本文選題：兒童 + EB病毒��； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：研究背景： EB病毒（Epstein-Barr，EBV）為雙鏈DNA病毒，是一種嗜人B淋巴細(xì)胞病毒，屬于皰疹病毒γ亞科家族中的一員。該病毒在人群中普遍易感，國(guó)外報(bào)道，成人血清EBV抗體陽(yáng)性率達(dá)95%以上~[1]，我國(guó)3~5歲兒童EBV抗體陽(yáng)性率達(dá)80.7~100%，10歲時(shí)100%的兒童血清EBV抗體陽(yáng)性轉(zhuǎn)化~[2]。EBV是兒童感染性疾病常見(jiàn)的病原體，感染時(shí)癥狀輕重不一，部分僅表現(xiàn)無(wú)癥狀的血清學(xué)轉(zhuǎn)換，其中50%發(fā)展為傳染性單核細(xì)胞增多癥（Infection mononucleosis，IM）~[2]。IM被認(rèn)為是一種免疫病理性疾�。↖mmunopathological disease）[13]，EBV原發(fā)感染后引起大量CD8~+T淋巴細(xì)胞增殖，一方面殺傷EBV感染的B細(xì)胞，一方面侵犯淋巴組織器官、釋放炎癥因子，主要是Th1型細(xì)胞因子，引起一系列的臨床癥狀。嬰幼兒免疫發(fā)育尚不成熟，因此大部分感染EBV后表現(xiàn)為血清學(xué)轉(zhuǎn)化或僅表現(xiàn)為輕癥或不典型病變。IM是自限性疾病，一般預(yù)后良好，病死率僅為1～2%[14]。在少數(shù)患兒可發(fā)生多個(gè)系統(tǒng)并發(fā)癥，發(fā)展為重癥IM。在極少數(shù)個(gè)體可發(fā)生EBV相關(guān)性噬血細(xì)胞淋巴組織細(xì)胞增生癥（Epstein-Barr virus-associatedhemophagocytic lymphphistiocytosis，EBV-AHS）本病在臨床上呈暴發(fā)性，如不及時(shí)診斷和治療，患者迅速死亡，病死率可達(dá)30～40%~[15]。 噬血細(xì)胞性淋巴組織細(xì)胞增生癥（Hemophagocytic lymphohistiocytosis，HLH）是多種原因造成自然殺傷細(xì)胞（natural killer cell，NK）和T細(xì)胞功能缺陷，引起大量炎癥細(xì)胞因子釋放，造成機(jī)體巨噬細(xì)胞活化，機(jī)體臟器、組織損傷的一組臨床綜合征。其臨床表現(xiàn)具有特征性但缺乏特異性。主要臨床表現(xiàn)為持續(xù)發(fā)熱，肝、脾大，全血細(xì)胞減少，骨髓、淋巴結(jié)等組織可見(jiàn)吞噬血細(xì)胞現(xiàn)象。多種原因可引起HLH：遺傳因素、感染、腫瘤、自身免疫系統(tǒng)疾病。EBV-AHS是感染所致HLH中的常見(jiàn)類型。無(wú)論是先天性還是后天性HLH，EBV都是最常見(jiàn)的觸發(fā)因素。不同病因所致HLH預(yù)后不同，非EBV感染所致HLH，60-70%隨原發(fā)病的治愈而緩解，而未經(jīng)治療的EBV-AHS常常是致命的[46]，特別是EBV-DNA定量分析持續(xù)增高者。目前對(duì)于極少數(shù)EBV感染后發(fā)展為EBV-AHS的患兒特別是無(wú)明確基因缺陷者，其體內(nèi)病毒與機(jī)體免疫力相互作用的研究尚不十分清楚。 EBV原發(fā)感染后產(chǎn)生病毒特異的體液免疫反應(yīng)和細(xì)胞免疫反應(yīng)，通過(guò)檢測(cè)血清中EBV特異性抗體滴度可以反映病毒感染后機(jī)體的體液免疫反應(yīng)情況。間接免疫熒光法（Indirect immunofluorescence assay，IFA）是檢測(cè)EBV特異性抗體的經(jīng)典方法，但該方法耗時(shí)耗力且敏感性低于酶聯(lián)免疫吸附試驗(yàn)（Enzyme-linked immunosorbent assay，ELISA），ELISA具有高效、快速、敏感性高等特點(diǎn)，現(xiàn)已廣泛用于EBV感染血清學(xué)檢查。實(shí)時(shí)熒光定量PCR技術(shù)（Real-time quantitative Polymerase Chain Reaction，Real-time PCR）是在定性PCR技術(shù)基礎(chǔ)上發(fā)展起來(lái)的核酸定量檢測(cè)技術(shù)，是目前確定樣本中DNA拷貝數(shù)最敏感、最準(zhǔn)確的方法，現(xiàn)已替代了其它定量PCR的方法廣泛用于測(cè)定EBV負(fù)載量。通過(guò)FQ-PCR檢測(cè)患者外周血中EBV-DNA拷貝數(shù)，能夠很好的反映體內(nèi)病毒的負(fù)荷量。因此我們采用ELISA和FQ-PCR的方法檢測(cè)EBV感染患兒外周血抗體譜表現(xiàn)和EBV-DNA負(fù)載量，分析普通EBV感染和EBV-AHS患兒機(jī)體免疫反應(yīng)和機(jī)體內(nèi)病毒活動(dòng)的特點(diǎn)。 目的： 了解EB病毒感染所致IM和EBV-AHS的臨床特點(diǎn)以及血清學(xué)EBV抗體譜和外周血病毒負(fù)載量的特點(diǎn)。 方法： 1EBV感染患兒臨床特點(diǎn)和實(shí)驗(yàn)室檢查分析 將EBV感染患兒分為IM組和EBV-AHS組，分析兩組病例的臨床特點(diǎn)和實(shí)驗(yàn)室檢查特點(diǎn)。 2EBV特異性抗體檢測(cè) 2.1標(biāo)本收集：所有患兒標(biāo)本在入院時(shí)采集，用促凝管收集外周靜脈血2ml。 2.2分離血清：收集的血標(biāo)本離心，2500rpm/min×15min，吸取上層血清，將血清等分裝于ep管中，放于-80℃冰箱保存。 