Clinica Chimica Acta 440 (2015) 97–103

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Sepsis biomarkers Miroslav Prucha a,⁎, Geoff Bellingan b, Roman Zazula c a b c

Department of Clinical Biochemistry, Hematology and Immunology, Hospital Na Homolce, Prague, Czech Republic University College London Hospitals, 235 Euston Rd, London NW1 2PG, United Kingdom 1 Department of Anesthesiology and Intensive Care, First Faculty of Medicine, Charles University in Prague and Thomayer Hospital, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 5 November 2014 Accepted 11 November 2014 Available online 18 November 2014 Keywords: Sepsis SIRS Biomarkers Sensitivity Specificity

a b s t r a c t Sepsis is the most frequent cause of death in non-coronary intensive care units (ICUs). In the past 10 years, progress has been made in the early identification of septic patients and in their treatment and these improvements in support and therapy mean that the mortality is gradually decreasing but it still remains unacceptably high. Leaving clinical diagnosis aside, the laboratory diagnostics represent a complex range of investigations that can place significant demands on the system given the speed of response required. There are hundreds of biomarkers which could be potentially used for diagnosis and prognosis in septic patients. The main attributes of successful markers would be high sensitivity, specificity, possibility of bed-side monitoring, and financial accessibility. Only a fraction is used in routine clinical practice because many lack sufficient sensitivity or specificity. The following review gives a short overview of the current epidemiology of sepsis, its pathogenesis and state-of-the-art knowledge on the use of specific biochemical, hematological and immunological parameters in its diagnostics. Prospective approaches towards discovery of new diagnostic biomarkers have been shortly mentioned. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . Definition of sepsis, SIRS and the PIRO concept . . . . . . . Mechanisms of SIRS and sepsis. . . . . . . . . . . . . . . Diagnostics of sepsis . . . . . . . . . . . . . . . . . . . 4.1. C-reactive protein and procalcitonin . . . . . . . . . 4.2. Cytokines . . . . . . . . . . . . . . . . . . . . . 4.3. Lipopolysaccharid binding protein . . . . . . . . . . 4.4. Surface markers of circulating leukocytes . . . . . . . 4.5. D-dimer. . . . . . . . . . . . . . . . . . . . . . 4.6. Presepsin and other novel markers . . . . . . . . . 5. The future of diagnostics of sepsis—genomics and proteomics . 6. Summary . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Abbreviations: ATB, antibiotics; ICU, Intensive care unit; SIRS, Systemic Inflammatory Response Syndrome; MODS, Multiple Organ Dysfunction Syndrome; RRT, renal replacement therapy; RA, rheumatoid arthritis; PCT, procalcitonin; LPS, lipopolysaccharide; LBP, lipopolysaccharide binding protein; BPI, bactericidal/permeability increasing protein; TNM system, tumor, nodes, metastasis ⁎ Corresponding author. E-mail address: [email protected] (M. Prucha). 1 Supported by UCLH NHS Biomedical research center.

http://dx.doi.org/10.1016/j.cca.2014.11.012 0009-8981/© 2014 Elsevier B.V. All rights reserved.

Sepsis is the most frequent cause of death in non-coronary intensive care units (ICUs). It is a serious disease with a high mortality and represents an immense financial burden on the health care system. In the past two decades, the incidence of sepsis has been on the rise not only in developing countries but also in the USA and countries of Western Europe [1]. In developed countries, the incidence of sepsis is 2% of all hospitalizations and 6 to 30% of ICU patients; even more dramatic is the situation in developing countries [2]. The problem is

