Sepsis is a complex clinical syndrome, caused by the body’s response to severe infection. Based on the heterogeneity of this disease process, significant confusion exists around the changing definition and diagnosis of sepsis¹.

The term, “Sepsis”, has long been interchangeably used with other terms such as, “infection”.

Infection is defined as the invasion of healthy tissues by pathogenic microorganisms. Sepsis, however, is a serious, potentially fatal, complication of an infection.

Sepsis can be thought of as existing along a continuum from infection to multiple organ dysfunction syndrome.

The three core components of sepsis are-infection, host response, and organ dysfunction. Sepsis is caused by the immune system’s response to a serious infection, most commonly bacteria, but also fungi, viruses and parasites Sepsis differs from other noninfectious insults, such as trauma or pancreatitis, which are triggered by endogenous factors.

1.1.1.1 The American College of Chest Physicians/Society of Critical Care Medicine Sepsis Definitions Conference, 1991

In 1991, a first consensus panel convened by the American College of Chest Physicians (ACCP) and the Society of Critical Care Medicine (SCCM) defined sepsis as a “systemic inflammatory response to infection”. This definition, however, did not clearly define “infection”, and whether it included bacterial, viral, fungal or parasitic infection as the underlying cause². Confusion around this definition arose from the fact that the clinical response observed in sepsis can also result from non-infective causes.

A systemic response to infection is more often than not a normal and adaptive process that helps the host to mount an effective attack and clear the infection. With this in mind, sepsis can be seen as either helpful or harmful. To address this confusion around the definition of sepsis, the conference suggested the term, severe sepsis, defined as “sepsis in association with organ dysfunction, hypoperfusion, or hypertension”, to differentiate those causes of harm outweighing the benefits of a response. Finally, the term, septic shock, was proposed to describe the harmful cases of sepsis in which hypotension was present, and tissue oxygenation was impaired. A result of these new definitions was the suggestion that the previously used term, “septicemia”, be abandoned as inaccurate. The conference also proposed the use of the terminology, multiple organ dysfunction syndrome (MODS).

The concept of Systemic Inflammatory Response Syndrome (SIRS) was also proposed by this consensus panel in 1991, describing the inflammatory response that accompanies sepsis, but also other etiologies. This consensus committee determined that when the cause of SIRS was confirmed to be an infection, the term “sepsis” is appropriate. SIRS was defined as the presence of more than one of the following conditions²:

Temperature >38c or <36c (Hyperthermia/hypothermia)
Tachycardia evidenced by heart rate >90 beats/min
Tachypnea evidenced by respiratory rate >20 breaths/min
White blood count >12000 cells/mm³ or <4000 cells/mm^3,or morethan 10% immature neutrophils

1.1.1.2 The Second Sepsis Definitions Conference, 2001

The next major review of sepsis was in 2001, when a further consensus panel convened to reconsider the definition of sepsis.

The group made no recommendations to change the existing definition of sepsis but provided an expanded list of diagnostic criteria ³. Again as with the 1991 Sepsis definition, this definition also did not distinguish between bacterial, viral, parasite infections and other causes of the clinical syndrome.

Perhaps the most important outcome from the 2001 Consensus Conference was the proposal for a “Predisposition, Infection, Response and Organ dysfunction” (PIRO) system for stratifying sepsis (Table 1). The concept of PIRO was analogous to the Tumor, Node, Metastasis (TNM) model widely used in staging cancer⁴. The underlying concept of the PIRO system is that a multidimensional approach is needed to stratify patients to guide the multimodal management of different stages of sepsis. The aim of this model is not prognosis and the edges of the stages are poorly defined. However, the aim of this PIRO staging was to foresee potential response to specific therapies and to recognize different characteristics of sepsis. It thus helps the clinician to identify the stage of sepsis in a patient and therefore inform treatment.

Table 1. Guidelines for Stratification of Sepsis Patients. Adapted from ⁵.

Domain	Laboratory Result	Clinical Findings	Advanced Testing	Rationale
Predisposition	Genetic abnormalities	Premorbid illness/ Comorbidities	Genetic polymorphisms in components of inflammatory response (e.g. TNF, IL-1, CD14); enhanced understanding of specific interactions between pathogens and host diseases.	Comorbidities of an acute insult is heavily dependent on genetic predisposition. Additionally, premorbid factors impact on the potential attributable morbidity and mortality of an acute insult.
Infection	Identification of causative micro-organisms	Site of infection; specific causative pathogen(s)	Assay of microbial products (LPS, mannan, bacterial DNA); gene transcript profiles.	Specific therapies directed against inciting insult require demonstration and characterization of that insult.
Response	WBC, coagulation profile, CRP levels, and lactate levels	Core temperature, heart rate, blood pressure and cardiac function	Nonspecific markers of activated inflammation (e.g., CD64, PCT or IL-6) or impaired host responsiveness (e.g. HLA-DR); specific detection of target therapy (e.g., protein C, TNF, PAF).	Both mortality risk and potential to respond to therapy vary with non-specific measures of disease severity.
Organ Dysfunction	PaO₂/FiO₂ratio, bilirubin, creatinine, and CSF chemistry	Glasgow Coma Scale, urine output, and capillary refill	Dynamic measures of cellular response to insult- apoptosis, cytopathic, hypoxia, cell stress.	Response to preemptive therapy (e.g. targeting microorganism) is not possible if damage is already present, therapies targeting the injurious cellular process require that it be present.

Abbreviations: CRP, C-reactive protein; WBC, White Blood Cell; HLA-DR, human leukocyte antigen-DR; IL, interleukin; LPS, Lipopolysaccharide; PAF, platelet-activating factor; PCT, procalcitonin; TNF, Tumor necrosis factor; CSF, cerebrospinal fluid.

1.1.1.3 SEPSIS-3

The most recent attempt to define sepsis was in 2016 when the scientific community questioned the concept of SIRS and found it to lack specificity, overly focusing on inflammation. At this event, Sepsis was defined as “life threatening organ dysfunction caused by a dysregulated host response to infection”⁶. This definition was previously used to describe the term “Sever sepsis”.

A detailed scoring system with five levels, namely, dysfunction of respiration, coagulation, liver, cardiovascular, central nervous system and renal functions, was introduced. This scoring system, known as, Sequential [Sepsis Related] Organ Failure Assessment Score (SOFA), predicts severity and thus outcome of sepsis (Table 2).

As noted by the 2016 task force, septic shock isolated a subset of a sepsis in which underlying circulatory and cellular metabolism abnormalities are profound enough to substantially increase mortality⁷. Septic shock thus reflects on a more severe illness with a much higher likelihood of death than sepsis alone.

The task force identified three variables- 1. altered mentation (Glasgow Coma Scale >15), 2. a respiratory rate greater than 22 breaths per minute, and a 3. systolic blood pressure less than 100mm Hg, that independently predicts a risk of in-hospital death or an ICU stay of 3 days or longer. The model was termed, quick sequential (sepsis-related) organ failure assessment (q-SOFA). The presence of any two or more ofthese variables suggests a high risk of poor outcome and an indication that the patient has sepsis and should have their lactate levels tested for evidence of organ dysfunction.

Sepsis-3 was clearer when compared to previous definitions of sepsis in term of defining q-SOFA and introducing MODS in the definition itself. Sepssi-3, however, has limitations. The new definition focusses on sepsis patients at the greatest risk of imminent deterioration as opposed to early diagnosis of infection. Finally, the task force panel set up to develop the definition has been criticized for the lack of diversity in the panel, in particular, the absence of women and clinicians working in low and middle-income countries.

	SOFA Score
	0	1	2	3	4
Respiration PaO₂/ FiO₂, mmHg (kPa)	≥400 (53.3)	<400 (53.3)	<300 (40)	<200 (26.7) with respiratory support	<100 with respiratory support
Coagulation Platelets, x10³/mm³	≥150	<150	<100	<50	<20
Liver Bilirubin, mg/dl (µmol/L)	<1.2 (20)	1.2-1.9 (20-32)	2.0-5.9 (33-101)	6.0-11.9 (102-204)	>12.0 (204)
Cardiovascular Hypotension	MAP ≥70mm Hg	MAP <70mm Hg	Dopamine <5 Or dobutamine (any dose)^a	Dopamine 5.1-15 Or epinephrine ≤0.1 Or norepinephrine ≤0.1^a	Dopamine >15 Or epinephrine >0.1 Or norepinephrine >0.1^a
Central nervous system Glasgow Coma Score^b	15	13-14	10-12	6-9	<6
Renal Creatinine, mg/dl (µmol/L) or urine output,ml/day	<1.2 (110)	1.2-1.9 (110-170)	2.0-3.4 (171-299)	3.5-4.9 (300-400) or <500 ml/day	>5.0 (440) or <200 ml/day

^a Catecholamine doses are administered for at least 1 hour (doses given are in µg/kg/min)

^b Glasgow Coma Scale scores range from 3-15; higher score indicates better neurological function

Abbreviations: PaO₂, partial pressure of oxygen; FiO_2,fraction of inspired oxygen; MAP, mean arterial pressure.

Table 2. The SOFA score Adapted from ⁸

1.1.2 Morbidity and Mortality associated with Sepsis

Despite advances in modern medicine, such as vaccines, antibiotics and acute care, sepsis remains a global healthcare problem. Globally, sepsis caused over 5 million deaths in 2016⁹, and the number of mortality cases that were due to septic shock was 46.5%¹⁰. The incidence of sepsis is rising, possibly due to growing elderly population, antibiotic resistance, immunosuppressive medication, and invasive surgery^11–14. In the US alone, the number of hospital admissions for sepsis following healthcare-associated as well as community-acquired infections increased three-fold over the last decade, while hospital admissions for stroke and myocardial infarction remained stable over the same period¹⁵. Sepsis is a more common condition than cardiac arrest, and claims more lives than any one form of cancer each year.

In the least developed countries, sepsis remains a leading cause of death. Despite all these worrying statistics, only 10-30% of patients with sepsis receive excellent care¹⁶. Mortality rates vary from country to country, and among patients of differing age and underlying comorbidities; but data from several studies suggest mortality from sepsis is typically between 20 to 30%¹⁷.

Early diagnosis of sepsis and the early starting of antibiotic treatment has been shown to make a massive difference to outcome, not just in terms of reducing mortality but also in reducing the disability that can result from a severe bacterial infection.

Survivors of sepsis have an increased risk of death in the following five years and may suffer from persistent physical and cognitive dysfunction^18,19.

According to World Health Organization (WHO), it is estimated around one million neonatal deaths are due to neonatal sepsis. An immature immune system, especially in preterm neonates, lacking the ability to produce pro-inflammatory cytokines, coupled with impaired neutrophil production and function, predisposesneonates to suffer from neutropenia and contributes to an increase in susceptibility to sepsis^20–22 .