2.3檢測(cè)血清中EBV抗體：待收集一定數(shù)量標(biāo)本后用ELISA法檢測(cè)患兒血清中EBV四項(xiàng)抗體，包括：抗EBV衣殼抗原（capsid antigen，CA）IgM（EBV-CA-IgM）/IgG（EBV-CA-IgG）抗體、抗EBV早期抗原（early antigen，EA）IgG(EBV-EA-IgG)抗體、抗EBV核抗原（nucler antigen，NA）IgG(EBV-NA-IgG)抗體。 2.4根據(jù)抗體檢測(cè)結(jié)果將EBV感染分為原發(fā)感染組和亞急性感染組，比較兩組的臨床特點(diǎn)和實(shí)驗(yàn)室檢查。 3EBV-DNA拷貝數(shù)檢測(cè) 3.1標(biāo)本收集：所有患兒標(biāo)本在入院時(shí)采集，用促凝管收集外周靜脈血2ml。 3.2分離血清：收集的血標(biāo)本離心，2500rpm/min×15min，吸取上層血清，將血清等分裝于ep管中，儲(chǔ)存于-80℃冰箱。 3.3提取DNA：使用QIAampDNA Mini kit（QIAGEN）提取血清中DNA，將提取的DNA等分儲(chǔ)存于-80℃冰箱。 3.4檢測(cè)EBV-DNA負(fù)載量：待收集一定數(shù)量標(biāo)本后用FQ-PCR檢測(cè)血清中EBV-DNA負(fù)載量。 4統(tǒng)計(jì)學(xué)方法 采用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)分析。計(jì)量資料用Kolmogorov-Smirnov正態(tài)性檢驗(yàn)，正態(tài)分布資料用均數(shù)±標(biāo)準(zhǔn)差（(?)±s）表示，采用兩獨(dú)立樣本的t檢驗(yàn)（Independent-samples T text）；偏態(tài)分布資料用中位數(shù)（范圍）表示，采用Mann-Whitney秩和檢驗(yàn)。計(jì)數(shù)資料以構(gòu)成比（%）表示，采用兩獨(dú)立樣本率的卡方檢驗(yàn)；兩變量間的相關(guān)分析用Peason相關(guān)分析，，當(dāng)P＜0.05時(shí)，認(rèn)為有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.本研究中IM組患兒男女比例1.8：1；發(fā)病年齡13個(gè)月～13歲，平均4.6歲；其中以學(xué)齡前兒童多見(jiàn)（60.5%）。 2.本研究IM發(fā)熱、咽峽炎發(fā)生率（97.7%），頸部淋巴結(jié)腫大（95.3%），眼瞼浮腫（48.8%），肝腫大（65.1%），脾腫大（46.5%），皮疹（20.9%），熱程中位數(shù)為5d，熱峰39.2℃，眼瞼浮腫發(fā)生率高于脾腫大和皮疹。EBV-HLH熱程中位數(shù)為27d，熱峰40.4℃，EBV-AHS熱程顯著長(zhǎng)于IM組（P=0.002），熱峰顯著高于IM組（P=0.001）。 3.EBV-AHS臨床特點(diǎn)為持續(xù)高熱、肝脾腫大，實(shí)驗(yàn)室檢查主要特點(diǎn)：白細(xì)胞計(jì)數(shù)、中性粒細(xì)胞計(jì)數(shù)、血紅蛋白、血小板計(jì)數(shù)減低，肝功能異常，以酶學(xué)改變?yōu)橹�，尤以LDH升高明顯（1690±759U/L），甘油三脂（TG）升高、血清鐵蛋白（SF）明顯升高（均＞2000ug/L），纖維蛋白原（Fb）均降低。 4.原發(fā)感染（抗VCA-IgM陽(yáng)性，抗EBNA-IgG陰性，抗VCA-IgG、EA-IgG陽(yáng)性或陰性）是IM抗體譜的主要類型（65.1%），入院時(shí)抗EBV-CA-IgM陽(yáng)性率（97.6%），其中1例患兒入院時(shí)抗EBV-CA-IgM陰性1周后復(fù)查轉(zhuǎn)陽(yáng)性。5例EBV-AHS患兒抗體譜均表現(xiàn)為既往感染。 5.比較EBV原發(fā)感染和亞急性感染臨床表現(xiàn)和主要實(shí)驗(yàn)室數(shù)據(jù)顯示，EBV原發(fā)感染眼瞼浮腫率（P=0.033）和肝臟腫大率(P=0.011)顯著高于EBV亞急性感染組；LDH前者較后者明顯升高（P=0.005）。比較兩組合并肝功能損害發(fā)生率顯示：EBV原發(fā)感染顯著高于亞急性感染（P=0.005）。 6.IM組入院時(shí)均行血清EBV-DNA負(fù)載量檢測(cè)，其中陽(yáng)性者24例（56%），均數(shù)為5.88×10~4copies/ml，最大值2.88×10~5copies/ml，最小值8.24×10~3copies/ml。其中有10例于住院治療7天后復(fù)查血清中EBV-DNA負(fù)載量，結(jié)果均轉(zhuǎn)陰。5例EBV-AHS血清中EBV-DNA拷貝數(shù)均陽(yáng)性，均數(shù)為1.86×10~8copies/ml，最大值9.25×10~8copies/ml，最小值2.88×10~5copies/ml。EBV-AHS組中EBV-DNA負(fù)載量顯著高于IM組（P=0.001）。 7.IM組根據(jù)血清EBV-DNA檢測(cè)結(jié)果分為EBV-DNA定量陽(yáng)性組和陰性組，比較入院時(shí)主要實(shí)驗(yàn)室數(shù)據(jù)，結(jié)果顯示EBV-DNA陽(yáng)性組肝功明顯異常，以酶學(xué)改變?yōu)橹鳎ˋLT、AST、LDH），ALT、LDH較EBV-DNA陰性組相比明顯升高，差異有統(tǒng)計(jì)學(xué)意義（ALT:P=0.013；LDH:P=0.003）。EBV-DNA負(fù)載量與AST(r=0.567， P=0.004)、LDH(r=0.885，P=0.004)呈正相關(guān)。 結(jié)論： 1．本研究中IM以男性多見(jiàn)，好發(fā)于學(xué)齡前兒童。發(fā)熱、咽峽炎、頸部淋巴結(jié)腫大、肝脾腫大是IM常見(jiàn)臨床表現(xiàn)，眼瞼浮腫與其它典型臨床表現(xiàn)一樣對(duì)IM具有診斷價(jià)值。