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Approaches combining mice and Drosophila melanogaster models to decipher human sepsis

R Ramy, S Garnier, Lydie Pradel, Laurence Röder, Béatrice B. Loriod, A

Defaye, Laurent Perrin, Pascal Rihet, C Nguyen

► To cite this version:

R Ramy, S Garnier, Lydie Pradel, Laurence Röder, Béatrice B. Loriod, et al.. Approaches combining mice and Drosophila melanogaster models to decipher human sepsis. Critical Care, 2014, 18 (S2), 10.1186/cc14070. doi: 10.1186/cc14070

HAL Id: hal-01619083

https://amu.hal.science/hal-01619083

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# Sepsis 2014

Paris, France. 3-5 December 2014

Published: 3 December 2014

These abstracts are available online at http://ccforum.com/supplements/18/S2

## POSTER PRESENTATIONS

### P1

**TLR-independent activation of NK cells during systemic inflammation.**

O Rasid¹, J-M Cavillon
Cytokines and Inflammation, Institut Pasteur, Paris, France
*Critical Care* 2014, **18**(Suppl 2):P1; doi:10.1186/cc14004

**Introduction:** During the course of systemic inflammation, most of the immune cell types get activated to a certain degree as part of, or contributing to, the cascade of physiopathological events. Whether for some cells, classically phagocytes of the innate immune system, it is clear that direct sensing of pathogen-associated molecular patterns leads to activation initiating systemic inflammation, the picture is not so clear for natural killer (NK) cells. While NK cells have been shown to express toll-like receptors (TLR), the role of these receptors on NKs during systemic inflammation has not been directly addressed.

**Methods:** To directly assess the role of TLR expression on NK cells we used an adoptive transfer model in which NKs purified from the spleens of WT, TLR4KO and TLR2/4DKO mice were transferred intravenously to RAG2<sup>−/−</sup>γc<sup>−/−</sup> (devoid of T, B and NK cells). Five days after reconstitution the mice were challenged intraperitoneally with conventional or TLR-grade lipopolysaccharide (LPS). Immune cell activation and production of IFNγ by NK cells was determined after 6 hours by FACS analysis.

**Results:** We observed no differences in reconstitution of the recipient mice with NK cells from different backgrounds suggesting no difference in trafficking and survival of the transferred cells. At 6 hours after LPS challenge, WT, TLR4KO or TLR2/4DKO NK cells recovered from the spleen and lungs of RAG2<sup>−/−</sup>γc<sup>−/−</sup> mice showed comparable levels of CD69 activation marker expression. Intracellular labeling for IFNγ in NK cells also revealed no significant differences.

**Conclusion:** Whether there is a role for direct TLR signaling on NK cells remains the objective of further investigations; however, our data show that in the course of a systemic inflammatory process, like endotoxemia, the expression of TLR2 and TLR4 by NK cells makes no difference in terms of their activation and secretion of IFNγ

### P2

**Role of 6-hour, 12-hour, and 24-hour lactate clearance in mortality of severe sepsis and septic shock patients.**

V Herwanto¹*, KC Lie², S Suwarto², CM Rumende³

¹Department of Internal Medicine, University of Indonesia, Jakarta, Indonesia;
²Division of Tropical Medicine and Infectious Diseases, Department of
Internal Medicine, University of Indonesia, Jakarta, Indonesia; ³Division of
Respirology and Critical Care, Department of Internal Medicine, University of
Indonesia, Jakarta, Indonesia

*Critical Care* 2014, **18**(Suppl 2):P2; doi:10.1186/cc14005

**Introduction:** Lactate is one of biomarkers used for risk stratification, resuscitation target, and death prediction in sepsis [1,2]. Interpretation of lactate clearance was proven more superior than single measurement to

evaluate resuscitation adequacy and to determine prognosis [3,4]. This study aimed to find out whether mean differences of 6-hour, 12-hour, and 24-hour lactate clearance were observed between nonsurvivors and survivors of acute phase mortality in severe sepsis and septic shock patients.

**Methods:** The study design was prospective cohort. Subjects were collected by consecutive sampling from the emergency department, hospital ward, and ICU at Cipto Mangunkusumo Hospital, Jakarta. Lactate levels were measured at 6, 12, and 24 hours, and subjects were subsequently followed to evaluate 3-day mortality. To determine their association with mortality, we used mean difference analysis of those three lactate clearance periods between nonsurvivors and survivors. In addition, to determine the cutoff value, we used receiver operator curve analysis.

**Results:** Eighty-one subjects were included in this study. Eighty of 81 were followed until 12 hours, and 72 out of 80 were followed until 24 hours. Twenty-five subjects (31%) did not survive within 3 days of hospitalization. Only 24-hour lactate clearance had significant median difference (-17.0% in nonsurvivor vs. 15.2% in survivor group; *P* = 0.034). The best cutoff value for 24-hour lactate clearance was -6.0% (AUC 0.744, sensitivity 62.5% and specificity 87.5%, positive predictive value 58.8% and negative predictive value 89.1%, relative risk 5.39). From multivariate analysis, 24-hour lactate clearance was proven to be an independent predictor of mortality.

**Conclusion:** Median of 24-hour lactate clearance was significantly lower in nonsurvivors of severe sepsis and septic shock patients. Its cutoff value was -6.0%.

**Acknowledgements:** Gratitude to Dr Imam Subekti, head of Internal Medicine Department, Faculty of Medicine University of Indonesia, and Dr Aida Lydia, head of Internal Medicine study program, Faculty of Medicine University of Indonesia for their guidance and useful critiques of this research work. Thanks also to Dr Kuntjoro Harimurti, Dr Esthika Dewiasty, and Ms Utami for their advice and assistance in doing methodology and statistic of this study

**References**

1. Okorie ON, Dellinger P: Lactate: biomarker and potential therapeutic target. *Crit Care Clin* 2011, 27:299-326.

2. Schuetz P, Haubitz S, Mueller B: Do sepsis biomarkers in the emergency room allow transition from bundled sepsis care to personalized patient care? *Curr Opin Crit Care* 2012, **18**:341-349.

3. Nguyen HB, Loomba M, Yang JJ, Jacobsen G, Shah K, Otero RM: Early lactate clearance is associated with improved outcome in severe sepsis and septic shock. *Crit Care Med* 2004, **32**:1637-1642.

4. Nichol A, Bailey M, Egi M, Pettila V, French C, Stachowski E: Dynamic lactate indices as predictors of outcome in critically ill patients. *Crit Care* 2011, **15**:R242.
P3

High frequency of myeloid-derived suppressor cells in sepsis patients, with the granulocytic subtype dominating in Gram-positive cases.

H Janols¹,², C Bergenfelz², R Allaoui², A-M Larsson³, L Rydén⁴, S Björnsson⁵,
S Janciauskiene⁶, M Wullt¹, A Bredberg⁷, K Leandersson²

¹Department of Infectious Diseases, Skane University Hospital, Lund University, Malmo, Sweden; ²Center for Molecular Pathology, Skane University Hospital, Lund University, Malmo, Sweden; ³Department of Laboratory Medicine, TCR, MV, Lund University, Lund, Sweden; ⁴Department of Surgery, Lund University Hospital, SUS, Lund, Sweden; ⁵Cytometry Laboratory and Department of Laboratory Medicine, Skane University Hospital, Lund University, Malmo, Sweden; ⁶Department of Pulmonology, Hannover Medical School, Hannover, Germany; ⁷Department of Medical Microbiology, Skane University Hospital, Lund University, Malmo, Sweden
*Critical Care* 2014, **18**(Suppl 2):P3; doi:10.1186/cc14006

**Introduction:** Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were originally identified in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity and even bacterial infections. Human MDSCs are commonly divided into monocytic (Mo-MDSCs) and granulocytic (PMN-MDSCs) subtypes. To what extent the *bona fide* cancer MDSCs are representative of the proposed MDSCs found in other diseases is not well known. PMN-MDSCs have previously been found to be enriched among low-density granulocytes (LDGs) in density gradient centrifuged blood.

**Methods:** In this study we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood as compared to density gradient centrifuged blood.

**Results:** We found a high frequency of typical CD14⁺HLADRlow Mo-MDSCs in all sepsis patients, whereas the typical PMN-MDSCs as well as a prominent CD14low PMN-MDSC-like population appeared preferentially in Gram-positive cases (Figures 1 to 3). The CD14low PMN-MDSC variant was demonstrated to suppress T-cell proliferation *in vitro* via a ROS-dependent

mechanism, to display an increased IL-10:TNFα ratio, and to present with signs of immaturity: blast morphology and low cytokine levels (Figures 4 and 5).

**Conclusion:** We conclude that a spectrum of cells with MDSC features are enriched in sepsis, and that microbial origin of sepsis contributes to the substantial interindividual patient variation in MDSC pattern.

P4

Selective decontamination using antibiotics in ICU patients: counterfactual protection versus contextual hazard toward bacteremia incidences.

JC Hurley

Melbourne Medical School, University of Melbourne and Ballarat Health
Services, Ballarat, Australia

*Critical Care* 2014, **18**(Suppl 2):P4; doi:10.1186/cc14007

**Introduction:** Among methods for preventing pneumonia and possibly also bacteremia in ICU patients, selective digestive decontamination (SDD; topical with or without protocolized parenteral antibiotic) appears most effective within randomized concurrent controlled trials (RCCTs) [1]. However, whether parenteral antibiotic is required, and whether SDD actually increases pneumonia incidences in SDD RCCTs versus the broader ICU pneumonia evidence base, remain unresolved [2,3]. The purpose of this analysis is to test for counterfactual and contextual effects of the topical and parenteral SDD components on the bacteremia incidence versus the broader evidence base related to the patient group at risk of VAP.

**Methods:** Bacteremia incidence proportion data were extracted from component (control and intervention) groups from studies investigating antibiotic (SDD) or nonantibiotic methods of VAP prevention. Both the counterfactual and the contextual effects of SDD factorized as topical or protocolized parenteral exposures were estimated using random-effects meta-analysis of study and group level data. Studies without any prevention methods under study constituted the reference category (benchmark groups).

**Results:** As a counterfactual within RCCTs, SDD when given as combined topical and parenteral antibiotic appears to halve the bacteremia incidence (odds ratio (OR) 0.59; 0.48 to 0.73; *n* = 18 studies). As a contextual however, the mean bacteremia incidence among 27 control groups (17.1%; 13.1 to 22.1%) and 12 intervention groups receiving topical antibiotic alone (16.2%; 9.1 to 27.3%) from SDD RCCTs is double that of 36 benchmark groups (8.3; 7.0 to 10.8%) and 19 control groups from studies of nonantibiotic methods (7.7%; 5.2 to 11.1). The upward dispersion in bacteremia incidence among component groups from SDD RCCTs away from this benchmark is striking with all but two of the 27 control groups and all but two of 12 SDD intervention groups that did not receive PPAP being above this benchmark.

**Conclusion:** The major contextual hazard of SDD toward bacteremia among ICU patients is inapparent within individual studies. The apparent protection in SDD RCCTs is spurious as the SDD counterfactual is conflated by the strong contextual effect with partial mitigation by SDD

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>Figure 2(abstract P3)</figcaption></figure>
Mononuclear fraction
Low density granulocytes

<figure><img src="image_5.png" /><figcaption>Figure 3(abstract P3)</figcaption></figure>

PMN-MDSCs

<figure><img src="image_6.png" /></figure>

low CD14 PMN-MDSCs

<figure><img src="image_7.png" /></figure>

Suppression of T cell proliferation

<figure><img src="image_8.png" /></figure>

inhibitors of
T cell suppression

<figure><img src="image_9.png" /><figcaption>Figure 4(abstract P3)</figcaption></figure>

Cytokine concentrations

<figure><img src="image_10.png" /></figure>

IL10:TNFα ratio

<figure><img src="image_11.png" /><figcaption>Figure 5(abstract P3)</figcaption></figure>
protocolized parenteral antibiotic. Not only is the safety of SDD within the ICU environment unclear, but this SDD contextual effect may conflate the apparent SDD counterfactual effect on the incidence of bacteremia, as with VAP.

### References

1. Hurley JC: Prophylaxis with enteral antibiotics in ventilated patients: selective decontamination or selective cross-infection? *Antimicrob Agents Chemother* 1995, 39:941-947.

2. Hurley JC: Ventilator associated pneumonia prevention methods using topical antibiotics: herd protection or herd peril? *Chest* 2014 in press.

3. Hurley JC: The perfidious effect of topical placebo: Calibration of Staphylococcus aureus ventilator-associated pneumonia incidence within selective digestive decontamination studies versus the broader evidence base. *Antimicrob Agents Chemother* 2013, 57:4524-4531.

## P5

### Thalidomide exerts protective immunomodulatory action during Klebsiella pneumoniae B5055-induced acute lung infection in BALB/c mice.

V Kumar*, S Chhibber

Department of Microbiology, Panjab University, Chandigarh, India

Critical Care 2014, **18**(Suppl 2):P5; doi:10.1186/cc14008

**Introduction:** Thalidomide (α-naphthylimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, anti-angiogenic, immunomodulatory properties through a mechanism that is not fully established. Keeping these properties in mind, we have tried to find out the anti-inflammatory and immunomodulatory properties of thalidomide in mouse model of acute inflammation by introducing Klebsiella pneumoniae B5055 in BALB/c mice via the intranasal route.

**Methods:** Acute lung infection (ALI) or pneumonia in BALB/c mice was induced via instillation of selected dose (10⁴ CFU/ml) of bacteria (that is, K. pneumoniae B5055) intranasally. Mice were observed for 7 days and lungs were isolated on designated days for studying difference in bacterial load and other proinflammatory mediators using standard biochemical methods and ELISA.

**Results:** The intranasal instillation of bacteria in this mouse model of acute pneumonia-induced inflammation led to significant increase in neutrophil infiltration into the lungs. This was further accompanied by an increased production of proinflammatory cytokines (that is, TNFα and IL-1α) and other mediators of inflammation (that is, malondialdehyde (MDA), myeloperoxidase (MPO) and nitric oxide (NO)) in the lung tissue. The animals, which received thalidomide alone orally or in combination with augmentin, 30 minutes prior to bacterial instillation into the lungs

via intranasal route, showed significant (P ≤ 0.05) decrease in neutrophil influx into the lungs. A significant (P ≤ 0.05) decrease in the production of proinflammatory cytokines (that is, TNFα and IL-1α) and other biochemical mediators of acute inflammation (that is, MDA, MPO, and NO) was also observed in this group. But the augmentin treatment alone did not decrease these proinflammatory mediators significantly (P ≥0.05) as compared to the control group.

**Conclusion:** We therefore conclude that thalidomide ameliorates lung inflammation induced by K. pneumoniae B5055 without significantly (P ≤ 0.05) decreasing the bacterial load in the lung tissue whereas augmentin takes care of bacterial proliferation. Hence, it can be used as an adjunct therapy along with antibiotics as an anti-inflammatory or an immunomodulatory agent in case of acute lung infection or pneumonia.

## P6

### Impact of purulent complications and sepsis on cardiovascular system in patients with type 2 diabetes.

E Shalaeva*, B Babadjanov, U Pulatov, N Dadabayeva, A Shalaeva
Republican Center of Purulent Surgery and Complications of Diabetes,
Tashkent Medical Academy, Tashkent, Uzbekistan
Critical Care 2014, **18**(Suppl 2):P6; doi:10.1186/cc14009

**Introduction:** Purulent complications in patients with type 2 diabetes are usually severe, often complicated by sepsis and require emergency surgery. Noncardiac surgery is associated with a 7 to 11% complication rate and mortality of 0.8 to 1.5% [1], up to 42% are cardiac reasons [2]. After surgery, 2% of patients suffer major cardiac complications [3], and 8% show evidence of significant myocardial injury [2]. The aim of this study was to identify the impact of purulent complications and sepsis on cardiovascular system in patients with type 2 diabetes.

**Methods:** We analyzed 112 consecutive patients (54 men and 58 women) aged 57.2 ± 8.4 years with purulent-necrotic complications (gangrene, phlegmon, and abscess) of type 2 diabetes and sepsis in 2013. We compared laboratory and instrumental data (blood tests, ECG, echocardiography and others), which were previously obtained in the same patients receiving inpatient treatment before sepsis (2011 to 2012).

**Results:** Gangrene of lower extremities in 59 (52.7%) prevailed among purulent complications. After the development of sepsis we detected in all patients significantly increased heart rate, respiratory rate per minute, leukocytosis, anemia, worse glucose metabolism and renal function (Table 1). Congestive heart failure became more severe. This was confirmed by decrease of left ventricle ejection fraction (55.2 ± 5.1% before sepsis vs. 49.3 ± 4.1% after) and increase brain natriuretic peptide (291.4 ± 34.5 ng/ml vs. 395.2 ± 28.1 ng/ml, P < 0.001). Prior sepsis in 66 (58.9%) of patients with arterial hypertension was observed, after in 88 (78.6%). After admission to

<table><caption>**Table 1(abstract P6) Hemodynamic parameters and blood tests in patients with purulent complications of type 2 diabetes and sepsis**</caption><thead><tr><th>Parameter</th><th>Before sepsis (n = 112)</th><th>After sepsis (n = 112)</th><th>P value</th></tr></thead><tbody><tr><td>Heart rate (beats/minute)</td><td>78.4 ± 15.2</td><td>112.5 ± 18.9</td><td>&lt; 0.001</td></tr><tr><td>Respiratory rate (breaths/minute)</td><td>18.0 ± 2.0</td><td>29.5 ± 5.5</td><td>&lt; 0.001</td></tr><tr><td>Systolic BP (mmHg)</td><td>155.7 ± 35.4</td><td>154.2 ± 58.5</td><td>n.s</td></tr><tr><td>Diastolic BP (mmHg)</td><td>90.4 ± 10.3</td><td>91.9 ± 8.6</td><td>n.s</td></tr><tr><td><strong>Left ventricle ejection fraction (%)</strong></td><td><strong>55.2 ± 5.1</strong></td><td><strong>49.3 ± 4.1</strong></td><td><strong>0.033</strong></td></tr><tr><td>Fasting plasma glucose (mmol/l)</td><td>8.4 ± 2.5</td><td>15.4 ± 4.8</td><td>&lt; 0.001</td></tr><tr><td>Two-hour plasma glucose (mmol/l)</td><td>10.2 ± 2,8</td><td>19.9 ± 3.3</td><td>&lt; 0.001</td></tr><tr><td><strong>HbA1c (%)</strong></td><td><strong>8.4 ± 0.5</strong></td><td><strong>12.1 ± 0.5</strong></td><td><strong>&lt; 0.001</strong></td></tr><tr><td>Hemoglobin (g/l)</td><td>121.5 ± 12.5</td><td>105.4 ± 11.7</td><td>0.04</td></tr><tr><td>White count (10<sup>3</sup>)</td><td>6.7 ± 1.2</td><td>14.4 ± 2.1</td><td>&lt; 0.001</td></tr><tr><td>Fibrinogen (mg%)</td><td>411.6 ± 103.6</td><td>715.4 ± 215.5</td><td>&lt; 0.001</td></tr><tr><td>Blood urea (mmol/l)</td><td>6.1 ± 2.9</td><td>8.8 ± 2.5</td><td>0.011</td></tr><tr><td>Blood creatinine (mmol/l)</td><td>88.4 ± 18.5</td><td>105.6 ± 17.3</td><td>0.02</td></tr><tr><td>Brain natriuretic peptide (ng/ml)</td><td>291.4 ± 34.5</td><td>395.2 ± 28.1</td><td>&lt; 0.001</td></tr></tbody></table>

BP, blood pressure; HbA1c, glycosylated hemoglobin A1c
<table><caption>**Table 2(abstract P6) Cardiovascular comorbidity in patients with type 2 diabetes before and after purulent-necrotic complications and sepsis**</caption><thead><tr><th>Parameter</th><th>Before sepsis (n = 112)</th><th>After sepsis (n = 112)</th></tr></thead><tbody><tr><td><strong>Insulin dependence</strong></td><td><strong>42 (37.5)</strong></td><td><strong>112 (100)</strong></td></tr><tr><td>Normal blood pressure (110 to 139 mmHg)</td><td>46 (41.1)</td><td>11 (9.8)</td></tr><tr><td><strong>Arterial hypertension</strong></td><td><strong>66 (58.9)</strong></td><td><strong>88 (78.6)</strong></td></tr><tr><td>First degree (140 to 159 mmHg)</td><td>33 (29.5)</td><td>21 (8.9)</td></tr><tr><td>Second degree (160 to 179 mmHg)</td><td>21 (18.6)</td><td>43 (38.4)</td></tr><tr><td>Third degree (&gt;180 mmHg)</td><td>12 (10.7)</td><td>24 (21.4)</td></tr><tr><td>Arterial hypotension (&lt;90 mmHg)</td><td>-</td><td>13 (11.6)</td></tr><tr><td>CAD, stable angina</td><td>108 (94.6)</td><td>82 (73.2)</td></tr><tr><td>FC I</td><td>18 (16.1)</td><td>-</td></tr><tr><td>FC II</td><td>29 (25.9)</td><td>18 (16.1)</td></tr><tr><td>FC III</td><td>52 (46.4)</td><td>45 (40.2)</td></tr><tr><td>FC IV</td><td>9 (8.0)</td><td>19 (17.0)</td></tr><tr><td><strong>CAD, unstable angina</strong></td><td><strong>4 (3.6)</strong></td><td><strong>17 (15.2)</strong></td></tr><tr><td><strong>Acute myocardial infarction</strong></td><td>-</td><td><strong>13 (11.6)</strong></td></tr><tr><td>Postinfarction cardiiosclerosis</td><td>7 (6.3)</td><td>7 (6.3)</td></tr><tr><td>Atrial fibrillation</td><td>7 (6.3)</td><td>7 (6.3)</td></tr><tr><td>Supraventricular arrhythmia</td><td>3 (2.7)</td><td>12 (10.7)</td></tr><tr><td>Ventricular arrhythmia</td><td>14 (12.5)</td><td>36 (32.1)</td></tr><tr><td>Congestive heart failure</td><td>112 (100)</td><td>112 (100)</td></tr><tr><td>FC II (NYHA)</td><td>76 (67.8)</td><td>26 (23.2)</td></tr><tr><td>FC III (NYHA)</td><td>36 (32.1)</td><td>65 (58)</td></tr><tr><td>FC IV (NYHA)</td><td>-</td><td>21 (18.7)</td></tr><tr><td>Abscesses of the lower extremity</td><td>-</td><td>22 (19.6)</td></tr><tr><td>Phlegmon of the lower extremity</td><td>-</td><td>31 (27.7)</td></tr><tr><td>Gangrene of lower extremity</td><td>-</td><td>59 (52.7)</td></tr></tbody></table>

Data presented as n (%). CAD, coronary artery disease; FC, functional class; NYHA, New York Heart Association

the centre, patients had no signs of septic shock. In 13 (11.6%) patients, the perioperative period was complicated by acute myocardial infarction, which was accompanied by a fall in blood pressure. We detected an increase of the functional class of stable angina, congestive heart failure, 4.2 times increased incidence of unstable angina, 2.6 times ventricular and four times supraventricular extra systole after septic complications (Table 2).

**Conclusion:** After the development of purulent complications and sepsis in patients with type 2 diabetes, we observed increased incidence of arterial hypertension, arrhythmias, worsened severity of coronary artery disease and congestive heart failure. Perioperative risk of acute myocardial infarction amounted to 11.6%.

