of glucocorticoid receptor isoforms by quantitative PCR. Additionally, effects of gene silencing of glucocorticoid receptor-β by siRNA transfection were determined. Secondly, microRNA-mediated silencing was evaluated by cloning of a glucocorticoid receptor-α-specific 3'-untranslatedregion reporter construct and subsequent transfection experiments in cell cultures. Effects of miRNA transfection on glucocorticoid receptor-α expression were analyzed in Jurkat T cells and in T cells from healthy volunteers (quantitative PCR and western blotting). Finally, expression of glucocorticoid receptor-α, glucocorticoid receptor-β, and miR-124 was tested in T cells of sepsis patients (n = 24). Results: Stimulation of T cells induced a significant upregulation of glucocorticoid receptor-α (not glucocorticoid receptor-β), thereby possibly rendering T cells more sensitive to glucocorticoids; this T-cell response was hindered by hydrocortisone. Silencing of glucocorticoid receptor-β doubled the inhibitory effects of glucocorticoids on IL-2 production. MicroRNA-124 was proved to specifically downregulate glucocorticoid receptor-α. Furthermore, a glucocorticoid-induced threefold upregulation of microRNA-124 was found. T cells of sepsis patients exhibited slightly decreased glucocorticoid receptor-α and slightly increased miR-124 expression levels, whereas glucocorticoid receptor-β expression was twofold upregulated (P < 0.01) and exhibited a remarkable interindividual variability. Conclusion: Glucocorticoid treatment induces expression of miR-124, which downregulates glucocorticoid receptor-α thereby limiting antiinflammatory effects of glucocorticoids. Steroid treatment might aggravate glucocorticoid resistance in patients with high glucocorticoid receptor-β levels.   Background: The true effectiveness of early antibiotic administration relative to the clinical identification of sepsis in a real-world setting is unknown. Previous studies described the impact of antibiotic timing within an isolated septic shock cohort. A novel electronic health record (EHR) screening tool, Clinical Vigilance™ for Sepsis, is able to identify the presence of sepsis and correlate with meaningful patient-centered outcomes. The objective was to define the impact of delayed antibiotic administration relative to the clinical identification of sepsis using an EHR alert system. Methods: A retrospective analysis of a prospectively compiled registry of patient EHR records in a single-center 300-bed community hospital. A consecutive assessment of all adult patients with suspected infection over a 3-month period in 2011. A physician order for intravenous antibiotics was used as a surrogate for the clinical suspicion of systemic infection (sepsis). The test variable was application of a comprehensive automated EHR screening tool, CV Alert, to identify high-risk sepsis patients based on a multifactor alert system including labs, vital signs, and treatment team documentation. The primary outcome was all cause in-hospital mortality, and a secondary outcome was hospital length of stay (LOS). Antibiotic delivery was defined a priori as the time a physician order was placed for intravenous antibiotics and outcomes were assessed every 12 hours prior to and subsequent to the CV Alert. Results: We identified 2,255 consecutive patients with suspected infection over a 3-month period from a total of 23,717 screened (9.5%). CV Alert was triggered in 867 of 2,255 (38%). Patients identified by CV Alert (n = 867) had an increased mortality rate (5.3% vs. 0.6%, P < 0.001) and increased hospital LOS (5 vs. 2 days, P < 0.001) compared with patients not triggering an alert (n = 1,388). Patients given antibiotics 0 to 12 hours after the alert had a significantly increased mortality rate (8.9% vs. 3.3%, P < 0.002) and longer LOS (6 vs. 4 days, P < 0.001) compared with patients given antibiotics 0 to 24 hours prior to alert. Conclusion: Among patients with suspected infection, those identified by the CV Alert had an increased mortality rate and hospital length of stay. Delayed antibiotics relative to the time of CV Alert were associated with a progressive increase in mortality rate and hospital LOS. An EHR-based screening tool applied to a real-time healthcare system could aid in the early identification of at-risk patients within a sepsis cohort.

