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§ÆT VÊN §Ò MINISTRY OF EDUCATION AND TRAINING MINISTRY OF NATIONAL DEFENCE MILITARY MEDICAL UNIVERSITY VU MANH CUONG STUDY ON PLASMA LEVELS OF TNF α, IL 6, IL 10 AND RELATIONSHIP WITH DISEASE SEVERITY[.]
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF NATIONAL DEFENCE MILITARY MEDICAL UNIVERSITY VU MANH CUONG STUDY ON PLASMA LEVELS OF TNF-α, IL-6, IL-10 AND RELATIONSHIP WITH DISEASE SEVERITY IN PATIENTS WITH GRAM-NEGATIVE SEPSIS Speciality: Infectious and tropical diseases Code: 9.72.01.09 SUMMARY OF THESIS DOCTOR OF MEDICINE HA NOI - 2022 WORK COMPLETED AT MILITARY MEDICAL UNIVERSITY Full name of supervisor: Associate Professor Ph.D Hoang Vu Hung Associate Professor Ph.D Vu Xuan Nghia Review 1: Professor Ph.D Nguyen Van Kinh Review 2: Associate Professor Ph.D Le Van Dong Review 3: Associate Professor Ph.D Do Tuan Anh The thesis will be defended in front of the school-level Thesis Judging Committee On: … time, date … month … year … The thesis can be found at: National Library Military Medical University Library …………………………….……… INTRODUCTION The urgency of the subject Sepsis is a life-threatening condition that arises when the body's overactive immune response to an infection leads to organ failure Sepsis was, is and remains one of the challenges facing the global health care system The causative agent of sepsis emerging today is Gram-negative bacteria because of its prevalence, clinical presentation is often severe and often accompanied by septic shock Mortality rates in sepsis caused by Gram-negative bacteria are higher than those caused by Gram-positive bacteria The treatment of sepsis caused by Gram-negative bacteria remains difficult due to resistance to existing antibiotics Lipopolysaccharide endotoxin is a major component of the cell wall of Gram-negative bacteria, which is released when the bacterial wall is broken down Endotoxins can stimulate the organism to synthesize and release the most potent inflammatory cytokines In sepsis, in the early stages, inflammation is primarily the release of pro-inflammatory cytokines, including IL-6 and TNF-α In the anti-inflammatory phade, immunosuppression is expressed by dysfunction of immune cells and increased IL-10 In recent years, the inflammatory role of TNF-α, IL-6 and the antiinflammatory role of IL-10 had been identified and further elucidated the pathogenesis of sepsis and multiorgan failure syndrome These cytokines are mostly produced within a few hours to 24 hours after the entry of the pathogen into the body Therefore, the quantification and monitoring of changes in the levels of these cytokines can help diagnose and classify at an early stage, helping to predict the disease in patients with sepsis In Vietnam, there have been a number of studies, recently by Pham Thi Ngoc Thao, on the prognostic value of TNF-α, IL-6, IL-10, but only focused on patients with severe sepsis The value of these cytokines in the progression of sepsis, especially Gram-negative bacteremia, and the relationship with disease severity had not been fully explored and evaluated system In order to contribute to improving the quality of treatment, as well as to elucidate the role of cytokines in infectious diseases, especially Gram-negative bacteremia, we conducted the topic: "Study on concentration plasma TNF-α, IL-6, IL-10 and their relationship to disease severity in patients with Gram-negative sepsis” Research objective + To determine of plasma concentrations of TNF-α, IL-6, IL-10 in patients with Gram-negative sepsis + To investigate the relationship between plasma levels of TNF-α, IL-6 and IL-10 with disease levels and some prognostic factors for shock and death in patients with Gram-negative sepsis New contributions of the thesis topic 2.1 First contribution * Evaluation of plasma levels of TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL-10/TNF-α in patients with Gram-negative sepsis in the group of patients with shock and without shock * Compared plasma levels of cytokines TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL-10/TNF-α between groups of patients with Gram-negative bacteremia with damage to or more organs, with mechanical ventilation and without mechanical ventilation, with vasopressors and without vasopressors, groups of patients alive and dead 2.2 Second contribution