| Neonatal sepsis refers to the clinical syndromes caused by bacteria,viruses,fungi and protozoa's invasion into the body circulation system and toxins production that cause serious infections in the whole body.It is an important cause of neonatal death.Especially in premature infants with sepsis,the onset is acute,and the mortality rate is high.If the infection process is not discovered or terminated in time,it can rapidly progress to septic shock,which can easily cause terminal organ failure and death.Therefore,early diagnosis and precise treatment of neonatal sepsis are of great significance in improving survival rate and improving long-term prognosis.Studies have shown that the incidence of septic shock in hospitalized children in NICU is 1.3%,of which the mortality rate of premature infants and low birth weight infants is high,and the mortality rate of ultra-low birth weight infants is as high as 71%.Preterm birth-related complications are the worldwide leading cause of death among children under the age of 5.Deaths from sepsis and other serious infections account for about 15% of total neonatal deaths,while studies on preterm infants with septic shock are rare.Therefore,It is urgent to further develop EBM evidence based on the study of premature sepsis and to continuously update the standard of diagnosis and treatment,to guide clinical diagnosis and related research.Because of the immature development of multiple systems in premature infants and the complex and variable hemodynamics,non-culture-based clinical diagnosis and sepsis prediction scores are still active research areas for neonatal sepsis diagnosis.The first part of this study was to retrospectively analyze 114 preterm infants with septic shock.We collected the general clinical data,perinatal related factors,laboratory tests,pathogens,complications and risk factors for prognosis in preterm infants with septic shock.The study showed that amniotic fluid contamination,coagulopathy,CRP level elevation and patent ductus arteriosus were the risk factors for death in preterm infants with septic shock;thrombocytopenia and persistently elevated CRP were of predictive value in the prognosis of septic shock preterm infants and further provided evidence-based support and important theoretical evidence for the treatment and larger epidemiological studies of premature infants.Based on previous findings and literature reports,thrombocytopenia(NT)is a common problem in neonatal sepsis and is the most independent predictive risk factor for sepsis-related mortality in very low birth weight infants.However,the correlation between NT and pathogen types,significant bleeding,and prognosis is unclear.In the second part of the study,a retrospective cohort study of 663 neonates with sepsis who had been confirmed to be positive by blood culture was performed to elucidate the relationship between NT occurrence,severity,duration,platelet transfusions(PT)and infection type to further analyze the risk factors for sepsis death in neonates.The results showed that the occurrence and severity of NT in sepsis neonates were related to different pathogen types.The incidence of NT in Gram-negative sepsis was high,duration was long,the condition was heavy and the proportion of PT and mortality rate were high;preterm birth,Low birth weight,small for gestational age infant,Gram-negative bacteria,and fungal infection were related to NT;small for gestational age infant,Gram-negative infection,NT and PT were independent risks of sepsis in neonates.The risk of death in children with NT is about 3 times that of non-infected children.The risk of death in children with Gram-negative infection is about 2 times that of positive bacterial infection.This provided new ideas for the treatment of neonates with sepsis and also provided a theoretical basis for the necessity of platelet transfusion and reducing inappropriate blood use.The clinical symptoms of neonatal sepsis are mostly atypical.Early recognition of sepsis plays an important role in reducing the mortality of children.At present,there is still a lack of specific indicator for judging the course of sepsis.Recent studies have reported that Nicotinamide Adenine Dinucleotide(NAD~+)is elevated in the acute inflammatory phase of sepsis compared with the initial state,while the amplitude of NAD~+ increase in re-infected mononuclear cells is lower than normal monocytes during the immunosuppressive phase,which suggests that the dynamic state of energy status of mononuclear phagocytic cells,especially the reflected NAD~+ level,is closely related to the progression of sepsis.On this basis,we used the human monocyte cell line THP-1,which could simulate the acute inflammatory state and immunosuppressive state of mononuclear macrophages in sepsis under LPS stimulation.We then detected the expression changes of NAD~+ major metabolic enzymes and found the expression of the NAD~+ metabolic enzyme CD38 was potentially associated with the course of sepsis,which provided a partial mechanism for the translation of the key metabolic indicator NAD~+ in sepsis.At the same time,the changes of CD38 were detected in peripheral blood mononuclear cells of septic neonates,and the clinical significance of CD38 expression changes was explored.Both results provided basic data for further clinical application of CD38 molecules in the diagnosis of sepsis.Part ? Clinical features and risk factors on prognosis of premature infants with sepsis shock: analysis of 114 casesObjective: To investigate the risk factors affecting the prognosis of preterm infants with septic shock.Methods A retrospective study was conducted on the clinical data of 114 preterm