This thesis is focused on the possibility of analysis of volatile organic compounds produced by monocytes during sepsis. Method of comprehensive two-dimensional gas chromatography with mass spectrometric detection was chosen for this purpose. Content of the first part was the optimization of the method of two-dimensional gas chromatography for the determination of volatile organic compounds. In this part were gradually adjusted parameters of the gas chromatography method to achieve the maximum efficiency. Further were adjusted conditions of samples preparation. Content of the second part was the usage of already optimized method for the analysis of the samples set of monocytes. Samples were subjected to the action of different inhibitors of the immune system and stimulators simulating bacterial or yeast infection. Based on this analysis were identified some compounds, which are produced by monocytes under condition simulating the infection.

The aim of the thesis was to determine the microbial colonization at the injection site of central venous catheter and its possible influence to maintain the lowest incidence of catheter sepsis. The culture results of skin swabs and taken blood cultures from two groups of patients were compared with each other using qualitative analysis methods. Those were Intensive Care Unit patients with established central venous catheter. The result was the discovery of microbial colonization of the skin and the representation of different pathogenic strains at the site of a central venous catheter for both established groups. Further showed that the higher the risk of microbial colonization with symptoms of catheter sepsis was seen where patients have central venous catheter established for longer period of time and have been treated under home parental nutrition program with stoma. The recommendation is the continuous education of health professionals to act in accordance with the latest preventive treatment recommendations, and the emphasis is mainly placed on hands cleaning and disinfecting as the hands of health professionals are one of the most common and risky way of transmission of pathogenic strains with potential nosocomial infection formation. Aseptic principles must be repeatedly communicated to all...

With increasing incidence rates aligned with poor prognosis; sepsis represents one of the biggest challenges in modern health care. It is a multifactorial syndrome defined as organ dysfunction caused by disturbed systemic response to an infection. Most of the inpatient sepsis are caused by Gram positive bacteria and one of its major constituents of the cell envelope: lipoteichoic acid (LTA). An adjuvant treatment that has gained prominence recently is extracorporeal blood removal therapies i.e., hemoperfusions. The concept is to remove the bacterial virulence factors that triggers immune responses and therefor stabilize the hemodynamic parameters of the patient. The dominating research of this method centres around adsorption of the Gram negative bacterias’ endotoxin lipopolysaccharide (LPS) but not LTA, whose biochemical and physiological properties resembles each other. The aim of this study was to determine whether LTA can be adsorbed using immobilized truncated derivates of antimicrobial peptides (AMPs). LTA was quantified using ELISA comparing before and after passage through columns with immobilized peptides. Further, the absorption abilities of LTA from two different solid phases with distinctive surfaces were investigated. This was of interest to elucidate the nature of the mechanisms behind LTA extractions. All results generated inconclusive data, except for one trial which demonstrated that peptide KEF-19 adsorbed most LTA and that the electrostatic force had the greatest influence of the adsorption. Future studies should however be carried out to validate these statements as well as feasibility and safety estimations for KEF-19 as the sorbent in hemoperfusions for Gram positive bacteria and LTA.

Sepsis is still a leading cause of death in the United States despite extensive research and modern advancement in technology. Early recognition of sepsis and timely management strategies are important for effective reduction of sepsis-related morbidity and mortality. Guided by the logic model, the purpose of this project was to evaluate the effectiveness of electronic reminders in enhancing clinical decision-making among 30 nurses in 3 medical-surgical units. The practice-focused question addressed the effectiveness of electronic reminders for early recognition and initiation of goal-directed treatment of sepsis in hospitalized patients on medical-surgical units in an effort to reduce sepsis mortality rates. Data were collected from a randomized convenience sample using a self-constructed questionnaire and through observation. The observations were aimed at assessing whether the nurses adhered to the sepsis protocol, while the questionnaire captured the participants' perceptions regarding the use of automated alerts measured on a 5-point Likert scale. Statistical analysis involved the use of frequencies and percentages, positive predictive value (PPV), and negative predictive value (NPV). The results indicated that all the nurses adhered to sepsis protocol. The sepsis-related mortality rate, mean response time, and rate of severe sepsis at the hospital were reduced by 17.2%, 14 minutes, and 11.1%, respectively. It was concluded that automatic alert systems improve nurses' ability to recognize early symptoms of sepsis and their ability to initiate Code Sepsis. However, replication of this study using a large sample size could provide findings that are more generalizable. Electronic reminders may promote positive social change because earlier recognition of sepsis by nurses may lead to a reduction of healthcare costs through improved management of sepsis patients in acute care settings.

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Antimicrobial resistance in bacteria is a global health challenge with a projected fallout of 10 million deaths annually and cumulative costs of over 1 trillion dollars by 2050. The currently available tools exploited in the detection of bacteria or their DNA can be expensive, time inefficient, or lack multiplex capabilities among others. The research work highlighted in this dissertation advances techniques employed in the phenotypic or genotypic detection of bacteria and their DNA. In this dissertation, I present polymethyl methacrylate-pressure sensitive adhesive microfluidic platforms developed using a time-efficient, inexpensive fabrication technique. Microfluidic devices were then equipped with functionalized monoliths and utilized for sequence-specific capture and detection of picomolar concentrations of bacterial plasmid DNA harvested from cultured bacteria. I then showed multiplex detection of multiple bacteria gene targets in these devices with an improved monolith column. Finally, I demonstrated a genotypic approach to studying single bacteria growth in water-in-oil droplets with nanomolar concentrations of a fluorescence reporter, and detection via laser-induced fluorescence after convenient room temperature 2-h incubation conditions. The systems and methods described herein show potential to advance tools needed to address the surging problems and effects of drug-resistant bacteria.