P104
Effects on outcome of patients with severe sepsis and septic shock admitted to the ICU after implementation cooperative sepsis management protocol R Champunot * , N Kamsawang, D Thimsri, P Tuandoung, S Tansuphaswasdikul Buddhachinaraj Phitsanulok Hospital, Phitsanulok, Thailand Critical Care 2012, 16(Suppl 3):P104 Background: The application in clinical practice of evidence-based guidelines for the cooperative management of patients with severe sepsis and septic shock between community hospitals and tertiary referral hospital is still poor. The purpose of this study was to examine the outcome of patients with severe sepsis and septic shock admitted to the ICU after implementation of a cooperative sepsis management protocol. Methods: From 1 October to 30 November 2009, patients with severe sepsis and septic shock admitted to the ICU received standard therapy (control group). From 1 December 2009 through 31 January 2010, patients with severe sepsis and septic shock (protocol group) were managed with a cooperative sepsis management protocol. The protocol included early recognition and the initiation of therapy by enabling nurses and physicians in community hospitals to mobilize institutional resources for the treatment of patients with severe sepsis and septic shock (Figures 1 and 2). Using goal-directed resuscitation protocols, early intensivist involvement and rapid transfer to the ICU from emergency department were implemented in tertiary referral hospital. Results: Sixty-two and 77 patients, respectively, were enrolled in the control and protocol groups. Rapid transfer of patients with severe sepsis and septic shock to the ICU from emergency department was observed in 1/62 (1.5%) of the control group and 45/77 (58.4%) of the protocol group (difference 56.9%; P = 0.01). The hospital mortality rate was 62.3% in the control group and 37.7% in the protocol group (P = 0.01). The protocol group had significant reductions in ICU length of stay, ICU cost and numbers of organ failure (P = 0.01). Conclusion: Empowerment of nurses and physicians in a community hospital to mobilize hospital resources for taking care of patients with severe sepsis and septic shock and implementation of a cooperative sepsis management protocol between community hospitals and tertiary referral hospital was temporally associated with improved outcomes. Acknowledgements: The authors thank the Phitsanulok Co-operative Sepsis Management Team.

P105
Saving 500 Lives Campaign: another way to improve the mortality rate of patients with severe sepsis and septic shock R Champunot * , N Kamsawang, P Tuandoung, S Tansuphaswasdikul Buddhachinaraj Phitsanulok Hospital, Phitsanulok, Thailand Critical Care 2012, 16(Suppl 3):P105 Background: In September 2010, 9 months after empowerment of caring for patients with severe sepsis and septic shock and implementation of a cooperative sepsis management protocol between community hospitals and tertiary referral hospital in Phitsanulok, the Phitsanulok Co-operative Sepsis Management (PCSM) team announced the Saving 500 Lives Campaign. This campaign aimed to encourage the unity and importance of caring for patients with severe sepsis and septic shock. Methods: From October 2010 to September 2011, eight community hospitals and one tertiary referral hospital in Phitsanulok established and promoted a set of achievable goals and interventions for patients with severe sepsis and septic shock (Figure 1). These interventions included: establishing sepsis team in all hospitals to coordinate and monitor the achievement goals; using a search out severity (SOS) score and a sepsis screening tool to help early and more accurate diagnosis; providing early resuscitation protocol and other measures as indicated by implementing a checklist for sepsis management (sepsis six bundles, rule of three, early goaldirected therapy); communication and providing important information during transportation using a protocol; and early intensivist involvement and rapid transfer to the ICU from the emergency department using a sepsis fast-track protocol. Physician and nurse leadership actively engaged in the data review and shared ideas in every hospital for improvement and closely tracked progress against those goals and interventions. The mortality rate of patients with severe sepsis and septic shock was used to measure the success of the campaign. Results: The Saving 500 Lives Campaign succeeded in efforts to saved 660 patients from 1,048 patients with severe sepsis and septic shock in Phitsanulok, Thailand. The total mortality rate was 37% (decreased from 47% in the past year). The group of patients from community hospitals that can be admitted directly to the ICU from the emergency department using a sepsis fast-track protocol had the lowest mortality rate (19%). Conclusion: Setting a numeric goal using the 500 Lives Campaign was another way to support and empower the cooperation of sepsis care in Phitsanulok, Thailand.