children with septic shock admitted to the neonatal intensive care unit(NICU)in the our Hospital from February 2014 to January 2017.According to the clinical outcomes,the cases were divided into the cured group and the death group(including those with ineffective treatment and death after discharge who abandoned treatment).Two groups of data including the general clinical data,perinatal risk factors,clinical features and prognosis of the two groups of children,as well as the occurrence of related complications were statistically analyzed by t test,Wilcoxon nonparametric test,?2 test or Fisher exact probability method.Logistic regression was used to analyze the influencing factors of disease prognosis.The predictive value of the indicator was evaluated using receiver operating characteristic(ROC)curve.Results:1.Of the 114 patients,87 were cured and 27 were dead.The proportion of amniotic fluid pollution,anemia and thrombocytopenia(referred to as platelet count <100×109/L)in the death group,and the C-reactive protein(CRP)level was higher than that in the cured group [29.6%(8/27)with 8.1%(7/87),2=6.618;22.2%(6/27)and 5.9%(5/87),2=4.665;59.3%(16/27)and 23.3%(20/87);36.0(1.0~80.0)and 7.5(1.0~25.0)mg/L,Z=2.400];hemoglobin levels were lower than that in the cured group [(122.2±43.3)and(140.5±34.4)g/L,t=2.260](P<0.05).2.The proportion of children with patent ductus arteriosus,pulmonary hemorrhage and coagulopathy in the death group was higher than that in the cured group [81.5%(22/27)and 60.9%(53/87),2=3.871;37.0%(10/27)with 12.6%(11/87),2=6.616;48.2%(13/27)and 20.7%(18/87),2=7.847;P<0.05].3.Multivariate logistic regression analysis showed that amniotic fluid contamination,coagulopathy,patent ductus arteriosus and C-reactive protein levels were risk factors for poor prognosis in neonates(P<0.05).4.A total of 77 pathogens were isolated from 114 children(66 cured and 11 death).The composition ratios of Gram-positive bacteria,Gram-negative bacteria and fungi in the two groups were 37.9%(25/66)and 3/11,37.9%(25/66)and 6/11,24.2%(16/66)and 2/11,respectively.There was no significant difference in the distribution of pathogen types between the 2 groups.5.The area under the ROC curve of CRP was 0.649(P=0.024).When CRP was 31 mg/L,the sensitivity and specificity of predicting adverse outcomes in preterm infants with septic shock were 0.802 and 0.556,respectively,and the Yoden index was 0.358;the area under the ROC curve of thrombocytopenia was 0.708(P<0.001).When the platelet level was 94×109/L,the sensitivity and specificity of predicting adverse outcomes in preterm infants with septic shock were 0.767 and 0.593,respectively,and the Yoden index was 0.360?Conclusion: Amniotic fluid contamination,coagulopathy,persistently elevated CRP and patent ductus arteriosus are important risk factors for death in preterm infants with sepsis shock.Thrombocytopenia and persistently elevated CRP have predictive value for prognosis of preterm infants with sepsis shock.Part ? Platelet index change and prognosis analysis in neonatal sepsisObjective: To investigate the relationship between thrombocytopenia(NT)occurrence,severity,duration,platelet transfusions(PT)and the type of infection as well as prognosis analysis in neonatal sepsis.Materials and Methods:1.A retrospective cohort design was used to collect 663 neonates with sepsis diagnosed in NICU from January 2015 to December 2017 in our hospital.All cases were confirmed to be culture-positive,and only single type of pathogen was confirmed during the course of the disease.The follow-up deadline was 28 days after admission.The perinatal related factors,laboratory tests,etiology type and diagnostic information,death status and time were collected.2.Perinatal related factors and platelet-related indicators were described according to different gestational ages;single factor analysis was used to study the relationship between NT and perinatal factors in neonates with sepsis;single factor analysis was used to study infection type and perinatal related factors.The relationship between platelet related indicators and prognosis was also explored.3.Log-rank test was used to analyze the effects of different gestational age,NT severity,infection type and PT on prognosis.4.Cox regression was used to study the risk factors for survival and death in 28 days.Results:1.In the 663 neonates with sepsis,345 cases(52.0%)had NT.Pathogen types in neonates with NT were 130 cases(38%)of Gram-positive bacteria,169 cases(49%)of Gram-negative bacteria,and 46 cases(13%)of fungi.There were 61 cases(20%)with small gestational age;55 cases died(15.9);the difference was statistically significant(P < 0.05).2.The proportion of NT in extremely preterm infants,preterm infants and full term infants were 82.9%(184/222),47.1%(65/138),30.1%(90/293),respectively.The average NT durations were 7.0 days(4.0-15.0),4.0 days(3.0-8.0)and 5.0 days(3.0-8.0),respectively.There was a statistically significant difference in the incidence,PT,and duration of NT among the three groups(P < 0.05).3.There were 107 infants(16.4%)with NT who received PT in sepsis.The proportion of PT treatment in Gram-negative bacteria and Gram-positive septic patients were 36.1%(51/141 cases)and 12.7%(21/166 cases);the male ratio were 71.6%(101/141 cases)and 56.0%(93/166 cases);Mechanical ventilation time: 11.0 days(5.0-25.0)and 8.0 days(4.0-14.0);neonatal asphyxia: 27.7% and 16.3%;the mortality rate were 22.7%(32/141 cases)and 9.0%(15/166 cases),the difference between groups was statistically significant(P < 0.05).4.Compared with Gram-positive bacteria,the incidence of NT in Gram-negative bacteria infected with sepsis was higher,the initial