Blood cultures were also evaluated and processed by a BacT/Alert 3D system (bioMérieux, Italy); CRP was measured by immunonephelometry (Siemens Healthcare Diagnostic, Italy) and PCT was assayed by an enzyme-linked fluorescent assay (VIDAS BRAHMS PCT, bioMérieux, Italy); presepsin levels were measured by rapid automated PATHFAST immunoanalyzer (kindly provided by GEPA SRL, Italy), based on chemiluminescent enzyme immunoassay. A statistical analysis was carried out by Mann-Whitney test.

**Results:** Presepsin and PCT levels were significantly higher in culture-positive subjects versus negative controls; such difference was found even at the admission time. The presepsin values in worsening/dead patients exhibited a significantly higher level at admission time. On the contrary, in the same group of patients, PCT exhibited a decrease of its level. In poor prognosis patients CRP showed a quite irregular kinetic, although in such a group the admission value was higher than the same marker in live subjects.

**Conclusion:** In this preliminary study, presepsin and PCT levels exhibited substantial higher values in culture-positive patients. The kinetic curves of presepsin, obtained from both survival and worsening/dead subjects, revealed the optimal performance of this biomarker, particularly in severely ill patients, as also shown in other studies. During sepsis, increase of presepsin levels may be a more reliable marker, indicating an unfavorable outcome [3,4]. Furthermore, high presepsin levels could alert clinicians not to suspend antibiotic treatments even after clinical symptoms have improved and PCT levels have returned to normal.

### References

1. Masson S, Caironi P, Spanuth E, Thomae R, Panigada M, Sangiorgi G, Fumagalli R, Mauri T, Isgrò S, Fanizza C, et al: Presepsin (soluble CD14 subtype) and procalcitonin levels for mortality prediction in sepsis: data from the Albumin Italian Outcome Sepsis trial. *Crit Care* 2014, 18:R6.

2. Yaegashi Y, Shirakawa K, Sato N, Suzuki Y, Kojika M, Imai S, Takahashi G, Miyata M, Furusako S, Endo S: Evaluation of a newly identified soluble CD14 subtype as a marker for sepsis. *J Infect Chemother* 2005, 11:234-238.

3. Ulla M, Pizzolato E, Lucchiari M, Loiacono M, Soardo F, Forno D, Morello F, Lupia E, Moiraghi C, Mengozzi G, et al: Diagnostic and prognostic value of presepsin in the management of sepsis in the emergency department: a multicenter prospective study. *Crit Care* 2013, 17:R168.

4. Endo S, Suzuki Y, Takahashi G, Shozushima T, Ishikura H, Murai A, Nishida T, Irie Y, Miura M, Iguchi H, et al: Presepsin as a powerful monitoring tool for the prognosis and treatment of sepsis: a multicenter prospective study. *J Infect Chemother* 2014, 20:30-34.

which produce less S1P, and wildtype mice treated with a SPHK1 inhibitor were used to characterize whether T cells in these mice have a higher rate of emigration compared to control mice. Thymic and serum S1P levels were quantified by LC-MS/MS. Apoptosis was determined by annexin V FACS staining. SPHK mRNA levels were determined by qPCR.

**Results:** The thymus of septic mice showed more CD3-positive cells but a decreased number of CD4/CD8 double-positive T cells, pointing to a thymic retention of single-positive mature T cells. In line with our assumption, CLP causes apoptosis of thymocytes and increases SPHK1 mRNA expression. Concomitantly S1P levels are increased in the thymus and consequently decreased in serum following CLP. The knockout of the S1P producing sphingosine-kinases SPHK1 or SPHK2 and the pharmacological inhibition of SPHK1 restores T-cell egress as indicated by an increase of double-positive immature T cells compared to wildtype mice.

**Conclusion:** Our data suggest that inhibition of SPHK1-mediated S1P generation during sepsis restores thymic T-cell egress, which might improve septic outcome. Therefore, understanding mechanisms of thymus involution during sepsis may help to reconstitute thymic function, finally improving immune reactions in the hypoinflammatory phase of sepsis.

### References

1. Soller M, Tautenhahn A, Brüne B, Zacharowski K, John S, Link H, von Knethen A: Peroxisome proliferator-activated receptor gamma contributes to T lymphocyte apoptosis during sepsis. *J Leuk Biol* 2006, 79:235-243.

2. Schmidt MV, Paulus P, Kuhn A-M, Weigert A, Morbitzer V, Zacharowski K, Kempf VAJ, Brüne B, von Knethen A: Peroxisome proliferator-activated receptor gamma-induced T cell apoptosis reduces survival during polymicrobial sepsis. *Am J Respir Crit Care Med* 2011, 184:64-74.

3. Verinaud V, de Souza Souto P, Niéto Brito V: Thymic atrophy in infectious diseases. *Braz J Morphol Sci* 2004, 21:111-116.

4. Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG: Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. *Nature* 2004, 427:355-360.

5. Spiegel S, Milstien S: Sphingosine-1-phosphate: an enigmatic signalling lipid. *Nat Rev Mol Cell Biol* 2003, 4:397-407.

6. Weigert A, Johann AM, von Knethen A, Schmidt H, Geisslinger G, Brüne B: Apoptotic cells promote macrophage survival by releasing the antiapoptotic mediator sphingosine-1-phosphate. *Blood* 2006, 108:1635-1642.

## P51

### Sphingosine-1 phosphate promotes thymic atrophy during sepsis progression.

L Kuchler¹*, LK Sha¹, AK Giegerich¹, T Knape², A Weigert¹, B Brüne¹, A von Knethen¹

¹Faculty of Medicine, Institute of Biochemistry I - Pathobiochemistry, Goethe-University Frankfurt, Germany; ²Project Group Translational Medicine & Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Frankfurt, Germany

*Critical Care* 2014, **18**(Suppl 2):P51; doi:10.1186/cc14054

**Introduction:** T-cell depletion is a marker of the hypo-inflammatory phase of sepsis [1,2]. Systemic T-cell loss could be caused i.a. by thymus involution during sepsis, thus contributing to immune paralysis [3]. Usually, thymic T-cell egress is mediated by a sphingosine-1-phosphate (S1P) gradient. That is, T cells leave the thymus towards an increased S1P-level in the periphery (blood/lymph) [4]. A disruption of this S1P gradient blocks T-cell emigration. S1P is produced by sphingosine kinases 1 and 2 (SPHK1 and SPHK2), which are ubiquitously expressed [5]. Apoptotic cells are known to release S1P [6]. We suggest that apoptotic events in the thymus during sepsis increase S1P levels, thereby disrupting the gradient and preventing egress of T cells from the thymus.

**Methods:** We used a murine polymicrobial sepsis model to analyze thymus involution. Sepsis is induced in wildtype mice, SPHK1⁻/⁻ or SPHK2⁻/⁻ mice by cecal ligation and puncture (CLP). Thymus involution was determined by analyzing the T-cell amount of single-positive mature (CD4⁺CD8⁻ vs. CD4⁻CD8⁺), double-positive late immature (CD4⁺/CD8⁺), and double-negative early immature cells by FACS analysis. T cells from SPHK1⁻/⁻ or SPHK2⁻/⁻ mice,

## P52

### Activation of the peroxisome proliferator activated receptor γ counteracts sepsis-induced T-cell cytotoxicity towards alloantigenic target cells.

A von Knethen¹*, LK Sha¹, T Knape², L Kuchler¹, AK Giegerich¹, M Schulz¹, IA Hauser³, B Brüne¹

¹Institute of Biochemistry I - Pathobiochemistry, Faculty of Medicine, Goethe-University Frankfurt, Germany; ²Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine & Pharmacology TMP, Frankfurt am Main, Germany; ³Department of Nephrology, Medical Clinic III, Frankfurt am Main, Germany
*Critical Care* 2014, **18**(Suppl 2):P52; doi:10.1186/cc14055

**Introduction:** Sepsis originates from an uncontrolled inflammatory response. Despite intensive research, sepsis remains a major cause of death in ICUs. Therefore, new therapeutic approaches are mandatory. Taking into account that during sepsis progression cytotoxic T cells (CTL) are activated in an autoimmune fashion contributing to multiorgan damage, it remains unclear whether CTL are activated towards alloantigenic cells as well. This is especially important for patients receiving an immune suppressive therapy to permit organ transplantation and thus known to be at high risk for developing sepsis. Therefore, we analyzed whether sepsis activates CTL towards alloantigenic target cells and whether this can be inhibited by PPARγ activation, known to block T-helper cell responses.

**Methods:** To characterize whether sepsis activates CTL and whether this can be inhibited by PPARγ activation, we used an *ex vivo* cytotoxicity assay to analyze CD8⁺ T-cell-dependent cytotoxicity. Responder CD8⁺ T cells were isolated from C57Bl/6N PPARγ wildtype (PPARγfl/fl) and T-cell specific knockout (Tc-PPARγ⁻/⁻) mice (haplotype H2Kb) following cecal ligation and
puncture (CLP) versus sham treatment. P815 mastocytoma cells, a cell line originally derived from DBA/2 mice (haplotype H2Kd), were used as alloantigenic target cells. Pharmacological inhibition and/or activation of PPARγ in vivo and ex vivo was performed to clarify the impact of PPARγ in blocking CTL-dependent cytotoxicity. In vivo, PPARγ activity in wildtype mice was pharmacologically inhibited by the irreversible antagonist GW9662 or induced by the thiazolidinedione rosiglitazone. Systemic application of both compounds was performed intraperitoneally. A classic splenocyte-driven stimulation protocol to activate CTL was carried out as control.

**Results:** CTL isolated from septic mice showed enhanced cytotoxicity towards alloantigenic P815 target cells. Enhanced cytotoxicity was effectively reduced by both PPARγ activation in vivo and ex vivo. In line, in CTL isolated from T-cell-specific PPARγ knockout (TC-PPARγ⁻/⁻) mice PPARγ activation was ineffective, strengthening a PPARγ-dependent mechanism. At the molecular level in vivo and ex vivo activation of PPARγ reduced Fas and granzyme B expression in activated CTL, which might explain reduced cytotoxicity.

**Conclusion:** Our study therefore suggests PPARγ activation in vivo to attenuate CTL-dependent alloantigenic cytotoxicity to possibly inhibit acute organ rejection.

P53

**Using process mapping to identify barriers to effective management of sepsis in a cancer hospital: lessons for successful implementation of a whole hospital pathway.**

K Thursky¹, G Haeusler¹, B Teh¹, D Comodo², N Dean², C Brown², J Willis², K Morris², S Lingaratnam³, M Slavin¹, L Worth¹

¹Department of Infectious Diseases, Peter MacCallum Cancer Centre, Melbourne, Australia; ²Nursing, Peter MacCallum Cancer Centre, Melbourne, Australia; ³Pharmacy Department, Peter MacCallum Cancer Centre, Melbourne, Australia

Critical Care 2014, **18**(Suppl 2):P53; doi:10.1186/cc14056

**Introduction:** Infection and sepsis are common problems in cancer management affecting up to 45% of patients. However, international guidelines focus on the management of neutropenic fever, and fail to address the recognition and resuscitation of patients who meet sepsis criteria. Peter MacCallum Hospital is a 100 inpatient-bed tertiary cancer hospital with hematology, medical oncology, cancer surgery and radiation oncology, as well as a medical and chemotherapy day unit, apheresis, and large ambulatory service but no emergency department. Records showed that up to 25% of all in-hospital Medical Emergency Team (MET) calls were attributable to sepsis with in-hospital mortality rates of up to 25%. We aimed to identify barriers to effective management of inpatient sepsis at Peter MacCallum Cancer Centre and to implement a hospital-wide sepsis pathway.

**Methods:** A sepsis working party was formed with the antimicrobial stewardship team, clinicians, and senior ambulatory and inpatient nurses. Each member undertook direct observation and focus group interviews in an allocated clinical area. Three key areas were examined: issues relating the identification of sepsis, issues relating to clinical review of the patient, and issues relating to timely administration of first dose of antibiotic. Inpatient and outpatient issues were graphically represented in a process map (Figure 1).