Evaluate the relationship between plasma levels of cytokines TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL10/TNF-α with levels of sepsis There by contributing to the prognosis of shock and death in patients with Gram-negative sepsis The structure of the thesis Thesis consists of 128 pages Introduction: 02 pages; Overview: 35 pages; Objects and research methods: 23 pages; Research results: 30 pages; Discussion: 35 pages; Conclusion and recommendations: 03 pages In the thesis, there are 36 tables, 05 charts, 05 diagrams, 02 drawings There are 166 references, including 15 Vietnamese documents and 151 English documents Chapter 1: OVERVIEW 1.1 Sepsis and the role of Gram-negative bacteria in bacteremia Sepsis: is a condition in which the body's (host's) response to an infection is out of control, causing life-threatening organ dysfunction of the organs This dysregulation results in the excessive and uncontrolled release of inflammatory mediators, setting off a chain of events that causes widespread tissue damage Septic shock: A bacteremia with hypotension, cellular and metabolic abnormalities severe enough to increase the risk of death compared with bacteremia alone, despite adequate fluid resuscitation vasopressors still require vasopressors to maintain a mean arterial pressure (MAP) ≥ 65 mmHg and a blood lactate > mmol/L (> 18 mg/dL) The causative agents of bacteremia are diverse: Gram-positive, Gramnegative bacteria and fungi Gram-negative bacteria are becoming an increasingly important cause of bacteremia The family of enteric bacteria (mainly E coli and K pneumoniae) remains the main cause among Gramnegative bacteria causing bacteremia Gram-negative bacteria are capable of producing many cytokines for host cells Endotoxins are a major component of the cell wall of Gram-negative bacteria that are released when the bacterial wall is broken down Endotoxins and bacterial components will stimulate the host body to produce cytokines Endotoxin shock is the result of an imbalance of several mediators with proinflammatory and anti-inflammatory effects The initial response may be local, once the local imbalance manifests as a systemic inflammatory response syndrome This uncontrolled process leads to organ dysfunction, including circulatory shock, multi-organ failure and death 1.2 Plasma cytokines TNF-α, IL-6, IL-10 and their role in Gramnegative sepsis A cytokine is a chemical secreted by one cell to activate or inhibit the action of another cell There are two types of cytokines: inflammatory cytokines and anti-inflammatory cytokines The imbalance of these two types determines the prognosis of the systemic infection syndrome The main inflammatory cytokines that regulate early responses, implicated in the pathogenesis of bacteremia, include TNF-α, IL-6, etc., These inflammatory cytokines stimulate the production of other mediators from arachidonic acid Host immunosuppression may be the cause of late death in patients with bacteremia Anti-inflammatory cytokines act primarily to inhibit inflammation and promote healing Inhibits the production of TNF-α and IL-6, which regulate inflammation The important antiinflammatory cytokines in bacteremia include IL-4, IL-10, IL-12, etc For Gram-negative bacteria, endotoxins stimulate cytokine production from cells of the immune system through receptors (Toll-like receptors) Lipopolysaccharide (LPS) - bacterial endotoxin - consists of components In which lipid A is released when bacteria multiply or die Lipid A binds to proteins to form protein complexes - LPS This complex activates the immune system by binding to the cell's TLR4 receptor When combined with LPS, the superantigen had an additive effect, increasing the susceptibility of immune cells to LPS, increasing the amount of cytokines many times Tumor necrosis factor: TNF-α stimulates an acute inflammatory phade response associated with systemic inflammation In bacteremia, TNF-α had a role in disease progression, is a biomarker, and predicts survival The concentration of TNF-α in bacteremia caused by Gram-negative bacteria is higher than in bacteremia caused by Gram-positive bacteria In a state of bacteremia, endotoxin shock, TNF-α is frequently elevated in the blood TNF-α activates myeloid leukemia cells and stimulates the synthesis of inflammatory cytokines, inflammatory proteins in the liver