1.1.3 Etiology of Sepsis

Our body’s survival depends on our physical barriers and also a rapid innate immune response. Host protection is provided by intact epithelial and mucosal surfaces as well as antimicrobial peptides that protect the body’s barrier²³. When the integrity of the physical barrier has been compromised or there is an impairment in the function of antimicrobial peptides, microorganisms can enter the body by directly contaminating tissue or via blood or lymphatic fluid leading to infection, this is sometimes complicated by development of sepsis. As sepsis with organ dysfunction worsens, hypotension and life-threatening metabolic changes (septic shock) can develop²⁴.

Bacteria account for nearly 90% of infections in cases of sepsis²⁵. The species of microorganism that causes sepsis may also affect mortality, as gram-positive sepsis tends to have higher mortality than gram-negative sepsis in nosocomial infections²⁶. However, Gupta and colleagues reported 47.1% of all severe sepsis admissions in the United States were for culture-negative sepsis²⁷. The data on the incidence of viral sepsis is scarce, some studies have reported it to be approximately 1-10%^28,29. Additionally the incidence of fungal sepsis has grown at an alarming rate of 207% through 2000³⁰. This may represent a general increase in nosocomial cases of sepsis, or it may reflect emergence of resistance to antifungal drugs³¹. However the exact impact of type of a given type organism on mortality due to sepsis is unclear ³².

It has also been reported that different bacteria elicit a different kind of immune response. Gram negative bacteria elicit a more pro-inflammatory cytokine profile (dominated by TNF-alpha, IL-1, IL-6, and IL-8), in contrast, gram positive bacteria induces a typical Th-1-type cytokine response, (dominated by TNF-beta (or lymphotoxin-alpha) and interferon-gamma³³^,³⁴). Conversely, another paper has found that the cytokine gene expression was similar in patients with gram-positive and gram-negative infection³⁵.

The most important pathogens for community-acquired sepsis are bacteria with high virulence factors such as Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitis, β-hemolytic streptococcus, Escherichia coli, and Klebsiella spp¹⁷^,³⁶. However, in about one-third of the cases, an etiological microbial agent is never found^36,37. Patients with culture-negative sepsis were reported to have fewer comorbidities and milder illness compared to culture-positive sepsis³⁸.

1.1.4 Symptoms and Signs of Sepsis

A recent evaluation of clinical criteria to identify sepsis in patients with suspected or confirmed infection used the SIRS criteria, the SOFA score and qSOFA score, as well as a modified LOD (Logistic Organ Dysfunction System) score in categorizing the severity of sepsis.

LODS score can be calculated using multiple logistic regression model. Points are allocated for neurological, cardiovascular, and renal dysfunction and for the pulmonary, hematologic, and hepatic systems and address both the relative severity among organ systems and the degree of severity within an organ system. The total number of points provides an estimated risk of mortality.

In patients admitted to the ICU the SOFA and LOD scores had the highest predictive value for mortality, whereas in patients outside ICU the qSOFA had the highest predictive validity for mortality³⁹.

Symptoms of sepsis are highly variable, depending on the age of the patient, the infecting organism underlying the development of sepsis, the presence of other comorbidities, and the level and site of associated organ dysfunction¹⁷. Classical symptoms and signs include fever, hypothermia, tachycardia, tachypnea, altered mental state, hypotension, and oliguria⁴⁰. Classical symptoms of sepsis, however, may be absent in elderly patients and neonates²⁸. Associated organ dysfunction or failure can affect cardiac, respiratory, renal, central nervous and hepatic systems.

There are two distinct phases associated with septic shock; namely, hyperdynamic phase (warm shock) and cold shock. The early onset of fever is the initial warm phase characterized by a bounding pulse, peripheral vasodilatation and a hyperkinetic circulation. Without treatment, the warm phase is followed by cold shock with poor peripheral perfusion, vasoconstriction and high mortality⁴¹. Septic shock is considered to be a distributive form of circulatory shock, with peripheral vasodilatation and inadequate blood supply to organs and tissues⁴².

1.1.6 Diagnosis and treatment of sepsis

Diagnosis of sepsis is challenging, given that there is no specific diagnostic test and the diagnostic criteria are regularly reviewed and changed. The diagnosis of sepsis is based on criteria obtained from an evaluation of the patient’s history, the clinical symptoms and signs, use of sepsis syndromic definitions, and biochemical abnormalities. As the clinical picture of sepsis can be due to conditions other than infection (e.g. pancreatitis, trauma, hemorrhagic shock), confirmation of infection as the etiology of sepsis through positive culture from blood or from another sterile site is helpful. Positive bloodstream microbiological detection of the underlying cause, with two or more blood cultures is recommended but can take days or even longer for a positive result and can be negative especially in patients who are given antibiotics prior to blood culture. The use of screening tools (e.g. qSOFA) can improve recognition of sepsis.

Novel laboratory methods, such as MALDI TOF, real-time multiplex PCR and calorimetry are used currently in laboratories; other non-culture methods are being developed⁴³. Physiological scoring systems such as APACHE and SOFA are used to provide a more accurate indication of disease severity and prognosis ⁴⁴. Biomarkers may help in the early detection and risk assessment of sepsis patients, and these are described in detail in the next section.

“The golden hour”, the time period within which rapid treatment can make an outcome difference between life and death, is a well-known term used to describe the current practice of early therapeutic intervention (within six hours of diagnosis) in order to significantly reduce mortality and also morbidity through early administration of antibiotics and early reversal of shock. In 2001, when Rivers et al. reported on early goal-directed therapy (EGDT), an aggressive resuscitation program was introduced. This led to significantly reduced mortality⁴⁵^,^46–48. Put simply, EGDT is an active surveillance program of high risk septic patients, providing crystalloid resuscitation to restore central venous pressure, vasopressors to maintain mean arterial pressure, inotropic drugs, blood transfusions as well as an invasive monitoring of central venous oxygenation, all undertaken within 6 hours of presentation at the emergency department⁴⁵.

The key items in the initial management of the patient with sepsis are microbiological culture and administration of broad-spectrum antibiotics. In a large cohort of sepsis patients inappropriate antimicrobial therapy was shown to be independently associated with increased mortality⁴⁹. Effective antimicrobial administration within the first hour of the initial onset of sepsis, which is broad spectrum and empiric, leads to significant improvement in the outcome of sepsis ^50,51. To prevent the emergence of resistant organisms, minimize the risk of drug toxicity, and reduce costs, a de-escalation of initial broad spectrum therapy to more specific therapy must take place as soon as possible⁵⁰.

Glucocorticoids are widely used as anti-inflammatory agents in treatment of several chronic conditions. Their use in the treatment of severe infections is however, greatly debated. Some studies have demonstrated a shorter time to shock reversal and improved survival⁴⁵^,⁵⁴, but, on the other hand, the incidence of hyperglycemia and superinfections was higher in corticosteroid-treated patients⁵³. The guidelines of today are to treat patients with refractory shock with hydrocortisone (200 to 300 mg per day for up to 7 days or until vasopressor support is no longer required), supported by a meta- analysis study⁵²^,⁵⁵. This therapy is given to substitute for a relative adrenal insufficiency, but the evidence for this has been questioned in a more recent publication¹⁷.

Different sepsis trials with immunomodulating drugs (e.g. anti-tumour necrosis factor-alpha-TNF-,IL-6) all appeared promising in animal studies, but have not translated well in human clinical trials^56,57. Recombinant activated protein C, a promising molecule with anti-inflammatory, anti-thrombotic and pro-fibrinolytic properties, was initially shown to reduce sepsis mortality⁵⁸, however, the side effects of bleeding⁵⁹ and the failure to reproduce the initial positive results has made the manufacturer withdraw it from the market⁶⁰.

Polyspecific intravenous immunoglobulin G (IVIG) is pooled from thousands of donors and exhibits high polyspecificity, ensuring coverage of different superantigens and bacterial serotypes. IVIG has been suggested as an adjunctive therapy in severe infections^61,62. In sepsis overall, results of trials on IVIG as adjunctive therapy have been conflicting, and IVIG treatment is still controversial^63,64. Additionally, the most recent guidelines of the Surviving Sepsis Campaign suggest against the use of IVIG in patients with sepsis⁶⁵. Only toxin-mediated bacterial diseases, such as severe Staphylococcus and Streptococcus infections, are currently given as examples of indications for IVIG therapy in infection.

1.1.7 Emerging point-of-care tests to diagnose sepsis

The World Health Organization seeks to reduce the global burden of sepsis (a disease that is responsible for millions of preventable deaths every year), targeted prevention, rapid diagnosis of sepsis, and immediately focused antibiotics would substantially address this problem. The process of early, accurate and rapid diagnosis of sepsis is one of the greatest challenges that is faced globally.

Traditional sepsis diagnostics include culture-based pathogen identification to verify the efficacy of antimicrobial treatment, whether administered before or after culturing. In addition to this, several biomarkers have been investigated to guide the clinician in this diagnostic approach. However, the heterogeneity of the immune response during sepsis has made research on a potential biomarker for sepsis difficult. There remains an unmet need for practical point-of-care (POC) test that can identify sepsis with a high degree of sensitivity, correlated with high negative predictive value; this should continue to be a major focus of work in the future. Two new approaches for the diagnosis of sepsis that are currently underway are the development of molecular diagnostics for the assessment of host response, and for pathogen detection.

1.1.7.1 Sepsis diagnostics based on pathogen identification

If sepsis is suspected or confirmed the next concern for clinicians is to identify the causative organism(s). Pathogens may differ widely, depending upon epidemiological and clinical factors. Need for rapid pathogen identification is particularly true if the patient has been rendered immune-compromised by treatments for cancer, auto-immune disease, or transplant, where opportunistic infections can be very rapidly fatal. To select the appropriate antibiotic, the organism(s) needs to be identified and its sensitivity to antibiotics determined. Blood culture is typically used in identification and is currently seen as the cornerstone in the investigation of any patient with severe infection or sepsis.

Although an infectious cause is fundamental to the definition of sepsis, in many cases isolation of specific organism by culture remains challenging. Various multicenter studies have reported the proportion of culture-negative severe sepsis to be between 28-49% of all patients with severe sepsis^28,66–68. It is difficult to discern the reason for culture negativity; however, some plausible explanation includes inappropriate culture technique, administration of antibiotics before obtaining cultures, infections with fastidious or fragile organisms (e.g., pneumococcus), viral etiology or no organism present.

