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37th International Symposium on Intensive Care and Emergency Medicine (part 3 of 3)

Brussels, Belgium. 21-24 March 2017

P349 Muscle mitochondrial function and N+/K+ -ATPase activity are unaffected by sepsis in pigs

M Von Seth, L Hillered, A Otterbeck, K Hanslin, A Larsson, J Sjölin, M Lipcsey

Uppsala University, Uppsala, Sweden

Introduction

Imbalance in cellular energetics has been suggested to be an important mechanism for organ failure in sepsis and septic shock. We hypothesized that such energy imbalance would either be caused by metabolic changes leading to decreased energy production or by increased energy consumption. Thus, we set out to investigate if mitochondrial dysfunction or decreased energy consumption alters cellular metabolism in muscle tissue in experimental sepsis.

Methods

We submitted anesthetized piglets to sepsis (n = 12) or placebo (n = 4) and monitored them for 3 hours. Plasma lactate and markers of organ failure were measured hourly, as was muscle metabolism by microdialysis. Energy consumption was intervened locally by infusing ouabain through one microdialysis catheter to block major energy expenditure of the cells, by inhibiting the major energy consuming enzyme, N+/K + -ATPase. Similarly, energy production was blocked infusing sodium cyanide (NaCN), in a different region, to block the cytochrome oxidase in muscle tissue mitochondria.

Results

All animals submitted to sepsis fulfilled sepsis criteria as defined in Sepsis-3, whereas no animals in the placebo group did. Muscle glucose decreased during sepsis independently of N+/K + -ATPase or cytochrome oxidase blockade. Muscle lactate did not increase during sepsis in naïve metabolism. However, during cytochrome oxidase blockade, there was an increase in muscle lactate that was further accentuated during sepsis. Muscle pyruvate did not decrease during sepsis in naïve metabolism. During cytochrome oxidase blockade, there was a decrease in muscle pyruvate, independently of sepsis. Lactate to pyruvate ratio increased during sepsis and was further accentuated during cytochrome oxidase blockade. Muscle glycerol increased during sepsis and decreased slightly without sepsis regardless of N+/K + -ATPase or cytochrome oxidase blocking. There were no significant changes in muscle glutamate or urea during sepsis in absence/presence of N+/K + -ATPase or cytochrome oxidase blockade.

Conclusions

These results indicate increased metabolism of energy substrates in muscle tissue in experimental sepsis. Our results do not indicate presence of energy depletion or mitochondrial dysfunction in muscle and should similar physiologic situation be present in other tissues, other mechanisms of organ failure must be considered.

P350 Pilot study showing reduced bone strength at 96 hours in rodent sepsis

ME Cove1, NS Chew2, LH Vu2, RZ Lim2, Z Puthucheary3

1National University Hospital, Singapore, Singapore; 2Yong Loo Lin School of Medicine, National University Singapore, Singapore, Singapore; 3University College London, London, United Kingdom

Introduction

Bone mineral density (BMD) is reduced in critical care survivors [1], and long-term follow up has shown increased fracture risk [2]. It is unclear if these changes are a consequence of acute critical illness, or reduced activity afterwards. Bone health assessment during critical illness is challenging, and direct bone strength measurement is not possible. We used a rodent sepsis model to test the hypothesis that critical illness causes early reduction in bone strength and changes in bone architecture.

Methods

20 Sprague-Dawley rats (350 ± 15.8g) were anesthetised and randomised to receive cecal ligation and puncture (CLP) (50% cecum length, 18G needle single pass through anterior and posterior walls) or sham surgery (cecum mobilised, no CLP), and then returned to their cages. 10 rodents (5 CLP, 5 sham) were sacrificed at 24 hours, and the remaining 10 at 96 hours. Femur bones were harvested and bone strength testing was conducted using the Instron 5543 (Instron Corp, USA). Trabecular bone strength was measured using a femoral neck break and cortical bone strength tested using a femoral shaft 3-point bending test. Bone architecture was assessed using micro-computerised tomography (microCT) imaging (PerkinElmer, USA), and images analysed with BoneJ [3].

Results

All 20 rats survived to the end of the protocol. The load required to fracture the femoral neck and shaft was not significantly different for CLP and sham groups at 24 hours (97 ± 19N vs 81 ± 10N p = 0.12 and 127 ± 8N vs 119 ± 18N p = 0.35, respectively). However, at 96 hours there was a significant reduction in the fracture force at both the femoral neck and shaft in the CLP group, compared to sham (75 ± 11N vs 97 ± 13N p = 0.02 and 102 ± 20N vs 139.9 ± 28N p = 0.04). In contrast, there were no bone architecture differences, as measured by bone volume/total volume, trabecular thickness/separation, connectivity density, anisotropy and BMD (all p > 0.20) using microCT at 24 or 96 hours.

Conclusions

In this rodent model of sepsis, there is a significant reduction in trabecular and cortical bone strength at 96 hours. In the absence of changes in bone architecture, these findings suggest sepsis may induce early biochemical changes affecting bone strength. We plan further rodent experiments to confirm these results, increase our power, assess nano-mechanics and complete a histological analysis.

References

1 Orford NR et al: Am J Resp Crit Care 2016,193:736–744

2 Rawal J et al: Crit Care 2015,19:165

3 Doube M et al: Bone 2010, 47:1076–1079

P351 Endotoxin clearance by the spleen is unaffected by pre-existing systemic inflammation in porcine septic shock

K Hanslin1, F Wilske2, P Skorup2, E Tano2, J Sjölin2, M Lipcsey1

1Uppsala University, Uppsala, Sweden; 2Uppsala University, Department of Medical Sciences, Uppsala, Sweden

Introduction

As part of the mononuclear phagocytic system, the spleen participates in bacterial and endotoxin clearance. In our previous study we saw decreased endotoxin clearance by the liver in pigs with pre-existing systemic inflammatory response (SIR). We therefore hypothesized that immunosuppression induced by SIR may also lead to decreased trans-splenic endotoxin clearance, and set out to investigate this in a porcine model of sepsis.

Methods

15 anesthetized pigs received an [i]E. coli[/i] infusion intravenously (i.v.) for 3 hours (h). In group Pre-existing SIR (n = 6), SIR was induced by 24 h of i.v. endotoxin infusion prior to the [i]E. coli[/i] infusion. Group Non-Pre-existing SIR (n = 6) received the bacterial infusion without prior endotoxin exposure. To study the effects of 24 h of anesthesia alone, “Controls” (n = 3) received saline instead of endotoxin for 24 h prior to the bacterial infusion (not included in the primary analysis). The kinetic chromogenic LAL-test was used to analyze endotoxin in arterial and splenic venous blood samples.

Results

All animals receiving endotoxin developed SIR prior to the [i]E. coli[/i] infusion. The amounts of [i]E. coli[/i] given to the groups were comparable. Endotoxin levels were similar at 3 h, just before the end of the [i]E. coli[/i] infusion, in the Pre-existing SIR and Non-Pre-existing SIR groups in arterial (Fig. 1) and splenic venous blood (2.40 (1.94-2.60) vs. 2.81 EU/mL (2.73-2.91) median (IQR)). Furthermore, endotoxin levels at 4 h, one hour after completed [i]E. coli[/i] infusion, were lower in Pre-existing SIR vs. Non-Pre-existing SIR group both in arterial (Fig. 1) and splenic venous blood (0.38 (0.31-0.42) vs. 0.51 EU/mL (0.47-0.71); p < 0.05). There was no difference in the ratio of splenic venous to arterial endotoxin levels between Pre-existing SIR and Non-Pre-existing SIR groups.

Conclusions

In our model, the endotoxin clearance by the spleen is not affected by pre-existing inflammatory response in porcine [i]E. coli[/i] septic shock.

Fig. 1 (abstract P351).
figure 1

See text for description

P352 The role of autophagy in critical illness-induced organ failure

I Derese, S Thiessen, S Derde, T Dufour, L Pauwels, Y Bekhuis, G Van den Berghe, I Vanhorebeek

University Hospital, Leuven, Belgium

Introduction

Increasing evidence implicates mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR), as contributors to critical illness-induced organ failure. Both can be alleviated by autophagy, a cellular defense mechanism. However, a phenotype of insufficiently activated autophagy has been observed during critical illness. We hypothesized that insufficient hepatic autophagy during critical illness aggravates liver damage/failure, hallmarked by mitochondrial dysfunction and ER stress.

Methods

In a centrally catheterized mouse model of critical illness, induced by cecal ligation and puncture, the effect of genetic inactivation of hepatic autophagy (via inducible deletion of autophagy gene 7 in liver) on survival, markers of organ damage, apoptosis, UPR and mitochondrial content and function was evaluated in the acute (30 hrs) and prolonged (3 days) phase. For each time point, 2 groups of critically ill mice and 2 groups of healthy pair-fed mice were included (at least 10 surviving mice per group), where each time autophagy was inactivated in one group but not in the other.

Results

Hepatic autophagy deficiency during critical illness did not affect survival, but increased hepatic damage/dysfunction. In the acute phase, this was illustrated by higher plasma ALT (P = 0.0001), and by elevated markers of apoptosis (P = 0.001) and more mitochondrial dysfunction (Complex V activity, P = 0.02) in liver. In the prolonged phase, hepatic autophagy inactivation increased apoptosis (P = 0.01) and aggravated mitochondrial dysfunction (Complex V activity, P = 0.005) in liver. Autophagy deficiency did not affect mitochondrial DNA content (day 1 P = 0.98, day 3 P = 0.57). Autophagy deficiency time-dependently modulated several branches of the UPR in liver. On day 1, it decreased activation of the IRE1alpha-XBP1s (P = 0.003) and ATF6-CREB3L3 pathway (P = 0.003), coinciding with a diminished inflammatory response as shown by lower C-reactive protein gene expression (P = 0.006), but did not affect the p-eIF2alpha pathway (P = 0.26). At day 3, autophagy deficiency increased the activation of the p-eIF2alpha pathway (P = 0.03), but not the IRE1alpha-XBP1s (P = 0.37) or ATF6-CREB3L3 (P = 0.14) pathway.

Conclusions

Insufficient hepatic autophagy during critical illness aggravates liver damage, coinciding with more mitochondrial dysfunction and a time-dependent modulation of the UPR, hereby likely aggravating liver failure.

P353 Role of leptin and proprotein convertase subtilisin/kexin type 9 in modulating pulmonary inflammation in a murine model of early sepsis

M Khan1, D Dwivedi1, J Zhou1, A Prat2, NG Seidah2, PC Liaw1, AE Fox-Robichaud1

1McMaster University, Hamilton, Canada; 2Montreal Clinical Research Institute, Montreal, Canada

Introduction

Obesity increases the risk of sepsis but how obesity shapes the immune responses to infection is unknown. Similar to patients, we previously demonstrated that Western diet fed obese mice have reduced lung inflammation during early sepsis. In this study we explore the potential mechanisms to explain this finding. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a protein involved in cholesterol homeostasis that is implicated in sepsis survival. Leptin is a hormone produced by adipocytes that regulates energy homeostasis and is increased in obesity and sepsis. We hypothesized that either PCSK9 and/or leptin contributes to the obesity-associated lung protection in sepsis.