signs, and/or maternal sepsis risk factors. The puncture site was sterilized with alcohol prior to phlebotomy for microbial and biomarker tests. Blood was collected in clotting activator tubes, and incubated at room temperature for 1 hour before centrifuging to separate out serum for later acute-phase protein quantification. For gold-standard tests, an immunoturbimetric CRP assay was performed using a Beckman analyser and a chemiluminescent Elecsys BRAHMS PCT immunoassay using a Cobas e602 analyser.
Results: The mean age of infant evaluation was 2.29 ± 10.37 days. The average gestational age at birth was 37.04 weeks (4.27), 30% of which were preterm. Eighteen out of 350 samples (5%) were blood culture positive. Thirteen samples met the criteria for a laboratory-confirmed bloodstream infection (LCBSI), the most common organisms being Escherichia coli (28%) and Streptococcus agalactiae (22%). CRP results (n = 350) ranged from ≤0.1 to 226 mg/l, sepsis-positive (≥5 mg/l) CRP tests occurred in 69% of LCBSI. PCT quantified samples to date (n = 100) Figure 1(abstract P104) Checklist for early recognition patients with severe sepsis and septic shock.
Critical Care 2012, Volume 16 Suppl 3 http://ccforum.com/supplements/16/S3 ranged from 0.03 to 67.23 ng/ml. Both analytes featured in higher cases that suggested sepsis than positive blood culture numbers: 56 out of 350 cases had CRP levels ≥5 mg/l, for PCT 23 out of 100 individuals tested positive at the cutoff (>0.5 ng/ml) for bacterial infection. Conclusion: Sepsis diagnosis requires accurate detection biomarkers; preliminary study findings show that CRP is present in the majority of bloodstream infections. Furthermore, results using both biomarkers suggest that blood culture results do not indicate all cases of true sepsis. Further research will correlate the completed CRP and PCT gold-standard dataset with that produced by ASCMicroPlat PCRbased assays to assess the clinical utility of this device for sepsis diagnostics. Background: Quantitative gene expression data are often normalised to the expression levels of control genes (that is, housekeeper genes). An assumption in the use of housekeeper genes is that the expression of the genes remains constant in the cells/tissues under investigation. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Recent studies, however, have shown that GAPDH expression is highly variable in different experimental settings. This study aimed to characterise GAPDH mRNA expression in severe sepsis, using whole blood from patients with severe sepsis and healthy controls. Methods: Patients admitted to the ICU within 24 hours of onset of severe sepsis were recruited. Healthy controls were also recruited. Exclusion criteria included patients on immunosuppressants or chemotherapy and those with a moribund status. Ethical approval (REC W06/Q1107/42) was obtained. Whole blood (1 ml) was taken from the patients and Quantigene RNA extraction techniques were used. The Quantigene plex assay was used in duplicate to determine GAPDH mRNA expression. The assay uses branched DNA signal amplification to enable the detection and quantification of multiple RNA targets simultaneously. It has been compared with other techniques favourably [1]. GAPDH mRNA expression was standardised to housekeeper genes succinate dehydrogenase (SDHA) and hypoxanthine-guanine phosphoribosyltransferase (HPRT). Results: Sixty-five patients with severe sepsis were included, of whom 27 died within 28 days. Seventeen control samples were included in the study. The groups were age and sex matched. In terms of GAPDH mRNA expression there was no significant difference between survivors and nonsurvivors. However, with regards to overall GAPDH mRNA expression in severe sepsis there was a marked increase in patients with severe sepsis. GAPDH mRNA expression showed a 16-fold mean increase in expression compared with control samples. Conclusion: GAPDH mRNA expression in patients with severe sepsis showed a marked increase compared with controls. These data question the suitability of GAPDH as a housekeeper gene in gene expression profiling studies in sepsis. The data also highlight GAPDH as another potential biomarker of sepsis that requires further evaluation. Background: In patients with severe sepsis, previous work has suggested that cytokine gene expression profiles may have diagnostic/prognostic potential as biomarkers. Cytokines operate within complex networks; their predictive value may be improved by the simultaneous measurement of multiple cytokines. Cytokines are key inflammatory proteins involved in sepsis. Gene expression profiling has been shown to have a clinical role in the diagnosis of haematological malignancies. Gene expression profiling