熱程、熱峰有助于判斷IM是否發(fā)生EBV-AHS。 2．兒童EBV-AHS血液系統(tǒng)和肝臟損害嚴(yán)重，酶學(xué)中以LDH升高更為顯著。血清鐵蛋白、甘油三脂和纖維蛋白原在EBV-AHS的早期階段即有改變，一旦臨床上懷疑EBV-AHS應(yīng)盡快完善以上檢查，便于早期診斷和治療。 3．本研究中IM入院診斷時(shí)血清抗體譜以原發(fā)感染為主（65.1%），EBV原發(fā)感染較EBV亞急性感染臨床癥狀更典型，病情更重。臨床懷疑IM入院時(shí)抗VCA-IgM陰性患兒應(yīng)予以復(fù)查，可提高陽(yáng)性率。 4．血清抗體檢測(cè)在診斷EBV-AHS的敏感性不及FQ-PCR，在診斷EBV-AHS時(shí)血清EBV抗體陰性是不能除外EBV感染，需完善外周血EBV-DNA熒光定量檢測(cè)以明確。 5．IM血清中EBV-DNA負(fù)載量與臨床嚴(yán)重程度呈正相關(guān)；檢測(cè)外周血EBV-DNA拷貝數(shù)能更好反映體內(nèi)EBV的活動(dòng)情況。EBV-AHS血清中EBV-DNA負(fù)載量顯著高于IM患兒，對(duì)于EBV感染特別是血清EBV-DNA呈高拷貝數(shù)的患兒，應(yīng)動(dòng)態(tài)觀察外周血中EBV-DNA負(fù)載量變化，以了解病情的變化和治療效果。
[Abstract]:Research background:
EB virus (Epstein-Barr, EBV) is a double stranded DNA virus. It is a human B lymphocyte virus and belongs to the family of herpes virus gamma subfamily. The virus is common in the population. The positive rate of serum EBV antibody in adult serum is above 95% ~[1]. The positive rate of EBV antibody in 3~5 years old children in our country is 80.7~100%, and the serum EBV of 100% children at the age of 10 Antibody positive transformation ~[2].EBV is a common pathogen of infective diseases in children. The severity of the infection is different, and some of them only show asymptomatic serological conversion. 50% of them are infectious mononucleosis (Infection mononucleosis, IM) ~[2].IM is considered as an immune pathological disease (Immunopathological disease) [13], EBV After primary infection, a large number of CD8~+T lymphocyte proliferation, on the one hand, EBV infected B cells, on the one hand invasion of lymphoid tissues and organs, the release of inflammatory factors, mainly Th1 type cytokines, causing a series of clinical symptoms. Infant immune development is not mature, so a large portion of the infection after EBV is serological transformation or only manifested as Light or atypical.IM is a self limiting disease with a good prognosis. The mortality rate is only 1 ~ 2%[14]. in a few children, and there are many systemic complications in a few children. The development of severe IM. can occur in a very few individuals with EBV related hemophagocytic lymphohistiocytosis (Epstein-Barr virus-associatedhemophagocytic lymphphistiocytosis, E). BV-AHS) the disease is fulminant in clinic. If it is not diagnosed and treated in time, the patient will die quickly. The fatality rate can reach 30 to 40%~[15]..