Methods

PCSK9 deficient, PCSK9 overexpressing and wild type mice on a C57/Bl6 background were fed either a high fat Western diet (WD) or a normal chow diet (NCD) for 15 weeks (n = 5/group). Sepsis was induced by cecal ligation and puncture (CLP). Tissues were harvested six hours post surgery. For the leptin studies mice were housed in static cages for 10-12 weeks. Mice were injected with recombinant leptin protein (1mg/kg)) one hour prior to CLP, then re-anesthetized and tissues collected at 6 hours. All mice were resuscitated with 2ml of lactated Ringers SQ pre surgery, and 1ml IV post surgery. Lung injury was assessed by myeloperoxidase (MPO in U/mg of tissue) assay of lung tissues and histopathology scores. Data are expressed as mean ± SEM and analyzed using ANOVA or t-test.

Results

Septic PCSK9 over expressing mice fed NCD had greater lung MPO levels (46.5 ± 4.5) compared to PCSK9 deficient mice (31.1 ± 1.7) on NCD (p < 0.01). In mice fed the WD for 15 wks the protection from the loss of PCSK9 was no longer present, however the injury was reduced. Septic PCSK9 deficient (14.4 ± 1.4), wildtype (17.6 ± 0.9) and overexpressing (17.9 ± 1.0) mice on WD had no significant differences in lung MPO levels. This correlated with histopathology scores for PCSK9 deficient (0.7 ± 0.2), wildtype (1.1 ± 0.2) and PCSK9 overexpressing (1.3 ± 0.7) septic mice. We found that leptin treated septic mice had lower lung MPO (32.6 ± 1.6) levels compared to saline treated septic mice (46.6 ± 3.5) (p < 0.001). Sham operated mice had significantly lower MPO levels (12.7 ± 1.7 for leptin and 11.8 ± 1.2 for saline) compared to septic counterparts.

Conclusions

Our data suggests that both lack of PCSK9 and increases in leptin contribute to the lung protection in early sepsis. However, when exposed to a WD the potential benefits of PCSK9 deficiency to further reduce lung injury are no longer evident. These findings have implications for potential therapeutic strategies to reduce sepsis-induced lung injury.

P354 The role of oxygen delivery on plasma lactate and organ failure in experimental septic shock

M Von Seth, P Skorup, L Hillered, A Larsson, J Sjölin, M Lipcsey

Uppsala University, Uppsala, Sweden

Introduction

The concept resuscitation of patients with septic shock, aiming at normalization of oxygen delivery (DO2), to limit tissue dysoxia and organ failure has not been confirmed in recent trials. Elevated plasma lactate in septic shock is considered as a key marker of inadequate DO2. We hypothesized that, apart from severely decreased levels, DO2 is not associated to plasma lactate in a model of septic shock.

Methods

We investigated the effects of circulatory shock and inflammation on plasma lactate in a retrospective analysis of 105 anesthetized endotoxemic (N = 61) or bacteremic (N = 44) piglets in shock. Tumor Necrosis Factor alpha (TNF-α) and Interleukin-6 (IL-6) were measured hourly during 6 hours (h) of shock. Muscle metabolism was monitored by microdialysis. The animals were stratified per degree of shock by DO2. The primary analysis was the breakpoint of insufficient DO2 to yield an elevated plasma lactate. ANOVA and regression models were used.

Results

All animals developed macrocirculatory shock, elevated plasma and muscle lactate levels, elevated levels of cytokines in plasma, as well as renal and pulmonary failure. At 3 h, DO2 was 289 ± 68 mL x min-1 x m-2 (mean ± SD) and plasma lactate levels were 2.7 (2.0-3.6) mmol x L-1 (median(IQR)). Mixed venous saturation (SvO2) decreased and oxygen extraction increased linearly with DO2 (p > 0.001). Oxygen consumption (VO2) was not DO2 dependent.

Plasma lactate increased at DO2 < 250 mL x min-1 x m-2 (p < 0.001). Urinary output decreased at DO2 < 250 mL x min-1 x m-2 (p < 0.01), but static lung compliance was not DO2-dependent. Muscle glucose, lactate and pyruvate, urea and glutamate were not DO2-dependent. Muscle glycerol was DO2-dependent without breakpoint.

Plasma lactate correlated to Mean Arterial Blood Pressure (MAP), DO2 and peak IL-6, but not Systemic Vascular Resistance Index (SVRI) and peak TNF-α.Urinary output correlated to DO2 and MAP. Static lung compliance did not correlate to any parameters above.

Over time, muscle pyruvate increased and muscle glycerol and glucose decreased but no changes in muscle lactate and glutamate were seen. Muscle pyruvate correlated to MAP. Muscle glycerol correlated to MAP and to TNF-α.

Conclusions

In porcine experimental sepsis, elevated plasma lactate was only associated with very low DO2 while oxygen consumption was unaffected by low DO2 despite development of organ failure. Tissue metabolism was associated with both inflammatory and circulatory changes. Our findings suggest that the current concepts of resuscitation focusing on restoration of oxygen delivery must be combined with measures to limit the inflammatory response.

P355 The lung is not a significant source of lactate in a pig model of sepsis

A Otterbeck, K Hanslin, M Lipcsey, A Larsson, M Von Seth

Uppsala Universitet, Uppsala, Sweden

Introduction

We used a porcine sepsis model to investigate pulmonary hypoxia as an explanation for lactate elevation in sepsis. We measured the pulmonary lactate production and shunt fraction during bacteremia. Sepsis is a condition characterized by severe organ failure as a result of a dysregulated response to infection. Central in many pathophysiological theories is the decreased delivery or utilization of oxygen by tissues. Normal physiology dictates that hypoxia leads to lactate production, a prognostic marker in sepsis. Thus, hypoxia has been used as an explanation for both organ dysfunction and elevated plasma (p-)lactate in sepsis. However, new research has implied other mechanisms. Previous studies have reported increased pulmonary lactate production in sepsis [1], the lungs being a generally well-oxygenated organ. However, these studies have not measured pulmonary shunt fraction and hence cannot estimate if the entire lung is ventilated.

Methods

We used 13 anesthetized pigs where 9 were randomized to a sepsis group and 4 were randomized to a sham group. All pigs received a pulmonary artery catheter and an arterial line. Pigs in the sepsis group were infused with live Escherichia coli for 3 hours (h) and in the sham group with NaCl. Blood cultures were used to confirm bacteremia. Blood gases, blood tests and physiological parameters were collected hourly. Lactate production was calculated by the p-lactate gradient from pulmonary artery to systemic artery and cardiac index. Shunt fraction was estimated at 3 h after ventilation with 100% O2 for 5 minutes.

Results

Sepsis occurred in all pigs in the sepsis group, according to the criteria from Sepsis-3, and in no pigs in the sham group (p = 0.03). Global oxygen delivery (DO2) remained equal in both groups. Arterial lactate was higher (p = 0.003) in the sepsis group after 1 hour with a median value of 2.3 mmol/L vs. 0.95 mmol/L. Negative and positive lactate production occurred over the lungs in both groups (Fig. 2). There was no difference in pulmonary lactate production or pulmonary shunt fraction between groups. Neither group had significant shunt formation.

Conclusions

In this study we found a high p-lactate in septic pigs despite a high DO2. In absence of pulmonary shunts, the lung was not a major source, nor a major scavenger, of plasma lactate.

Reference

1. Garcia-Alvarez, M. et al. Sepsis-associated hyperlactatemia. Crit. Care Lond. Engl. 18, 503 (2014).

Fig. 2 (abstract P355).
figure 2

See text for description

P356 Association between inflammation, mitochondrial function and lactate clearance

T Correa1, J Pereira1, J Takala2, S Jakob2

1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction

Systemic lactate clearance (LaCl) higher than 10% during the first hours of sepsis resuscitation is associated with better outcomes, but the mechanisms are unclear. We aimed to investigate the relationship between lactate clearance, inflammatory response, and mitochondrial respiration.

Methods

Original data from two previously published studies were re-analyzed [1,2]. In cohort 1, pigs were randomized to be resuscitated for 48h starting 6, 12 and 24h, respectively, after fecal peritonitis induction (n = 8, each) [1]. Hemodynamics, inflammatory parameters, and mitochondrial function were analyzed. In cohort 2, 16 pigs with fecal peritonitis were immediately resuscitated for 24h [2]. Regional lactate exchange was measured. Animals of both groups were categorized according to LaCl > =10% or <10% during 6h of resuscitation.

Results

Overall mortality was 20% (4/20) for animals with LaCl > =10% and 60% (12/20) for animals with LaCl > =10% (p = 0.022). In cohort 1, systemic hemodynamics were similar in LaCl > =10% (n = 13) and LaCl < 10% (n = 11) groups. Plasma interleukin-6 levels were lower at study end in LaCl > =10% [45 (37-204) vs. 166 (128-310; median, IQR), p = 0.047]. Complex 1 state 3 [586 (386-688) vs. 353 (242-483), p = 0.026] and state 4 [157 (117-247) vs. 122 (89-151), p = 0.045], and Complex 2 state 3 [1156 (828-1401) vs. 761 (664-932), p = 0.041] and state 4 [376 (281-451) vs. 269 (225-326), p = 0.032] isolated brain but not hepatic, myocardial or skeletal muscle mitochondrial respiration were higher at study end in LaCl > =10% compared to LaCl < 10%. In cohort 2, mesenteric, total hepatic and renal blood flows were higher at study end in LaCl > =10% (n = 7) vs. LaCl < 10% group (n = 9), despite similar cardiac output. Hepatic lactate influx and uptake were approximately 1.5 and 3 times, respectively, higher in LaCl > =10% vs. LaCl < 10% (p = 0.066, both).

Conclusions

Systemic lactate clearance > =10% vs. <10% during early resuscitation after abdominal sepsis was associated with lower plasma interleukin-6, and higher brain mitochondrial respiration. Blood flow redistribution to abdominal organs in animals with high systemic lactate clearance increases the potential to deliver and extract lactate.

References

1. Correa TD et al. Effect of treatment delay on disease severity and need for resuscitation in porcine fecal peritonitis. Crit Care Med 40:2841–9, 2012.

2. Brandt S et al. Effect of fluid resuscitation on mortality and organ function in experimental sepsis models. Crit Care 13:R186, 2009.

P357 Dynamics of endotoxin, interleukin-6 and organ dysfunction after treatment with antibiotics in an E.coli porcine intensive care sepsis model

P Skorup1, L Maudsdotter2, E Tano1, M Lipcsey3, M Castegren1, A Larsson1, J Sjölin1

1Dept. of Medical Sciences, Uppsala, Sweden; 2Dept. of Molecular Biosciences, Stockholm, Sweden; 3Dept. of Surgical Sciences, Uppsala, Sweden

Introduction

Endotoxin released during Gram-negative bacterial infections induces the production of pro-inflammatory cytokines and may accentuate the development of septic shock [1]. Gram-negative bacteria exposed to â-lactam antibiotics in vitro release endotoxin but in a minor extent when the â-lactam antibiotic is combined with an aminoglycoside [2]. The primary purpose of the study was to investigate the dynamics in endotoxin and interleukin-6 (IL-6) concentration as well as leukocyte activation and subsequent organ dysfunction in a large animal intensive care sepsis model in order to explore the relevance of antibiotic-induced endotoxin liberation and inflammatory response in vivo. Whether the addition of an aminoglycoside to a â-lactam antibiotic results in a reduced endotoxin release and systemic inflammation constituted a secondary aim.