**Results:** Process mapping revealed significant gaps in knowledge in medical and nursing staff and structural barriers to rapid resuscitation of patients. There were significant knowledge gaps in the awareness of sepsis diagnostic criteria, the role of lactate, effective fluid resuscitation, and the need for early clinical review and referral to the ICU. Examples of structural barriers to effective resuscitation included lack of availability of nurse cannulators, availability of antibiotics, rapid intravenous fluid infusers and sufficient after-hours medical and ICU liaison support. Knowledge and structural barriers were systematically addressed during the implementation of the clinical sepsis pathway. The sepsis pathway was designed as a medical record form to be used across all clinical areas, and supports nurse initiation. Following pathway implementation in March 2013 there have been substantial improvements in the number of patients who have had a lactate taken, received appropriate fluid resuscitation and time to first dose of antibiotics. Administrative data shown in Figure 2 demonstrate increased ascertainment of cases, and a fall in all-cause sepsis mortality after the pathway was commenced.

<figure><img src="image_38.png" /><figcaption>Figure 1(abstract P53) Portion of process map identifying barriers to timely administration of antibiotics in the ambulatory setting.</figcaption></figure>

RTx, radiotherapy department.

**Conclusion:** Identifying knowledge gaps and structural barriers using process mapping led to the successful design and implementation of a sepsis program. The figures show that despite increased rates of coded sepsis cases, mortality and ICU admission rates have not increased. Mortality
<figure><img src="image_39.png" /><figcaption>Figure 2(abstract P53) Increased rate of sepsis at Peter MacCallum Cancer Centre from March 2013, coinciding with improved identification of patients with sepsis.</figcaption></figure>

of patients coded for sepsis March to October 2012 (13/56 (23.2%)) compared with those for March to October 2013 (10/121 (8.3%)).

P54

Dopamine mediates vagal modulation of the immune system by electroacupuncture.

R Torres Rosas¹,², P Morcillo², L Ulloa²,³

¹Medical Research Unit on Immunochemistry, National Medical Center Siglo XXI, Mexico City, Mexico; ²Laboratory of Anti-inflammatory Signaling, Department of Surgery, Rutgers University New Jersey Medical School, Newark, NJ, USA; ³Center of Immunology and Inflammation, Rutgers University New Jersey Medical School, Newark, NJ, USA
*Critical Care* 2014, **18**(Suppl 2):P54; doi:10.1186/cc14057

**Introduction:** Previous anti-inflammatory strategies against sepsis, a leading cause of death in hospitals, had limited efficacy in clinical trials, in part because they targeted single cytokines and the experimental models failed to mimic clinical settings [1-3]. Neuronal networks represent physiological mechanisms, selected by evolution to control inflammation, that can be exploited for the treatment of inflammatory and infectious disorders [3].

**Methods:** Animal procedures were approved by the Institutional Animal Care & Use Committee of the New Jersey Medical School of Rutgers University. All animal experiments were performed in 6-week-old to 8-week-old (~25 ± 5 g) male mice without any exclusion criteria. Experimental sepsis: endotoxemia and CLP were performed as we previously described. LPS was dissolved in sterile pyrogen-free PBS and sonicated for 30 minutes immediately before use. Mice received a LD₅₀ dose of LPS (6 mg/kg body weight i.p.). LPS was added to the whole blood to a final concentration of 250 ng/ml for the *in vitro* procedures. Selective neurectomies and electrical stimulations: all selective neurectomies and electrical stimulations were performed in mice anesthetized with ketamine and xylazine. The electrical

stimulation in electroacupuncture and direct nerve stimulation (sciatic and vagus nerves) was performed with a continuous-mode stimulation for 15 minutes with an electrical potential difference of 4 V, an electric current of 40 mA, a pulse width of 50 µs and a frequency of 10 Hz using an electrostimulator.

**Results:** Here, we report that sciatic nerve activation with electroacupuncture controls systemic inflammation and rescues mice from polymicrobial peritonitis. Electroacupuncture at the sciatic nerve controls systemic inflammation by inducing vagal activation of aromatic L-amino acid decarboxylase, leading to the production of dopamine in the adrenal medulla. Experimental models with adrenolectomized mice mimic clinical adrenal insufficiency [4], increase the susceptibility to sepsis and prevent the anti-inflammatory effects of electroacupuncture. Dopamine inhibits cytokine production via dopamine type 1 (D1) receptors. D1 receptor agonists suppress systemic inflammation and rescue mice with adrenal insufficiency from polymicrobial peritonitis. Our results suggest a new anti-inflammatory mechanism mediated by the sciatic and vagus nerves that modulates the production of catecholamines in the adrenal glands.

**Conclusion:** From a pharmacological perspective, the effects of selective dopamine agonists mimic the anti-inflammatory effects of electroacupuncture and can provide therapeutic advantages to control inflammation in infectious and inflammatory disorders. Preliminary results in human clinical trials indicate that electroacupuncture attenuates the postsurgical inflammatory response decreasing the serum levels of inflammatory cytokines.

References

1. Angus DC, van der Poll T: Severe sepsis and septic shock. *N Engl J Med* 2013, **369**:840-851.

2. Ulloa L, Tracey KJ: The 'cytokine profile': a code for sepsis. *Trends Mol Med* 2005, **11**:56-63.

3. Ulloa L: The vagus nerve and the nicotinic anti-inflammatory pathway. *Nat Rev Drug Discov* 2005, **4**:673-684.

4. Annane D: Adrenal insufficiency in sepsis. *Curr Pharm Des* 2008, **14**:1882-1886.
P55

Immunomodulation and infection: identification of small molecule TLR3 blockers to combat deleterious inflammation in pneumonia.

F Sónego¹*, B Pouzet¹, F Pecceu², L Leotard¹, B Martin¹, P-E Rouby¹, L Gouillieux¹, C Ravinet-Trillou¹, J-M Bras³, C Serradeil-Le Gal¹
¹Sanofi TSU ID, Toulouse, France; ²SCP LGCR, Toulouse, France; ³Biologics, Toulouse, France
*Critical Care* 2014, **18**(Suppl 2):P55; doi:10.1186/cc14058

**Introduction:** Pneumonia is a common cause of death worldwide and the leading cause of sepsis and shock in the ICU. Controlling excessive immune stimulation with deleterious consequences for the host organs is a major strategy in severe infections. In addition to viral dsRNA, the immune Toll-like receptor 3 (TLR3) senses RNA released from injured tissues and necrotic cells promoting excessive inflammatory cytokine release in pulmonary epithelial, endothelial and immune cells. Thus, as a potent regulator of the lung immune response, TLR3 represents a specific target to control damaging inflammation in severe lung infections in combination with antimicrobials. Here, we aimed to characterize potent small molecules displaying *in vitro* and *in vivo* TLR3 blocker activities and develop several preclinical models to investigate the role of TLR3 in pneumonia.

**Results:** We identified compounds with anti-TLR3 activity (IC₅₀ = 50 nM) through random screening and structure-activity relationship analysis by testing small molecule libraries on recombinant hTLR3-HEK293 cells. Selected compounds interacted with mouse and human TLR3, were devoid of effect on TNFα-induced activation at 1 μM on HEK293 cells and exhibited good early-ADME properties. Hit molecules also counteracted PolyAU (PAU)/Poly(I:C) (PIC)-induced cytokine release (IL6, IL8, IP-10) on human bronchial epithelial cells (BEAS-2B) expressing native TLR3 receptors. Interestingly, sustained stimulation of BEAS-2B by PAU/PIC (0.1 to 10 μM) to mimic viral activation induced overexpression of TLR3 mRNA, which was dose-dependently inhibited by these compounds, as evidenced by RT-qPCR. *In vivo*, we confirmed the anti-TLR3 activity (3 to 30 mg/kg i.p.) against PAU/PIC (100 μg/mouse i.v.) but not flagellin-induced plasma cytokine release in CD1 mice. To study these TLR3 inhibitors under pathological situations, we developed models of *Streptococcus pneumoniae* (ATCC6303) or *Pseudomonas aeruginosa* (PAO-1)-induced pneumonia post influenza A virus (IAV PR/8/34 H1N1) infection. Mice previously challenged with intranasal IAV showed enhanced susceptibility to lung bacterial infections with a dramatic mortality rate, whereas separately these pathogens are not lethal. In addition, IAV/S. pneumoniae co-infected mice showed increased lung and blood bacterial loads with severe inflammation signs evidenced by elevated systemic IL-6 and KC levels and important lung tissue damages. Currently, TLR3 blockers are ongoing evaluation in these models.

**Conclusion:** Immunomodulation and personalized treatments are becoming relevant approaches to manage severe infections. Here, we discovered the first small molecule probes targeting mouse and human TLR3. These compounds efficiently control TLR3-triggered proinflammatory response *in vitro* and *in vivo* and shape TLR3 overexpression in human lung epithelial cells. They represent valuable pharmacological tools to study the contribution of TLR3 in pneumonia and to propose new adjuvant immunotherapies.

**Acknowledgements:** The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007-2013/ under REA grant agreement number 316682

P56

Similarity of inflammatory response in epileptic seizures and sepsis: does the sensitivity to sepsis in epileptic patients increase?.

G Üzüm¹*, K Akgün-Dar², A Kandil², N Bahçekapılı¹, I Albeniz²

¹Department of Physiology, Medical Faculty of Istanbul, Istanbul University, Istanbul, Turkey; ²Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey
*Critical Care* 2014, **18**(Suppl 2):P56; doi:10.1186/cc14059

**Introduction:** It is known that the systemic response during sepsis is caused by proinflammatory mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα). The inflammatory response in sepsis

causes disorders in the brain in addition to multiorgan dysfunctions including the kidneys and liver [1]. Inducible nitric oxide synthase (iNOS) is induced in hepatocytes by sepsis and mediates hepatic injury [2]. Matrix metalloproteinases (MMPs) play an important role in the formation of sepsis and mediate inflammatory response and tissue damage [3]. On the other hand, there are some cases in which systemic inflammatory response occurs without the presence of infection such as epilepsy. Cytokines are well-known inflammatory mediators in the brain, and they increase following seizures. We previously demonstrated that pentyleneterazol (PTZ)-induced generalized epileptic seizures significantly increased inflammatory markers (TNFα, IL-1β, IL-6) in the brain and S100B in serum [4]. In this preliminary study, we aimed to investigate the MMP2, MMP9, NOS, and myeloperoxidase activity in the liver and kidney and levels of serum proinflammatory cytokines following PTZ-induced generalized clonic-tonic seizures.

**Methods:** Adult Sprague-Dawley rats were divided into two groups as Control and PTZ groups. The Control group was given saline and the PTZ group was given 80 mg/kg PTZ i.p. Two hours after seizures, the rats were decapitated and a cardiac blood sample was drawn, and liver and kidneys were removed. Proinflammatory markers (IL-1βN, TNF-α, IL-6) were investigated in serum by ELISA. eNOS, iNOS, MMP2, and MMP9 levels were analyzed immunohistochemically in the liver and kidney.

**Results:** Proinflammatory markers significantly increased in the serum of rats after PTZ-induced seizures (Table 1). iNOS reaction was markedly increased while eNOS reactions were decreased (Figure 1) in the liver of rats after PTZ-induced seizures. MMP2 in the central vein of the liver and connective tissue areas of liver and kidney tissues in the PTZ group were markedly increased (Figure 2). MMP9 immune reaction in the PTZ group slightly increased in the kidney and liver (Figure 3). MPO reactions, which are an indicator of inflammatory activity, were markedly increased in both tissues (Figure 4).

**Conclusion:** The first findings show that long-term generalized clonic-tonic seizures markedly increase markers that mediate inflammation (iNOS, especially MMP2, MMP9, MPO) in the liver and kidney such as sepsis. In addition, proinflammatory markers (TNFα, IL-1β, IL-6) were found significantly high in serum. Thus, it is concluded that it will be worthwhile to determine whether epileptic seizures cause sensitivity to sepsis.

References

1. Chen Q, Yu W, Shi J, Shen J, Gao T, Zhang J, Xi F, Li J, Li N: *J Inflamm* 2014, 11:18.

2. Zhang B, Crankshaw W, Nesemeier R, Patel J, Nweze I, Lakshmanan J, Harbrecht BG: *J Surg Res* 2014 in press.

3. Al-Azri AR, Gibson RJ, Bowen JM, Stringer AM, Keefe DM, Logan RM: *J Oral Pathol Med* 2014 in press.

4. Bahçekapılı N, Akgün-Dar K, Albeniz I, Kapucu A, Kandil A, Yağiz O, Uzüm G: *Int J Neurosci* 2014, **124**:762-770.

P57

Early phases of sepsis: effects of simvastatin on mitochondrial enzyme activities in kidney tissue in rats.

