Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children

BACKGROUND: Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. METHODS: A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. RESULTS: The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all had positive PCR results. No sample contained PCR inhibitors. CONCLUSIONS: A real time TaqMan PCR assay allows rapid identification of patients with primary EBV infection and those with EBV infectious mononucleosis.     

传染性单核细胞增多症患儿B细胞刺激因子 mRNA表达水平研究

目的：B淋巴细胞增殖是传染性单核细胞增多症免疫变化的始发环节,该实验采用实时荧光定量PCR(RFQ-PCR) 技术检测儿童传染性单核细胞增多症患儿外周血人B细胞刺激因子（BLyS）mRNA表达水平,为临床诊断该病提供新的实验依据。方法：在BLyS基因的高保守区设计相应引物和荧光探针,实时检测PCR产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中靶基因中BLyS的水平。结果：RFQ-PCR检测靶基因mRNA含量的线性范围为109 ng/L ～101 ng/L,批内和批间重复性测定的变异系数v分别为1.88%~5.89%和6.32%~12.34%。18例传染性单核细胞增多症患儿样本的BLyS mRNA水平的表达水平均显著高于正常儿童（P<0.01）。结论：成功建立RFQ-PCR检测BLyS mRNA含量的方法,具有较好的检测灵敏度和重复性;而且发现BLyS在儿童传染性单核细胞增多症发病过程中可能起了重要作用,BLyS可以作为儿童传染性单核细胞增多症的一个参考指标。［中国当代儿科杂志,2007,9（6）：553-556］     

Serodiagnosis of infectious mononucleosis in children

Abstract Background: Although anti-viral capsid antigen (VCA)-immunoglobin M (IgM) is the most reliable serological marker of primary Epstein-Barr virus (EBV) infection, it could only be detected in limited cases of infectious mononucleosis in children. We analyzed anti-EBV antibodies by an enzyme-linked immunosorbent assay (ELISA), a sensitive method for detecting IgM antibody and compared these results with those obtained by a conventional indirect immunofluorescence (IF) method.
Methods: Anti-Epstein-Barr virus early antigen (EA)-IgM and nuclear antigen 1 (EBNAl)-IgG were examined by an ELISA assay in 180 sera from 70 infants and children with infectious mononucleosis, diagnosed serologically by standard IF methods.
Results: Although by IF, VCA-IgM was detected in only 37 of 70 (52.9%) of the sera from the acute phase of the disease, by ELISA, EA-IgM was detected in 65/70 (92.9%) of these sera. Among infants less than 12 months of age, EA-IgM was positive in 11/13 cases (84.6%) while VCA-IgM was detected in only 3/13 cases (23.1%). Anti-Epstein-Barr virus nuclear antigen 1-IgG was not detected by ELISA in the sera from the acute phase of infectious mononucleosis. Anti-EBNA was not detected by IF in about one-third of the sera during6–8 months after onset of the disease, whereas by ELISA, EBNAl-IgG was detected in 93.0%. Sera that were positive or negative for both EA-IgM and EBNAl-IgG by ELISA were observed in several cases after the patients recovered from the disease.
Conclusions: Although serodiagnosis by the combination of ELISA for EA-IgM and EBNAl-IgG was more sensitive than IF methods, especially in the case of infants and young children, several patients during convalescence and recovery might be judged as seronegative or as being in highly reactivated states.     

Infectious Mononucleosis and Mononucelosis-like Illnesses in Children and Adults in Saudi Arabia

The diagnosis of heterophil antibody positive, heterophil negative infectious mononucleosis and mononucleosis-like illnesses as made in 58 children and adult patients with clinical features suggestive of the mononucleosis syndrome and significant number of atypical lymphocytes. Epstein-Barr virus (EBV) specific serological tests revealed that six children and 23 adults had primary heterophil positive EBV infections based on the detection of IgM to EB-viral capsid antigens (IgM-VCA), presence of IgG to DB-early antigens (EA-D) and absence of antibodies to EB-nuclear antigen (EBNA). A further nine cases (five children, four in adults) of heterophil negative EBV infectious mononucleosis were likewise detected by EBV-specific serologic tests. Fourteen cases including one in children were due to active cytomegalovirus (CMV) infections as evident by positive CMV-IgM; they were all heterophil negative. Of the remaining heterophil negative cases, one was due to T. gondii with positive Toxo-IgM and five cases were of undetermined aetiology.     

