Study on the change of TNF-α, IL-6 AND IL-10 concentrations in gram-negative sepsis patients

T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 109 STUDY ON THE CHANGE OF TNF-α, IL-6 AND IL-10 CONCENTRATIONS IN GRAM-NEGATIVE SEPSIS PATIENTS Vu Manh Cuong1, Hoang Vu Hung2, Vu Xuan Nghia2 SUMMARY Objectives: To evaluate the change of TNF-α, IL-6, IL-10 concentrations in patients with Gram-negative sepsis. Subjects and methods: 124 patients who were diagnosed with Gram-negative sepsis based on blood culture results at Military Hospital 103 and E Hospital. TNF-α, IL-6, and IL-10 concentration were quantified by ELISA technique at T0, T24. Results: TNF-α and IL-6 level increased at T0, then decreased significantly at T24 (p < 0.001). IL-10 concentration had the highest increasing rate compared to the normal concentration at all timepoints of follow-up and tended to increase at T24 (80.44 times) compared to T0. However, this difference was not statistically significant with p > 0.05. Among Gram-negative bacteria, E. coli accounted for the highest proportion (44.4%), followed by Kleb. pneumoniae (21.8%) and P. aeruginosa (5.6%). In case of E. coli sepsis at T0, IL-6 concentration had the highest increasing rate, followed by TNF-α and IL-10 (28.36, 12.09, and 11.28 times, respectively). At T24, TNF-α concentration had the highest increasing rate compared to the normal concentration, followed by IL-6 and IL-10 (15.28, 9.15, and 3.58 times, respectively). Conclusion: In Gram- negative sepsis, TNF-α and IL-6 level increased at T0 and decreased sharply at T24. IL-10 concentration had the highest increasing rate compared to the normal concentration at all timepoints of follow-up and tended to increase at T24 (80.44 times) compared to T0. * Keywords: Gram-negative sepsis; TNF-α; IL-6; IL-10. INTRODUCTION Sepsis is a serious systemic infection caused by bacteria and toxins of bacteria circulating in the blood. Sepsis has a high risk of mortality caused by septic shock and multiple organ dysfunction [1]. The common causative agent of sepsis is Gram-negative bacteria due to its prevalence, severe clinical manifestations, and associated septic shock. The mortality rate in patients with septic shock can be up to 80%. Moreover, the treatment of Gram-negative sepsis is very difficult due to their high resistance to antibiotics [4]. The pathogenesis of sepsis is a complex chain of inflammatory and anti-inflammatory responses, as well as humoral and cellular responses, etc. In particular, the interaction between inflammatory and anti-inflammatory mediators can be seen as a struggle between two opposing sides: pathogens and the body's protective responses [5]. 1E Hospital 2Vietnam Military Medical University Corresponding author: Vu Manh Cuong (vumanhcuongbve@gmail.com) Date received: 14/8/2020 Date accepted: 22/9/2020 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 110 Recently, the inflammatory roles of TNF-α and IL-6, as well as the anti-inflammatory role of IL-10 have been identified and helped to elucidate the pathogenesis of sepsis and multi-organ failure syndrome. These cytokines are mostly produced quickly within a few hours to 24 hours after the entry of a pathogen into the body. Therefore, quantifying and monitoring changes of these cytokine levels not only helps to diagnose and evaluate severity at an early stage, but also helps to prognostic of sepsis patients [3]. From these issues, we conducted this study: To assess the change of TNF-α, IL-6, and IL-10 concentrations in patients with Gram-negative sepsis. SUBJECTS AND METHODS 1. Subjects 124 inpatients treated at the Clinical Departments of E Hospital and Military Hospital 103, who were diagnosed as Gram-negative sepsis based on the positive blood cultures results from December 2016 to June 2018. * Inclusion criteria: + Aged ≥ 18, regardless of gender and occupation. + Two positive blood cultures with the same Gram-negative bacteria. + TNF-α, IL-6, IL-10 concentrations were measured at two time points of study: T0(time of blood culture) and T24. * Exclusion criteria: + Pregnancy. + End-stage cancer, end-stage chronic kidney failure, Child-C cirrhosis, immunodeficiency such as HIV/AIDS, etc. + Disagreed to participate in the study. 