E Özkök¹*, H Yorulmaz², G Ateş³, İ Aydın⁴, AŞ Tamer³

¹Department of Neuroscience, The Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey; ²School of Health of Science, Halic University, Istanbul, Turkey; ³Department of Physiology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey; ⁴Department of Histology and Embryology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
*Critical Care* 2014, **18**(Suppl 2):P57; doi:10.1186/cc14060

**Introduction:** Acute kidney injury (AKI) is a frequent and serious complication of sepsis. Moreover, there is strong evidence that AKI in patients with severe sepsis is associated with a higher mortality rate. The

<table><caption>Table 1(abstract P56) Proinflammatory cytokines in control versus experimental groups</caption><thead><tr><td>pg/ml</td><td>Control</td><td>PTZ</td></tr></thead><tbody><tr><td>IL-1β</td><td>0.117 ± 0.042</td><td>0.775 ± 0.064</td></tr><tr><td>TNFα</td><td>0.062 ± 0.010</td><td>0.783 ± 0.044</td></tr><tr><td>IL-6</td><td>0.160 ± 0.012</td><td>0.0654 ± 0.026</td></tr></tbody></table>

<footnote>*P < 0.001, compared to control one-way ANOVA and Benferroni for post hoc were used. n = 7</footnote>
<figure><img src="image_40.png" /><figcaption>Figure 1(abstract P56) Immunohistochemical detection of iNOS and eNOS staining (arrows) in liver sections in control and experimental groups (bar: 50 µm)</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>Figure 2(abstract P56) Immunohistochemical detection of MMP2 staining (arrows) in liver (A) and kidney (B) sections in control and experimental groups. PT, proximal tubule; GL, glomerulus (bar: 50 µm)</figcaption></figure>
<figure><img src="image_42.png" /><figcaption>**Figure 3(abstract P56) Immunohistochemical detection of MMP9 staining (arrows) in liver (A) and kidney (B) sections in control and experimental groups.** PT, proximal tubule; GL, glomerulus (bar: 50 µm).</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>**Figure 4(abstract P56) Immunohistochemical detection of MPO staining (arrows) in liver (A) and kidney (B) sections in control and experimental groups.** PT, proximal tubule; GL, glomerulus (bar: 50 µm).</figcaption></figure>
devastating effects of Gram-negative sepsis are largely based on the effects of lipopolysaccharide (LPS), also known as endotoxin. Mitochondrial dysfunction has been suggested to contribute to the development of organ dysfunction and failure in sepsis. The mitochondrial electron transport chain consists of four complexes (CI to CIV) and its function can be assessed with different approaches. Statins, such as simvastatin and atorvastatin, are hypocholesterolemic drugs that possess pleiotropic effects, including antioxidant and anti-inflammatory properties, that are either dependent on or independent of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) inhibition. In addition, *in vitro* and *in vivo* studies have demonstrated that simvastatin has an anti-inflammatory effect in patients with predialytic chronic kidney disease, and may play an important role in counteracting the mechanisms involved in pathogenesis of inflammation. We aimed to investigate the effects of prior simvastatin on mitochondrial enzyme activities in kidney tissue of the early phase of sepsis.

**Methods:** We used male adult Wistar albino rats weighing 200 to 250 g in the experiments. The rats were divided into four groups, each composed of eight rats: control group, LPS group, Simvastatin group, Simvastatin + LPS group. Lipopolysaccharide (LPS) from *Escherichia coli* O127:B8 (Sigma, St. Louis, MO, USA) was injected intraperitoneally at a daily dose of 20 mg/kg, Simvastatin (20 mg/kg) was given p.o. via oral gavage for 5 days. In the Simvastatin + LPS-treated group, LPS was given 1.5 hours after the fifth dose of simvastatin. Mitochondrial electron transport chain enzymes citrate synthase, NADH-cytochrome c reductase (complex I + III), and NADH dehydrogenase (complex I) were measured kinetically in spectrophotometer from kidney tissue homogenate. The kidney tissue samples were fixed in 10% buffered formalin and embedded in paraffin for hematoxylin and eosin (H&E) staining. Data were expressed as mean ± standard deviation (SD) and analyzed during analysis of variance.

**Conclusion:** There were no changes in activities of citrate synthase, complex I, complex I + III in tissue homogenate (*P* >0.05) (Figures 1 to Figure 3). In the LPS + Simvastatin group, we found decreased activity of complex I compared with those of the LPS and Simvastatin groups (*P* = 0.05, *P* = 0.07, respectively). As a result of the light microscopic examination with H&E stained sections, we observed tubule lumens widened and partially damaged in the epithelium. In the Simvastatin group was seen partially widened tubular structure and even in some areas tubular structure was found the same as control sections. Moreover, the damage in tubular width and proximal epithelium was observed to continue (Figure 4).

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

References

1. Oppert M, Engel C, Brunkhorst FM, et al: Acute renal failure in patients with severe sepsis and septic shock - a significant independent risk factor for mortality: results from the German Prevalence Study. *Nephrol Dial Transplant* 2008, **23**:904-909.

2. Cohen J: The immunopathogenesis of sepsis. *Nature* 2002, **420**:885-891.

3. Crouser ED, Julian MW, Blaho DV, Pfeiffer DR: Endotoxin-induced mitochondrial damage correlates with impaired respiratory activity. *Crit Care Med* 2002, **30**:276-284.

<figure><img src="image_46.png" /></figure>
<figure><img src="image_47.png" /><figcaption>Figure 4(abstract P57) Hematoxylin and eosin staining in all experimental groups</figcaption></figure>

4. Jeger V, Djafarzadeh S, Jakob SM, Takala J: Mitochondrial function in sepsis. Eur J Clin Invest 2013, 43:532-542.

P58

Characterization of a murine model of septic cachexia.

D Restagno¹*, J-M Bonnet¹, K Mouchain², P Brune², C Paquet¹, T Delale³, V Louzier¹

¹VetAgro sup, campus vétérinaire, Marcy L'Etoile, France; ²Bertin Pharma, CEA Saclay, Gig sur Yvette, France; ³Alizé Pharma, Espace Européen, Ecully, France
*Critical Care* 2014, **18**(Suppl 2):P58; doi:10.1186/cc14061

**Introduction:** Muscular loss is a characteristic phenomenon induced by a massive inflammation as occurring in sepsis. Indeed, the early phase of sepsis is responsible for the expression of proinflammatory cytokines, namely TNFα and IL-6, known to be effectors of cachexia. Although cachexia is a morbidity factor in human, there is to date no animal model of septic cachexia. The goal of this study is to create and characterize a murine model of septic cachexia and evaluate endogenous ghrelin variations. Among the factors involved in both sepsis and muscular protection, current research highlights a potential role for ghrelin in muscle protection but there are conflicting data regarding its variations
during experimental sepsis. In this study we evaluate its two circulating forms (AG, acylated ghrelin and UAG, unacylated ghrelin).

**Methods:** Sixty male C57Bl6 mice (20 to 25 g, 6 to 8 weeks) were used for these experiments. Sepsis is induced by a mild CLP (survival ≈ 80%). After anesthesia, cecum is isolated, ligated at 1/3, and punctured twice (21G needle). A laparotomy is performed without ligation or puncture. Mice weight is followed every day, blood collections and hind limb muscles dissection are realized at 2 hours, 6 hours, 1, 2, 3, 5 and 13 days after CLP. Plasmatic cytokines (TNFα, IL-6) and ghrelin levels are evaluated via the Luminex technique and ELISA assays respectively and muscles are weighed.

**Results:** After surgery, weight loss in CLP mice significantly decreases from D1 to D11 with a peak at D3 (20% vs. 10% for the Sham). On D12, septic mice reach their original weight, which becomes identical to the Sham on D13. Despite a similar weight, CLP mice muscles are lighter. Circulating TNFα and IL-6 levels increase 2 hours after CLP and remain higher than for the Sham operated mice. Ghrelin concentrations, regardless of its form, increase after the surgery: AG rises on D1 and D2 while UAG increases earlier, from 2 to 24 hours.

**Conclusion:** We finalize a model of muscular and weight loss due to septic conditions. Wasting syndrome and Inflammation are two of the three conditions for cachexia. The next step is to determine proteolysis in our model to confirm that we performed a murine model of septic cachexia. The role of increased circulating levels of ghrelin in this model remains to be elucidated.

P59
Mice survival in a two-hit model of sepsis depends on intratracheal P. aeruginosa bacterial load.

D Restagno¹*, J-M Bonnet¹, A Kodjo¹, F Venet², C Paquet¹, L Freyburger¹, V Louzier¹

¹VetAgrO sup, campus vétérinaire, Marcy l'Etoile, France; ²Groupement Hospitalier Edouard Heriot, Laboratoire d'immunologie cellulaire, Lyon, France

Critical Care 2014, **18**(Suppl 2):P59; doi:10.1186/cc14062

**Introduction:** Sepsis is a systemic reaction in the presence of an infection characterized by an early systemic inflammatory response syndrome followed by a compensatory anti-inflammatory response syndrome. The first side of this typical immune response is responsible for widespread damage organ whereas the second part leads to an immunoparalysis increasing patients' susceptibility to secondary infections. The goal of this study is to finalize a murine model of sepsis to understand the physiopathology of sepsis and eventually test new therapeutic approaches.

**Methods:** Male C57Bl6 mice (20 to 25 g, 6 to 8 weeks) were used. Sepsis is induced by a mild CLP (survival ≈ 80%). Cecum is isolated, ligated (1/3), and punctured twice (21G needle). A laparotomy is performed without ligation or puncture. Blood is collected 2 hours, 6 hours, 1, 2, 3, 5 and 13 days after the CLP. Plasmatic cytokines (TNFα, IL-6, IL-10) are evaluated via the Luminex technique. Five days after CLP, splenocytes are collected and used for immunological assays. Mice are intratracheally instilled with *P. aeruginosa* 5 days after CLP (5 × 10⁶, 2 × 10⁷ and 10⁸ CFU) to evaluate survival.

**Results:** Circulating TNFα, IL-6 and IL-10 levels increase 2 hours after CLP and remain high until the end of the experiment, as compared to Sham operated mice. Five days after CLP, total T cells (T-CD3⁺) population and proliferation significantly decrease as compared to the nonseptic condition whereas the T-reg cell (T-CD4⁺/CD25⁺) population significantly increases whereas it remains low in the Sham operated animals. TNFα induction by LPS in cultured splenocytes increases both for CLP and Sham operated mice but remains lower in septic mice. Survival following a secondary infection decreases while the quantity of instilled *P. aeruginosa* increases. No mortality is observed for 5 × 10⁶ CFU, 50% of CLP die with 2 × 10⁷ CFU and 100% of mortality is observed for both CLP and Sham with 10⁸ CFU. Two days after instillation, *P. aeruginosa* is detected in the lungs of CLP and Sham but in the spleen and liver only for CLP mice.

**Conclusion:** This study demonstrates that immunosuppression following CLP increases mice susceptibility to secondary *P. aeruginosa* intratracheal infection. However, this susceptibility depends on the bacterial load.

P60
Tyrosine metabolism disorder and the potential capability of anaerobic microbiota to decrease the value of aromatic metabolites in critically ill patients.

N Beloborodova*, V Moroz, A Osipov, A Bedova, Y Sarshor, A Vlasenko, A Olenin

Negovsky Research Institute of General Reanimatology, Russian Academy of Medical Science, Moscow, Russia

Critical Care 2014, **18**(Suppl 2):P60; doi:10.1186/cc14063

**Introduction:** Metabolism of tyrosine can be switched in conditions of hemodynamic instability and tissue hypoperfusion. The violation of the oxygen-dependent metabolism of tyrosine must be accompanied by the activation of alternative pathways (Figure 1). Previously, we found a high level of content in the blood of aromatic intermediates p-hydroxyphenyllactic acid (p-HPLA) and p-hydroxyphenylacetic acid (p-HPAA) in patients with sepsis [1,2]. We assume that the anaerobic microbiota can take an important part in biodegradation of excess alternative metabolites of aromatic amino acids [3,4].