Methods

A prospective placebo-controlled study was conducted on anesthetized pigs in an intensive care setting. All pigs were administered Escherichia coli as a 3h intravenous infusion. At 2h the animals were subjected to antibiotic treatment (n = 18) receiving either cefuroxime alone (n = 9) or the combination of cefuroxime and tobramycin (n = 9), whereas controls received saline (n = 18). During 4h after administration of antibiotics/saline, plasma endotoxin, IL-6, leukocytes and organ dysfunction variables were recorded hourly and differences to the values before treatment were calculated.

Results

All animals developed sepsis. Antibiotic-treated animals demonstrated a higher IL-6 response (p < 0.001), stronger leukocyte activation (p < 0.001) and more pronounced deterioration in pulmonary static compliance (p < 0.01) over time in comparison with controls. Animals treated with the combination demonstrated only a trend towards less inflammation in comparison with animals treated with cefuroxime alone. In plasma no differences in endotoxin concentration were observed between the groups.

Conclusions

Treatment with antibiotics elicits an inflammatory IL-6 response which is associated with leukocyte activation and pulmonary organ dysfunction, whereas no observable differences were seen in plasma endotoxin concentration. The reduction in cefuroxime-induced endotoxin release after the addition of an aminoglycoside in vitro could not be reproduced in vivo.

References

1. Bozza FA et al.: Crit Care 2007; 11: 1.

2. Goscinsky G et al.: Scand J Infect Dis 2003; 35: 40–6

P358 Persisitent shift of th1 to th2 in one week after diagnosis of community-acquired severe sepsis predicts mortality

M Xue, JY Xu, L Liu, YZ Huang, FM Guo, Y Yang, HB Qiu

Southeast University, Nanjing, China

Introduction

Recent studies have revealed that inflammation mediated by CD4+ T cells may contribute to the pathogenesis of sepsis. The role of the Th(T helper)1/Th2 balance in sepsis remains largely unknown. The aim of this study was to investigate the th2/th1 pattern and its impact on disease severity and outcomes in patients with new onset community-acquired severe sepsis.

Methods

This was a prospective observational study. Patients with community-acquired severe sepsis admitted to ICU within 24 hours were included. Blood sample was collected on day of admission(Day0, D0), 3rd Day (D3) and 7th Day (D7) after admission. Th2 and Th1 in lymphocyte were tested by flow cytometry. The increase of th2/th1 (>0.22) indicated immunosuppression. According to the change of th2/th1, patients were divided into 3 groups: immunosuppression recovered in early stage (th2/th1 began to decrease on D3, Group 1), immunosuppression recovered in late stage (th2/th1 began to decrease on D7, Group 2), immunosuppression worsened (th2/th1 kept increasing in a week, Group 3). The organ dysfunction, hospital-acquired infection(HAI) and 28-day prognosis was recorded. All patients or their legal representatives provided written informed consent. The study is registered with ClinicalTrials.gov, NCT 02883218.

Results

Seventy-four patients were eligible for study during Sept 18, 2014 to Sept 30, 2016. There were 34 cases in Group 1, 19 cases in Group 2, and 21 cases in Group 3. Baseline characteristics(age, sex, source of bacteremia, presence of comorbidities) of the population in different groups were similar.

(1) Compared with Group1(11.8%), Group3(52.4%) showed a significantly higher 28-day mortality(P = 0.001), while group 2 showed a higher 28-day mortality of 31.6% with no significant difference (Fig. 3).

(2) There were no significant differences in terms of incidence of HAI and organ dysfunction among groups.

(3) The areas under the receiver operating characteristic (AUC) curves of value of th2/th1 on D7 was of great value(0.875)(Fig. 4). Using a th2/th1 on day7 cutoff value of >2.74 to determine 28-day mortality, the sensitivity was 76.2% with 96.1% specificity.

Conclusions

Persisitent shift of th1 to th2 in one week after diagnosis of community-acquired severe sepsis may be a predictor for immunosuppression. Patients with persistently increasing th2/th1 have poor outcome.

References

[1] Leentjens J, Kox M, Rebecca M, et al. Reversal of immunoparalysis in humans in vivo. Am J Respir Crit Care Med. 2012, 186: 838–845.

[2] Monneret G, Venet F, Pachot A et al. Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony. Mol Med. 2008, 14: 64–78.

[3] Inoue S, Suzuki-Utsunomiya K, Okada Y, et al. Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly. Crit Care Med. 2013, 41:810–819.

Fig. 3 (abstract P358).
figure 3

28-day mortality in different groups

Fig. 4 (abstract P358).
figure 4

Receiver operating characteristics (ROC) curves comparing the ability of value of th2/th1 on Day0, Day3, Day7 as well as change of th2/th1 in one week. D3-0, change of th2/th1 between Day3 and Day0; D7-0, change of th2/th1 between Day7 and Day0; D7-3, change of th2/th1 between Day7 and Day3. Legend 2 : Figure 4. Receiver operating characteristic (ROC) curves of value of th2/th1

P359 Genetic variants of intron region of aquaporin AQP5 gene and development of pulmonary edema in lung infection complicated by septic shock

A Kuzovlev, V Moroz, A Goloubev, A Myazin, A Chumachenko, V Pisarev

V.A. Negovsky Research Institute of General Reanimatology, Moscow, Russia

Introduction

Product of AQP5 gene belongs to a family of aquaporins (AQPs), membrane proteins, responsible for the selective transmembrane transport of water. However, the value of polymorphic variants AQP5 in the development and progression of pul_monary edema in severe lung infection was studied so far. The aim of the investigation was to determine the value of genetic variants of a single nucleotide polymorphic site rs3736309 of intron 3 of aquaporin_5 (AQP5) gene in the course of critical illness in patients with documented pulmonary infection.

Methods

Patients with critical illness admitted to the intensive care units were examined during the course of treatment (n = 86, age 27 to 82 years, mean age 53.20 ± 14.34 years). Main diagnosis included malignancies (15%), peritonitis (16%) and necrotizing pancreatitis (37%). Patients developed nosocomi_al pneumonia (55%), acute respiratory distress syndrome (ARDS) (54%), septic shock (48%), ARDS combined with septic shock (33%). DNA genotyping was carried out using tetra_primer polymerase chain reaction (PCR). Statistical processing was performed using GraphPad InStat program (GraphPad, USA).

Results

The distribution of frequencies of genotypes AA, GA and GG (AQP5, rs3736309) in cohort of patients corresponded to Hardy_Weinberg equilibrium (P = 0.923) and was similar to frequencies of the alleles determined in healthy Caucasian individuals (literature data) (P > 0.05). In a subgroup of patients with septic shock and AQP5 AA (rs3736309) genotype the lower EVLWI values were found compared to patients with geno_types GG and GA with septic shock in spite of the same approach to treatment. Genetic variant AQP5 G+ (rs3736309) contributed to the development of pulmonary edema resistant to treatment (odds ratio, OR = 6,75; P = 0.032). Only the subgroup of patients with septic shock and geno_type G + (but not all patients or the subgroup of patients without septic shock of the same genotype) were char_acterized by significantly elevated levels of surfactant protein SP_D in plasma compared to patients of genotype AQP5 AA with septic shock (P < 0.05).

Conclusions

In septic shock, the presence of homozygous variant allele A (AA) of AQP5 rs3736309 is a favor_able factor for patients developing the pulmonary edema. The presence of allele AQP5 G (rs3736309) is a risk fac_tor for developing severe pulmonary edema and unfavorable prognosis in spite of treatment.

P360 Withdrawn

P361 Altered T cell repertoire diversity and increased PD-1 expression predict mortality in patients with septic shock

N Takeyama, M Tsuda, H Kanou, R Aoki, Y Kajita, M Hashiba, T Terashima, A Tomino

Aichi Medical University, Aichi, Japan

Introduction

Sepsis causes impairment of innate and adaptive immunity by multiple mechanisms, including depletion of immune effector cells and T cell exhaustion. Although lymphocyte dysfunction is associated with increased mortality and potential reactivation of latent viral infection in patients with septic shock, the relation between viral reactivation and lymphocyte dysfunction is obscure. The objectives of this study were 1) to determine the relation of lymphocyte dysfunction to viral reactivation and mortality, and 2) to evaluate recovery of lymphocyte function during septic shock, including T cell receptor (TCR) diversity and the expression of programmed death 1 (PD-1).

Methods

In 18 patients with septic shock and latent cytomegalovirus infection, serial blood samples were obtained on days 1, 3, and 7 after the onset of shock, and immune cell subsets and receptor expression were characterized by flow cytometry. TCR diversity of peripheral blood mononuclear cells was analyzed by Multi-N-plex PCR, and cytomegalovirus DNA was quantified using a real-time PCR.

Results

Monocytes showed a decrease of TCR diversity and HLA-DR expression in the early stage of septic shock, while CD4+ T cells displayed an increase of PD-1 expression. Normalization of TCR diversity and PD-1 expression was observed by day 7, except in patients who died. cytomegalovirus reactivation was detected in 3 of the 18 patients during the first week of their ICU stay and all 3 patients died.

Conclusions

These changes are consistent with the early stage of immune cell exhaustion and indicate the importance of normal lymphocyte function for recovery from septic shock. Ongoing lymphocyte dysfunction is associated with cytomegalovirus reactivation and dissemination, as well as with unfavorable outcomes.

P362 Vasopressin alone and with noradrenaline attenuates TNF-α production in an in-vitro model of monocyte priming and deactivation

R Davies, KP O´Dea, S Soni, JK Ward, DJ O´Callaghan, M Takata, AC Gordon

Imperial College, London, United Kingdom

Introduction

Vasopressin is a safe and effective ‘catecholamine-sparing’ vasopressor in septic shock [1]. It also has anti-inflammatory properties, including inhibition of endotoxin-induced inflammatory cytokine release from macrophages in-vitro [2]. To further assess vasopressin’s anti-inflammatory effects in sepsis, we developed an in-vitro assay of monocyte priming and deactivation to model the pro- and anti-inflammatory responses, respectively.

Methods

Healthy volunteer CD14+ monocytes were cultured at 37°C for 20hrs with lipopolysaccharide (LPS, 100pg/ml) or IL-10 (10ng/ml), in combination with noradrenaline (5000pg/ml) and ‘high’ (300pmol/L) or ‘low’ (100pmol/L) dose vasopressin. Monocytes were analysed for HLA-DR and CD86 (T-cell co-stimulatory ligand) expression by flow cytometry, or stimulated with a ‘second-hit’ of LPS (10ng/ml) and TNF release measured by ELISA.

Results

Pre-treatment of monocytes with LPS resulted in a primed phenotype of higher HLA-DR expression and TNF release on stimulation, whereas IL-10 reduced HLA-DR, CD86, and TNF release indicating a deactivated phenotype. Under normal and priming conditions, co-incubation with vasopressin alone or with noradrenaline significantly reduced TNF release (Fig. 5), but not HLA-DR/CD86 expression. In contrast, neither vasopressin nor noradrenaline affected the IL10-induced deactivated phenotype.

Conclusions

The vasopressin-mediated suppression of TNF release in normal or primed monocytes, but not in deactivated monocytes, suggests a selective immune-modulatory activity that may be beneficial in septic patients and warrants further investigation.