2. Methods * Study design: Descriptive, prospective follow-up. * Study material: - Blood culture: using BacT/Alert 3D automatic bacteria detection system at Department of Microbiology, E Hospital and Military Hospital 103. - Quantitative testing of cytokine TNF-α, IL-6 and IL-10: + Using TNF-α, IL-6 and IL-10 kits of AviBion - Orgenium (Finland) to quantify TNF-α, IL-6 and IL-10 concentrations in plasma samples of patients. + The necessary supplies for technical implementation: Normal pipettes, multi- channel pipettes, pipette tip, 2 times distilled water. + Reading the ELISA results using BECKMAN-COULTER-DTX 880 system of Beckman-Coulter (USA) at D3, Institute of Biomedicine and Pharmacy - Vietnam Military Medical University. + The normal ranges: TNF-α (< 11 pg/mL); IL-6 (< 1.23 pg/mL); IL-10 (< 1.9 pg/mL) [2]. * Procedures: - Sampling: Measure the concentrations of TNF-α, IL-6, and IL-10 at T0 (time of blood culture) and T24 (after 24h). - Sample preparation: + 2 mL of heparin anticoagulant blood was taken, centrifuged to separate the plasma, and stored in the freezer; after that, samples were transferred to laboratory and stored at the temperature of -80°C T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 111 before testing at Institute of Biomedicine and Pharmacy - Vietnam Military Medical University. + Reagents and samples were stored at room temperature (18 - 25°C) before use. The sample was thawed before the test. + Dilution test standards: According to the diagram instructions from the manufacturer. + Reading the results on an optical machine with a wavelength of 450 nm. The time for correct results within 20 minutes of completing the reaction. The optical density (OD) was directly proportional to the concentration of cytokines present in the samples. * Data processing: Using SPSS 20.0 software. RESULTS AND DICUSSIONS Tabe 1: Distribution by age group and gender Gender Age group Total n (%) Male n (%) Female n (%) p ≤ 40 years 14 (11.3) 7 (9.7) 7 (13.5) 41 - 60 years 38 (30.6) 25 (34.7) 13 (25) > 60 years 72 (58.1) 40 (55.6) 32 (61.5) Total 124 (100) 72 (58.1) 52 (41.9) > 0.05 X̅ ± SD 63.49 ± 16.76 years (Min: 21 years, max 94 years) The mean age of study group was 63.49 ± 16.76 years old, with the minimum age of 21 years and maximum age of 94 years. The age group of 60 years and above accounted for the highest proportion (58.1%), followed by the age group of 41 - 60 years (30.6%). The age group of 40 and below accounted for the lowest proportion (11.3%). Male accounted for 58.1% and female accounted for 41.9%. The proportion of male and female in general and in each age group was not significantly different with p > 0.05. The mean age and the common age group in our study were higher than study by Dung N.T.H. et al. on 62 Gram-negative patients. In this study, the mean age was 43 ± 16.8 years old (11 - 84 years old). The most common age group was from 18 to 40 years old (48.4%), followed by the age group of 41 - 60 years old (27.4%) [3]. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 112 Table 2: Some major clinical manifestations in study group (n = 124). Manifestations X̅ ± SD Min - max Glasgow (score) 14.11 ± 2.20 4 - 15 Temperature (0C) 38.62 ± 0.74 36.8 - 40.3 Yes 93 (75.0) Signs of chills (n, %) No 31 (25.0) Respiratory rate (breaths per minute) 20.56 ± 3.33 14 - 35 SpO2 (%) 96.53 ± 2.77 78 - 100 APACHE II score 10.81 ± 5.20 0 - 35 SOFA score 4.66 ± 3.52 0 - 15 The majority of patients with Gram-negative sepsis did not have much change in neurological status and some indices such as respiratory rate and SpO2. 75% of patients showed signs of chills at the time of blood culture. Most patients had higher normal values for temperature, APACHE and SOFA scores. Table 3: Blood culture results in study group. Bacterial identification Number of patients Percentage (%) Escherichia coli 55 44.4 Klebsiella pneumoniae 27 21.8 Pseudomonas aeruginosa 7 5.6 The remaining bacteria 35 48.2 Total 124 100.0 Among the isolated bacteria, E. coli accounted for the highest proportion (44.4%), followed by Kleb. pneumoniae (21.8%) and P. aeruginosa (5.6%). The remaining bacteria accounted for 48.2%. In a study on 165 patients with severe abdominal infection, Maja Surbatovic et al. found than among Gram-negative bacteria, the most common were Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. Less common Gram-negative pathogens were Escherichia coli, Proteus vulgaris and Citrobacter [7]. Table 4: TNF-α, IL-6, and IL-10 concentrations at T0 and T24 (n = 124). Cytokines Time Median (IQR) Min - Max p T0 32.25 (10.72 - 129.1) 0.94 - 24,586.54 TNF-α T24 16.81 (6.1 - 36.45) 1.65 - 23,960.70 < 0.001 T0 12.37 (4.69 - 58.28) 1.13 - 3,675.73 IL-6 T24 6.61 (3.31 - 24.08) 1.38 - 3,770.82 < 0.001 T0 137.75 (19.92 - 259.24) 0.84 - 1,003.92 IL-10 T24 152.83 (20.36 - 266.27) 0.59 - 819.10 > 0.05 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 113 Concentrations of TNF-α and IL-6 increased at T0, then decreased significantly at T24. This difference was statistically significant with p < 0.001. IL-10 concentration tended to increase at T24, however, this difference was not statistically significant with p > 0.05. According to Bah I et al in 2018, when studying sepsis in mice, IL-6 level increased rapidly and then significantly decreased a week after sepsis. In contrast, IL-10 level increased gradually and slightly during the first week after sepsis [8]. Median values of TNF-α, IL-6, and IL-10 at T0 and T24 in Thao PTN's study on severe sepsis patients were 12.48 pg/mL and 8.81 pg/mL; 530.0 pg/mL and 211.4 pg/mL; 45.84 pg/mL and 13.36 pg/mL, respectively. We found that TNF-α and IL-10 concentrations in our study were higher, while IL-6 concentration was lower than this study [2]. The study by Maja Surbatovic et al. showed that in the group of severe abdominal infections caused by Gram- negative bacteria, TNF-α had a mean value of 0.97 pg/mL (min: 0.11 pg/mL; max: 9.22 pg/mL) and IL -10 had a mean value of 11 pg/mL (min: 0 pg/mL; max: 302 pg/mL) [7]. In 2016, Liu K.T. et al. when studying on 32 patients with Gram-negative sepsis showed that there were 16 patients with E. coli infection, and 7 patients with K. pneumoniae infection. The mean IL-6 concentrations were 2.552 ± 4.004 pg/mL and 632.43 ± 829 pg/mL, respectively [9]. Table 5: The relationship between the concentration of cytokine and some common Gram-negative sepsis-causing bacteria. E. coli (n = 55) Kleb. Pneumoniae (n = 27) P. aeruginosa (n = 7) Bacteria Cytokine Median (IQR) p Median (IQR) p Median (IQR) p T0 34.88 (10.82 - 131.89) 59.84 (8.84 - 339.2) 8.14 (4.29 - 23.79) IL-6 T24 11.26 (5.4 - 34.35) < 0.001 17.07 (11.97 - 60.91) < 0.01 12.1 (4.89 - 17.15) > 0.05 T0 21.44 (6.56 - 69.18) 16.96 (8.28 - 227.74) 10.6 (2.83 - 47.02) IL-10 T24 6.8 (3.25 - 29.53) < 0.001 9.3 (3.94 - 24.39) < 0.05 8.4 (4.86 - 44.63) > 0.05 T0 133.06 (62.5 - 259.5) 150.57 (37.54 - 236.22) 240.77 (3.12 - 504.66) TNF- α T24 168.13 (81.99 - 273.52) > 0.05 142.26 (8.43 - 195.03) > 0.05 171.57 (19.69 - 245.5) > 0.05 Among common