**Methods:** Serum samples were collected from critically ill patients (n = 65) with surgical diseases (n = 32), brain injury (n = 22), and lung diseases (n = 11). Patients were included in the study on the day of admission to the ICU. The median of age was 62 (IR 42 to 77) years, the APACHE II score was 17 (IR 11 to 29). The level of p-HPLA and p-HPAA were measured in serum using GC-FID. Anaerobic bacteria (Figure 2) were cultured in Shedler media, and the level of aromatic metabolites were measured before and after 48 hours of incubation using GC-MS. Data were compared by Mann-Whitney *U* test, *P* < 0.05 considered significant (IBM SPSS Statistics 22).

**Results:** In surviving patients (n = 24) the total level of p-HPLA and p-HPAA (4.47; 3.24 to 8.35 µM) was less (*P* < 0.001) than in patients who died (n = 41) (13.67; 5.78 to 52.26 µM). The severity of organ dysfunction on a SOFA scale correlates (*r*<sub>s</sub> = 0.7, *P* < 0.001) with the total level of the p-HPLA and p-HPAA. Also the total level of aromatic compounds correlates with lactate (*r*<sub>s</sub> = 0.6; *P* < 0.001), BE (*r*<sub>s</sub> = -0.5, *P* < 0.001) and perfusion blood pressure (*r*<sub>s</sub> = -0.5, *P* < 0.001). ROC analysis revealed that p-HPLA has the largest area under the curve (0.78; CI 0.67 to 0.90, *P* < 0.001). In experimental studies, anaerobic bacteria significantly reduced the level of p-HPAA and p-HPLA (Figure 2).

**Conclusion:** High level of p-HPLA and p-HPAA correlate with severity and mortality of patients. Hypoxia can be one of the leading mechanisms of tyrosine metabolism disorders in critically ill patients. *Bacteroides* spp. are able to consume p-HPLA and p-HPAA and consequently may be involved in the elimination of these intermediates from the human body mutually with endogenous mechanisms of detoxification.

**Acknowledgements:** This work was supported by the Russian Foundation for Basic Research (project number 1304-01758/13)

**References**

1. Khodakova A, Beloborodova N: Microbial metabolites in the blood of patients with sepsis. *Crit Care* 2007, 11(Suppl 4):P5.

2. Beloborodova NV, Khodakova AS, Olenin AJ: Are phenylcarboxylic acids really markers in severe sepsis? *Crit Care* 2009, 13:41.

3. Beloborodova NV, Khodakova AS, Bairamov IT, Olenin AYu: Microbial origin of phenylcarboxylic acids in the human body. *Biochemistry (Mosc)* 2009, 74:1350-1355.

4. Beloborodova NV: Integration of metabolism in man and his microbiome in critical conditions. *General Reanimatol* 2012, 8:42-54.

P61
RADICAL study: rapid diagnosis of suspected bloodstream infections from direct blood testing using PCR/ESI-MS.

D Brealey¹*, N Libert², J Pugin³, M O'Dwyer⁴, K Zacharowski⁵, M Mikaszewska-Sokolewicz⁶, MP Maureau⁷, DJ Ecker⁷, R Sampath⁷, M Singer¹, J-L Vincent⁸

¹University College Hospital, London, UK; ²Military Hospital du Val-de-Grâce, Paris, France; ³Hôpitaux Universitaires de Genève, Geneva, Switzerland; ⁴The Royal London Hospital Barts, London, UK; ⁵Universitätsklinikum Frankfurt, Germany; ⁶Child of Christ Hospital, Warsaw, Poland; ⁷Ibis Biosciences, Abbott,
<figure><img src="image_48.png" /></figure>

Carlsbad, CA, USA; ªErasme University Hospital, Université Libre de Bruxelles, Belgium, Belgium
*Critical Care* 2014, **18**(Suppl 2):P61; doi:10.1186/cc14064

**Introduction:** Survival from sepsis and bloodstream infections (BSI) often depends upon rapid identification of the infecting pathogen and expeditious antimicrobial therapy. We report findings from the final analysis of RADICAL, a multicenter observational study that compared results from direct blood specimen testing using PCR/ESI-MS to standard microbiology in critically ill patients.

**Methods:** Eight ICUs in six European countries participated. Patients with suspected infection plus ≥2 new onset SIRS criteria were enrolled and had an extra blood specimen taken for PCR/ESI-MS direct analysis. Results were compared to standard of care microbiology. All patients were followed up to 28 days or until inpatient death or discharge. A panel of three independent physicians reviewed a summary of each case to determine the potential impact upon patient management if results had been available for decision-making.

**Results:** A total of 609 direct blood specimens from 543 patients meeting inclusion criteria were tested. Patient demographics and organ failure criteria observed were consistent with previously published sepsis studies.
<figure><img src="image_49.png" /><figcaption>Figure 2(abstract P60)</figcaption></figure>

Culture/PCR comparisons were as follows: +/+ 54; +/- 13; -/+ 169; and -/393, respectively, for a sensitivity of 81%, specificity of 69%, PPV of 24% and NPV of 97%. The distribution of the organisms in the culture-/PCR+ group was similar to the culture+/PCR+ group and was reproducible in replicate testing with an independent blood draw. Analysis of additional sample types (lower respiratory tract or sterile fluids and tissues) corroborated direct blood findings in 58% of cases where multiple specimens from the same patients were analyzed. The independent expert panel would have considered a different course of care in 57% of the cases when PCR/ESI-MS was positive, 41% of which would have resulted in altered, instituted, or ceased antibiotic therapy, earlier.

**Conclusion:** These results suggest that PCR/ESI-MS accurately detects the infecting pathogen in critically ill patients with BSI, and that its use might often lead to a different treatment. The test's ability to rule in or rule out infection approximately within 8 hours of the blood draw has added potential clinical and economic benefit, as it might minimize unnecessary use of antibiotics.

**Acknowledgements:** RADICAL Study Team

P62

## Subgroup analysis of the lipid infusion and patient outcomes in sepsis trial (LIPOS) reveals benefit in a subgroup not treated with stress replacement doses of corticosteroids.

TS Parker<fnref n="1" />,²*, DM Levine<fnref n="1" />,², BR Gordon<fnref n="1" />,³, SD Saal<fnref n="1" />,³

Critical Care 2014, **18**(Suppl 2):P62; doi:10.1186/cc14065

**Introduction:** Lipidose (formerly GR270773) is a protein-free phospholipid emulsion intended for the treatment of hospitalized patients with suspected or confirmed Gram-negative severe sepsis. Lipidose contains phosphatidylcholine, triglyceride and sodium cholate formulated to optimize delivery of the phospholipid component to the surface of high-density lipoprotein (HDL) and other lipoproteins, thereby enhancing the capacity of the patient's circulating lipoprotein pool to bind and neutralize microbial toxins. When Lipidose is infused into blood, the cholic acid is adsorbed onto serum albumin and the phospholipid selectively associates with lipoproteins. Bound and neutralized toxins are removed from the circulation by the liver and excreted along with the cholic acid into the bile. The LIPOS trial enrolled 1,400+ patients at 235 study centers in 31 countries to access Lipidose treatment at two dose levels. The LIPOS headline data presented only a small mortality benefit for the lower dose and no benefit from the higher dose [1]. A subgroup analysis was carried out to test the hypothesis of benefit in the subgroup with adequate liver function, using serum albumin levels as a measure of liver function, and adequate pre-existing HDL or total lipoprotein to accept phospholipid as predicted by the mechanism of action.

**Methods:** Albumin, cholesterol and HDL were measured in stored serum samples. The response to treatment and interactions with baseline covariates specified in LIPOS were tested after exclusion of subjects in the lowest biomarker quartiles (AlbTC25 and AlbHDL25).

**Results:** Subjects above the lowest quartile of albumin cleared Lipidose significantly faster than those in the lowest quartile (*P* < 0.003). Interactions between treatment and planned use of intravenous stress replacement doses of corticosteroids (IVCST) were found in the AlbTC25 and AlbHDL25 subgroups (*P* < 0.05). Exclusion of these subjects revealed strong relationships between treatment benefit and cholesterol or HDL that were used to select optimal biomarker thresholds. Requiring albumin ≥1.5 g/dl and either cholesterol ≥1 mM or HDL ≥0.5 mM selected 59% and 36%, respectively, of the LIPOS population. Treatment with Lipidose reduced mortality in these subgroups by 6.6% (*P* < 0.025) or 10.8% (*P* < 0.005) respectively. The treatment benefits persisted for at least 1 year.

**Conclusion:** A strong negative interaction with IVCST may have masked a significant treatment benefit in LIPOS. This interaction may be related to the ability of bile acids to slow clearance and raise concentrations of corticosteroids [2,3]. Biomarkers can be used to select subjects with early severe septic shock responsive to treatment with Lipidose.

**Acknowledgements:** TSP, DML, BRG and SDS are listed as inventors on patents filed by and/or assigned to Sepsicure, L.L.C

**References**

1. Dellinger RP, Tomayko JF, Angus DC, Opal S, Cupo MA, McDermott S, Ducher A, Calandra T, Cohen J: Efficacy and safety of a phospholipid emulsion (GR270773) in Gram-negative severe sepsis: results of a phase II multicenter, randomized, placebo-controlled, dose-finding clinical trial. *Crit Care Med* 2009, **37**:2929-2938.

2. Boonen E, Vervenne H, Meersseman P, Andrew R, Mortier L, Declercq PE, Vanwijngaerden YM, Spriet I, Wouters PJ, Vander Perre S, et al: *Reduced cortisol metabolism during critical illness*. *N Engl J Med* 2013, **368**:1477-1488.

3. Vanwijngaerden YM, Wauters J, Langouche L, Vander Perre S, Liddle C, Coulter S, Vanderborght S, Roskams T, Wilmer A, Van den Berghe G, et al: *Critical illness evokes elevated circulating bile acids related to altered hepatic transporter and nuclear receptor expression*. *Hepatology* 2011, **54**:1741-1752.

P63

## Presence of infection in patients with presumed sepsis at the time of ICU admission.

PMC Klein Klouwenberg<fnref n="1" />,²,³*, OL Cremer<fnref n="1" />, LA van Vught⁴, DSY Ong<fnref n="1" />,²,³, JF Frencken<fnref n="1" />,³, MJ Schultz⁵, MJ Bonten²,³, T van der Poll⁴

<footnote n="1">¹The Rogosin Institute, New York, NY, USA; ²Department of Biochemistry, New York Presbyterian Hospital and Weill Cornell Medical College, New York, NY, USA; ³Department of Medicine, New York Presbyterian Hospital and Weill Cornell Medical College, New York, NY, USA</footnote>

<footnote n="1">¹Department of Intensive Care Medicine, University Medical Center Utrecht, the Netherlands; ²Department of Medical Microbiology, University Medical Center Utrecht, the Netherlands; ³Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, the Netherlands; ⁴Center for Experimental and Molecular Medicine and Division of Infectious Diseases, Academic Medical</footnote>
Center, University of Amsterdam, the Netherlands; ⁵Department of Intensive Care Medicine, Academic Medical Center, University of Amsterdam, the Netherlands
*Critical Care* 2014, **18**(Suppl 2):P63; doi:10.1186/cc14066

**Introduction:** A clinical suspicion of infection is mandatory for diagnosing sepsis in patients with a systemic inflammatory response syndrome. Yet the accuracy of categorizing critically ill patients presenting to the ICU as being infected or not is unknown. We therefore assessed the likelihood of infection in patients who were treated for sepsis upon admission to the ICU, and quantified the association between plausibility of infection and mortality.

**Methods:** We studied a cohort of critically ill patients admitted with clinically suspected sepsis to two tertiary ICUs in the Netherlands between January 2011 and December 2013. The likelihood of infection was categorized as none, possible, probable or definite by *post hoc* assessment. We used multivariable competing risks survival analyses to determine the association of the plausibility of infection with mortality.

**Results:** Among 2,579 patients treated for sepsis, 13% had a *post hoc* infection likelihood of 'none', and an additional 30% of only 'possible'. These percentages were largely similar for different primary suspected sites of infection. In crude analyses, the likelihood of infection had no impact on ICU mortality, but was associated with increased length of stay and complications. In multivariable analysis, however, patients with an unlikely infection had a higher mortality rate compared to patients with a definite infection (subdistribution hazard ratio 1.23; 95% confidence interval 1.03 to 1.49).

**Conclusion:** This study is the first prospective analysis to show that the clinical diagnosis of sepsis upon ICU admission corresponds poorly with the presence of infection on *post hoc* assessment. A higher likelihood of infection does not adversely influence outcome in this population.