Innovative techniques such as exhaled breath analysis are being developed that analyses volatile organic compounds (VOC) are a non-invasive easily applied way to reduce diagnosis time. VOCs in the exhaled breath were measured by gas chromatography and mass spectrometry. This is being taken forward by the multicenter prospective observational study BreathDx (Molecular Analysis of Exhaled Breath as Diagnostic Test for Ventilator-Associated Pneumonia), where exhaled breath is to be analyzed using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). This study will also potentially put together patterns of VOCs in exhaled breath indicative of specific microorganisms⁶⁹. Non-invasive sampling of breath is a useful for mechanically ventilated patients and could potentially help our understanding of the pathogenesis of acute respiratory distress syndrome (ARDS). In saying that, this technique does not allow for antimicrobial-sensitivity testing which would lead to more effective use of antibiotics and reduction in antibiotic duration.

BioMérieux E-test is a method of rapidly detecting antibiotic susceptibility via a plastic strip containing a pre-defined gradient of antibiotic which can be directly applied to critically ill patients. Results from this test are provided in less than 24 hours which is faster compared to time taken for standard blood culture (48-72 hours).

Common methodologies applied in the area of culture-dependent pathogen identification are matrix-assisted laser desorption/ionization- time-of-flight mass spectrometry (MALDI-TOF MS) and multiplex real-time molecular assays.

Database dependent “fingerprint” methods such as Matrix Assisted Laser Desorption-Time Of Flight Mass Spectrometry (MALDI-TOF MS) are now adapted for clinical microbiology by exploiting ‘soft ionization’ of bacterial or fungal isolates to preserve components key for detection and analysis. MALDI-TOF MS takes minutes compared to hours or days for biochemical identification methods or blood culture and costs are considerably less per isolate⁷⁰. While the expense of running the testing is minimal, the upfront cost of the instrument is not. Most institutions from resource constrained settings will not be able to afford an instrument and those from developed countries will most likely be able to have 1 instrument and, therefore, would be affected if the instrument breaks down for any reason. In the U.S. two FDA-cleared MALDI-TOF MS systems are the Vitek-MS (bioMérieux) and the BioTyper (Bruker Daltronics). The other disadvantage with MALDI-TOF MS is the lack of susceptibility data. Clinicians might keep the patient on broad-spectrum antibiotics regardless of the identification until the susceptibility results from conventional methods are released. To solve this problem BD Kiestra system, available from Becton Dickinson, integrates mass spectrometers with automated antimicrobial susceptibility systems, thus allowing partial or complete automation of routine microbial characterization⁷¹. MALDI-TOF MS is a rapidly emerging technology for the identification of microorganisms however significant effort needs to be made to reduce cost and optimize databases and algorithms for the upcoming instruments to undergo thorough validation.

Multiplex real-time molecular assays in a sample-to-answer format are being increasingly adopted in clinical settings to improve time to pathogen identification. Biomérieux’ (USA) FilmArray Blood CultureIdentification (BCID) Panel enables accurate automated detection of a set of 24 gram positive, gram negative, yeast pathogens, and 3 resistance genes associated with bloodstream infections^72,73. The turnaround time for this FDA approved and CE marked test is about an hour. Other options are the FDA-cleared Verigene (Nanosphere) Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, respectively) and assays that use Gold/Ag nanoparticle probes and micro-array for detection of bacterial pathogens and several resistance markers. Both platforms allow random access testing and showed consistent results. The accuracy of the BC-GP and BC-GN assays as well as the FilmArray has been reported to be relatively low for the direct identification of microorganisms in polymicrobial cultures as compared to monomicrobial cultures^74,75.

Several approaches to culture-independent direct pathogen detection include most advanced technologies such as Abbott’s Iridica device and T2 biosystems’ magnetic resonance technology. Capable of detecting a large number of microorganisms, Abbott’s Iridica couples broad range PCR to electro-spray ionisation mass spectrometry (PCR/ESI-MS). However, in April 2017, Abbott ceased producing IRIDICA instruments and IRIDICA test kits. In 2014 this was partly achieved by the bench top T2Dx instrument with its inaugural T2Candida assay (T2 Biosystems). T2 relies on changes in a sample’s T2 magnetic resonance (T2MR) signal caused by hybridization of PCR-amplified pathogen DNA to capture probe-decorated nanoparticles⁷⁶. T2Candida requires 1 mL of uncultured whole blood and provides results in about 3 hours with a claimed limit of detection as low as 1 colony forming unit/ml. However, T2Candida assay is currently limited to detecting only the five most common Candida species (accounting for 95% of candidemia) and does not provide antifungal susceptibility data

In the last decade, a number of nucleic acid amplification tests for detection of bacteria in blood (e.g., SepsiTest test [Molzym], SeptiFast test [Roche], MagicPlex test [Seegene], and VYOO test [SIRS-Lab]) have been evaluated in clinical studies and become CE-marked and commercialized in Europe⁷⁷. Challenges have included long hands-on time, cost, and varying results in performance studies. In addition, outcome studies surrounding their use are lacking.

1.1.7.2 Sepsis diagnostics based on biomarker identification:

Biomarkers such as C-reactive protein (CRP), white blood cell count, lactate and procalcitonin (PCT) have classically been used to assist in the diagnosis and prognostication in patients with sepsis. However, these biomarkers only offer a moderate diagnostic performance and predictive abilities that do not differ greatly from readily available clinical scoring systems.

Several biomarker-guided antibiotic treatment strategy trials in sepsis have been published over the last years, mostly based upon PCT and CRP. The recently updated guidelines of the Surviving Sepsis Campaign guidelines incorporates PCT as the only other sepsis biomarkers (apart from lactate) to play a minor role in clinical practice. The guidelines stress the point that a clinical decision to initiate, alter or stop antimicrobial treatment should never be based solely on changes in any current biomarker, including PCT.

Several qualitative and quantitative assays for measurement of PCT are commercially available; however, these assays vary in their performance characteristics, and detection limits. The PCT assays currently available in the market in the US (FDA approved) are Thermo Fisher Scientific, Inc., B•R•A•H•M•S PCT sensitive KRYPTOR, Roche ELECSYS B•R•A•H•M•S PCT and BioMérieux VIDAS⁷⁸^,⁷⁹. These assays are based on the principle of time-resolved amplified cryptate emission (TRACE) technology (KRYPTOR®, BRAHMS). All BRAHMS PCT assays use a ‘sandwich ELISA’ principle to quantify procalcitonin by forming antibody–procalcitonin–antibody complexes. The main difference between these assays are the mechanism of detection of these complexes.

Different PCT algorithms have been developed to help decision making regarding the start and duration of antibiotic treatment. Levels of PCT between 0.5 and 2 g/L suggest the presence of bacterial infection and >2 g/L confirms bacterial sepsis for which antimicrobial therapy is required⁸⁰. No consensus has been reached about the correct cut-off value for PCT in this decision making. The current data on the use of PCT for antibiotic stewardship in critically ill patients with sepsis are also not consistent. The most positive evidence to date has been the recent prospective, multicentre, randomized controlled, open-label intervention trial in 15 hospitals in Netherlands, that was able to demonstrate a reduction in the median duration of antibiotic treatment from 7 to 5 days as well as 5% improvement in mortality⁸¹. In contrast, a recent investigation determined the use of PCT and associate outcomes in ICUs in United States, about 18% patients out of 20,000 critically ill patients in 107 hospitals had their PCT levels checked however, the use of PCT was not associated with improved antibiotic use or other clinical outcomes⁸². The main drawback for the current PCT tests available in the market is the cost. At an average unit price of £13.79, the PCT-guided antibiotic therapy was found to be costly and therefore difficult to implement in a resource-limited setting. In addition to this, PCT is not completely specific to bacterial infection and can be raised following severe trauma or in para-neoplastic syndromes and auto-immune conditions⁸³.

The New Surviving Sepsis Campaign (SSC) guidelines recommends incorporating lactate as a biomarker for detection of septic patients, with the lactate cut-off set at 4mmol/L. A number of portable lactate analyzers are available commercially and are primarily used in sports science for longitudinal monitoring and progression of athletes. Nova Biomedical’s StatStrip Lactate Test Strips are FDA approved. The device comprises of a single-use test strips containing an enzyme-coated electrode. The result turnover time is only 13 seconds and uses only 0.6l of blood. However, lactate is fairly nonspecific as it can be elevated due to various reasons, such as such as cardiac arrest, trauma, seizure or excessive muscle activity, although serial measurement to calculate lactate clearance has shown to have prognostic value in sepsis. But studies suggest that this may be a false assurance, for example, in a study by Dugas et. al., 45% of patients in vasopressor-dependent septic shock did not have lactate levels > 2.4 mmol/L initially, but their mortality remained high⁸⁴. The reasons why some patients have elevated lactate levels compared to others is not well understood.

C-Reactive protein (CRP) is a general acute phase protein and its plasma concentration increases significantly in response to inflammation and/or infection, mediated by cytokine stimulation. Although some studies in the literature suggested that high plasma CRP concentration may help in distinguishing bacterial from viral and other infections, the clinical utility of such diagnostic approach remains ambiguous^85,86. Several point-of-care tests for CRP are available commercially, however, studies directly comparing all available devices are scarce. Quick Read Go (Orion Diagnostica, Finland) has been both FDA approved (approved for use in USA) and CE-IVD certified (approved for use in the EU). Several CE-IVD certified devices are currently available in the market, such as, AQT90 Flex (Radiometer Medical ApS), iChroma (Boditech Med), NycoCard Reader II (Alere) as well as Smart analyser (Eurolyser Diagnostica). A significant overlap in CRP values exists between patients with and without infection, and it is particularly apparent for ICU patients in whom other causes of inflammation may be present concomitant to sepsis. Therefore, despite the continued and widespread use of CRP in various clinical situations, its lack of sensitivity and specificity cannot support a major diagnostic role for this biomarker in sepsis.

Finally, two other host biomarker based sepsis-diagnostic tests currently available are ImmunoXpert (MeMed Diagnostics, Israel), and SeptiCyte (Immuneexpress, USA). These tests measure proprietary molecular markers from the patient’s immune system and uses an algorithm to diagnose sepsis rapidly.

Septicyte has advanced commercially having received its 510(k) clearance from the U.S.FDA. The device uses the white cell transcriptome to delineate infection related inflammation from other inflammatory causes (e.g., surgery). In a published study (adult and paediatric) cohorts the device was shown to be able to differentiate infection from other forms of inflammation with an AUC of 0.89–0.95 with an improved performance compared to PCT^87,88.