TNF-α is the primary inflammatory mediator involved in the systemic inflammatory response and multi-organ dysfunction syndrome TNF-α had an important role in endotoxin shock and tissue damage During LPSinduced shock, TNF-α, in addition to inducing anti-inflammatory cytokines such as IL-10 and IL-4 it also activates the expression of inflammatory cytokines such as IL-1, IL-6 and IL-8 TNF-α is the major cytokine that causes septic shock and bacteremia-induced multi-organ damage TNF-α had become the most studied inflammatory cytokine in sepsis Interleukin 6: IL-6 is an important factor in local and systemic immune responses IL-6 stimulates inflammatory and autoimmune processes IL-6 participates in the human body's early immune response to bacterial infections, had a role in stimulating the immune response IL-6 also plays a role in fighting infection, an important part of the systemic inflammatory response In bacteremia, IL-6 is associated with disease severity, mortality, and serves as a biomarker IL-6 is the major cytokine in the pathophysiology of severe bacteremia In addition, increased IL-6 was associated with the highest risk of death in patients with bacteremia Among the cytokines produced in bacteremia, plasma IL-6 had the best association with mortality In patients with severe bacteremia, a decrease in IL-6 levels after admission (24h, 48h or 72h) had a prognostic role and was a predictor of survival while changes in TNF-α and IL-10 is not a predictor of survival Therefore, in terms of clinical application, in patients with bacteremia and septic shock, IL-6 should be measured on admission and 24h thereafter Interleukin 10: IL-10 is also known as an inhibitor of inflammatory cytokine synthesis IL-10 had multiple effects on immunoregulatory and inflammatory processes The important role of IL-10 is to inhibit the production of proinflammatory cytokines by activating the monocyte system IL-10 had direct anti-inflammatory effects on many different cells IL-10 can exert potent anti-inflammatory activities IL-10 is considered to be a key cytokine in anti-inflammatory responses, with an important role in the compensatory anti-inflammatory response syndrome IL-10 is one of the important cytokines in the pathophysiology of bacteremia IL-10 had a role in diagnosis rather than survival in bacteremia Increased serum IL-10 levels were associated with bacteremia scores and mortality Furthermore, the overproduction of IL-10 is the major risk factors for the seriousness of bacteremia and the fatal outcome indicate that the patient with bacteremia is in a state of profound immuno suppression The concentrations of TNF-α, IL-6, IL-10 in patients with septic shock were significantly higher than in patients with severe bacteremia The ratio of cytokine levels: The ratio of IL-6/IL-10 and IL-10/TNF-α levels reflects the balance in the body's pro-inflammatory and antiinflammatory responses Elevated IL-6/IL-10 and IL-10/TNF-α ratios are associated with mortality in patients with septic shock and multi-organ dysfunction Increased levels of cytokines (TNF-α, IL-6, IL-10) and an imbalance in the pro-inflammatory response are important factors associated with mortality The extent of burn injury was inversely correlated with the ratio of TNF-α/IL-10 concentrations This early plasma ratio may be a novel biomarker for predicting increased risk for multiple episodes of infection After stimulation with Lipopolysaccharide (LPS), the concentration of TNF-α was significantly higher, while the concentration of IL-6 tended to be lower There is a relationship between the severity of bacteremia and the concentration of some cytokines: + The concentrations of TNF-α and IL-1β were elevated in patients with bacteremia compared with controls + Levels of TNF-α, IL-1β and IL-6 were elevated in patients with fatal bacteremia compared with the living group + The concentration of some circulating cytokines was increased in patients with bacteremia in shock compared with the group not in shock 1.3 Studies on the role of plasma TNF-α, IL-6, IL-10 in sepsis and Gram-negative sepsis In the world, there have been many studies on the role and change of cytokines TNF-α, IL-6, IL-10 in early diagnosis and prognosis of bacteremia Most studies showed that the concentration of these cytokines appeared early in bacteremia, increased rapidly