**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).

## P64
### Aetiology of community-acquired pneumonia in the ICU setting and its effect on mortality, length of mechanical ventilation and length of ICU stay: a 1-year retrospective review.

D Ryan*, R Connolly, J Fennell, G Fitzpatrick
The Adelaide and Meath Hospital, incorporating the National Children's Hospital (AMNCH), Tallaght, Dublin, Ireland
*Critical Care* 2014, **18**(Suppl 2):P64; doi:10.1186/cc14067

**Introduction:** Community-acquired pneumonia (CAP) is the most common infectious reason for admission to ICUs and has mortality of up to 37% [1]. Highest mortality rates are in Gram-negative infections with lower rates in *Streptococcus pneumoniae* and viral infections [2]. Microbiology is difficult to establish with most prospective studies identifying agents in only 50% of cases [3]. We analysed microbial aetiology of CAP in ICU over 1 year and assessed its effects on inpatient mortality, length of mechanical ventilation and length of ICU stay.

**Methods:** We retrospectively reviewed admissions to AMNCH ICU between February 2013 and February 2014 catalogued as having pneumonia from chest radiograph and clinical findings on the internal audit system (n = 91). After chart review, 28 were excluded as hospital-acquired pneumonias, 12 due to insufficient information and 21 due to primary diagnosis other than pneumonia. Thirty patients were selected on the basis that CAP was the likely reason for ICU admission.

**Results:** Pathogens were detected in 73% of patients by culture, antigen detection or molecular methods: *S. pneumoniae* (n = 6; 20%), influenza (n = 4; 13%), viral with superimposed bacterial infection (n = 4; 13%), viruses other than influenza (n = 3; 10%), Gram-negative bacilli (n = 2; 7%), *Legionella* spp. (n = 2; 7%) and *Haemophilus* spp. (n = 1; 3%). No organism was identified in 27%. Gram-negative infection had highest mortality (100%), average length of mechanical ventilation (57 days) and average ICU stay (64 days). The mean age of affected patients was 68.2. Mortality was lower in influenza/other viral infections (75/50%), patients spent less time mechanically ventilated (mean 6.6/13.3 days) and less time in ICU (mean 4/16.3 days), reflecting the younger mean age of patients (53.2/51.4). Of patients infected with *S. pneumoniae*, *Legionella* spp. or *Haemophilus* spp. alone, all survived to discharge. However, of note, when superimposed on viral infection, these pathogens carried a higher mortality (50%).

Infection with pneumococcus alone occurred in younger patients (mean age 50.6) and was associated with a shorter ventilation period (mean 2.66 days) and ICU stay (mean 6 days).

**Conclusion:** Microbial aetiology was identified in a high proportion of patients (72%) admitted to ICU with CAP, reflecting timely collection of appropriate specimens. Infection with Gram-negative organisms had the highest mortality, length of mechanical ventilation and length of ICU stay, while the pathogens usually seen in CAP were associated with more favourable outcomes.

## References

1. Fine MJ, Smith MA, Carson CA, Mutha SS, Sankey SS, Weissfeld LA, Kapoor WN: Prognosis and outcomes of patients with community-acquired pneumonia. A meta-analysis. *JAMA* 1996, 275:134.

2. Kothe H, Bauer T, Marre R, Suttorp N, Welte T, Dalhoff K: Competence Network for Community-Acquired Pneumonia study group: Outcome of community-acquired pneumonia: influence of age, residence status and antimicrobial treatment. *Eur Respir J* 2008, 32:139.

3. Miyashita N, Shimizu H, Ouchi K, et al: Assessment of the usefulness of sputum Gram stain and culture for diagnosis of community-acquired pneumonia requiring hospitalization. *Med Sci Monit* 2008, 14:CR171-CR176.

## P65
### *In vivo* study of endothelial barrier-related GTPase expression in the kidney and liver during the acute phase of nonlethal sepsis.

BC Zychar¹, MA Cenezede², AMA Liberatore¹, NOS Câmara², IH Koh¹
¹Department of Surgery, UNIFESP/ΕΜΡ, São Paulo, Brazil; ²Laboratory of Cellular Immunology Experimental, UNIFESP/ΕΜΡ, São Paulo, Brazil
*Critical Care* 2014, **18**(Suppl 2):P65; doi:10.1186/cc14068

**Introduction:** Maintenance of the integrity of the endothelial barrier is crucial in pathological inflammatory/infectious conditions [1]. The endothelial barrier dysfunction leading to increased vascular permeability and leukocyte transmigration in the systemic inflammatory state has been intrinsically related to the multiple organ dysfunction syndrome. The Rho GTPases family regulates the organization of the actin cytoskeleton, and plays a fundamental role in maintaining homeostasis and function of the endothelial barrier [2]. The study of the mechanisms of intracellular signaling related to the integrity of the endothelial barrier may help in understanding the hemodynamic changes during systemic infection. Thus, this study aimed to correlate the pattern of genic expression of GTPases during the initial periods of sepsis in order to understand the kinetic of these molecular mechanisms in organs often affected in sepsis.

**Methods:** Wistar rats weighting 200 to 250 g were submitted to: nonlethal sepsis (2 ml *Escherichia coli* 10⁷ UFC/ml i.v. inoculation [3,4], n = 5, S7 group); minor trauma (cervical incision with catheter implantation in jugular, injection of 2 ml saline, n = 5, SHAM group); without any procedure (n = 5, NAIVE group). After 2 and 6 hours, the liver and kidney were collected to determine RhoA, Rac1 and Cdc42 gene expression by quantitative real-time PCR.

**Results:** Low expression of RhoA was observed in both organs (Figures 1A and Figure 2A). Rac1 and/or Cdc42 showed higher expression in both organs in animals submitted to systemic infection (S7 group) compared to the SHAM and NAIVE groups in both periods (Figures 1B, C and Figure 2B, C). Although both organs showed a similar pattern, the kidney showed a statistically significant increase of Cdc42 and Rac1 as compared to the liver, showing that GTPase expression might differ, possibly due to endothelial heterogeneity of each organ to perform its specific function. These results suggest that the infectious process leads to a higher gene expression of Rac1 and/or Cdc42 in relation to a minor surgical trauma inflammation. The RhoA and Rac1 gene expression were inversely correlated, as reported in the literature [5].

**Conclusion:** Rac1 and Cdc42 possibly act in favor of preserving the integrity of the endothelial barrier by their attributed stabilization and protection of microvessel endothelial cells, by gathering the cell junctions during the infectious state. Studies are being conducted to better understand the mechanisms of intracellular signaling of the endothelial barrier in different organs of animals submitted to varying degrees of systemic infections.

**Acknowledgements:** FAPESP.
<figure><img src="image_50.png" /><figcaption>**Figure 1(abstract P65) Gene expression of GTPases RhoA (A), Rac1 (B) and Cdc42 (C) in the liver of rats.** Animals were subjected to nonlethal sepsis 2 ml *Escherichia coli* 10⁷ UFC/ml i.v. inoculation (S7 group), minor trauma 2 ml saline i.v. inoculation (SHAM group), or animals without inoculation were used as control group (NAÍVE group). After 2 or 6 hours the kidney was isolated. Total RNA was extracted and transcribed to cDNA. Gene expression was quantified by real-time PCR. Graph bars show the relative concentration of each cDNA compared with the NAÍVE group and the housekeeping gene HPRT. Results are mean ± SEM (n = 5 animals/group) and analyzed in triplicate. \*P < 0.05 compared to NAÍVE group</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>**Figure 2(abstract P65) Gene expression of GTPases RhoA (A), Rac1 (B) and Cdc42 (C) in the kidney of rats.** Animals were subjected to nonlethal sepsis 2 ml *Escherichia coli* 10⁷ UFC/ml i.v. inoculation (S7 group), minor trauma 2 ml saline i.v. inoculation (SHAM group), or animals without inoculation were used as control group (NAÍVE group). After 2 or 6 hours the kidney was isolated. Total RNA was extracted and transcribed to cDNA. Gene expression was quantified by real-time PCR. Graph bars show the relative concentration of each cDNA compared with the NAÍVE group and the housekeeping gene HPRT. Results are mean ± SEM (n = 5 animals/group) and analyzed in triplicate. \*P < 0.05 compared to NAÍVE group</figcaption></figure>

## References

1. Nourshargh S, Hordijk PL, Sixt M: Breaching multiple barriers: leukocyte motility through venular walls and the interstitium. *Nat Rev 2010*, **11**:366-378.

2. Ramiro-Marcos B, Weber-Garcia D, Millán J: TNF-induced endothelial barrier disruption: beyond actin and Rho. *Thromb Haemost* 2014, **112**:1-15.

3. Koh IH, Liberatore AM, Menchaca-Diaz JL, Ruiz-Silva M, Vilela-Oliveira L, Watanabe AY, Salomao R, Fagundes-Neto U, Silva RM: Bacterial

translocation, microcirculation injury and sepsis. *Endocr Metab Immune Disord Drug Targets* 2006, **6**:143-150.

4. Koh IH, Menchaca-Diaz JL, Koh TH, Souza RL, Shu CM, Rogerio VE, Liberatore AMA: Microcirculatory evaluation in sepsis: a difficult task. *Shock* 2010, **34**(7, Suppl 1):27-33.

5. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. *Nature* 2002, **420**:629-635.
P66

Direct bacterial identification from blood culture by matrix-assisted laser desorption-ionization time of flight mass spectrometer using a simplified protocol.

A Garcia-Tapia¹, I Guerrero, T Trujillo, F Galán, P Marín, C Fernández, M Rodriguez-Iglesias

Clinical Microbiology Laboratory, Puerta del Mar University Hospital, Cadiz, Spain

*Critical Care* 2014, **18**(Suppl 2):P66; doi:10.1186/cc14069

**Introduction:** Throughout the world, the number of patients at risk for bloodstream infections (BSIs) continues to rise. BSIs are associated with high rates of morbidity and mortality, and they markedly increase the costs of hospital care. Prompt identification of the causative agent(s) and rapid initiation of appropriate antimicrobial therapy are critical for reducing mortality, especially in patients with septic shock. Matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) equipment is increasingly used in the microbiological laboratory. The goal of the present investigation was to apply mass spectrometry directly from positive blood cultures in order to detect and identify bacterial strains using a simplified homemade protocol.

**Methods:** One-hundred and sixty-six positive blood cultures (Bactec FX; Becton Dickinson) were analyzed. Gram and conventional plates were used and the identification was done by Wider System (Soria Melguizo). In the MALDI-TOF protocol, positive blood cultures were processed as follow: 5 ml sample of positive broth was extracted and centrifuged at 1,000 rpm for 10 minutes in order to remove blood cells. The supernatant was collected and centrifuged at 4,000 rpm for 15 minutes, then the supernatant was removed and the pellet was placed directly onto a MALDI-TOF target plate and dried at room temperature, subsequently adding 1 µl HCCAMatrix solution (10 mg/ml cyano-4-hydroxycinnamic acid) and drying. Mass spectra were acquired with a Microflex LT mass spectrometer using FlexControl 3.3 software (Bruker Daltonics GmbH).

**Results:** One-hundred and forty-nine (89.7%) blood cultures were correctly detected. Thirty-three bacterial species were identified as being *Escherichia coli* (47), the most frequent, followed by *Pseudomonas aeruginosa* (14), *Enterobacter* spp. (11), *Klebsiella* spp. (9), *Staphylococcus aureus* (9), coagulase-negative staphylococci (9), *Enterococcus* spp. (8), *Bacteroides* spp. (7) and streptococci (6). Bacteria fastidious as *Burkholderia*, *Raoultella*, *Delftia*, *Listeria*, *Acinetobacter* or *Stenotrophomonas* were correctly identified. Only one strain of *Streptococcus pyogenes* and 14 strains of coagulase-negative staphylococci cannot be identified. In hemolyzed samples, it was not possible to identify two strains of *S. aureus*.

**Conclusion:** Using MALDI-TOF MS for bacteria identification directly from blood cultures represents an advance in the treatment of septic patients. Compared with conventional culture-based methods, this approach can improve species-level identification of bloodstream isolates in terms of time, accuracy, and costs. We conclude that this technique is simple, fast and reliable for direct bacterial detection from positive blood cultures.