may allow early, aggressive management in patients predicted to fare badly. This study examined cytokine gene expression profiles in patients with severe sepsis. Methods: Patients admitted to the ICU within 24 hours of onset of severe sepsis were recruited. Healthy controls were also recruited. Exclusion criteria included patients on immunosuppressants or chemotherapy and those with a moribund status. Ethical approval (REC W06/Q1107/42) was obtained. Whole blood (1 ml) was taken from the patients and Quantigene RNA extraction techniques were used. The following cytokines were measured in duplicate using the Quantigene plex assay: IL-6, IL-8, IL-10, IL-12p40, IL-12p35, IL-13, IL-17, MCP-1 and MIF. The assay uses branched DNA signal amplification to enable the detection and quantification of multiple RNA targets simultaneously. It has been compared with other techniques favourably [1]. Cytokine mRNA expression was standardised to housekeeper genes succinate dehydrogenase (SDHA) and hypoxanthine-guanine phosphoribosyltransferase (HPRT). Results: Sixty-five patients with severe sepsis were included, of whom 27 died within 28 days. Seventeen control samples were included in the study. The groups were age and sex matched. Three cytokines showed marked upregulation in severe sepsis compared with controls; IL-10 (15-fold), IL-18 (15-fold), IL-15 (twofold). However, IL-6, IL-8, IL-12p40, IL-12p35, IL-13, IL-17, MCP-1 all showed a greater than twofold downregulation in severe sepsis. There was no significant difference in profiles between survivors and nonsurvivors of severe sepsis. Conclusion: These results show a distinct cytokine gene expression profile in severe sepsis. This includes the upregulation or downregulation of both proinflammatory and anti-inflammatory cytokines. Cytokine gene expression profiling using the Quantigene plex assay is able to demonstrate distinct profiles in patients with severe sepsis. This has the potential to be developed into a diagnostic/prognostic tool with larger studies.

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Quantified temporal changes of heart rate variability when developing SIRS N Matsumaru 1* , K Shirai 1 , Y Kawamura 2 , Y Yokota 2 , K Kumada 1 , I Toyoda 1 , S Ogura 1 1 Advanced Critical Care Center, Gifu University Hospital, Gifu, Japan; 2 Faculty of Engineering, Gifu University, Gifu, Japan Critical Care 2012, 16(Suppl 3):P109 Background: Heart rate variability (HRV) has been reported to gradually decrease long before the onset of sepsis [1,2]. Since the development of systemic inflammatory response syndrome (SIRS) has been included in the definition of sepsis, we compared temporal changes of HRV depending on the SIRS state coming after. Methods: Vital data of patients admitted in ICU were downloaded from bedside monitoring systems. No specific registration criteria for disease were defined. Vital signs regarding SIRS evaluation -that is, hear rate, respiration rate, and core temperature -are recorded every 10 seconds, 0.1 Hz. By taking the median of 60 data, the SIRS state was determined for each hour, ignoring the count of white blood cells. The sampling rate of ECG data is 500 Hz, and the HRV for each minute (0.017 Hz) was calculated from that. Abnormal heartbeats and trends were eliminated using a stochastic model, and the power spectrum was derived with an autoregressive model [3]. We considered the integral value within 0.04 to 0.4 Hz as an estimate of HRV. Temporal linear trends of HRV were quantified as Spearman's correlation coefficient. Note that the target period for HRV trend evaluation is immediately before the SIRS evaluation period. The following software was used: IBM SPSS version 19, R version 2.15.0, and G*Power Version 3.1.4. Results: Forty-nine patients were registered, and 34 patients (69.4%) suffered from sepsis. The average age was 64.3 years, and the average ICU stay was 31.4 days, ranging from 3 to 113 days. The number of data where a SIRS state was identified was 21,919, and 56.5% (12,380) were evaluated as SIRS-positive. Immediately before SIRS-positive states, the mean value of the correlation coefficients was -0.00234 (SD 0.321). The mean value before SIRSnegative states was 0.0201 (SD 0.309). The mean difference was 0.0224 (P < 0.05), and the statistical power was 99.9%. Conclusion: Our analysis shows that the mean trend of the HRV value immediately before developing SIRS was negative, and the mean trend of HRV immediately before a non-SIRS state was positive. This result does support previous results. For sepsis monitoring, however, further investigation is mandatory. This result only suggests a stronger negative trend of HRV when developing SIRS for the next period, regardless of the current state.