Gram-negative bacteria, the majority of IL-6, IL-10 concentrations, and TNF-α increased at T0 and then tended to decrease at T24. However, only IL-6 and IL-10 concentrations in E. coli and Kleb. Pneumoniae bacteria had a significant T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 114 difference with p < 0.05. TNF-α concentrations in sepsis caused by E. coli and IL-6 in sepsis by P. aeruginosa tended to increase at T24 compared to T0. However, these differences were not statistically significant. Compared to the total of 124 Gram-negative sepsis patients in this study, the change of TNF-α and IL-6 concentrations in sepsis caused by E. coli and P. aeruginosa, and the change of IL-10 concentrations in sepsis caused by three above bacteria did not follow the general rule because the proportion of sepsis caused by these three bacteria (51.8%) could not represent for all 124 patients in the study group. Table 6: Differences in cytokine concentration at the time of study compared to the normal value. TNF-α (< 11 pg/mL) IL-6 (< 1.23 pg/mL) IL-10 (< 1.9 pg/mL) Bacterial identification Cytokine value T0 T24 T0 T24 T0 T24 Median 32.25 16.81 12.37 6.61 137.75 152.83 Total (n = 124) Number of increases 2.93 1.53 10.06 5.37 72.50 80.44 Median 133.06 168.13 34.88 11.26 21.44 6.8 Sepsis caused by E. coli (n = 55) Number of increases 12.09 15.28 28.36 9.15 11.28 3.58 Median 150.57 142.26 59.84 17.07 16.96 9.3 Sepsis caused by K. pneumoniae (n = 27) Number of increases 13.69 12.93 48.65 13.88 8.93 4.89 Median 240.77 171.57 8.14 12.1 10.6 8.4 Sepsis caused by P. Aeruginosa (n = 7) Number of increases 21.89 15.60 6.62 9.84 5.58 4.42 In 124 Gram-negative sepsis patients, IL-10 concentration had the highest increasing rate compared to the normal concentration at all the time of follow-up. IL-10 concentration was higher at T24 (80.44 times) than at T0 (72.5 times), followed by TNF-α and IL-6 concentrations (both were higher than normal values at all time of follow-up and tended to decrease over time). For sepsis caused by E. coli, at T0, IL-6 concentration had the highest increase compared to normal values, followed by TNF-α and IL-10 (28.36, 12.09, and 11.28 times, respectively). At T24, TNF-α concentration had the highest increase compared to normal values, followed by IL-6 and IL-10 (15.28, 9.15, and 3.58 times, respectively). For sepsis caused by K. pneumoniae, all cytokine concentrations increased compared to normal values and tended to decrease over time. IL-6 concentration had the highest increase compared to normal values, followed by TNF-α and IL-10. For sepsis caused by P. aeruginosa, TNF-α concentration had the highest increase compared to normal values, followed by IL-6 and IL-10. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 115 Surbatovic M et al. found that Gram- negative bacteria produce more IL-10 and TNF-α than Gram-positive pathogens. TNF-α and IL-10 concentrations were 2 times and 1.83 times higher in Gram- negative sepsis than in Gram-positive sepsis [7]. CONCLUSIONS Study on the change of TNF-α, IL-6, and IL-10 concentrations in 124 Gram- negative sepsis patients, we found that: - Concentrations of TNF-α and IL-6 increased at T0, then decreased sharply at T24. The median concentration of IL-10 had the highest increasing rate compared to the normal concentration at all time of follow-up and tended to increase at T24 compared to T0. However, this difference was not statistically significant with p > 0.05. - Common Gram-negative bacteria caused sepsis in the order of E. coli, Kleb. pneumoniae, and P. aeruginosa. For sepsis caused by E. coli, at T0, IL-6 concentration had the highest increase compared to normal values, followed by TNF-α and IL- 10. 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