**Methods:** Female C57BL6/J mice received an intravenous oleic acid (OA) injection to induce a controlled inflammation. Lung, brain and peripheral blood mononuclear cell (PBMC) expression patterns were analyzed using an Agilent 60K cDNA mouse microarray. The enrichment of canonical pathways revealed marked changes in pathways involving the immune and inflammatory responses. The inflammatory process in *D. melanogaster* shares common mechanisms in innate immunity with mammals (Toll pathway/TLR). We decided to set up a model, called the double-hit model, where drosophila females aged from 7 to 10 days have been injured with a needle (inflammatory hit) before being infected with a needle that has been dipped in a bacterial solution of *Pseudomonas entomophila* (infectious hit). The next step will be to investigate the transcriptome in order to highlight the differences and have a global view of biological mechanisms involved; and also look for genetic factors involved in this experiment. The DGRP (Drosophila Genomic Reference Panel) project is a panel of 200 inbred fly lines that have been fully sequenced.

**Results:** Strikingly, in mice models, all significant pathways identified in the brain were also significant in the lung. The inflammatory responses oscillated between proinflammatory and anti-inflammatory response in both the lung and brain, the time course, however, being different in the two organs. In PBMC, we observed a significant response delay after OA injection and the pathways identified differed from those identified in the lung and brain. Our second objective will be to use this model of the expression to set up a tool to analyze precisely the effect of an infection in a second hit during the sterile inflammation activation/repression time courses. In *D. melanogaster*, we recorded their survival in comparison with flies that only received the infectious hit and observed a difference of ~40%. Flies that have received a non-infectious double-hit showed a 10% decrease in survival rate. We will perform the double-hit experiment with all the lines of the DGRP and then through genome-wide association study. We expected to identify single nucleotide polymorphisms that are associated with resistance or susceptibility to the double-hit.

**Conclusion:** In mice, we assessed gene expression profiles in mouse lung, brain and PBMC associated with a lung inflammation during a 24-hour time course. Overall, our microarray analysis provides a global and detailed overview of critical transcriptional events occurring in the blood, brain and lung. The analysis of gene functional annotation revealed several major features. First, many genes were upregulated or downregulated over the time in the lung, brain, and blood. Second, the analysis revealed pathways clearly related to inflammation mainly in the lung and brain. The strong inflammation observed in the brain, and a limited inflammation in the blood, indicates that blood gene expression profiles poorly reflect those observed in the lung and brain. Third, all the pathways identified in the brain were also identified in the lung, although a few common genes were characterized; it should also be stressed that there was a delay response in the brain.

P67

Approaches combining mice and *Drosophila melanogaster* models to decipher human sepsis.

R Ramy¹, S Garnier, L Pradel, L Röder, B Loriod, A Defaye, L Perrin, P Rihet, C Nguyen

Aix-Marseille Université, Inserm, UMRS 1090, TAGC, Parc scientifique de Luminy, Marseille, France

*Critical Care* 2014, **18**(Suppl 2):P67; doi:10.1186/cc14070

**Introduction:** Sepsis is a complex and heterogeneous syndrome in which inflammatory and infection mechanisms are implicated. Thus, it is difficult to differentiate those mechanisms in mammals where inflammation is a highly complex biological process. In our laboratory, besides studying sepsis in human blood biological samples, we choose to investigate a murine model in ordered to describe a global transcriptome overview of critical events occurring in the blood, brain and lung during an induced non-infection inflammation. We decided to complete our view with another animal model where the inflammatory process is less complex and mainly achieved through innate immunity *Drosophila melanogaster*.

P68

Role of nonpneumoniae mycoplasma in the pathogenesis of ventilator-associated pneumonia: an *in vitro* assessment.

TJ Nolan¹, AC Morris, A Rossi, T Walsh

Centre for Inflammation Research, Queens Medical Research Centre, Little France, Edinburgh, UK

*Critical Care* 2014, **18**(Suppl 2):P68; doi:10.1186/cc14071

**Introduction:** Mycoplasma organisms are the smallest bacteria capable of self-replication [1] and include species capable of causing disease (for example, *Mycoplasma pneumoniae*, *Mycoplasma genitalium*) as well as those that are generally thought to exist synergistically with their human host (for example, *Mycoplasma salivarium*). The Edinburgh critical care group (Prof TW/ ACM) has recently identified a high prevalence of *M. salivarium* in the bronchoalveolar lavage washings from patients with confirmed and suspected ventilator-associated pneumonia (VAP) (Figure 1) [2]. The aim of this study was to examine the effect of *M. salivarium* on human immune cells in *in vitro*. Specifically, we measured cytokine production and phagocytosis activity in response to *M. salivarium* exposure.

**Methods:** Whole human blood was obtained from healthy donor volunteers and cell types were isolated using diffusion gradients and magnetic labeling as appropriate. Monocytes and macrophages were incubated with *M. salivarium* for 24 hours before a subsequent LPS stimulus.
<figure><img src="image_52.png" /><figcaption>Figure 1(abstract P68) Combined mycoplasma detection by PCR in the prospective and clinical groups</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>Figure 2(abstract P68) Twenty-four-hour exposure of macrophages to *Mycoplasma salivarium*.</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>Figure 3(abstract P68) *Mycoplasma salivarium* (30,000) for 24 hours prior to 100 ng/ml LPS exposure</figcaption></figure>

Macrophage phagocytosis assays were conducted after exposure times of 60 minutes and 24 hours to *M. salivarium*. Cytokines were measured using ELISA and human cytokine bead array kits.

**Results:** There was a statistically significant decrease in phagocytosis between control cells and the macrophages exposed to both a low titer of *M. salivarium* (P value 0.018) and a medium titer of *M. salivarium*

<figure><img src="image_55.png" /><figcaption>Figure 4(abstract P68)</figcaption></figure>

(P value 0.011) after 24 hours of exposure (Figure 2). There was a statistically significant decrease in phagocytosis activity between the macrophages exposed to the medium titer of *M. salivarium* for 24 hours versus 60 minutes (P value 0.013). Exposure of macrophages to mycoplasma resulted in decreased release of TNFα after a subsequent LPS stimulus (Figure 3). To our knowledge, this is the first time extracellular traps have been induced in macrophages in response to *M. salivarium* (Figure 4).

**Conclusion:** Although further research is needed, it is interesting that the presence of *M. salivarium* caused an anti-inflammatory effect as well as impaired antigen presentation secondary to impaired phagocytosis. This could be consistent with the better outcome in mechanically ventilated patients that did not have *M. salivarium* bacteria detected in their bronchoalveolar lavage washings. Extracellular traps contribute to microbial containment by forming a physical barrier composed of chromatin and cytoplasmic proteins to enhance antimicrobial synergy while minimizing damage to host tissues [3]. It is interesting that *M. salivarium* induced extracellular traps.

**Acknowledgements:** Thanks to supervisors ACM and Prof AR for their support and expertise. Thanks also to all the staff in the Centre for Inflammation Research for their help throughout the year and for being so willing to help when help was needed

## References

1. Razin S, Yogev D, Naot Y: Molecular biology and pathogenicity of mycoplasmas. *Microbiol Mol Biol Rev* 1998, 62:1094-1156.

2. Wilkinson TS, et al: Ventilator-associated pneumonia is characterized by excessive release of neutrophil proteases in the lung. *Chest* 2012, 142:1425-1432.

3. Papayannopoulos V, Zychlinsky A: NETs: a new strategy for using old weapons. *Trends Immunol* 2009, 30:513-521.

## P69

### Probiotic pretreatment improves survival and prevents gut mucosal barrier dysfunction in sepsis.

KL Calisto*, ACAP Camacho, FC Mittestainer, MCS Mendes, AC Santos, MJA Saad

Department of Internal Medicine, Faculty of Medical Sciences, UNICAMP Campinas, Brazil

*Critical Care* 2014, **18**(Suppl 2):P69; doi:10.1186/cc14072

**Introduction:** The gut is the largest immune organ and plays a central role in the promotion of systemic inflammatory responses [1]. Perturbations of intestinal epithelial homeostasis during sepsis include increased proinflammatory cytokine production, increased intestinal permeability and apoptosis [2-6]. Healthy gut is essential to promote host health and prevent organ dysfunction in sepsis. Probiotics seem to keep gut homeostasis through different pathways, such as the modulation of microbial activity, energy regulation, anti-inflammatory cytokine production, gene expression
<figure><img src="image_56.png" /><figcaption>Figure 1 (abstract P69)</figcaption></figure>

and cell differentiation [7]. Probiotics have been shown an effective treatment in various clinical conditions, although the potential benefits of probiotic treatment in sepsis remain largely undefined. The aim of the present study was to investigate the effect of probiotic treatment on gut dysfunction and inflammatory signaling in septic rats.

**Methods:** Sepsis was induced by cecal ligation and puncture (CLP) in Wistar male rats (8 weeks old). They were pretreated with probiotics or vehicle once a day during 7 days before CLP. The chosen probiotic mixture contained 10 × 10⁷ CFU *Bifidobacterium longum*, 10 × 10⁶ CFU *Lactobacillus bulgaricus*, 10 × 10⁶ CFU *Lactobacillus acidophilus*. Colonic tissue and serum samples were collected 24 hours after CLP for ELISA and protein expression analysis by western blotting.

**Results:** Our data demonstrate that probiotic pretreatment improved survival of septic rats (Figure 1) and this effect is accompanied by a marked decrease of IL-1β and TNFα (Figure 2A,B). Sepsis leads to severe intestinal epithelial damage with a decrease in claudin 2 and occludin protein expression (Figure 3A,B); probiotic pretreatment reversed these alterations in parallel with an increase in Hsp72 and Hsp25 activation (Figure 4A,B). In intestinal epithelial cells, the inducible Hsp have been shown to preserve tight junction and barrier function. The maintenance of epithelial barrier integrity induced by probiotic pretreatment, in parallel with an activation of cytoprotective pathway, may culminate in the restoration of the intestinal epithelial function.

**Conclusion:** Our results show that probiotics pretreatment fulfills a dual function at the intestinal mucosa: in addition to preventing intestinal permeability disruption, it also attenuates proinflammatory cytokine release, diminishing the exacerbate host's reaction to infection and offering a novel prophylactic strategy to sepsis.

References

1. Clark JA, Coopersmith CM: Intestinal crosstalk: a new paradigm for understanding the gut as the 'motor' of critical illness. *Shock* 2007, 28:384-393.

2. Dominguez JA, Coopersmith CM: Can we protect the gut in critical illness? The role of growth factors and other novel approaches. *Crit Care Clin* 2010, 26:549-565.

3. Hassoun HT, Kone BC, Mercer DW, Moody FG, Weisbrodt NW, Moore FA: Post-injury multiple organ failure: the role of the gut. *Shock* 2001, 15:1-10.

4. Yu P, Martin CM: Increased gut permeability and bacterial translocation in Pseudomonas pneumonia-induced sepsis. *Crit Care Med* 2000, 28:2573-2577.

5. Neal MD, Leaphart C, Levy R, Prince J, Billiar TR, Watkins S, Li J, Cetin S, Ford H, Schreiber A, et al: Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier. *J Immunol* 2006, 176:3070-3079.

6. Clark JA, Gan H, Samocha AJ, Fox AC, Buchman TG, Coopersmith CM: Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis. *Am J Physiol Gastrointest Liver Physiol* 2009, 297:G471-G479.

7. Sartor RB: Probiotic therapy of intestinal inflammation and infections. *Curr Opin Gastroenterol* 2005, 21:44-50.

P70

**Positive fluid balance and prognostic factors of ICU mortality in patients admitted with septic shock.**

W Koonrangsesomboon¹*, B Khwannimit²

¹Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand; ²Division of Critical Care Medicine, Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla, Thailand

*Critical Care* 2014, **18**(Suppl 2):P70; doi:10.1186/cc14073

**Introduction:** The amount of fluid during resuscitation of septic shock is important. Too little fluid may result in tissue hypoperfusion; however, too much fluid may result in volume accumulation. Several recent studies have demonstrated that a positive fluid balance in critical illness is associated with deteriorating outcomes. However, some studies have shown opposite results. The objective of this study was to determine whether initial fluid balance in septic shock patients is correlated with ICU mortality.

**Methods:** This is a retrospective study of septic shock patients admitted to a mixed medical-coronary care unit of Songklanagarin hospital from 2005 to 2011. Multivariate logistic regression analysis was used to identify predictors of mortality.