Background: Initially described in endothelial cells from the lung, endothelial cell specific molecule 1 (ESM-1), also called endocan, is a circulating proteoglycan of 50 kDa that can be easily quantified in biological fluids (sera, plasma). This robust macromolecule is upregulated in the presence of proinflammatory agents and has been described as a biomarker of endothelial dysfunction in several disease conditions. Methods: We here compared and analyzed the results obtained from studies using a sandwich-type ELISA (DIYEK H1, Lunginnov) for evaluating whether ESM-1 could be useful to predict organ failure in ICU patients such as septic patients and polytrauma patients (n = 67). Data baseline characteristics, APACHE II score, procalcitonin (PCT) and C-reactive protein (CRP) levels and ICU course such as ICU length of stay were also evaluated for each ICU patient admitted and compared with ESM-1 levels.
Results: In all studies we observed that elevated blood levels of ESM-1 correlated with the severity of sepsis and the poor outcome in patients with severe sepsis or in septic shock at ICU admission. We furthermore evaluated the presence of ESM-1 and ESM-1 degradation products in the biological fluids (sera, plasma and urines) from septic patients by ELISA, immunoprecipitation and western blotting procedures. When compared with data baseline characteristics, ESM-1 levels were shown to not correlate with CRP and PCT levels. Interestingly we may suggested from our analysis that blood ESM-1 >3 ng/ml represents an additional criterion of severity in a context of SIRS that may be useful in the ICU. At 72 hours, ESM-1 exhibited a clear predictive value for acute lung injury (sensitivity 85%; specificity 100%) in septic patients. In comparison, lower levels of serum ESM-1 in polytrauma patients were associated with development of acute lung injury and reflect respiratory failure. Conclusion: No circulating molecule was up to now described as an indicator of respiratory failure in septic patients and in polytrauma patients. In a context where respiratory failure is still the first cause of death in sepsis, our study analysis suggests that blood levels of ESM-1 may be a useful early biomarker of lung tissue injury and respiratory failure in ICU patients.

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A microbiome approach to sepsis: development and case-study application of novel methods for detection and isolation of microbes from whole blood M Faria Crowder 1* , JM Conly 2 , MG Surette 3 Background: The application of molecular profiling methods to a wide variety of infections suggests that a polymicrobial community is much more common than suggested by standard clinical culture [1]. Our goal was to develop methods, using a microbiome approach, to improve culture and molecular diagnostics for bacterial sepsis.