Hemophagocytic lymphohistiocytosis (Hemophagocytic lymphohistiocytosis, HLH) is a group of clinical syndromes that cause a variety of reasons for the functional defects of natural killer cells (natural killer cell, NK) and T cells, causing a large number of inflammatory cytokines to release, resulting in the activation of macrophages, organs and tissue damage in the body. Its clinical table It is characterized by lack of specificity. The main clinical manifestations are persistent fever, liver, splenomegaly, total hemocytosis, bone marrow, lymph nodes and other tissues. A variety of causes can cause HLH: genetic factors, infection, tumor, and autoimmune disease.EBV-AHS are common types of HLH caused by infection. It is acquired HLH and EBV are the most common triggers. Different causes of HLH have different prognosis, HLH and 60-70% are cured with non EBV infection, and untreated EBV-AHS is often fatal [46], especially in EBV-DNA quantitative analysis. There is no clear genetic defect, and the interaction between virus and immunity is not clear.
After the primary infection of EBV, the virus specific humoral immune response and cell immune response are produced. The detection of EBV specific antibody titer in serum can reflect the humoral immune response of the body after the virus infection. The indirect immunofluorescence (Indirect immunofluorescence assay, IFA) is the classic method for detecting specific antibodies of EBV, but this prescription The method is time-consuming and energy consuming and is less sensitive than Enzyme-linked immunosorbent assay (ELISA). ELISA has the characteristics of high efficiency, rapid and high sensitivity. It has been widely used in the serological examination of EBV infection. Real time fluorescent quantitative PCR (Real-time quantitative Polymerase Chain Reaction) is a qualitative technique. The nucleic acid quantitative detection technology developed on the basis of operation is the most sensitive and accurate method to determine the number of DNA copies in the sample, and has now replaced the other quantitative PCR method for measuring the load of EBV. The detection of EBV-DNA copies in the peripheral blood of patients by FQ-PCR can reflect the load of the virus in the body very well. Therefore, we can well reflect the load of the virus in the body. The ELISA and FQ-PCR methods were used to detect the peripheral blood antibody spectrum and the amount of EBV-DNA load in children with EBV infection. The characteristics of the immune response and the virus activity in the body of children with common EBV infection and EBV-AHS were analyzed.
Objective:
To understand the clinical characteristics of IM and EBV-AHS caused by EB virus infection and the characteristics of serological EBV antibody spectrum and the amount of viral load in peripheral blood.
Method錛

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