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high mortality of severe sepsis, which is higher than that of myocardial infarction at the moment and even worse outcomes are revealed when looking at patients with septic shock [3]. In the past 10 years, progress has been made in the early identification of septic patients and in their treatment and these improvements in support and therapy means the mortality is gradually decreasing but it still remains unacceptably high. Moreover, due to the ever growing incidence of sepsis, the overall number of patients who die of sepsis is continuing to increase [4]. The study by Martin et al. from 2009 analyzed more than 11 000 patients with severe sepsis from the international register [5]. In this study, 57% of patients suffered from Gram-negative, 44% from Gram-positive, and 11% from mycotic infections. The lungs were the primary source of infection in 47% of patients, abdominal infection was found in 23%, and urinary tract infection in 8%. The total mortality reached almost 50%. The European Sepsis Occurrence in Acutely Ill Patients (SOAP) study, with a 35% incidence of sepsis in patients at ICUs showed an overall mortality of 27% [6]. It is not surprising thus that the priority continues to be timely diagnosis of sepsis and active surveillance in hospitalized patients `with a high risk of sepsis development. The objective of diagnostics is finding the focus and targeted surgical intervention along with targeted antibiotic (or other) therapy. Our review gives a short overview of the current epidemiology of sepsis, its pathogenesis and state-of-the-art knowledge on the use of specific biochemical, hematological and immunological parameters in its diagnostics. Prospective approaches towards discovery of new diagnostic biomarkers have been shortly mentioned. 2. Definition of sepsis, SIRS and the PIRO concept One of the important points in the understanding of pathogenesis of sepsis developed from the consensus conference in 1991 with the definition of the Systemic Inflammatory Response Syndrome (SIRS), sepsis, severe sepsis, septic shock, and Multiple Organ Dysfunction Syndrome—MODS [7]. This practical approach with a clinical definition of SIRS, represented a fresh understanding of interactions of pro- and anti-inflammatory mechanisms and of the mediators in inflammatory response of the host to a range of insults. The term—SIRS—was thus seen to be relevant to a wide spectrum of etiological agents besides infection, with non-infectious causes of SIRS such as burn, surgery trauma, pancreatitis being recognized. Clinical practice has since shown the SIRS concept to be too sensitive and not specific enough. [8]. Patients defined on the basis of SIRS were very heterogeneous and, where used alone to define inclusion criteria for clinical trials, this heterogeneity has likely contributed to some of the many well recognized failures of new therapeutic strategies. In 2003, a new concept of sepsis-related conditions was defined [9], the PIRO concept. The abbreviation PIRO refers to: Predisposition (P), Infection (I), Response (R), and Organ dysfunction (O). The PIRO concept can better define septic patients and evaluate their staging relating to the severity of infection—similarly to TNM system in cancer patients. 3. Mechanisms of SIRS and sepsis The syndrome of systemic inflammatory response and sepsis is characterized by inflammation and defects of homeostasis [10]. The concept of a hyper-inflammatory syndrome that dominated the past two decades has been challenged and at present, sepsis is seen more as a dynamic syndrome characterized by many often antagonistic phenomena [11]. Hence sepsis should be viewed as spanning a hyperinflammatory response and anergy or immunoparalysis response. These responses will evolve with different time courses; however it is increasingly understood that the old idea of a pro-inflammatory process followed by a compensatory anti-inflammatory phase does not represent the common picture. Rather the two processes progress with a significant degree of synchrony although not necessarily with the same time courses; commonly systemic immunosuppression

dominates [12]. A unique study documenting immunosuppression in sepsis patients was published in 2011 [13]. Boomer et al. conducted a functional study designed to assess the immune system status in patients who had died due to sepsis. In a neat study the authors isolated the lung and spleen cells from these patients and compared them with cells from trauma victims. They immunophenotyped the cells and elucidated their functional status as defined by cytokine production following stimulation with lipopolysaccharide. The study demonstrated a state of functional immunosuppression in patients dying with sepsis, manifesting itself largely by decreased production of both pro- and anti-inflammatory cytokines after stimulation with lipopolysaccharide, reduced monocyte HLA-DR expression, and a decreased immunocompetent cell count. Of particular interest was the fact that the sepsisinduced immunosuppression state could be reversed by removing the cells from a sepsis “milieu”. Typically, the immune response of sepsis patients is a hyper-inflammatory systemic response and/or a state of “immunoparalysis“, characterized by low monocyte HLA-DR expression and decreased TNF-α production ex vivo after lipopolysaccharide stimulation. Characteristic features of the hyper-inflammatory phase include increased production of cytokines, i.e., tumor-necrosis factor α (TNF-α), macrophage migration-inhibiting factor (MIF), and highmobility group box 1 (HMGB-I) protein [14]. By contrast, the compensatory anti-inflammatory response syndrome (CARS), which can yield immunoparalysis, typically involves the production of IL-4, IL-10, IL-11, IL-13, transforming growth factor-β (TGF-β), granulocyte, and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF, respectively), soluble TNF-α receptors and IL-1 receptor antagonists. Clinical findings in these patients include skin test anergy to memory cell-specific antigens, leukopenia, and increased susceptibility to infection [15]. In addition to infection, immunoparalysis can be induced by non-infectious causes such as trauma, ischemia, and burns. A summary of mechanisms responsible for the status of immunosuppression in sepsis: 1. Increased/enhanced apoptosis of cells of the innate and adaptive immune systems 2. Exhaustion of the T-cell phenotype 3. Monocyte deactivation with decreased HLA-DR expression 4. Increase in T-regulatory cell count 5. Increase in negative and decrease in positive co-stimulation molecules 6. Switch of Th 1 to Th2 response 7. Regulatory effect of CNS on the immune system Detailed description of the pathogenesis of sepsis exceeds the scope of this text, therefore we refer the reader to synoptic reviews [16,17]. 4. Diagnostics of sepsis Since there is no such thing as “a magic bullet” in the diagnosis of sepsis, the clinical diagnosis [with general variables, inflammatory variables, haemodynamic variables, organ dysfunction variables and tissue perfusion variables] remains the gold standard [18]. Nevertheless, there are a number of parameters across laboratory branches that are used in diagnostics with different utility and penetration. The high mortality of sepsis can be seen as an indication of insufficient laboratory diagnostics. Recent studies have shown that each hour of delay in diagnosis increases mortality of septic patients and that inappropriate and delayed antibiotic therapy is a negative prognostic factor for the clinical outcome [19,20]. The situation is even more complicated as not only the “patient's state” but also the causal agent and its susceptibility to antibiotics have to be determined. Leaving clinical diagnosis aside, the laboratory diagnostics represent a complex range of investigations that can place significant demands on the system given the speed of response required. Bed-side monitoring of selected parameters is already a routine part of laboratory diagnostics for many critically ill patients, the sensitivity and specificity of the applied methods being