Neutrophil CD64 expression is one of the most frequently studied sepsis biomarker. CD64 is a high affinity Fc receptor for immunoglobulin G (IgG) that is expressed on neutrophils during an infectious or inflammatory state. In a bacterial infection, an increase in the number or the density of CD64 antigens as well as an increase in the percentage of neutrophils showing increased CD64 expression, has been reported⁸⁹. In contrast to a viral infection, only an increase in the percentage of neutrophils showing CD64 expression is typically observed. CD64 expression is also increased in patients who have non-infectious inflammatory systemic conditions such as sickle cell crisis⁹⁰ as well as localized inflammatory conditions such as the synovial fluid of rheumatoid arthritis patients⁹¹. The utility of neutrophil CD64 expression extends over other traditional hematologic markers in predicting clinically determined sepsis or infection⁹² as well as to the neonatal population, a population in which the diagnosis of sepsis is more difficult and associated with greater morbidity and mortality⁹³. Its main drawback is likely related to the use of flow cytometry. Recently, the Accellix system was developed for use at the bedside or in emergency labs.⁹⁴ It is a fully automated table top cytometer requiring only introduction of the patient sample into a cartridge. Sample preparation and reading are performed in a dedicated disposable cartridge. A microfluidic based cartridge measuring neutrophil CD64 manufactured by LeukoDx Ltd (Jerusalem, Israel) recently received FDA approval. However, in recent meta-analysis it was found that neutrophil CD64 expression alone cannot be a satisfactory marker for diagnosing neonatal sepsis with relatively low sensitivity of 76% in adults and 80% in neonates^95,96.

Thus, a biomarker(s) with a high degree of sensitivity and specificity is still required to enable the clinician to make or refute a diagnosis of a severe infection or sepsis with relative certainty. In this thesis, I describe the studies conducted to validate two novel biomarkers of sepsis, Total CD64 and total Neutrophil Elastase and undertake preliminary lateral flow experiments for proof of concept to develop a rapid, immunochromatographic test to detect sepsis at point of care.

1.2 Introduction to the Immune System

1.2.1 Myelopoiesis

Leukocytes originate from haematopoietic stem cells (HSCs) in the bone marrow by a process in which a complex network of different cytokines, intracellular interactions, and an intricate regulation of transcription factors orchestrate the lineage commitment processes of the HSCs⁹⁷. HSCs subsequently develop along distinct differentiation pathways into multipotent progenitors and into lineage-restricted progenitors^98,99.

Cells of myeloid lineage normally present within the peripheral blood are neutrophils, eosinophils, Natural killer (NK) cells, basophils, monocytes, erythrocytes, dendritic cells, and platelets.

During severe infections, such as sepsis, there is an increased consumption of and subsequent requirement of myeloid cells. Both immature and mature cells of the myeloid lineage are mobilized from the bone marrow into the peripheral blood. This is called emergency myelopoiesis resulting in a “left shift” of immature myeloid cells within the periphery, a sign of underlying severe pathology¹⁰⁰. A shift to the left occurs when immature neutrophils, at a stage of differentiation in between metamyelocytes and neutrophils (called “band forms” as the nucleus has not completely segmented into lobes), are found within the blood.

1.2.2 The adaptive and the innate immune systems

To fully comprehend the pathogenesis of sepsis in severely ill patients, the pathways of the “normal” immune system and its defense mechanisms to combat infections need to be understood. Traditionally, the immune system is divided into two categories, the innate and the adaptive immune systems¹⁰¹.

The innate system depends on pattern recognition and constitutes the first line of hosts’ defense against pathogens. It is phylogenetically well conserved and present in both vertebrates and invertebrates¹⁰². This innate immune system (including the complement system, the coagulation pathway and the fibrinolytic system, cytokines, antimicrobial peptides, and acute phase proteins), can be readily mobilized within minutes or hours of infection and serves to attack and eliminate the microbes invading the body. It has, however, no memory function, and thus always reacts the same way to a given stimulus. It is orchestrated mainly by cells of myeloid origin i.e. neutrophils, monocytes/macrophages, natural killer cells, mast cells and dendritic cells. These immune cells induce phagocytosis of microorganisms, remove debris, and activate the complement cascade and subsequently the adaptive immune system¹⁰³.

The adaptive immune systems, which is centered around lymphocytes, arose approximately 500 million years ago in our vertebrate ancestors¹⁰⁴. The adaptive immune system serves as a second line of defense, with a memory function, responding more powerfully after each new exposure to a particular pathogen. It is built of lymphocyte interactions to provide recognition of foreign invaders, i.e. antigens with specificity and diversity and provides a long-lasting immunological memory¹⁰³^,¹⁰⁵. The adaptive immune response is however delayed¹⁰⁵. Different lymphocyte populations play a part in the adaptive immune response, viz. B- and T- lymphocytes and antigen presenting cells (APCs)¹⁰¹.

The immune system combats microbial infections, but in the case of sepsis, its untoward activity seems to contribute to organ dysfunction. One of the inflammatory criteria for sepsis include neutrophil count. High numbers of blood neutrophils, or neutrophilia (>12×10⁹cells per liter), could be due to excessive recruitment from the bone marrow, the return of marginated cells into the circulatory pool, or both. Overwhelming activation of neutrophils is known to elicit tissue damage. However, neutropenia (<4×10⁹ cells per liter), as can be observed after treatment such as chemotherapy, also increases susceptibility to infection and to sepsis.

In the studies in this thesis I have focused largely on innate immune responses, in particular neutrophil responses. What, then, are neutrophils?

1.2.3 Overview of Neutrophils

White blood cells or leukocytes can be divided into mononuclear cells and polymorphonuclear cells (PMN), also known as granulocytes. The neutrophils are short-lived PMN cells that die within one hour after they have engulfed bacteria. Under normal conditions neutrophils account for 40 to 70 % of all leukocytes in the circulation. Neutrophils play a crucial role in the first line of defense against pathogens.

During an infection, the number of neutrophils rises due to increased synthesis and release from the bone marrow, as well as mobilization from the marginating pool¹⁰⁰^,¹⁰⁶. However, leukopenia with concomitant neutropenia can occur in severely ill patients and is caused by sepsis-mediated bone marrow suppression as well as in neonates with an immature immune system². In sepsis, a subset of neutrophils with suppressive functions has been discovered and these neutrophils produce significant amounts of IL-10¹⁰⁷. Another study also found a subset of mature neutrophils that suppressed T cell-function when healthy volunteers were injected with low-dose endotoxin¹⁰⁸.

1.2.3.1 Role of Neutrophils in Sepsis

Neutrophils have been regarded as a double-edged sword in sepsis. Several studies have shown that neutrophil responses in sepsis are aberrant with respect to their survival, migratory capacity and functionality^109,110. Neutrophils are essential for the eradication of microorganisms, but the release of anti-microbial peptides often leads to organ injury¹⁰⁹.

Neutrophils become activated during sepsis and kill microorganisms via antimicrobial peptides contained within their granules, by oxidative burst and the release of reactive oxygen species (ROS), or through release of neutrophil extracellular traps (NETs), a process called NETosis. These NETs are comprised of extracellular or cell free neutrophil-derived DNA (cf DNA) and histones, as well as bactericidal granule proteins such as elastase, cathepsin G, and myeloperoxidase^111–113. In activated neutrophils ROS production results in the release of elastase from azurophilic granules. Following translocation of elastase to the nucleus, it partially degrades nucleosomal histones leading to chromatin decondensation. Myeloperoxidase then binds to the degraded chromatin, resulting in neutrophil rupture and release of NET¹¹⁴. It has been shown that NET formation can precede phagocytosis, and NETs seem to be able to encounter and trap far more bacteria simultaneously than phagocytosis alone^115,116. Although their functions are not completely understood, NETs are thought to augment the killing of microorganisms and to limit dissemination of bacteria via abscess formation. NET formation has also been linked with dysregulated coagulation¹¹⁷, endothelial damage, organ dysfunction and sepsis severity¹¹⁸ in septic patients. NET formation has been shown to be impaired in newborn infants, both pre-term and term, and may be associated with increased risk of sepsis¹¹⁹.

A distinct feature of infection and sepsis is the recruitment of immature neutrophils from the bone marrow into circulation. In addition, activated tissue macrophages and endothelial cells express proinflammatory cytokines, such as TNF, IL-1, IL-8 and nitric oxide (NO), which attract neutrophils. A complex process is initiated involving adhesion of circulating neutrophils to the activated endothelium in the blood vessel wall, followed by the extravasation and migration of neutrophils to the place of tissue injury and the subsequent elimination of microbes through phagocytosis, generation and rapid release of reactive oxygen species (ROS), and degranulation and release of microbicidal molecules¹²⁰.

Proinflammatory cytokines give neutrophils a prolonged lifespan, which is attributed to delayed apoptosis. This prolonged inflammatory response augments cell injury. Studies have discovered that these neutrophils have an altered function, which includes impaired chemotactic activity, reduced clearance of microbes and reduced production of ROS¹⁰⁹.

Neutrophil granules can be classified as primary, secondary or tertiary. The primary granules are formed in the promeylocytic stage, and the secondary granules form in the myelocytic stage. In a later stage, called the metamyelocytic stage in which the neutrophils differentiate further, the tertiary granules are formed. The stages of development determine the specific granule contents in different granula. In addition to the granules, the secretory vesicles are characterized by their immediate release when contact is established between the neutrophil and the endothelium¹²¹. Upon activation of the neutrophils, the secretory vesicles translocate and cover the neutrophil surface membrane with β-integrins (membrane-associated receptors) which mediate endothelial adhesion and initiate transmigration of the neutrophils through the endothelium¹²².

Neutrophils store a reservoir of different proteins and proteases, as well as membrane-bound receptors for endothelial adhesion molecules, extracellular matrix proteins and soluble mediators of inflammation. Most of the above-described steps followed by neutrophil-activation are dependent and due to the mobilization of cytoplasmic granules and secretory vesicles stored within the neutrophils. They enable the destruction of internalized pathogens, but also they cause local tissue damage¹⁰⁹. The neutrophils contain more than 300 different proteins released in a hierarchical order during the movement of the neutrophils from the blood stream to the tissue of infection¹²⁰^,¹²³^,^124,125.

A recent report found circulating neutrophils in sepsis patients to have suppressed apoptosis, a longer life-span and pro-inflammatory phenotype with increased TNF-α/IL-10 ratio¹⁰⁶. In most cases of sepsis and septic shock, the total number of circulating neutrophils increases, although some patients, in contrast, have low neutrophil counts or immature forms of neutrophils. It seems likely that rather than the total number of neutrophils, it is a subset of neutrophils that plays an important role and is engaged in tissue insult, since even in neutropenic patients, immature neutrophils degranulate and cause neutrophil-mediated lung injury¹⁰⁹.

1.3 Immunopathology in Sepsis

1.3.1 The Inflammatory Response in Sepsis

When microbes invade the body, the first line of defense of the innate immune system is activated very rapidly. This event is initiated through pathogen-associated molecular patterns (PAMPs), evolutionarily conserved outer components or patterns on the surface of the microbes, such as the endotoxin lipopolysaccharide (LPS) of the Gram-negative bacteria, as well as peptidoglycan and lipoteichoic acid of the Gram-positive bacteria¹²⁶. The PAMPs are discovered by immune cells (such as neutrophils and macrophages) by pattern recognition receptors (PRR) on their surface. Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors and retinoic acid-inducible gene-1 (RIG-1)–like receptors are examples of PRRs¹²⁶.