Atypische Epstein-Barr-Virus(EBV)-Infektionen im Kindes- und Jugendalter

The clinical syndrome of acute infectious mononucleosis is predominantly a disease of older children and adolescents. Primary EBV infection in younger infants is often subclinical. Complications may affect any organ system and are usually mild. In the majority of cases acute infectious mononucleosis has an excellent prognosis. Severly immuncompromised children and adolescents (i. e. under immunosuppressive therapy, after stem cell transplantation) may develop EBV⁺ B-cell lymphoproliferative disorders and malignant B-cell lymphoma. In this review, mainly the following forms of atypical EBV infections are described in detail: Fulminant, mostly fatal acute infectious mononucleosis following primary EBV infection may occur 1) sporadically (approx. 1 per 3000 cases of acute infectious mononucleosis), 2) in aprox. 60% of boys with X-linked lymphoproliferative disease (XLP), and 3) in very rare cases of a fulminant EBV+ T-cell lymphoproliferative disorder. No efficient therapy exists so far. Early allogeneic stem cell transplantation in boys with XLP may prevent fatal acute infectious mononucleosis and other complications. Chronic active EBV (CAEBV) infection is characterized by recurrent clinical episodes of severe infectious mononucleosis over months or years and additional unusual clinical signs and complications such as coronary artery aneurisms, hypersensitivity to mosquito bites and hydroa vacciniforme, as well as an markedly increased risk for malignant lymphoma, mostly of a T-cell type. In general, prognosis of CAEBV infection is poor. Allogeneic stem cell transplantation may lead to clinical remission. EBV-associated hemophagocytic syndrome may occur as an independent disorder [EBV-related hemophagocytic lymphhistiocytosis (EBV-HLH)] or as a serious complication of fatal infectious mononucleosis or CAEBV infection. Early treatment with etoposide, cyclosporine A and corticosteroids may improve the otherwise poor prognosis. The pathogenesis of atypical EBV infections is not known in most cases. Further molecular and immunologic studies may help to characterize these severe disorders and to develop more specific and more efficient therapies.     

A specific IgM test for the diagnosis of Epstein-Barr virus infections (author's transl)]

IgM antibodies specific for Epstein-Barr virus (EBV) were measured in 302 patients with high IgG antibody titers to study whether EBV was the cause of disease in children having one or more symptoms of classical infectious mononucleosis. IgM antibodies specific for EBV were found in all patients with the defined clinical picture of infectious mononucleosis. In addition the majority of cases with clinical suspicion of the disease had also specific IgM titers. Besides infectious mononucleosis EBV can also be the cause of other diseases like hepatitis, and lymphadenitis: we found IgM antibodies specific for EBV in 48% of patients with nonbacterial lymphadenitis and in 64% of patients with hepatitis not due to hepatitis A or B virus. In contrast to observations by others we were able to show heterophile antibodies in cases with incomplete features of infectious mononucleosis. IgM antibodies to EBV were found in 4 out of 85 controls only. We conclude that untypical features of infectious mononucleosis can be caused by EBV also. Therefore the determination of specific IgM antibodies to EBV can be helpful in the diagnosis of uncharacteristic EBV infections.     

