The Infectionof Cytomegalovirus Resulted In Abnormal Brain Development Inneonates And Its Regulating Effects On Cytokines

Objective:Human cytomegalovirus?HCMV?infection is prevalent throughout the world.The infection rate of women of childbearing age can reach 50%-80%and is specific to the species.HCMV infection can be divided into symptomatic and asymptomatic infection according to clinical signs.Neonatal infection with HCMV can lead to abnormal development of central nervous system,hepatitis,interstitial pneumonia,congenital deafness,thrombocytopenia,biliary malformation,and so on.Damage to the development of central nervous system represents the most serious consequence of neonatal HCMV infectionincludingpolymicrogyria,schizencephaly,pachygyria/lissencephaly,hydranencephaly,cleft cortical dysplasia and cerebellar hypoplasia.It is the main cause of neonatal retardation,mental retardation and sensorineural hearing loss,which seriously affects the quality of the birth population,and brings huge spiritual and economic burden to family and society.The early diagnosis of neurodevelopmental abnormalities caused by HCMV infection and the regulation of cytokines are not clear,so that the clinical intervention of neonatal neurological deficit resulted from infection is also facing challenges.In recent years,various laboratory methods for detecting HCMV have been developed and used,but they have their own advantages and disadvantages.Positive HCMV DNA in urine samples can predict the early existence of HCMV,but simply relying on the number of HCMV-DNA copies in urine cannot distinguish between symptomatic and asymptomatic infection.There are many factors affecting the test result,so it is necessary to check the urine for many times.Serum HCMV-IgM antibody positive suggests the existence of HCMV active infection in the near future.However,its detection is limited by its own metabolic rules and detection methods,which cannot fully meet the clinical needs.And the immune system of the neonates is not perfect,so the clinical diagnosis of neonatal symptomatic HCMV infection is not easy to be clear.Previous studies have suggested a novel mechanism by which HCMV is vacuolized in mononuclear macrophage,avoiding virus degradation and scavenging from cells in order to survive in mononuclear macrophages.Therefore,the detection of HCMV-DNA copies in mononuclear macrophages may provide a new method for the early diagnosis of neonates with HCMV infection.In neonates with brain anomalies caused by HCMV infection,compared with serum and other body fluids,the level of cytokines in Cerebro-Spinal Fluid?CSF?can directly reflect the immune response of central nervous system.Studies have shown that cytokines can be changed in CSF after infection of central nervous system.Therefore,a fast and effective method to detect cytokines in CSF is urgently needed to help diagnose the disease,guide treatment and improve prognosis.Because the protein content of CSF itself is very low,and most of them are albumin and immunoglobulin,the traditional method is difficult to separate,purify and identify.Protein chip is a high-throughput protein immunoassay and analysis technology,it is a new technology developed after gene chip in recent years.Miniaturization,integration,high-throughput,high sensitivity and easy operation are the main advantages.Preliminary progress has been made inprotein chips in detectingtarget proteins or the change of proteins related to diseases such as tumors,endocrine diseases,infectious diseases,cardiovascular systems and other diseases.Weevaluated the diagnosis value of peripheral blood mononuclear cells?Peripheral Blood Mononuclear Cell,PBMC?,urine quantitative detection of HCMV-DNA and HCMV-IgM in serum in neonatal HCMV infection,analyzed the correlation between the testing results and abnormal brain development and other clinical symptoms.We also detected cytokines in serum and cerebrospinal fluid in neonates with HCMV infection using protein chip technology,analyzed the effect of HCMV infection on nervous system related cytokines in neonates with abnormal brain development.This study is expected to provide evaluation index for central nervous system damage caused by HCMV infection,clinical curative effect and prognosis,and provide a theoretical basis for further elucidation of the pathogenic mechanism of central nervous system injury caused by HCMV infection.Methods:1.MaterialsSpecimens were collected from Neonatal Ward,the Affiliated Shengjing Hospital of China Medical University.HCMV-DNA quantitative detection kit was purchased in Shanghai star Yao medical science and Technology Development limited company.The serum HCMV-IgM antibody test kit was purchased in Italy Sorin Company.AAH-CUST-G chip kit,Human Matrix Metalloproteinase-3?MMP-3?ELISA kit,Human Acrp30 ELISA kit and Human IL-1?ELISA kit were purchased in Guangzhou RayBiotech limited company.Lymphocyte separation