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comparable to classical methods such as ELISA or turbidimetry and nephelometry [21]. Unfortunately, despite many advances, the majority of laboratory investigations are not sufficiently sepsis-specific. A number of overlapping individual disciplines typically have combined to help in the laboratory diagnosis of sepsis with clinical biochemistry, hematology, immunology, microbiology, and molecular biology now all playing a part. To diagnose infection means to find the focus, to isolate the causal microorganism and identify its sensitivity to antibiotics. However, we have to assess the severity of the clinical status of the patient, which is expressed, in part, by the scale of their inflammatory response. There are hundreds of biomarkers which could be potentially used for diagnosis and prognosis in septic patients [22]. The main attributes of successful markers would be high sensitivity, specificity, possibility of bed-side monitoring, and financial accessibility. Only a fraction are used in routine clinical practice because many lack sufficient sensitivity or specificity, others represent an expression of the dysfunction of individual organs or systems rather than sepsis markers and others are only in developmental stages still. Laboratory diagnosis of sepsis is thus a mosaic of different technological and methodological approaches with the subsequent integration of the clinical picture of the patient. The objective is timely and more reliable diagnostics which would lead to faster and more effective therapy of these patients. In patients with the syndrome of systemic inflammatory response or sepsis, answers have to be found to the question of: i) whether the inflammation is of infectious or non-infectious origin; both infectious and non-infectious insult can trigger a cascade of inflammatory response which ends up in multiorgan failure ii) whether the biomarker makes it possible to determine a prognosis for the patient; iii) whether the biomarker will influence the therapy and/or provide information on the success in treatment. 4.1. C-reactive protein and procalcitonin C-reactive protein is an archetype acute phase protein found in 1930 by Tillett and Francis in patients with pneumococcal pneumonia, whose sera precipitated somatic C-polysaccharide fraction of pneumococci. C-reactive protein is produced by the liver with the maximum production 24-38 hours after inflammation onset. Studies using liver tissue cultures show that IL-6 is a massive inducer of CRP mRNA. IL-1 is not active alone but will do so in synergy with IL-6 [23]. CRP binds Gram-positive and Gram-negative bacteria and stimulates their adhesion and complement dependent phagocytosis by leukocytes. Generally it can be said that the main function of CRP is to bind heterogeneous structures of both endo- and exogenous origin (altered membranes, cell debris, bacteria, and parasites) and with this bond, trigger defense mechanisms of the macro-organism—adherence and modulation of phagocytic cells, activation of complement system, stimulation of opsonization and phagocytosis [24]. The concentration of CRP in healthy subjects is lower than 5 mg/l; its level is diagnostically used to distinguish viral and bacterial infections [25,26]. It is necessary to emphasize that CRP is not a specific parameter for the presence of infectious inflammation as it is also elevated in certain systemic autoimmune diseases of rheumatoid arthritis (RA) type, some oncological diseases, with significant trauma, or after surgery and tissue damage [27]. It is a parameter independent of the use of therapeutic intervention methods such as renal replacement therapy (RRT), as well as of the presence of neutropenia [28]. On the other hand, its concentration decreases with the use of systemic corticosteroids [29]. In the past 15 years another protein called procalcitonin has gained ground in clinical practice [30]. Procalcitonin (PCT) is a protein of 116 amino acid sequence and a prohormone of calcitonin. In physiological conditions, calcitonin is secreted by C cells of thyroid gland, where it is

