Clinical Research And Virological Features Of Epstein-Barr Virus Associated Infectious Mononucleosis And Hemophagocytic Lymphohistiocytosis

BackgroundEpstein-Barr virus （EBV） belongs to the genus B Lymphocryptovirus, subfamilyGammaherpesvirinae, family Herpesviridae, EBV virions have a dobule-stranded.Sero-epidmiologic surveys indicate that over95%of adults worldwide are infectedwith EBV^[1]. Previous investigations revealed that in our countries,80-100%ofchildren were serpopositive of EBV-specific antibodied by3to5of age, and thealternate with100%of seropositive by10of age. EBV is one of the most commonpathogens of the infectious diseases in childhood, primary EBV infection are oftenmanifested as nonspecific illnesses, about50%of primary infection may result inIM^[2]. IM is believed to be an immunopathological disease, and it is characterized bydramatic antigen-driven clonal expansions of CD8T lymphocytes in response toprimary EBV infection, the activated CD8T lymphocytes killer EBV infection B celland infringe upon lymphoid tissue organ, release cytokines that are predominantly ofa Th1type[13]. Because of relative immature of immune system in infants and young children, primary EBV infections usually occurs more common in these age groups.IM is usually self-limiting and resolves spontaneously after the emergence ofEBV-specific immuno-responses, the mortality rate observed in only1to2%of casesof IM[14]. Severe acute complications may be seen in a few IM cases, fulminantinfectious mononucleosis（FIM）may occur. In a limited number of individuals, IMmay develop into a lifethreatening disease, EBV-associated hemophagocyticsyndrome（EBV-AHS）. EBV-AHS is an extremely dangerous state with highmortality, the mortality rate rangs from30to40%^[15].Hemophagocytic lymphohistiocytosis is a rare and often fatal disorder, this life-threatlening condition is a type of immune dysregulation characterized by an impairedor absent function of natural kille（rNK）cells and cytotoxic T cells, and the release ofproinflammatory cytokines. HLH is a distict clinical entity characterized by a highfever, splenomegaly, pancytopenia, and the pathological finding of hemophagocytosisin bone marrow, lymph nodes and other tissues. HLH has since been associated with avariety of genetic defects, infection, tumor, autoimmune system disease. Althoughmany viruses can trigger HLH, EBV is the most common triggering agent for HLH.For patients with reactive HLH associated with pathogens other than EBV, supportivecare and treatment of the underlying infection is associated with recovery in60-70%.EBV-AHS in most is universally fatal if untreated[46]. Patients with sporadic HLH,however, usually have no gene mutations, the relationship between EBV infection andthe function of immune system deserves to be futher investigation in the future.In an effective humoral and celluar response during primary EBV infection,historically, the humoral response to EBV infection was determined by a set ofimmunofluorescence assays measuring EBV-specific serological antibody titers.Indirect immunofluorescence assay （IFA） is the classical technique, but the enzymeimmunoassay technique is often used in routine determination of EBV infectionbecause of its reliability in high-throughput analyses and high sensitivity. Real-time quantitative polymerase chain reaction（Real-time PCR）, which is developed fromqualitative PCR assays to be the most sensitive and technique for the determination ofEBV-DNA. It has replaced the other PCR assays and widely used in EBV detection.Measuring the cell-free EBV-DNA copy number in peripheral blood by real-timePCR may reflect virus activitese. So we examined EBV anyibody and EBV-DNAcopy numbers in sera by ELISA and real-time PCR respectively, to explorevirological features of Epstein-Barr virus associated infectious mononecleosis andhemophagocytic lymphohistiocytosis in Childhood.Objectives:To observe clinical manifestations and virological features of Epstein-Barrvirus associated infectious mononecleosis and hemophagocytic lymphohistiocytosisin Childhood.Methods:1. Clinical characteristics and laboratory analysis of Epstein-Barr Virus Infection inChildhood Patients with EBV infectious were separated into IM and EBV-AHS，clinical and laboratory characteristics were analysed.2. EBV antibody tests2.1Peripheral blood（2ml）was collected from each subject into an serum tube.2.2Blood samples were centrifuged at2500rpm×15min, and serum was carefullyremoved from the serum tube and transferred into plain polypropylene tubes. Thesample were stored at-80℃in aliquots until further processing.2.3Four antibodys, incuding EBV-IgM/IgG anti-VCA, EBV-IgG anti-EA, EBV-IgGanti-NA, were detected in sera of all of cases by ELISA with a commercial kit.2.4Patients were divided into primary EBV infection group and subacute EBVinfection group according to the sero-EBV antibody result, to investigate clinicalcharacteristics and laboratory analysis of two groups.3. EBV-DNA assays 3.1Peripheral blood（2ml）was collected from each subject into an serum tube.3.2Blood samples were centrifuged at2500rpm×15min, and serum was