| BackgroundSepsis is a systemic, deleterious host response to infection leading to severe sepsis (acute organ dysfunction secondary to a documented or suspected infection), and possibly septic shock (severe sepsis plus hypotension that cannot be reversed by fluid resuscitation) as defined by the Surviving Sepsis Campaign guidelines.Thrombocytopenia is a common finding in sepsis, which is associated with adverse outcomes in patients admitted to the intensive care unit (ICU). In patients with sepsis, thrombocytopenia may not merely be a marker of disease severity but even play a causative role in increased sepsis morbidity and mortality. Thus, the correction of thrombocytopenia becomes a key issue for clinicians during the treatment of sepsis.The mechanisms of septic thrombocytopenia are complicated. The infectious pathogens leading to sepsis include bacteria, viruses, fungi, rickettsia, etc. A persistent viral or bacterial infection, with pathogens including the hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human parvovirus B19, can cause chronic thrombocytopenia,common clinical bacterial infection such as escherichia coli, klebsiella pneumonia bacillus Often merge thrombocytopenia; however, its pathogenesis has not been fully elucidated. Currently, with the exception of acute platelet transfusion when necessary, there is no effective method to prevent thrombocytopenia.Salic acid is a natural sugar acid compound that exists widely across organisms. The ends of glycoprotein chains on platelet membranes are all covered by sialic acid to protect the platelets from being destroyed. Sialidases, also known as neuraminidases, are sialic acid-releasing exoglycosidases that catalyze the removal of terminal sialic acids from sialosides and sialoglycoconjugates in nature. Sialidase widely exists in viruses, bacteria and mammalian cells. Additionally, resting platelets contain an internal pool of sialidase activity, which is up-regulated after activation due to infection or after a pathogenic bacterium-released new sialidase is introduced into the serum. The endogenous and exogenous sialidase hydrolyze terminal sialic acid moieties from platelet glycoproteins. Desialylation targets platelets for removal, a process that could possibly be circumvented by adding sialidase inhibitors during sepsis.Oseltamivir, also known as Tamiflu, is an extensively used and clinically effective anti-influenza drug. It is a viral sialidase inhibitor that prevents the release of progeny virions, thus limiting the spread of infection.23 Recently, we reported the successful treatment with oseltamivir phosphate of a patient with chronic immune thrombocytopenia (ITP).24 Hence, we hypothesized that thrombocytopenia was associated with increased platelet desialylation in septic patients and that the addition of oseltamivir to an antibiotic therapy would improve the clinical outcome in severe sepsis patients with thrombocytopenia.Objective:This study was to observe the association between platelet desialylation and thrombocytopenia in patients with sepsis, so as to interpret a new sepsis thrombocytopenia pathogenesis. At the same time to evaluation the important role of sialidase inhibitors oseltamivir in inhibiting the the sialidase activity, promoting platelet derived growth in severe septic patients with thrombocytopenia.Then for the treatment of sepsis thrombocytopenia provide new treatment goals and countermeasures.Methods:1. Object of study:The studies were carried out in the Departments of Internal Medicine, Hematology, and Medical ICUs at Yantai Yuhuangding Hospital, Yantaishan Hospital, Infectious Disease Hospital of Yantai, and Qilu Hospital, Shandong University, Shandong, China, from May 2014 to February 2016.2. The research group:In Study I, healthy adults were the negative control group; The septic patients were eligible for enrollment if they were 18 to 80 years of age; According to with or without thrombocytopenia(platelet counts less than 100 ×109/L), they were divided into:(1) sepsis with thrombocytopenia group; (2)sepsis without thrombocytopenia group. In Study II, we only included the severe sepsis patients whose platelet counts were less than 50 × 109/L, According to the computer random serial number, they were randomly divided into; (1) the odd number:the oseltamivir application group; (2) the even number:the control group.3. The eligibility criteria:The diagnostic criteria for sepsis, severe sepsis, and septic shock were based on the Surviving Sepsis Campaign guidelines.Briefly,sepsis was defined by a documented or suspected infection plus at least two the following conditions:temperature higher than 38.3? or lower than 36?, heart rate more than 90 beats/min, tachypnea, altered mental status, white blood cell (WBC) count greater than 12 × 109/L or less than 4 x 109/L, or elevated plasma procalcitonin (PCT) levels. Severe sepsis was defined as sepsis plus organ dysfunction. Septic shock was defined as sepsis