**References**

1. Haynes AB, Weiser TG, et al: A surgical safety checklist to reduce morbidity and mortality in a global population. N Engl J Med 2009, 360:491-499.

2. Devereaux PJ, Chan MT, et al: Association between post-operative troponin levels and 30-day mortality among patients undergoing noncardiac surgery. JAMA 2012, 307:2295-2304.

3. Devereaux PJ, Goldman L, et al: Perioperative cardiac events in patients undergoing noncardiac surgery: a review of the magnitude of the problem, the pathophysiology of the events and methods to estimate and communicate risk. CMAJ 2005, 173:627-634.

P7

**Severity of sepsis in patients with acute purulent destructive pulmonary disease depending on the presence of type 2 diabetes: impact on the forecast.**

A Babobekov, B Babadjanov, S Atakov, E Shalaeva
Republican Center of Purulent Surgery and Complications of Diabetes, Tashkent Medical Academy, Tashkent, Uzbekistan
*Critical Care* 2014, **18**(Suppl 2):P7; doi:10.1186/cc14010

**Introduction:** Lung abscesses and gangrene are the most severe clinical manifestation and outcome among acute purulent destructive pulmonary disease (APDPD). Mortality ranges from 10 to 35%, and in the presence of diabetes increases up to 30 to 90% [1]. The main reason for this is the generalization of infection (sepsis), leading to the development of multiple organ failure [2,3]. The aim of this study was to identify the severity of sepsis in patients with APDPD depending on the presence of type 2 diabetes, and the impact on the forecast.

**Methods:** During the period 2012 to 2013, we examined 408 patients aged 48.5 ± 12.5 years (258 men/150 women) who underwent surgical treatment for APDPD. The patients were divided into two groups: 144 patients with type 2 diabetes, and controls (n = 246). We carried out computed tomography, ECG, echocardiography, laboratory biochemical testing, and bacteriological analysis of pathologic material and blood samples.

**Results:** Patients with type 2 diabetes had much more complications and cases of severe sepsis and septic shock (Table 1). Bacteriological analysis of the pathologic material showed Gram-positive bacteria in 35 to 45%, anaerobic association in 55 to 65%, pathological fungi in 50 to 60%. The patients with type 2 diabetes had much more time from the onset of the first symptoms of lung disease prior to admission (12.5 ± 3.5 vs. 7.5 ± 2.5 days, P = 0.002), and the duration of inpatient treatment was significantly longer (13.8 ± 5.5 vs. 7.1 ± 3.4 days, P = 0.001). Only 53 (36.8%) of patients with type 2 diabetes and 68 (29.5%) without it had bacteriological positive blood culture. The analysis of the distribution of pathogens in groups is presented in Figure 1. Patients with diabetes had more Candida spp.

<table><caption>**Table 1(abstract P7) Clinical symptoms and severity of sepsis in patients with acute purulent destructive pulmonary disease depending on the presence of type 2 diabetes**</caption><thead><tr><th>Data</th><th>Type 2 diabetes (n = 144)</th><th>Control (n = 264)</th></tr></thead><tbody><tr><td>Acute lung abscess</td><td>59 (40.9)</td><td>122 (46.2)</td></tr><tr><td>Necrotizing pneumonia</td><td>47 (32.6)</td><td>98 (37.1)</td></tr><tr><td>Lung gangrene</td><td>38 (26.4)</td><td>44 (16.7)</td></tr><tr><td>Empyema</td><td>88 (61.1)</td><td>81 (30.7)</td></tr><tr><td>Pyopneumothorax</td><td>16 (11.1)</td><td>9 (3.4)</td></tr><tr><td>Mediastinitis</td><td>34 (23.6)</td><td>16 (6.1)</td></tr><tr><td>Body temperature &gt;38°C/&lt;36°C</td><td>98 (68.1)/21 (14.6)</td><td>261 (98.9)/3 (1.1)</td></tr><tr><td>Respiratory rate &gt;20/minute</td><td>144 (100)</td><td>264 (100)</td></tr><tr><td>Heart rate &gt;90 beats/minute</td><td>138 (95.8)</td><td>242 (91.7)</td></tr><tr><td>PaCO₂ &lt;32 mmHg</td><td>144 (100)</td><td>264 (100)</td></tr><tr><td>Leukocytes &gt;12,000/&lt;4,000 cells/mm³</td><td>111 (77.1)/13 (9.1)</td><td>202 (76.5)/11 (4.2)</td></tr><tr><td>Renal failure, oliguria</td><td>42 (29.2)</td><td>34 (12.9)</td></tr><tr><td>Increase liver enzymes</td><td>34 (23.6)</td><td>45 (17.1)</td></tr><tr><td>Systolic blood pressure &lt;90 mmHg</td><td>33 (22.9)</td><td>51 (19.3)</td></tr><tr><td>Sepsis</td><td>101 (70.1)</td><td>223 (84.5)</td></tr><tr><td>Severe sepsis</td><td>25 (17.4)</td><td>29 (11.0)</td></tr><tr><td>Septic shock</td><td>18 (12.5)</td><td>12 (4.5)</td></tr></tbody></table>

Data presented as n (%)

A Babobekov, B Babadjanov, S Atakov, E Shalaeva
Republican Center of Purulent Surgery and Complications of Diabetes, Tashkent Medical Academy, Tashkent, Uzbekistan
*Critical Care* 2014, **18**(Suppl 2):P7; doi:10.1186/cc14010
<figure><img src="image_12.png" /><figcaption>Figure 1(abstract P7)</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>Figure 2(abstract P7) Man, 42 years old, with right lung abscess after admission to the centre</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Figure 3(abstract P7) The same patient on the fourth day after drainage of the abscess</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>Figure 4(abstract P7) The same patient before discharge, 14 days in dynamics</figcaption></figure>

(Figure 1). Figures 2, 3 and Figure 4 present the X-ray dynamics of a 42-year-old man with lung abscess. Clinical recovery in patients with type 2 diabetes was significantly worthy compared to controls (45 (31.2%) vs. 153 (57.9%)), mortality rate 48 (33.3%) versus 39 (14.7%), respectively.

**Conclusion:** In patients with acute purulent destructive pulmonary disease and type 2 diabetes, severe sepsis and septic shock more often prevailed, inpatient mortality rate was 2.27 times higher, compared to patients with normal glucose metabolism.

**References**

1. Defraigne JO, et al: Cavernostomy: an old but effective technique in the treatment of pulmonary abscess. *Rev Med Liege* 2007, 52:498-501.

2. Refaely J, Weissberg D: Gangrene of the lung: treatment in two stages. *Ann Thorac Surg* 1997, **64**:970-973.

3. Rice TW, Ginsberg PJ, Todd TR: Tube drainage of lung abscesses. *Ann Thorac Surg* 1987, **44**:356-359.

P8

Risk factors and incidence of mediastinitis in patients with lung abscess and sepsis.

E Shalaeva¹, B Babadjanov, B Janabaev, A Shalaeva
Republican Center of Purulent Surgery and Complications of Diabetes,
Tashkent Medical Academy, Tashkent, Uzbekistan
*Critical Care* 2014, **18**(Suppl 2):P8; doi:10.1186/cc14011

**Introduction:** Mediastinitis is a life-threatening condition, which is accompanied by high rates of mortality in cases of delayed diagnosis and inadequate treatment. The aim of the study was to identify the risk factors and incidence of mediastinitis in patients with lung abscess and sepsis.

**Methods:** In 2013, 218 consecutive patients (83 women and 135 men) with lung abscess and sepsis aged 45.8 ± 13.2 years were operated. They had a full range of laboratory and instrumental examinations, including echocardiography and computed tomography.

**Results:** Aerobic-anaerobic association in sputum was revealed in all patients with lung abscess and sepsis, *Candida* spp. in 34 (15.6%). Blood culture was positive only in 59 (27%) patients, which had not previously received antibacterial therapy (polymicrobial flora including *Staphylococcus* and *Streptococcus* specimen). Empyema was diagnosed in 123 patients (56.4%), 31 (14.2%) of them were complicated by mediastinitis. The main clinical symptoms of mediastinitis were hyperthermia (100%), dysphagia (83.8%), dyspnea (80.6%), chest pain (61.3%), orthopnea
<table><caption>Table 1(abstract P8) Risk factors for mediastinitis in patients with lung abscess and sepsis</caption><thead><tr><th></th><th>With mediastinitis (n = 31)</th><th>Without mediastinitis (n = 187)</th><th>P value</th></tr></thead><tbody><tr><td>Gender, male/female, n (%)</td><td>7 (22.6)/24 (77.4)</td><td>128 (68.5)/59 (31.5)</td><td>0.001</td></tr><tr><td>Type 2 diabetes mellitus, n (%)</td><td>26 (83.9)</td><td>42 (22.5)</td><td>0.001</td></tr><tr><td>Body mass index</td><td>32.3 ± 5.3</td><td>27.8 ± 6.1</td><td>0.031</td></tr><tr><td>Hemoglobin (g/l)</td><td>87.9 ± 9.2</td><td>128.4 ± 18.4</td><td>&lt; 0.001</td></tr><tr><td>Fibrinogen (mg%)</td><td>800 ± 200</td><td>533 ± 166</td><td>&lt; 0.001</td></tr><tr><td>End-diastolic volume of the left ventricle (ml)</td><td>139 ± 27</td><td>108 ± 28</td><td>0.024</td></tr><tr><td>End-systolic volume of the left ventricle (ml)</td><td>66.1 ± 8.2</td><td>46.8 ± 10.4</td><td>&lt; 0.001</td></tr><tr><td>Left ventricular ejection fraction (%)</td><td>52.4 ± 2.2</td><td>57.2 ± 3.4</td><td>0.002</td></tr></tbody></table>

(61.3%), and tachycardia (87.1%). The computer tomography revealed an increase in mediastinum size with accumulation of fluids and fluid in the pleural cavities (100%), free gas in the mediastinum (45.1%), enlarged mediastinal lymph nodes (45.1%), and fluid in the pericardium cavity (48.4%). To analyze the risk factors, we include 31 patients with lung abscess and sepsis complicated by mediastinitis in the first group, and 187 patients without mediastenitis in the second group. Groups were similar in age (46.1 ± 8.2 years vs. 45.8 ± 13.2 years). A total 77.4% of patients with mediastinitis were women who suffered from type 2 diabetes (HbA1c = 9.7 ± 1.4%), congestive heart failure and anemia. Significant differences in the groups according to the data of laboratory and instrumental studies are presented in Table 1.

**Conclusion:** In total, 14.2% of patients presented with lung abscess and sepsis complicated by mediastinitis, more commonly in women with diabetes mellitus, obesity, anemia and reduced ejection fraction of the left ventricle.

P9

Impact of KDO in biological activity of Re-LPS.

I Prokhorenko*, S Zubova, D Kabanov, S Grachev
Institute of Basic Biological Problems, Pushchino, Moscow Region, Russia
*Critical Care 2014, 18(Suppl 2):P9; doi:10.1186/cc14012

**Introduction:** The minimal biological active structure of endotoxins (lipopolysaccharides (LPS)) is Re-LPS (KDO2-lipid A), which consist of lipid A and two (or three) molecules of 3-deoxy-D-manno-2-octulosonic acid (KDO) [1,2]. Biological activity of endotoxins is defined in general by the number and distribution of acyl residues on the lipid A backbone [3]. Recently it has been reported that KDO-treated RAW 264.7 cells exhibited a gene expression pattern similar to that in LPS-treated cells. These authors revealed that free KDO participated in crosstalk between Toll-like receptors (TLR) and G protein-coupled receptors and so that regulated activators and repressors of immune signaling [4]. LPS-dependent TLR4-triggered activation of target cells leads to specific changes in the levels of surface receptors and induces synthesis of proinflammatory cytokines [5]. However, the dependence of these processes on the structural composition of LPS is not well understood. To extend our knowledge in this field, the effects of free KDO as well as KDO as covalently linked to lipid A constituent of Re-LPS on expression of TLR4, CD11b and CD14 receptors and TNFα synthesis in whole human blood have been investigated.

**Methods:** Human blood was incubated with Re-LPS from *Escherichia coli* JM103 or *Salmonella enterica* sv *Typhimurium* SL1181 (100 ng/ml) or with lipids A from *E. coli* F583 or *S. enterica* sv Minnesota R595 (80 ng/ml) or with ammonium salt of KDO (20 ng/ml) at 37°C in 5% CO₂-humidified atmosphere for 2 or 6 hours to determine receptor expression or TNFα release, respectively. Receptor expression was monitored by EPICS XL-MCL flow cytometer using Alexa Fluor 488 anti-TLR4 (HTA125), anti-CD11b (ICRF44) and anti-CD14 (HCD14) antibodies. Human TNF-α ELISA Kit II was exploited to TNFα determination.

**Results:** Re-LPS *E. coli* or Re-LPS *S. enterica* differentially affected receptor expression in comparison to their respective lipids A. Free KDO in the equimolar concentration as it exists in KDO2-lipid A (Re-LPS) did not influence the level of CD14 but downregulated the expression of TLR4 and CD11b (Figure 1). Tenfold increased KDO concentration did not affect further the receptor expression. The addition of KDO2 to lipid A *E. coli* - that

is, applying KDO as covalently linked constituent of Re-LPS - led to upregulation of CD14 and TLR4 but downregulated CD11b expression. The expression of TLR4 was most pronounced upregulated by Re-LPS *S. enterica* but in the case of CD14 and CD11b this Re-LPS had an opposite effect in comparison to *E. coli* endotoxins (table in Figure 1). Lipid A *S. enterica* was a less potent TNFα inductor than that from *E. coli* (Figure 2). This may be explained by the differences in lipid A composition determining lipid A affinity to target receptor(s). LPS *E. coli*, as had been shown early, caused MyD88-dependent fast NF-κB degradation (rapid TNFα response) whereas LPS *S. enterica* induced MyD88-independent signaling (delayed TNFα response) [5]. In our study, free KDO did not stimulate TNFα release. KDO2 as a constituent of Re-LPS *S. enterica* increased significantly the TNFα-inducing activity of lipid A *S. enterica* but this effect was not so distinguished between Re-LPS *E. coli* and lipid A *E. coli* (Figure 2).

**Conclusion:** Free KDO in the used concentration was inactive in regulation of TLR4, CD11b and CD14 expression and did not induce TNFα release but its impact in biological activity was detected when KDO was applied as constituent of Re-LPS. This may be explained by the effect of KDO on the spatial conformation of Re-LPS.

**Acknowledgements:** The work was supported by Grant 16.N08.12.1014 established by the Russian Ministry of Education and Science

References

1. Olsthoorn M, Peterson B, Schlecht S, Haverkamp J, Bock K, Thomas-Oates J, Holst O: Identification of a novel core type in *Salmonella* lipopolysaccharide. *J Biol Chem* 1998, 273:3817-3829.

2. Fregolino E, Fugazza G, Galano E, Gargiulo V, Landini P, Lanzetta R, Lindner B, Pagani L, Parrilli M, Holst O, et al: Complete lipooligosaccharide structure of the clinical isolate Acinetobacter baumannii, strain SMAL. *Eur J Org Chem* 2010, 7:1345-1352.

3. Rietschel E, Kirikae T, Schade U, Mamat U, Schmidt G, Loppnow H, Ulmer A, Zahringer U, Seydel U, Di Padova F, et al: Bacterial endotoxin: molecular

<figure><img src="image_16.png" /><figcaption>Figure 1(abstract P9) Expression of TLR4, CD11b and CD14 on monocytes after incubation of whole blood with Re-LPS, lipid A or KDO. Presented are the results of six independent experiments. Alteration in receptor expression was calculated according to the control level that had been expressed as 100%. *Changes in receptor expression were calculated as %MnIX [KDO2-lipid A] - %MnIX [lipid A]</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure 2 (abstract P9) Production of TNFα after incubation of whole blood with lipid A or Re-LPS</figcaption></figure>

relationships of structure to activity and function. *FASEB J* 1994, 8:217-225.

4. Krishnan J, Choi S: Systems biological approaches reveal non-additive responses and multiple crosstalk mechanisms between TLR and GPCR signaling. *Genomics Inform* 2012, 10:153-166.

5. Zugaier S, Zimmer S, Datta A, Carlson R, Stephens D: Differential induction of Toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins. *Infect Immun* 2005, 73:2940-2950.

P10

Two novel formulae are superior to procalcitonin for prediction of sepsis in trauma patients.

H-p Liang*, H Jin, Y Xiao, Z Liu
State Key Laboratory of Trauma, Burns and Combined Injury, Research
Institute of Surgery, The Third Military Medical University, Chongqing,
People's Republic of China
*Critical Care* 2014, **18**(Suppl 2):P10; doi:10.1186/cc14013

**Introduction:** The purpose of this study was to verify the predictive value of two novel formulae and compare them with that of procalcitonin (PCT) for predicting sepsis in trauma patients.

**Methods:** We performed a retrospective study of trauma patients treated at Daping Hospital in Chongqing, China and Affiliated Hospital of Zunyi Medical College between 2010 and 2013. Patients ≥16 years old, admitted to hospital after injury within 24 hours and with length of hospital stay ≥48 hours were included. Predictive ability of two formulae based on LD₅₀ values of the Injury Severity Score (ISS) and New Injury Severity Score (NISS) were verified: ISS/LD₅₀ISS+SIRS score and NISS/LD₅₀NISS+SIRS score, and then were compared with the most common used biomarker PCT. LD₅₀ values for different age groups and genders were obtained in our former study. The statistical performance of the two formulae and PCT to predict sepsis was evaluated using receiver operating characteristic (ROC) curve analysis.

**Results:** Two hundred and twenty-one trauma patients' data were enrolled in the study, including 44 females and 177 males. The average age of the patients was 44.77 ± 15.01 years. The performance of the ISS/LD₅₀ISS+SIRS score and the NISS/LD₅₀NISS+SIRS score was equivalent (area under the ROC curve (AUC) = 0.816 vs. 0.819, *P* >0.05) and both performed better than PCT (AUC = 0.592, *P* < 0.05) in predicting posttraumatic sepsis. For the ISS/LD₅₀ISS+SIRS score, the cutoff value was 2.38, with a positive predictive value of 78.08%, a negative predictive value of 81.33%, a sensitivity of 89.06%, a specificity of 65.59%, a positive likelihood ratio of 2.59, a negative likelihood ratio of 0.17, a Youden index of 0.5465, an odds ratio of 15.52, and an accuracy of 79.19%. For the NISS/LD₅₀NISS+SIRS score, the cutoff value was 2.4677, with a positive predictive value of 79.70%, a negative predictive value of 75.00%, a sensitivity of 82.81%, a specificity of 70.97%, a positive likelihood ratio of 2.85, a negative likelihood ratio of 0.24, a Youden index of 0.5378, an odds ratio of 11.78, and an accuracy of 77.83%.

**Conclusion:** The two novel formulae ISS/LD₅₀ISS+SIRS score and NISS/LD₅₀NISS+SIRS score performed well and were both better than PCT in predicting sepsis post trauma. The value of the two formulae can be easily calculated in real time and can identify the high-risk patients susceptible to sepsis. This method may become an effective way to guide the early assessment and treatment in trauma patients.

P11

Inhibitory effects of evodiamine on zymosan-induced inflammation: inactivation of NF-κB by inhibiting IκBα phosphorylation.

X Fan*, J-y Zhu, Z Liu, J Yan, Q Ma, H-p Liang
State Key Laboratory of Trauma, Burns and Combined Injury, Research Institute of Surgery, The Third Military Medical University, Chongqing, People's Republic of China
*Critical Care* 2014, **18**(Suppl 2):P11; doi:10.1186/cc14014

**Introduction:** Inflammation is a host defense reaction against pathogenic infection. In this process, inflammatory cytokines contribute to combat against infection, but excess cytokines will lead to tissue damage. Nonsteroidal anti-inflammatory drugs and corticosteroids are commonly used for regulating these inflammatory mediators and treatment of inflammatory disorders. But these drugs are not sufficient for clinical practice due to their adverse effects, so new anti-inflammatory drugs are still needed. Evodiamine (EVO), an important alkaloidal component extracted from the fruit of Evodiae fructus, possesses the property of analgesia, antiemesis, and vascular dilatation. Its anti-inflammatory effect and underlying mechanism were investigated using a zymosan-induced inflammatory model.

**Methods:** *In vitro*, RAW264.7 cells and primary peritoneal macrophages were treated with different doses of EVO (25, 50 or 100 µM) for 1 hour prior to incubation with zymosan (100 µg/ml), and the effects of EVO on protein and mRNA levels of proinflammatory cytokines were determined by ELISA and qRT-PCR, respectively. *In vivo*, peritonitis was induced in C57BL/6 mice by zymosan (500 mg/kg) injection, and the effects of EVO (10 mg/kg) on plasma cytokine levels and organ injury were evaluated. Activation of the NF-κB signal pathway was investigated by ELISA-based Trans-AM transcription factor NF-κB p65 kit, immunocytochemistry and western blotting.

**Results:** EVO effectively suppressed the expression of IL-1β, IL-6 as well as TNFα in both protein and mRNA level *in vitro*. It can also attenuate zymosan-induced DNA-binding activity of NF-κB, which was achieved through the inhibitory effects on the phosphorylation of inhibitory kappa B α (IκBα) and p65 nuclear translocation, but there was little association with mitogen-activated protein kinase activation. *In vivo*, treatment with EVO could ameliorate inflammatory cell infiltration and vascular ectasia induced by zymosan in both lung and intestine tissues. EVO can markedly decrease the level of TNFα and IL-6 in plasma, and effectively downregulate expression of IL-6, TNFα and myeloperoxidase in both lung and intestine. Moreover, cell apoptosis in organs was also attenuated by treatment of EVO. The underlying mechanism that a decrease in the phosphorylation of IκBα and the subsequent transcription activity of NF-κB was also confirmed.

**Conclusion:** Taken together, our data demonstrate that EVO displays anti-inflammatory actions *in vitro* and *in vivo* by suppressing the phosphorylation of IκBα and inactivating NF-κB, which suggests that EVO is a potential therapeutic agent against inflammatory disorder.

P12

Imaging in severe sepsis and septic shock: is early radiological identification of occult sources of infection needed?.

A Creamer*, J Keep
Emergency Department, Kings College Hospital, London, UK
*Critical Care* 2014, **18**(Suppl 2):P12; doi:10.1186/cc14015

**Introduction:** The importance of imaging in establishing the focus of infection is recognised in current guidelines for the management of severe sepsis [1], with decisions regarding timing and modality of imaging left to the physicians' clinical judgement. In the emergency department (ED), clinical assessment combined with bedside investigations of chest X-ray
<figure><img src="image_18.png" /></figure>

( CXR) and urine dip can be used to confirm the two most common sources [2]. However, they may fail to identify occult sources of infection, such as intraabdominal collections and abscesses, the treatment of which may require alteration of empirical treatment or be refractory to antibiotic therapy alone. Further imaging is necessary to confirm the focus so that optimal treatment can be achieved.

**Methods:** The study cohort was composed of 50 consecutive patients who met the criteria for severe sepsis [1] attending the ED in 2013. Electronic and paper patient records and radiology results were analysed. All radiological studies done in the first 72 hours following attendance were included in the study.