Bakgrund: Sepsis är ett akut sjukdomstillstånd med en hög mortalitet världen över.Tidig identifiering och snabb behandling är viktig för patientens överlevnad.Sjuksköterskan har i sitt patientnära arbete en unik position att snabbt identifierasepsis och initiera behandling. För att möjliggöra en säker vård för patient, kollegoroch vårdansvarig sjuksköterska ska sjuksköterskan arbeta förebyggande ochpatientsäkert. Syfte: Att kartlägga åtgärder som främjar sjuksköterskans förmåga attidentifiera sepsis på sjukhus. Metod: En litteraturöversikt har genomförts för attskapa en överblick av det aktuella kunskapsläget. För att besvara syftet användesartiklar med en kvantitativ metod. Med hjälp av Polit och Becks niostegsmodellanalyserades artiklar som sedan presenterades i resultatet. Resultat: Resultatetvisade att sjuksköterskorna använde sig utav utbildning, riktlinjer samtscreeningverktyg för att främja identifieringen utav sepsis. Signifikanta skillnader sågsi relation till de olika åtgärderna. Skillnaderna visade bland annat en minskadmortalitet och minskad vårdtid. Slutsats: Sjuksköterskan har en central roll i attidentifiera sepsis. Resultatet förväntas identifiera åtgärder som i den kliniskavardagen kan hjälpa framtida sjuksköterskor eller annan vårdpersonal. Genom attsjuksköterskan är ödmjuk för den egna kompetensen, tar hjälp av screeningverktygoch vidhåller de riktlinjer som finns kan en grund för säker vård byggas. / Background: Sepsis is an acute illness with a high mortality rate worldwide. It istherefore important to act quickly as rapid identification and treatment can be crucialfor the patient's survival. The nurse has, with her close work with the patient a uniqueposition to quickly identify sepsis and initiate treatment. In order to enable safe carefor both the patient, colleagues and healthcare providing nurse, the nurse must workpreventively and in a patient safe manner. Aim: To map interventions that promotethe nurse's identification of sepsis in a hospital setting. Method: A literature reviewhas been carried out to create an overview of the current state of knowledge. To answerthe purpose, articles with a quantitative methodology were chosen. Using Polit andBeck's nine-step model articles were analyzed and then presented in the results.Results: The results showed that the nurses used training, protocols and screeningtools to promote the identification of sepsis. Significant differences were seen inrelation to the various interventions. The differences showed for instance a decreasedmortality and abbreviated the hospital stay. Conclusion: The nurse has a central rolein identifying sepsis. The results are expected to identify measures that can help futurenurses or other healthcare professionals in the clinic. By the nurse being humble abouther own competence, using screening tools and maintaining the existing protocols, afoundation for safe care can be built.

Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to assess cell surface Dectin-1 expression. C. albicans sepsis resulted in an increase in the percentage of Dectin-1 positive (Dectin+) blood and splenic leukocytes by increasing the percentage of neutrophils. C. albicans infection increased the percentage of Dectin+ splenic T cells. CLP decreased the percentage of highly Dectin-1 positive leukocytes in the blood by decreasing the percentage of Dectin+ neutrophils. GP treatment in sepsis further decreased the percentages of Dectinhigh blood leukocytes and Dectin+ neutrophils. CLP decreased the percentage of Dectin+ splenic leukocytes by decreasing the percentage of splenic macrophages. GP administration to CLP mice further decreased the percentage of Dectin+ splenocytes by decreasing the percentage of Dectin+ macrophages. Administration of GP resulted in a prolonged decrease in the percentage of Dectinhigh blood leukocytes. The changes in Dectin-1 expression with GP were because of decreases in the percentage of Dectin+ neutrophils and monocytes. In the trafficking studies, macrophages were incubated with fluorescent labeled glucans and then stained for intracellular organelles and signal transduction molecules. Cells were imaged using confocal microscopy. GP is internalized by clathrin and trafficked to the Golgi apparatus. GP internalization is regulated but not dependent on caveolin-1. GP co-localized with SRA, TLR2, and PI3K/p85. The trafficking of laminarin and particulate glucan is similar. We speculate that loss of cell surface Dectin-1 may be important in the protection conferred by glucans in sepsis. Additionally, intracellular trafficking and interaction with signaling components may be important steps in modulation of cellular function by glucan-pattern recognition receptor complexes.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

Circulating lactate levels are a critical biomarker for sepsis and are positively correlated with sepsis-associated mortality. We investigated whether lactate plays a biological role in causing endothelial barrier dysfunction in sepsis. We showed that lactate causes vascular permeability and worsens organ dysfunction in CLP sepsis. Mechanistically, lactate induces ERK-dependent activation of calpain1/2 for VE-cadherin proteolytic cleavage, leading to the enhanced endocytosis of VE-cadherin in endothelial cells. In addition, we found that ERK2 interacts with VE-cadherin and stabilizes VE-cadherin complex in resting endothelial cells. Lactate-induced ERK2 phosphorylation promotes ERK2 disassociation from VE-cadherin. In vivo suppression of lactate production or genetic depletion of lactate receptor GPR81 mitigates vascular permeability and multiple organ injury and improves survival outcome in polymicrobial sepsis. Our study reveals that metabolic cross-talk between glycolysis-derived lactate and the endothelium plays a critical role in the pathophysiology of sepsis.