value was lower,the duration was longer,the PT ratio was higher,and mortality was higher.The difference was statistically significant(P < 0.05).5.After Cox regression analysis,the independent risk factors for 28-day mortality in neonatal sepsis were: small gestational age(HR=1.97),Gram-negative infection(HR=2.00),NT(HR=2.86),and PT(HR=1.95).Conclusions:1.preterm birth,Low brith weight,Smaller gestational age,Gram-negative bacteria and fungal infections are associated NT;2.The occurrence and severity of NT in sepsis neonates were related to the type of infection.The incidence of NT in Gram-negative sepsis was higher,the duration was longer,the condition was more serious,the proportion of PT treatment was higher and the mortality rate was higher.3.Independent risk factors for neonatal sepsis death were small gestational age,Gram-negative infection,NT and PT.Among which the risk of death in infants with NT was about 3 times that without sepsis,and the risk of death from Gram-negative infections was about 2 times that of Gram-positive bacteria.Part ?: Basic and clinical studies on the expression changes of NAD~+ metabolic enzyme CD38 in neonatal sepsisObjective:1.In the human monocyte cell line THP-1,the expression of NAD~+ major metabolic enzymes under LPS stimulation was detected,and the potential related NAD~+ metabolic enzyme with the course of sepsis was screened and verified,which was the key metabolism in sepsis.The change in indicator NAD~+ provided a partial mechanism explanation.2.To detect the change of NAD~+ digestive enzyme CD38 in peripheral blood of septic neonates,and to explore the clinical significance of CD38 expression in peripheral blood mononuclear cells in neonatal sepsis,and provide potential indicators for the diagnosis of sepsis.Materials and Methods:1.According to literature reports and bioinformatics database tips,we listed the relevant NAD~+ metabolic enzyme family members to be tested,and designed the corresponding quantitative PCR primers(referring to the primerbank website).2.LPS was used to stimulate human monocyte cell line THP-1.After stimulation,the cells were harvested and RNA was extracted and reverse transcribed into c DNA.Quantitative PCR was used to detect the changes of inflammatory factors and NAD~+ metabolic enzymes.3.The metabolic enzyme molecule CD38,which was statistically different from the initial value before stimulation,was screened by t-test.After extracting the protein from the THP-1 cell LPS stimulation model,Western Blot was used to detect changes in CD38 protein expression levels.4.Further,in the THP-1 cell endotoxin tolerance model,the changes of CD38 m RNA and protein were detected by quantitative PCR and Western Blot.5.The peripheral blood samples of newborns in the control group and the sepsis group were collected and extracted with RNA,and c DNA was obtained by reverse transcription,and the medical records were recorded.Quantitative PCR was used to detect the expression of CD38 m RNA.6.Flow cytometry was used to detect the distribution and expression of CD38 in peripheral blood leukocytes of control neonates and neonates in the sepsis group.The role and clinical significance of CD38 changes in the course of neonatal sepsis were analyzed.Results:1.The quantitative PCR results of THP-1 cells stimulated by LPS showed that the m RNA levels of CD38 and Sirtuin6 were significantly increased after 24 hours of LPS stimulation in the three types of NAD~+ digestive enzymes,and the CD38 m RNA level was the most dramatic.There were no significant increase in other NAD~+ digestive enzyme m RNA levels.2.THP-1 cells stimulated by LPS showed a significant increase in the protein level of CD38 by Western Blot detection.3.THP-1 cells in endotoxin tolerance stage could still increase the amount of CD38 protein after LPS re-stimulation,and under the stimulation of LPS,the m RNA and protein levels of CD38 in endotoxin-tolerant THP-1 cells were higher than normal THP-1 cells.4.Quantitative PCR and flow cytometry showed that the ratio of CD38 mRNA and CD38+ cells in leukocytes of neonates with sepsis was significantly higher than that of normal controls.5.The CD38-positive cells in the peripheral blood leukocytes of neonates and sepsis groups in the control group were mainly distributed in monocytes,and the positive rate of CD38 in neonatal mononuclear cells of sepsis was significantly higher than that in the control group.Conclusions: The m RNA and protein levels of NAD~+ digestive enzyme CD38 in normal THP-1 cells were increased under LPS stimulation.The expression of CD38 in endotoxin-tolerant stage THP-1 cells stimulated by LPS can be further increased.The phenomenon reported can be partially explained by previous literature: when the mononuclear cells were re infected during immunosuppression,the increase in NAD~+ level was lower than that of normal monocytes.Quantitative PCR and flow cytometry to detect clinical blood samples found that the expression of CD38 in peripheral blood leukocytes of neonates with sepsis was significantly higher than that of the control group.The rate of CD38 positive monocytes in septic neonates was significantly higher than that of the control group.These results suggested that the increased expression of CD38 on the surface of monocytes may be a potential indicator for the diagnosis and course judgment of sepsis.It was sufficient to clarify the changes and effects of the expression of metabolic related enzymes represented by NAD~+ metabolic enzyme CD38 in the pathological process of sepsis,which could provide a new theoretical basis for the diagnosis of sepsis. |
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