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formed from its precursor i.e. procalcitonin. This occurs under specific intracellular proteolysis in which N-terminal area PCT (57 AK), calcitonin (32 AK), and catacalcin (21 AK) are formed from the precursor peptide i.e. preprocalcitonin composed of 141 (AK) amino acids. In sepsis the main producers of PCT are macrophages and monocytic cells of different organs, especially liver. Experimental models of infection showed synthesis of PCT and other tested tissues, or organs including adipocytes [31,32]. Tests performed on volunteers have demonstrated that following the injection of bacterial endotoxin synthesis of PCT can be identified in plasma within 2–3 h. Its concentration increases to reach a maximum typically after 12–48 h. The concentration of induced PCT in sepsis fluctuates in the range of tens to hundreds of ng/ml. This plasma PCT is not converted in the form of active calcitonin and it is very stable. The way of elimination of PCT from the organism is not known yet though it is probably degraded, as is the case with many other plasma proteins, by proteolysis. Procalcitonin acts as chemokine and modulates induction of anti-inflammatory cytokines and induces production of nitric oxide synthase [iNOs]. Its effect on mortality of experimental animals is also of great interest. The injection of procalcitonin increased mortality of experimental animals with sepsis, but, did not have this effect in animals without the presence of sepsis [33]. In viral infections, there is a minimum elevation of PCT concentration. This is caused by the production of interferon alpha which inhibits synthesis of TNF-α necessary for the production of PCT in tissues. In recent years, a number of studies have been published comparing the usefulness of determination of C-reactive protein and procalcitonin in differential diagnostics of infectious and non-infectious inflammation. Meta analyses showed higher specificity and sensitivity of procalcitonin in comparison with C-reactive protein; however neither procalcitonin nor C-reactive protein fulfills the role of an ideal biomarker in the diagnostics of sepsis [34–37]. We know that its sensitivity is not sufficient in patients with abscesses, with invasive mycotic infections or in patients with tuberculosis [38–40]. Procalcitonin is also not fully specific for sepsis with elevated levels being found after surgery and in those with florid autoimmune disease [41,42]. A Czech study also focused on the specific group of patients with organ transplantation and immunosuppression where the diagnosis of sepsis is vital. They showed that those who were given the monoclonal antibody OKT3 during peri-operative treatment exhibited a significant increase of procalcitonin levels without any infectious complications [43]. The normal range for procalcitonin usually b0.5 ng/ml, surgery and autoimmune diseases can elevate this though usually not much more than 2 ng/ml while sepsis will increase it often much more. Practically the negative predictive value of procalcitonin is also of importance as if PCT levels are lower than 0.2 ng/ml, the negative prediction related to bacteremia is higher than 90%. Monitoring of the levels of procalcitonin provides useful information on the efficacy of antibiotic therapy and this is a grade 2C recommendation in the latest surviving sepsis guidelines. Not only can procalcitonin guide when to start or stop antibiotic therapy, but it also has been useful in supporting the use of shorter courses of antibiotics without any impact on morbidity and mortality and thus, in some studies, in lowering the cost of antibiotic therapy [44–46]. Its biological half-life means that sampling at 24 hour intervals is convenient. It is important to recognize that not all studies demonstrate this [47]; in a recent Dutch study 32 out of 132 patients with sepsis and septic shock had a PCT value of b 0.5 ng/ml and 8 of these (25%) have a positive blood culture [48]. At present we have several published studies at our disposal comparing CRP a PCT in different target groups of patients [49–52]. The results are sometimes controversial. How should we approach their interpretation then? The use of any biomarker or interpretation of the results has to be based on the evaluation of the patient's clinical state. One of the controversial topics is use of PCT to determine antibiotic therapy. Individual studies published various algorithms suggesting a positive impact on the duration of antibiotic therapy and a