**Results:** A total of 1,048 patients admitted to ICU for septic shock was divided into two groups: in-ICU survivors ($n = 555$ (53%)) and nonsurvivors ($n = 493$ (47%)). Median survival time was 10 days (95% CI: 8 to 12 days). The respiratory tract was the most common site of infection (47.6%). Community-acquired infections accounted for 59.6%. Survivors were older than nonsurvivors (62 vs. 56 years, $P = 0.016$). Nonsurvivors were more severely ill and had shorter ICU stays (2 vs. 5 days, $P < 0.001$). Nonsurvivors received albumin and steroid more than survivors. Median cumulative fluid at 24, 48 and 72 hours of septic shock onset were 4.2, 7.7 and 10.5 l respectively. Nonsurvivors had significantly larger median cumulative fluid intake at 24 hours (4.6 vs. 3.9 l, $P < 0.001$), at 48 hours (8.2 vs. 7.1 l, $P < 0.001$) and at 72 hours (11.4 vs. 9.9 l, $P < 0.001$). Nonsurvivors also had

<figure><img src="image_57.png" /></figure>
<figure><img src="image_58.png" /><figcaption>Figure 3 (abstract P69)</figcaption></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /><figcaption>Figure 4 (abstract P69)</figcaption></figure>

<figure><img src="image_61.png" /></figure>

significantly larger fluid balance (5.4 vs. 4.4 l, *P* < 0.001) and mean fluid balance (2.8 vs. 1.6 l, *P* < 0.001) within 72 hours. In multivariate logistic regression analysis, factors significantly associated with ICU mortality were mean fluid balance, APACHE II score, SOFA score, length of ICU stay, ARDS, steroid use, parenteral nutrition use and source of infection.

**Conclusion:** A more positive cumulatively fluid balance over 3 days is associated with ICU mortality in septic shock. Multivariate analysis found not only nonmodifiable factors such as severity score, source of infection, length of ICU stay and ARDS, but also modifiable factors such as parenteral nutrition use, steroid use and mean fluid balance were significantly associated with mortality.

P71

**Alpha lipoic acid attenuates oxidative stress-induced damage macromolecules in the brain of rats with sepsis-associated encephalopathy.**

LG Daniëlski¹, M Michels, D Florentino, A Viera, A Lauriano, Fabrícia Petronilho Laboratório de Fisiopatologia Clínica e Experimental (LAFICEXP), Programa de Pós, Graduação em Ciências da Saúde, Universidade do Sul de Santa Catarina (UNISUL), Tubarão, SC, Brazil

Critical Care 2014, **18**(Suppl 2):P71; doi:10.1186/cc14074

**Introduction:** Pathophysiological mechanisms of sepsis-associated encephalopathy involve oxidative stress. This imbalance between the pro-oxidant and antioxidant causes damage to macromolecules such as lipids and proteins, thus the employment of antioxidants becomes an attractive proposition. Alpha lipoic acid (LA), a potent antioxidant, is able to cross

the blood-brain barrier, and is an important cofactor in enzymatic and cellular energy metabolism. We aimed to determine the use of AL in oxidative damage and neutrophil infiltration in rat brain 12 and 24 hours after induction of sepsis model by cecal ligation and puncture (CLP).

**Methods:** Male Wistar rats (250 to 350 g) were subjected to CLP model, with sham control. Groups were divided into sham + saline, sham + AL, CLP + saline and CLP + AL (200 mg/kg orally with single administration after CLP), *n* = 10. At 12 and 24 hours, rats were euthanized, the hippocampus, striatum, cerebellum, cortex and prefrontal cortex removed, lipid peroxidation assessed by TBARS, damage to proteins by protein carbonylation, myeloperoxidase activity (MPO) and the formation of nitrite and nitrate. Data were analyzed by ANOVA with *post hoc* Tukey test and log-rank test with *P* < 0.05.

**Results:** In 12 hours compared with the CLP group, the CLP + AL group showed a reduction in lipid peroxidation in the striatum, in the protein carbonylation in the cortex and hippocampus, in the MPO activity in the striatum and hippocampus, and decreased formation of nitrite and nitrate in the hippocampus and cortex.

**Conclusion:** While differences were not observed in 24 hours, TBARS found protein carbonylation in a reduction of damage to the CLP + AL group over the cerebellum, MPO in the striatum, hippocampus and prefrontal, and hippocampus, cerebellum, and prefrontal to nitrite/nitrate. AL may be an important therapeutic target in reducing neurologic complications in animal models of sepsis.

**Acknowledgements:** Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Universidade do Sul de Santa Catarina (UNISUL)
## P72
### Effects of mesenchymal stromal cells on human umbilical vein endothelial cells in *in vitro* sepsis models.

K Lund¹*, J Peltzer¹, F Montespan¹, N Oru², E Vicaut², J Duranteau³, J-J Lataillade¹

¹Unité de Thérapie Cellulaire et Réparation Tissulaire, Institut de Recherche Biomédicale des Armées/Centre de Transfusion Sanguine des Armées, Clamart, France; ²Laboratoire d'étude de la microcirculation, Université Paris - Diderot, Paris, France; ³Service d'Anesthésie-Réanimation Chirurgicale, Centre Hospitalier Universitaire de Bicêtre, Le Kremlin-Bicêtre, France
*Critical Care* 2014, **18**(Suppl 2):P72; doi:10.1186/cc14075

**Introduction:** Septic shock is a medical emergency that, despite the medical advances that have been made, still remains a major cause of hospital deaths. Cell therapy is an innovative field of research that could provide a therapy for sepsis. Mesenchymal stromal cells (MSC) are promising in cell therapy and more importantly for sepsis because of their immunosuppressive capabilities [1]. MSC have been shown by several groups to have a positive effect against sepsis *in vivo* [2-4]. It has been predicted that the MSC interact with macrophage to release IL-10 that in turns reduces inflammation [4]. Other groups have focused on the use of stimulated MSC to ameliorate their immunosuppressive capabilities [5]. The main stimulation of MSC has been the use of inflammatory stimulants like IFNγ. Our work focuses on the identification of effective MSC donors, whether primed with IFNγ or naïve, and the development of *in vitro* models that will predict how an MSC donor will act *in vivo*. We also want to eliminate the use of cells completely and use their secreted microvesicles as a therapy. The hypothesis is that the *in vitro* models will eliminate a noneffective MSC donor and allow us to identify the MSC donor that will have the greatest effect.

**Methods:** We developed two *in vitro* models that are similar to what happens *in vivo* with WBC as they circulate in a septic patient. The first test is the adherence of WBC to a layer of HUVECs in the presence of MSC or microvesicles. The second is a permeability test to determine MSC ability to block the permeability of a HUVEC layer.

**Results:** Our preliminary results have shown that we are able to identify, using our two *in vitro* models, which MSC donor would be an effective MSC for cell therapy.

**Conclusion:** MSC and their paracrine factors have to the potential to be an effective therapy for sepsis, but one needs to identify an effective donor before use in cell therapy.

### References

1. Meirelles L, Fontes AM, Covas DT, et al: Mechanisms involved in the therapeutic properties of mesenchymal stem cells. *Cytokine Growth Factor Rev* 2009, **20**:419-427.

2. Gonzalez-Rey E, Anderson P, Gonzalez MA, et al: Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. *Gut* 2009, **58**:929-939.

3. Hall SR, Tsoyi K, Ith B, et al: Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils. *Stem Cells* 2012, **31**:397-407.

4. Nemeth K, Leelahavanichkul A, Yuen PS, et al: Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. *Nat Med* 2009, **15**:42-49.

5. Polchert D, Sobinsky J, Douglas G, et al: IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. *Eur J Immunol* 2008, **38**:1745-1755.

## P73
### Endotoxin Activity Assay levels correlate with the microbiological results of Gram-negative organisms in septic patients.

M Tsunoda*, M Kang, M Saito, N Saito, M Namiki, T Harada, M Takeda, A Yaguchi

Department of Critical Care and Emergency Medicine, Tokyo Women's Medical University, Tokyo, Japan

*Critical Care* 2014, **18**(Suppl 2):P73; doi:10.1186/cc14076

**Introduction:** The Endotoxin Activity Assay (EAA™; Spectral Diagnostics Inc., Toronto, Canada) is a useful diagnostic test for sepsis due to Gram-negative

infection and is based on the reaction of neutrophils to endotoxin complexed with an anti-endotoxin antibody. However, the relations between values of EAA and microbiological data have not been elucidated. Our hypothesis is that EAA values correlate to the results of microbiological cultures and also severity.

**Methods:** From July 2008 to July 2013, all adult patients with suspected sepsis admitted to our medico-surgical ICU in whom EAA was measured were included in this study. Data collected included age, gender, ICU mortality, white blood cell (WBC) count, C-reactive protein (CRP), procalcitonin (PCT), EAA levels, SOFA score and results of microbiological culture. Patients with no microbiological data were excluded. Data were analyzed by Kruskal-Wallis test, Mann-Whitney U test and multivariate logistic regression. *P* < 0.05 was considered significant.

**Results:** Of 569 patients (353 men and 216 women; mean age 66.0 ± 17.4 years), 283 patients had Gram-negative infection and 286 patients had no Gram-negative infection. Of 283 patients with Gram-negative infection, 65 patients had Gram-negative organisms in blood. EAA levels were significantly different between patients with Gram-negative blood, in other infectious sites and no Gram-negative infection (0.45 ± 0.21 vs. 0.39 ± 0.17 vs. 0.36 ± 0.15, *P* = 0.03). The odds ratio (95% confidence interval (CI)) of EAA levels for Gram-negative infection and Gram-negative bacteremia were 3.89 (1.44 to 10.4) (*P* = 0.007) and 3.36 (2.16 to 40.6) (*P* = 0.003), respectively. The odds ratio and CI of age and SOFA score for ICU mortality were 1.03 (1.01 to 1.04) (*P* = 0.0003) and 1.33 (1.26 to 1.41) (*P* < 0.0001), respectively, while gender, WBC, CRP, PCT and EAA levels had no relations with ICU mortality. SOFA score was significantly higher in patients with Gram-negative infection than in patients with no Gram-negative infection (8.0 ± 4.6 vs. 6.7 ± 4.2, *P* = 0.0003).

**Conclusion:** EAA levels related to the detections of Gram-negative organisms in cultures. Thus, a high EAA level may show the existence of Gram-negative organisms in patients' sites. EAA levels had relations with SOFA score but no relations with ICU mortality.

## P74
### Early diagnosis of sepsis due to Gram-negative infection with the Endotoxin Activity Assay.

M Kang¹, M Tsunoda, M Saito, N Saito, M Namiki, M Takeda, T Harada, A Yaguchi

Department of Critical Care and Emergency Medicine, Tokyo Women's Medical University, Tokyo, Japan

*Critical Care* 2014, **18**(Suppl 2):P74; doi:10.1186/cc14077

**Introduction:** The Endotoxin Activity Assay (EAA™; Spectral Diagnostics Inc., Toronto, Canada) is a rapid *in vitro* diagnostic test of the neutrophil reaction to endotoxin and reflects the endotoxemia. A higher value of EAA (>0.60) has been shown to correlate with developing severe sepsis and a high mortality in other previous studies. We hypothesize that a value of EAA more than 0.55, not >0.60, may be useful to earlier diagnose sepsis due to Gram-negative organisms and to assess the severity.

**Methods:** The present study is a single-center retrospective observational analysis of adult septic patients in whom EAA was performed from July 2008 to July 2013. Patients were divided into two groups: (1) EAA >0.55 and (2) EAA < 0.54. Age, sex, days of ICU stay, ICU mortality, body temperature, WBC, CRP, procalcitonin (PCT), SOFA score, and microbiological data were compared between two groups. Values are expressed as mean ± SD. Data were analyzed by chi-square test and Mann-Whitney U test. *P* < 0.05 was considered significant. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and odds ratio were also evaluated.