**Results:** CXR was performed as the initial investigation in 49 of the 50 patients (98%). The median time from arrival at the ED to initial imaging was 1 hour:00 minutes (range 0 hours:04 minutes to 4 hours:25 minutes). Initial investigations in the ED of CXR and urine dip identified a septic focus in 30 of 50 patients (60%). Fourteen of the remaining 20 went on to have one or more further imaging studies. Figure 1 outlines the second-line and third-line radiological investigations performed (number where a septic focus was identified in parentheses). The median time to first positive imaging was 0 hours: 50 minutes (range 0 hours:04 minutes to 40 hours), with the source remaining unidentified by imaging and urine testing in 15 of the 50 patients.

**Conclusion:** Our results indicate that simple bedside investigations are able to identify a focus of infection in 60% of patients presenting to the ED with severe sepsis. Our results support the continued use of CXR as the initial imaging modality in severe sepsis, but also demonstrate the benefit of further imaging in confirming the focus of infection and to guide definitive treatment. Instances where further imaging was delayed by several days highlight the need for guidelines detailing which investigations should be done and in what time frame.

References

1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. *Intensive Care Med* 2013, 39:165-228.

2. Perman SM, Goyal M, Gaieski DF: Initial emergency department diagnosis and management of adult patients with severe sepsis and septic shock. *Scand J Trauma Resusc Emergency Med* 2012, 20:41.

P13

**Proinflammatory versus anti-inflammatory response in sepsis patients: looking at the cytokines.**

D Anand¹*, S Ray², S Bhargava¹, S Das¹, A Garg², S Taneja², D Dhar², L Mohan Srivastava¹

¹Department of Biochemistry and Critical Care Medicine, Sir Ganga Ram Hospital, New Delhi, India; ²Department of Critical Care and Emergency Medicine, Sir Ganga Ram Hospital, New Delhi, India

*Critical Care* 2014, **18**(Suppl 2):P13; doi:10.1186/cc14016

**Introduction:** Despite improvements in supportive care, mortality rates in sepsis remain substantially high. Sepsis exhibits phases of enhanced inflammation, alternating with immune suppression with a resultant variant time point of mortality; yet no human study has correlated levels of cytokines to the timeline of mortality. Our study attempts to analyze the association of levels of proinflammatory and anti-inflammatory cytokines in sepsis with the timeline of death in terms of early (<5 days) versus late (>5 days) mortality, and day of death. We also assessed correlation of these cytokines with length of stay.

**Methods:** The study protocol was approved by Institutional Ethics Committee. Subjects were 74 consecutive patients with community-acquired severe sepsis/septic shock admitted to the ICU of a tertiary care superspecialty hospital. Blood samples drawn on days 1, 3 and 7 of admission were analysed for proinflammatory cytokine (TNFα) by chemiluminescent immunometric assay and anti-inflammatory cytokine (IL-10) by ELISA. Subjects were segregated on basis of: ratio of proinflammatory and anti-inflammatory mediators on day 1 of admission into patients with predominant proinflammatory or predominant anti-inflammatory response. Survival and time point of mortality into survivor, early mortality and late mortality groups. Statistical analyses were performed using SPSS version 17.

**Results:** There were 37 patients each in predominant proinflammatory and predominant anti-inflammatory groups. The number of deaths was 11 and 17 respectively in these groups. However, there was significantly higher early mortality in the proinflammatory group as compared to the anti-inflammatory group (7 vs. 1, *P* = 0.0247). On the contrary, late deaths were significantly higher in the anti-inflammatory group as compared to the proinflammatory (16 vs. 4 *P* = 0.0017). The ratio of proinflammatory/anti-inflammatory cytokines (TNF/IL-10) on day 1 was significantly lower in patients of late death (*n* = 20) as compared to patients of early death (*n* = 8) and survivors (*n* = 46) as shown in Table 1. Further, the median day of death
<table><caption>Table 1(abstract P13) TNF/IL-10 ratio in study groups at different time points</caption><thead><tr><th></th><th>Early death (≤ 5 days) (n = 8)</th><th>Late death (>5 days) (n = 20)</th><th>Survivors (n = 46)</th><th>P value</th></tr></thead><tbody><tr><td>Day 1</td><td>1.81 (1.00 to 3.44)</td><td>0.50 (0.31 to 0.90)</td><td>1.22 (0.43 to 3.91)</td><td>0.020*</td></tr><tr><td>Day 3</td><td>1.12 (0.50 to 3.91)</td><td>1.01(0.20 to 2.21)</td><td>2.5 (0.90 to 3.91)</td><td>0.158</td></tr><tr><td>Day 7</td><td>-</td><td>1.25 (0.59 to 2.38)</td><td>1.79 (0.75 to 3.90)</td><td>0.256</td></tr></tbody></table>

Data presented as median (IQR). Kruskal-Wallis test was performed for significance between three groups. *P < 0.05 considered significant

was significantly delayed in patients in the anti-inflammatory as compared to the proinflammatory group (20 vs. 5, *P* < 0.001). Length of hospital stay amongst survivors was significantly longer in the anti-inflammatory as compared to the proinflammatory group (23 vs. 10 *P* < 0.001).

**Conclusion:** Our preliminary data suggest that in sepsis, the ratio of proinflammatory/anti-inflammatory cytokines on day 1 is significantly associated with time point of mortality; hence, this ratio can be used to particularize management. Further studies are in progress to substantiate the role of proinflammatory and anti-inflammatory cytokines in this subset of patients. Moreover, since predominant anti-inflammatory response was associated with later death, role of immunomodulators in sepsis needs to be explored.

P14

**Understanding heterogeneity in the host response to *Staphylococcus aureus* infection for prognostic biomarker discovery.**

JB Dinoso¹*, J Gutierrez², DF Choy¹, S Kummerfeld³, A Baruch², HF Chambers⁴, CM Rosenberger¹

¹ITGR Diagnostic Discovery, Genentech Inc., South San Francisco, CA, USA;

²Pharmacodynamic Biomarkers, Genentech Inc., South San Francisco, CA, USA;

³Bioinformatics, Genentech Inc., South San Francisco, CA, USA; ⁴Division of Infectious Disease, University of California San Francisco, San Francisco, CA, USA
*Critical Care* 2014, **18**(Suppl 2):P14; doi:10.1186/cc14017

**Introduction:** Invasive *Staphylococcus aureus* infections remain an unmet medical need with the issues of drug resistance (MRSA) and mortality. Understanding clinical trial data in the development of antibiotics to *S. aureus* is complicated by heterogeneous clinical outcomes (that is, length of hospitalization, mortality, treatment response), which makes interpretation of drug efficacy challenging. Identification of prognostic biomarkers of different biological processes that associate with clinical outcomes would aid in clinical development of novel therapies for *S. aureus* infections.

**Methods:** In an effort to discover biomarkers that differentiate patient populations based on clinical outcomes following *S. aureus* infection, we retrospectively analyzed published gene expression datasets of *S. aureus* infection and sepsis.

**Results:** We identified a leukocyte gene expression signature that positively correlated with *S. aureus* disease severity [1]. This severity signature was enriched for genes associated with neutrophils but was not solely explained by increased percentage of neutrophils. This set of genes was also associated with severity in sepsis, with higher expression in patients with septic shock compared with sepsis [2] and in nonsurvivors compared with survivors of septic shock [3]. Our *in vitro* studies revealed that the severity signature may reflect an increase in circulating immature neutrophils or band cells which has been previously reported in the context of bacterial stimuli and sepsis [4,5]. This line of evidence is consistent with a recent report by Guerin and colleagues that demonstrated that quantification of immature neutrophils by flow cytometry was prognostic for sepsis mortality [6].

**Conclusion:** To extend the insight gained from our retrospective analysis and *in vitro* studies, we are conducting a longitudinal, non-interventional clinical study of patients with *S. aureus* bacteremia. The goal of the study is to associate molecular metrics (gene expression, plasma protein levels, immune cell subsets) with clinical outcomes (length of hospitalization, mortality, treatment response, readmission for recalcitrant infection). Our preliminary data show an association between grade of sepsis or infection localization and increased immature neutrophils as well as monocyte subsets that can promote inflammation or immune exhaustion. Ongoing experiments are designed to understand the impact of these cellular phenotypes on disease progression and to identify robust protein or RNA biomarkers that are prognostic for clinical outcomes.

References

1. Banchereau R, Jordan-Villegas A: Host immune transcriptional profiles reflect the variability in clinical disease manifestations in patients with *Staphylococcus aureus* infections. *PLoS ONE* 2012, 7:e34390.

2. Wong HR, Cvijanovich N: Genomic expression profiling across the pediatric systemic inflammatory response syndrome, sepsis, and septic shock spectrum. *Crit Care Med* 2009, 37:1558-1566.

3. Wynn JL, Cvijanovich NZ: The influence of developmental age on the early transcriptomic response of children with septic shock. *Mol Med* 2011, 17:1146-1156.

4. Taneja R, Sharma AP: Immature circulating neutrophils in sepsis have impaired phagocytosis and calcium signaling. *Shock* 2008, 30:618-622.

5. Pillay J, Ramakers P: Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia. *J Leukoc Biol* 2010, 88:211-220.

6. Guerin E, Orabona M: Circulating immature granulocytes with T-cell killing functions predict sepsis deterioration. *Crit Care Med* 2014, 42:2007-2018.

P15

**miR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in acute kidney injury.**

I-K Wang¹,²*, C-Y Li²,³

¹Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan; ²Division of Nephrology, China Medical University Hospital, Taichung, Taiwan; ³Department of Anesthesiology, China Medical University Hospital, Taichung, Taiwan

*Critical Care* 2014, **18**(Suppl 2):P15; doi:10.1186/cc14018

**Introduction:** Autophagy could be induced under stress conditions, including starvation, infection, and hypoxia. The microRNA (miRNA) network may be critical in the regulation of autophagy. Upregulation of autophagy may be a protective response for cell survival in ischemic kidney injury. The aim of this study was to evaluate whether miRNA regulates autophagy in ischemic kidney injury and renal proximal tubular cells under hypoxic conditions.

**Methods:** Ischemic kidney injury was performed by clamping bilateral renal pedicles for 60 minutes in male mice. Human kidney proximal tubular (HK2) cells are incubated in a hypoxic chamber with 0.3% O₂. Bioinformatics analyses were used to select the candidate miRNA. Gain-of-function and loss-of-function methods were employed to evaluate the effects of miRNA on autophagy. Chromatin immunoprecipitation analyses and promoter luciferase reporter assays were used to evaluate the interaction of transcriptional factors with miRNA.

**Results:** Increase of LC3 and ATG16L1, autophagy-related proteins, and down expression of miR-20a-5p were detected in kidneys after ischemic injury and in HK2 cells under hypoxic conditions. The 3'-untranslated region luciferase reporter assays indicated that miR-20a-5p targeted ATG16L1 messenger RNA. Overexpression of miR-20a-5p reduced the expression of LC3-II and ATG16L1 in HK2 cells under hypoxic conditions, whereas antagomiR-20a reversed the inhibition. Using RNAi against hypoxia-inducible factor-1α (HIF-1α) in HK2 cells, we confirmed the inhibitory binding of HIF-1α to miR-20a-5p.

**Conclusion:** The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury.
P16

Evaluating the sensitivity and specificity of a severe sepsis tool utilized
at a community hospital in Miami, FL.

J Hirigoyen

4 Tower Medical-Surgical Unit, Baptist Hospital of Miami, Miami, FL, USA

*Critical Care* 2014, **18**(Suppl 2):P16; doi:10.1186/cc14019

**Introduction:** Since the initial development of the Surviving Sepsis Campaign guidelines outlining the management of severe sepsis, there has been an absolute discount on the management of septic patients in medical surgical units. In efforts to improve severe sepsis, a community hospital in Miami adopted a severe sepsis screening tool (SSST) to rapidly identify severe septic patients in medical surgical units. A pilot study was conducted to evaluate the sensitivity and specificity of the SSST.

**Methods:** A descriptive retrospective study. There were two phases. Phase 1 evaluated the percentage of patients with sepsis criteria utilizing the SSST. Patients admitted to 4 Tower during 2013 presenting with a diagnosis of sepsis syndrome and admitted to 4 Tower presenting without sepsis syndrome were reviewed. Phase 2 evaluated the sensitivity and specificity of SSST from August 2013 to January 2014. Total number of patients admitted to 4 Tower: of those patients, total number with discharge diagnosis of sepsis, total number who screened positive >1 time during hospital stay, and total number who screened negative during hospital stay; there were five missing cases. The receiver operating curve (Figure 1) and the respective area under the curve were calculated. Utilizing a 2 × 2 design, the sensitivity and specificity of the tool was calculated.

**Results:** Phase 1: a total of 220 patients records were reviewed, a frequency distribution was utilized (Table 1), demonstrating that the SSST identified those patients with sepsis criteria 76% (n = 167) of the time. Phase 2: a total of 1,555 patients were included during phase 2. A 2 × 2 design (Table 2) was utilized: 78 patients were identified as true positive and 1,233 patients were identified as true negative. The study yielded a sensitivity of 41.49% and a specificity of 90.53%. The positive predictive value of the tool was estimated at 37.68%, negative predictive value was estimated at 91.81% and disease prevalence was 12.13%. Area under the receiver operating curve (Table 3) was 0.66.

**Conclusion:** A two-phase retrospective chart review study demonstrated that the SSST utilized at a community hospital in Miami had a sensitivity value of 41.49% and a specificity value of 90.53% when evaluating medical

surgical patients. These results indicate the tool is accurate in detecting patients that are not septic; however, it is not reliable in identifying patients who are truly septic. Further studies need to be conducted to validate the sensitivity and specificity of the SSST; changes will be recommended in an effort to improve sensitivity.

**Acknowledgements:** Thanks to Eve Butler and Andrea Prentiss from Baptist Research Department, 4 Tower team. Special thanks to Melanie Santos, Luz Lorenzo, Disney Granado, Katiuska Diaz, Sandra Benitez, Magdely Perez, Viviana Castillo and Ofelia Cabrera.

P17

Surviving Sepsis Campaign 2012 3-hour bundle in the emergency department: compliance and impact of pathway of care before and after implementation.

J Masse¹, A Filali², O Nigeon¹, N Van Grunderbeeck², P Gosselin³, L Tronchon², J Mallat², D Thevenin²

¹Emergency Department, Hospital Center of Lens, France; ²Intensive Care Unit, Hospital Center of Lens, France; ³Emergency Department, Lille

University Hospital, Lille, France

*Critical Care* 2014, **18**(Suppl 2):P17; doi:10.1186/cc14020

**Introduction:** Compliance with the Surviving Sepsis Campaign 2012 (SSC) bundle in the emergency department (ED) is a key point to improve outcome of severe sepsis and septic shock [1,2]. Before and after education of ED staff, we registered compliance and timing of lactate dosing, blood culture sampling, empiric antibiotic therapy (ATB) and fluid resuscitation, the 3-hour (H3) bundle. Survival and compliance according to the initial pathway of care were also studied.

**Methods:** A monocentric study before and after education of ED staff about SSC bundles (courses, posters, pocket guides). We looked at compliance of the H3 bundle items in a retrospective and a prospective cohort, timing of realisation, day 28 survival, overall severity (SAPS2, SOFA and RISCC scores), impact of prehospital medical management, and initial pathway of care. Statistical analysis was performed with Fisher exact test and Mann-Whitney test. Multivariate analysis of factors associated with survival was made through logistic regression.

**Results:** Eighty-nine patients were included in the prospective cohort, 65 in the retrospective cohort. Patterns of patients in the retrospective and prospective cohort were respectively: sex ratio M/F 29/39 and 39/47 (NS);

<figure><img src="image_19.png" /><figcaption>Figure 1(abstract P16) ROC curve</figcaption></figure>
<table><caption>**Table 1(abstract P16) Frequency distribution**</caption><thead><tr><th>Statistics</th><th>Diagnosis on admission</th><th>Sepsis tool identifies sepsis</th></tr></thead><tbody><tr><td>Valid</td><td>220</td><td>220</td></tr><tr><td>Missing</td><td>0</td><td>0</td></tr><tr><th>Frequency table</th><th>Frequency</th><th>Percent</th><th>Valid percentage</th><th>Cumulative percentage</th></tr><tr><td>Diagnosis on admission</td><td></td><td></td><td></td><td></td></tr><tr><td>Valid</td><td>220</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Sepsis tool identifies sepsis</td><td></td><td></td><td></td><td></td></tr><tr><td>Not valid</td><td>53</td><td>24.1</td><td>24.1</td><td>24.1</td></tr><tr><td>Valid</td><td>167</td><td>75.9</td><td>75.9</td><td>100.0</td></tr><tr><td>Total</td><td>220</td><td>100.0</td><td>100.0</td><td></td></tr></tbody></table>

<table><caption>**Table 2(abstract P16) The 2 × 2 design**</caption><thead><tr><th></th><th>Sepsis present</th><th>Sepsis absent</th></tr></thead><tbody><tr><td>Positive</td><td>78 true positive</td><td>129 false positive</td></tr><tr><td>Negative</td><td>110 false negative</td><td>1,233 true negative</td></tr></tbody></table>

<table><caption>**Table 3(abstract P16) Area under the curve**</caption><tr><td>Test result variable(s):screen</td></tr><tr><td>Area</td></tr><tr><td>0.660</td></tr></table>

median age 63.29/61.38 (NS); SAPS2 44/40 (P = 0.019); SOFA 4/3 (P = 0.005); RISCC 9/12.5 (P = 0.002). Compliance with the H3 bundle items before and after intervention was: lactate 72.1% vs. 81.4% (NS); blood cultures 61.8% vs. 67.4% (NS); ATB 29.3% vs. 52.3% (P = 0.005); fluids 52.9% vs. 59.3% (NS). Median delays before and after implementation were (in minutes): lactate 56 vs. 40 (P = 0.024); blood cultures 68 vs. 75 (NS); ATB 229 vs. 160 (NS); fluids 100 vs. 74 (NS). Survival was superior after intervention 67.6 vs. 81.4% (P = 0.049), and associated with a low SAPS2 score in multivariate analysis. Admission through a prehospital medical team was associated with a stronger H3 ATB compliance before intervention (P = 0.032). Within the ED, initial orientation to the acute care unit was associated with a better H3 ATB compliance compared to standard care before and after staff education (P = 0.001; P = 0.003), and with better overall compliance (P = 0.004; P = 0.026).

**Conclusion:** Compliance with the SSC H3 bundle was increased but still needs to be improved. There is an impact of the initial pathway of care on compliance of the bundle, and on timing of ATB injection. Differences in healthworker/patient ratio in the units of care could explain these disparities [3]. Improvement could be obtained through optimizing early screening, correct initial guidance, or with dedicated teams.

**Acknowledgements:** The authors would like to thank the members of the Unit of Clinical Research of the Hospital Center of Lens for their great help in data recovery

**References**

1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb S, Beale RJ, Vincent JL, Moreno R, Surviving Sepsis Campaign Guidelines Committee including The Pediatric Subgroup: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. *Intensive Care Med* 2013, **39**:165-228.

2. van Zanten AR, Brinkman S, Arbous MS, Abu-Hanna A, Levy MM, de Keizer NF, Netherlands Patient Safety Agency Sepsis Expert Group: Guideline bundles adherence and mortality in severe sepsis and septic shock. *Crit Care Med* 2014, **42**:1890-1898.

3. Shin TG, Jo IJ, Choi DJ, Kang MJ, Jeon K, Suh GY, Sim MS, Lim SY, Song KJ, Jeong YK: The adverse effect of emergency department crowding on compliance with the resuscitation bundle in the management of severe sepsis and septic shock. *Crit Care* 2013, **17**:R224.

P18

**Benefit of achieving lactate clearance versus central venous oxygen saturation target as microcirculation end point resuscitation in severe sepsis and septic shock.**

R Sinto*, S Suwarto, KC Lie, D Widodo, HT Pohan

Division of Tropical and Infectious Disease, Department of Internal Medicine, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia

*Critical Care* 2014, **18**(Suppl 2):P18; doi:10.1186/cc14021

**Introduction:** In severe sepsis and septic shock patients, lactate clearance >10% and central venous oxygen saturation (ScvO₂) >70% are accepted parameters of tissue oxygenation adequacy. There is controversy of which parameters better associate with early mortality, and thus should be implemented as the microcirculation end point resuscitation [1-3]. This study was aimed to address the association of achieving either one or two targets of microcirculatory end point resuscitation and early mortality in severe sepsis and septic shock patients.

**Methods:** A retrospective cohort study was conducted in severe sepsis and septic shock patients (aged 18 years and older) hospitalized in the ICU, Cipto Mangunkusumo Hospital, Indonesia. Patients' early outcomes were observed during first 120 hours of hospitalization. Cox's regression analysis was used to analyse risk of early mortality in subject groups achieving lactate clearance target only, ScvO₂ target only, both targets, and not achieving any target in 6 hours after onset of resuscitation.

**Results:** Subjects consisted of 268 patients. Early mortality developed in 70 subjects. Fifty-four subjects achieved lactate clearance target only, 16 achieved ScvO₂ target only, 138 achieved both targets, 60 did not achieve any target. Subjects who achieved both targets had a significant lowest early mortality risk (P = 0.104 compared with subjects achieved lactate clearance target only and P = 0.000 compared with remaining subject groups) (Figure 1). In subgroup analysis of subjects who achieved lactate clearance or ScvO₂ target only, failure to achieve lactate clearance target associated with higher early mortality risk (hazard ratio 5.92; 95% CI 2.18 to 16.01).

**Conclusion:** Achieving both lactate clearance and ScvO₂ targets in 6 hours after onset of resuscitation associates with lowest early mortality risk in severe sepsis and septic shock patients. Patients who achieve lactate clearance target only have a significant lower early mortality risk compared with those who achieve ScvO₂ target only.

**References**

1. Jones AE, Shapiro NI, Trzeciak S, Arnold RC, Claremont HA, Kline JA: Lactate clearance vs. central venous oxygen saturation as goals of early sepsis therapy: a randomized clinical trial. *JAMA* 2010, **303**:739-746.

2. Jones AE: Point: should lactate clearance be substituted for central venous oxygen saturation as goals of early severe sepsis and septic shock therapy? Yes. *Chest* 2011, **140**:1406-1408.

3. Rivers EP, Elkin R, Cannon CM: Counterpoint: should lactate clearance be substituted for central venous oxygen saturation as goals of early severe sepsis and septic shock therapy? No. *Chest* 2011, **140**:1408-1413.
<figure><img src="image_20.png" /><figcaption>Figure 1(abstract P18) Survival analysis of groups based on target achievement</figcaption></figure>

P19

**The PRESEP score: an early warning scoring system to identify septic patients in the emergency care setting.**

O Bayer<fnref n="1" />*, CS Hartog<fnref n="1" />,², D Schwarzkopf², C Stumme<fnref n="1" />, A Stacke<fnref n="1" />, F Bloos<fnref n="1" />,², C Hohenstein<fnref n="3" />, B Kabisch<fnref n="1" />, C Weinmann<fnref n="1" />, J Winning<fnref n="1" />, Y Sakr<fnref n="1" />, JK Reinhart<fnref n="1" />,²

¹Department of Anaesthesiology and Intensive Care Medicine, Jena
University Hospital, Jena, Germany; ²Center for Sepsis Control & Care, Jena
University Hospital, Jena, Germany; ³Emergency Department, Jena University
Hospital, Jena, Germany

*Critical Care* 2014, **18**(Suppl 2):P19; doi:10.1186/cc14022

**Introduction:** Many patients present with sepsis through emergency services [1]. Their outcome could be improved if sepsis could be detected already in the prehospital setting. This study aims to develop and evaluate a score to detect prehospital early sepsis.