after a short time, then gradually decreased over time, increasing gradually in the normal bacteremia group then to the group of bacteremia with shock and the group of fatal bacteremia, the group of bacteremia caused by Gram-negative bacteria was higher than the group caused by Gram-positive bacteria In Vietnam, there have been some initial studies evaluating the role of cytokines including TNF-α, IL-6, IL-10 on some groups of patients with bacteremia However, mainly focused on nhóm bacteremia in general, not focusing on the group of bacteremia caused by Gram-negative bacteria More over, the results of the study on bacteremia above showed that although the etiology is Gram-negative bacteria, there were differences in the pathogenic roles of these bacteria CHAPTER 2: RESEARCH SUBJECTS AND METHODS 2.1 Research subjects The study was carried out on 110 patients diagnosed with Gramnegative bacteremia, including 80 patients without shock and 30 patients with septic shock, treated at Hanoi E Hospital and 103 Military Hospital from December 2016 to June 2018 * Patient selection criteria + Patients ≥ 18 years old + Blood culture results at sites, at the same time, were positive for only type of Gram-negative bacteria + The patient was diagnosed with bacteremia and classified according to the criteria mentioned in Sepsis-3 (2016) *Exclusion criteria + Pregnant women + End-stage cancer, end-stage chronic renal failure, Child C cirrhosis + Immunodeficiency: HIV/AIDS infection, + Patients were not sampled for TNF-α, IL-6 and IL-10 cytokines at the time of study or samples were rejected due to poor test quality + Patients or family members not agree to participate in the study 2.2 Research Methods 2.2.1 Study design: prospective, cross-sectional, comparative 2.2.2 How to choose a template: Calculate the sample size according to the following formula: - Z: value from the normal distribution (Z = 1.96 for 95% confidence interval) - α: probability of type error (when rejecting hypothesis H 0) Choose α = 0.05 for the value Z1- α/2 = 1.96 - ∆: allowable error In this study, we choose ∆ = 0.05 - The mortality rate in the previous author's Gram-negative bacteremia was 7% (p = 0.07) Calculated according to the formula, the study must have at least 101 patients (in fact, in the study, there were 110 eligible patients selected) 2.2.3 Research content and steps Patients admitted to the hospital were asked about their illness, examined, according to the research medical record, the following criteria were collected: * Clinical: + Age, gender, history of underlying medical conditions and physical condition + Clinical examination: Assess the whole body (consciousness, fever, ) parts (circulation, respiratory, digestive, renal, urinary, hematological, ) + Find primary and secondary bacterial contamination + Time from the patient's symptom onset to the diagnosis of sepsis, septic shock (into shock, out of shock) + Interventional procedures: mechanical ventilation, vasopressors, + Treatment results: Survived, discharged from hospital, seriously asked to return or died in hospital * Subclinical: + Hematology - Coagulation: number of red blood cells, hemoglobin, white blood cells, platelets, prothrombin, APTT + Biochemistry: Glucose, kidney function, liver enzymes, CRP, PCT + Blood gases: pH, Pa02, PaC02, HC03-, lactate + Pictures: X-ray, ultrasound, computed tomography, + Blood culture: At the Department of Microbiology - E Hospital, 103 Military Hospital Blood was taken at the time when the patient showed signs of chills (or started to have a high fever, within 30 minutes), before using antibiotics bacterial identification and antibiotic preparation using the BACT/ALERT 3D 60, BACTEC FX 40 system + Culture for bacteria from other body fluids: sputum, tracheal fluid, urine, feces * Quantitative testing of cytokines TNF-α, IL-6 and IL-10 in plasma (ELISA technique): + Principle: This is a technique used according to the principle of immunology to determine the presence of a cytokine in a patient sample by a pair of antibodies specific to that cytokine in a Sandwich style Conducted at the laboratory of the Military Medical Research Institute Military Medical University + Kit used: of Multiscienses (Lianke) Biotech - China Each different cytokine is used 01 different Kit + Sampling time: - For the group of patients