Sepsis 2019

P2 Epigenetic changes are reported in animal model of sepsis Monique Michels, Mariane Rocha Abatti, Andriele da Silva Vieira, Heloisa Borges, Amanda Indalécio Goulart, Roger Varella, Samira Valvassori, Felipe Dal-Pizzol Laboratory of Experimental Pathophysiology, Extreme University South of Santa Catarina, Criciúma, Brazil; Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde (PPGCS), Universidade do Extremo Sul Catarinense (UNESC), Criciúma, SC, Brazil Correspondence: Monique Michels (moniquemichels@hotmail.com) Intensive Care Medicine Experimental 2019, 7(Suppl 2):P2


Background
The presence of oxidative stress and inflammatory mediators in sepsis may lead to epigenetic changes [1,2]. Epigenetic alterations of histones, such as methylation, acetylation and phosphorylation may direct the folding or unfolding of DNA through mechanisms still unknown, thus altering gene transcription [3]. Once gene transcription in sepsis is altered the response may be further exacerbated. Our objective was to report epigenetic changes in brain structures in animal model of sepsis. Materials and Methods Male Wistar rats were subjected to sham or CLP and cerebral structures were removed in 24h, 72h, 10, 30 and 60 days after sepsis. HAT, HDAC and DNMT enzymes activities were measured in frontal cortex and hippocampus in different times.

Results
No changes found in HAT activity (Fig. 1). Increased HDAC (Fig. 2) and DNMT activity (Fig. 3) was observed 72h, 10 and 30 days after sepsis and a significant reduction 60 day after. Conclusions It's possible observe epigenetic alterations and deregulation in gene transcription in animal model of sepsis. Since in sepsis the presence of oxidative stress and the release of inflammatory mediators are well reported, these insults can lead to epigenetic changes, such as gene transcription, which may be related to exacerbation of the inflammatory response. Environmental influences can modulate the epigenetic response and therefore, be a therapeutic strategy for the treatment of sepsis.
Background Some aromatic derivatives of tryptophane -5-hydroxyindoleacetic acid (5-HIAA) and 3-indoleacetic acid  are under direct or undirect control of the gut microbiota and may play an important role in gut-brain axis. It was found that in serum high levels of some aromatic microbial metabolites (AMM) of phenolic structure are associated with severity of infection in critically ill patients with sepsis. Neuron specific enolase and S100 protein are biomarkers that reflect the neurotrophic and neurotoxic effects of neuron and glial cells. Procalcitonin (PCT) is a biomarker which is elevated in serum in the case of bacterial infection, however, the usefulness of PCT measurement in CSF has shown conflicting results. Cut-off value of PCT in CSF for bacterial meningitis was lower than serum level of PCT [1]. High level of PCT in CSF may indicate the loss of integrity of the blood-brain barrier, but some microbial metabolites may also penetrate the barrier in critical conditions. The aim of our study was to identify and qualify indolic and phenolic metabolites and evaluate correlation of certain biomarkers in the CSF in critically ill patients.

Materials and Methods
The study included 37 CSF samples taken from neurosurgical critically ill patients with CNS infection, traumatic brain injury. The levels of AMM and 3-IAA were measured in the CSF using GC-MS (Thermo Scientific), biomarkers (PCT, S100, NSE, IL6) were measured by Elecsys immunoassay, 5-HIAA was measured by ELISA (Cloud-Clone Corp).

Results
The median level of the sum of 6 aromatic metabolites (benzoic, phenyllactic (PhLA), p-hydroxybenzoic (p-HBA), phydroxyphenilacetic, homovanilic (HVA) and p-hydroxyphenillactic (p-HPhLA) acid) in the CSF was 4.4 μM. The direct Spearman`s correlation between the level of PCT and sum of AMM (0.35, p<0.05) was revealed. The correlations between neurological biomarkers and some phenolic and indolic metabolites were also revealed (table 1). Conclusions Connection between aromatic metabolites and neurological biomarkers (S100, NSE) indicates the potential involvement of phenolic and indolic metabolites in the pathogenesis of brain dysfunction. The impact of infection site in patients with sepsis on hospital mortality have not been reliably estimated1. We aimed to determine, in patients presenting to the emergency department with sepsis or septic shock, the association between the infection site and in-hospital mortality.

Materials and Methods
Multicenter prospective cohort in three emergency departments and critical care units of high complexity hospitals in Medellín (Colombia). We recruited patients older than 18 years admitted with sepsis or septic shock as the main diagnosis. The exposure variable was site of infection according to standardized CDC definitions and the primary outcome variable was in-hospital mortality. A hierarchical logistic regression model was fitted for adjusting for acknowledged prognostic factors as comorbidities, organ dysfunction and emergency treatments.

Results
From 5022 eligible patients, 2510 were included in the study. The most frequent site of infection was the urinary tract with 27.8% of the cases, followed by pneumonia with 27.5% and intra-abdominal focus with 10.8% of the patients. In the 5.4% of the cases there was not a clear site of infection at admission. Using hierarchical logistic regression models with urinary tract as the reference, there were significant differences in mortality (Table 1).