**Results:** Five hundred and ninety-four patients (377 men and 217 women; mean age 65.0 ± 17.5 years) were studied. There were (1) 104 patients with EAA >0.55 and (2) 490 patients with EAA < 0.54. ICU mortality (39.4 vs. 26.6 %, *P* = 0.01), PCT (17.2 ± 36.0 vs. 11.8 ± vs. 30.6 ng/ml, *P* = 0.04), SOFA score (8.1 ± 4.9 vs. 6.2 ± 4.6, *P* = 0.04) and positive Gram-negative organisms in cultures (57.7 vs. 45.5 %, *P* = 0.02) were significantly higher in group (1) than group (2). Age, sex, body temperature, WBC and CRP were not significantly different between two groups. Using detections of Gram-negative organisms in cultures, the sensitivity, the specificity, the PPV and the NPV were 22.1, 85.2, 57.7 and 54.6%, respectively. The odds ratio was 1.64.

**Conclusion:** ICU mortality and severity were higher in patients with EAA >0.55 than in patients with EAA < 0.54. There is a possibility that an EAA
value more than 0.55 is a meaningful value for early diagnosis of sepsis due to Gram-negative infection.

## P75
### Effect of estimated glomerular filtration rate and fluid balance on clinical course and outcomes of children admitted with severe dengue.

N Vijayakumar¹,²*, S Kandasamy², T Sangaralingam¹, V Varadarajan², N Krishnamoorthi¹,³

¹Department of Pediatrics, Mehta Children's Hospital, Chennai, India;

²Department of Pediatric Intensive Care, Mehta Children's Hospital, Chennai, India; ³Department of Academics and Research, Mehta Children's Hospital, Chennai, India

*Critical Care* 2014, **18**(Suppl 2):P75; doi:10.1186/cc14078

**Introduction:** Dengue fever is one of the most important seasonal epidemics in Asia. Case fatality rates vary from 1 to 5% [1]. Mortality from severe dengue may range from 26% in DHF to as high as 47% in DSS [2,3]. The pathogenesis of shock in dengue fever (DF) is centered on increased capillary permeability in the critical phase leading to hypovolemia and shock in severe dengue. There have been multiple studies that compare fluid regimens in the management of dengue [4,5]. These studies do not assess the child's renal function and ability to handle the fluid loads. GFR <60 ml/ minute indicates a significant decrease in the renal functioning and there are no pediatric studies that examine their association with in-hospital stay and outcomes in children with severe dengue. With this introduction we formulated this study protocol to examine that association. The objectives were to measure the estimated glomerular filtration rate (eGFR) at admission and fluid balance in the first 36 hours of ICU stay and assess their effect on disease course and outcomes in severe dengue.

**Methods:** This was designed as a retrospective descriptive study in a tertiary-level pediatric ICU in South India. Case records of all children fulfilling the WHO case definition of severe dengue were included, those who received intravenous fluid for less than 12 hours were excluded. Primary parameters measured included fluid balance in the first 36 hours measured every 12 hours, durations of oxygen requirement, mechanical ventilation, ICU stay and total hospital stay. Outcomes measured were death and survival.

**Results:** Twenty-six children were enrolled, 14 boys and 12 girls. The median duration of ICU stay was 60 hours, and that of hospital stay 109 hours. eGFR was less than 60 ml/minute in six patients (83.3% expired and 16.7% survived). eGFR, measured by modified Schwartz's formula, at the time of admission correlated inversely with requirement of oxygen therapy and mechanical ventilation (P < 0.05) and fluid balance in the first 36 hours. Positive fluid balance (FO > 15%) in the first 36 hours was significantly higher in children who expired (P = 0.011). eGFR <90 ml/minute at admission had 100% sensitivity and 79% specificity to predict the possible occurrence of fluid overload >15% (area under curve = 0.882).

**Conclusion:** Fluid balance in the first 36 hours had a significant positive correlation with mortality and negative correlation with eGFR. Children with admission eGFR <90 ml/minute may require restrictive fluid therapy to improve survival.

## References

1. Dengue Hemorrhagic Fever: Diagnosis, Treatment, Prevention and Control. Geneva: World Health Organization (WHO) and Special Programme for Research and Training in Tropical Diseases (TDR), 3 2009.

2. Agarwal R, Kapoor S, Nagar R, et al: A clinical study of patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow, India. Southeast Asian J Trop Med Public Health 1999, 30:735-740.

3. Ranjit S, Kissoon N, Jayakumar I: Aggressive management of dengue shock syndrome may decrease mortality rate: a suggested protocol. Pediatr Crit Care Med 2005, 6:412-419.

4. Dung NM, Day NP, Tam DT, et al: Fluid replacement in dengue shock syndrome: a randomized, double-blind comparison of four intravenous-fluid regimens. Clin Infect Dis 1999, 29:787-794.

5. Ngo NT, Cao XT, Kneen R, et al: Acute management of dengue shock syndrome: a randomized double-blind comparison of 4 intravenous fluid regimens in the first hour. Clin Infect Dis 2001, 32:204-213.

## P76
### Audit of antibiotic prophylaxis at a district general hospital.

K McGrath*, J King-Robson, A Hayes, F Dashti, S Patel
Department of Microbiology, Kingston Hospital NHS Trust, Kingston-upon-
Thames, UK

*Critical Care* 2014, **18**(Suppl 2):P76; doi:10.1186/cc14079

**Introduction:** This audit evaluates the adherence to the prophylactic surgical antibiotic policy at Kingston Hospital. With a greater understanding of the current use of prophylactic surgical antibiotics comes the ability to improve patient care while minimising the development of antibiotic resistance. Surgical site infections are a major source of hospital-acquired infections, causing significant morbidity and mortality. In appropriate cases, surgical antibiotic prophylaxis is essential in preventing such infections; however, this comes with increased risks of antibiotic resistance and antibiotic-associated diarrhoea. Consequently, this institution has extensive guidelines as to the cases in which antibiotic prophylaxis is required and what antibiotics should be administered. This audit examines the adherence to these guidelines. We audited against our local antibiotic prescribing policy, named the Blue Book guidelines.

**Methods:** Notes were audited retrospectively for 80 patients undergoing surgery between 19 August and 18 December 2013. These were audited against local antibiotic guidelines (Blue Book guidelines) and analysed using descriptive statistics.

**Results:** Only 57% of operations were compliant with Blue Book antibiotic prophylaxis guidelines. For operations where administration of antibiotics was appropriate, 24% of patients received the incorrect choice, 19% were given the incorrect dose, 28% were given antibiotics for an inappropriate time relative to the procedure, and 38% received antibiotics for the incorrect duration. For operations not requiring prophylaxis, 48% of patients incorrectly received antibiotics.

**Conclusion:** Compliance to local guidelines for prophylactic antibiotics is extremely poor. This not only risks increased morbidity and mortality from surgical site infections but also risks the development of antibiotic resistance and Clostridium difficile colitis.

We suggest enhancement of the antibiotic guidelines, and education of those using them. This will improve the provision of antibiotic prophylaxis in the Trust, and ultimately improve patient care.

## P77
### Presepsis biomarker: high-density lipoprotein.

S Al-Zaidawi
Surgery Department, Burn Unit, Al-Sadr Teaching Hospital, Maysan, Iraq
*Critical Care* 2014, **18**(Suppl 2):P77; doi:10.1186/cc14080

**Introduction:** Delay in diagnosis and initiation of antibiotic treatment has been shown to increase mortality. Biomarkers can play an important role in diagnosis and prognosis of sepsis. We aimed to evaluate the correlation between septicemia and high-density lipoprotein (HDL) level in burned patients.

**Methods:** A prospective study conducted at Al-Sadr teaching hospital, Maysan, Iraq, during the period from April to September 2013. Blood samples were collected from patients every other day to measure the level of HDL and triglycerides. Other blood samples were collected in blood culture tubes for culturing to verify septicemia depending on the clinical evidence.

<table><caption>**Table 1(abstract P77) Characteristics of patients**</caption><thead><tr><td>Patient characteristic</td><td>Average</td><td>Range</td></tr></thead><tbody><tr><td>Age (years)</td><td>17</td><td>1 to 85</td></tr><tr><td>TBSA%</td><td>33.5%</td><td>15 to 95%</td></tr><tr><td>Sex of patients</td><td>Female 61%</td><td>Male 39%</td></tr><tr><td>Burn type</td><td>Scalds 48%</td><td>Flame 52%</td></tr></tbody></table>

Most patients were female (61%) with average age 17 years and a wide range of burned surface area (15 to 95%)
<table><caption>Table 2(abstract P77) Lipid profile for all 75 patients at the onset of thermal injury during the first day of admission: all patients were with a normal range of HDL, triglycerides and cholesterol</caption><thead><tr><td>Lipid profile</td><td>Range (mg/dl)</td><td>Average (mg/dl)</td><td>Mode (mg/dl)</td><td>Normal range (mg/dl)</td></tr></thead><tbody><tr><td>HDL</td><td>30 to 56</td><td>39</td><td>38</td><td>39 to 59</td></tr><tr><td>Triglycerides</td><td>37 to 148</td><td>70</td><td>58</td><td>0 to 149</td></tr><tr><td>Cholesterol</td><td>46 to 155</td><td>78</td><td>86</td><td>0 to 199</td></tr></tbody></table>

<table><caption>Table 3(abstract P77) Lipid profile at the onset of sepsis showed that HDL level dropped to less than 15 mg/dl with range (4 to 13 mg/dl): elevation in triglyceride level out of normal range with no significant change in cholesterol level</caption><thead><tr><td>Lipid profile</td><td>Range (mg/dl)</td><td>Average (mg/dl)</td><td>Mode (mg/dl)</td><td>Normal range (mg/dl)</td></tr></thead><tbody><tr><td>HDL</td><td>4 to 13</td><td>7.6</td><td>4</td><td>39 to 59</td></tr><tr><td>Triglycerides</td><td>133 to 435</td><td>214.5</td><td>180</td><td>0 to 149</td></tr><tr><td>Cholesterol</td><td>56 to 139</td><td>82.8</td><td>76</td><td>0 to 199</td></tr></tbody></table>

<table><caption>Table 4(abstract P77) Levels of urea, creatinine, albumin, WBC, platelets during onset of sepsis: most patients developed hypoalbuminemia and thrombocytopenia</caption><thead><tr><td></td><td>% TBSA</td><td>WBC (x1,000/μl)</td><td>Platelet count (x1,000/μl)</td><td>Blood urea (mg/dl)</td><td>Serum creatinine</td><td>Serum albumin (g/dl)</td><td>Time to get sepsis (days)</td></tr></thead><tbody><tr><td>Average</td><td>55</td><td>11.86</td><td>154.9</td><td>14</td><td>0.51</td><td>2.0</td><td>7</td></tr><tr><td>Mode</td><td>45</td><td>9.04</td><td>121</td><td>12</td><td>0.37</td><td>1.8</td><td>10</td></tr><tr><td>Minimum</td><td>27</td><td>2.27</td><td>32</td><td>9</td><td>0.32</td><td>1.4</td><td>2</td></tr><tr><td>Maximum</td><td>95</td><td>15.03</td><td>535</td><td>21</td><td>0.73</td><td>3.1</td><td>20</td></tr></tbody></table>

<table><caption>Table 5(abstract P77) Comparison between level of lipid profile before and after sepsis showed the significant dropping in HDL level during onset of sepsis</caption><thead><tr><td></td><td>HDL</td><td>Triglycerides</td><td>Cholesterol</td><td>P value</td></tr></thead><tbody><tr><td>Burn onset</td><td>39</td><td>70</td><td>78</td><td>&lt; 0.01</td></tr><tr><td>Sepsis onset</td><td>7.6</td><td>214.5</td><td>76</td><td>&lt; 0.01</td></tr></tbody></table>

**Results:** Seventy-five patients (Table 1) were admitted consecutively into the burn unit, 35 of them (46%) developed septicemia and 11 of the 35 patients died. All dead patients had HDL value <5 mg/dl 1 or 2 days before dying since our blood samples were collected every 2 days (Tables 2 and 3). Other laboratory tests such as WBCs, platelet account, albumin level, and so forth were made to confirm sepsis (Table 4). A comparison between the level of lipid profile before and after sepsis showed a significant drop in HDL level during the onset of sepsis (Table 5). We also found that patients with HDL value <15 mg/dl were at high risk of developing sepsis.

**Conclusion:** There was a strong correlation between HDL level and septicemia in burn patients. The HDL value is a good biomarker for sepsis; it decreases below normal level and continues to diminish and reach an immeasurable level at the advanced stage of septicemia.

Cite abstracts in this supplement using the relevant abstract number, e.g.: Al-Zaidawi: Presepsis biomarker: high-density lipoprotein. Critical Care 2014, 18(Suppl 2):P77