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significant economic effect [53,54]. Use of the so-called sensitive PCT where it is possible to measure values b0.2 ng/ml has also been suggested to be helpful but this required a different assessment methodology which raises the cost further making the economic case less clear. In 2013 recommendation from an expert panel on the use PCT for decision making to start, interrupt or stop antibiotic therapy was published [55]. The results are as follows:Four clinical situations were evaluated: 1. Acute pancreatitis—good predictive value of PCT for the presence of infection but the existing data are not sufficient for the decision to administer ATB. 2. Community acquired infection of lower respiratory tract in adults (LRTI). Discontinuation of ATB at the value of PCT b 0.25 ng/ml in patients with nosocomial infection LRT; there are no sufficient data for the decision to start antibiotic therapy on the basis of one or repeated measurements. 3. Children with suspected bacterial meningitis—Bacterial Meningitis Score or the Meningitest is recommended as a therapeutic decisions; a single PCT level N0.5 ng/ml may be used, but false-negatives can occur. 4. ICU patients with suspected of community-acquired infection—a threshold serum PCT value is not recommended for the decision to start ATB therapy. In non-immunocompromised patients treated for the infection of respiratory tract, ATB can be discontinued, if at day 3 PCT level is b 0.25 ng/ml or has decreased by N80–90%, whether or not microbiological documentation has been obtained. It is also important to distinguish between the use of the parameter for the diagnosis of systemic infection and prognosis of the patient. Generally we find that the absolute value of the parameter is less relevant than trend changes and monitoring of the dynamics of the infective process or its response to therapy. For example a decrease of 30% or more in procalcitonin levels in 24 h suggests that you have selected the antibiotic therapy correctly. We have to keep in mind that from the patient's point of view a falsely negative result is more dangerous than a falsely positive one. The results of SAPS study (Stop Antibiotics on guidance of Procalcitonin Study), which should be published the next year, will surely be of great interest [56]. 4.2. Cytokines Cytokines are pleiotropic regulators of the immune response, which have a role in the complex pathophysiology underlying sepsis [57]. Some of the cytokines used to diagnose sepsis, evaluate current level of the inflammatory response and help determine a prognosis for the patient, are interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-10 (IL-10). IL-6 is a prototype of proinflammatory cytokine, IL-8 is the main chemokine, and IL-10 represents an important antiinflammatory cytokine. IL-6 is produced by various types of cells including monocytes, fibroblasts, endothelial cells, keratinocytes, T-lymphocytes and tumor cells. IL-6 acts as a differentiating factor for B-lymphocytes and an activating factor for T-lymphocytes. After the administration of lipopolysaccharide (LPS) or live bacteria, IL-6 is released into the bloodstream for 4–6 h, with levels subsequently decreasing over the next 24–48 h. The chemokine IL-8 is produced by macrophages and also by endothelial cells. IL-10 is an anti-inflammatory cytokine produced by monocytes, macrophages, T and B lymphocytes, neutrophils and mesangial cells. From experimental studies we understand that IL-10 plays a fundamental role in reducing the intensity of inflammatory response triggered by the administration of LPS. Experimental models in animals where IL-10 was administered intravenously or intraperitoneally show that they were protected against the effects of LPS. This protective role of IL-10 results from the inhibition of proinflammatory mediators including TNF-α and IL-1β,