Conclusions
There is an association between the different sites of infection and inhospital mortality in patients with sepsis and septic shock and this should be considered in the prognostic models for these conditions. With regard to the PIM score, patients with sepsis (19.4%) had it between 0 to 1,1; and among those without sepsis (34.5%) were at the same score range (p-value = 0.025). Therefore showing correlation of higher scores and the possibility of the sepsis diagnosis. Conclusions Sepsis is a serious public health issue; the diagnosis is clinical and the faster and aggressive the treatment begins, it alters drastically the prognosis and development of the disease. The primary objective of the first hours of diagnosis is lower the death risk and restore signs of hypoperfusion, lowering the death risk and time of internship. Background Chronic suppurative otitis media is a chronic inflammation of the middle ear and mastoid cavity, with more than 2 weeks of otorrhea. Various studies have shown that both gram-positive and gramnegative bacteria, which differ according to the sites, are responsible for infection of middle ear. The knowledge of the prevailing flora and their susceptibility to antibiotics is an important step for an appropriate treatment Materials and Methods The current study was cross sectional survey involving enrolled 110 patients who consulted ENT Department at KUTH with active chronic suppurative otitis media or its complication, from November 2014 up to January 2015. The patient demographics, clinical presentation, microbiology and antibiotic sensitivity were collected using data collection sheet.

Results
The age of our population ranged between 2 and 89 years, the maximum was in the age range of 16-30 years (55.5%). The proportion of male to female was almost similar, male constituted 50. 9% while females were 49.1%. The majority had discharge for more than 5 years. For the results of culture and sensitivity, 65.5% showed significant microbial growth of single organism, with majority being Staphylococcus aureus 35%, followed by Klepsiella spp 15%, and Pseudomonas aeruginosa together with Enterobacter spp accounting for 10 % for each. S.aureus showed high sensitivity to ciprofloxacin and clindamycin, but it were resistant to penicillin. For overall of antimicrobial used, ciprofloxacin was revealed to be most effective antimicrobial drug against many organisms at 51.8%. Chloremphenicol was effective at 14.5% while cefotaxim and augmentin showed to be effective at 10% and 8.2% respectively. Conclusions There is variation in isolated organisms as well as antimicrobial drugs. For this reason, to know the exact sensitive antibiotic to a certain ear infection treated without success, it is advisable to do culture of discharge and sensitivity.

Background
Globally, approximately 2.5 million babies die in the first month of life [1]. Nearly all (99%) of these neonatal deaths occur in low income countries. The aim of this study was to describe the bacterial etiology and the antimicrobial resistance patterns of the isolated bacteria among newborns clinically suspected of having sepsis Materials and Methods A cross-sectional study was conducted at the Mulago national referral hospital in Kampala, Uganda. Venous blood for culture was collected from 305 newborns with clinical signs of sepsis. Validated questionnaires on mobile devices were used to obtain sociodemographic characteristics. An automated blood culture system was used (BD BactecTM) plus other conventional culture methods. Kirby Bauer disk diffusion method was used for antimicrobial susceptibility testing according to clinical laboratory standard institute. mecA PCR was conducted for confirmation of methicillin resistant Staphylococcus aureus (MRSA) Results The mean birth weight of the neonates was 3.1 kg (SD 0.6), 32% of them were ≤7 days old and 55% were males. The proportion of patients with a bacterial pathogen known to cause sepsis was 14% (95% CI; 10%-19%). This included 27 Staphylococcus aureus isolates, Escherichia coli (6), Klebsiella pneumoniae (5), Streptococcus pneumoniae (1), Neisseria spp (1), Enterobacter spp (1) and Citrobacter freundii (1). All the 5 K.pneumoniae isolates, 5/6 E.coli isolates and 26/27 S.aureus isolates were resistant to ampicillin. Resistance to the most commonly used aminoglycoside varied between species in that 6 (22%) of the S. aureus, one of the E. coli and two of the K. pneumoniae isolates were resistant to gentamicin. Among the twenty seven S.aureus isolated, 20(74%) were MRSA, 19 (70%) were resistant to erythromycin, 10 (37%) were resistant to ciprofloxacin, 7 (26%) were resistant to trimethroprim-sulphamethoxazole and 8(30%) displayed erythromycin inducible clindamycin resistance (D-test positive). However all S.aureus isolates were sensitive to vancomycin. Three Gramnegative enteric bacterial isolates were extended broad spectrum beta lactamase producers; 1 E. coli and 2 K. pneumoniae but were sensitive to imipenem. Conclusions S. aureus was the most common bacterial isolate among newborns with clinical signs of sepsis at the national referral hospital. The high frequency of MRSA among these isolates is worrisome and questions the empirical management of neonatal sepsis. Erythromycin inducible clindamycin resistance further limits treatment options for MRSA infections  Background Sepsis is a potentially fatal organ dysfunction caused by a dysregulated host response to infection. Acute kidney Injury is the most frequent complication in patients with septic shock and is an independent risk factor for death. Patients diagnosed with Sepsis-3 were included in a prospective observational protocol with the following objectives: 1) Mortality at 28 and 90 days, 2) Acute Renal Injury and causes of non-recovery at 7 days and 3) Type-5 Cardiorenal Syndrome.