References

1. Gordon AC et al. JAMA.316:509–18,2016

2. Peng T-C et al. Tzu Chi Medical Journal.25:150–4,2013

Funding: Intensive Care Foundation

Fig. 5 (abstract P362).
figure 5

Effect of vasoprissin & noradrenaline on second-hit LPS induced monocyte TNF release. n = 4, **p < 0.01; ***p < 0.001

P363 Relationship between lymphocyte subset expression and serum concentrations of pd-1/pd-l1 in sepsis – pilot study

J Wilson1, Y Zhao1, M Singer2, J Spencer1, M Shankar-Hari1

1King’s College London, London, United Kingdom; 2Bloomsbury Institute of Intensive Care Medicine, London, United Kingdom

Introduction

Programmed death antigen (PD-1) and ligand (PD-L1) are inducible negative regulators on leukocyte surface. The PD-1/PD-L1 pathway contributes to lymphocyte exhaustion and immunosuppression in sepsis [1]. A clinical trial is currently evaluating the safety of an anti-PD-L1 antibody in sepsis patients [2]. However, serum levels and differences in PD-1/PD-L1 expression by B and T cell subsets are unknown and are likely to influence the efficacy of this intervention. We tested the hypothesis that surface PD-1/PD-L1 expression will differ in B and T cell subsets, and that serum PD-1/PD-L1 levels will be high in sepsis.

Methods

A prospective observational cohort study in 22 critically ill adult sepsis patients, excluding those with immune deficiency states, was done with ethics approval and informed consent. Blood was taken on ICU admission day and PBMCs isolated. Patients (11 survivors & 11 non-survivors) were compared with contemporaneously collected and analysed samples from 11 healthy controls. Cell surface staining was performed with antibodies to CD3, CD19 [BD Biosciences], CD4, CD27, PD-1, PD-L1 and PD-L2 [Biolegend], and live dead stain Amcyan [Invitrogen]. FACS analyses were performed on a FACScalibur flow cytometer [BD Biosciences] and using Tree Star FlowJo software. Serum PD-1 and PD-L1 in the same cohort were measured by ELISA [Proteintech]. Data was analysed using PRISM.

Results

The percentages of B and CD4+ T cells expressing PD-1 and PD-L1 were significantly higher in survivors and non-survivors of sepsis compared to healthy controls. There were no significant differences between survivors and non-survivors in expression of PD-1 or PD-L1 by any lymphocyte subset. CD4 + CD27- memory T cells had significantly higher mean fluorescence intensity (MFI) and percentage positivity of PD-1 than CD4 + CD27+ T cells. The PD-1 MFI was significantly higher in CD19 + CD27+ memory B cells than CD19 + CD27- B cells. Serum PD-1 and PD-L1 concentrations were not different in sepsis patients compared to controls and values did not correlate with surface expression of PD-1 or PD-L1 by any lymphocyte subset in sepsis patients.

Conclusions

We show higher expression of PD-1 and PD-L1 by B cells and CD4+ T cells in sepsis compared to health, and higher expression of PD-1 by memory compared to naïve B cells and CD4+ T cells. Further research to identify patients likely to benefit from PD-1/PD-L1 blockade in sepsis is required.

References

1. Hotchkiss R et al. Nat Rev Imm; 13(12):862–74, 2013.

2. https://clinicaltrials.gov/ct2/show/NCT02576457

3. Singer M et al. JAMA; 315(8):801–10, 2016.

P364 Pcsk9 loss-of-function genotype is associated with better short and long-term outcomes in sepsis

K Roveran Genga1, C Lo1, M S. Cirstea1, K R. Walley1, J A. Russell1, A Linder2, J H. Boyd1

1Center for Heart Lung Innovation - University of British Columbia (UBC), Vancouver, Canada; 2Lund University, Lund, Sweden

Introduction

Reduced activity of proprotein convertase subtilisin/kexin type 9 (PCSK9), by increasing the density of low density lipoprotein (LDL) receptors on hepatic cells, may decrease the systemic inflammatory response to sepsis due to increased clearance of pathogen lipids incorporated into LDL [1]. The purpose of this study was to determine the relationship between PCSK9 loss-of-function (LOF) variants and the risk of short and long-term death and/or hospital readmission(s) within a year following an episode of sepsis, in two distinct cohorts.

Methods

This was a retrospective observational study involving two cohorts from St. Paul’s Hospital in Vancouver, Canada: Cohort 1 was composed by 189 patients with septic shock admitted to intensive care unit between July 2000 and January 2004 and who survived at least 60 days post- hospitalization; Cohort 2 included 185 patients admitted to the Emergency Department from January 2011 to July 2013, with clinical diagnosis of sepsis. R46L, A53V, and I474V PCSK9 missense LOF SNPs were genotyped in all patients, who were classified in 3 groups: WT, 1 LOF, and 2 or more LOF according to the number of LOF alleles.

Results

Cohort 1: Time to event curves for 5-year mortality showed a trend to statistically lower probability of death within 5 years in patients with PCSK9 LOF (overall p = 0.062) and the presence of 1 PCSK9 LOF allele (Cox model) was independently associated with decreased hazard-ratio for 5-year mortality (0.578, 95% CI = 0.36-0.93, p = 0.024).

Cohort 2: Patients from the 2 or more LOF group had lower probability of death within 90 days in comparison to WT (p = 0.010) or 1 LOF patients (p = 0.028), and the presence of 1 PCSK9 LOF allele (Cox model) was independently associated with decreased HR for 90-day mortality (0.46, 95% CI 0.23-0.92, p = 0.030). Patients from the 2 or more PCSK9 LOF group also had lower probability of death or infection related readmissions when compared to WT (p = 0.015) or 1 LOF allele (p = 0.002), and the presence of 2 or more PCSK9 LOF alleles had the lowest adjusted HR for this outcome (HR = 0.32, 95% C.I. 0.11-0.92, p = 0.035).

Conclusions

PCSK9 LOF genotype is associated with decreased risk of 5-year and 90-day mortality, and all-cause 1-year death or readmissions due to infection after an episode of sepsis.

Reference

1. Walley KR et al.: Science translational medicine Sci Transl Med. 6(258):258ra143, 2014

P365 Monocyte surface marker expression profile and cytokine secretion of critically ill patients with pseudomonas aeruginosa induced sepsis

A Sedlag1, C Riedel1, M Georgieff2, E Barth2, H Bracht2, A Essig2, D Henne-Bruns2, F Gebhard2, K Orend2, M Halatsch2, M Weiss2

1University, Ulm, Germany; 2University Hospital Medical School, Ulm, Germany

Introduction

Some critically ill patients are at high risk of severe sepsis due to infections with Pseudomonas aeruginosa (PSA) in the lung or abdomen, which is difficult to treat. The present study was performed to find out whether these critically ill patients on a surgical intensive care unit with sepsis due to PSA have a characteristic monocyte surface receptor expression and cytokine secretion patterns.

Methods

The surface markers CD163 (hemoglobin scavenger receptor; clearance of hemoglobin, adhesion to endothelial cells, tolerance induction, tissue regeneration; soluble form: antiinflammation), CD206 (mannose receptor for mannose on surface on microorganisms), intracellular levels of IFN-γ, CXCR1 (IL-8α chemokine receptor) and CXCR2 (IL-8β chemokine receptor) of monocytes of critically ill patients with PSA sepsis and of healthy controls were analyzed by flow cytometry. Furthermore, the IL-8 secretion levels in vivo and ex vivo after LPS stimulation were determined by ELISA.

Results

20 surgical patients with severe sepsis / septic shock with underlying PSA infections and of 22 healthy controls were monitored. The monocytes of the patients showed differences in the IL-8 secretion level. In line with high IL-8 or low IL-8 secretion in serum (965 ± 139 pg/ml vs. 232 ± 15 pg/ml) as well as in culture after LPS stimulation (2838 ± 259 pg/ml vs. 1097 ± 356 pg/ml), the expression of surface markers IFN-γ, CXCR1, CXCR2 and CD163 was high or low, respectively, however, always above those of the healthy control group (p < 0.05). CD206, only, showed the opposite behavior in that CD206 was highly expressed (3657 ± 279 MFI) on IL-8 low cells, whereas a low expression (17 ± 6 MFI) could be observed on IL-8 high cells (p < 0.001). The IL-8 low group had markedly higher severity of disease scores (SAPSII 34 ± 8) than the IL-8 high group (SAPSII 17 ± 6), and worse outcome (p < 0.001).

Conclusions

Different patterns of monocyte surface pattern expressions are associated with low or high IL-8 secretion of monocytes in patients with PSA sepsis. Low IL-8 expression and the respective surface pattern on monocytes may be associated with worse outcome.

P366 Identification of a distinct metabolomic profile in acute influenza infection

M Chase1, E Freinkman2, A Uber1, X Liu1, MN Cocchi1, MW Donnino1

1Beth Israel Deaconess Medical Center, Boston, MA, United States; 2Whitehead Institute, Cambridge, MA, United States

Introduction

The goal of this investigation is to characterize the effect of acute influenza infection on the host metabolome. Metabolomics is an emerging field of research studying small molecules or metabolites providing a profile of the physiologic status of an organism at a point in time. The human metablomic response to acute influenza infection is not well-characterized. We hypothesized that acute influenza infection will induce a distinct metabolic response in the host which may enable the identification of host mediators in influenza pathogenesis.

Methods

We are conducting a randomized clinical trial administering atorvastatin or placebo to patients with acute influenza infection. As an exploratory aim, we assessed the metabolomic profile of enrolled patients at baseline, prior to study drug administration, compared to healthy controls. T-tests were used to compare 117 metabolites. Raw data were entered into MetaboAnalyst statistical software for analysis. We report findings based on Partial Least Squares-Discriminant Analysis (PLS-DA) which uses multivariate regression techniques to predict class membership based on original variables.

Results

We performed metabolomic analysis on serum samples of 49 statin-naïve patients with acute influenza and 25 healthy controls. We found 17 individual metabolites that were significantly different between groups at a threshold of p = 0.05. PLS-DA score plot demonstrated a distinct difference between influenza subjects and controls (Fig. 6) and PLS-DA model validation by permutation tests was highly significant (p < 5e-04). The most significant discriminating metabolites between influenza patients and controls were phosphocholine, phosphoethanolamine, nicotinamide, taurine, ADP, tryptophan, threonine, proline, citrulline (lower in flu samples) and kynurenine, acetoacetate, 3-hydroxybutyrate, hypoxanthine (higher in flu samples). Each of these metabolites had a VIP score of >1.4

Conclusions

In our metabolomics analysis of ED patients with acute influenza, we found a statistically significant difference in 17 metabolites as compared to healthy controls. We believe these data represent a potentially unique metabolic fingerprint in influenza infection. Further study is needed to elucidate potential metabolic pathways and host mediators that may contribute to influenza pathogenesis.

Fig. 6 (abstract P366).
figure 6

See text for description

P367 Von Willebrand factor and ADAMTS-13 influence the outcome of s. aureus bacteremia

M Peetermans1, L Liesenborghs2, J Claes2, T Vanassche2, M Hoylaerts2, M Jacquemin2, K Vanhoorelbeke2, S De Meyer2, P Verhamme1

1UZ Leuven, Leuven, Belgium; 2KU Leuven, Leuven, Belgium

Introduction

The size and functionality of the multimeric von Willebrand factor (VWF) molecule is regulated by its cleaving protease ADAMTS-13. While VWF and ADAMTS-13 levels correlate with disease course and outcome in the heterogenous population of septic patients, animal models have been inconclusive and mainly focused on gram-negative abdominal sepsis. It is unknown if modulation of the VWF/ADAMTS-13 balance can modulate the outcome of [i]S. aureus[/i] sepsis.