plus hypotension (refractory to intravenous fluids) or hyperlactatemia Thrombocytopenia was defined as a peripheral blood platelet count less than 100,000/?L.4. The exclusion criteria were as follows:(1) patients diagnosed with malignancies or bone marrow stem cell disorders within the last two years; (2) patients who received chemotherapy or immunosuppressive agents within the prior 6 months; (3) patients who had a history of ITP or a similar autoimmune disease; (4) patients who had status-post cardiopulmonary resuscitation (CPR); (5) patients who were pregnant; (6) patients who had an acute gastrointestinal hemorrhage; (7) patients who had a history of bone marrow, lung, liver, kidney, or small bowel transplantation; (8) patients with end-stage hepatic or renal failure; (9) patients with disseminated intravascular coagulation (DIC); and (10) patients with drug (e.g., antibiotic or heparin)-associated thrombocytopenia.5. Study end points:The primary outcomes were platelet desialylation level, platelet overall response, and all-cause mortality within 28 days post-randomization. Secondary outcomes included platelet recovery time, and the amount of platelets transfused. Platelet overall response was defined as platelet counts returning to or above a normal level (100 × 109/L). Platelet recovery time was calculated as the date of randomization to the date when platelet counts were> 100 × 109/L.6. Data collection:For each enrolled patient, the following variables were recorded: 1) general characteristics including age, gender, and primary diseases; 2) severity of illness as assessed by the Acute Physiology and Chronic Health Evaluation (APACHE) II, and the SOFA score; 3) platelet desialylation level; 4) laboratory data upon admission including hematologic and chemistry tests, blood coagulation assays and arterial blood gas analysis; 5) screening tests for virus infection, serum galactomannan and (1?3)-?-D-glucan assays, and bacterial culture from clinical specimens such as sputum, blood, urine, secretions, and drainage fluid; 6) interventions including mechanical ventilation and continuous renal replacement therapy (CRRT); 7) the development of complications such as acute respiratory distress syndrome (ARDS), DIC, and bleeding events; and 8) the amount of platelets transfused. Time from sepsis onset for measurement of all laboratory parameters was limited within 24 hours.7. Application of oseltamivir:Both groups received appropriate antimicrobial agents and standard medical support based on the guidelines issued by the Surviving Sepsis Campaign. The oseltamivir group additionally received 5 full days of oseltamivir therapy. The oseltamivir was administered orally or through a feeding tube at a dose of 75 mg once every 12 hours. Time from randomization to the administration of oseltamivir was less than 24 hours. The control group did not administere oseltamivir. Platelet counts of both groupswere were measurederery day after randomization. The antimicrobial agents were continuously administered until 3 days after the resolution of the physiological abnormalities related to the systemic inflammatory response syndrome (SIRS).8. Platelet preparation:2 ml blood samples were obtained from subjects and were put into the vacuum of anticoagulant (EDTA or heparin anticoagulation) pipe, rich in platelet plasma were get after centrifugal, then 20 to 50 ul plasma to join centrifugal flow tube, centrifugal, platelet precipitation were separated. Platelet counts were measured.9. Assessment of platelet desialylation:To detect platelet desialylation, PE-Cy5-labelled anti-human CD41 monoclonal antibodies (20 ?L per test) were used to label human platelets. FITC-labelled RCA-I (5 ?g/mL), ECL (10?g/mL;) and sWGA (0.1?g/mL) were used to analyze salic acid exposure on platelet surfaces. Platelets (1 × 106) were incubated with anti-CD41 and RCA-I, ECL or sWGA, respectively for 20 minutes, washed twice and resuspended. The percentages of platelets positive for RCA-I, ECL or sWGA represented the levels of platelet desialylation.10. Data analysis:It was performed using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL, USA). Normally distributed variables were expressed as the mean ± standard deviation (SD), while skewed variables were expressed as the median (interquartile range). Between-group comparisons were evaluated by the 2-tailed unpaired Student's t-test (for normally distributed data) and Mann-Whitney U-test (for skewed data). The comparison of categorical variables was processed by the chi-squared(x2) test. The Spearman or partial correlation test was used to assess the association between platelet count and the other variables, such as age, SOFA, APACHE ?, and so on, while independent variables correlating with the 28-day mortality were determined by a multiple logistic regression analysis. Survival analysis was conducted based on whether oseltamivir was used, and differences in survival rates between groups