When PRRs recognize PAMPs, an intracellular signal-transduction pathway activates and releases transcription factors, such as, nuclear factor-κB (NF-κB) – thereby inducing transcription of genes coding for numerous cytokines including TNF, IL-1, IL-6 and an initiation of the innate immune response¹²⁷. In addition, the PRRs sense endogenous molecules released from the dying host cells, the danger associated molecular patterns (DAMPs), or alarmins. The alarmins (eg high mobility group box 1 (HMGB-1) protein and S100 proteins) are also released during sterile injury such as trauma, giving rise to the concept that the pathogenesis of multiple organ failure in sepsis is not totally different from that in noninfectious critical illness¹²⁸.

In conclusion, both PAMPs and DAMPs elicit the inflammatory response seen in sepsis. Thus, a cascade of various complex processes is initiated: phagocytosis of the microbes, release of mediators, activation of the complement system and the coagulation cascade, release of cytokines and other mediators.

The cause of sepsis-associated multi organ failure remains largely unknown. One postulated mechanism is that impaired tissue oxygenation leads to an imbalance between oxygen delivery and oxygen consumption causing global tissue hypoxia⁴⁸^,¹²⁹. Many factors contribute to the decreased oxygen utilization, including cytopathic hypoxia, myocardial depression, increased metabolism, impaired red-cell deformability, microcirculatory dysfunction, as well as tissue hyperfusion ^129,130. The associated systemic inflammatory response associated with sepsis causes microvascular leak and macrocirculatory endothelial lesions, resulting in intravascular depletion and subcutaneous oedema^131,132. The dysfunctional epithelial barriers also predispose to secondary infections. Severe sepsis and septic shock is associated with coagulation abnormalities, impaired fibrinolysis and disseminated intravascular coagulation (or DIC)^133,134. Inflammation leads to activation of coagulation and deposition of fibrin. The fibrin removal is impaired because of down regulation of endogenous fibrinolysis¹³⁴. The modulation of fibrinolysis is mediated by the intrinsic coagulation pathways and the alternate pathway by neutrophil elastase and results in widespread clotting and subsequent bleeding¹³³.

1.3.2 The Pro and Anti-Inflammatory Phase in Sepsis

The assumption that sepsis was an overwhelming inflammatory reaction of the patient to microbes was widely accepted for a long time¹³⁵^. For many years, two phases of sepsis have been described: initially the systemic inflammatory response syndrome (SIRS), with production of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β, and IFN-γ), from which the term “cytokine storm” arose¹³⁶. This phase is followed by a secondary compensatory anti-inflammatory response syndrome (CARS) with secretion of anti-inflammatory cytokines (IL-10, TGF-β and IL-1 receptor antagonist (IL-1ra)) in an attempt to restore immunological equilibrium^137–139. The prolonged state of immune suppression is nowadays referred to as sepsis- induced immunoparalysis, which is characterized by an impaired innate and adaptive immune response¹⁴⁰^.

This sepsis model of SIRS and CARS has been modified with time, and now there is growing consensus that the production of pro-inflammatory (IL-1, IL-6, TNF- α) and immunosuppressive cytokines (IL-1 receptor antagonists, IL-4 and IL-10) begins immediately after the onset of sepsis, to balance the host’s need to maintain defense while minimizing self-induced tissue damage¹³⁷^,^135,141. However, the net effect of these two competing responses is usually an initial dominant hyper-inflammatory phase and a secondary immunosuppressive phase¹³⁷.

Regulatory anti-inflammatory responses induce leukocyte anergy, reduction and apoptosis of lymphocytes, decreased pro-inflammatory cytokine response by monocytes to stimulation, loss of monocyte HLA-DR expression, decreased antigen-presentation by monocytes, as well as increased expression of the above immunosuppressive cytokines¹⁴²^,¹⁴³^,¹⁴⁴. A recent study strengthens the hypothesis that the pro- and anti-inflammatory responses act in concert, by the induction of both pro- and anti-inflammatory genes in critically ill patients¹⁴⁵.

In best case scenarios, the innate responses of activated immune cells lead to a balanced response contributing to the elimination of pathogens and tissue recovery¹⁴⁶. In worst case scenarios (e.g. in cases of septic shock), the responses lead to an imbalanced inflammation and tissue injury (mostly the pro- inflammatory response) or to a state of immune suppression (mostly the anti- inflammatory reactions)¹⁴⁶. The current understanding is that the inflammatory response in these patients is too strong in the initial stages, while at later stages patients have a reduced responsiveness of blood leukocytes to pathogens, and are left more fragile and highly susceptible to secondary infections^147,148.

In addition to these responses, neural mechanisms are initiated, that can inhibit inflammation¹⁴⁹. In the neuroinflammatory reflex, sensory input is relayed through the afferent vagus nerve to the brain stem, from which the efferent vagus nerve activates the splenic nerve in the celiac plexus, resulting in norepinephrine release in the spleen and acetylcholine secretion by a subset of CD4+ T cells. The acetylcholine release targets α7-cholinergic receptors on macrophages, suppressing the release of pro-inflammatory cytokines¹⁵⁰.

The detrimental effects of a dysregulated inflammatory response and its contribution to systemic toxicity, as seen in severe sepsis/septic shock, were illustrated by the unfortunate outcome of the of the first phase 1 clinical trial of TGN1412, a novel superagonist anti-CD28 monoclonal antibody that directly stimulates T cells151. After the first dose of TGN1412, all six human volunteers faced life-threatening conditions involving multi-organ failure. Another interesting observation was that TGN1412 elicited a much higher cytokine response in humans compared to that noted in non-human primates, clearly illustrating the difference in cytokine responses between human and non-human primates and the obvious importance of careful research before conducting a clinical trial¹⁵². The failure of former sepsis trials has led to the development of novel strategies where immunostimulatory rather than immunosuppressive drugs could play a role. A requirement for applying immunotherapy is a proper selection of patients, and there is a need for therapeutic biomarkers.

1.4 Immunometabolism in Sepsis

Immunometabolism can be defined as an interdisciplinary field that is at the crossroads of diverse disciplines such as immunology and metabolism¹⁵³^,¹⁵⁴. It can further be categorized into two main branches: (1) Cellular immunometabolism dealing with the research on the metabolic pathways in immune cells determining their fate under diverse conditions^155,156. (2) Tissue immunometabolism mainly focuses on the effect of immune cells on tissue and systemic metabolism supporting the organismal adaptation to environmental changes(infection or trauma)¹⁵⁴.

The pathogenesis of sepsis involves a complex interaction between host and infecting microorganism, including an initial hyper-metabolic state followed by a hypo-metabolic state¹⁵⁷. In the initial acute phase response to infection (hyper-metabolic state), there is an increase in inflammatory, immune, hormonal, and metabolic response. In the late hypo-metabolic state, however, an altered bioenergetics function of the innate immune cells are observed, decreased mitochondrial respiration, ATP production, as well as down-regulated and/or hormone-resistant endocrine pathways are manifested¹⁵⁷. Understanding the phenotype of innate immune cells during different stages of sepsis will potentially inform the development of host-specific targeted therapeutic interventions to mitigate clinical consequences of human sepsis. During different immunological states of sepsis, the predominant circulating cells of the innate immune system- neutrophils, monocytes, and CD4 T cells- are at different metabolic states. A better understanding of the mechanisms responsible for transition from the hyper- to the hypo-inflammatory phase will likely lead to a newer phase of specific therapeutic targets and improve the survival of patients with sepsis.

1.4.1 Metabolic changes during hyper-inflammation

During the early acute phase response to infection /hyper-inflammatory phase of sepsis, the myeloid cells, such as, such as dendritic cells, macrophages, natural killer cells, neutrophils and non-immune cells, such as, epithelial cells¹⁵⁸^,¹⁵⁹^,^160,161 are recruited to sites of inflammation during immune responses. These early responders to infecting pathogens, have three specific requirements: (1) energy (2) capacity to kill microbial pathogens and (3) rapid immune cell regeneration or the capacity of immune cells to support regeneration in order to kill pathogens despite widespread immune cell apoptosis^162,163. Immune cells, similar to proliferating cancer cells, fulfil these three requirements by undergoing aerobic glycolysis also described as the “Warburg effect”¹⁶⁴. Otto Warburg observed that the proliferating cancer cells need energy (ATP) and nucleotide synthesis for rapid regeneration¹⁶⁵. To fulfil these, the cancer cells preferentially undergo glycolysis under aerobic conditions. Evidence suggests that similarly, the effector immune cells also undergo aerobic glycolysis as described below.

(1) Energy requirements:

Effector immune cells need energy in the form of ATP for phagocytosis^166,167. ATP generation during the process of phagocytosis and microbial killing is dependent on glycolysis. To achieve this, glucose enters immune cells via upregulation of GLUT1¹⁶⁸, and GLUT1 expression increases during hyper-inflammation^168,169^,¹⁷⁰. Once inside the cell, glucose metabolizes via glycolysis to pyruvate through a series of enzymatic steps, generating two molecules of ATP¹⁷¹.

Many intermediates of the glycolytic pathway act as a source of carbon and gets diverted into various pathways for the synthesis of biomolecules that support biosynthetic processes.

For instance, in mammalian cells, pyruvate, the pre-mitochondrial product of glycolysis, is converted to lactate and secreted from the cell. Of note, during inflammation and/or depending on the redox state of the cytosol, ATP production is derived by breakdown of glucose due to glycolysis and pyruvate being routed toward lactate instead acetyl-CoA (Kreb’s cycle intermediate)¹⁷².

Even in septic conditions, glycolysis appears to be going on perfectly and pyruvate is abundantly generated in the cytoplasm of cells. Under well-oxygenated conditions, pyruvate enters the mitochondria via a heterodimeric mitochondrial pyruvate carrier protein (MPC) complex. The next step for pyruvate breakdown involves the role of the enzyme complex, pyruvate dehydrogenase complex (PDC), which is responsible for the transformation of pyruvate into acetyl‐CoA and CO₂. PDC is found to be heavily compromised in sepsis. Several explanations for this has been provided in the literature. One of them is that thiamine: the cofactor necessary for PDH complex, is reduced in sepsis samples¹⁷³. However, results from clinical trials using thiamine to prevent lactic acidosis and cellular energy deficit were not convincing and future research requires to be conducted¹⁷⁴. A different explanation based on the regulation mechanism of PDC activity have been also provided by recent studies^175,176^,¹⁷⁷.PDC loses activity due to phosphorylation by a set of kinases known as pyruvate dehydrogenase kinase, PDKs. There are four such kinases, PDK1 to PDK4¹⁷⁸. Among the four PDK isoenzymes, the effect of PDK4 on the regulation of PDC activity has been extensively studied. Transcriptional regulation of these kinases and the pathways involved in the expression of PDK have been elegantly summarized in the review by Jeong et al¹⁷⁹. Further studies evaluating the efficacy of controlling PDC activity by down-regulating PDK expression with PDK inhibitors are warranted in this field. This will then provide us an opportunity to identify new therapeutic targets for treating metabolic and inflammatory diseases.