Methods

We aimed to study the role of VWF and ADAMTS-13 in [i]S. aureus[/i] sepsis, using patient data and an animal model.

Results

In patients with [i]S. aureus[/i] bloodstream infection, high VWF levels correlated with inflammatory parameters and inversely with kidney function. Low ADAMTS-13 levels were associated with disease severity and DIC parameters.

In a severe sepsis model, mice deficient in VWF showed improved survival, compared to wildtype mice. In contrast, [i]Adamts13-/-[/i] mice had increased mortality. Immediate clearance of bacterial load in blood was better in VWF deficient mice. The differences in mortality for the studied genotypes were associated with differential loads of organ microthrombi in liver and kidneys. Further experiments will study if administration of ADAMTS-13 can alleviate the course of [i]S. aureus[/i] sepsis.

Conclusions

In conclusion, this is the first study that consistently shows the relation of VWF, ADAMTS-13 and their ratio to disease severity in patients and mice with [i]S. aureus[/i] sepsis. Not only is the balance of VWF and its cleaving protease implicated in primary adhesion and bacterial retention, but VWF/ADAMTS-13 ratio also regulates the amount of organ microthrombi containing platelets, neutrophils and bacteria – and thus potentially end organ failure.

Targeting VWF multimers and/or the relative ADAMTS-13 deficiency that occurs in sepsis, should be explored as a potential new therapeutic target in [i]S. aureus[/i] endovascular infections.

P368 Admission levels of asymmetric and symmetric dimethylarginine predict long-term outcome in patients with community-acquired pneumonia

A Vögeli1, M Ottiger1, M Meier1, C Steuer1, L Bernasconi1, A Huber1, M Christ-Crain2, C Henzen3, C Hoess4, R Thomann5, W Zimmerli3, B Müller1, P Schütz1

1Kantonsspital Aarau, Aarau, Switzerland; 2University Hospital Basel, Basel, Switzerland; 3Kantonsspital Luzern, Lucerne, Switzerland; 4Kantonsspital Münsterlingen, Münsterlingen, Switzerland; 5Bürgerspital Solothurn, Solothurn, Switzerland

Introduction

During infection, there is an activation of the L-arginine-nitric-oxide pathway, with a shift from nitric oxide synthesis to a degradation of L-arginine to its metabolites, asymmetric and symmetric dimethylarginine (ADMA and SDMA). We investigated the association of L-arginine, ADMA, and SDMA with adverse clinical outcomes in a well-defined cohort of patients with community-acquired pneumonia (CAP).

Methods

We measured L-arginine, ADMA, and SDMA in 268 CAP patients from a Swiss multicenter trial by mass spectrometry and used Cox regression models to investigate associations between blood marker levels and disease severity as well as mortality over a period of 6.1 years.

Results

Six-year mortality was 44.8%. Admission levels of ADMA and SDMA (μ mol/L) were correlated with CAP severity as assessed by the pneumonia severity index (r = 0.32, p < 0.001 and r = 0.56, p < 0.001 for ADMA and SDMA, respectively) and higher in 6-year non-survivors versus survivors (median 0.62 vs. 0.48; p < 0.001 and 1.01 vs. 0.85; p < 0.001 for ADMA and SDMA, respectively). Both ADMA and SDMA were significantly associated with long-term mortality (hazard ratios [HR] 4.44 [95% confidence intervals (CI) 1.84 to 10.74]) and 2.81 [95%CI 1.45 to 5.48], respectively). No association of L-arginine with severity and outcome was found.

Conclusions

Both ADMA and SDMA show a severity-dependent increase in patients with CAP and are strongly associated with mortality. This association is mainly explained by age and comorbidities.

Fig. 7 (abstract P368).
figure 7

Metabolism of nitric oxide

Fig. 8 (abstract P368).
figure 8

Kaplan-Meier survival estimates for SDMA

P369 Decreased functional protein c levels may be predictive of the severity of sepsis associated coagulopathy and DIC

D Hoppensteadt1, A Walborn1, M Rondina2, K Tsuruta3, J Fareed1

1Loyola University, Chicago, IL, United States; 2University of Utah School of Medicine, Eccles Institute of Human Genetics, Salt Lake City, UT, United States; 3Asahi Kasei Pharma America Corporation, Waltham, United Kingdom

Introduction

Sepsis is a severe systemic inflammatory response to infection that manifests with widespread inflammation as well as endothelial and coagulation dysfunction that may lead to hypotension, organ failure, shock, and death. Disseminated intravascular coagulation (DIC) is a complication of sepsis involving systemic activation of the fibrinolytic and coagulation pathways that can lead to multi-organ dysfunction, thrombosis, and bleeding, with a two-fold increase in mortality. Several studies have reported that low levels of protein C predict outcome in patients with severe sepsis. Protein C helps to regulate coagulation by controlling the activation of factors Va and VIIIa. In addition, activated protein C has anti-inflammatory functions. The purpose of this study was to determine the functional protein C levels in this cohort of patients over the 8 day study period and to correlate protein C levels with survival.

Methods

De-identified serial plasma samples from patients diagnosed with sepsis-associated coagulopathy (n = 137) were obtained from the University of Utah under an IRB approved protocol. The citrated plasma samples were collected from adult patients in the ICU upon admission and ICU days 4 and 8. In addition, plasma samples from healthy volunteers (n = 50) were purchased from George King Biomedical (Overland, KS). P). Patients were assigned a DIC score based on the International Society of Thrombosis and Hemostasis (ISTH)criteria and categorized as having sepsis and no DIC, non-overt DIC and overt DIC. Plasma samples were analyzed for functional protein C levels using a clot-based method (Diagnostica Stago, Parsippany, NJ).

Results

The functional protein C levels on day 0 and day 4 were decreased in the septic patients compared to normal controls (p < 0.0010). In addition, the functional protein C levels decreased with an increase in severity of DIC. On day 8, there was a decrease in the functional protein C levels in both the non-overt and overt DIC groups compared to normal and patients with sepsis and no DIC. There also was a significant decrease in functional protein C levels in survivors compared to non-survivors (p < 0.010).

Conclusions

These results underscore the importance of functional protein C levels in the regulation of hemostasis. Furthermore, these studies demonstrate that decreased functional protein C contributes to the pathogenesis of sepsis associated coagulopathy as evident by the observed relationship with the severity of sepsis and decreased protein C levels. Thus functional protein C levels may be a useful prognostic marker to risk stratify patients with sepsis and DIC.

P370 Cholesterol is a marker of multiple organ dysfunction syndromes after abdominal surgeries.

S Tachyla

Mogilev Regional Hospital, Mogilev, Belarus

Introduction

According to the third international consensus sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. The aim of the study was to investigate the possibility of using additional laboratory marker cholesterol for early detection of multiple organ dysfunction syndromes (MODS) after abdominal surgeries.

Methods

After approval the ethics committee of the Mogilev Regional Hospital in a prospective observational study included 58 patients aged 18 to 85 years. All patients underwent laparotomy abdominal surgeries after which hospitalized in the intensive care unit. In R group (n = 30) of patients in the postoperative period was followed by the development of MODS, in the C group (n = 28) - patients without MODS. Determination of cholesterol produced daily by AU 680 biochemistry analyzer.

Results

Patients on the R group marked decrease in the level of cholesterol on the 4th day after surgery with 158.7 (96.8; 189.6) mg / dL to 127.7 (112.2; 174.1) mg / dL (Wilcoxon test, p = 0.0035). In group C was no significant differences in cholesterol levels. In R group value of cholesterol on 4th day was significantly higher in patients (n = 17) with recovery 147.1 (112.2; 170.3) mg / dL, than in patients (n = 13) with a fatal outcome 116.1 (100.6; 127.7) mg / dL (Mann-Whitney U test, p = 0.034). In the surviving patients reduced cholesterol level normalization was observed after 14 days after the operation.

We performed ROC-analysis to determine the diagnostic significance of cholesterol as a marker MODS. The area under the curve was 0.726 (p <0.001) 95% confidence interval from 0.684 to 0.769, sensitivity 73.6%, specificity 63.8%. The optimal threshold level of cholesterol as a predictor of MODS was determined of 130.0 mg / dL.

Conclusions

Determining the level of cholesterol, together with an assessment of clinical symptoms and blood formula, will provide early diagnosis of MODS after abdominal surgeries.

P371 Relationship between white blood cell count and sepsis-related biomarkers in critically ill patients

T Ikeda, S Ono, T Ueno, S Suda, T Nagura

Hachiouji medical center, Tokyo medical university, Tokyo, Japan

Introduction

Antibiotic therapy is a very important treatment for critically ill patients, but long-term administration can lead to antimicrobial resistance. Procalcitonin (PCT) has been used as a biomarker to monitor the effectiveness of antibiotic therapy with the aim of shortening the administration period. This study aimed to clarify the relationship of WBC counts to sepsis-related biomarkers (procalcitonin [PCT], endotoxin activity assay [EAA], interleukin-6 [IL-6], and presepsin) and 28-day mortality rate in critically ill patients.

Methods

We studied 422 patients (L-group with WBC counts <4000: 79 patients; H-group with WBC counts >12,000: 343 patients). Blood biochemistry and PCT, EAA, IL-6, and presepsin were measured immediately after ICU admission. Results were expressed as the mean ± SD (median). The Mann-Whitney U-test and chi-square test or Fisher’s exact test were used for statistical analysis. A p-value of <0.05 was considered to indicate a statistically significant difference.

Results

Regarding background factors, the APACHE2 scores in the L-group and H-group were 24.9 ± 9.2 (23.5) and 22.4 ± 9.1 (23.0), and the SOFA scores were 9.3 ± 3.9 (9) and 12.9 ± 3.4 (8), respectively. These values showed no significant differences between groups. PCT levels in the L-group and H-group were 65 ± 96 (25) and 20 ± 51 (3.2) respectively; EAA levels in the L-group and H-group were 0.59 ± 0.26 (0.62) and 0.36 ± 0.22 (0.32), respectively. IL-6 levels in the L-group and H-group were 61,728 ± 10,8417 (15,150) and 2440 ± 3422 (217), respectively. All of these values in the L-group tended to be higher than in the H-group. On the other hand, presepsin levels in the L-group and H-group were 1277 ± 943 (882) and 2440 ± 422 (1210), respectively. The 28-day mortality rate in the L-group was 34%, and 14.7% in the H-group. There was a significant difference between groups (p < 0.05).

Conclusions

In critically ill patients who entered the ICU, most sepsis-related biomarkers (PCT, EAA, IL-6) tended to be higher in the low leukocyte count (WBC < 4000) group, but only presepsin tended to be higher in the high WBC (>12,000) group. Further study to explain this difference is necessary.

P372 Neutrophil-lymphocyte ratio and mortality during critical illness

E Damiani, R Domizi, C Scorcella, S Tondi, S Pierantozzi, S Ciucani, N Mininno, E Adrario, P Pelaia, A Donati

Università Politecnica delle Marche, Ancona, Italy

Introduction

Immunologic alterations are common during critical illness and may determine outcome. We evaluated whether lymphopenia (lymphocyte count <1000*106/mmc) or a higher neutrophil-lymphocyte ratio (NLR, as a marker of inflammation) were associated with mortality in critically ill patients.