were compared using the log-rank test. A multivariate analysis with the Cox proportional hazards regression model was used to assess the influence of each variable on the response to oseltamivir treatment. A P value less than 0.05 was considered statistically significant.Results:1. During the study period,276 patients were admitted, and 261 patients met the criteria of Study I. Among them,134 were septic patients without thrombocytopenia, and 127 were patients with thrombocytopenia. Among the 127 septic patients with thrombocytopenia,110 severe sepsis patients whose platelet counts were less than 50 × 109/L were enrolled in Study II. After randomization,52 patients were treated with antibiotics alone (the control group), whereas the other 54 patients were treated with antibiotics plus oseltamivir (the oseltamivir group). The 28-day follow-up was completed in 96.4% of the patients.2. Patients with thrombocytopenia had higher SOFA scores [10 (5,12) vs 5 (3.25, 7), P< 0.001], PCT levels [6.69 (1.0,38.9) vs 1.55 (0.32,5.36), P< 0.001], and creatinine levels [88 (63.3,225) vs 81 (56,119.0) ?mol/L, P= 0.036], but had a lower mean arterial pressure (79.6± 14.7 vs 86.8 ± 15.5 mmHg, P< 0.001) than patients without thrombocytopenia. Patients with thrombocytopenia had a higher occurrence of renal replacement (25.2% vs 14.2%, P= 0.029) and septic shock (18.9% vs 8.2%, P= 0.017) than patients without thrombocytopenia. The 28-day mortality rate was higher in patients with thrombocytopenia than in patients without thrombocytopenia (36.2% vs 14.2%, respectively; P< 0.001).3. About the risk factors for the 28-day mortality rate, It was demonstrated that platelet count was negatively associated with the 28-day mortality rate (OR= 0.963,95% CI:0.930-0.997, P= 0.033).4. The study revealed that bloodstream infections and the infection pathogens of Escherichia coli and New Bunia virus were risk factors for thrombocytopenia in sepsis patients. In contrast, lung infection and the infection caused by Pseudom onas aeruginosa and Staphylococcus aureus occurred more frequently in patients without thrombocytopenia.5. To compare the platelet desialylation levels between septic patients with or without thrombocytopenia,The data demonstrated that platelet desialylation increased significantly in septic patients with thrombocytopenia compared to those without thrombocytopenia, as detected with ECL (5.70 ± 4.37% vs 2.33± 1.90%; P< 0.001), RCA-I (95.64 ± 4.19% vs 90.14± 8.88%; P< 0.05) or sWGA (0.50±0.293% vs 0.15±0.95%; P< 0.001) lectins6. The responses and outcomes of patients in the oseltamivir group and the control group. Within 15 days after randomization, platelet response was achieved in 45 of the 54 patients in the oseltamivir group (83.3%) compared with 34 of the 52 patients in the control group (65.4%; P= 0.045). In patients who had a platelet response, the platelet recovery time was 5 days with quartiles of 4-6 days in the oseltamivir group compared with 7 days with quartiles of 5-10 days in the control group (P< 0.01). The amount of platelets transfused decreased significantly in the oseltamivir group (0.676 ± 1.36 apheresis units) compared to the control group (1.35 ± 1.98 apheresis units; P= 0.044; Table 2)7. The changes in the platelet counts of the oseltamivir and control groups over time. At baseline, the platelet counts were 41.81 ± 10.29× 109/L in the oseltamivir group and 46.69 ± 9.13 × 109/L in the control group, with no significant difference between these 2 groups (P= 0.560). Platelet counts in both groups increased over time and reached 91.30 ±21.51 × 109/L in the oseltamivir group and 58.17± 32.99 × 109/L in the control group on the fourth day (P= 0.031). From day 4 through day 9, the difference continued to be statistically significant between the groups, and on the 10th day, platelet counts were 198.8± 82.6 × 109/L in the oseltamivir group and 147.7 ± 81.78× 109/L in the control group (P= 0.684).8. Multivariate Cox proportional hazards models were used to estimate the variables that were associated with the responses in the oseltamivir and control groups. It was revealed that the SOFA score, platelet recovery time, mechanical ventilation, and continuous renal replacement therapy were independent indicators of oseltamivir therapy.Conclusion:1. the platelet desialylation increased significantly in septic patients with thrombocytopenia compared to those without thrombocytopenia as detected with ECL, RCA-I or sWGA lectins, indicating that thrombocytopenia was associated with increased platelet desialylation in septic patients.2. thrombocytopenia is associated with the severity of sepsis and is an important risk factor for the death of patients with sepsis.3. The data demonstrated that the addition of oseltamivir to antibiotic therapy could significantly increase the platelet response rate, shorten the platelet recovery time and reduce platelet transfusion. |
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