In sepsis, along with the increased glycolytic effect, and increased reduction of pyruvate, as mentioned earlier, it is observed that there is also an increase in production of lactate and the major pathways to remove lactate are blocked. Lactate is a biomarker of poor prognosis in sepsis, since lactate levels strongly correlate with disease severity, morbidity, and mortality in sepsis. Studies have shown that the increase in blood lactate levels during sepsis is a sign of impaired lactate clearance and not evidence of lactate overproduction¹⁸⁰.

Alternatively, following the transportation of pyruvate into the mitochondria, it is further metabolized to CO₂ by the Krebs cycle, which drives oxidative phosphorylation converting ADP to ATP, generating up to 34 ATP per molecule of glucose. Extra-mitochondrial aerobic glycolysis generates two ATP, and intra-mitochondrial oxidative phosphorylation generates 36 molecules of ATP per molecule of glucose¹⁷¹.

Appropriate inflammatory responses are essential for pathogen elimination, but excessive inflammation may result in overstimulation, organ damage, and even death. Studies are being conducted targeting key mediators of aerobic glycolysis, such as, pyruvate kinase M2 (PKM2), hypoxia inducible factor-1 α (HIF-1 α), mammalian target of rapamycin (mTOR), 2-deoxy-D-glucose (2-DG), to limit exaggerated immune and inflammatory responses during sepsis, while at the same time ensuring an optimum amount of immunity for the clearing of the ongoing infection¹⁸¹. This is crucial because patients that survive the acute hyper-inflammatory phase of sepsis remain at an increased risk for secondary infections, and consequently late stage mortality¹⁸².

(2) Pathogen-killing capacity:

Immune cells resist infection by killing or containing microorganisms. Production of reactive oxygen species support the “killing capacity” of immune cells, particularly phagocytes^183,184. Activated innate and adaptive immune cells increase glycolysis and divert glucose-6-phosphate into the pentose phosphate pathway to support nucleotide synthesis. The pentose phosphate pathway fuels NADPH (which is essential for lipid synthesis) to activate NADPH oxidase and generate the ROS-dependent “killing capacity” needed by the immune cells¹⁶². Moreover, evidence also suggests that glucose-6-phosphate dehydrogenase (G-6PD) is essential for neutrophil extracellular trap formation to further assist with phagocytosis and killing of pathogens¹⁸⁵. Deficiencies in G-6PD is found to be linked to worsened survival and mortality by sepsis possibly due to decreased phagocytosis via impairment of the pentose phosphate pathway and NADPH activity as a result^186,187.

(3) Rapid immune cell regeneration:

Immune cells may die by apoptosis during early sepsis; a major pathophysiologic mechanism in sepsis is lymphocyte apoptosis. Studies have shown profound lymphopenia is observed in patients who died of sepsis and other acute inflammatory injuries^188–190^,¹⁹¹. Evidence suggests that T lymphocytes from septic shock patients exhibit decreased glycolysis, as well as glucose uptake along with lymphopenia via the mTOR-HIF-1α pathway. Earlier this year a study published from a small randomized clinical trial comprising of seventeen sepsis patients on IL-7 treatment shows an increase in CD4+ and CD8+ T lymphocytes¹⁹². Similar to IL-7, anti-PD-1 (nivolumab), a second immunotherapeutic agent that acts primarily to restore the number and function of T cells is currently in clinical trials in patients with sepsis¹⁹³^,¹⁹⁴. Both IL-7 and anti–PD-1 represents a transformational therapeutic approach by targeting a hallmark of sepsis, which is massive loss in T cells and the accompanying defect in adaptive immunity in sepsis.

Immunometabolism among different T cell subsets has a significant impact on the immune phenotype of these cells during sepsis. While pro-inflammatory T cell population, which includes Th1, Th2, and Th17 cells exhibits increased glycolysis but les OXPHOS, it is known that naive T cells depend on OXPHOS for their metabolism. Evidence suggests that CD4+ cells depend on GLUT1 receptors, while CD8+ cells depend on upregulation of other members of glucose transporter family, GLUT3 and GLUT6, on their surface for glucose uptake; however, the importance of GLUT3 and GLUT6 in regulating CD8+ cell function is unclear¹⁹⁵. Interestingly, regulatory T cells (T-regs) depend on fatty acid oxidation for their energy requirement and proliferation¹⁹⁵.

Unlike T-lymphocytes which are more prompt to undergo apoptosis, there have been conflicting reports of increased or delayed apoptosis observed in neutrophils during sepsis. While it is unclear whether neutrophil apoptosis is protective or harmful to the host^196–198, early immune cell activation and increased cell death require a marked increase in cell biomass and in lymphocyte replication to rapidly replete those cells. Glucose-6-phosphate generated during aerobic glycolysis feeds into the pentose phosphate pathway (as discussed earlier) to generate ribose phosphate, a building block necessary for nucleic acid synthesis in a manner similar to proliferating cancer cells.

Thus, glycolysis fulfils all three “needs” of the activated immune cell during the hyper-inflammatory phase: the energy requirement (ATP), pathogenic killing capacity (NADPH) and nucleotide synthesis for cell proliferation. Glycolysis also fuels fatty acid synthesis and protein synthesis.

While efficient in ATP-generation, oxidative phosphorylation is slower than glycolysis. Glycolysis, on the other hand, although inefficient for ATP generation per molecule of glucose, can be ramped up rapidly. Thus, glycolysis is able to meet the anabolic “energy requirement” to accommodate altered functional outputs of innate immune cells by simply utilizing more molecules of glucose to undergo aerobic glycolysis^162,199,200.

1.4.2 Metabolic changes during hypo-inflammation

The early hyper-inflammatory phase of sepsis, while supporting pathogenic clearance, is cytotoxic for the host immune cells and tissue/organs. Energy depletion for biosynthetic processes, cell injury and mass cell death processes cause immune cells to lower their metabolic demands and enter a “cell hibernation” mode accompanied by the hypo-inflammatory phase during late sepsis²⁰¹. This transition from hyper- to hypo-inflammation is seen in macrophages and T cells^202,203. The pro-inflammatory macrophages transition to the endotoxin-tolerant hypo-inflammatory phenotype during this phase. Interestingly, while the cytokine profile of these macrophages changes from the hyper- to hypo-inflammatory phenotype, these cells are fundamentally different from the traditional “M2” type macrophages. The chemokine expression profile, including chemokine (C-C motif) ligand 17 (CCL17), CCL22, and CCL24, suggested a shift to the M1 phenotype, which is related to T cell recruitment. The results may explain the underlying mechanism of immunosuppression after sepsis²⁰⁴. These macrophages exhibit endotoxin tolerance, a well-accepted indicator and a marker of hypo-inflammation/immunosuppression^205,206.

As noted previously, Singer et al published their hypothesis that multiple organ failure induced by critical illness is primarily a functional, rather than structural, abnormality²⁰⁷.

This does make biological sense in the context of an acute systemic infection. A high energy and maximal effector response is mounted to clear the pathogen after which the immune and inflammatory response switches to a lower energy restorative state that rebalances immunity and inflammation and regains homeostasis. The later adaptive state of sepsis with modified bioenergetics may, however, contribute to immunosuppression.

Fatty acid metabolism:

In immune cells there have been well-characterized shifts in metabolic pathways, based on substrate availability, which promote maintenance of immune system function under stress²⁰⁸. Fatty acid oxidation is an important source of ATP for many tissues, particularly when supplies of other nutrients may be compromised. The prime pathway for the degradation of fatty acids is mitochondrial fatty acid β-oxidation (FAO).

Increased levels of fatty acid translocase (CD36) and CPT-I (the gene that converts acyl-CoA into an acylcarnitine) during hypo-inflammation in cell models of sepsis and leukocytes from septic mice support the notion that there is increased fatty acid uptake in cytosol and transfer into mitochondria^209,210. Evidence suggests that signal transducer and activator of transcription 6 (STAT6) and PPARγ-coactivator-1β (PGC-1β )prime macrophages for alternative activation to mute pro-inflammatory phenotype, thus linking mitochondrial oxidative metabolism with the anti-inflammatory phenotype in immune cells²⁰⁹. PGC-1 knockdown decreases fatty acid oxidation while increasing glycolysis and the pro-inflammatory effector properties of macrophages²⁰⁹.

In a metabolomics and proteomics study on plasma on over 1000 sepsis patients and in non-human primates, fatty acid metabolism was deﬁned as one of the most promising metabolic predictors for survival in sepsis^211,212. These studies show that dysregulated fatty acid oxidation with increased long and short chain acyl carnitine fatty acids within 48 hours of sepsis onset are predictors of sepsis survival. Specifically, six carnitine metabolites increased in survivors, while 16 carnitine esters and four fatty acids were elevated in non-survivors of sepsis. Interestingly, fatty acid CPT1 transporters decreased in non-survivors, further supporting this relationship²¹¹. Increased CD36 expression levels were observed during apoptosis-induced immunosuppression in peripheral blood mononuclear cells²¹³. Together, these data support abnormal management of glucose and lipid nutrient substrates during life-threatening sepsis with profound immune suppression and organ failure.

Mitochondrial dysfunction:

A series of clinical observation in both patients and animal models with sepsis and septic shock show a seemingly paradoxical combination of organ dysfunction occurring in the absence of structural damage to tissues despite functional dysregulation^214,215. The absence of severe organ cell damage during sepsis except mitochondrial structural changes (e.g., liver) suggests a crucial role for metabolic dysregulation that impairs cellular bioenergetics. Like starvation and hibernation, oxygen consumption and nutrient anabolism decrease during sepsis²¹⁶. Thus, increased oxygen consumption rates for anabolism during the hyper-inflammatory phase switch to decreased oxygen consumption during the hypo-inflammatory phase in animal, human and cell models²¹⁷. If the cellular metabolism feeding biomass expansion and cellular replication continue without enough ATP (an imbalance in energy demand and supply), this apparent ATP deficit can activate apoptotic pathways. To avoid this, the hibernating cells during the hypo-inflammatory phase of sepsis compensate by switching off the metabolic processes that are not directly involved in cell survival²¹⁸. The rebound increase in oxygen consumption occurs in resolving sepsis and ischemia reperfusion injury^217,218.

It is critical to understand how the immunometabolism transitions between these immunometabolic and bioenergy phenotypes during life threatening sepsis. New tools to study cellular metabolism, such as extracellular flux assays or mass spectrometry-based metabolomics, have recently boosted the field by allowing researchers to study metabolic dynamics of immune cells upon activation.