medium,PCR reagent,cell lysate,double distilled water,plastic centrifuge tube?2-5m L,50m L?,shaking table,plastic wrap,aluminum foil,adjustable multichannel pipette?1-25mL?,super-clean workbench,PCR quantitative amplification instrument,stable pressure electrophoresis apparatus,desktop centrifuge,LIAISON^?XL automatic chemiluminescence immunoassay analyzer,Philips Intera Achieva 1.5T and 3.0T magnetic resonance machine,Philips Brilliance CT?128?,InnoScan 300 Microarray Scanner fluorescence scanner,Thermo Scientific Wellwash Versachip washing machine.Primer synthesis and sequencing were accomplished by Invitrogen2.Methods:?1?Collection of specimen and separation of peripheral blood mononuclear cells?PBMC?The whole blood and urine specimens of 102 neonates with HCMV infection were collected from Neonatal Ward in Shengjing Hospital affiliated to China Medical University.Ficoll density gradient centrifugation was used to separate PBMC.Fluorescence Quantitative PCR?FQ-PCR?was used to detect PBMC and urine HCMV-DNA.Serum HCMV-IgM antibody was detected by chemiluminescence immunoassay?CLIA?.?2?Processing of dataClinical data such as image of nervous system of 102 neonates with HCMV infection were collected and analyzed.According to clinical signs,they were divided into symptomatic HCMV infection group?55 cases?and asymptomatic HCMV infection group?47 cases?,which accorded with the diagnostic criteria and exclusion criteria of symptomatic HCMV infection and asymptomatic HCMV infection respectively.Diagnostic criteria for symptomatic HCMV infection:HCMV infection was confirmed by urinary HCMV PCR or serum HCMV IgM antibody.At the same time,there was at least one following signs:intrauterine growth retardation,hepatosplenomegaly,thrombocytopenia(less than 100 x 10¹²/L platelet),skin rash,elevated direct bilirubin?direct bilirubin greater than 34umol/L?,elevated transaminases?alanine aminotransferase greater than 40U/L?,interstitial pneumonia,imaging abnormalities of the central nervous system?intracranial calcification,ventricular dilatation,brain cyst,agyria,etc.?.Diagnostic criteria for asymptomatic HCMV infection:HCMV infection was confirmed by urinary HCMV PCR or serum HCMV IgM antibody.There were no clinical signs at the same time.Exclusion criteria:Patients with hypoxic ischemic encephalopathy,bilirubin encephalopathy,intracranial hemorrhage?more than grade 2?,hypoglycemia encephalopathy;combined with Toxoplasma,rubella virus,herpes simplex virus,EB virus,hepatitis virus,syphilis infection in children;neonates with other diseases of central nervous system infection;congenital chromosome disease?3?Screening differential cytokines by protein chip methodFourneonates diagnosed as symptomatic HCMV infectionwere selected as the HCMV infection group.Four neonates without central nervous system infectionwere selected as control group.A protein chip containing 29 central nervous system related cytokines was used to screen differentially expressed cytokines in serum and cerebrospinal fluid between HCMV infected group and control group.HCMV infection group:neonates with symptomatic HCMV infection together with abnormal imaging of central nervous system?in accordance with exclusion criteria?were divided into HCMV infection group.The control group:neonates without HCMV infectionor central nervous system infection were divided into control group.?4?Verification by ELISA methodIn order to validate the results of protein chip,cerebrospinal fluid of 30 neonates with HCMV infection,30 neonates diagnosed as neonatal purulent meningitis and 30 cases in the control group were detected by ELISA.The HCMV infection group and the control group were defined as mentioned in?3?.Purulent meningitis group:neonates diagnosedwith purulent meningitis were divided into this group.?5?Statistical analysisStatistical analysis method for clinical data of neonates infected with HCMV:Median?25-75 percentiles?was described because quantitative data was not satisfied with normality hypothesis.Wilcoxon rank sum test was used for comparison between groups.Qualitative data was described by frequency?constituent ratio?,and Pearson chi square test or Fisher exact probability method was used for comparison between groups.Spearman grade correlation coefficient was used to measure the degree and direction of the two quantitative indexes.The difference of bilateral P<0.05 was statistically significant.Binary logistic regressionregression was used to analyze the relationship between the clinical symptoms and the results of head imaging by stepwise regression analysis.Using the area of ROC curve evaluate the model fitting result.The data of protein chip was analyzed by AAH-CUST-G's data analysis software.The drawing method of the protein chip result was heatmap.2/ggplot function,packet gplots,from R/bioconductor.ELISA test results were drawn by sigma plot 12.0.Data analysis was made by SPSS version 17.?6?This study was