production of IL-8, interferon-γ, nitric oxide, IL-6 and metabolites of prostaglandins. High levels of IL-6 and IL-10 predict higher mortality for septic patients [58]. IL-8 is considered to be a good predictive marker of the severity of septic patients although this has only been shown in children and has not been confirmed in adults [59,60]. These cytokines also allow a quantitative assessment of the severity of sepsis and this may relate to outcome. A meta-analysis by Eichacker et al., showed that the efficacy of anti-inflammatory therapy depended on the severity of the underlying clinical state and that the more severe the sepsis, the greater the efficacy of the anti-inflammatory therapy [61]. This can provide a window of opportunity for assessment of cytokine levels and determination of selected cytokines in septic patients has been shown in a number of studies [62,63]. Nevertheless, the problem remains how to implement these outcomes into routine clinical practice. As regards the diagnosis of sepsis itself, determinations of CRP or PCT are more sensitive and more specific than cytokines in most studies whilst determination of cytokines is valuable in monitoring the intensity of the inflammatory response although elevated levels are also present in SIRS of noninfectious origin. There are currently no studies which would prove that the treatment of sepsis based on these markers influences the treatment strategy or improves the clinical result. In the future the development of a bedside test for cytokine levels could help for targeting and monitoring therapeutic interventions rather than diagnostic purposes. 4.3. Lipopolysaccharid binding protein Lipopolysaccharide binding protein (LBP) is 58 kD protein, which is produced by the liver as part of the acute phase inflammatory response. Under physiological conditions, its role rests in facilitating binding to lipid A or bacterial lipopolysaccharide binding to CD 14, which is present on monocytes and macrophages. Also as a consequence of LPS binding to the cells of monocyte–macrophage system, pro-inflammatory cytokines IL-1 and TNF-α are induced [64]. The concentration of LBP in serum under physiological conditions fluctuates between 5 and 15 μg/ml and increases several times during the acute phase response. LBP belongs to lipids binding proteins along with “bactericidal/ permeability increasing protein” (BPI) and transport proteins for cholesterol esters. Experimentally, a high concentration of LBP has been shown to block the effect of lipopolysaccharide and significantly reduced lethality for mice in which sepsis had been stimulated [65,66]. Under “in vitro” conditions, at high concentration of LBP, lipopolysaccharide was not capable of inducing the production of cytokines by mouse macrophages [67]. On the basis of the current findings LBP seems to exhibit double function: in lower concentrations it increases the effect of lipopolysacharides, and in high concentration it weakens or inhibits the effect of LPS. A number of studies have been published exploring the importance of LBP in diagnostics of sepsis. In general, LBP adds little value when trying to distinguish between infectious and noninfectious etiology of SIRS; however its prognostic importance has been demonstrated for patients with proven sepsis [68–71]. 4.4. Surface markers of circulating leukocytes Flow cytometry is another diagnostic method used in the investigation of sepsis; this includes evaluating CD64 expression on neutrophils. CD 64 is a high affinity receptor for, IgG (Fcgamma RI) and represents marker of neutrophil activation. It is constitutively expressed by macrophages and monocytes only, whereas its expression on neutrophils occurs after activation by cytokines i.e. interferon γ and G-CSF (Granulocyte-colony stimulating factor). A number of studies have been published on the importance of CD64 for diagnostics of sepsis. The Bhandar et al. study demonstrated sensitivity of CD64 at 80% and specificity of 79% with neonatal sepsis [72]. In adults, Icardi et al.

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showed a very good predictive value of CD64 for clinical and laboratory (microbiological) diagnosis of sepsis with sensitivity of 94.6% and specificity of 88.7% [73]. Similar results were confirmed in other studies [74–76]. Currently we have commercial kit Trillium Diagnostic's Leuko64 assay at our disposal; the use of which has removed nonstandard use of different types of diagnostics. The determination of the expression of CD64 on neutrophils thus represents a positive step in the sepsis mosaic especially in the differential diagnosis of SIRS of an infectious vs noninfectious etiology [77]. 4.5. D-dimer Severe sepsis is associated with defects in hemostasis and with the development of disseminated intravascular coagulation (DIC). D-dimer is a product of degradation of fibrin by the process of fibrinolysis. It is known especially for its association with thrombosis; nevertheless there are studies concerned with its diagnostic value in septic patients. As early as in 1990, a study was published on the significant predictive value of D-dimer for the presence of bacteremia in septic patients [78]. The marked elevation of D-dimer in patients with sepsis was confirmed by the PROWESS study [79]. One recently published study demonstrated the prognostic importance of D-dimer in relation to the severity of the patient's state [80]. On the contrary however, Japanese and American studies have not confirmed a significant diagnostic value for patients in early phases of sepsis [81,82]. The D-dimer is an easily accessible assay in common routine practice, but its role in the diagnostics for sepsis will have to be more fully validated by future studies. 4.6. Presepsin and other novel markers 10 years ago, a new biomarker sCD14-ST was discovered and named as presepsin [83,84]. CD14 is a glycoprotein on membrane surfaces of monocytes/macrophages, and acts as a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). CD14 activates the toll-like receptor 4 proinflammatory cascade in the presence of infectious agents. Two forms of CD14 are present—membrane CD14(mCD14) and soluble CD14(sCD14). Complex LPS-LPBP-CD14 is shed into the circulation and plasma protease generates sCD14 molecule called sCD14 subtype(sCD14-ST)-presepsin. Recently some clinical studies were published concerning the relationship between presepsin and sepsis. Presepsin is increased in septic patients and is not different significantly between patients with Gram-positive and Gram negative infection. Presepsin seems to be very early biomarker of sepsis whose place in the clinical diagnostic armory still needs clarification [85–87]. TREM-1 is a cell surface receptor expressed on the myeloid cells and a member of the immunoglobulin superfamily. In addition to mTREM-1, which is expressed on monocytes, sTREM-1 can be detected in the serum or bronchoalveolar lavage. In recent years, TREM-1 has been studied as a factor mediating the inflammatory response of the body to infection. The determination of the levels of its soluble form (sTREM-1) or determination of expression of TREM-1 on monocytes (mTREM-1) has been investigated as a perspective diagnostic method to distinguish infectious from non-infectious etiology of the inflammation [88,89]. Actually TREM-1 has not been adopted formally in the diagnosis or tracking of sepsis but is worth watching for future developments. CD73, an enzyme expressed on vascular endothelium and other cells, acts to dephosphorylate adenosine monophosphate. The product is adenosine which binds to its A2B receptor that is widely expressed and mediates anti-inflammatory effects including prevention of vascular leak and leucocyte recruitment. CD73, like CD14, can be shed into the circulation and levels measured. The role of CD73 levels as a prognostic or tracking marker for sepsis or lung injury remains to be clarified but is of potential interest to this field [90].