with bacteremia caused by Gram-negative bacteria without septic shock: when the patient was diagnosed with bacteremia according to Sepsis-3 - For the group of patients with bacteremia caused by Gram-negative bacteria with septic shock: the time when the patient enters the shock (also the time when the patient is diagnosed with bacteremia according to Sepsis-3) and the time when the patient is out of shock + Sample preparation: Collect mL of peripheral venous blood in a test tube anticoagulant with EDTA (Ethylene Diamine Tetracetic Acid), citrate or heparin Centrifuge at 1000 rpm for 30 minutes, proceed the plasma is separated, refrigerated, then transferred to the laboratory of the Military Medical Research Institute - Military Medical University and stored at - 800C Plasma samples were thawed prior to testing Reagents and samples are kept at room temperature (18 - 25 0C) before use Perform the technique immediately or store the sample at ≤ 20 0C in case it had not been done + Sample dilution: According to the manufacturer's instruction chart + How to calculate the result: - Using ELISA reader (Diganostic Autmation Inc., ELX800DA) - USA - According to the manufacturer's recommendations, the sensitivity or minimum detectable dose (Minimum Detectable Dose: MDD) of TNF-α is < 0.42 pg/mL, of IL-6 is < 0.37 pg/mL mL and IL-10 were < 0.43 pg/mL Specificity for natural and recombinant TNF-α, IL-6, and IL-10, with no cross-reactivity between humans, mice and rats The range of determination of the Kit powder (Non Detactable: ND) is 1000 pg/mL 11 3.2.2 Comparison of TNF-α, IL-6, IL-10 10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL-10/TNF-α levels between the group of patients with sepsis alive and dead Table 3.11 Comparison of plasma levels of TNF-α, IL-6, IL-10 between two groups of patients with sepsis alive and dead Cytokine IL-6 (pg/mL) IL-10 (pg/mL) TNF-α (pg/mL) Death (n = 21) Alive (n = 89) 60.91 (18.16-169.93) 17.07 (6.58 - 35.4) 24.67 (6.57 - 84.24) 6.92 (3.25 - 21.72) 163.13 (7.58 - 394.2) 168.13 (21.04-271.23) p < 0.005* < 0.01* > 0.05* IL-6/IL-10 ratio 4.11 (1.17 - 7.1) 1,89 (0.61 - 7.47) > 0.05* IL-6/TNF-α ratio IL-10/TNF-α ratio 0.49 (0.1 - 6.94) 0.28 (0.03 - 1.41) 0.09 (0.02 - 1.11) 0.06 (0.02 - 0.42) < 0.05* > 0.05* * Mann-Whitney U test The median values of IL-6, IL-10 concentrations and the ratio of IL-6/TNF-α were statistically significantly higher in the group of patients who died than in the group of patients who were alive (p-values ranged from p < 0.005 to p < 0.05) The ratio of IL-6/IL-10, IL-10/TNF-α in the dead group was also higher than the living group, but the difference was not statistically significant with p > 0.05 Table 3.12 Comparison of plasma concentrations of TNF-α, IL-6, IL-10 between two groups of non-shock sepsis patients alive and dead Cytokine Sepsis death (n = 11) Sepsis alive (n = 69) p IL-6 (pg/mL) IL-10 (pg/mL) TNF-α (pg/mL) IL-6/IL-10 ratio IL-6/TNF-α ratio IL-10/TNF-α ratio 36.45 (14.82 - 88.85) 13.14 (4.9 - 26.67) 23.15 (3.88 - 70.57) 5.62 (2.95 - 12.98) 103.26 (5.13-556.43) 146.4 (39.14-257.19) 2.77 (0.56 - 7.52) 1.41 (0.56 - 7.14) 0.86 (0.09 - 7.10) 0.06 (0.02 - 0.81) 0.67 (0.04 - 1.53) 0.04 (0.02 - 0.18) < 0.05* > 0.05* > 0.05* > 0.05* < 0.05* < 0.05* * Mann-Whitney U test In the group of patients with bacteremia without shock, survival and death: Median values of IL-6 concentration, IL-6/TNF-α ratio and IL-10/TNF-α ratio were higher with statistical significance in the dead group compared to the surviving group (p value < 0.05) The median value of IL-10 concentration and the ratio of IL-6/IL-10 in the dead group were also higher than in the surviving group, but the difference was not statistically significant with p > 0.05 12 Table 3.13 Comparison of plasma levels of TNF-α, IL-6, IL-10 between two groups of septic shock patients alive and dead Cytokine IL-6 (pg/mL) IL-10 (pg/mL) TNF-α (pg/mL) IL-6/IL-10 ratio IL-6/TNF-α ratio IL-10/TNF-α ratio Septic shock death Septic shock alive (n = 10) (n = 20) 110.58 (31.07-617.49) 58.21 (19.49-127.67) 31.52 (7.66 - 163.84) 26.78 (4.95 - 56.05) 183.84 (88.62 - 379.6) 190.81 (4.74 - 452.5) 4.54 (1.17 - 6.9) 2.54 (0.91 - 10.7) 0.41 (0.15 - 40.40) 0.27 (0.05 - 13.96) 0.18 (0.02 - 2.38) 0.2 (0.02 - 1.78) p > 0.05* > 0.05* > 0.05* > 0.05* > 0.05* > 0.05* * Mann-Whitney U test In the group of patients