**Methods:** A retrospective study of 375 patients admitted to Jena University Hospital emergency department (ED) through emergency medical services (EMS). Sepsis was present in the ED in 93 (24.8%) patients, of which 60 (16.0%) had severe sepsis and 12 (3.2%) had septic shock. The following predictors for sepsis based on consensus criteria were extracted from the EMS protocol: body temperature (T), heart rate (HR), respiratory rate (RR), oxygen saturation (SaO₂), Glasgow Coma Scale, blood glucose and systolic blood pressure (BP). Sepsis predictors were determined based on inspection of loess graphs. Backward model selection was performed to select risk factors for the final model. The PRESEP score was calculated as the sum of simplified regression weights. Its predictive validity was compared to the modified Early Warning Score (MEWS) [2], the Robson screening tool [3] and the BAS 90-30-90 [4].

**Results:** Backward model selection identified T, HR, RR, SaO₂ and BP for inclusion in the PRESEP score. Its AUC was 0.93 (CI 0.89 to 0.96). The cutoff based on maximum Youden's Index was ≥4 (sensitivity 0.85, specificity 0.86, PPV 0.66, NPV 0.95). The PRESEP score had a larger AUC than the MEWS (0.93 vs. 0.77, *P* < 0.001) and surpassed MEWS and BAS 90-60-90 concerning sensitivity (0.74, 0.62), specificity (0.75, 0.83), PPV (0.45, 0.51) and NPV (0.91, 0.89). The Robson screening tool had a higher

sensitivity and NPV (0.95, 0.97) was better, but its specificity and PPV lower (0.43, 0.43).

**Conclusion:** The PRESEP score can be easily applied in the emergency setting and could be a valuable tool to identify septic patients in the case of suspected infection.

**Acknowledgements:** OB was supported, in part, by an unrestricted grant of the Thuringian Ministry of Cultural Affairs (Landesprogramm ProExzellenz; PE 108-2), the Foundation of Technology, Innovation, and Research Thuringia (STIFT), and the German Sepsis Society. JKR and CSH receive CSCC research grants. DS is funded in full by the Center for Sepsis Control and Care (CSCC). The CSCC is funded by the German Federal Ministry of Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1002.

**References**

1. Seymour CW, Rea TD, Kahn JM, Walkey AJ, Yealy DM, Angus DC: Severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome. *Am J Respir Crit Care Med* 2012, 186:1264-1271.

2. Subbe CP, Kruger M, Rutherford P, Gemmel L: Validation of a modified Early Warning Score in medical admissions. *QJM* 2001, 94:521-526.

3. Robson W, Nutbeam T, Daniels R: Sepsis: a need for prehospital intervention? *Emerg Med J* 2009, 26:535-538.

4. Ljungstrom L: A challenge to doctors of infectious disease: make the management of patients with acute severe bacterial infection as good as the management of acute coronary syndromes. [http://www.mediahuset.se/Infektionslakaren/il405/en_utmaning.htm].

P20

**Simultaneous targeting of interleukin-1 and interleukin-18 is required for protection against inflammatory and septic shock.**

T Vanden Berghe<fnref n="1" />,²*, D Demon<fnref n="1" />,²,<fnref n="3" />, P Bogaert<fnref n="1" />,², B Vandendriessche<fnref n="1" />,²,

A Goethals<fnref n="1" />,², B Depuydt<fnref n="1" />,², M Vuylsteke⁴, R Roelandt<fnref n="1" />,²,

E Van Wonterghem<fnref n="1" />,², J Vandenbroecke<fnref n="1" />,², SM Choi<fnref n="1" />,², E Meyer<fnref n="3" />,

S Krautwald⁵, W Declercq<fnref n="1" />,², N Takahashi<fnref n="1" />,², A Cauwels<fnref n="1" />,², P Vandenabeele<fnref n="1" />,²

<footnote n="1">¹Inflammation Research Center, VIB, Ghent, Belgium; ²Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium;</footnote>

<footnote n="3">³Department of Plant Systems Biology, VIB, Ghent, Belgium; ⁴Lab</footnote>
Aquaculture & Artemia Reference Center, Faculty Bioscience Engineering,
Ghent University, Belgium; ⁵Department of Nephrology and Hypertension,
University Medical Center Schleswig-Holstein UKSH, Kiel, Germany
*Critical Care* 2014, **18**(Suppl 2):P20; doi:10.1186/cc14023

**Introduction:** Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and still insufficiently understood. The objective is to clarify the long disputed hierarchical contribution of several central inflammatory mediators, namely IL-1β, IL-18, CASP7, CASP1 and CASP11, in septic shock, and to explore their therapeutic potential.

**Methods:** LPS-induced and TNF-induced lethal shock, as well as cecal ligation and puncture (CLP), were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation.

**Results:** Interestingly, deficiency of both IL-1β and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1β and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11 or CASP11/11 deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist Anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality.

**Conclusion:** Our data point towards the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

**Acknowledgements:** DD and PB contributed equally to this work. AC and PV share senior authorship. The authors are grateful to R Flavell (Howard Hughes Medical Institute, Chevy Chase, USA) and A Zychlinsky (Max Planck Institute, Berlin, Germany) for respectively the CASP1 knockout mice and the IL-1β/IL-18 double knockout mice. They thank B Lambrecht for fruitful discussions. TVB holds a postdoctoral fellowship from the FWO, PB is paid by VIB. Research in the Vandenabele group is funded by European grants (FP6 AopTrain, MRTN-CT-035624; FP7 EC RTD Integrated Project, Apo-Sys, FP7-200767; Euregional PACT II), Belgian grants (Interuniversity Attraction Poles, IAP 7/32), Flemish grants (Research Foundation Flanders, FWO G.0875.11, FWO G.0973.11 and FWO G.0A45.12N, FWO G.0787.13N, Methusalem), Ghent University grants (MRP, GROUP-ID consortium), a grant from the Foundation against Cancer, F94 and grants from Flanders Institute for Biotechnology (VIB). PV holds a Methusalem grant (BOF09/01M00709) from the Flemish Government. The authors declare no conflict of interest.

P21

**Sepsis electronic surveillance and clinical outcomes: impact over mortality of a sepsis early detection electronic rule implemented in the emergency department.**

P Martin-Rico<fnref n="1" />*, A Valdivia-Perez², MD Marco-Lattur<fnref n="1" />, J Chorda-Ribelles<fnref n="1" />, N Lozano-Cortell<fnref n="1" />, P Olcina-Lloret<fnref n="1" />, R Andres-Navarro<fnref n="1" />, E Mateo-Sanchis<fnref n="1" />, M Jordan-Lluch<fnref n="1" />, O Esparcia-Rodriguez², J Magrane-Egea<fnref n="3" />, J Lacalle-Martinez<fnref n="4" />, A Barcelo-Lopez⁵

**Introduction:** Severe sepsis and septic shock (SS/SS) have a high mortality. Therapeutic guidelines can improve mortality, but early recognition and timely implementation of these requires a proactive attitude that can be electronically supported.

**Methods:** From May 2013 our hospital implemented a Sepsis Code (SC) based on an early detection electronic rule developed by our multiprofessional sepsis team: clinicians and IT engineers (EMR Cerner Millennium platform) and a standardized order set plus systematic follow-up by our sepsis team. We performed a before-after study to assess the impact over mortality of this strategy. Time-series analysis of sepsis admissions and

mortality from January 2011 to December 2013, before and after SC implementation. (Analysis by STATA.) All urgent admissions recorded in the minimum basic data set in patients over 14 years from 1 January 2011 to 31 December 2013 were included. Inclusion criteria: patients with ICD-9 sepsis-associated codes in the principal diagnosis or patients with infection-associated codes in the principal diagnosis together with sepsis-associated codes in secondary diagnosis. Medical records were manually reviewed by clinicians to confirm SS/SS diagnosis. Temporal series analysis (Poisson regression). First analysis: sepsis admissions in relation to total urgent admissions. Second analysis: deaths due to SS/SS related to admissions in this group. In both cases we compared results before SC activation (28 months) and after that (first 2 transitional months and 6 consolidated months). The multivariate adjustment in both analyses included year, month of the year, and months with activated rule. Graphic analysis estimated predictions for the last 8 months based on the previous 28 months, comparing both observed and predicted sepsis and deaths.

**Results:** A total of 24,118 urgent admissions were included, 5,657 in the postalert period. Mean monthly admissions: 652 (SD 47) (570 to 740). In total, 408 and 178 SS/SS were identified in the prealert and postalert period, respectively. After SC implementation we observed no significant changes in sepsis admission risk but a clear downward trend in sepsis mortality: in the first 2 transitional months we did not observe major changes, while in the last 6 months the risk of death does fall 36% reaching statistical significance (IRR 0.64 (95% CI 0.43 to 0.97, *P* = 0.036)) (Table 1 Figures 1 and Figure 2). Both antibiotic door-to-needle time and adequacy significantly improved in sepsis cases where the alert was triggered.

**Conclusion:** Implementation of a SC triggered by an electronic detection alert, compared to the prealert period, decreased mortality risk by 36% (IRR 0.64 (95% CI 0.43 to 0.97, *P* = 0.036)) with the rule fully implemented.

P22

**Assessing the value of a real-time electronic screening algorithm for early detection of severe sepsis in the emergency department.**

P Martin-Rico<fnref n="1" />*, A Valdivia-Perez², JM Lacalle-Martinez², J Chorda-Ribelles<fnref n="1" />, MD Marco-Lattur<fnref n="1" />, N Lozano-Cortell<fnref n="1" />, M Jordan-Lluch<fnref n="1" />, E Mateo-Sanchis<fnref n="1" />, R Andres-Navarro<fnref n="1" />, P Olcina-Lloret<fnref n="1" />, JD Garcia-Pedro<fnref n="1" />, O Esparcia-Rodriguez<fnref n="4" />, J Magrane-Egea<fnref n="4" />, A Barcelo-Lopez⁵

**Introduction:** In severe sepsis/septic shock (SS/SS), early recognition and timely implementation of treatment is critical for survival, and this could be electronically supported. We assess the value of an electronic automatic algorithm based on EMR data as a screening tool for early detection of sepsis.

**Methods:** Our multiprofessional sepsis team (clinicians and IT engineers) developed an electronic algorithm using data from our EMR (Cerner Millennium platform) aimed at the automatic, real-time recognition of two or more systemic inflammatory response syndrome components + one or more organ failure parameter (according to sepsis definition) in every patient attended in the ER. The firing of this sepsis rule issues an alert to the responsible clinician to confirm an infectious etiology and opens an electronic standardized order set according to sepsis bundles. The alert database (from its start in May 2013 to December 2013) was cross-matched with the minimum basic data set for urgent admissions (>14 years) during this same period. We selected, based on an *ad hoc* syntaxes, those admissions due to sepsis. Medical records were manually reviewed for confirmation of SS/SS. We assessed sensibility, specificity, negative and positive predictive value of the electronic rule, considering the confirmed clinical diagnosis at discharge as the gold standard.

**Results:** In total, 37,323 patients were seen in the ER, 5,657 emergency admissions took place and 178 were due to SS/SS. Alert fired in 1,190 (3.2%) total emergencies and in 754 emergency admissions (13.3%). Data analysis after alert implementation identifies a global sensitivity of 80%, which improved after the first 2 months of transition. In the last 6 months (consolidated period) it was between 85 and 90%. Global specificity 89%, NPV of 99% and PPV of 19% for a global prevalence at admission of 3.2 cases/100 (Table 1). The mean door-to-alert time was 167 minutes (SD 193).

<footnote n="1">¹Internal Medicine (Infectious D. Unit), Hospital de Denia, Denia Alicante, Spain; ²Preventive Medicine, Hospital de Denia, Denia Alicante, Spain;</footnote>

<footnote n="3">³Microbiology Department, Hospital de Denia, Denia Alicante, Spain;</footnote>

<footnote n="4">⁴IT Department, Hospital de Denia, Denia Alicante, Spain; ⁵Emergency Department, Hospital de Denia, Denia Alicante, Spain</footnote>

<footnote>*Critical Care* 2014, **18**(Suppl 2):P21; doi:10.1186/cc14024</footnote>

<footnote n="1">¹Internal Medicine (Infectious D. Unit), Hospital de Denia, Denia Alicante, Spain; ²Preventive Medicine, Hospital de Denia, Denia Alicante, Spain;</footnote>

<footnote n="3">³IT Department, Hospital de Denia, Denia Alicante, Spain; ⁴Microbiology Department, Hospital de Denia, Denia Alicante, Spain; ⁵Emergency Department, Hospital de Denia, Denia Alicante, Spain</footnote>

<footnote>*Critical Care* 2014, **18**(Suppl 2):P22; doi:10.1186/cc14025</footnote>
<table><caption>Table 1(abstract P21) Risk of death in sepsis admissions</caption><thead><tr><th>Variable</th><th>Category</th><th>IRR</th><th>95% CI</th><th>P value</th></tr></thead><tbody><tr><td rowspan="10">Month</td><td>January</td><td>2.11</td><td>0.80 to 5.55</td><td>0.130</td></tr><tr><td>February</td><td>2.55</td><td>1.04 to 6.24</td><td>0.040</td></tr><tr><td>March</td><td>2.45</td><td>1.00 to 6.01</td><td>0.051</td></tr><tr><td>April</td><td>1.0 (reference)</td><td></td><td></td></tr><tr><td>May</td><td>2.24</td><td>0.86 to 5.88</td><td>0.100</td></tr><tr><td>June</td><td>1.45</td><td>0.53 to 4.01</td><td>0.470</td></tr><tr><td>July</td><td>2.15</td><td>0.82 to 5.66</td><td>0.121</td></tr><tr><td>August</td><td>2.62</td><td>1.00 to 6.85</td><td>0.050</td></tr><tr><td>September</td><td>3.74</td><td>1.47 to 9.53</td><td>0.006</td></tr><tr><td>October</td><td>2.31</td><td>0.89 to 6.02</td><td>0.085</td></tr><tr><td rowspan="3">Alert</td><td>November</td><td>2.69</td><td>1.09 to 6.65</td><td>0.032</td></tr><tr><td>December</td><td>1.89</td><td>0.71 to 5.01</td><td>0.203</td></tr><tr><td>Previous</td><td>1.0 (reference)</td><td></td><td></td></tr><tr><td rowspan="3">Alert</td><td>Transition</td><td>1.26</td><td>0.64 to 2.47</td><td>0.506</td></tr><tr><td>Implemented</td><td>0.64</td><td>0.43 to 0.97</td><td>0.036</td></tr></tbody></table>

Median door to needle (antibiotic) time in the alert-sepsis group was 259 minutes vs. 417 minutes in the group where alert did not fire. In the alert + order set group, antibiotic treatment was adequate in 70% vs. 35% in the nonalert sepsis group (Fisher's exact = 0.001). Overall mortality among alert-fired admissions not due to sepsis was 15% (RR for death during admission: 5.33, 95% CI: 3.73 to 7.59; *P* < 0.0001).

**Conclusion:** This pioneering sepsis algorithm identified 80% of SS/SS. The alert prompted adequate antibiotic treatment and shortened door to needle time from 7 to 4 hours. Furthermore, in patients without sepsis the algorithm identifies a poor-prognosis subset (RR for death 4.75 (95% CI: 3.72 to 6.07).

P23

Histidine-rich glycoprotein prevents septic lethality through neutrophil regulation.

M Nishibori¹*, H Wake¹, S Mori², K Liu¹, Y Morioka¹, K Teshigawara¹, M Sakaguchi³, K Kuroda⁴, H Takahashi⁵, A Ohtsuka⁶, T Yoshino⁷, H Morimatsu⁴

¹Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan;

²Department of Pharmacology, Faculty of Pharmaceutical Science, Shujitsu University, Okayama, Japan; ³Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; ⁴Department of Anesthesiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; ⁵Department of Pharmacology, Faculty of Medicine, Kinki University, Osakasayama, Japan; ⁶Department of Human Morphology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; ⁷Department of Pathology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan

Critical Care 2014, **18**(Suppl 2):P23; doi:10.1186/cc14026

**Introduction:** Although there have been many clinical trials for treatment of sepsis, no drugs are available at present. Sepsis is thought to be complex disorders; activation/lesion of vascular endothelial cells, accelerated coagulation and platelet aggregation, leukocyte activation or paralysis, and hypercytokinemia. To treat the pathological status of sepsis, it must be necessary to control these plural disorders simultaneously or sequentially. In the present study, we identified and characterized a plasma protein histidine-rich glycoprotein (HRG) as a factor that decreases dramatically in septic condition and maintains neutrophil's shape and functional quiescence. We clarified the involvement of HRG in septic pathogenesis and propose a novel therapy for sepsis based on that.

**Methods:** Sepsis was induced in mice by cecal ligation and puncture. The mice were treated with HRG purified from human plasma after confirming the marked decrease in plasma HRG. We evaluated the beneficial effects of HRG administration on survival rate, lung inflammation, and the state of circulating neutrophils using *in vivo* imaging. Purified neutrophils from human blood were treated with HRG and analyzed with respect to neutrophil shape, adhesiveness to vascular endothelial cells, passage through microcapillaries, production of reactive oxygen species, and cytoskeleton rearrangement with relevant signal transduction.

**Results:** Supplementary treatment of septic mice with exogenous HRG for the decrease in plasma HRG improved the survival of mice remarkably. HRG treatment reduced the number of neutrophils in the lung, on which platelet aggregation and fibrin deposit were observed. HRG also inhibited the expression of mRNAs of IL-6, TNFα, iNOS, and PAI-1 in the lung. In contrast, knockdown of HRG by siRNA exacerbated lethality. Purified human HRG reversibly induced morphological changes in human neutrophils *in vitro*; induction of spherical shape with reduced microvilli and adhesiveness to vascular endothelial cells. HRG maintained the passage of neutrophils through microcapillaries and abolished the production of reactive oxygen species whereas HRG had no effect on the expression of adhesion molecules including CD11b and CD62L.

**Conclusion:** We show that plasma protein HRG is a crucial factor that keeps the circulating neutrophils quiescent and prevents unnecessary activation in bloodstream using a cecal ligation and puncture model in

<figure><img src="image_21.png" /></figure>
<figure><img src="image_22.png" /><figcaption>Figure 2(abstract P21) Mortality in emergency admissions for sepsis</figcaption></figure>

<table><caption>Table 1(abstract P22) Prevalence of SS/SS upon admission and sensitivity, specificity, and predictive values of the sepsis rule</caption><thead><tr><td>Month</td><td>Prevalence</td><td>Sensitivity</td><td>Specificity</td><td>PPV</td><td>NPV</td></tr></thead><tbody><tr><td>May</td><td>3.1 (1.8 to 4.5)</td><td>72.7 (51.8 to 93.6)</td><td>88.3 (85.8 to 90.7)</td><td>16.7 (8.7 to 24.6)</td><td>99.0 (98.1 to 99.9)</td></tr><tr><td>June</td><td>3.0 (1.6 to 4.3)</td><td>55.0 (30.7 to 79.3)</td><td>89.2 (86.7 to 91.6)</td><td>13.4 (5.4 to 21.4)</td><td>98.5 (97.4 to 99.6)</td></tr><tr><td>July</td><td>3.0 (1.7 to 4.3)</td><td>86.4 (69.8 to 100)</td><td>89.8 (87.5 to 92.1)</td><td>20.7 (11.8 to 29.5)</td><td>99.5 (89.9 to 100)</td></tr><tr><td>August</td><td>1.8 (0.8 to 2.8)</td><td>78.6 (53.5 to 100)</td><td>87.8 (85.4 to 90.2)</td><td>10.7 (4.2 to 17.1)</td><td>99.6 (99.0 to 100)</td></tr><tr><td>September</td><td>2.2 (1.0 to 3.4)</td><td>86.7 (66.1 to 100)</td><td>88.0 (85.4 to 90.5)</td><td>14.0 (6.4 to 21.6)</td><td>99.7 (99.1 to 100)</td></tr><tr><td>October</td><td>4.3 (2.7 to 5.9)</td><td>82.8 (67.3 to 98.2)</td><td>90.6 (88.3 to 92.9)</td><td>28.2 (18.1 to 38.4)</td><td>99.2 (98.3 to 100)</td></tr><tr><td>November</td><td>5.4 (3.5 to 7.2)</td><td>84.9 (71.1 to 98.6)</td><td>88.9 (86.2 to 91.5)</td><td>30.1 (20.3 to 40.0)</td><td>99.1 (98.1 to 100)</td></tr><tr><td>December</td><td>2.9 (1.6 to 4.2)</td><td>90.5 (75.5 to 100)</td><td>88.4 (85.9 to 90.8)</td><td>19.0 (10.8 to 27.2)</td><td>99.7 (99.1 to 100)</td></tr><tr><td>Total</td><td>3.2 (2.7 to 3.6)</td><td>80.1 (73.9 to 86.3)</td><td>88.8 (88.0 to 89.7)</td><td>19.0 (16.1 to 21.8)</td><td>99.3 (99.0 to 99.5)</td></tr></tbody></table>

Results of each period are indicated in percentages with confidence intervals at 95%

mice and human neutrophils. The attachment of neutrophils on the vascular wall under reduced concentration of HRG was accompanied by microthrombus formation in the lung, leading to ARDS. Supplementary therapy with HRG may thus provide a novel strategy for the treatment of septic patients through neutrophil regulation.

**Acknowledgements:** This work was supported by grants from the Scientific Research from the Ministry of Health, Labor and Welfare of Japan (WA2F2547) and from the Japan Society for the Promotion of Science (No. 2567046405).

P24

**Usefulness of intravenous immunoglobulin administration to sepsis-induced coagulopathy in ICU patients.**

N Matsumoto*, H Ishikura, Y Nakamura, J Tanaka, M Mizunuma, Y Kawano, S Morimoto, H Kaneyama, A Murai
Department of Emergency and Critical Care Medicine, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
*Critical Care* 2014, **18**(Suppl 2):P24; doi:10.1186/cc14027

**Introduction:** Intravenous immunoglobulin (IVIG) has been used as adjuvant therapy for severe sepsis patients expecting the anti-inflammatory effect. However, the effects of IVIG have been controversial. The majority of ill patients with SIRS had coagulation abnormalities. In addition, inflammation and coagulation play pivotal roles in the pathogenesis of sepsis. Moreover, the evidence of extensive cross-talk between these two systems has been increasing. The aim of this study is to investigate the effects of IVIG treatment for inflammation and hemostatic abnormality in sepsis patients.

**Methods:** This prospective single-center observational study was conducted in our ICU between January and July 2013. We enrolled 41 patients (≥18 years, admitted to the ICU diagnosed for sepsis, and more than 7 days of ICU stay) and divided them into two groups: IVIG-treated group (IVIG group) and non-IVIG-treated group (non-IVIG group). After that, we compared inflammatory molecule markers (WBC, CRP, procalcitonin (PCT), and interleukin-6 (IL-6)), coagulation/fibrinolysis markers (platelet counts, PT-INR, APTT, D-dimer, TAT, PIC, soluble fibrin (SF), and plasminogen activator inhibitor-1 (PAI-1)), and Japanese Association for Acute Medicine Disseminated Intravascular Coagulation (JAAM DIC) score and positive rate at the admission period (day 1) and days 4 to 7 between two groups. Moreover, the 28-day mortality rate was investigated.