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5. The future of diagnostics of sepsis—genomics and proteomics The genotype affects the incidence and severity of infectious disease. Numerous studies have demonstrated an association between the genomic variability of an individual with the incidence and outcome in inflammatory and infectious diseases. One of the first such studies was that conducted by Sorensen et al. who, while investigating the incidence of cancer and role of the genetic background, revealed an association between infectious disease and the death of children whose biological parent died of infection at an age below 50 years (relative risk 5.8) [91]. Further evidence was furnished by studies with twins, designed to determine the incidence of tuberculosis, leprosy, poliomyelitis and hepatitis B [92]. With further progress in knowledge of the human genome, questions have now been focused on understanding the immune response in sepsis for example whether the gene expression differs with infectious and non-infectious etiologies. New methodologies— DNA and RNA microchips have brought about the possibility of complex investigations to try to get to such answers. Studies from the turn of millennium showed the immune response to be stereotypical with infection but different with various types of infectious agent [93]. Our study was one of the first to point to the exclusiveness of the immune response in systemic inflammation of infectious etiology [94]. Studies of genetic polymorphism of the innate immune system and cytokines have not produced any unequivocal results so far and are not being used in clinical practice. Similar to many other fields of medicine, it has become evident that for the immune system the genotype does not actually correspond to the phenotype. In recent years significant efforts have focused on proteomics followed by genomics and transcriptomics with the objective of defining proteins in the cells, tissues,or organism, and their relation to the septic process. Through this approach it is hoped to find new biomarkers that could diagnose or track progression of sepsis or predict its incidence or outcome. The use of gene expression in diagnostics of sepsis could be vital, given the complexity of this syndrome and represents a beginning of the so called personalized medicine in septic patients [95–97]. Transcriptomics provides information about the amount of messenger RNA, the transcriptome being the complete set of RNA transcripts produced by the genome at any one time. The technique used most commonly for this purpose is that of DNA microarray. Another modality available besides DNA microarray is multi-gene transcription profiling with mRNA quantification using PCR. Techniques for gene expression determination have been shown to be able to distinguish between patients with SIRS of infectious vs non-infection etiology. In addition, these techniques are employed to identify predictive biomarkers in sepsis patients [98]. The effort to discover novel diagnostic markers that could identify well in advance patients with developing sepsis or those with a potentially more serious course of disease has necessarily led to the introduction of proteomic approaches to this area of medicine. Proteomic analysis has been successful in identifying new diagnostic proteomic biomarkers in medical specialties other than in the field of sepsis, for example of ovarian cancer and the search is well underway now for such markers in sepsis [99]. Both human and animal models have been employed for proteomic analyses with the use of LPS challenge providing a most elegant opportunity to monitor the host response in individual cell subpopulations or tissues [100]. The substrates available from patients in the clinical setting include plasma, cells, and urine. The expression of diagnostic biomarkers from individuals varies significantly and this enormous inter-individual variability in the intensity of the inflammatory response is further affected by the site samples and the patients age, sex and other co-morbidities. This heterogeneity has thus far made it impossible to successfully translate any of these into relevant biomarkers for clinical practice. Another major factor in this respect is that of the technologies employed with a number of technologies employed in basis research being able to analyze trace amounts of