Methods

Prospective observational study on 221 consecutive adult patients admitted to our 14-bed Intensive Care Unit (ICU). Neutrophil and lymphocyte counts were recorded every day. Receiver operating characteristics (ROC) curves and binary logistic regression analysis were used to test the association between lymphopenia/NLR and ICU-mortality.

Results

83% of patients showed lymphopenia at least once in the ICU stay. The mean lymphocyte count was a weak discriminant of ICU-mortality (area under the ROC curve: 0.63 [95% confidence interval 0.52-0.74]). A mean NLR [>=] 12 was able to discriminate Non-survivors with a sensitivity of 64% and specificity of 71% (ROC: 0.71 [0.61-0.80], Fig. 9). A mean NLR [>=] 12 was associated with higher mortality independently of age, presence of infection, days of mechanical ventilation and the Simplified Acute Physiology Score (adjusted odds ratio: 3.597 [1.760-7.354]).

Conclusions

A higher mean NLR was independently associated with mortality.

Fig. 9 (abstract P372).
figure 9

NLR and mortality

P373 Leukocyte activity assessment using magnetic levitation in septic patients

M Schou Andersen1, S Lu1, G Lopez1, AT Lassen2, I Ghiran1, NI Shapiro1

1Beth Israel Deaconess Medical Center, Boston, MA, United States; 2Odense University Hospital, Odense, Denmark

Introduction

In sepsis, chemotactic factors induce swelling and activation of leukocytes. We have invented a novel portable bedside device based on the principles of magnetic levitation (two opposing magnets with a capillary tube in between that suspend cells) to image and quantify morphological properties of circulating leukocytes using whole blood. The device separates blood cells based on their mass and magnetic properties. Our aim was to determine whether magnetic levitation technique, by measuring leukocyte size and morphology parameters can accurately identify Emergency Department(ED) patients with sepsis.

Methods

Single-center, prospective, observational cohort study of a convenience sample of adult (>17y) patients from a 56.000 visit ED or affiliated out-patient lab between 3/2016-11/2016. Inclusion criteria: Sepsis: patients admitted to the hospital with suspected or confirmed infection. Non-infected Controls: ED or outpatient clinic patients without infection or acute illness. Procedures: Half a microliter of whole blood collected in EDTA tube, mixed with a paramagnetic gadolinium solution, transferred to a capillary tube, and placed between magnets for imaging and data analysis. Primary analysis: comparison of sepsis patients vs non-septic controls. Covariates of interest: leukocyte area, length, width, roundness, standard distribution(SD) of levitation height (measure of mass/charge dispersion). Means reported with t-test comparisons and calculation of area under the curve for assessment of diagnostic accuracy.

Results

We enrolled 41 non-infected controls and 21 sepsis patients: sepsis(6), severe sepsis(9) and septic shock(6). Our analyses identified a significant increase in the size of the circulating leukocytes in sepsis patients versus controls, as seen by the following morphology parameters: mean cell area, 570 pixels (SD) (±115) vs 411 (±46), p < 0.0001 with AUC = 0.89(0.80-0.99); cell length, 30 pixels (±2.5) vs 25 (±1.9), p < 0.0001, AUC = 0.89(0.81-0.98); and cell width, 27 (±2.4) vs 23 (±1.5), p < 0.0001, AUC = 0.93(0.86-1.00). Cell roundness was 2.2 (±1.1) vs 2.2 (±1.2), p = 0.9, AUC = 0.55(0.40-0.71). For mass:charge, levitation height SD was 71 (±26) vs 47 (±16), p = 0.0012, AUC = 0.80(0.67;0.93).

Conclusions

Septic patients had increased area, length, and width with strong diagnostic accuracy. Sepsis patients had leukocytes with a pattern of more variable mass and magnetic properties. Cell roundness was less promising. Trends towards these differences were associated with increased severity. This bedside technique shows promise as a novel diagnostic test for infection.

P374 Intensive care patients undergoing tracheostomy have different patterns of cytokine and biomarker response

U Trahtemberg1, S Sviri1, M Beil2, Z Agur3, P Van Heerden1

1Hadassah University Hospital, Jerusalem, Israel; 2Marienhaus-Kliniken, Saarlouis, Germany; 3IMBM, Bene Ataroth, Israel

Introduction

Bedside tracheostomies are standardized, repeated procedures performed on relatively stable patients. The effects of tracheostomies at the biochemical level have not been studied before.

Methods

5 blood samples were obtained from patients undergoing bedside tracheostomies at t = 0, 4, 8, 12 and 24 hrs. Vital signs and clinical lab measures were also recorded. The blood samples were assayed for analytes shown in Fig. 10.

Results

We report results for 23 patients from this ongoing study. Parametric and nonparametric tests showed few statistically significant changes between the time points. Clearly discernible patterns of response were observed, which were different between patients. Using a self-organizing map clustering algorithm, we were able to cluster the responses over time, for every analyte eg GCSF showed 3 possible patterns of response: peak at t = 2 and then a steady decrease (4 pts); peak at t = 2 and then a plateau starting at t = 3 (12 pts); and peak at t = 2 with an increase at t = 4 (5 pts; 2 pts were excluded by the algorithm). The other cytokines showed between 3 to 5 clusters of response patterns each.

Conclusions

Results were not amenable to parametric or non-parametric approaches. We could classify the patients according to response patterns. This highlights the need for the development of individual-level measures and the personalization of clinical care

Fig. 10 (abstract P374).
figure 10

Clusters of cytokine patterns after tracheostomy.

P375 The h3 haplotype of the EPCR gene determines high sEPCR levels in critically-ill patients

E Jahaj1, A Vassiliou1, Z Mastora1, SE Orfanos2, A Kotanidou1

1Medical School of Athens University, Evangelismos, Athens, Greece; 2Medical School of Athens University, “Attikon” Hospital, Athens, Greece

Introduction

The endothelial protein C receptor (EPCR) is a protein that regulates the protein C anticoagulant and anti-inflammatory pathways. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits activated protein C (APC) activities. The clinical impact of sEPCR and its involvement in the septic process is under investigation. This study investigated possible association of EPCR haplotypes with sEPCR levels in critically-ill patients with suspected infection.

Methods

Two polymorphisms in the EPCR gene were previously genotyped in 239 Caucasian critically-ill patients, hospitalized in the intensive care unit (ICU) of &#8220;Evangelismos&#8221; Hospital, Athens, Greece. The old [1-2] and new [3] sepsis definitions were used to divide patients in groups. Patients were further divided according to their genotype. Plasma sEPCR levels were measured using a dedicated ELISA assay in all patients at the time of admission to the intensive care unit (ICU).

Results

Patients were categorized using both old and new sepsis definitions. With the old definitions patients were divided in two groups: severe sepsis/septic shock-positive (SS/SS + ve) and severe sepsis/septic shock-negative (SS/SS-ve). sEPCR levels were slightly higher in the SS/SS-ve group (p < 0.05). The patients were also divided according to their qSOFA score. In the three groups of patients, sEPCR levels were comparable (p > 0.05). However, when patients were divided according strictly to their genotype, plasma levels of sEPCR differed between genotypes (p < 0.0001) and between H3 and non-H3 carriers (p < 0.0001), with higher sEPCR levels in the H3 carriers.

Conclusions

Frequencies of SNPs determining EPCR haplotypes were in concordance with Caucasian frequencies. Critically-ill patients carrying at least one H3 allele had significantly higher levels of sEPCR than patients with no H3 alleles. Using both classification systems, sEPCR levels were not associated with sepsis severity.

References

1. ACCP/SCCM Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Critical care medicine 1992, 20(6):864–874.

2. Levy MM et al.: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive care medicine 2003, 29(4):530–538.

3. Singer M et al.: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Jama 2016, 315(8):801–810.

P376 The vasopressin surrogate marker copeptin reflects stress and predicts adverse clinical outcomes in unselected emergency patients: results of a multinational prospective cohort study

Y Wirz1, R Sager1, D Amin2, A Amin2, S Haubitz1, P Hausfater3, A Huber1, A Kutz1, B Mueller1, P Schuetz1

1Kantonsspital Aarau, Aarau, Switzerland; 2Morton Plant Hospital, Clearwater, FL, United States; 3Pitié-Salpêtrière, Paris, France

Introduction

Copeptin (pro-vasopressin) is a hormone derived from the same precursor protein as Vasopressin (AVP). Both hormones are secreted equimolar in response to physical stress and correlate with adverse clinical outcomes. In contrast to AVP, copeptin can be easily quantified by a sandwich immunoassay. Therefore, it serves as a surrogate marker for AVP and has emerged as a new prognostic marker in different settings including cardiovascular and infectious disease. The aim of this study was to evaluate the association between copeptin plasma concentrations and adverse outcomes in a large unselected study population seeking for emergency department (ED) care like under “real life” conditions.

Methods

This multicenter, observational cohort study consecutively included adult medical patients seeking for ED care in Switzerland, France and the USA during March 2013 to October 2014. We recorded initial clinical parameters and batch-measured the copeptin levels in a study population involving 7039 patients with available copeptin measures. We used logistic multivariate regression models (expressed in odds ratio, OR; 95% confidence interval, CI) and area under the receiver operating characteristic curve (AUC) to investigate the association of copeptin Plasma concentrations and different adverse outcomes (30-day all-cause mortality, intensive care unit [ICU] admission, initial treatment priority, main diagnoses and abnormal vital signs).

Results

During a 30-day follow-up 329 (4.7%) participants reached the primary endpoint of death. In our logistic regression models adjusted for age and gender copeptin levels (log transformed with a base of ten) significantly predicted the 30-day risk of death (OR 3.35, 95%CI 2.78-4.04, AUC 0.78), ICU admission (OR 2.89, 95%CI 2.48-3.36, AUC 0.69) and high initial treatment priority (OR = 2.23, 95%CI = 2.04-2.43, AUC = 0,66). Furthermore we found significant associations between copeptin levels and abnormal vital signs including low blood pressure (OR = 2.71, 95%CI = 2.35-3.11, AUC = 0.70). Stratifying by main admission diagnoses, subgroup analyses showed best performance of copeptin levels for 30-day mortality prediction in metabolic disorders (OR 11.00, 95%CI 2.46-49.20, AUC 0.87) and acute infections (OR 6.90, 95%CI 4.13-11.53, AUC 0.80).

Conclusions

Based on our findings copeptin serves as a strong prognostic marker on ED admission and might help to improve risk stratification in unselected medical ED patients.

P377 The association of admission procalcitonin levels and adverse clinical outcome across different medical emergency patient populations: results from the multi-national, prospective, observational TRIAGE study

RS Sager1, YW Wirz1, DA Amin2, AA Amin2, PH Hausfater3, AH Huber1, S Haubitz1, A Kutz1, B Mueller1, P Schuetz1

1University Department of Medicine, Kantonsspital Aarau, Aarau, Switzerland; 2Morton Plant Hospital, Clearwater, FL, United States; 3Groupe Hospitalier Pitié-Salpêtrière, Paris, France

Introduction

Although Procalcitonin (PCT) has been extensively studied in infectious conditions, the clinical relevance of PCT in unselected emergency department (ED) patients remains incompletely understood. We investigated association of admission serum PCT levels and adverse clinical outcomes in a large ED cohort from a previous multicenter study.