**Results:** Nineteen patients were treated with IVIG (IVIG group) and 22 patients were without (non-IVIG group). In the IVIG group, PCT and IL-6 were significantly higher than that in the non-IVIG group at day 1 (*P* < 0.01, and *P* < 0.01). In this group, CRP, PCT, and IL-6 were significantly decreased at days 4 to 7 rather than that at day 1 (*P* < 0.01, *P* < 0.01, *P* < 0.01, respectively). Moreover, the JAAM DIC score was decreased at days 4 to 7 than that at day 1 significantly (*P* < 0.05) in this group. We also confirmed that PT-INR, APTT, TAT, SF, and PAI-1 were significantly improved between day 1 and days 4 to 7. On the other hand, in the non-IVIG group there was significantly decreased IL-6 and TAT only, but not CRP, PCT, PT-INR, APTT, SF, PAI-1 and JAAM DIC score between day 1 and days 4 to 7. For the 28-day mortality rate, the IVIG group was lower than that of the non-IVIG group (IVIG group, 5.3%; non-IVIG group, 18.2%).

**Conclusion:** Our study demonstrated that IVIG treatment significantly improved the inflammatory and hemostatic abnormalities in sepsis patients.
P25

### Impact of fluid management during the three ICU days after admission in patients with ARDS.

A Murai¹, H Ishikura, N Matsumoto, Y Nakamura, D Ohta, K Muranishi, Y Izutani, T Nishida

Department of Emergency and Critical Care Medicine, Faculty of Medicine, Fukuoka University, Fukuoka, Japan

Critical Care 2014, **18**(Suppl 2):P25; doi:10.1186/cc14028

**Introduction:** The optimal evaluation of fluid resuscitation in patients with acute respiratory distress syndrome (ARDS) remains to be clearly elucidated. The purpose of this study was to clarify the influence of fluid overload in patients with ARDS.

**Methods:** Two hundred and seven ARDS patients admitted to the ICUs of 23 tertiary referral hospitals in Japan were enrolled in this study. These patients were divided into survivor (survival group, *n* = 137) or nonsurvivor (nonsurvival group, *n* = 70) groups according to the 28-day mortality. All patients received mechanical ventilation and also underwent transpulmonary thermodilution monitoring. The data for analysis were collected for three consecutive days from time of admission.

**Results:** On the second hospital day, the extravascular lung water index of the nonsurvival group was significantly higher than that of the survival group (18.6 ± 9.4 ml/kg vs. 15.4 ± 6.3 ml/kg, *P* = 0.03). Moreover, regarding the first 3-day cumulative fluid balance, the nonsurvival group was significantly higher than survival group (5.1 ± 4.3 l vs. 3.5 ± 0.4 l, *P* = 0.015). We suspected that these results might be related to the cardiovascular and/or renal function. We therefore excluded any patients with a score of three or more regarding the cardiovascular and/or renal criteria about the Sequential Organ Failure Assessment score. Thereafter, we confirmed the results to be similar for the first 3-day cumulative fluid balance between the two groups (3.8 ± 1.6 l vs. 2.2 ± 4.0 l, *P* = 0.0339). A stepwise logistic regression analysis identified the 3-day cumulative fluid balance to be an independent risk factor for the 28-day mortality (adjusted odds ratio: 1.0001, 95% CI: 1.000017 to 1.000217, *P* = 0.0252).

**Conclusion:** Our findings suggest that excessive fluid resuscitation may therefore have a negative impact on the 28-day mortality for patients with ARDS.

**Acknowledgements:** In this study, we retrospectively analyzed the database of a multicenter observational study (The PiCCO Pulmonary Edema Study)

P26

### Candida albicans versus Mycobacterium tuberculosis: infection-specific human immune responses.

JPLF Saraiwa¹,²*, R König¹,²

¹Network Modelling, Hans-Knöll-Institut, Jena, Germany; ²Centre for Sepsis Control and Care, Jena University Hospital, Jena, Germany

Critical Care 2014, **18**(Suppl 2):P26; doi:10.1186/cc14029

**Introduction:** Systemic infection of the human host can arise from several pathogenic bacteria as well as from fungal species, and it is of high clinical relevance to trace back the nature of infection from the host response. Amongst these systemic infections are sepsis inducers *Candida albicans* and *Mycobacterium tuberculosis*, responsible for a great amount of worldwide deaths [1]. Sepsis, however, does not necessarily originate from infection. Traumas and non-infection-based injuries can also trigger an unbound inflammatory response from the human host most commonly known as systemic inflammatory response syndrome. Both cases, infection and non-infection, nevertheless display similar clinical symptoms with significantly better recovery times of patients included in the latter. Despite the clinical similarities, studies have suggested that distinct infection-induced host responses from different pathogens occur, namely at the signalling pathway level [2].

**Methods:** Studies have been performed focused on common gene expression profiles between several pathogens [3]. However, understanding the difference in immune responses between these two pathogens, but not exclusively, might lead to better diagnostic tools and treatment decision-making. Relying on systems biology concepts and bioinformatics tools, we use gene expression data to distinguish *C. albicans* and *M. tuberculosis*

infections in the human host; for example, cellular regulation and communication between host immune cells and pathogens [4,5].

**Results:** We have, in a first analysis, focused on the cytokine-cytokine signalling pathway due to its role in inflammation response towards infections. Genes belonging to the IL-2 cytokine family are only expressed when facing infection by *C. albicans* in comparison to *M. tuberculosis* suggesting a tendency towards B-cell proliferation and production of antibodies. However, during infection caused by *M. tuberculosis*, no significant changes in gene expression occur in this pathway that indicates a specific immune response for this pathogen. Further analysis of additional signalling pathways might highlight other human infection-specific immune responses in regards to the pathogens considered.

**Conclusion:** Next we will focus on developing a mathematical model capable of simulating such immune responses and possibly identifying genes and pathways which might clarify how these inflammation responses can be targeted, countered and moderated [6].

### References

1. Angus, et al: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. *Crit Care Med* 2001, 29:1303.

2. Smeekens SP, et al: Functional genomics identifies type I interferon pathway as central for host defense against Candida albicans. *Nat Commun* 2013, 4:1342.

3. Jenner RG, Young RA: Insights into host responses against pathogens from transcriptional profiling. *Nat Rev Microbiol* 2005, 3:281-294.

4. Väremo L, Nielsen J, Nookaew I: Enriching the gene set analysis of genome-wide data by incorporating directionality of gene expression and combining statistical hypotheses and methods. *Nucleic Acids Res* 2013, 41:4378-4391.

5. Luo W, Brouwer C: Pathview: an R/Bioconductor package for pathway-based data integration and visualization. *Bioinformatics* 2013, 29:1830-1831.

6. Mogensen TH: Pathogen recognition and inflammatory signaling in innate immune defenses. *Clin Microbiol Rev* 2009, 22:240-273.

P27

### Vasoactive intestinal peptide inhibits the production of Salmonella-induced inflammatory cytokines by human monocytes.

B Askar*, P Barrow, N Foster

Animal Infection and Immunity, School of Veterinary Medicine and Science, University of Nottingham, UK

Critical Care 2014, **18**(Suppl 2):P27; doi:10.1186/cc14030

**Introduction:** Systemic salmonella infection is a frequent cause of Gram-negative sepsis. Bacterial lipopolysaccharide in blood triggers immune response by monocytes, which results in overwhelming production of proinflammatory cytokines and pathology in peripheral organs such as the liver, heart and lungs. The mortality rate due to sepsis remains high even after chemotherapeutic clearance of pathogens, due to sustained production of inflammatory mediators. Therefore, anti-inflammatory therapy as an adjunct to antibiotics could reduce the mortality from sepsis.

**Methods:** To date, several studies have evaluated the role of vasoactive intestinal peptide (VIP) as a therapeutic agent in sepsis both *in vivo* and *in vitro* since it possesses several desirable biological properties. The peptide, acting via VIP cell receptors, mediates effects by altering production and secretion of inflammatory mediators such as cytokines. VIP has been shown to decrease production of proinflammatory cytokines, ameliorate histopathological changes and inhibit mortality in mice rendered septic by LPS administration. Nothing is known about the effect of VIP on production of proinflammatory cytokines in human monocytes infected by virulent Salmonella.

The aim of the current study, therefore, was to investigate the effect of vasoactive intestinal peptide on the production of inflammatory cytokines in human monocytes exposed to *S. Typhimurium* 4/74.

**Results:** Our finding demonstrates that freshly isolated human monocytes produce proinflammatory cytokines such as TNFα, IL-6, IL-1β and also anti-inflammatory cytokines such as IL-4 and IL-10 following bacterial challenge.

**Conclusion:** However, co-culture of infected monocytes with VIP (10-7 M) significantly reduced (*P* < 0.05) production of TNFα, IL-6, IL-1β but significantly increased (*P* < 0.05) concomitant production of anti-inflammatory IL-10.
P28

Mortality reduction in patients with severe sepsis and septic shock through a comprehensive sepsis initiative.

K Nguyen¹*, L Cook², EP Greenlee³

¹Performance Improvement Department, El Camino Hospital, Mountain View, CA, USA; ²Emergency Department, El Camino Hospital, Mountain View, CA, USA; ³Clinical Effectiveness, El Camino Hospital, Mountain View, CA, USA

*Critical Care* 2014, **18**(Suppl 2):P28; doi:10.1186/cc14031

**Introduction:** The Integrated Nurse Leadership Program (INLP), a regional quality improvement initiative to reduce deaths from sepsis, began in 2008. Nine participating hospitals were provided networking opportunities and training on: care of the septic patient, program and leadership development, and data collection and analysis. El Camino Hospital (ECH) applied these concepts during implementation of the early goal-directed therapy bundle as recommended by the Surviving Sepsis Campaign and the Institute for Healthcare Improvement. This paper describes the implementation process and presents data analysis from the 22-month project, focusing on severe sepsis and septic shock mortality.

**Methods:** A multidisciplinary team approach guided the sepsis initiative. Lean methodologies such as: root cause analysis, process mapping, data collection and direct observation were applied. Frequent meetings with executive personnel, frontline staff and physician leaders occurred to evaluate current practices. Policies and procedures were created, including standardized screening tools and order sets for early identification and management. Training materials (modules, lectures, handouts and simulations) were developed. Extensive training occurred at all levels, with experts presenting to large groups of clinicians. Ongoing data analysis included screening tool and bundle element compliance as well as ICD-9-based mortality among patients with severe sepsis and septic shock (excluding patients <18 years old, pregnant, or designated 'Do Not

Resuscitate' within 24 hours of presentation). Length of stay (LOS) data were compared with data from a large healthcare collaborate quality alliance (Premier) using US Medicare Severity Diagnosis-Related Group sepsis codes (MSDRG 870, 871, 872).

**Results:** Prior to implementation of the program (April 2009) through April 2014, ECH achieved a relative reduction of 68% in the mortality rate among those with severe sepsis and septic shock (P = 0.03). This equates to 1,456 lives saved (Figure 1) [1-3]. During the initiative ECH maintained a lower than expected average LOS compared to other hospitals within the Premier database (Figure 2).

**Conclusion:** The significant and sustained decrease in mortality among those with severe sepsis and septic shock was achieved through the structure and support of multisite collaboration with the INLP and robust internal operations [1,3]. The focus is now on sustainability; including key elements pertaining to accountability, affordability, compassionate care and systematic excellence [4].

References

1. Agency for Research Healthcare and Quality: Nine-hospital collaborative uses patient screening criteria, fast-track diagnosis, and treatment protocols to reduce sepsis mortality by approximately 50 percent. 2014 [http://www.innovations.ahrq.gov/content.aspx?id=3424], Retrieved June 30.

2. Colliver V: Sepsis: Bay Area hospitals sharply cut death rates. SF Gate 2014 [http://www.sfgate.com/bayarea/article/Sepsis-Bay-Area-hospitals-sharply-cut-death-rates-2374402.php], Retrieved June 30.

3. Nguyen K: How a great team can save 900 lives. Center for the Health Professions 2014 [http://www.futurehealth.ucsf.edu/Public/Publications-and-Resources/Content.aspx?topic=Kelly_Nguyen_MS_RN:How_a Great Team Can Save 900 Lives], Retrieved June 30.

4. Mullins JM: The performance imperative. The Public Manager 2010, Summer: 60-65.

<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /><figcaption>Figure 2(abstract P28) Average length of stay for patients with sepsis codes (MSDRG 870, 871, 872), comparing El Camino Hospital to expected LOS from the Premier database. Red line, Premier database expected LOS for sepsis patients; blue line, El Camino Hospital LOS for sepsis patients.</figcaption></figure>

## P29

### Presence of bacterial infection and duration of antibiotic therapy in patients with standardized sepsis detection in the emergency department.

TC Minderhoud¹*, C Spruyt¹, M Lomax¹, S Huisman¹, SCE Schuit², M-D Levin¹
¹Department of Internal Medicine, Albert Schweitzer Ziekenhuis, Dordrecht, the Netherlands; ²Department of Internal Medicine and Emergency Medicine, Erasmus University Medical Center, Rotterdam, the Netherlands
*Critical Care* 2014, **18**(Suppl 2):P29; doi:10.1186/cc14032

**Introduction:** In 2002 the international Surviving Sepsis Campaign was initiated. Following this, Dutch authorities introduced a nationwide safety campaign, encouraging screening for sepsis and advocating early treatment. In 2010, the Albert Schweitzer hospital, a large community hospital, introduced a screening program for sepsis in the emergency department (ED) [1]. The goal of this study was to evaluate the bacterial outcome in patients targeted with this campaign.

**Methods:** All patients 18 years and older visiting the ED were screened using the criteria of systemic inflammatory response syndrome (SIRS). Patients with more than two SIRS criteria and a clinical suspicion for infection were eligible for prompt antibiotic administration, after a short assessment by an ED physician. Patient data were collected prospectively, but a retrospective analysis was conducted using a cohort of patients presenting in the ED in the first 6 months of 2011. The definitions for sepsis severity were derived from the guidelines of the Surviving Sepsis Campaign in 2008 and criteria to define bacterial infection were derived from an article by Limper and colleagues [2].

**Results:** A total of 269 patients were included in the study. Review of infectious outcomes showed no evidence of bacterial disease in 79 (29.4%) patients. Of these patients, 70.9% were in the lowest category of sepsis (SIRS and clinical suspicion of infectious disease). Patients in the lowest category were less likely to suffer from bacterial infection than patients (P = 0.046, see Table 1). In the patients without objective bacterial infection, the median

duration of antibiotic treatment was 7 days (IQR 4 to 10). Overall mortality was 7.8 %, which is low compared to current literature regarding (severe) sepsis [3,4], but comparable to literature addressing SIRS and fever in the ED settings [5-8].

**Conclusion:** Much effort has been put into promoting early antibiotic treatment for (bacterial) sepsis. However, overtreatment has hardly been addressed and no optimal screening strategy has been identified. Evaluation of our screening protocol using SIRS criteria showed that almost 30% of patients did not suffer from bacterial infection but did receive antibiotic treatment for a median duration of 7 days. Future investigations should address the possible negative effects of overtreatment.

### References

1. Rivers E, Nguyen B, Havstad S, Early goal-Directed Therapy Collaborative Group, et al: Early goal-directed therapy in the treatment of severe sepsis and septic shock. *N Engl J Med* 2001, 345:1368-1377.

2. Limper M, Eeftinck Schattenkerk D, de Kruif MD, van Wissen M, Brandjes DP, Duits AJ, van Gorp EC: One year epidemiology of fever at the emergency department. *Neth J Med* 2011, 69:124-128.

3. Angus DC, Linde-Zwirble WT, Lidicker J: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. *Crit Care Med* 2001, 29:1303-1310.

4. Pittet D, Rangel-Fausto MS, Li N: Systemic inflammatory response syndrome, sepsis, severe sepsis and septic shock: incidence, morbidities and outcomes in surgical ICU patients. *Int Care Med* 1995, 21:302-309.

5. Comstedt P, Storgaard M, Lassen AT: The systemic inflammatory response syndrome (SIRS) in acutely hospitalised medical patients: a cohort study. *Scand J Trauma Resusc Emerg Med* 2009, 17:67.

6. Shapiro N, Howell MD, Bates DW: The association of sepsis syndrome and organ dysfunction with mortality in emergency department patients with suspected infection. *Ann Emerg Med* 2006, 48:583-590.

7. Heffner AC, Horton JM, Marchick MR, Jones AE: Etiology of illness in patients with severe sepsis admitted to the hospital from the emergency department. *Clin Infect Dis* 2010, 50:814-820.

<table><caption>Table 1(abstract P29)</caption><thead><tr><th></th><th>No bacterial infection (n = 79)</th><th>Bacterial infection proven/probable (n = 190)</th><th>P value</th></tr></thead><tbody><tr><td>Culture-proven infection</td><td>-</td><td>97 (51.1%)</td><td>-</td></tr><tr><td>Positive blood cultures</td><td>-</td><td>51 (26.8%)</td><td>-</td></tr><tr><td>Probable bacterial infection</td><td>-</td><td>93 (48.9%)</td><td>-</td></tr><tr><td>Sepsis category</td><td></td><td></td><td></td></tr><tr><td>Sepsis</td><td>56 (70.9%)</td><td>110 (57.9%)</td><td>0.046<sup>a,b</sup></td></tr><tr><td>Severe sepsis</td><td>20 (26.3%)</td><td>57 (30.0%)</td><td>0.549<sup>a</sup></td></tr><tr><td>Sepsis-induced hypotension</td><td>2 (2.6%)</td><td>18 (9.5%)</td><td>0.053<sup>a,b</sup></td></tr><tr><td>Septic shock</td><td>1 (1.3%)</td><td>5 (2.6%)</td><td>0.506<sup>a</sup></td></tr><tr><td>Antibiotic treatment duration (days)<sup>c</sup></td><td>7 (4 to 10)</td><td>10 (7 to 14)</td><td>0.000<sup>b,d</sup></td></tr></tbody></table>

<footnote>aChi-square test. bTest reached statistical significance. cData provided as median (IQR). dMann-Whitney U test</footnote>
8. Rangel-Fausto MS, Pittet D, Costigan M: The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. *JAMA* 1995, **273**:117-123.

P30

Clinical audit system in implementing Surviving Sepsis Campaign guidelines in patients with peritonitis.

RC Valiveru¹*, NK Maroju¹, K Srinivasan¹, A Cherian²

¹Department of Surgery, Jawaharlal Institute of Postgraduate Medical Education and Research, Dhanvantari Nagar, Pondicherry, India; ²Department of Anesthesiology and Critical Care, Jawaharlal Institute of Postgraduate Medical Education and Research, Dhanvantari Nagar, Pondicherry, India
*Critical Care* 2014, **18**(Suppl 2):P30; doi:10.1186/cc14033

**Introduction:** Sepsis is the predominant cause of morbidity and mortality in patients with peritonitis [1-6]. The Surviving Sepsis Campaign (SSC) is an international effort in reducing mortality based on evidence-based guidelines [7-13]. This study aims to assess the impact of audit-based feedback in a Plan-Do-Study-Act (PDSA) format on improving implementation of the SSC guidelines in patients with generalised peritonitis at our centre.

**Methods:** This prospective observational study was conducted in four audit cycles in PDSA format. Multidepartmental inputs were taken to suggest modifications in practice. A questionnaire-based analysis of reasons for noncompliance was done to find out the opinions and reasons for noncompliance. Morbidity, mortality, the ICU and hospital stay among these patients were also analysed.

**Results:** The baseline compliance with i.v. bolus administration, CVP-guided fluids and inotrope supports when indicated were 100%. Over the course of the three audit cycles, statistically significant improvement in compliance was noted for antibiotic administration within 3 hours of presentation (46% to 90%) (Table 1 Figure 1), obtaining blood cultures before antibiotics (13.8% to 72.5%) (Table 1 Figure 2) and serum lactate measurement (0% to 78.2%) (Figure 3). Overall bundle compliance improved from 9.2% to 64.7% (Table 2 Figure 4) by the end of Audit III. The mortality rate decreased from 32.3% to 20% (Table 2 Figure 5).

**Conclusion:** This study demonstrates that audit-based feedback is a dependable means of improving compliance with SSC guidelines. It brings about improvement by educating users, by modifying their behaviour through feedback and also enhances process improvement by identifying and correcting systemic deficiencies in the organisation.

**References**

1. Afriji SP, Malik F, Ur-Rahman S, Shamim S, Samo KA: Spectrum of perforation peritonitis in Pakistan: 300 cases Eastern experience. *World J Emerg Surg* 2008, **3**:31.

2. Chakma SM, Singh RL, Parmekar MV, Singh KHG, Kapa B, Sharatchandra KH, Longkumer AT, Rudrappa S: Spectrum of perforation peritonitis. *J Clin Diagn Res* 2013, **7**:2518-2520.

3. Jimenez MF, Marshall JC: International Sepsis Forum: source control in the management of sepsis. *Intensive Care Med* 2001, **27**(Suppl 1):S49-S62.

4. Bali RS, Verma S, Agarwal PN, Singh R, Talwar N: Perforation peritonitis and the developing world. *ISRN Surg* 2014, **2014**:105492.

5. Søreide K, Thorsen K, Søreide JA: Strategies to improve the outcome of emergency surgery for perforated peptic ulcer. *Br J Surg* 2014, **101**:e51-e64.

6. Noguiera C, Silva AS, Santos JN, Silva AG, Ferreira J, Matos E, Vilaça H: Perforated peptic ulcer: main factors of morbidity and mortality. *World J Surg* 2003, **27**:782-787.

7. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb S, Beale RJ, Vincent JL, Moreno R, Surviving sepsis Campaign Guidelines Committee including The Pediatric Subgroup: Surviving

Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. *Intensive Care Med* 2013, **39**:165-228.

8. Marshall JC, Dellinger RP, Levy M: The Surviving Sepsis Campaign: a history and a perspective. *Surg Infect* 2010, **11**:275-281.

9. Castellanos-Ortega A, Suberviola B, García-Astudillo LA, Holanda MS, Ortiz F, Llorca J, Delgado-Rodríguez M: Impact of the Surviving Sepsis Campaign protocols on hospital length of stay and mortality in septic shock patients: results of a three-year follow-up quasi-experimental study. *Crit Care Med* 2010, **38**:1036-1043.

10. Zambon M, Ceola M, Almeida-de-Castro R, Gullo A, Vincent JL: Implementation of the Surviving Sepsis Campaign guidelines for severe sepsis and septic shock: we could go faster. *J Crit Care* 2008, **23**:455-460.

11. Kang MJ, Shin TG, Jo IJ, Jeon K, Suh GY, Sim MS, Lim SY, Song KJ, Jeong YK: Factors influencing compliance with early resuscitation bundle in the management of severe sepsis and septic shock. *Shock* 2012, **38**:474-479.

12. Wang Z, Xiong Y, Schorr C, Dellinger RP: Impact of sepsis bundle strategy on outcomes of patients suffering from severe sepsis and septic shock in china. *J Emerg Med* 2013, **44**:735-741.

13. Li Z-Q, Xi X-M, Luo X, Li J, Jiang L: Implementing surviving sepsis campaign bundles in China: a prospective cohort study. *Chin Med J (Engl)* 2013, **126**:1819-1825.

P31

Introduction of bundle of care and effect on surgical site infections in patients taken for elective surgical procedures.