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sample and such are simply not ready for use in prime time clinical practice which still relies on studying proteins produced in relatively easily detectable quantities [101]. 6. Summary The diagnostics in critically ill patients in non-coronary intensive care units are characterized by the search for new biomarkers which will enable the timely diagnosis of infectious inflammation and determine patient's prognosis. It has been demonstrated that the timely and accurate diagnosis of sepsis significantly influences accuracy in the choice of antibiotic therapy and the patient's prognosis. The laboratory diagnosis of sepsis is thus a mosaic of different technological and methodological approaches which are vital to the subsequent integration of the clinical picture and outcome of the septic patient. References [1] Vincent JL. EPIC II: Sepsis around the world. Minerva Anestesiol 2008;74:293–6. [2] Jawad I, Luksic I, Rafnsson SB. Assessing available information on the burden of sepsis: global estimates of incidence, prevalence and mortality. J Global Health 2012;2:1–9. [3] Esper A, Martin GS. Is severe sepsis increasing in incidence and severity? Crit Care Med 2007;35:1414–5. [4] Martin GS. Sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. Expert Rev Anti-Infect Ther 2012;10:701–6. [5] Martin G, Brunkhorst FM, Janes JM, et al. The international PROGRESS registry of patients with severe sepsis: drotrecogin alfa (activated) use and patients outcomes. Crit Care 2009;13:R 103. [6] Vincent JL, Sakr Z, Sprung CL, et al. Sepsis occurrence in sepsis in European intensive care units: results of the SOAP study. Crit Care Med 2006;34:344–53. [7] American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992;20:864–74. [8] Vincent JL. Dear SIRS, I'm sorry to say that I don't like you. Crit Care Med 1997;25: 372–4. [9] Levy MM, Fink MP, Marshall JC, et al. SCCM/ESICM/ACCP/ATS/SIS. 2001 SCCM/ ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med 2003;29:530–8. [10] Deutschman CS, Tracey KJ. Sepsis: current dogma and new perspectives. Immunity 2014;40:463–75. [11] Cavillon JM, Eisen D, Annane D. Is boosting the mmune system in sepsis appropriate? Crit Care 2014;8:216–26. [12] Hotchkiss RS, Monneret G, Payen D. Immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach. Lancet Infect Dis 2013; 13:260–8. [13] Boomer JS, To K, Chang KC, et al. Immunosuppression in patients who die of sepsis and multiple organ failure. JAMA 2011;306:2594–605. [14] Huang W, Tang Y, Li L. HMGB1, a potent proinflammatory cytokine in sepsis. Cytokine 2010;51:119–26. [15] Tschaikowsky K, Hedwig-Geissing M, Schiele A, Bremer F, Schywalsky M, Schüttler J. Coincidence of pro- and anti-inflammatory responses in the early phase of severe sepsis: longitudinal study of mononuclear histocompatibility leukocyte antigen— DR expression, procalcitonin, C-reactive protein, and changes in T-cell subsets in septic and postoperative patients. Crit Care Med 2002;30:1015–23. [16] van der Poll T, Opal SM. Host–pathogen interaction in sepsis. Lancet Infect Dis 2008;8:32–43. [17] Giamarellos-Bourboulis EJ, Raftogiannis M. The immune response to severe bacterial infection. Expert Rev Anti Infect Ther 2012;10:369–80. [18] Dellinger RP, Levy MM, Rhodes A, et al. Surviving Sepsis Campaign Guidelines Committee including The Pediatric Subgroup. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med 2013;39:165–228. [19] Kumar A, Roberts D, Wood KE, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;341:589–96. [20] Kumar A, Ellis P, Arabi Y, et al. Cooperative Antimicrobial Therapy of Septic Shock Database Research Group. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest 2009;136:1237–48. [21] Schefold JC, Hasper D, von Haehling S, Meisel C, Reinke P, Schlosser HG. Interleukin-6 serum level assessment using a new qualitative point-of-care test in sepsis: a comparison with ELISA measurements. Clin Biochem 2008;41:893–8. [22] Pierrakos C, Vincent JL. Sepsis biomarkers: a review. Crit Care 2010;14:R15. [23] Arnaud C, Burger F, Steffens S, et al. Statins reduce interleukin-6 i induced Creactive protein in human hepatocytes: new evidence for direct antiinflammatory effects of statins. Arterioscler Thromb Vasc Biol 2005;25:1231–6. [24] Peisajovich A, Marnell L, Mold C, Du Clos TW. C-reactive protein at the interface between innate immunity and inflammation. Expert Rev Clin Immunol 2008;4: 379–90.

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