carefullyremoved from the serum tube and transferred into plain polypropylene tubes. Thesample were stored at-80℃in aliquots until further processing.3.3DNA from serum samples was extracted using a QIAamp DNA Mini kit（QIAGENGermany）. Serum samples（200ul/column）were used for DNA extraction. DNA extr-action from serum samples were stored at80℃in aliquots until further processing.3.4ALL were run in a Bio-Rad IQ5instrument（Bio-Rad，America）.4. Statistical analysisDate were analysed using the SPSS17.0for windows （SPSS Inc., Chicago IL,USA）. The Kolmogorov-Smirnov test was used to verify the normality of distributionof continuous variables. Nomal distribution date are presented as mean（StandardDeviation；SD）. Skewness distribution date are presented as median（range）.Twoindependent-samples T text and Mann-Whitney U test was used to test for differencesof quantitative variables. Two sample rate compared with a chi-square test. Peasoncorrelation test was used to examine associations between2quantitative variables. Pvaule less than0.05were considered significant.Results:1.In our investigatious of all IM cases, the ratio of boys versus girls was1.8:1; the ageof onset ranged from13months to13years,60.5%of the patients were preschoolers.2.The main symptoms and signs of the IM included feve（r97.7%）, tonsillopharyngitis（97.7%）, cervical lymphadebopathy（95.3%）, eyelid edema（48.8%）, hepatomegaly（65.1%）, splenomegaly （46.5%） and rash（20.9%）; the percentage of eyelid edemawas higher than splenomegaly and rash in IM group. Longer febrille duration（P=0.002）and higher peak temperature（P=0.001）were found in EBV-AHS.3. EBV-AHS is a distinct clinical entity characterized by persistent high fever, hepat- osplenomegaly, significantly reduced WBC, ANC, Hb and PLT, live dysfunction andthe increased level of LDH was the most obvious, elevated levels of triglyceride（TG）and serum ferritin（SF）, reduced levels of fibrinogen （Fb）could be observed.4. Primary EBV infection[viral capsid antige immunoglobulin M（IgM）-（⁺）,viral nucleantige IgG-（-）, viral capsid antigen IgG and viral early antigen IgG-（⁺）/（-）]was themost common sero-antibody type in IM（65.1%）. At the initial visit, the VCA-IgMpositive ELA was seen in42of43patients with IM （97.6%）, and that in1case theVCA-IgM turned positive one week later from the first assay.5serum samples fromEBV-AHS with previously acquired EBV infections.5. Comparison of clinical manifestation and laboratory findings between primaryEBV infection and subacute EBV infection, the percent of eyelid edema and enlagedliver was significantly higher in primary EBV infection than those of subacute EBVinfection（P1=0.003; P2=0.011）; LDH were higher in primary EBV infection groupthan in subacute EBV infection, the differences were statistically significant betweenthe two groups（P=0.005）. The incidence of liver dysfunction in primary EBVinfection was significantly higher than subacute EBV infection group（P=0.005）.6. The serum EBV-DNA level in24of43patients with IM was in the range of8.24×10³～2.88×10⁵copies/ml with a mean value of5.88×10⁴copies/ml. Five HLH caseswith positive of EBV-DNA loads in sere were diagnosed EBV-AHS, the EBV-DNAloads were in the range of2.88×10⁵～9.25×10⁸copies/ml with a mean value of1.86×10⁸copies/ml. There were significant differences of EBV-DNA loads in sere ofpatients betwween IM and EBV-AHS（P=0.001）.7.43pediatric patients with IM were seperated into EBV-DNA loads positive groupand negative group, ALT、LDH were statistically significant higher in EBV-DNApositive group than negative group(P_ALT=0.013; P_LDH=0.003), EBV-DNA loads weresignificantly correlated with levels of AST and LDH(r=0.567, P_AST=0.004; r=0.885, PLDH=0.004).Conclusion:1. In our study EBV-associated IM is more common in preschool male children, themajority of presentations were fever, tonsullopharyngitis, cervical lymphadebopathy、hepatomegaly and splenpmegaly, enlarged eyelid edema had the same diagnositicvalue as the other typical manifectious in IM. The total febrile duration and peaktemperature may be hepful for the decision of EBV-AHS.2. Impairments of hemotopoietic system and liver were more serious in EBV-AHS.SF, TG and Fg were noticeable abnormal in EBV-AHS. Therefore, SF、TG and Fgshould be examined when a child was suspected a diagnosis of EBV-AHS.3. In Our study primary EBV infection was the most common type in IM, the clinicalmanifestations of primary EBV infection were more severe than subacute EBVinfection.4. The test of qEBV was more sensitive than sera EBV antibody, so if EBVsero-antibody assays are negative, EBV infection can not be excluded and qPCRshould be perfromed.5.EBV-DNA load in the serum correlates with diesase severity in IM; quantitativeanalysis for sero-EBV-DNA may be a useful research tool in EBV infection.Therewere significant differences of EBV-DNA loads in serum of patients between IM andEBV-AHS, Monitor of EBV-DNA loads in serum maybe useful for determinations ofprogression, therapeutic response or prognosis of the primary EBV infection andEBV-AHS, and possibly for predicting the progression to EBV-AHS and evaluatingthe therapeutic response.