A Waheed¹*, S Bhat², M Parvez³

¹Department of Anesthesia, District Hospital Doda (Deemed Medical College), Jammu and Kashmir, India; ²Department of Gynecology, District Hospital Doda (Deemed Medical College), Jammu and Kashmir, India;

³Department of Surgery, District Hospital Doda (Deemed Medical College), Jammu and Kashmir, India

*Critical Care* 2014, **18**(Suppl 2):P31; doi:10.1186/cc14034

**Introduction:** Surgical site infection (SSI) previously termed postoperative wound infection is defined as that infection presenting up to 30 days after a surgical procedure if no prosthetic is placed and up to 1 year if a prosthetic is implanted in the patient. Several recommendations have been published (skin preparation, surgical antibiotic prophylaxis, control of the OR environment and improvements in the surgical technique) to improve the patient safety and quality of care in the OR. Use of the bundle of care to improve patient outcomes is becoming more widespread; however, their use is more common internationally than in India.

**Methods:** A surveillance study for SSI after routine surgical procedures was conducted from September 2012 until August 2014. A bundle of care consisting of five elements covering the surgical process was introduced in September 2013. The elements of the bundle were perioperative antibiotic prophylaxis, hair removal before surgery, perioperative normothermia, perioperative euglycemia and operating room discipline. Normothermia was defined as a temperature between 36.0 and 38.0°C. Euglycemia was defined as blood glucose <180 mg/dl. Antibiotic prophylaxis was given 15 minutes before the incision. Hair removal whenever needed was done with clippers. Theatre discipline was counted for following points: permanent wearing of scrub suits, surgical cap and mask covering the nose and mouth by all persons in the OR during the surgical procedure. The incidence of SSI was studied as a primary outcome. Morbidity/mortality for next 3 months was studied as the secondary outcome.

**Results:** Implementing the bundle of care led to a decline in infection rate from 15% before the intervention to 11.4% after the introduction of bundle of care, a fall of 27%, which is significant (P < 0.001). No significant difference in 3-month mortality was found. The compliance to this bundle of care also steadily increased from 10% in September 2013 to 55% in August

<table><caption>Table 1(abstract P30) Compliance with obtaining blood cultures before antibiotics and antibiotic administration within 3 hours</caption><thead><tr><th>Number of patients</th><th>Pre audit (n = 65)</th><th>Audit I (n = 55)</th><th>Audit II (n = 50)</th><th>Audit III (n = 51)</th></tr></thead><tbody><tr><td>Blood cultures obtained before antibiotics</td><td>9 (13.6%)</td><td>18 (32.7%)<sup>a</sup></td><td>30 (60%)<sup>b</sup></td><td>37 (72.54%)<sup>b</sup></td></tr><tr><td>Antibiotics given within 3 hours</td><td>30 (46.1%)</td><td>30 (67.2%)<sup>c</sup></td><td>40 (80%)<sup>d</sup></td><td>46 (90.1%)<sup>b</sup></td></tr></tbody></table>

<footnote>aP = 0.016. bP < 0.0001. cP = 0.463. dP = 0.0002</footnote>
<figure><img src="image_25.png" /><figcaption>Figure 1(abstract P30) Compliance with antibiotic administration within 3 hours of presentation</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>Figure 2(abstract P30) Compliance with obtaining blood cultures before antibiotic administration</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>Figure 3(abstract P30) Compliance with measurement of serum lactate</figcaption></figure>
<table><caption>Table 2(abstract P30) Total bundle compliance</caption><thead><tr><th>Total number of bundle components performed</th><th>Pre audit (n = 65)</th><th>Audit I (n = 55)</th><th>Audit II (n = 50)</th><th>Audit III (n = 51)</th></tr></thead><tbody><tr><td>6</td><td>0</td><td>0</td><td>0</td><td>33<sup>a</sup> (64.7)</td></tr><tr><td>5</td><td>6 (9.2%)</td><td>13 (23.6%)</td><td>27 (54%)</td><td>3 (5.8%)</td></tr><tr><td>4</td><td>26 (40%)</td><td>29 (52.7%)</td><td>16 (32%)</td><td>11 (21.5%)</td></tr><tr><td>3</td><td>33 (50.7%)</td><td>13 (23.6%)</td><td>7 (14%)</td><td>4 (7.8%)</td></tr><tr><td>2</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>

<figure><img src="image_28.png" /><figcaption>Figure 4(abstract P30) Overall bundle compliance</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>Figure 5(abstract P30)</figcaption></figure>

<table><caption>Table 3(abstract P30) Overall mortality</caption><thead><tr><th></th><th>Pre audit (n = 65)</th><th>Audit I (n = 55)</th><th>Audit II (n = 50)</th><th>Audit III (n = 51)</th></tr></thead><tbody><tr><td>Overall mortality in percentage</td><td>21 (32.3%)</td><td>16 (29%)</td><td>12 (24%)</td><td>11 (20%)</td></tr><tr><td>P value (compared with the initial cycle)</td><td></td><td>0.843</td><td>0.407</td><td>0.143</td></tr></tbody></table>

<footnote>ªSerum lactate was available in the hospital only during audit cycle III</footnote>
2014. For 542 surgical procedures during the study period, 62 SSI (11.4%) occurred as compared to 102 cases for 680 in the control period (15%). The adjusted odds ratio of the SSI rate was 0.7319 and was found to be 27% lower post intervention.

**Conclusion:** The implementation of the bundle was associated with improved compliance over time and a significant reduction of the SSI rate. This makes the bundle an important tool to improve patient safety.

P32

**More effective use of polymyxin-B hemoperfusion for nonoperation cases.**

M Yahata<sup>1*</sup>, Y Sakamoto, S Inoue, T Iwamura, R Fujita, H Koami, T Miike, H Imahase, S Narumi, A Goto, M Ohta
Emergency and Critical Care Medicine, Saga University, Saga City, Japan
*Critical Care* 2014, **18**(Suppl 2):P32; doi:10.1186/cc14035

**Introduction:** Direct hemoperfusion with polymyxin-B has been reported to improve hemodynamics in postsurgical patients. In 2012, the Japanese Guidelines for the Management of Sepsis were published and mention the efficacy of polymyxin-B direct hemoperfusion. But how to use and the target patients are varied by facilities. We investigated the effective use of polymyxin-B direct hemoperfusion in nonsurgical patients.

**Methods:** We analyzed retrospectively all septic shock patients who were treated with polymyxin-B hemoperfusion between January 2008 and December 2012. We checked their mean arterial pressure (MAP), and vasopressor requirement every 30 minutes until stopping treatment.

**Results:** There were 32 patients under treatment and 11 patients did not need surgical treatment. Even in the nonsurgical group, hemodynamic states and vasopressor requirement was improved after polymyxin-B hemoperfusion started. And the effects were continued over 120 minutes. A second polymyxin-B hemoperfusion treatment underwent in nine patients. In second treatment, MAP increased in the nonsurgical group greater than in the postsurgical group.

**Conclusion:** Polymyxin-B direct hemoperfusion improves hemodynamic status even in nonsurgical patients. A second polymyxin-B direct hemoperfusion is effective especially in nonsurgical septic shock patients. And if its hemodynamic effect was significantly, long-time treatment should be considered.

P33

**Sepsis modulates the human hematopoietic stem cell compartment in peripheral blood and bone marrow.**

T Skirecki<sup>1,2*</sup>, U Zielińska-Borkowska<sup>2</sup>, J Kawiak<sup>1</sup>, G Hoser<sup>1</sup>

¹Laboratory of Flow Cytometry, The Center of Postgraduate Medical Education, Warsaw, Poland; ²Department of Anesthesiology and Intensive Care, The Center of Postgraduate Medical Education, Warsaw, Poland
*Critical Care* 2014, **18**(Suppl 2):P33; doi:10.1186/cc14036

**Introduction:** Efficient fight with infection requires robust production of immunocompetent cells. This response is called emergency hematopoiesis and depends on the proliferation of progenitor cells and awakening dormant hematopoietic stem cells (HSCs) into cycling. As during sepsis an altered immune system is often observed, it seems important to reveal the impact of this syndrome on HSCs. Recent discoveries have shown that HSCs circulate in the peripheral blood and may boost local immune response via paracrine mechanisms and differentiation into myeloid cells. Altogether, these rationales led us to investigate the circulating HSCs in septic patients and in the bone marrow (BM) of septic 'humanized mice' transplanted earlier with human HSCs.

**Methods:** Samples of peripheral blood were collected from 23 patients with sepsis (on days 1 and 3) and 20 healthy volunteers. The following antigens were analyzed by flow cytometry: CD34, CD38, Ki-67, CD133, Lin and CD45. In order to investigate HSCs in their microenvironment, a model of cecum ligation and puncture (CLP) was performed on the NOD.Cg-Prkdc/scidIL2ry mice that were transplanted with human cord blood CD34⁺ cells 8 weeks earlier. BM cells were analyzed 24 hours after CLP by colony-forming unit assay with medium supporting growth of human cells.

**Results:** Septic patients had a significantly increased (threefold, *P* < 0.01) number of CD34⁺CD38⁻ HSCs on the third day of the disease. Also, the CD133⁺ HSC number was increased in septic patients, while CD34⁺CD45⁺Lin⁻ progenitors were detected at much lower level than in controls. Interestingly, Ki-67⁺CD34⁺Lin⁻ cells were fourfold higher in septic patients. Patients with higher number of CD133⁺ HSCs had significantly lower likelihood of 60-day survival (*P* < 0.05). Analysis of human HSCs from BM of septic mice revealed significantly compromised hematopoietic colonies output (248 vs. 125 in sham animals). CLP caused also expansion of CD34⁺CD38⁻ HSCs in BM and absolute increase of Ki-67⁺CD34⁺Lin⁻ cells (1.5-fold).

**Conclusion:** In this work we have observed significant changes in circulating HSCs during sepsis. During the disease, dormant HSCs enter the cell cycle (measured by Ki-67 expression) and are mobilized to the peripheral blood. However, the progenitor cells disappear from circulation. Novel use of humanized mice confirmed expansion of early human HSCs in BM during the sepsis model. Despite expansion of the HSC pool, the amount of functional progenitors in BM is decreased in this model. We suggest that HSCs play a significant role in the course of sepsis and may become a new prognostic and therapeutic target.

**Acknowledgements:** Supported by 'Innovative Methods of Stem Cell Applications in Medicine', EU Innovative Economy Operational Program, POIG 01.02-00-109/09.

P34

**Physiological changes after fluid bolus therapy in sepsis: a systematic review of the contemporary literature.**

NJ Glassford<sup>1,2*</sup>, GM Eastwood<sup>1,3</sup>, R Bellomo<sup>1,2</sup>

¹Department of Intensive Care, Austin Hospital, Heidelberg, VIC, Australia;

²ANZICS Research Centre, School of Public Health and Preventative Medicine, Monash University, Melbourne, VIC, Australia; ³School of Nursing and Midwifery, Faculty of Health, Deakin University, Burwood, VIC, Australia
*Critical Care* 2014, **18**(Suppl 2):P34; doi:10.1186/cc14037

**Introduction:** Fluid bolus therapy (FBT) is a ubiquitous intervention in intensive care. However, the physiological effects in the critically ill are poorly understood. Therefore, we systematically reviewed the contemporary literature to determine the current practice and effect of FBT in the management of severe sepsis and septic shock.

**Methods:** We interrogated the MEDLINE, CENTRAL and EMBASE electronic reference databases using a combination of terms to define a set of records of studies of fluid administration in patients with severe sepsis or septic shock. To achieve contemporary relevance, results were limited to English-language studies in adults between 2010 and 2013.

**Results:** We identified 22 prospective observational studies, four retrospective observational studies, two quasi-experimental studies, and five randomised controlled trials (RCTs), 41 boluses in total. No RCT compared FBT with alternative interventions. The median fluid bolus was 500 ml (range: 100 to 1,000 ml) administered over 30 minutes (range: 10 to 60 minutes) and 0.9% sodium chloride solution was the most commonly administered. Although 17 studies describe the temporal course of physiological changes after FBT in 31 patient groups, only three studies describe the physiological changes at 60 minutes, and only one study beyond this point (Figure 1). No studies related the physiological changes after FBT with clinically relevant outcomes.

**Conclusion:** There is a need for obtaining randomised controlled evidence for the physiological effects of FBT in patients with severe sepsis and septic shock beyond the period immediately following its administration.

P35

**Forty percent of hospitalizations after severe sepsis are potentially preventable.**

HC Prescott<sup>1*</sup>, KM Langa<sup>1,2,3</sup>, TJ Iwashyna<sup>1,2,3</sup>

¹University of Michigan, Department of Medicine and Institute of Healthcare Policy and Innovation, Ann Arbor, MI, USA; ²VA Center for Clinical
<figure><img src="image_30.png" /><figcaption>Figure 1(abstract P34) Haemodynamic changes following FBT at 1, 2, 3 and 4 hours</figcaption></figure>

Management and Research, Ann Arbor, MI, USA; ³Institute of Social Research, Ann Arbor, MI, USA

Critical Care 2014, **18**(Suppl 2):P35; doi:10.1186/cc14038

**Introduction:** Patients are frequently rehospitalized in the 90 days after severe sepsis. The rate of readmission exceeds patients' baseline rate of hospitalization, and also exceeds the rate after matched nonsepsis hospitalizations [1]. We sought to determine the most common readmission diagnoses after severe sepsis, the extent to which readmissions may be preventable, and whether the pattern of readmission diagnoses differs from that of nonsepsis hospitalizations.

**Methods:** We studied participants in the US Health and Retirement Study with linked Medicare claims (1998 to 2010) [2]. Using validated methods [3,4], we identified severe sepsis and nonsepsis hospitalizations, then measured 90-day readmissions in each cohort. Using Healthcare Cost & Utilization Project's Clinical Classification Software [5], we determined the 10 most common readmission diagnoses after severe sepsis. We measured rates of 'potentially preventable' readmissions using published definitions [6]. We compared rates of all-cause, potentially preventable, and cause-specific hospitalizations between survivors of severe sepsis and nonsepsis hospitalizations using chi-squared tests.

**Results:** We identified 3,703 severe sepsis and 44,840 nonsepsis hospitalizations, of which 3,036 (82.0%) and 43,539 (93.1%) survived to discharge, respectively. In the next 90 days, 43.6% of severe sepsis survivors were rehospitalized, compared to 34.8% of nonsepsis survivors, *P* < 0.001. The top readmission diagnoses following severe sepsis (Table 1) included several recognized potentially preventable diagnoses: heart failure, pneumonia, exacerbation of chronic obstructive pulmonary disease (COPD), and urinary infection. Also common were readmissions for sepsis, acute renal failure, and aspiration pneumonitis, diagnoses that could

plausibly be prevented or treated early to prevent hospitalization. Patterns of readmission differed in severe sepsis survivors; rates of readmission for sepsis, renal failure, respiratory failure, device complication, and aspiration pneumonitis were higher and accounted for a greater proportion of the total readmissions. Potentially preventable hospitalizations - infection (sepsis, pneumonia, urinary tract, and skin or soft tissue), heart failure, COPD exacerbation, acute renal failure, and aspiration pneumonitis - accounted for 40.5% of all readmissions after severe sepsis (compared to 25.8% following nonsepsis admission, *P* < 0.001), and 19.6% of severe sepsis survivors experienced a readmission for one of these diagnoses (compared to 9.5% following a nonsepsis admission, *P* < 0.001).

**Conclusion:** Forty percent of hospitalizations after severe sepsis occur for diagnoses that may be preventable. A few disease categories account for a relatively large proportion of the hospitalizations after severe sepsis, suggesting the feasibility of tailoring postdischarge interventions to patient's personalized risk for these common postsepsis diagnoses.

**Acknowledgements:** The authors declare financial support from the US National Institutes of Health and the US Department of Veteran's Affairs. There are no potential conflicts of interest. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the US government

**References**

1. Prescott HC, Langa KM, Liu V, Escobar GJ, Iwashyna TJ: Increased 1-year healthcare use in survivors of severe sepsis. *Am J Respir Crit Care Med* 2014, 190:62-69.

2. Sonnega A, Faul JD, Ofstedal MB, Langa KM, Phillips JWR, Weir DR: Cohort profile: the Health and Retirement Study (HRS). *Int J Epidemiol* 2014, 43:576-585.

<table><caption>Table 1(abstract P35) Top ten hospitalization diagnoses in the 90 days following severe sepsis</caption><thead><tr><td>Rank</td><td>Diagnosis category</td><td>Proportion of all 90-day admissions (%)</td><td>Survivors with 90-day admission (%)</td></tr></thead><tbody><tr><td>1</td><td>Congestive heart failure, nonhypertensive</td><td>10.4</td><td>5.7*</td></tr><tr><td>2</td><td>Septicemia</td><td>9.5*</td><td>6.5*</td></tr><tr><td>3</td><td>Pneumonia</td><td>5.4</td><td>3.5*</td></tr><tr><td>4</td><td>Rehabilitation care</td><td>5.1</td><td>3.2</td></tr><tr><td>5</td><td>Acute and unspecified renal failure</td><td>4.6*</td><td>3.2*</td></tr><tr><td>6</td><td>Respiratory failure</td><td>4.1*</td><td>2.5*</td></tr><tr><td>7</td><td>Complication of device, implant, or graft</td><td>3.5*</td><td>2.3*</td></tr><tr><td>8</td><td>COPD and bronchiectasis</td><td>3.1</td><td>1.8</td></tr><tr><td>9</td><td>Urinary tract infection</td><td>3.1</td><td>1.8*</td></tr><tr><td>10</td><td>Aspiration pneumonitis</td><td>2.8*</td><td>1.8*</td></tr></tbody></table>

<footnote>*Value greater than that of nonsepsis survivors, *P* ≤ 0.001 for each comparison</footnote>
3. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. *Crit Care Med* 2001, **29**:1303-1310.

4. Iwashyna TJ, Odden A, Rohde J, Bonham C, Kuhn L, Malani P, Chen L, Flanders S: Identifying patients with severe sepsis using administrative claims: patient-level validation of the angus implementation of the international consensus conference definition of severe sepsis. *Med Care* 2014, **52**:e39-e43.

5. Elixhauser A, Steiner C, Palmer L: Clinical Classifications Software (CCS), 2013. US Agency for Healthcare Research and Quality [http://www.hcup-us.ahrq.gov/toolssoftware/ccs/ccs.jsp], (Accessed 29 June 2014).

6. Culler SD, Parchman ML, Przybylski M: Factors related to potentially preventable hospitalizations among the elderly. *Med Care* 1998, **36**:804-817.

## P36
### Aryl hydrocarbon receptor activation increases survival in polymicrobial sepsis.

A de Freitas<fnref n="1" />, P Barbim Donate, F Vargas e Silva Castanheira, V Borges, D Carvalho Nascimento, J Talbot, JC Alves-Filho, F de Queiróz Cunha Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil

*Critical Care* 2014, **18**(Suppl 2):P36; doi:10.1186/cc14039

**Introduction:** Sepsis is a systemic inflammatory response resulting from the inability of the host to restrict the infection locally. Studies realized in our laboratory demonstrated that the high mortality observed in severe sepsis (S-CLP) correlates with the failure of the neutrophil migration to infectious focus and infection dissemination. However, animals subjected to a model of local infection (NS-CLP) present an efficient neutrophil recruitment that constrains the spreading of infection. In this context, we demonstrated a direct action of IL-17 mediating the neutrophil recruitment [1]. Recently, it was demonstrated that aryl hydrocarbon receptor (AhR) activation has a role in immune response. The AhR is expressed by Th17 cells and also by innate immune system cells and is important for their effectors functions, including IL-17 and IL-22 production [2]. However, the function of the AhR in sepsis remains uncertain. Herein, we investigate the role of AhR in polymicrobial sepsis induced by cecal ligation and puncture (CLP).

**Methods:** C57BL/6 mice were subjected to NS-CLP or S-CLP sepsis. The protein expressions of AhR, CYP1A1 (indicator of AhR activation) and AhRR (AhR repressor) were determined in the spleen, liver and lung by western blot, 18 hours after sepsis. AhR and CYP1A1 were also evaluated, 6 hours after surgery, by RT-PCR in peripheral blood mononuclear cells (PBMC). Mice were pretreated subcutaneously with 30 µg/kg FICZ, high-affinity AhR agonist, 12 hours and 1 hour before the induction of moderate sepsis (M-CLP). Intraperitoneal neutrophil migration, bacteremia, kidney function and AhR activation were determined 6 hours after surgery. The survival rate was assessed every 12 hours up to 120 hours.

**Results:** We observed reduced expression of AhR, CYP1A1 and increased AhRR expression in all analyzed organs of the S-CLP group compared with the NS-CLP group. Moreover, AhR and CYP1A1 were not detected in PBMC after S-CLP. Our results also demonstrated that activation of AhR, through FICZ pretreatment, increased the neutrophil recruitment to the peritoneal cavity of mice subjected to M-CLP. Consequently, these animals presented reduced bacteremia and kidney injury, resulting in increased survival rate.

**Conclusion:** These data suggest that during severe infection the AhR expression and activity is reduced and can contribute to the mortality observed in this process.

**Acknowledgements:** Financial support by #2012/04076-9 and 2013/08216-2 from FAPESP

### References

1. Freitas A, Alves-Filho JC, Victoni T, Secher T, Lemos HP, Sônego F, Cunha FQ, Ryffel B: IL-17 receptor signaling is required to control polymicrobial sepsis. *J Immunol* 2009, **182**:7846-7854.

2. Esser C, Rannug A, Stockinger B: The aryl hydrocarbon receptor in immunity. *Trends Immunol* 2009, **30**:447-454.

## P37
### Epidemiology, management and clinical outcomes of ICU-acquired enterococcal bacteraemias.

DSY Ong<fnref n="1" />,²,<fnref n="3" />, K Safdari<fnref n="4" />, PMC Klein Klouwenberg<fnref n="1" />,²,<fnref n="3" />, C Spitoni<fnref n="1" />,<fnref n="4" />, JF Frencken²,<fnref n="3" />, E Witteveen<fnref n="5" />, J Horn<fnref n="5" />, MJM Bonten<fnref n="1" />,<fnref n="3" />, OL Cremer²

*Critical Care* 2014, **18**(Suppl 2):P37; doi:10.1186/cc14040

**Introduction:** Enterococcal bacteraemias are associated with high mortality rates, ranging from 23 to 48% in critically ill patients. However, it remains uncertain whether these events are causes or merely markers of disease severity and mortality. Our aim was to describe the epidemiology, management and clinical outcomes of ICU-acquired enterococcal bacteraemia in comparison to other frequently isolated pathogens. Furthermore, we aimed to estimate the population attributable fraction of ICU mortality caused by enterococcal bacteraemia.

**Methods:** Between January 2011 and March 2013 we included consecutive patients with an ICU length of stay of at least 3 days in two tertiary care centres in the Netherlands. ICU-acquired bacteremia was defined as a first positive blood culture occurring at least 3 days after ICU admission. Enterococcal bacteraemias were compared to other frequently isolated pathogens with respect to patient characteristics, their management and outcomes. We used competing risk survival regression, a multistate model and cumulative incidence functions to estimate the population attributable fraction of ICU mortality due to enterococcal bacteraemia.