Materials and Methods
All patients with Sepsis-3 were were included in the study (December 2017-December 2018.) Epidemiological data, SOFA, Nt-proBNP, proinflamatory cytokines, procalcitonin, lactate, primary site of infection, microbiological culture, days of ventilación and standard care were determined. To identify the subgroup of patients with ARF, we used sepsis as an initial insult and the KDIGO criteria to determine creatinine increase ≥ 0.3 mg / dl or 50% of the previous lower value within 48 hours of admission to the protocol, or urine volume <0.5 ml/kg/H in the same period. Patients with CKD or hemodialysis before admission were excluded. Septic shock was established in the initial protocol and NA drugs (5 μg/minute) were administrated. Aditionally, Nt-proBNP and echocardiographic were determined. Blood samples were collected and mRNA of proinflammatory cytokines were measured by RT-qPCR. Background Sepsis is a global public health problem representing a leading cause of morbidity and mortality and increased costs in many countries [1]. According to a previous study, implementation of a national sepsis program resulted in improved adherence to sepsis bundles in severe sepsis and septic shock patients and was associated with reduced adjusted in-hospital mortality [2]. Another recent study reported that implementation of multidisciplinary emergency department (ED) sepsis bundle was associated with improved time to achieve key therapeutic interventions and a reduction in 30-day mortality [3]. In other study, the use of a sepsis triage screening tool significantly decreased the time to antibiotics in patients presenting to the ED [4]. Surviving Sepsis Campaign (SSC) 2016 recommends that hospitals have a performance improvement program for sepsis [5]. We have newly developed Intelligent Sepsis Management System (i-SMS) which help clinicians to screen, diagnose, and manage septic patients. The purpose of the present study was to assess the effect of i-SMS on compliance with the SSC bundles and survival outcome in patients with sepsis and septic shock diagnosed in ED. In addition, we tried to determine risk factors for 28-day mortality.

Materials and methods
We performed a pre-post study in patients with sepsis and septic shock. During the pre-period (January 1, 2016-Setember 25, 2017), patients were managed with routine customary process. During the post period (September 26, 2017-July 10, 2018), patients were managed with assistance from i-SMS upon arrival to ED. Results A total of 548 patients were included; 317 in preperiod and 231 in postperiod (Table 1 and Figure 1). After implementation of i-SMS, overall compliance with SSC recommendations improved from 26.8% to 52.8% (P <0.001) ( Figure 2). There was no significant difference in 28-day mortality between preperiod (37.2%) and postperiod (32.0%) (P = 0.08) ( Table 1). In Kaplan-Meier survival analysis and Log-rank test, there was no significant difference of survival curves between preperiod and postperiod (P = 0.666) ( Figure 3). SOFA score and lactate levels were independent risk factors for 28-day mortality in all enrolled patients (Table 2). Conclusions Implementation of i-SMS significantly improved compliance with SSC recommendations among patients with sepsis and septic shock in accordance with Sepsis-3 definitions, but did not improve short-term survival outcome. Background Sepsis is a global public health problem. Despite the advances in modern medicine, more than 5.3 million people die from sepsis annually, with an estimated overall mortality of about 30% [1][2][3]. Early diagnosis and appropriate treatment can improve survival outcome in patients with sepsis [4]. Despite pre-existing diagnostic criteria for sepsis, early diagnosis is usually challenging due to unknown source of infection and the vague definitions of sepsis syndrome [5]. Creactive protein (CRP) and procalcitonin (PCT) have been widely used to facilitate the diagnosis of sepsis, but the clinical values of these are limited [6,7]. A recent study reported that IL-6 level is a diagnostic marker of infection and a prognostic marker, in patients with organ dysfunction [8]. Another study showed that PTX-3 discriminates sepsis and septic shock patients from healthy controls in a medical intensive care unit (ICU) setting [9]. However, evidence concerning the clinical value of IL-6 and PTX-3 has been controversial in several studies. The purpose of the present study was to investigate both the diagnostic and prognostic value of IL-6, PTX-3, and PCT among patients with sepsis and septic shock diagnosed at an emergency department (ED) using the latest Sepsis-3 definitions.

Materials and Methods
This study investigated biomarkers' clinical value among patients with sepsis and septic shock. Among 143 enrolled subjects (51 with sepsis, 46 with septic shock, and 46 healthy volunteers), serum levels of IL-6, PTX-3, and PCT were measured (Table 1). Follow-up IL-6 and PTX-3 levels were measured among patients with initial septic shock within 24 hours of hospital discharge. Optimal cut-off values were obtained for sepsis and septic shock. Prognostic value was evaluated by Cox regression analysis and Kaplan-Meier survival analysis.