Methods

We prospectively enrolled 7132 adult medical patients seeking ED care in three tertiary care hospitals in Switzerland, France and the United States. We used adjusted multivariable logistic regression models to examine association of admission PCT levels and 30-day mortality, stratified by principal medical diagnoses and concomitant comorbidities. We calculated regression models across different clinically-established PCT cut-offs (0.05, 0.1, 0.25 and 0.5ng/ml).

Results

During a 30-day follow-up 328 (4.9%) participants died. Mortality rate in the cut-off stratified groups were 1%, 3%, 7%, 14 and 17%, respectively. PCT had a high prognostic power for 30-day mortality with an area under the receiver operating characteristic curve of 0.74 (95%CI 0.72- 0.77; SE 0.0133). After adjustment for age, gender and main diagnoses, PCT cut-off levels were associated with a step-wise increase in risk of 30-day mortality with an adjusted odds ratio of 1.9, 4.5, 8.6 and 11.0 for pulmonal disease; 1.9, 4.7, 8.7 and 11.0 for cardiovascular disease and 1.9, 4.6, 8.8 and 11.4 for infectious disease, respectively. These associations were similar among different types of patients in regard to main diagnoses, comorbidities and age of patients.

Conclusions

In this large medical ED patient cohort, admission PCT was a strong and independent outcome predictor for 30-days mortality across different medical diagnoses. PCT may help to improve risk assessment in undifferentiated medical ED patients.

P378 Procalcitonin level in klebsiella pneumoniae MDR infections in icu

L Gottin, C Dell´amore, G Stringari, G Cogo, M Ceolagraziadei, M Sommavilla, F Soldani, E Polati

University Hospital, Verona, Italy

Introduction

Infections due to K.Pneumoniae carbapenemase (KPC) are increasing [1]. Trials analyzing the levels of Procalcitonin (PCT) in KPC infections are lacking. Aim of this study was to evaluate the levels of PCT in KPC infections compared with other Gram negative and verify if PCT levels correlate with sepsis severity.

Methods

From March 2012 to May 2013 133 adult patients with documented Gram negative bacteria infection, admitted to intensive care department were observed. In 53 patients KPC was isolated (KPC Group, KPCG), while in 80 patients other Gram negative bacteria caused the infection (Control Group, CG). All the patients were classified daily according to sepsis severity. Organ dysfunction was evaluated daily according to the SOFA score. PCT levels were detected daily by means of a quantitative method (Liaison). Microbiological coltures and imaging exams were performed according to clinicians decision. Statistical analysis was performed by means of the SPSS11.0 software.

Results

As regards demographic data significant differences were showed except ICU LOS that was longer in KPCG. The main site of isolation of KPCs was the respiratory tract. According to the different sepsis severity diagnosis we found the following PCT levels (ng/ml). Sepsis: KPCG 0.9 ± 1.9, CG 6.6 ± 11.1 (p < 0.01); Severe Sepsis: KPCG 3.4 ± 6, CG 11.6 ± 19.8 (p < 0.01); Septic Shock: KPCG 8.9 ± 15.9, CG 30.5 ± 35.7 (p < 0.01) In KPC Group the mean PCT level in non-survivors was 7ng/ml, while in survivors was 2.7 ng/ml (p < 0.01). PCT mean values increased according to SOFA score increases.

Conclusions

Our data demonstrated that in septic patients with KPC infection PCT levels were significantly lower that those in septic patients with infection due to other Gram negative species. As regards outcome PCT levels confirmed to be related to severity of the disease with a mean level significantly higher in non-survivor compared to survivor. Mortality rate was higher in KPC Group compared to Control Group, but the difference was not significant.

References

1. NadKami AS, et al. Am J Infect Control 2009; 30: 1180–5

Table 1 (abstract P378). See text for description

P379 Prognostic value of procalcitonin or lactate clearance, or both, for risk assessment in sepsis patients

M Meier1, T Baumgartner1, G Zurauskaité1, S Gupta2, B Mueller1, A Devendra2, P Schuetz1

1Kantonsspital Aarau, Aarau, Switzerland; 2Morton Plant Hospital, Clearwater, FL, United States

Introduction

Several prognostic scores and biomarkers have been assessed for risk stratification in septic patients in intensive care units (ICUs). Serum lactate is a routinely used biomarker for the management of patients with sepsis and correlates with hypoperfusion and fluid resuscitation. Procalcitonin (PCT) is a bacterial infection marker and kinetics indicates the response to antimicrobial management. There is insufficient data comparing these two markers regarding outcome prediction. Herein, we compared the prognostic accuracy of lactate and PCT kinetics, and the combination of them, in a large, well defined US sepsis patient population.

Methods

This is an observational cohort study of adult patients with confirmed severe sepsis or septic shock included in a sepsis database from 14 different BayCare hospitals (Florida). All patients had PCT and lactate measurements on admission and during follow-up based on the treatment protocol. We used logistic regression and area under the curve (AUC) as a measure of discrimination of lactate and PCT with in-hospital mortality.

Results

The in-hospital mortality rate of the 1075 included patients (mean age 66.9 years) was 18.8%. Concerning prognosis, the initial lactate level was a better mortality predictor (AUC 0.64) compared to PCT (AUC 0.55). For follow-up measurements, PCT (AUC 0.75) showed a better discrimination than lactate (AUC 0.73). When looking at biomarker kinetics, PCT increase was more strongly associated with fatal outcomes compared to initial levels alone (AUC 0.74) and was a better predictor compared to lactate kinetics (AUC 0.63). A joint logistic regression model combining follow-up measurements of lactate and PCT-kinetics showed a superior prognostic accuracy (AUC 0.82) compared to these markers alone.

Conclusions

Both biomarkers, PCT, and lactate, provide prognostic information in ICU patients with sepsis, particularly when looking at kinetics.

Fig. 11 (abstract P379)
figure 11

Kaplan-Meier curve over 30-days stratified by score.

P380 Impact of CRP, procalcitonin and CRP on mortality related with sepsis in ICU

D Mandaci, G Eren, F Ozturk, N Emir, O Hergunsel

Bakirkoy Dr.Sadi Konuk Training and Research Hospital, Istanbul, Turkey

Introduction

Sepsis is known to be the leading cause of morbidity and mortality of ICU patients. APACHE 2, SAPS 2 ve SOFA scores are most frequently used for predicting mortality. Herein, it’s aimed to search for the effect of C-reactive protein (CRP), procalcitonin (PCT) and mean platelet volume (MPV) on mortality of sepsis in the ICU.

Methods

Retrospectively, 25 sepsis patients admitted to our ICU on May 2016 with at least 5 days of stay in the ICU were searched for APACHE 2, SAPS 2 and SOFA scores. The effect on mortality of CRP, procalcitonin and MPV values on the 1st, 5th and 10th days were analyzed with SPSS version 15 through receiver operating characteristic (ROC) curve analysis. Sensitivity, specificity, positive and negative predictive values were calculated.

Results

Patients were between the ages of 18-79 years with a median of 53 and a female-male ratio of 56-44%. ROC analysis revealed that the last day MPV values were highly predictive for mortality (AUC: 0.99, p < 0.01). The cut-off for this value was calculated as 7.73, with a sensitivity of 90%, specificity of 100%, and positive predictive value as100%, negative predictive value as 53%. CRP values on the last day of ICU stay were predictive for mortality as well (AUC: 0.85, p < 0.01) with a cut-off value of 7.55, sensitivity of 92.3% but a specificity of 87.5% (positive predictive value: 92.3% and negative predictive value: 63.6%). Procalcitonin values of neither the first nor the last day of ICU stay were predictive of mortality. APACHE 2, SAPS 2 and SOFA were found to be well correlated with MPV values and the last day CRP values, but not with any of the procalcitonin values.

Conclusions

Together with APACHE 2, SAPS 2 and SOFA scores, CRP and MPV values are good predictors of mortality of sepsis in ICU. We consider a further study with a larger sample size to evaluate better the effect of procalcitonin in this issue.

References

1. Faix James D. Biomarkers of sepsis. Crit Rev Clin Lab Sci. 2013 Jan; 50(1): 23–36.

2. Tajarernmuang P, Phrommintikul A, Limsukon A, et al. The Role of Mean Platelet Volume as a Predictor of Mortality in Critically Ill Patients: A Systematic Review and Meta-Analysis. Critical Care Research and Practice, 2016. doi:10.1155/2016/4370834

P381 Serum procalcitonin in cirrhotic patients: a reliable biomarker of bacterial infection?

S Azaiez, S Khedher, A Maaoui, M Salem

Charles Nicolle Hospital, Tunis, Tunisia

Introduction

Bacterial infection often leads to decompensated liver cirrhosis in cirrhotic patients. Its diagnosis should be swift, otherwise it is associated with a poor prognosis.

Our aim is to quantitate the various inflammatory markers in cirrhotics with and without obvious infectious disease.

Methods

A total of 80 patients admitted due to decompensated liver cirrhosis were included in this study. We explored the key epidemiologic, clinical and biological features of these patients.

Results

54 patients (67,5%) were assigned to the infection group of which nine patients (16,6%) had positive blood culture. In this group, the following diagnoses were made : 34 lung infections, 11 spontaneous bacterial peritonitis, nine urinary tract infections, three skin infections, and two gastroenteritis. Three patients had an infection without an identified source.

The mean age for the infected patients (group 1) was 62.8 years and for the uninfected ones 63.4 years (group 2). There were higher levels of C reactive protein (CRP) and procalcitonine (PCT) in group 1 than in group 2 (a mean of 52.7 mg/ L and 1.3 ng/ mL versus 13,3 mg/l and 0,1 ng/mL respectively). The median of CRP for group 1 was 43,15 mg/ L. Infection at admission was associated with significantly higher levels of these proteins (p = 0.000). Leucocyte count was also higher in group 1 (p = 0.04).

49 patients had CRP levels below 43.15 mg/ L, wherein 25 (51%) had infections. In this particular group, CRP was significantly higher in infected patients (p = 0.000), unlike PCT (p = 0,06) and leucocyte count (p = 0.23).

Conclusions

Elevated PCT appear only in inflammations of an infectious etiology with systemic signs. Therefore in cirrhotic patients, procalcitonin determination seems appropriate for the diagnosis of severe infections but does not seem more reliable than CRP in other situations.

P382 The assessment of bacterial load in ICU nosocomial pneumonia

E Chernevskaya1, N Beloborodova1, A Bedova1, YU Sarshor1, A Pautova1, V Gusarov2

1Negovsky Research Institute of General Reanimatology, Moscow, Russia; 2Pirogov National Medical and Surgical Center, Moscow, Russia

Introduction

Development of new microbial load biomarkers proceeds, but still - there is no perfect one. Procalcitonin (PCT) is commonly used and its level correlates with the extent of microbial invasion. Earlier it was found that aromatic microbial metabolites (AMM) - are associated with the severity and mortality of ICU patients [1, 2]. Also the effectiveness of the treatment can be evaluated by the AMM level [1]. Experimental studies have shown their biological activity [3,4]. The aim of this work is to analyze different criteria of bacterial load in ICU patients with nosocomial pneumonia.