approved by the ethics committee of the Shengjing Hospital affiliated to China Medical UniversityResults:1.Collection of specimen and separation of PBMC?1?General information and clinical data of 102 neonates with HCMV infection were collected,55 of which were symptomatic HCMV infected neonates,and 47 were asymptomatic HCMV infected neonates.?2?102 cases of HCMV infected neonates'PBMC were successfully separated.?3?The results of PBMC,HCMV-DNA in urine and serum HCMV-IgM antibody titer showed:The median of PBMC HCMV-DNA quantitative was 888.0,and the 25-75percentile was 0.0-10500.0,quantitative detection of greater than 10 of PBMC HCMV-DNA was taken as a negative and positive critical value,PBMC HCMV-DNA were quantitatively negative in 43 cases?42.16%?,59cases were positive?57.84%?,the median of serum HCMV-IgM antibody titer was 0,the 25-75 percentile was 0.00-29.95,the serum HCMV-IgM antibody titers no less than 22 was taken as the positive and negative critical value,serum HCMV-IgM antibody were negative in 65 cases?63.73%?,37 cases were positive?36.27%?,the median of urinary HCMV-DNA quantitative detection was 12600,the 25-75 percentile was 2517.5-183000.0,the urinary HCMV-DNA level no less than 1000 was regarded as negative and positive critical value,quantitative HCMV-DNA were negative in 13 cases?12.75%?,89 cases were positive?87.25%?.2.Comparison and analysis of three kinds of experimental diagnostic methods?1?In a total of 102 neonates with HCMV infected,the sensitivity of PBMC HCMV-DNA quantitative detection?70.9%?in symptomatic HCMV infected neonateswas higher than that of serum HCMV-IgM antibody,and the specificity?57.45%?was higher than that of urine HCMV-DNA.PBMC HCMV-DNA quantitative detection and magnetic resonance results had good consistency,with high sensitivity?80.0%?and specificity?48.39%?.?2?Compared with the PBMC negative group,the abnormal rate of head MRI increased significantly,the gestational age was smaller,and the birth weight was lowerin PBMC positive group.The value of alanine aminotransferase and total bilirubin were significantly higher in the HCMV-IgM positive group than those in the negative group.The abnormal rate of alanine aminotransferase and brainstem auditory evoked potential was higher?P<0.05?.3.Protein chip screening differential expression cytokinesCompared with the control group,3 kinds of differential expression of cytokines in cerebrospinal fluid were found in HCMV infection group among the screening of 29kinds of nerve factors:adipocyte compiement-reiated protein of 30 kD?Acrp30?,matrix metalloproteinase-3?MMP-3?and interleukin-1??IL-1??.In the expression of serumcytokines,no difference was found in symptomatic HCMV infection group and the control group.4.Verification by ELISA methodELISA results showed that the content of Acrp30 in cerebrospinal fluid of 30 cases of HCMV infection in neonatal brain abnormalities is?39.76�2.01pg/m L?,it in 30 cases with non HCMV infection and disease of central nervous system infection is?7.75�0.10pg/m L?and?40.86�2.28pg/m L?in cerebrospinal fluid of neonatal purulent meningitis.Acrp30 in cerebrospinal fluid of symptomatic HCMV infection compared with non infectious diseases of the nervous system in neonates were significantly increased?P<0.01?.The content of MMP-3 were HCMV infection group?0.18�0.45ng/m L?and control group?1.40�2.13ng/mL?and purulent meningitis group?1.15�1.37ng/m L?.The levels of MMP-3 in cerebrospinal fluid of neonates with symptomatic HCMV infection were significantly lower than those of control neonates?P<0.01?and neonatal cerebrospinal fluid?P<0.01?of purulent meningitis.The IL-1?content was respectively HCMV infection group?2.36�0.99pg/mL?and control group?2.91�0.78pg/m L?and purulent meningitis group?2.41�0.47pg/m L?.The content of IL-1?in the cerebrospinal fluid of HCMV infection group was significantly lower than that of the control group?P<0.05?.Conclusion:1.For the neonates with symptomatic HCMV infection,the quantitative detection of PBMC HCMV-DNA has good sensitivity and specificity.2.PBMC HCMV-DNA quantitative detection and magnetic resonance results have good consistency.The abnormal rate of head MRI in PBMC positive children is significantly higher,and the gestational age is smaller.The values of alanine aminotransferase and total bilirubin were significantly increased in children with positive HCMV-IgM antibody.The abnormal rate of alanine aminotransferase and brainstem auditory evoked potential was higher.3.PBMC virus load and symptomatic HCMV infection of the neonatal nervous system injury has a certain correlation.4.Compared with the control group,the expression of 3 cytokines in the cerebrospinal fluid?CSF?of the neonates with symptomatic HCMV infection was different.5.ELISA method identified 3 differentially expressed cytokines in cerebrospinal fluid?CSF?,Acrp30,MMP-3 and IL-1?,which were consistent with the results of protein chip screening.