Results
The median values (IQR) of IL-6 in controls, sepsis, and septic shock were 0.6 (0.    Figure  2). Twenty-eight-day mortality was significantly higher in the high IL-6 (≥53.59 pg/ml) group than in the low IL-6 (<53.59 ng/ml) group (p=0.002) (Figure 3). IL-6 was an independent risk factor for 28-day mortality among patients with sepsis and septic shock (HR, 1.0004; 95% CI, 1.0003-1.0005; p=0.024) ( Table 3). PTX-3 level was not significant in the multivariate Cox regression analysis (HR, 1.003; 95% CI, 0.998-1.008; p=0.095) ( Table 3). Both initial and follow-up PTX-3 levels of septic shock patients who died during admission were consistently significantly higher than those of septic shock patients who recovered (initial: p=0.004, follow-up: p<0.001) (Figure 4). Conclusions IL-6 level was superior to PTX-3 and PCT levels in both diagnostic and prognostic value for sepsis and septic shock diagnosed in the emergency department using Sepsis-3 definitions. IL-6 level was an independent risk factor for 28-day mortality.       The detection of sepsis-specific biomarkers ameliorate recognition and management of sepsis by improvement: diagnosis, monitoring response to treatment, and stratifying patients based on prognosis or underlying biological response. The diagnostic and prognostic role of novel biomarkers of sepsis is the aim of our study. In particular, we have investigated the kinetic of IL-35, presepsin and procalcitonin in critical septic patients with poor prognosis. Materials and Methods Fifty-nine critical septic patients admitted to ICU of the University Hospital of Catanzaro (Italy) were enrolled; a group of healthy volunteers were also included. All studied subjects were stratified into survivors and nonsurvivors, based on 28 days survival and according to microbiological results in blood culture positive and blood culture negative groups. Clinical data and blood samples were collected for quantitative analysis of IL-35, sCD14-ST and PCT at: i) time 0 (T0): time at which the first blood culture for analysis was collected; ii) time T minus 7 (T-7): 7 days before exitus or hospital discharge; iii) time T minus 2 (T-2): 2 days before exitus or hospital discharge. Data were subjected to statistical analysis by ANOVA plus PLSD test. A p value < 0.05 was considered statistically significant.

Results
Interleukin-35 levels were found significantly (p< 0.05) increased in the group of eventually dead patients, both at T0 and at T-2 times. By contrary, such different behavior between alive and dead patients did not achieve any significant difference at T-7; although the group of patients with a poor prognosis still exhibited a light increase of IL-35. RegardingsCD14-ST, our data confirmed that presepsin is a valuable prognostic biomarker for the studied patients (p< 0.05). PCT exhibited a significantly lower levels in the eventually non-survivors only at time T0; by contrary both at times T-7 and T-2 PCT levels of dead patients were found significantly higher than survivors. Conclusions Serial increase of IL-35 and sCD14-ST or presepsin reveals prognostic value for in hospital mortality compared to PCT as observed at T0. Recent study shown the intrisic multimodal co-existing pro-and antiinflammatory mechanisms reflect specific immune reprogramming [1]. Background Listeria monocytogenes (L. monocytogenes) is a facultative intracellular Gram-positive bacterium that infects humans through food. The broad clinical spectrum of Listeria monocytogenes infections includes frequent clinical forms, such as meningitis or bacteremia, and uncommon manifestations, such as septic arthritis. Osteoarticular infections due to L. monocytogenes have been reported very rare. We report on a case of osteoarticular abscess, which developed into a bacteremia both caused by L. monocytogenes in an older adult, using a rapid identification by innovative technology.

Materials and Methods
An 83-year-old man was admitted to our university hospital with a retroperitoneal abscess and acute low back pain suggestive of spondylodiscitis. He had no sign of meningoencephalitis or fever. A clinical sample from vertebral joint abscess and some blood cultures were sent to our laboratory for microbiological diagnosis. Sepsis biomarkers (procalcitonin and sCD14-ST) were also required. All of the samples were analyzed by conventional methods. Moreover, vertebral joint abscess was analyzed by ePlex device, that identifies a broad panel of pathogens and resistance genes, using BCID-GP Panel (GenMarkDx, USA). The system relies on electrowetting technology to perform multiplexed nucleic acid extraction, amplification and digestion followed by the detection of analyte targets using eSensor technology. ePlex device, has been approved and commercialized for positive blood cultures only. We used this molecular technology directly on the sample of vertebral joint abscess. Positive blood cultures were assessed by molecular method using the FilmArray system (Bio-Merieux, Italy). Serum procalcitonin and presepsin were tested by ELFA and CLEIA methods respectively. Results ePlex system identified L. monocytogenes in about 90 minutes directly on sample of vertebral joint abscess. Moreover, L. monocytogenes was identified using the FilmArray blood culture ID Panel in about one hour on positive blood cultures. Sub-cultures, processed using conventional and proteomic methods, confirmed molecular diagnosis results.

Conclusions
Rapid detection of L. monocytogenes by innovative technology allowed the clinicians to establish an early antibiotic therapy. This, as well as other molecular techniques, may represents an integrated approach to conventional methods for etiological diagnosis.

P17
After recovery from severe-sepsis, the lung carries significant quiescent lesions with potential recurrent pathogenicity

Background
The causal role of sepsis on the long-term impairment and survival remains unclear, but is a fact that survivors of sepsis are profoundly functionally impaired. The lung is the organ often affected in sepsis and course with high lethality by ARDS. Thus, this study aimed to evaluate the pulmonary status in survivors of a severe sepsis in search of remaining lesions with potential recurrence of pulmonary disease.

Materials and Methods
Under general anesthesia, Wistar rats (250g) were submitted to severe sepsis (iv. inoculation of 2 mL Escherichia coli 108 CFU/ mL), that course with hypotension within 4-6 hours and with 50-60% mortality within 26 hours. Lung of the survivals was collected at 30 and 90 days under general anesthesia and were sacrificed after. (n=4/ period). The samples were submitted to histological study using HE dye and results were compared to lung of naïve (T0) and septic animals of 6hours after challenge (n=4/period).