Methods

In prospective study 46 patients with nosocomial pneumonia admitted to ICUs were observed in the first day. After liquid-liquid extraction from serum samples 9 phenylcarboxylic acids (benzoic (BA), phenylpropionic, cinnamic, phenyllactic (PhLA), p-hydroxybenzoic, p-hydroxyphenyllactic, p-hydroxyphenylacetic (HPhAA), p-hydroxyphenylpropionic and homovanillic (HVA)) were measured using GC-MS (Trace1310-ISQ, Thermo). DNA was extracted from bronchoalveolar lavage (BAL) samples from 5 of 46 patients for the quantitative detection of nosocomial bacteria by the PCR-real time (IQ5, BIORAD), PCT and presepsin were measured on the 1, 3, 7-9 days after the diagnosis of pneumonia. Spearman’s correlation coefficient was found, data presented as medians with interquartile range (IR, 25-75%) using STATISTICA 10.

Results

In serum samples of 46 patients the total concentration of 9 AMM was 3.4 (2.2-17.4) μ M that was higher (p < 0.05) than in healthy donors - 1.59 (1.46-1.85) μ M (n = 20). It correlated with APACHE II - 10 (6-18), SOFA - 3 (1-8) scores and mortality (41%): rs = 0.645, 0.666 and 0.717 respectively, p < 0.01. A closer analysis of 5 patient samples revealed the following correlations: the total concentration of 9 AMM correlated with PCT - 0.580, HVA with PCT and presepsin – 0.810 and 0.709, respectively, PhLA with presepsin-0.770, p-HPhAA with total DNA of bacteria and DNA Enterobactereacea in BAL– 0.635 and 0.724, p < 0.01.

Conclusions

The level of aromatic microbial metabolites in blood serum of ICU patients with nosocomial pneumonia reflects the microbial load as well as the quantity of bacterial DNA, PCT and presepsin.

Supported by Russian Science Foundation Grant 115-15-00110

References

1. Moroz VV et al. General Reanimatology 12(4): 37–48, 2016

2. Rogers AJ et al. PLoS One 9(1): e87538, 2014

3. Jankowski J et al. J. Am. Soc. Nephrol. 9(7): 1249–57, 1998

4. Beloborodova NV et al Anest. I rean. 61(3): 202–8, 2016

P383 Procalcitonin elimination during cytokine adsorption therapy in septic shock: a spin-off study of the ACESS trial

N Öveges1, I László1, M Forgács1, T Kiss1, P Hankovszky1, P Palágyi1, A Bebes1, B Gubán1, I Földesi1, Á Araczki1, M Telkes1, Z Ondrik1, Z Helyes2, Á Kemény2, Z Molnár1

1University of Szeged, Szeged, Hungary; 2University of Pécs, Pécs, Hungary

Introduction

According to in vitro data, levels of pro- and anti-inflammatory mediators can be markedly decreased in septic shock by hemoperfusion using a novel cytokine adsorbent therapy (CytoSorb). However, the capacity and performance of the absorber over time has not yet been investigated. Our aim was determine elimination of procalcitonin (PCT) by the adsorbent in vivo, in patients with septic shock treated with CytoSorb for 24 hours.

Methods

The current study is a spin-off part of the ongoing “Adsorption Cytokines Early in Septic Shock”, the ACESS-trial. CytoSorb therapy was commenced early (<48h) after the onset of septic shock and performed for 24 hours. Blood samples were taken from the systemic circulation in every 6 hourly from the beginning (T0) to the end of CytoSorb therapy (T6, T12, T18, T24). Serum PCT, CRP, IL-1, IL-1ra, IL-6, IL-8, IL-10, TNF-α levels were measured. PCT levels were determined from blood taken simultaneously from the pre-, and post-adsorbent samples. The efficacy of PCT elimination was defined at every step by subtracting post-adsorbent (PCTpost) values from pre-adsorbent (PCTpre) values: Δ PCT = PCTpre-PCTpost.

Results

Data were obtained from 7 patients and 8 treatments. Pre-adsorbent PCT levels showed a significant decline throughout the study (T0 = 12.2 [5.6-84.2], T6 = 6.8 [4.8-36.5], T12 = 6.7 [4.8-33.2], T18 = 5.9 [4.5-27.3], T24 = 5.1 [3.5-15.7] ng/ml; p = 0.011). Post-adsorbent PCT levels showed a similar pattern (p = 0.012). The efficacy of net PCT elimination (Δ PCT) was most effective at T0 = 11.7 [5.3-78.1] ng/ml, and showed a significant decline over time: T6 = 2.5 [1.4-14.9] ng/ml; T12 = 1.2 [0.3-6.1] ng/ml; T18 = 1.2 [0.5-4.8] ng/ml; T24 = 0.5 [-0.9-3.5] ng/ml; p = 0.004. This corresponded of a median of 90% elimination at T0, 30% at T6 and only 10% at T12 with no further change from T12-T24. This pattern showed consistency in every patient and was independent of the serum levels of PCT.

Conclusions

This study is the first to examine PCT elimination over time during CytoSorb therapy. According to these results, PCT elimination showed an exponential decline from 90% to a negligible degree after 12 hours. This phenomenon, on the one hand, shows the early efficacy of CytoSorb therapy, while on the other hand, it raises the question of changing the adsorbent earlier than the current practice of 24 hours.

Acknowledgements

NKFIH K116689

ClinicalTrials.gov ID: NCT02288975

P384 qSOFA (quick SOFA) score, presepsin and procalcitonin for severity assessment in initial sepsis

E Spanuth1, H Ebelt2, B Ivandic1, R Thomae3, K Werdan2

1DIAneering GmbH, Heidelberg, Germany; 2Department of Medicine III, University Clinics Halle (Saale), Martin-Luther-University, Halle-Wittemberg, Germany; 3Mitsubishi Chemical Europe, Düsseldorf, Germany

Introduction

The SOFA score is associated with an increased probability of mortality in sepsis. Assessment at admission in the ED requires several laboratory variables. The Third International Consensus Definitions for Sepsis and Septic Shock defined the qSOFA, which does not require laboratory tests and can be assessed at patient admission.

Objective: To compare sepsis biomarkers with SOFA and qSOFA to discriminate sepsis, severe sepsis or septic shock and to evaluate the association with increased risk of mortality.

Methods

66 Patients admitted to the ED with signs of sepsis and =/>2 SIRS-criteria were included. Severe sepsis and septic shock were defined according to current guidelines. qSOFA score was calculated using the recommended thresholds: respiratory rate =/>22/min, GCS score <15, stystolic blood pressure >100mmHG. Presepsin and procalcitonin were determined using the POC assay PATHFAST Presepsin (PSEP), LSI Medience, Japan and the BRAHMS luminescence immune assay (PCT).

Results

SOFA and qSOFA, differentiated significantly between patients with sepsis (n = 30, mortality = 6.6%) and the high-risk group with severe sepsis or septic shock (n = 36, mortality = 36.1%). Discrimination between the groups revealed AUC values of 0.621, 0.627, 0.731, 0.740 and 0.781 for lactate, PCT, qSOFA, PSEP, and the combination qSOFA + PSEP, respectively. 15 patients died during hospitalization. AUC values of mortality prediction were 0.715, 0.558, 0.734, 0.758 and 0.803 for lactate, PCT, qSOFA, PSEP and qSOFA + PSEP, respectively. qSOFA scores =/>2 should identify greater risk of death or prolonged ICU stay. Discrimination between qSOFA <2 and =/>2 revealed AUC values of 0.756, 0.669 and 0.606 for PSEP, lactate and PCT.

Using the threshold =/>2 of qSOFA and =/>500 ng/L of PSEP, the combination qSOFA + PSEP detected 14 non-survivors (93%) and 33 (92%) patients of the high-risk group (n = 36), whereas qSOFA alone detected only 10 non-survivors (67%) and 21 patients of the high-risk group (58%).

Conclusions

The results demonstrated that the qSOFA score is not a standalone criterion for risk stratification in sepsis at admission. Simultaneous assessment by combining qSOFA and PSEP improved the validity significantly. The POC assay PATHFAST Presepsin showed superior performance compared to lactate and PCT.

References

Singer M et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315(8):801–810.

P385 Role of presepsin compared to c-reactive protein in sepsis diagnosis and prognostication

M. El-Shafie1, K Taema1, M El-Hallag1, A Kandeel2

1Cairo University, Cairo, Egypt; 2El-Sahel Teaching Hospital, Cairo, Egypt

Introduction

Early identification of sepsis and its differentiation from non-infective SIRS are important for sepsis outcome. We intended to evaluate the use of presepsin in differentiating sepsis from non-infectious SIRS and its prognostic value compared to CRP.

Methods

We included 31 patients (median age 60 year old, 16 males) admitted with SIRS to El-Sahel Teaching Hospital, Egypt after excluding 21 patients with preadmission corticosteroids therapy, blood transfusion, immunosuppressive illness, and ICU length of stay (ICU-LOS) less than 24-hours. Patients were classified into non-infective SIRS group (13 patients) and sepsis group (18 patients). Presepsin, CRP and SOFA score were measured on admission and on days 2 and 4 of admission. The outcome parameters studied were ICU length of stay (ICU-LOS) and in-hospital survival.

Results

Apart from temperature and AST which were significantly higher in sepsis group, the two groups were comparable. All the presepsin levels and CRP on days 2 and 4 were significantly higher in sepsis than in SIRS groups. The ICU-LOS was positively correlated with all the presepsin levels and with the CRP levels on days 2 and 4. All Presepsin values were significantly higher in survivors while none of the CRP levels were significantly different in survivors and non-survivors. The decrease of presepsin over time was significantly associated with better survival. It was found to be 70% sensitive and 91% specific for predicting survival in SIRS patients. This relation was not found in CRP levels.

Conclusions

We concluded that the presepsin may be used for early differentiation between sepsis and non-infectious SIRS and predict higher mortality.

P386 Urinary albumin/creatinine ratio as an early predictor of outcome in critically-ill septic patients

O Tayeh1, K Taema1, M Eldesouky1, A Omara2

1Cairo University, Cairo, Egypt; 2Electricity Hospital, Cairo, Egypt

Introduction

Several cumbersome scoring systems were developed for prognosis and outcome prediction in sepsis. We intended in this study to evaluate the urinary albumin/creatinine ratio (ACR) as a prognostic predictor in sepsis.

Methods

We included 40 adult septic patients in a prospective observational study. We excluded patients with preexisting chronic kidney disease or diabetes mellitus. After clinical evaluation, urine spot samples were collected on admission and 24 h later for ACR1 and ACR2. Admission APACHE IV score and the highest recorded SOFA score of their daily estimation were considered. We also evaluated the need for mechanical ventilation, inotropic and/or vasoactive support, renal replacement therapy (RRT), and in-hospital mortality.

Results

In a population with 63 (55–71) year old with 29 (72.5%) males, we found that the ACR2 is correlated with the SOFA score (r =0.4, P = 0.03). SOFA was higher in patients with increasing ACR [14(4.8–16.8) vs 5(3–8), P = 0.01]. None of the ACR measures was correlated with APACHE IV score. ACR2 was higher in patients who needed mechanical ventilation and inotropic and/or vasoactive support [140(125–207) and 151(127–218) mg/g respectively] compared to [65(47–174) and 74(54–162) mg/g], P = 0.01 and 0.009. None of the measured parameters was related to the need of RRT. ACR1, ACR2, APACHE IV and increasing ACR were predictors of mortality. The AUC for mortality prediction was largest for APACHE IV (0.90) then ACR2 (0.88). ACR2 of 110.5