with septic shock who are alive and dead: Median values of IL-6, IL-10 concentrations, IL-6/IL-10 ratio, IL-6/TNF-α ratio in the septic shock group bacteriocins died higher than the group of bacteriophages alive, but the difference was not statistically significant with p > 0.05 3.3 Relationship between plasma levels of TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL-10/TNF-α with disease levels in patients with Gram-negative sepsis 3.3.1 The relationship between plasma levels of TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6/IL-10, IL-6/TNF-α, IL-10/TNF-α with some characteristics of patients with Gram-negative sepsis Table 3.14 Comparison of plasma concentrations of TNF-α, IL-6 and IL-10 in the sepsis group without shock at the time of diagnosis Cytokine First 24 hours (n = 10) After 24 hours (n = 70) IL-6 (pg/mL) 17.76 (10.99 - 49.96) 14.21 (5.05 - 30.27) IL-10 (pg/mL) 9.17 (4.05 - 31.24) 6.00 (2.82 - 17.00) TNF-α (pg/mL) 144.15 (58.87-188.26) 146.24 (17.16-279.18) IL-6/IL-10 ratio 4.66 (0.33 - 6.63) 1.52 (0.57 - 7.3) IL-6/TNF-α ratio 0.21 (0.06 - 0.65) 0.07 (0.02 - 2.89) IL-10/TNF-α ratio 0.08 (0.04 - 0.37) 0.04 (0.02 - 0.44) * Mann-Whitney U test p > 0.05* > 0.05* > 0.05* > 0.05* > 0.05* > 0.05* Median values of IL-6, IL-10 concentration, IL-6/IL-10 ratio, IL-6/TNF-α ratio and IL-10/TNF-α ratio in the non-shock bacteremia group were diagnosed early within the first 24 hours of the disease was higher than the group diagnosed after 24 hours of the disease However, the difference was not statistically significant with p > 0.05 13 Table 3.15 Comparison of plasma concentrations of TNF-α, IL-6 and IL-10 in the sepsis group with shock at the time of diagnosis Cytokine IL-6 (pg/mL) First 48 hours (n = 10) After 48 hours (n = 20) p 148.8 (45.56 - 995.2) 54,6 (19.49 - 112.77) > 0.05* IL-10 (pg/mL) 89.46 (20.88 - 298.12) TNF-α (pg/mL) 186.64 (4.75 - 569.71) IL-6/IL-10 ratio 3.15 (0.67 - 7.37) IL-6/TNF-α ratio 0.54 (0.03 - 165.92) IL-10/TNF-α ratio 0.62 (0.02 - 19.71) 12.23 (4.56 - 42.6) < 0.05* 187.68 (8.51-254.04) 2.92 (1.19 - 12.58) 0.27 (0.07 - 10.98) 0.16 (0.02 - 1.19) > 0.05* > 0.05* > 0.05* > 0.05* * Mann-Whitney U test The median value of IL-10 concentration in the group of patients with septic shock entering shock within the first 48 hours of illness was statistically significantly higher than that of patients with septic shock entering shock after 48 hours of illness (p < 0.05) Median values of IL-6 concentration, IL-6/IL-10 ratio, IL-6/TNF-α ratio and IL-10/TNF-α ratio in the group of patients with septic shock into shock within the first 48 hours of the disease were higher than the group of patients with septic shock and shock after 48 hours of illness However, the difference was not statistically significant (p > 0.05) Table 3.16 Comparison of plasma levels of TNF-α, IL-6, IL-10 between the group of patients with more than organs dysfunctional Cytokine ≥ organs (n = 35) < organs (n = 75) p IL-6 (pg/mL) 34.35 (16.55 - 174.15) 13.61 (5.23 - 36.45) < 0.001* IL-10 (pg/mL) 16.3 (5.62 - 70.57) 6.36 (3.44 - 19.37) < 0.01* TNF-α (pg/mL) 124.45 (6.74 - 374.48) 171.57 (25.17-268.88) > 0.05* IL-6/IL-10 ratio 4.98 (0.81 - 7.61) 1.84 (0.63 - 7.06) > 0.05* IL-6/TNF-α ratio IL-10/TNF-α ratio 0.49 (0.06 - 7.1) 0.17 (0.03 - 1.28) 0.09 (0.02 - 0.86) 0.04 (0.02 - 0.32) < 0.05* < 0.05* * Mann-Whitney U test The group of patients with ≥ dysfunctional organs had statistically significant higher levels of IL-6, IL-10, IL-6/TNF-α ratio and IL-10/TNF-α ratio compared with the group of patients with less than dysfunctional organs (with values from p < 0.001 to < 0.05) 14 Table 3.17 Comparison of plasma levels of TNF-α, IL-6, IL-10 between must be on a ventilator and non-ventilated patients IL-6 (pg/mL) Must be on a ventilator (n = 36) 32.18 (14.88 - 172.04) Not on a ventilator (n = 74) 17.07 (5.19 - 36.45) < 0.005* IL-10 (pg/mL) 23.91 (5.19 - 46.55) 6.36 (3.01 - 17.00) < 0.005* TNF-α (pg/mL) 165.24 (54.00-414.89) 157.26 (12.77-261.07) > 0.05* 2.55 (0.89 - 7.51) 0.27 (0.04 - 4.18) 0.08 (0.03 - 1.08) 1.89 (0.61 - 7.25) 0.09 (0.02 - 3.05) 0.05 (0.02 - 0.47) > 0.05* > 0.05* > 0.05* Cytokine IL-6/IL-10 ratio IL-6/TNF-α ratio IL-10/TNF-α ratio p * Mann-Whitney U test The group of patients requiring mechanical ventilation had higher concentrations and ratios of cytokines than the non-ventilated