Results
The 6h sepsis group showed a vascular congestion of the alveolar wall, alveolar wall thickening and neutrophils infiltration, and BALT hyperplasia, in a focal manner. (Figure 1). After 30 days of sepsis was observed multifocal thickening of alveoli walls with intense and broad congestion of capillaries. The BALT hyperplasia also was more intense as compared to 6h findings. These results showed a continuing inflammatory process and suggested that a state of an acute inflammatory reaction coexists with apparent clinical normality. In 90 days after sepsis induction, although all animals remained clinically asymptomatic, significant pulmonary lesions were seen, with intense capillaries congestion, broad atelectasis, severe thickening of the alveolar wall, infiltrate of mono and polynuclear cells in most of alveoli, multiple areas with hemorrhage, peri-bronchial leukocyte infiltrates, atherosclerosis, and intense BALT hyperplasia associated to obstruction of bronchioles and multiple areas of atelectasis. These findings demonstrated that healthy-looking animals were concealing a functionally impaired lung structure combined to an intense inflammatory reaction state in course, denoting an impaired lung physiological condition to face with new deleterious stimuli in sepsis survivors. Conclusions A severely compromised state of lung following sepsis recovery might explain the ease for higher susceptibility for recurrent pulmonary diseases. These events are possibly related to the postsepsis syndrome that courses with a persistent immunosuppression, inflammation and catabolism. Clinical studies are needed to confirm these animal experimental findings. In the future, understanding of mechanisms supporting these persistent lung injuries may afford intervention targets to ameliorate post-sepsis high mortality.

Background
In the United Kingdom 70% of sepsis cases are derived from an infection developed in the community [1]. It is estimated that there is potential to reduce deaths by up to 10,000 per year by the optimization of care. Mortality review of sepsis deaths in Royal Derby Hospital for November 2017 revealed that 50% of patients died in the first couple of hours to 1 day following admission. Of these, 85% were previously treated in the community with antibiotics for different periods of time, some of them with repeated visits to their General Practitioner The findings of the reviews suggested communication deficits and inconsistent pathways between primary and secondary care. Materials and Methods A Derbyshire wide project group was set up at the authors suggestion, including commissioners, the ambulance service, out of hours service, community providers, primary and secondary care. The final aim of the project was improving sepsis outcomes by adopting a standard approach to screening and treatment countywide, at all levels of care. We have conducted process mapping of the pathways and procedures each provider followed when dealing with a potential sepsis case and identified where delays/ inconsistencies/ issues existed. The agreed action plan:

Results
Important steps have been achieved following our intervention (Table 1) starting with agreement on using same new early warning scores (NEWS 2) and paediatric observation priority score (POPS) throughout Derbyshire. It is now possible for sepsis treatment to be initiated in the community long before the patient reaches hospital through education of community nurses and general practitioners' agreement. While the initiative is ongoing due to the massive ramifications of the project, intermediate data showed that crude mortality from sepsis is on a continuous downward trend (Graph 1). Conclusions Developing a unified approach and increasing communication between providers generated benefits for patient, practitioners and local health economy. While raw data analysis makes it easy to measure patient outcomes, the benefits of creating a local peer learning resource and support network are not easily quantifiable and can only be predicted as positive. Background Sepsis is a life-threatening condition caused by a dysregulated host response to infection. If unimpeded, sepsis can progress to septic shock, characterized by refractory hypotension and unresponsive vasculature (i.e. vasoplegia) resulting in tissue hypoperfusion and eventually organ failure [1]. The overall mortality rate of septic shock is more than 50%. The vascular dysfunction and refractory hypotension in septic shock are caused, at least in part, by excess reactive oxygen species (ROS) generation and dysregulated nitric oxide (NO) production (via inducible NO synthase upregulation and increased scavenging by excess ROS) [2,3,4]. Moreover, excess ROS oxidizes and hence damages soluble guanylate cyclase (sGC), the downstream mediator of NO, resulting in the impaired organ blood flow [5,6]. We believe the loss of sGC-mediated vasodilation is a critical determinant of reduced organ blood flow in sepsis. The objective of this study is to assess the state of vascular dysfunction and altered blood flow patterns as sepsis progresses to septic shock. We also sought to assess the effi-cacy of (i) the sGC activator cinaciguat or (ii) the superoxide dismutase mimetic tempol in improving hemodynamics and survival in a murine model of sepsis.

Materials and Methods
The experimental protocols described herein have been approved by the University of Alberta Animal Care and Use Committee in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education. Male C57Bl/6 mice were instrumented with fiber-optic pressure sensors for direct blood pressure monitoring. Flow probes were placed around the left common carotid, superior mesenteric, and right renal arteries to monitor blood flow to the brain, gut, and kidney, respectively. After baseline recordings, sepsis was induced by an intraperitoneal injection of a fecal slurry. Thirty minutes after induction of sepsis, mice were treated with the cinaciguat (15μg/kg IV) or tempol (30mg/kg, IV).