However, a comprehensive investigation of cellular metabolism of leukocytes, along with other innate immune cells, in sepsis and its effect on immunological function and outcome has not been performed. Therefore, there is a significant need to investigate the metabolic changes that parallel the inflammatory state in sepsis. The last chapter in this thesis is focused on the role of neutrophils, monocytes and CD4 T cells and their immunometabolic stages in sepsis.

1.5 Biomarkers

Sepsis is still defined by non-specific changes in clinical parameters and laboratory tests rather than specific diagnostic biomarkers combined with evidence (e.g. blood culture) or strong suspicion of infection.

1.5.1 Definition of a Biomarker

A biomarker, as defined by the National Institutes of Health Biomarkers Definitions Working Group in 2001, is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”²¹⁹.

There are general and overlapping categories of biomarkers, which have been reviewed and discussed in detail elsewhere^220–223

Biomarker categories include the following²²³: screening biomarkers to identify patients at risk of adverse outcome, to perform prophylactic intervention or further diagnostic test; diagnostic biomarkers to establish the presence or absence of disease and inform treatment decisions more reliably, more rapidly, or more inexpensively than with current methods; stratification biomarkers to stage severity and outcome of disease and help identify subgroups of patients who may benefit or be harmed by therapy; monitoring biomarkers to inform on the response to interventions and effectiveness of a therapy; and surrogate biomarkers to inform on disease progression and potential outcomes of therapy.

A multitude of biomarkers have been proposed in the field of sepsis, many more than in most other disease processes, largely due to the complex pathophysiology of sepsis²²⁴. Sepsis is a dynamic disease and a biomarker may reflect the immune status only at a given moment, and as such biomarker expression must be interpreted cautiously. The heterogeneity of the immune response during sepsis has made research on a potential biomarker difficult. More than 170 different molecules have been proposed as biomarkers for sepsis^220,225. It remains difficult to differentiate sepsis from other non-infectious causes of systemic inflammatory response syndrome, and there is a continuous search for better biomarkers of sepsis.

1.5.2 Biomarkers in Sepsis

The International Sepsis Forum Colloquium on Biomarkers of Sepsis was convened in 2005 to develop a systematic framework for the identification and validation of biomarkers of sepsis²²⁶. The team members for this report proposed that the use of biomarkers would help in therapeutic decision-making and improving the prognosis for septic patients. In order for sepsis biomarkers to be useful, there needs to be better standardization of assay methodologies, clearly defined and detailed biomarker studies, wider collaboration between scientists, clinicians, pharmaceutical industry, biomarker industry, and regulatory agencies.

Ideal Characteristics of a Sepsis Biomarker²²⁷

Fast kinetics
Differentiate bacterial from viral and fungal infection
Differentiate systemic sepsis from local infection
High sensitivity and specificity
Accurate and reproducible
Easy to implement/interpret
Fully automated technology
Short turn-around time
Availability as point-of-care tests
Low cost

The ideal characteristics of a sepsis biomarker are provided above. A biomarker with these characteristics should enable rapid and accurate diagnosis of sepsis. A number of excellent recent reviews have been published on the potential diagnostic and prognostic value of biomarkers in sepsis²²⁵^,²²⁰^,^228,229.

1.5.3 Biomarkers of the pro-inflammatory phase

1.5.3.1 Acute-phase mediators

White blood cell count, neutrophil count, C-reactive protein (CRP), and procalcitonin (PCT) are biomarkers in peripheral blood that are established biomarkers for infection and inflammation. They are often used together with other parameters to support a diagnosis of sepsis and to evaluate treatment response, but they have not achieved the ideal characterization, either alone or used in combination⁸³.

Leukocytosis with predominant neutrophil levels is often found early in patients with sepsis. However, leukopenia can occur in severely ill patients and is related to increased mortality². Both CRP and PCT rapidly increase in septic patients, and the debate continues as to which is the most useful biomarker for sepsis⁸³. CRP and/or PCT are used in some clinics as a tool to determine when antibiotics may be discontinued²³⁰^,²³¹^,²³².

Serum amyloid A protein was found to strongly correlate with CRP in a prospective study evaluating 29 patients in septic shock²³³. The level of plasma ceruloplasmin may provide new insights to predict septic hepatobiliary dysfunction^234,235. Ferritin and alpha1-acid glycoprotein were able to distinguish survivors from non-survivors of septic shock at 28 days (also correlated with SOFA score), suggesting that these two acute phase reactants can provide a prognostic value for the outcome of septic shock²³⁶^,²³⁷. Others, such as hepcidin and lipopolysaccharide binding protein (LBP) are increased in sepsis groups when compared with healthy controls and non-infected patients^238,239. Additionally, Pentraxan 3, which belongs to the same family as CRP and serum amyloid P protein, was reported to correlate with disease severity in patients critically ill with sepsis²⁴⁰.

However, thus far, no single mediator was able to precisely determine the immune/inflammatory status during sepsis. Therefore, further studies and many field trials, including in LMIC countries are warranted.

1.5.3.2 Cytokines

Cytokines, secreted by a wide range of immune cells including monocytes, macrophages, and lymphocytes—but also by endothelial cells, fibroblasts, and stromal cell, play an important part in the features of sepsis and are potential biomarkers. Cytokines act through cell surface receptors and are important for intracellular signaling, which influences the immune responses in complex ways. They have a short half-life of a few minutes to a few hours.

TNF-α is one of the most studied pro-inflammatory pleiotropic cytokines. It is an acute-phase reactant and is rapidly released by activated macrophages and other immune cells, and its levels in blood are increased in sepsis^241,242. Increased levels of TNF-α are associated with mortality in sepsis²⁴³.

IL-1β shares many properties with TNF-α²⁴¹. IL-1β levels are increased in septic patients compared with non-septic individuals and can be used as an indicator of early mortality in sepsis²⁴³.

IL-6 is a pleotropic cytokine with both pro-inflammatory and anti-inflammatory properties. IL-6 is associated with increased severity and mortality in sepsis^156,²⁴⁶. A direct correlation was observed between the levels of IL-6, TNF-α and CRP²⁴⁴.

IL-12 regulates innate immune responses and induces T cells and NK cells to produce IFN-γ, which directly activates macrophages²⁴⁷. IL-12 also stimulates the differentiation of naïve CD4 T cells into Th cells and increases the proliferation of haematopoietic progenitors²⁴⁷. Increased levels of IL-12 have been found in neonatal sepsis, but the role of IL-12 in sepsis still remains unclear²⁴⁸. Selective preoperative defects in monocyte IL-12 production have shown to be predictive of a lethal outcome from post-operative sepsis²⁴⁹.

IL-18 is a pro-inflammatory cytokine and appears to play a pivotal role in cytokine-induced organ failure during systemic inflammation in animal models²⁵⁰. Increased levels of IL-18 in the plasma have been seen in patients with gram-positive sepsis which may be suitable to discriminate between the presence of gram-positive and gram-negative sepsis²⁵⁰. High serum IL-18 was also found to be an early predictive factor for the lethal outcome from post-operative sepsis²⁵¹.

Multiplex cytokine approaches have been employed in research studies and although researchers have been able to associate distinct cytokine profiles with severity, organ failure and mortality in sepsis, larger studies with variable patient population and large sample sizes will be useful in order to gain a more comprehensive strategy for sepsis diagnostics.

1.5.3.3 Chemokines

Chemokines are known to be key determinants of inflammatory reactions and immunity^252,253. These belong to a family of more than 40 small peptides, and these peptides are secreted by tissue cells, leukocytes and activated epithelial cells²⁵⁴. Four different subfamilies can be identified based on the highly conserved presence of the first two cysteine residues, which are either separated or not by other amino acids: the CC chemokines, the CXC chemokines, the CX3C chemokines and the XC chemokines²⁵⁵.

CXC chemokines, which include growth-related gene product alpha (GRO-α) and IL-8 are potent neutrophil chemotactic factors, because they promote migration of primarily neutrophils to the site of infection and also induce phagocytosis²⁵⁶^,²⁵⁷. Higher levels of GRO-α was observed in septic shock patients than in sepsis patients²⁵⁶. Increased levels of IL-8 are observed in sepsis and in other inflammatory conditions²⁵⁸^,^259,260.

CC chemokines, including monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1α& β (MIP-1α and MIP-1 β), attract monocytes, lymphocytes, basophils, eosinophils and natural killer cells. Both MIP-1α and MIP-1 β are elevated in sepsis compared with healthy controls^261,262. Moreover, MCP-1 was reported to be significantly associated with sepsis and to correlate strongly with disease severity²⁶³^,²⁶³.

Due to their pivotal role in pathogenesis, cytokines and chemokines are sensitive and specific markers of an acute infection, such as sepsis. Currently, the analysis of these markers are expensive and can also be technically demanding if reproducible and accurate results are to be obtained.

1.5.3.4 Cell-surface markers

Many markers are used to determine the activation status of neutrophils, monocytes, and T cells.

The most intensively studied myeloid marker in sepsis is arguably the neutrophil CD64 index (nCD64i), which has been proposed to be an early biomarker for neonatal sepsis²⁶⁴. However, it has been shown to have low sensitivity and specificity in distinguishing between bacterial and viral infections²⁶⁵. CD64, a potential biomarker of particular relevance to this thesis will be described in more detail shortly. Until now, flow cytometry has been the gold standard for quantifying this cell surface marker, but limitations associated with its use as a biomarker include the requirement that the blood sample is analyzed shortly after collection in a flow cytometry laboratory and cannot be stored for later handling, thus complicating its clinical usage and turnaround time. Therefore, one of the aims for this thesis has been to develop a point of care rapid test to measure nCD64i.

CD11b is a member of the integrin family and regulates leukocyte adhesion and migration to the sites of inflammation. CD11b is myeloid marker on neutrophils and monocytes and increases in sepsis²⁶⁶. Other monocyte activation markers have also been studied, including CD14, the lipopolysaccharide-binding protein (LBP), and the receptor for advanced glycation end-products (RAGE)²²⁷. Expression of CD40, CD11c, CD163, and soluble CD163 are other myeloid activation markers used in the context of sepsis²⁶⁷^,^268–270. To measure T cell activation, different markers can be used (e.g. CD45RA/CD45RO ratio), CD69, CD71, and HLA-DR or co-stimulatory molecules (CD152, CD27, CD28, and CD134)²⁷¹.