**Results:** Out of a total of 3,108 patients, 222 (7.1%) of patients were responsible for 272 events of ICU-acquired bacteraemia, of which 76 were due to enterococci, 124 to coagulase-negative staphylococci and 40 to Gram-negative bacteria. Patients with enterococcal bacteraemia were more severely ill compared to patients with bacteraemia caused by other pathogens. In comparison to patients with coagulase-negative staphylococci, those with enterococci were more frequently managed with renal replacement therapy and also had more intravascular or orthopaedic hardware. Although crude ICU mortality was higher in patients with bacteraemias due to enterococci compared to coagulase-negative staphylococci, this association disappeared after adjustment for confounders (subdistribution hazard ratio 1.04, 95% confidence interval 0.65 to 1.68). The population attributable fraction of ICU mortality due to enterococcal bacteraemia was 4.7%.

**Conclusion:** Bacteraemias with enterococci occur in more severely ill patients, but their virulence seems comparable to that of coagulase-negative staphylococci. Furthermore, the population attributable fraction of ICU mortality due to enterococcal bacteraemia is low. Therefore, enterococcal bacteraemias are more likely to be markers than causes of increased disease severity and mortality.

**Acknowledgements:** This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (http://www.ctmm.nl), project MARS (grant 04I-201).

## P38
### Systemic inflammatory response in the pediatric emergency department: a common phenomenon that does not predict severe illness.

CPZ Foo<fnref n="1" />*, G Sangha<fnref n="1" />,², J Seabrook²,<fnref n="3" />, J Foster<fnref n="1" />,²

*Critical Care* 2014, **18**(Suppl 2):P38; doi:10.1186/cc14041

**Introduction:** Systemic inflammatory response syndrome (SIRS) was defined in pediatrics primarily to aid in research subject identification. There is a paucity of data on the clinical utility of SIRS in the pediatric population. We aimed to determine the incidence of SIRS in children presenting to the pediatric emergency department (PED) of a mid-sized Canadian tertiary care

<footnote n="1">¹Department of Medical Microbiology, University Medical Center Utrecht, the Netherlands; ²Department of Intensive Care Medicine, University Medical Center Utrecht, the Netherlands; ³Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, the Netherlands;</footnote>

<footnote n="4">⁴Department of Mathematics, Utrecht University, the Netherlands;</footnote>

<footnote n="5">⁵Department of Intensive Care, Academic Medical Center, University of Amsterdam, the Netherlands</footnote>

<footnote n="1">¹Department of Paediatrics, Western University, London, Canada; ²Children's Health Research Institute, London Health Sciences Centre, London, Canada;</footnote>

<footnote n="3">³Division of Food and Nutritional Sciences, Brescia University College, London, Canada</footnote>
center and, secondarily, to examine the sensitivity and specificity of SIRS for predicting infection, the range of clinical entities presenting with SIRS, and outcomes of children with SIRS.

**Methods:** We conducted a prospective cohort study of all children from birth to 18 years presenting to the PED on 16 days distributed evenly over 1 year. Charts of all patients presenting to the PED on study days were reviewed to determine the presence of SIRS and sepsis. Three pediatricians adjudicated all cases of presumed infection. All patients were followed for 1 week to determine outcomes.

**Results:** The incidence of SIRS was 14.3% (n = 202 of 1,416). The rate of documented or presumed infection in the entire population was 37.1% (n = 525), and in SIRS patients was 81.2% (n = 164). Therefore, 11.6% of the population (n = 164) met criteria for sepsis, one patient had severe sepsis, and there were no cases of septic shock. Sensitivity and specificity of SIRS for predicting infection was 31.2% (95% CI: 27.3 to 35.4%) and 95.7% (95% CI: 94.2 to 97.0%) respectively. No difference in sensitivity or specificity was seen when cases were separated by age. Patients with SIRS had a higher risk of requiring hospital admission with a relative risk of 2.8 (95% CI: 2.0 to 4.1, *P* < 0.0001). Although children with SIRS stayed in hospital statistically longer (*P* < 0.001), the median length of stay for both groups was ≤1 day. However, 28.7% (n = 47) of patients meeting definition of sepsis had nonsevere infection types (for example, acute otitis media, viral upper respiratory tract infection); 72.3% of patients with SIRS and 75% of patients meeting sepsis criteria were discharged home from the PED without return visits.

**Conclusion:** While SIRS has a high specificity for infection, its poor sensitivity suggests a risk of missing infection if used as a screening tool. Although patients with SIRS have an increased risk of admission, the current definition of sepsis may result in over-inclusion of patients with nonsevere disease.

**Acknowledgements:** JF received a New Investigator grant, Children's Health Foundation, LHSC. CF received a Resident research grant, Department of Paediatrics, Western University.

P39

Early bacterial spreading and inflammatory profile in a pneumosepsis model.

M de Carvalho Gonçalves*, V Horewicz, J Assreuy
Department of Pharmacology, Universidade Federal de Santa Catarina,
Florianopolis, Brazil
*Critical Care* 2014, **18**(Suppl 2):P39; doi:10.1186/cc14042

**Introduction:** Pneumonia is the major cause of sepsis, responsible for almost one-half of all sources of infection [1]. Sepsis and septic shock lead to organ failure and death. Spreading of microorganisms and their toxins through the blood could contribute to the organ dysfunction. The heart, liver and kidney are examples of organs damaged during the systemic infection and organ failure predicts poor prognosis in patients with sepsis [2]. However the correlation, if any, between bacterial spreading and organ injury is unclear. Thus, the aim of this study was to study the bacterial systemic spreading from a localized infection along with time and the target organ inflammatory profile using proinflammatory cytokines as surrogate markers.

**Methods:** Pneumosepsis was induced by Klebsiella pneumoniae as described in [3]. The number of viable bacteria inoculated was 10⁹ colony-forming units to achieve a mortality rate of ~50% by 48 hours. Blood, lung, heart, liver, spleen, kidney and brain were aseptically harvested, homogenized and plated on Müller-Hinton agar dishes, for 18 hours at 37°C. Plasma and the tissues homogenates were assayed for interleukin-6 (IL-6) and interleukin-1β (IL-1β) using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's recommendations (PeproTech Inc., Rocky Hills, NJ, USA).

**Results:** Lung inoculation with K. pneumoniae evolved to systemic spreading of bacteria to all organs, mostly the liver and kidney. Surprisingly, bacteria were found as early as 30 minutes in vital organs such as the brain and heart. The infection in the organs rose steadily up to 24 to 48 hours. Significant increases in IL-6 and IL-1β were found in the plasma, 24 hours after infection. However, cytokine levels in the organs were as high as fivefold the plasma levels.

**Conclusion:** Our data show that the early bacterial dissemination may be important for the onset of organ inflammation. Assuming that the higher

organ cytokine level is a marker of an ongoing inflammation, this may explain organ dysfunction during sepsis. Thus, our study suggests that systemic (meaning blood) parameters may not reflect the severity of inflammation/dysfunction in target organs, and that might be the determinant to sepsis outcome.

**Acknowledgements:** Brazilian financial support from CNPq, CAPES, FAPESC and FINEP

References

1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. *Crit Care Med* 2001, 29:1303-1310.

2. Martin GS, Mannino DM, Eaton S, Moss M: The epidemiology of sepsis in the United States from 1979 through 2000. *N Engl J Med* 2003, 348:1546-1554.

3. Sordi R, Menezes-de-Lima O, Della-Justina AM, Rezende E, Assreuy J: Pneumonia induced sepsis in mice: temporal study of inflammatory and cardiovascular parameters. *Int J Exp Pathol* 2013, 94:144-155.

P40

Effects of splenectomy and GTS-21, a selective α7 nicotinic acetylcholine receptor agonist, on the development of septic ileus in mice.

S Nullens¹*, JG De Man¹, PA Pelckmans¹,², BY De Winter¹

¹Laboratory for Experimental Medicine and Pediatrics, Division of Gastroenterology and Hepatology, University of Antwerp, Belgium;

²Department of Gastroenterology and Hepatology, Antwerp University Hospital, Antwerp, Belgium

*Critical Care* 2014, **18**(Suppl 2):P40; doi:10.1186/cc14043

**Introduction:** Sepsis remains a leading cause of mortality in our ICUs. Ileus, defined as the inhibition of the propulsive motility in the gastrointestinal (GI) tract, together with mucosal barrier dysfunction will maintain sepsis by the translocation of intestinal bacteria. Preliminary data in our group showed that the administration of GTS-21, a selective alpha7 nicotinic acetylcholine receptor (α7nAChR) agonist, ameliorates inflammation and GI motility disturbances during sepsis [1]. Activation of the α7nAChR is the final step in the vagal anti-inflammatory pathway, a spleen-dependent and macrophage-dependent pathway that dampens inflammation. We aimed to study the effects of splenectomy (SPLX) combined with GTS-21 on GI motility and on local colonic and systemic inflammation, as the role of the spleen in the vagal anti-inflammatory pathway is currently under debate.

**Methods:** Septic ileus was induced in Swiss-OF 1 mice by cecal ligation and puncture (CLP). One week prior to CLP, mice underwent SPLX or sham surgical incision (sham). GTS-21 (8 mg/kg) or vehicle was administered intraperitoneally 1 hour before CLP, and once every 24 hours after the procedure. Mice were sacrificed 48 hours following CLP. Motility was assessed by measurement of GI transit of beads, with calculation of percentage of gastric emptying (%GE) and geometric center (GC). Serum samples were analyzed with cytometric bead array (CBA) for the proinflammatory cytokines IL-6 and TNFα. Supernatants of centrifuged homogenized colonic samples were assessed with CBA for IL-6 and TNFα.

**Results:** Administration of GTS-21 resulted in a significant improvement in GI motility in septic mice, as was demonstrated by an increase in the %GE and GC (Table 1). GTS-21 significantly decreased the serum concentration of IL-6, but not TNFα, in septic mice treated with GTS-21 compared to vehicle-treated mice. Prior SPLX protected mice from developing ileus following CLP. SPLX + vehicle mice demonstrated a significantly lower serum concentration of IL-6 and TNFα compared to sham + vehicle, and colonic TNFα levels declined significantly following SPLX. Administering GTS-21 in SPLX mice did not result in an additional benefit on GI motility, nor on systemic or local colonic inflammation (Table 1).

**Conclusion:** Splenectomy protects mice from developing ileus following CLP, an animal model of polymicrobial sepsis, as does the preventive administration of the selective α7nAChR agonist GTS-21. Splenectomy resulted in a reduction of systemic TNFα and IL-6 levels, indicating that the spleen is a major source of proinflammatory cytokines. GTS-21 ameliorates CLP-induced septic ileus, but did not lead to a complimentary benefit in addition to splenectomy.
<table><caption>**Table 1(abstract P40) %GE, GC, serum cytokine levels (pg/ml) and colonic cytokine levels (pg/g colon) in mice that underwent splenectomy or sham surgery, followed by the CLP procedure and administration of vehicle or 8 mg/kg GTS-21**</caption><thead><tr><th>GI transit study (n = 11)</th><th>%GE</th><th>GC</th></tr></thead><tbody><tr><td>Nonseptic animals</td><td>88.96 ± 8.13</td><td>4.86 ± 0.45</td></tr><tr><td>Sham + vehicle + CLP</td><td>53.27 ± 10.32</td><td>2.53 ± 0.42</td></tr><tr><td>Sham + GTS-21 8 mg/kg + CLP</td><td>83.62 ± 5.91*</td><td>4.26 ± 0.35*</td></tr><tr><td>SPLX + vehicle + CLP</td><td>79.49 ± 8.98*</td><td>3.86 ± 0.44*</td></tr><tr><td>SPLX + GTS-21 8 mg/kg + CLP</td><td>77.64 ± 11.81*</td><td>3.55 ± 0.52</td></tr><tr><th>Cytokine level in serum (n = 8 to 11)</th><th>IL-6</th><th>TNFα</th></tr><tr><td>Nonseptic animals</td><td>2.57 ± 0.48</td><td>4.95 ± 0.29</td></tr><tr><td>Sham + vehicle + CLP</td><td>243.56 ± 54.89</td><td>63.72 ± 21.25</td></tr><tr><td>Sham + GTS-21 8 mg/kg + CLP</td><td>90.26 ± 31.31*</td><td>54.23 ± 25.14</td></tr><tr><td>SPLX + vehicle + CLP</td><td>74.31 ± 17.45*</td><td>11.52 ± 1.99#</td></tr><tr><td>SPLX + GTS-21 8 mg/kg + CLP</td><td>64.98 ± 7.15*</td><td>12.81 ± 3.91#</td></tr><tr><th>Cytokine levels in colonic tissue (n = 5 to 11)</th><th>IL-6</th><th>TNFα</th></tr><tr><td>Nonseptic animals</td><td>20.43 ± 4.87</td><td>41.30 ± 4.47</td></tr><tr><td>Sham + vehicle + CLP</td><td>1281.85 ± 932.10</td><td>137.15 ± 61.36</td></tr><tr><td>Sham + GTS-21 8 mg/kg + CLP</td><td>134.88 ± 36.15</td><td>46.46 ± 5.03*</td></tr><tr><td>SPLX + vehicle + CLP</td><td>214.22 ± 75.88</td><td>29.97 ± 8.60*</td></tr><tr><td>SPLX + GTS-21 8 mg/kg + CLP</td><td>487.49 ± 343.87</td><td>46.03 ± 17.22*</td></tr></tbody></table>

Data presented as mean ± SEM. Two-way ANOVA or one-way ANOVA as appropriate with SNK post hoc testing; \*P ≤ 0.05 versus sham + vehicle, \#P ≤ 0.05 significant effect of splenectomy (SPLX)

### Reference

1. Nullens S, et al: GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates septic ileus in a mouse model of cecal ligation and puncture [abstract]. Presented at the First Neurogastroenterology and Motility Meeting 2014. *Neurogastroenterol Motil* 2014 in press.

## P41

### Biobanking in the emergency department: implementation of the Mayo Clinic Emergency Department Sepsis Biorepository.

CM Clements, JR Anderson, J Uhl, MI Rudis, FR Cockerill
Department of Emergency Medicine, Mayo Clinic, Rochester, MN, USA
*Critical Care* 2014, **18**(Suppl 2):P41; doi:10.1186/cc14044

**Introduction:** Biomarker discovery research has not focused on the emergency department (ED) due to perceived lack of access to ED patients for study, difficult patient identification, and preemption by time-critical clinical needs. The aim of this study was to use an automated process to accrue, within the ED, a biobank of patients presenting at risk for severe sepsis.

**Methods:** Our group has previously derived an algorithm for identification of at-risk patients of which about 45% will be severely septic [1]. Here, we utilize that algorithm to identify a prospective cohort to be included in our ED sepsis biobank. Patients are excluded if they are pregnant, <18 years old, a vulnerable adult, or imprisoned. An electronic notification system identifies patients and automatically pages phlebotomy and laboratory processing personnel directly. Blood draws occur immediately after patient identification and at 6 and 24 hours into hospitalization. We use an IRB-approved delayed consent model that minimizes the time between identification and blood draw. Plasma extracts and cellular isolates are processed and stored immediately. Clinical data are extracted for relevant patient outcomes, severe sepsis, and indicators of critical illness.

**Results:** In 1 year of accrual, the alert algorithm identified 1,773 eligible patients for screening. Blood was drawn from 873 patients based on phlebotomy availability and laboratory processing staffing. A total of 642 patients completed blood draws and consented for inclusion, with the balance of patients having been discharged, transferred, or deceased with no identifiable legally authorized representative for consent. The median

time from alert to first blood draw was 25 minutes (range 5 to 60 minutes). In an observation period, during which 227 consecutive patients had blood drawn, only 12 patients or families (5.2%) declined consent after the initial blood draw.

**Conclusion:** We have successfully developed and implemented a biobank in the ED that functions within practice limitations and has high patient accrual. Automation is key for efficient implementation in the ED. Using automated design improvements, we obtain blood samples from sepsis patients presenting to the ED faster than previous studies. Prompt biobank collection within the ED allows research into mechanisms of sepsis earlier in a patient's hospital course than has heretofore been possible and enables biomarker discovery for testing development directly relevant to emergency providers.

### Reference

1. Attwood RJ, Smith VD, Russi CS, Hess EP, Escalante P, Baudoin MR, Frazee EN, Rudis MI: An automated electronic notification system as a screening tool for severe sepsis and septic shock. *Ann Emerg Med* 2010, 56:S64-S65.

## P42

### Defining fever: likelihood of infection diagnosis as a function of body temperature in the emergency department.

E Small, CM Clements
Department of Emergency Medicine, Mayo Clinic, Rochester, MN, USA
*Critical Care* 2014, **18**(Suppl 2):P42; doi:10.1186/cc14045

**Introduction:** Fever predicts an infection cause of SIRS/sepsis more specifically than other commonly used hemodynamic criteria. Traditionally, a body temperature of 38.0°C has been used to define fever, but this cutoff is based on limited research. Furthermore, it has been proposed that fever responses are blunted in older adults, limiting the utility of fever in infection. This study determines the likelihood that a body temperature will predict diagnosis of infection in the emergency department (ED).

**Methods:** This was a retrospective cohort analysis of adult patients (>18 years old) presenting to a large academic emergency department from September 2010 to December 2012. Patient age, emergency physician diagnosis, final disposition, and initial body temperature were examined for each patient. Likelihood of a diagnosis of infection was calculated for all temperature ranges. Sensitivity and specificity of fever thresholds for a
diagnosis of infection were calculated and receiver operating characteristic (ROC) curves were generated. Confidence intervals were determined using Newcombe-Wilson hybrid scoring with continuity correction.

**Results:** We identified and analyzed records from 121,587 patients, including 37,933 persons >65 years old. Overall, 15.9% of patients received a diagnosis of infection in the ED with those >65 years old having a higher rate of infection at 18.3%. Likelihood of infection varied by temperature in a nonlinear relationship (see Figure 1). In adults of all ages, temperature >38.0°C had a specificity >99% for a diagnosis of infection in the ED although sensitivity was relatively poor. Specificity decreases only slightly with lower fever cutoffs (37.2 to 37.9°C) while sensitivity is increased to a greater degree (see Table 1).

**Conclusion:** The likelihood of a diagnosis of infection increases at the extremes of body temperature and is higher overall among older adults (≥65 years old). In our population, elevated temperature was at least as sensitive for a diagnosis of infection in older people as in younger adults. The use of body temperature for predicting diagnosis of infection has an overall low sensitivity but high specificity. Changing the current practical definition of fever from ≥38.0°C to ≥37.5°C significantly increases sensitivity of predicting infection without greatly impacting the specificity. Only 2.3% of all patients had a temperature between 37.5 and 37.9°C, and these patients were four times more likely to have a diagnosis of infection than those with temperature of 36.0 to 36.9°C.

P43

**Ghrelin: an anti-inflammatory theurapeutic agent in septic rats.**

GA Uluçay¹*, E Özkök², H Yorulmaz³, İ Aydın⁴, Ş Tamer¹

¹Department of Physiology, Faculty of Medicine, University of Istanbul, Turkey; ²Institute of Experimental Medicine Research, Department of Neuroscience, University of Istanbul, Turkey; ³Faculty of Health Science, University of Halic, Turkey; ⁴Department of Histology, Faculty of Medicine, University of Istanbul, Istanbul, Turkey

*Critical Care* 2014, **18**(Suppl 2):P43; doi:10.1186/cc14046

**Introduction:** Sepsis is a life-threatening systemic inflammatory syndrome (SIRS), which affects many organ systems, that leads to hemodynamic changes in the presence of suspected or proven infection, advancing to organ dysfunction and failure [1-3]. In recent studies, 377 out of 100,000 cases of sepsis were observed while $14,600,000,000$ has been determined as the average annual hospital costs. Although there are many studies, the molecular mechanism is not yet clearly elucidated [2,3]. Lipopolysaccharide (LPS) is the lipid molecule which the outer membrane of Gram-negative bacteria used in designing the experimental sepsis model [4]. Ghrelin was discovered in 1999 as a specific ligand for growth hormone secretagogue receptor and then pleiotropic effects such as anti-inflammatory, antioxidant, and so forth were found. Ghrelin was released in many tissues and organs in which there also was its receptor. The liver is an organ which has ghrelin receptors and was affected by sepsis primary [5,6].

**Methods:** In our study, male Wistar albino rats of average body mass 200 to 250 g were separated into four groups including: Control ($n = 10$), LPS (E. coli 055:B5, 5 mg/kg, $n = 10$), ghrelin (10 nmol/kg i.v., $n = 10$), LPS + ghrelin LPS

<figure><img src="image_31.png" /><figcaption>Figure 1(abstract P42) Likelihood of infection diagnosis as a function of initial body temperature</figcaption></figure>

<table><caption>Table 1(abstract P42) Sensitivity and specificity of body temperature for a diagnosis of infection</caption><thead><tr><th rowspan="2"></th><th colspan="2">18 to 64 years of age</th><th colspan="2">≥65 years of age</th></tr><tr><th>Sensitivity (95% CI)</th><th>Specificity (95% CI)</th><th>Sensitivity (95% CI)</th><th>Specificity (95% CI)</th></tr></thead><tbody><tr><td>≥37.0</td><td>33.5% (32.6 to 34.3)</td><td>81.9% (81.6 to 82.2)</td><td>33.3% (32.2 to 34.4)</td><td>85.7% (85.3 to 86.1)</td></tr><tr><td>≥37.1</td><td>25.8% (25.0 to 26.6)</td><td>89.4% (89.1 to 89.6)</td><td>27.9% (26.8 to 29.0)</td><td>91.2% (90.8 to 91.5)</td></tr><tr><td>≥37.2</td><td>20.4% (19.7 to 21.2)</td><td>93.4% (93.2 to 93.5)</td><td>23.4% (22.4 to 24.4)</td><td>94.0% (93.7 to 94.3)</td></tr><tr><td>≥37.3</td><td>17.1% (16.4 to 17.8)</td><td>95.6% (95.5 to 95.8)</td><td>20.3% (19.3 to 21.2)</td><td>95.7% (95.5 to 95.9)</td></tr><tr><td>≥37.4</td><td>14.6% (14.0 to 15.3)</td><td>96.9% (96.8 to 97.0)</td><td>17.8% (16.9 to 18.7)</td><td>96.6% (96.4 to 96.8)</td></tr><tr><td>≥37.5</td><td>12.6% (12.0 to 13.2)</td><td>97.8% (97.7 to 97.9)</td><td>15.9% (15.0 to 16.7)</td><td>97.4% (97.2 to 97.6)</td></tr><tr><td>≥38.0</td><td>6.48% (6.06 to 6.93)</td><td>99.3% (99.2 to 99.3)</td><td>8.77% (8.12 to 9.47)</td><td>99.0% (98.9 to 99.1)</td></tr></tbody></table>
(5 mg/kg, ghrelin 10 nmol/kg i.v., n = 10). Rats were decapitated 24 hours after first injection. We aimed in this study to investigate effects of ghrelin in sepsis which is created by LPS with sepsis descriptive parameters such as body temperature and leukocyte count with proinflammatory cytokine TNFα and anti-inflammatory cytokines IL-10 by ELISA, and hematoxylin and eosin stain for observed morphological changes.

**Results:** We detected the increase of leukocyte number, hypothermia, proinflammatory and anti-inflammatory cytokines such as TNFα and IL-10 as developing inflammatory reactions, resulting in hemodynamic and metabolic changes in rats treated with LPS. Also in groups with ghrelin treatment, ghrelin affects leukocyte numbers, body temperature, proinflammatory and anti-inflammatory cytokine levels and histological changes in controls and the LPS group (Figures 1 to 4). As a result of histologic examination, the curative effect of ghrelin partially on liver tissue damage is observed (Figure 5).