Results
The blood pressure in septic mice reduced significantly over time (44% ± 4% reduction after 4 h of fecal slurry injection) compared to control mice ( Figure 1A), but no significant changes in heart rate were noted ( Figure 1B). Blood flow in the carotid, superior mesenteric, and renal arteries reduced at 47 ± 4%, 71 ± 8%, and 57 ± 13% respectively, 4 hours after fecal slurry injection (Figures 2A, B, and C, respectively). Vessel reactivity, assessed by monitoring constrictor and relaxation responses to bolus doses of phenylephrine (10 μg/kg body weight) and sodium nitroprusside (5 μg/kg body weight) was reduced by 36 ± 9% and 53 ± 7% respectively, in septic mice compared to controls, suggesting impaired regional vascular function. These data suggest certain organs are more susceptible to vascular dysfunction and hypoperfusion with the progression of sepsis. While a low dose (15μg/kg IV) of cinaciguat provided the best survival outcomes in our model, our preliminary data have also shown that administration of tempol (30mg/ kg, IV), 30min after injection of fecal slurry, prolongs survival and mitigates the decline in blood pressure and superior mesenteric artery blood flow.

Conclusions
The proposed therapeutic is expected to reduce organ damage and improve blood flow to organs without causing systemic vasodilation and hypotension in sepsis.

Background
We present the case of a 55-year-old Argentinian woman, with a clinical of peritonitis with 24h of evolution to sepsis, characterized initially by acute diffuse abdominal pain associated with diarrhea without mucus or blood. Patient denied nausea, evolving to abdominal distention, absence of peristalsis and signs of peritoneal irritation, negative catharsis and absence of flatulence with hemodynamic decompensation. Case Report In the physical examination, she was oriented in time and space, cooperative, Glasgow 15/15, no fever (36ºC), hypotensive (80/ 60mmHg), tachycardic 112 bpm, with respiratory rate at 18 breaths per minute, abdomen distended, painful at superficial palpation and deep peritoneal resistance, absence of intestinal sounds, symmetric extremities and venous return lower than 2 seconds. The following complementary exams were made: electrocardiogram and chest X-Ray, with the imaging of the X-Ray being interpreted as septic shock and validated by an intensive care unit professional [1]. During the 10 weeks hospitalized, the patient underwent various procedures following the diagnosis, including reanimation with vasopressors and this medication caused ischemia and necrosis of phalanges later, exploratory laparotomy, bilateral pleural effusion, pericardial effusion, acute cholecystitis, distal ileum fistula, blood transfusion, non-invasive ventilation and nephrostomy. She also presented various complications such as intestinal ischemia, surgical wound infection, respiratory insufficiency and hepatic dysfunction. The patient started with piperacillin, tazobactam and vancomycin, then imipenem with vancomycin. 7 weeks later, the patient was disconnected from the non-invasive ventilation, with no intercurrences and slowly evolving to improve. Two weeks after that she started oral feeding, with associated k108 tube for better caloric income. The patient was treated with antibiotics for 8 days (Colistine), 35 days (Linezolid), 26 days (Ciprofloxacin) and was suspended from the parenteral nutrition since 10 weeks of admission.
In the context of sepsis in abdominal focus with SOFA 2, tomography showed delayed right renal excretion associated with retropielocalicial dilation.

Background
Adenosine is considered as a potent metabokine with potential immunoregulatory function generated during acute inflammatory and infectious conditions. It is produced during conditions causing metabolic stress including hypoxia and inflammation. In the current study, we have investigated the effect of 2-cholroadenosine (2-CADO) on the pulmonary innate immune response including macrophage function in mice infected with Klebsiella pneumoniae B5055-induced pneumonia. Materials and Methods Acute lung infection/Pneumonia leading to the development of acute lung inflammation or injury (ALI) was induced by intranasal instillation of K. pneumoniae B5055 into mice. Subsequently, mice were treated with 2-CADO (10 μg/kg/day/iv) using a treatment schedule. Mice were observed for mortality and other inflammatory damage. Various proinflammatory cytokines (TNF-alpha and IL-1) and antiinflammatory cytokines including IL-10 were determined by ELISA.
Macrophage functions were evaluated in terms of Hydrogen peroxide release, macrophage spreading, and macrophage phagocytic potential. Neutrophil infiltration into the lungs was also evaluated by histopathologic examination of lungs, and myeloperoxidase (MPO) estimation in bronchoalveolar lavage fluid (BALF).

Results
No mortality was observed in both the control and treated groups. 2-CADO treatment modulated the pro-inflammatory function of alveolar macrophages by significantly (p≤0.05) decreasing their phagocytic activity (both phagocytic uptake and intracellular killing of the pathogen), nitric oxide (NO) and hydrogen peroxide (H2O2) release. 2-CADO also significantly (p≤0.05) decreased the neutrophil infiltration into the lungs. Levels of pro-inflammatory cytokines (IL-1α and TNFα) were decreased significantly (p≤0.05) decreased. However, levels of IL-10 were found to be significantly (p≤0.05) elevated. Conclusions Adenosine, a metabokine has promising immunomodulatory action during Gram-negative bacterial pneumonia and associated inflammation.