Perhaps the most promising monocyte marker is Presepsin, the soluble N-terminal fragment of CD14, which has been identified as a new, emerging marker for sepsis and severity of sepsis^272–274. In a study of 92 patients with suspected sepsis, presepsin levels were significantly higher in bacteremia versus non-bacteremic patients, and in those with circulating DNA levels²⁷⁵. Presepsin levels have been reported to be higher in infants than adults²⁷⁶. In a systematic review of eight studies comprising 1757 patients and meta-analysis of the value of presepsin for the diagnosis of sepsis, the pooled sensitivity, specificity and diagnostic odds ratio were 0.77 (95%CI 0.75-0.80), 0.73 (95%CI 0.69-0.77) and 14.25 ((5%CI 8.66-23.42)²⁷⁷. A separate meta-analysis reported similar results²⁷⁸. Of importance is the significant heterogeneity between different studies included in these analyses. A further caveat is that presepsin levels increase in patients with renal dysfunction²⁷⁹, a finding present in many patients with sepsis thus increasing the chances of false positive sepsis diagnosis.

1.5.4 Biomarkers of the immunosuppressive phase

1.5.4.1 Cytokines

IL-6 has also been shown to have anti-inflammatory characteristics, whereby it inhibits the release of TNF-α and IL-1β and stimulates the release of IL-10 and cortisol²⁸⁰. As mentioned previously, IL-6 is associated with increased sepsis severity and mortality²⁴⁵^,²⁴⁴.

IL-10 is a potent macrophage-deactivating cytokine that inhibits LPS-induced TNF production in vitro and in vivo²⁶⁷. IL-10 also inhibits the expression of HLA-DR and co-stimulatory molecules on monocytes and macrophages. IL-10 promotes the differentiation of regulatory T cells (T regs) and inhibits the proliferation of CD4+ T cells. However, its effect is not universally anti-inflammatory, as it enhances B cell proliferation and immunoglobulin secretion, and can also enhance the development of CD8+ T cells^281,282. Higher IL-10 levels are found in septic shock patients than in septicemic patients without shock^267,283,284. Increased levels of IL-10 correlate with sepsis mortality²⁸⁵.

Tissue inhibitors of metalloproteinase-1 (TIMP-1) is a glycoprotein with cytokine-like activities; it is expressed by a variety of cell types including neutrophils and monocytes. It functions as an inhibitor of matrix metalloproteinases (MMPs), which degrade the extracellular matrix and play a role in facilitating recruitment of leukocytes from the circulation to sites of infection in sepsis²⁸⁶. Recent studies have identified higher levels of TIMP-1 in patients dying from sepsis than in sepsis survivors, but the role of MMPs/TIMP-1 in sepsis is still unclear²⁸⁷.

1.5.4.2 Cell-surface markers

Expression levels of HLA-DR on monocytes remain one of the best studied immunosuppressive biomarkers; they provide valuable clinical information, such as risk of secondary infection and death^288,289. The expression levels of HLA- DR can be measured either as mean fluorescence intensity (MFI) or as percentage of a certain cell population showing expression. Down-regulation of activation markers is also a sign of immunosuppression (i.e. CD14, CD40, and CD11c)²⁹⁰^,²⁷⁰^,²⁹¹. Monocytes also have increased expression of the negative co-stimulatory molecule PD-1L on the cell surface, with their counterparts PD-1 and CTLA-4 on T cells^292–294. T cells often show reduced expression of the co-stimulatory molecule CD28¹³⁷.

1.5.4.3 Other biomarkers of interest

At the moment, the most frequently used biomarker for determination of organ dysfunction in sepsis is blood lactate^295,296. Many hospitals use lactate levels with a cut-of level of 4 mmol/L to screen for sepsis. However, new reports have revealed that this level is probably set too high, and that sepsis patients with lactate levels above 1.5 mmol/L have an increased mortality rate²⁹⁶.

In some studies, microarray analysis comparing sepsis patients with healthy controls has been able to identify gene expression markers associated with innate and adaptive immune function specific for early sepsis, but the clinical significance of these findings needs to be studied further^297,298. Other biomarkers have been suggested as possible diagnostic markers of sepsis, but they are not yet used in clinical practice⁸³^,²⁹⁹.

It will be challenging to find reliable biomarkers that can be used easily in routine clinical practice. As clinical studies have revealed that expression of both pro- and anti-inflammatory cytokines is elevated early in sepsis, therefore it is possible that a combination of specific biomarkers reflecting both phases would be most suitable³⁰⁰.

1.6 Neutrophil CD64 and Neutrophil CD64 index

Human cells of myeloid lineage contains three classes of receptors for the Fc portion of IgG, designated as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)³⁰¹. Human CD64 or FcγRI consists of α- and γ-chains. The α-chain alone is capable of mediating endocytosis, and calcium signaling, and forms a complex with the ITAM-containing homodimeric γ-chain that is indispensable for both surface membrane expression, and phagocytic function of CD64 in vivo³⁰²^, ³⁰³^,³⁰⁴. Interactions between the human CD64 α- and γ-chains are mediated solely via 21 amino acids in the transmembrane domain of the α-chain³⁰⁵.

In healthy subjects, antigen-presenting cells (monocytes, macrophages and dendritic cells) express CD64 on their surface, whereas only a very low level is found on non-activated neutrophils³⁰⁶. Neutrophil CD64 is upregulated in sepsis as well as in other conditions including lymphoma³⁰⁷, acute pancreatitis³⁰⁸, and adult onset Still’s disease³⁰⁹. Levels of CD64 are upregulated on monocytes in many conditions including sepsis, psoriatic arthritis³¹⁰, Kawasaki’s disease³¹¹, systemic lupus erythematosus³¹² and pregnancy³¹³.

In preterm neonates, neutrophils have a moderately increased level of CD64 expression, which normalizes during the first month of their life to the level of term newborn infants and adults³¹⁴. It is stimulated by pro-inflammatory cytokines [interferon-gamma (IFN-γ) and TNF-α] and granulocyte colony stimulating factor (G-CSF)³¹⁵^,³¹⁶.

An upregulated expression of CD64 (5- to 10-fold in comparison with baseline concentration) on the surface of granulocytes is detected within 1–6 h after a bacterial invasion, which is a huge advantage in comparison with CRP (usually increase after 24 h) in terms of being a potential early biomarker of sepsis. The increase in surface density of CD64 is associated with the intensity of cytokine stimulus³¹⁷.

In most published studies, CD64 expression is presented as either (i)an absolute number of CD64 molecules, (ii) a mean CD64 (abnormal >3 standard deviations above the levels seen in controls), (iii) an nCD64 index (ratio of the mean fluorescent intensity of the cell populations to that of the beads) or as (iv) the CD64 score point (quantitative analysis of CD64 expression on both neutrophils and monocytes)^265,318,319.

CD64 has potential both as a diagnostic and prognostic sepsis biomarker in adults, children and neonates²⁶⁹^,^320–322. A recent meta-analysis by Cid et al. reported that the overall pooled sensitivity of (all methods to measure) CD64 as a sepsis diagnostic biomarker was 79% and specificity was 91%³²³. The authors did note, however, a high degree of variability in the literature and concluded that the methodological quality of the included studies was suboptimal and there is a lack of standardization between many studies in terms of the cut-offs used to determine baseline levels of CD64 in healthy (non-sepsis) population.

CD64 index is not specific for sepsis. It has also been reported to be moderately elevated in patients undergoing coronary bypass surgery, although the elevations in CD64i in these patients were lower than in patients with sepsis³²⁴. CD64i is elevated not only in bacterial infections but also in some viral infections³²⁵ including HIV and hepatitis C virus³²⁶ and in other conditions including acute exacerbation of chronic obstructive pulmonary disease³²⁷.

Furthermore, it would be interesting to study neutrophil CD64 as a tool for monitoring therapy, like is currently being done for procalcitonin. However, implementation of CD64 in clinical practice might be cumbersome because it is measured by flow cytometry. Thus, as mentioned above, this thesis focuses mainly on the development of an alternate method to measure nCD64i using immunoassays for simultaneous measurement of neutrophil CD64 and a neutrophil-specific protein in whole blood, the details of which will discussed in later chapters.

1.7 Aims and objectives of the thesis:

The primary aim of the thesis is to validate the potential for total CD64 in whole blood as a novel biomarker for diagnosis of sepsis at point-of-care. Ultimately, the findings of this thesis will inform the development of a suitable lateral-flow based immuno-chromatographic test, which is amenable to rapid diagnostic platforms for point-of-care use.

The secondary aim of the thesis is to investigate the cellular metabolism of inflammatory process occurring within innate immune cells during sepsis.

Outline of this thesis:

The work presented in this thesis describes the proof-of-concept for the development of a simple lateral immunoassay that measures host biomarkers to affirm sepsis infection as an alternative to currently used gold standard for sepsis diagnosis- blood culture test, which is time-consuming and not always accurate.

To develop a proof-of-concept study towards the development a rapid test to diagnose sepsis, I have validated a biomarker of sepsis, CD64 and a surrogate neutrophil marker, Neutrophil Elastase (NE). I have demonstrated proof to show that the simple ELISA-based method of detection of these two proteins (CD64 and NE) can also be translated into a lateral flow diagnostic assay format.

Chapter 2 describes in details ELISA- based and lateral flow immunochromatographic techniques as well as flow-cytometric methods using to conduct the studies described in this thesis.

Chapter 3 will describe the close relationship between NE (our preferred surrogate neutrophil marker) and granulocyte counts showing that the NE component could potentially be used independently of CD64 for enumeration of neutrophil cell count at point of care. It describes a novel non-linear relationship between NE and CD64 in healthy controls which forms an important part of our proposed test development.

Chapter 4 will explore the potential utility of our approach of measuring CD64/NE for diagnosis of sepsis with good sensitivity and specificity. It will demonstrate the detection of elevated CD64 levels in sepsis patients as well as reveal novel aspects of the expression of CD64 in neutrophils (forming the basis of our IP WO2018/018095). The results from this chapter confirm the potential utility of our approach, and demonstrate better sensitivity for diagnosis of sepsis, compared to other sepsis biomarkers.

Chapter 5 will describe the immuno-metabolic changes that occur in sepsis patients and characterize these changes in innate immune cells according to patient survival and underlying infection. It will demonstrate the clinical, cellular activation, inflammation and immuno-metabolic changes across four groups- Sepsis, HIV, ICU controls and healthy controls. The results from the descriptive study presented in this chapter indicates that the metabolic changes parallel the inflammatory state in sepsis.

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Table . Sequential [Sepsis Related] Organ Failure Assessment Score (Abbreviations FiO_2,fraction of inspired oxygen; MAP, mean arterial pressure; PaO₂, partial pressure of oxygen.) *Glasgow Coma Scale scores range from 3-15; higher score indicates better neurological function.

A new term, qSOFA (for quick SOFA) was introduced by the taskforce. qSOFA incorporated altered mentation, systolic blood pressure of 100mm Hg or less, and respiratory rate of 22/min or greater, provides simple bedside criteria to adult patients with suspected infection who are likely to have poor outcomes.

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Contents

Towards a point-of-care test for sepsis- biomarkers of inflammation

1.1 Introduction

1.1.1 Definition of sepsis