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

**Conclusion:** We think the results of ghrelin may be affected depending on the dose and duration. Also partial healing effects of ghrelin in our results on this topic at the molecular level will contribute to other studies.

**References**

1. Angus DC, van der Poll T: Severe sepsis and septic shock. *N Engl J Med* 2013, **369**:2063.

2. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G, SCCM/ESICM/ACCP/ATS/SIS: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. *Crit Care Med* 2003, **31**:1250-1256.

3. Schorr CA, Zanotti S, Dellinger RP: Severe sepsis and septic shock: management and performance improvement. *Virulence* 2014, **1**:190-199.
<figure><img src="image_36.png" /><figcaption>Figure 5(abstract P43)</figcaption></figure>

4. Ramachandran G: Gram-positive and gram-negative bacterial toxins in sepsis: a brief review. *Virulence* 2014, 5:213-218.

5. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth-hormone-releasing acylated peptide from stomach. *Nature* 1999, 402:656-660.

6. Chang L, Zhao J, Yang J, Zhang Z, Du J, Tang C: Therapeutic effects of ghrelin on endotoxic shock in rats. *Eur J Pharmacol* 2003, 473:171-176.

P44

**Restrictive parenteral fluid therapy in infants and children presenting with acute severe viral pneumonia in the PICU: a single-center experience.**

S Kandasamy*, N Vijayakumar, T Sankaralingam, V Varadarajan,
N Krishnamoorthy
Division of Pediatric Intensive Care, Department of Pediatrics, Mehta
Children's Hospital Pvt Ltd, Chennai, India
*Critical Care* 2014, **18**(Suppl 2):P44; doi:10.1186/cc14047

**Introduction:** Acute viral (RSV and non-RSV) pneumonia is one of the most common causes of lower respiratory tract infection in infants and children. Although only 2 to 3% of infants with bronchiolitis require hospitalized management based on published literature, in recent years we see more

children requiring hospitalization in high-dependency units. Most of these children require intravenous fluid therapy and some form of oxygen supplementation. As there is no standardized evidence-based parenteral fluid therapy protocol specific for acute severe viral pneumonia available, we decided to retrospectively analyze the result of our restrictive fluid therapy protocol.

**Methods:** All children less than 5 years age admitted to our PICU from June 2013 to December 2013 with etiological evaluation confirmed acute severe viral (RSV and non-RSV) pneumonia who received a minimum 48 hours of parenteral fluid therapy were enrolled and the data retrieved from the case sheet. The data analyzed were duration of PICU stay, hospital stay, oxygen therapy requirement, duration of oxygen therapy and their relationship to fluid balance on restrictive fluid regimen.

**Results:** A total of 32 children met the criteria for inclusion in the study period, 23 boys (71.9%) and nine girls (28.1%). The median age of children was 9.5 months (IQR 3.3 to 27.3). Of 32 children, 4/32 (12.5%) required invasive mechanical ventilation; 5/32 (15.6%) required NP-CPAP; 12/32 (37.5%) were on HHFNC; and nasal prong oxygen supplementation in 26 (81.3%) children. The median duration of ICU stay was 68.9 hours (IQR 39.2 to 80.2), that of hospital stay was 116.5 (IQR 92 to 179.2). Fluid balance at the end of 72 hours of ICU stay did not significantly differ the need for ventilation (P = 0.45) or the duration of ventilation (P = 0.60). Fluid balance in the first 72 hours correlated positively with change in serum sodium
levels indicating a fall in sodium levels with more positive fluid balance (Spearman coefficient of 0.452 (P = 0.16)). There was no significant correlation between fluid balance and duration of PICU/hospital stay (P = 0.58/0.75). eGFR calculated using modified Schwartz formula did not correlate with treatment parameters like duration of ICU/hospital stay, mechanical ventilation, and CPAP.

**Conclusion:** Positive fluid balance may not influence the duration of PICU stay, hospital stay, or oxygen therapy in children with severe viral pneumonia when initiated on restrictive (70% of Holliday Segar calculation) parenteral fluid therapy. eGFR at admission did not influence the fluid balance when they were uniformly initiated on restrictive parenteral fluids. Further studies with calculated sample size may help to confirm the observations.

P45

**Blood sugar variation during the first 48 hours of hospitalization for patients with sepsis was associated with in-hospital mortality.**

K-F Chen*, C-C Wu

Chang Gung University, Taoyuan, Taiwan and Chang Gung Memorial

Hospital at Keelung and Linkou, Taiwan

Critical Care 2014, **18**(Suppl 2):P45; doi:10.1186/cc14048

**Introduction:** Blood sugar control for patients with sepsis remains controversial. We aimed to test the hypothesis that the variation of blood sugar level is associated with patient outcome in this study.

**Methods:** A retrospective cohort study on nontraumatic adult patients who visited the ED of a tertiary hospital in 2010 and had a clinical diagnosis of severe sepsis was conducted. Patients with two sets of blood culture ordered by emergency physicians and at least two blood sugar tests results available during the first 48 hours of hospitalization were included. The coefficients of variation (CoV, the ratio of the standard deviation to the mean) of the blood sugar level were analyzed with multivariate logistic regression models to test the association between in-hospital mortality.

**Results:** Of the 1,537 patients included, most were older than 70 years of age (median; 71, IQR: 59 to 80), male (54%), without a diagnosis of severe sepsis (63%) and had a previous diagnosis of diabetes (84%). The initial blood sugar levels of patients with and without previously diagnosed diabetes were 259 ± 9.9 and 154 ± 5.7, respectively (mean ± SEM). The CoV of the consecutively monitored blood sugar level during the first 48 hours of admission for patients with and without previously diagnosed diabetes were 29.4 ± 0.5% and 21.0 ± 0.5%, respectively. Patients with CoV lower than 10% and higher than 30% tended to have higher mortality rate, compared to patients with 10 to 30% CoV level (11% vs. 12% and 7%, respectively, Figure 1). In the multivariate logistic regression model adjusting for age, initial blood sugar level, severity of sepsis, previous diagnosis of diabetes and diagnosis of severe sepsis, the higher CoV level (>30%) was found to be associated with 60% increased odds of in-hospital mortality (aOR: 1.61 ± 0.34); while the previous diagnosis of diabetes was found to be associated with 45% lower odds of in-hospital mortality (aOR: 0.55 ± 0.13, Table 1).

<figure><img src="image_37.png" /><figcaption>Figure 1(abstract P45) Lowess smoother plot for mortality versus coefficient of variation</figcaption></figure>

<table><caption>Table 1(abstract P45)</caption><thead><tr><td></td><td>Odds ratio</td><td>Standard error</td><td>P value</td><td>95% CI</td></tr></thead><tbody><tr><td>Sugar CoV &lt;10%</td><td>1.51</td><td>0.44</td><td>0.15</td><td>0.86 2.67</td></tr><tr><td>Sugar CoV &gt;30%</td><td>1.61</td><td>0.34</td><td>0.02</td><td>1.06 2.43</td></tr><tr><td>Initial sugar level &lt;100</td><td>0.98</td><td>0.01</td><td>0.00</td><td>0.97 0.99</td></tr><tr><td>Initial sugar level ≥ 100 and &lt;500</td><td>1.00</td><td>0.00</td><td>0.67</td><td>1.00 1.00</td></tr><tr><td>Initial sugar level higher ≥ 500</td><td>1.00</td><td>0.00</td><td>0.54</td><td>1.00 1.00</td></tr><tr><td>Severe sepsis</td><td>2.43</td><td>0.48</td><td>0.00</td><td>1.65 3.56</td></tr><tr><td>Previous diagnosis of diabetes</td><td>0.55</td><td>0.13</td><td>0.01</td><td>0.34 0.88</td></tr><tr><td>Severity of sepsis</td><td>1.14</td><td>0.04</td><td>0.00</td><td>1.07 1.21</td></tr><tr><td>Age</td><td>1.01</td><td>0.01</td><td>0.49</td><td>0.99 1.02</td></tr></tbody></table>
**Conclusion:** In this retrospectively cohort study, we found that increased blood sugar variation was associated with worse patient outcome. However, further study is merited to test the possible causal relationship between variation of blood sugar level and patient outcome.

P46

**Association of initial intracellular signalling pathway and cytokine level with early mortality in severe sepsis patients.**

KC Lie¹*, D Widoło¹, S Lardo², Eppy³, R Sinto¹

¹Division of Tropical and Infectious Diseases, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia;

²Department of Internal Medicine, Gatot Subroto Indonesia Central Army Hospital, Jakarta, Indonesia; ³Department of Internal Medicine, Persahabatan Hospital, Jakarta, Indonesia

*Critical Care* 2014, **18**(Suppl 2):P46; doi:10.1186/cc14049

**Introduction:** Sepsis occurs as a result of systemic inflammatory process. The release of bacterial components to the systemic circulation leads to activation of inhibitor kappa B kinase (IKK)-β and nuclear factor kappa B (NF-κB) through phosphorylation and ubiquitination. Excessive inflammatory process with maladaptive host's immune response leads to organ dysfunctions and death [1,2]. This study was designed to investigate association of the initial level of intracellular signalling pathway and cytokine involved in sepsis pathophysiology (that is, IKK-β, NF-κB, tumour necrosis factor (TNF)α) and 72-hour (early) mortality in severe sepsis patients.

**Methods:** A prospective cohort study was conducted in severe sepsis patients (aged 18 years and older) admitted to the Emergency Unit of Cipto Mangunkusumo Hospital, Persahabatan Hospital, and Gatot Subroto Indonesia Central Army Hospital, Jakarta, Indonesia. All blood samples for intracellular signalling pathway (that is, IKK-β, total NF-κB, phospho NF-κB) and cytokine (that is, TNFα) were collected and measured using the ELISA method during first 24 hours of inclusion. Patients' outcome was observed during first 72 hours after inclusion. Mann-Whitney test was

<table><caption>**Table 1(abstract P46) Baseline characteristics of the subjects**</caption><thead><tr><th>Characteristic</th><th>Survival subjects (n = 63)</th><th>Death subjects (n = 27)</th></tr></thead><tbody><tr><td>Mean age (years)</td><td>52.98 ± 15.38</td><td>50.19 ± 13.46</td></tr><tr><td>Sex</td><td></td><td></td></tr><tr><td>Male</td><td>23 (36.5)</td><td>12 (44.4)</td></tr><tr><td>Female</td><td>40 (63.5)</td><td>15 (55.6)</td></tr><tr><td>Sepsis severity</td><td></td><td></td></tr><tr><td>Severe sepsis</td><td>42 (66.7)</td><td>13 (48.1)</td></tr><tr><td>Septic shock</td><td>21 (33.3)</td><td>14 (51.9)</td></tr><tr><td>Mean APACHE II score</td><td>13.03 ± 5.27</td><td>14.37 ± 6.55</td></tr><tr><td>Comorbidity</td><td></td><td></td></tr><tr><td>Without comorbidity</td><td>4 (6.3)</td><td>2 (7.4)</td></tr><tr><td>With comorbidity(s)</td><td>59 (93.7)</td><td>25 (92.6)</td></tr><tr><td>Source of infection</td><td></td><td></td></tr><tr><td>Respiratory tract</td><td>45 (71.4)</td><td>24 (88.9)</td></tr><tr><td>Intraabdominal</td><td>4 (6.3)</td><td>1 (3.7)</td></tr><tr><td>Skin and soft tissue</td><td>5 (7.9)</td><td>1 (3.7)</td></tr><tr><td>Urinary tract</td><td>2 (3.1)</td><td>1 (3.7)</td></tr><tr><td>≥ 2 sources</td><td>7 (11.3)</td><td>0</td></tr><tr><td>Mean procalcitonin (pg/ml)</td><td>21.47 ± 44.30</td><td>30.26 ± 17.29</td></tr><tr><td>Mean baseline lactate (mmol/l)</td><td>3.10 ± 1.77</td><td>4.89 ± 2.57</td></tr></tbody></table>

Data presented as n (%) or mean ± standard deviation. APACHE, Acute physiology and chronic health evaluation

<table><caption>**Table 2(abstract P46) Initial intracellular signalling pathway and cytokine level based on 72-hour survival**</caption><thead><tr><th>Parameter</th><th>Survived subjects (n = 63)</th><th>Death subjects (n = 27)</th><th>P value</th></tr></thead><tbody><tr><td>IKK-β<sup>a</sup></td><td>0.10 (0.03)</td><td>0.12 (0.03)</td><td>0.025</td></tr><tr><td>Total NF-κB<sup>a</sup></td><td>0.08 (0.08)</td><td>0.12 (0.08)</td><td>0.036</td></tr><tr><td>Phospho NF-κB<sup>a</sup></td><td>0.10 (0.38)</td><td>0.33 (0.38)</td><td>0.579</td></tr><tr><td>TNFα<sup>b</sup></td><td>9.10 (7.80)</td><td>10.91 (11.74)</td><td>0.393</td></tr></tbody></table>

<sup>a</sup>Median (interquartile range) in optical density, Mann-Whitney test. <sup>b</sup>Mean (standard deviation) in pg/ml, t test

used to analyse the median difference of IKK-β, total NF-κB, phospho NF-κB level in two groups based on 72-hour survival. The t test was used to analyse the mean difference of TNFα levels in both groups.

**Results:** Subjects consisted of 90 patients. Early mortality developed in 27 subjects. Baseline characteristics of survival and death subjects are shown in Table 1. The initial intracellular signalling pathway and cytokine parameters level are shown in Table 2. There was a significant higher median first 24 hours IKK-β and total NF-κB level in survival subjects compared with death subjects (P = 0.025 for median IKK-β and P = 0.036 for median total NF-κB). There was no significant difference of initial phospho NF-κB and TNFα level in survival and death subjects.

**Conclusion:** There is a significant lower initial IKK-β and total NF-κB level in severe sepsis patients surviving on 72-hour observation. There is a tendency of lower initial phospho NF-κB and TNFα level in severe sepsis patients surviving on 72-hour observation.

References

1. Coldewey SM, Rogazzo M, Collino M, Patel NSA, Thiemermann C: Inhibition of IkB kinase reduces the multiple organ dysfunction caused by sepsis in the mouse. *Dis Mode Mech* 2013, 6:1031-1042.

2. Liu SF, Malik AB: NFκB activation as a pathological mechanism of septic shock and inflammation. *Am J Physiol Lung Cell Mol Physiol* 2006, 290: L622-L645.

P47

**Sepsis and neutropenia in hematological malignancies.**

Y Labiad¹*, L Farnault², B Loriod¹, R Costello¹,², C Nguyen¹

¹Technological Advances for Genomics and Clinics U1090, INSERM, Marseille, France; ²Service d'hématologie de La Conception, Assistance publique - Hôpitaux de Marseille, France

*Critical Care* 2014, **18**(Suppl 2):P47; doi:10.1186/cc14050

**Introduction:** The pre-existing inflammatory state influences the behavior of infection, a first peak of inflammation may be the risk factor during infection and predispose to a severe form of the disease [1,2]. This hypothesis is frequently accepted but has never been tested. Patients with hematological malignancies treated by chemotherapy in case of autologous stem cell transplantation (ASCT) are exposed to a neutropenic state: an abnormal low number of neutrophils. In the neutropenic patient, sepsis is more frequent than in the non-neutropenic patient. The severity of infection depends on many factors, particularly the pre-existing inflammatory state, the genetic factors, and the duration and the depth of neutropenia. In the literature, the factors involved in inflammation and infection are often confused. On the other hand, no predictive transcriptomic signature of sepsis was found. Our objective is to identify predictive genes that influence the outcomes of the infection in neutropenic patients.

**Methods:** To test our hypothesis, high-throughput transcriptomic analysis and bioinformatics will be combined, laying on the modulation of gene expression of the peripheral blood mononuclear cells of patients with hematological malignancies treated by chemotherapy in case of ASCT.

**Results:** A preliminary study on a reduced number of patients has allowed us to assess the feasibility of the project. After a statistical analysis, 615 genes are differently expressed between patients who developed sepsis and those who have not developed with a false discovery rate of 5%. These 615 genes are potentially predictive of sepsis 2 days before the development of the infection. Furthermore, 28 genes are differentially expressed between patients who developed sepsis and those who have
not developed with a false discovery rate of 5%, and these 28 genes are potentially predictive of sepsis 7 days before the development of the infection.

**Conclusion:** The ongoing transcriptomic study with a larger effect will allow us to test our hypothesis and confirm the results of our preliminary study. The microarray results should be strengthened with quantitative PCR assays, than validated with protein assays. Finally, we will transfer the experimental data to clinical practices.

P48

**Histopathological changes in septic acute kidney injury in critically ill children: an observational analytical study of postmortem renal biopsies.**

R Rameshkumar¹*, S Mahadevan¹, RN Ganesh², P Narayanan¹, V Bhat¹

¹Department of Pediatrics, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India; ²Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India

*Critical Care* 2014, **18**(Suppl 2):P48; doi:10.1186/cc14051

**Introduction:** Critically ill children often manifest acute kidney injury (AKI) as part of their disease process with incidence up to 82% and it is independently associated with poor outcome [1]. Sepsis is the most common contributing factor and little is known about the pathogenesis of septic AKI. There are no consistent histopathological changes in human or experimental septic AKI [2]. Hence, understanding of the structural changes associated with its occurrence is therefore important in the management of AKI in critically ill children.

**Methods:** Children aged less than 12 years who died with septic AKI were screened for percutaneous USG-guided kidney biopsy after obtaining written informed consent from July 2012 to June 2014. Three cores of kidney tissue were taken for histopathological evaluation by light, immunofluorescence and electron microscopic examination. Sepsis and AKI was defined using international pediatric sepsis consensus conference and pRIFLE criteria respectively. Events related to death, laboratory parameters and microbiological details 24 hours preceding death were recorded.

**Results:** A total of 40 kidney biopsies were done during the study period. Median (IQR) age was 12 (2 to 36) months and PRISM-III was 14 (12 to 18). The most common change was normal histology in 37.5% (n = 15) followed by tubular change alone in 22% (n = 10), glomerular change alone in 5% (n = 2) and blood vessel change in 2.5% (n = 1) of specimens. Twelve specimens were showing a combination of changes (tubular + glomerular + interstitium = 5; tubular + glomerular + blood vessels = 3; tubular + glomerular = 2; tubular + interstitium = 1; tubular + glomerular + interstitium + blood vessels = 1). All tubular changes were consistent with acute tubular necrosis (ATN) and changes involved from 5 to 15% of cortex. Thrombotic microangiopathy was diagnosed in 12.5% (n = 5) of specimens. There were no significant difference between ATN versus non-ATN groups with respect to baseline characteristics and events related to mortality.

**Conclusion:** The most common change in septic AKI in critically ill children is normal histology followed by ATN. This is consistent with published literature [2].

**References**

1. Fortenberg JD, Paden ML, Goldstein SL: Acute kidney injury in children: an update on diagnosis and treatment. *Pediatr Clin North Am* 2013, 60:669-688.

2. Langenberg C, Bagshaw SM, May CN, Bellomo R: The histopathology of septic acute kidney injury: a systematic review. *Crit Care* 2008, 12:R38.

P49

**Impact before and after introduction of a multifaceted quality improvement intervention on device-related infections in a pediatric ICU in India: a single-centre experience.**

R Rameshkumar¹*, A Jagadeesh¹, M Kedarnath¹, S Mahadevan¹, P Narayanan¹, KN Harikrishnan¹, S Sistla²

¹Department of Pediatrics, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India; ²Department of Microbiology,

Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India

*Critical Care* 2014, **18**(Suppl 2):P49; doi:10.1186/cc14052

**Introduction:** Healthcare-associated infections (HAI) are a significant problem in the pediatric intensive care unit (PICU). Apart from contributing to mortality, they also increase the coast of PICU care [1]. Surveillance and prevention of HAI by multifaceted quality improvement intervention among critically ill children remain part of the standard of care [2].

**Methods:** The study was conducted in the 19-bed PICU of a tertiary care referral academic institute. Data regarding ventilator-associated pneumonia (VAP) and central line associated bloodstream infection (CLABSI) using the CDC definition were collected prospectively from July 2013 to June 2014. Multifaceted quality improvement intervention consisting of infection control nurse and physician and hand hygiene education module and wearing a gown and mask during the care of critically ill children was introduced from January 2014. Incidence of VAP and CLABSI was compared before (July to December 2013) and after (January to June 2014) introduction of the intervention.

**Results:** Before the intervention period, the incidence of VAP was 28.5 per 1,000 ventilation-days and CLABSI was 13.7 per 1,000 catheter-days. After the intervention period the incidence of VAP was 13.3 per 1,000 ventilation-days and CLABSI was 8.3 per 1,000 catheter-days. The proportion of patients ventilated for more than 48 hours who had VAP was significantly less after intervention as compared to before the intervention period (14.2%, n = 25/176 vs. 25.2%, n = 29/155; P = 0.012, odds ratio (OR), 95% CI 0.49, 0.28 to 0.86). Both groups were similar with respect to age, sex ratio, severity (PRISM-III), device utilization rate and grade of infection. No significant difference occurred in overall PICU mortality before and after intervention (28.2% vs. 28.9%).

**Conclusion:** Multifaceted quality improvement intervention results in significant reduction of the healthcare-associated infection rate although it was higher than reported from developed countries [1-3].

**References**

1. Muszynski JA, Sartori J, Steele L, Frost R, Wang W, Khan N, et al: Multidisciplinary quality improvement initiative to reduce ventilator-associated tracheobronchitis in the PICU. *Pediatr Crit Care Med* 2013, 14:533-538.

2. Esteban E, Ferrer R, Urrea M, Suarez D, Rozas L, Balaguer M, et al: The impact of a quality improvement intervention to reduce nosocomial infections in a PICU. *Pediatr Crit Care Med* 2013, 14:525-532.

3. Flodgren G, Conterno LO, Mayhew A, Omar O, Pereira CR, Shepperd S: Interventions to improve professional adherence to guidelines for prevention of device-related infections. *Cochrane Database Syst Rev* 2013, 3:CD006559.

P50

**Diagnostic and prognostic evaluation of soluble CD14 subtype for sepsis in critically ill patients: a preliminary study.**

A Foca*, C Peronace, V Marano, GS Barreca, AG Lamberti, N Marascio, A Giancotti, R Puccio, MC Liberto

Department of Health Sciences, Institute of Microbiology, University 'Magna Graecia' of Catanzaro, Italy

*Critical Care* 2014, **18**(Suppl 2):P50; doi:10.1186/cc14053

**Introduction:** Sepsis, a leading cause of death in critical care patients, is the result of complex interactions between the infecting microorganisms and the host responses that influence clinical outcomes [1]. Reliable biochemical markers that enable early diagnosis are still needed. A new marker, presepsin (soluble CD14 subtype, sCD14-ST) is a circulating fragment of 13 kDa of soluble CD14 that originates from the cleavage of the CD14 expressed on the surface membranes of monocytes/macrophages. sCD14-ST has been shown to increase significantly in patients with sepsis [2]. In our study we compare diagnostic performance of presepsin, C-reactive protein (CRP) and procalcitonin (PCT).

**Methods:** Critical patients with suspected sepsis admitted to the Unit of Intensive Care of the University Hospital of Catanzaro (Italy) were recruited into this study; healthy volunteers were also included as controls. Plasma samples in EDTA from each patient were collected at multiple time points; samples were tested for CRP, PCT and presepsin.