P22
Incidence and impact of sepsis and septic shock after subarachnoid Background Aneurysmal subarachnoid hemorrhage (SAH) is an acute cerebrovascular disease that can lead to devastating consequences, including high mortality as well as long-term functional impairment among survivors [1]. Approximately 30% of patients hospitalized with SAH will develop some type of nosocomial infection [2]. The diagnosis of sepsis is difficult in patients with SAH due to the high incidence of early systemic inflammatory response syndrome (SIRS), due to the primary bleed, and the lack of reliable biomarkers that can be used to differentiate between SIRS and sepsis [3,4]. Therefore, there is a risk of sepsis misdiagnosis, which can potentially lead to under and overtreatment, in SAH patients. Our objective was to define the incidence of sepsis and septic shock, diagnosed prospectively with new sepsis criteria, and its impact on mortality and functional out-comes of patients with SAH.

Materials and Methods
We prospectively included all adult patients (≥ 18 years) admitted to the Neu-rological Intensive Care Unit (ICU) of the Paulo Niemeyer State Brain Institute (Rio de Janeiro, Brazil) with aneurysmal SAH between April 2016 and May 2018. Daily clinical and laboratory follow-up were analyzed during the first 14 days of hospitalization or up to ICU discharge. Main outcome was the functional outcome, using the Modified Rankin Scale (mRs), prospectively assessed at hospital discharge and after 3, 6 and 12 months. Results 148 patients were enrolled in the study. 56 (38%) developed 60 infectious events. SIRS was present in 82% and sepsis or septic shock in 28%. Inhospital mortality was 17% Multivariate analysis revealed that death or functional dependence (defined as mRs 4-6) at hospital discharge were independently associated with sepsis/septic shock (OR 3.4, 95% CI 1.16 -9.96, p = 0.026) ( Table 1). SIRS was not independently associated with poor outcome. Mortality, on long-term follow-up, on septic patients was 52.5%, and on non-septic, 16%. Figure 1 shows the Kaplan-Meier survival curve. Figure 2 shows long-term functional outcomes both groups Conclusions Sepsis plays a significant role on the outcome of patients with SAH, affecting both mortality and long-term functional outcomes. Combining high-level neurocritical care management of neurological complications and optimal diagnosis and management of sepsis and septic shock may effectively reduce secondary brain injury and improve patients' outcomes after SAH, especially in low-and middle-income countries. Background Altered microvascular tone contributes to distributive shock and many evidences supports endothelial damage role on organ dysfunction. Vasopressor drugs requirement is related to vascular reactivity [1]. In other hands, it's known that altered insulin signaling by differences on its receptor isoforms expression, specifically insulin receptor-A (IR-A) isoform, is related to endothelial dysfunction [2,3]. To our knowledge, there is no previous report of IR-A expression in sepsis.

Materials and Methods
We describe two septic shock (SS) patients, and the association between vasopressor drug requirement and IR-A mRNA expression on mononuclear blood cell at first, second/third and fifth/seventh days after admission. We choose studied IR at monocytes because they are a known model to study IR in humans [4]. IR-A mRNA expression assessed by quantitative polymerase chain reaction and it was as fold of changes by ΔΔCt method analysis. Results 62 years male (Patient A) and 52 years female (Patient B) with SS criteria of abdominal focus. In both cases, besides antibiotic therapy, crystalloids solutions and vasopressor support to maintain PAM above 65mmHg were necessary. In patient A, the vasopressor requirements were higher at the admission and progressively decreased until its suspension at second day, and the IR-A expression at the beginning was 15,6x and it was progressive decreasing until 1,9x. Patient B required less vasopressor than patient A, but they were necessary for more days. Interestingly, IR-A expression of patient B was lower at the beginning (4,5x) but it remained elevated throughout the observation time (21,5x) as well as vasopressor support. Conclusions This is the first report of a plausible association between IR-A expression and vasoactive drugs requirements in SS, probably secondary to endothelial dysfunction. The alternative splicing of exon 11 human IR gene generates two isoforms [5] and, while alterations on IR-B expression are associated to metabolic processes, IR-A upregulated expression has been related to alterations on vascular function [6]. Although IR-A underlying mechanisms are poorly understood, it has been associated to gestational diabetes mellitus endothelial dysfunction [2,3] and cancer angiogenesis [7]. This report shown a higher IR-A expression seems to be associated to higher need for vasopressors. Further research is necessary to establish the IR-A role on vascular reactivity in sepsis.

Background
Herpes simplex virus-1 (HSV-1) is the most common infectious cause of sporadic encephalitis in older adults and young children. There are fatal prognostic factors like lower level of consciousness at presentation, older age, severe comorbid disease, immunodepression and delay in start of treatment. This is a potentially lethal illness with a high risk of morbidity and death and it is necessary to do an early diagnosis with a good clinical history, examination, Computed tomography (CT) scan and/or Magnetic Resonance Imaging (MRI), and lumbar puncture. Prompt treatment using acyclovir therapy can give a better prognosis, however, sometimes the disease tends to progress rapidly causing septic shock and multiorgan failure.