group, but the difference was statistically significant only in IL-6 and IL-10 (with p < 0.005) Table 3.18 Comparison of plasma concentrations of TNF-α, IL-6, IL-10 between groups of patients with vasopressors and without vasopressors Cytokine IL-6 (pg/mL) IL-10 (pg/mL) Have use (n = 48) 54.6 (16.68 - 127.67) 18.87 (4.56 - 57.08) Don't use (n = 62) 10.91 (4.64 - 28.52) 5.63 (3.25 - 11.19) TNF-α (pg/mL) 148.33 (5.13-329.87) 169.85 (59.49-270.05) p < 0.001* < 0.005* > 0.05* IL-6/IL-10 ratio 2.88 (1.17 - 7.51) 1.34 (0.53 - 7.1) > 0.05* IL-6/TNF-α ratio IL-10/TNF-α ratio 0.41 (0.07 - 11.45) 0.2 (0.03 - 1.54) 0.05 (0.01 - 0.46) 0.04 (0.02 - 0.14) < 0.001* < 0.005* * Mann-Whitney U test The group of patients who had to take vasopressors had a statistically significant higher IL-6, IL-10, IL-6/TNF-α ratio and IL-10/TNF-α ratio than the group of patients without vasopressors (with p ranging from p < 0.001 to p < 0.005) 15 Table 3.19 The relationship between plasma levels of TNF-α, IL-6, IL-10 and leukocytosis IL-6 (pg/mL) White blood cells > 10.5 G/L (n = 64) 25.45 (8.92 - 72.21) White blood cells ≤ 10.5 G/L (n = 46) 16.65 (5.64 - 32.77) > 0.05* IL-10 (pg/mL) 10.19 (4.09 - 36.83) 5.22 (2.98 - 22.29) < 0.05* TNF-α (pg/mL) 148.48 (10.65-275.63) 170.35 (23.8 - 230.57) > 0.05* 2.33 (0.57 - 10.07) 0.09 (0.03 - 0.99) 0.04 (0.02 - 0.41) > 0.05* > 0.05* > 0.05* Cytokine IL-6/IL-10 ratio 1.99 (0.85 - 6.49) IL-6/TNF-α ratio 0.18 (0.02 - 6.27) IL-10/TNF-α 0.09 (0.02 - 0.98) ratio * Mann-Whitney U test p In the group of patients with leukocytosis, the median value of IL-10 concentration was statistically significantly higher than in the group of patients without leukocytosis (p value < 0.05) Table 3.21 The relationship between plasma levels of TNF-α, IL-6, IL-10 with elevated PCT status IL-6 (pg/mL) PCT ≥ ng/mL (n = 78) 25.45 (8.35 - 80.63) PCT < ng/mL (n = 32) 17.07 (7.11 - 27.29) > 0.05* IL-10 (pg/mL) 9.72 (3.89 - 39.91) 5.63 (2.4 - 9.6) < 0.05* Cytokine TNF-α (pg/mL) 156.85 (22.33-265.79) 167.74 (12.11-272.4) p > 0.05* IL-6/IL-10 ratio 1.99 (0.62 - 7.37) 2.24 (0.78 - 9.21) > 0.05* IL-6/TNF-α ratio IL-10/TNF-α ratio 0.18 (0.03 - 4.13) 0.08 (0.02 - 0.84) 0.07 (0.02 - 3.78) 0.06 (0.02 - 0.37) > 0.05* > 0.05* * Mann-Whitney U test The group of patients with an increase in PCT ≥ ng/mL had a statistically significant higher IL-10 concentration than the group of patients with a PCT < ng/mL (p < 0.05) Table 3.22 Relationship between plasma levels of TNF-α, IL-6, IL-10 with hyperlactate status IL-6 (pg/mL) Lactate > mmol/L (n = 52) 54.6 (16.31 - 120.32) Lactate ≤ mmol/L (n = 93) 12.0 (4.94 - 26.19) < 0.001* IL-10 (pg/mL) 17.11 (6.10 - 43.73) 5.22 (2.64 - 9.96) < 0.001* Cytokine TNF-α (pg/mL) IL-6/IL-10 ratio IL-6/TNF-α ratio 183.04 (24.54-378.14) 133.06 (17.16-210.15) 2.7 (0.78 - 7.31) 1.45 (0.55 - 8.71) 0.23 (0.03 - 4.18) 0.07 (0.02 - 3.05) p > 0.05* > 0.05* > 0.05* 16 IL-10/TNF-α ratio 0.1 (0.02 - 1.0) 0.04 (0.02 - 0.44) > 0.05* * Mann-Whitney U test The group of patients with hyperlactateemia > mmol/L had higher concentrations and ratios of cytokine concentrations than the group of patients with blood lactate ≤ mmol/L, but the difference was statistically significant only in IL-6 and IL-10 (with p < 0.001) Table 3.25 Multivariate linear regression of factors affecting IL-6 Indicators SOFA Direct Bilirubin pH FiO2 IL-6 (n = 110) R2 corrected 0.20 Durbin - Watson p ANOVA 2.03 < 0.001* p VIF < 0.05 < 0.05 > 0.05 > 0.05 1.828 1.397 1.092 1.488 * Multivariate linear regression From the influencing factors of the univariate linear regression model, in multivariate analysis, only SOFA score and direct bilirubin were the factors that were actually associated with IL-6 levels in bacteremia patients Gram negative Table 3.27 Multivariate linear regression of factors affecting IL-10 Indicators APACHE II SOFA Platelet count APTT time Direct Bilirubin Protein PaCO2 Lactate HCO3- R2 corrected 0.285 IL-10 (n = 110) Durbin p Watson ANOVA 1.434 < 0.001* p VIF > 0.05 > 0.05 > 0.05 > 0.05 < 0.001 < 0.05 > 0.05 > 0.05 > 0.05 1.907 2.931 1.347 1.198 1.409 1.102 2.109 1.753 2.432 * Multivariate linear regression From the influencing factors of the univariate linear regression model, in multivariate analysis, only direct bilirubin and blood protein are the factors that are actually related to IL-10 levels in bacteremia patients