Methods: Culture and DNA extraction protocols were evaluated using synthetic bacterial communities inoculated into whole blood. Disruption of blood cells with a blood cell lysing detergent, with or without hypotonic osmotic shock, was carried out and evaluated for the ability to recover the community. Viable bacterial cells were recovered on solid media. Total DNA was examined by terminal-restriction fragment-length polymorphism (TRFLP) profiling. Efficiencies of recovery and limits of detection were determined. The optimized methodology was applied to clinical samples collected from consented patients in both the ICU and ED from two Calgary hospitals. Cultured organisms were identified by 16S rRNA gene sequencing. Molecular profiling was carried out using TRFLP and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Results: Treatment of synthetic community organisms with a 5% wt/vol detergent added at a 1:1 or 1:5 ratio did not significantly impact their viability. TRFLP analysis indicated that the DNA from these communities could be recovered from whole blood following lysis and removal of host cells. Whole blood samples were analysed from septic patients. In three case studies we identified two to 16 bacterial species in the primary infection samples using direct culture and molecular methods. Conventional diagnostics only reported one organism. Molecular profiling of blood samples from these patients also identified correlating polymicrobial communities. Blood cultures for these samples were either negative (two of three cases) or monomicrobial (one of three cases), thereby underestimating the diversity seen with the TRFLP and bTEFAP analysis. These methods have been applied to 88 adult ICU blood samples, 20 primary infection samples, 36 adult ED samples, and seven pediatric ED samples with analysis ongoing. Conclusion: We have successfully developed a novel method to analyse whole blood in order to characterize the microbiome of sepsis infections.
Preliminary results indicate that sepsis infections are polymicrobial in nature.
Acknowledgements: All samples were collected in collaboration with the Alberta Sepsis Network through the Critical Care Epidemiologic and Biologic Tissue Resource. Methods: There were 114 newborns in the first group, from 1992 to 2007. We determined the antibodies in blood to pathogenic microflora extracted from joint bursas and in 52 newborns -extracted from the abdominal cavity with peritonitis. These investigations were made using the agglutination reaction to staphylococci as well as the latex-based test for streptococci. The antibiotics were prescribed according to the results of the investigations. In the second group from 2005 to 2010, we undertook microbiological investigations of pathological material from lesion foci in 43 newborns with surgical sepsis. These investigations are devoted to the study of susceptibility and resistance of pathogenic microflora to antibiotics. Results: In the first group of infants, according to the data of serological investigations in the blood of newborns with bone and joint sepsis, we determined the antibodies to staphylococci in 80.3% cases, in 45.4% to staphylococci and streptococci, in 54% cases in newborns with peritonitis we determined the antibodies to Pseudomonas aerogenes, in 60.8% cases to Enterobacter spp. and in 62.5% to Staphylococcus epidermidis. In newborns of this group the bacterial flora were 70% and fungal in 30%. Our investigations determined the susceptibility to the limited range of antibacterial and antifungal medicines. In the second group we selected two subgroups of infants: one with sepsis as a complication of surgical deceases, and the second with bone and joint sepsis. In the first subgroup the bacterial microflora was detected in 94.7% of examined newborns and represented in 52.6% as microbial associations. In the second subgroup of newborns the bacterial flora was detected in 37.5% of cases, in the rest of the cases the flora consisted of fungi and protozoa. The analysis of susceptibility to pathogenic microflora showed that two of seven cultures of staphylococcus were methicillin-resistant Staphylococcus aureus, in five of eight cultures of staphylococcus they were resistant to four to 10 antibiotics, and the highest resistance to the antibiotics was detected in P. aerogenes (in nine of 18 cultures). Conclusion: Microbiological and serological diagnostics are a compulsory condition of adequate anti-inflammatory therapy for perinatal sepsis.

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Use of intravenous and intramuscular immunoglobulin in the practice of treatment for purulent and septic deaths in newborns G Khanes 1* , L Nazarchuk 2 , I Maksakova 1 , O Bakaieva 1 Conclusion: Based on the clinical and laboratory findings VL could be considered a systemic inflammatory response. Maybe in this disease we have not evaluated an inflammatory test like C-reactive protein or procalcitonin to be convicted if higher than the normal value, but we think we need to start a study about markers of sepsis in this systemic parasitic disease, because our opinion is that VL is a SIRS plus parasitic systemic infection -which does mean sepsis. In malaria, PCT levels are elevated in both severe and uncomplicated Plasmodium falciparum malaria, but how useful is this inflammatory test in VL [4]?