Materials and Methods
A 71-year-old woman was admitted to the Emergency department presenting with a two-day history of fever and speech impairment. She was previously healthy with no past medical history. On initial examination she was disorientated in time, space and person and her speech was unintelligible. Computed tomography CT) scan showed left temporal lobe hypodense lesion (Fig.1) indicating viral encephalitis. After few hours, she started to be drowsy with low Glasgow Coma Scale (GCS) requiring intubation and was transferred to the High Dependency Unit (HDU). Her chest X-ray was suggestive of aspiration pneumonia (Fig.2). Due the possibility of viral encephalitis and pneumonia, early empiric antibiotics and acyclovir, were started. Lumbar puncture was performed and the cerebrospinal fluid (CFS) analysis revealed lymphocytic pleocitosis, elevated protein, normal glucose and positive polymerase chain reaction (PCR) for herpes simplex virus (HSV)-1.

Results
The patient continued to deteriorate and septic shock was suspected. She was tachycardic (heart rate 140/min) and had severe arterial hypotension (blood pressure 60/30) despite aggressive intravenous fluid resuscitation and norepinephrine. Arterial blood gas revealed metabolic acidosis with hyperlactatemia. She had an ongoing progressive organ dysfunction with coagulopathy, acute renal failure and severe hypoxia, and was unresponsive to resuscitation measures and high doses of amines, finishing with completely irreversible organ failure.

Conclusions
Herpes simplex virus is one of the most common causes of encephalitis. It is important to suspect, diagnose and treat as soon as possible in order to reduce the mortality and morbidity rates. The use of dexamethasone is unclear but can be considered when there is a progression even when acyclovir has been started. Despite the early treatment, this severe infection has the potential to cause severe neuropsychiatric deficits, or septic shock and multiorgan failure with death, like in our case report. Consent Written, informed consent for publication was obtained from the patient for publication of this case report. Background Klebsiella pneumoniae is a common bacterial pathogen that can cause different diseases like pneumonia, bloodstream and urinary tract infections, liver abscess, necrotizing fasciitis, meningitis, sepsis. It is an unusual cause of community acquired pneumonia except in alcoholics. An important problem is the increasing number of strains resistant to antibiotics.

Materials and Methods
A 63-year-old male, with a history of alcohol abuse, presented to the Emergency Department with fever, dyspnea and productive cough with greenish sputum. He complained of nausea and vomiting for the last four days. Clinical examination revealed a conscious, oriented but distressed patient. He was clammy, sweaty, tachycardic (128 beats/min), hypotensive (85/50 mmHg), tachypneic (30 breaths/min), and his oxygen saturation was 88%. Lung auscultation showed crackles and his chest X-ray an extensive right upper lobe consolidation (Fig.1). Arterial blood gas displayed a mixed acidosis with pO2 56mmHg. Blood, urine, and sputum cultures were obtained and empiric antibiotics were initiated (piperacillin-tazobactam and azithromycin). He was immediately admitted to the High Dependency Unit (HDU) with the diagnosis of pneumonia and sepsis. High flow nasal cannula was started but the patient was using accessory muscles and showed a gradually worsening shortness of breath, requiring intubation and connection to mechanical ventilation. Blood test results revealed leucopenia, lactic acidosis, acute renal failure and high C-Reactive Protein (CRP).

Results
During his admission, the patient was hypoxic and in septic shock, requiring high FiO2 and fluid resuscitation plus vaso-active amine infusion, trying to achieve hemodynamic stabilization. Thoracic computed tomography (CT) scan exhibited the presence of extensive alveolar consolidation in right upper, middle and lower lobe. A Klebsiella pneumoniae was detected in sputum and blood cultures, and ceftazidimeavibactam was started. Even though the correct treatment was used, the patient was not improving. After 10 days, a second CT demonstrated worsening consolidation, abscess in right upper lobe and empyema (Fig.2). Progressive septic shock led to multiple organ failure not responding to the intensive management. Consent Written, informed consent for publication was obtained from the patient for publication of this case report.

Conclusions
Klebsiella pneumoniae is an opportunistic pathogen that can cause different nosocomial infections and must be considered in alcoholic patients with severe pneumonia. This pathogen always requires prior treatment due his virulence and multiple antimicrobial resistance. We have to be very cautious with invasive infections produced by this microorganism that are strongly associated with immunocompromised populations. Background Static values of central venous pressure (CVP) have limitations to guide fluid management although dynamic changes are considered useful [1][2][3]. We evaluated if CVP changes after a fluid challenge and the baseline cyclic respiratory variation in CPV amplitude curve (ΔCVP) could discriminate responders from non-responders. Materials and Methods This is a prospective, observational study conducted in two mixed intensive care. We included adult patients under mechanical ventilation, adequately sedated and with acute circulatory failure. They received a fluid challenge with crystalloids (Ringer's lactate or sodium  chloride 0.9% solution, 500 mL) infused over 15 minutes. We determined the CVP at baseline (CVPT0) and the changes at 5 (ΔCVPT5), 10 (ΔCVPT10) and 15 (ΔCVPT15) minutes during fluid infusion. We also measured ΔCVP at baseline. Fluid responsiveness was defined by a cardiac index increased ≥ 15%.

Results
We included 30 patients (11 responders, 19 non-responders). There was a significant increase in mean CVP over time in both responders and non-responders (0.12 mmHg; standard error: 0.02; P<0.001), although there were no statistically significant differences between the groups in the mean CVP (-2.7 mmHg; standard error: 1.37; p=0.06) and in the mean CVP changes up to 15 minutes (