Gram negative Table 3.28 Univariate linear regression of factors affecting TNF-α Element β TNF-α (n = 110) α R R2 p 17 APTT time -1820.29 71.56 0.341 0.107 < 0.005* * Univariate linear regression When investigating univariate linear regression, only APTT time was a factor related to TNF-α levels in patients with Gram-negative sepsis 3.3.2 Prognostic value of septic shock and mortality in patients with Gram-negative sepsis 3.3.2.1 Prognosis of septic shock Table 3.30 ROC curve predicts septic shock of cytokines in patients with Gram-negative sepsis Variables IL-6 (pg/mL) IL-10 (pg/mL) TNF-α (pg/mL) IL-6/IL-10 ratio IL-6/TNF-α ratio IL-10/TNF-α ratio AUC 0.775 0.715 0.553 0.578 0.628 0.608 p < 0.001* < 0.005* > 0.05* > 0.05* < 0.05* > 0.05* Cut point 37.47 36.56 0.17 - Sensitivit Specificity y 66.7% 82.3% 46.7% 88.8% 70% 60% - * ROC curve model Based on ROC curve model, IL-6, IL-10 levels and IL-6/TNF-α ratio have prognostic value for septic shock in 110 patients with Gram-negative sepsis In which, IL-6 had the best predictive value with AUC of 0.775; followed by IL-10 with an AUC of 0.715; finally, the ratio of IL-6/TNF-α with AUC of 0.628 (p values ranged from p < 0.001 to p < 0.05) Table 3.31 Multivariable logistic regression model of prognostic factors septic shock status in patients with Gram-negative sepsis Variables SpO2 Hemoglobin Protein PCT PaO2 OR 1.29 1.03 0.88 0.96 1.02 95% CI 0.99 - 1.67 1.00 - 1.05 0.81 - 0.95 0.94 - 0.99 1.00 - 1.03 p > 0.05* < 0.05* < 0.005* < 0.01* < 0.005* Lactate 1.82 1.35 - 2.46 < 0.001* * Multivariate Logistic Regression Over 110 patients studied, based on analysis of multivariable logistic regression model, hemoglobin, protein, PCT, PaO2 and lactate were independent predictors of septic shock in patients with Gram-negative sepsis 18 3.3.2.2 Mortality prognosis Table 3.33 ROC curve predicts mortality of cytokines in patients with Gram-negative sepsis Variables IL-6 (pg/mL) IL-10 (pg/mL) TNF-α (pg/mL) IL-6/IL-10 ratio IL-6/TNF-α ratio IL-10/TNF-α ratio AUC 0.723 0.685 0.504 0.564 0.647 0.628 p Cut point < 0.005* 35.4 14.37 < 0.01* > 0.05* > 0.05* < 0.05* 0.095 > 0.05* - Sensitivity 66.7% 66.7% - Specificity 75.3% 70.8% - 81% - 51.7% - * ROC curve model Based on ROC curve model, IL-6, IL-10 levels and IL-6/TNF-α ratio have prognostic value in mortality in 110 patients with Gram-negative bacteremia In which, IL-6 had the best predictive value with AUC of 0.723; followed by IL-10 with AUC of 0.685; the last is the ratio of IL-6/TNF-α with AUC of 0.647 (p value ranges from p < 0.005 to p < 0.05) Table 3.34 Multivariable logistic regression model prognostic factors for mortality in patients with Gram-negative sepsis Variables More than dysfunctional organs Use vasopressors IL-6 TNF-α IL-6/TNF-α ratio IL-10/TNF-α ratio PCT ≥ ng/mL * Multivariate Logistic Regression OR 21.57 14.47 1.00 1.00 0.94 1.13 0.18 95% CI 4.08 - 114.03 2.19 - 95.65 1.00 - 1.01 0.99 - 1.00 0.89 - 0.99 0.92 - 1.39 0.03 - 1.16 p < 0.001* < 0.01* < 0.05* > 0.05* < 0.05* > 0.05* > 0.05* Based on analysis of multivariate logistic regression model (110 patients), > organs, vasopressors, IL-6, IL-6/TNF-α ratio are independent prognostic factors mortality in patients with Gram-negative sepsis Chapter 4: DISCUSSION During the period from December 2016 to June 2018, 110 patients with Gram-negative bacteremia were selected for the study, of which E Hospital had 60 patients (54.5%) and 103 Military Medical Hospital had 50 patients (45.5%) 4.1 Characteristics of patients with Gram-negative bacteremia + Research results showed that the mean age was 63.65 ± 17.15 years old The distribution of age groups was uneven, focusing mainly on the age group > 60 (58.2%), followed by the age group from 41 to 60 ... Other Gram- negative bacteria accounted for 17.3% 10 3.2 Plasma concentrations of TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6 /IL-10, IL-6 /TNF-α, IL-10/ TNF-α in patients with Gram- negative... on the role of plasma TNF-α, IL-6, IL-10 in sepsis and Gram- negative sepsis In the world, there have been many studies on the role and change of cytokines TNF-α, IL-6, IL-10 in early diagnosis... Comparison of TNF-α, IL-6, IL-10 and the ratio of concentrations of IL-6 /IL-10, IL-6 /TNF-α, IL-10/ TNF-α levels between sepsis and septic shock Table 3.9 Comparison of plasma concentrations of TNF-α,