4例EB病毒感染相关急性肝功能衰竭患儿的临床特点分析

4例EB病毒感染相关急性肝功能衰竭患儿中男2例、女2例,年龄10（8.5~44）个月。3例诊断为传染性单核细胞增多症（IM）,其中2例符合噬血细胞淋巴组织细胞增生症（HLH）诊断标准;1例诊断为EBV既往感染。4例患儿的血EBV-DNA载量均阳性。4例患儿入院时均有发热、肝大、黄疸,3例患儿具有脾大、腹水或者呕吐症状,2例患儿有颈淋巴结大、皮疹或胸水,1例患儿出现消化道出血或者2期肝性脑病。4例患儿的异型淋巴细胞计数均 < 10%,仅1例出现白细胞升高和血小板减少;4例患儿的转氨酶10~100倍升高、以直接胆红素为主的总胆红素3~5倍升高、乳酸脱氢酶数10倍升高、凝血酶原时间显著延长。4例患儿均静脉给予阿昔洛韦或更昔洛韦抗病毒、促肝细胞生长因子促进肝细胞生长以及激素减轻炎症反应等治疗;2例加用血浆置换治疗,其中1例联合连续性静脉-静脉血液透析滤过治疗;2例HLH患儿按照HLH 2004方案化疗。3例存活,1例HLH因多脏器功能衰竭死亡。EB病毒感染可以引起儿童急性肝功能衰竭,早期给予包括血液净化治疗在内的综合治疗手段可能对预后有益。     

匹多莫德口服液辅助治疗传染性单核细胞增多症的疗效分析

目的 观察匹多莫德口服液辅助治疗传染性单核细胞增多症的疗效及对T淋巴细胞亚群的影响。方法 选取2016年7月至2017年6月收治的传染性单核细胞增多症患儿共76例为研究对象,随机分为常规治疗组和匹多莫德组各38例,常规治疗组给予注射用更昔洛韦抗病毒及常规对症治疗,匹多莫德组在此基础上加用匹多莫德口服液,疗程2周。分别比较两组临床指标的恢复情况及外周血T淋巴细胞亚群的变化。结果 匹多莫德组患儿热退时间、扁桃体炎消退时间、肿大淋巴结缩小时间、肿大肝脾缩小时间及住院时间均较常规治疗组显著缩短（P < 0.05）。匹多莫德组治疗后CD3⁺、CD8⁺水平较治疗前及常规治疗组治疗后下降（P < 0.001）;CD4⁺水平、CD4⁺/CD8⁺比值较治疗前及对照组治疗后上升（P < 0.001）。对照组在治疗2周后T淋巴细胞亚群较治疗前无明显变化（P > 0.05）。结论 匹多莫德口服液辅助治疗传染性单核细胞增多症有良好的临床疗效,并能改善细胞免疫功能,值得临床推广应用。     

重组人干扰素α1b辅助治疗传染性单核细胞增多症疗效的前瞻性随机对照研究

目的 探究重组人干扰素α1b辅助阿昔洛韦治疗对传染性单核细胞增多症（IM）患儿免疫功能、炎症因子及心肌酶谱的影响。方法 将2018年1~12月收治入院的182例IM患儿通过随机数字表法分为观察组（n=91）及对照组（n=91）。对照组接受静脉滴注阿昔洛韦治疗,观察组在对照组基础上雾化吸入重组人干扰素α1b。比较两组患儿临床症状、免疫功能、炎症反应、心肌酶谱及不良反应情况。结果 观察组体温恢复正常、咽峡炎消失、颈部淋巴结肿大消失、肝大消失、脾大消失时间均短于对照组（P < 0.05）。治疗后两组患儿CD4⁺、CD4⁺/CD8⁺、CD19⁺水平均较治疗前升高,且观察组高于对照组（P < 0.05）。治疗后两组患儿CD8⁺、肿瘤坏死因子α、白细胞介素6、肌酸激酶、肌酸激酶同工酶水平均较治疗前降低,且观察组低于对照组（P < 0.05）。治疗后两组患儿不良反应发生情况比较差异无统计学意义（P > 0.05）。结论 重组人干扰素α1b辅助阿昔洛韦治疗能有效改善IM患儿免疫功能,抑制机体炎症反应,减轻心肌损伤,进而缓解患儿临床症状。     

Splenic infarction in a child with primary Epstein–Barr virus infection

Described herein is the case of a previously healthy 7‐year‐old girl who had splenic infarction. This lesion was identified 1 day after the first presentation of peri‐umbilical and right upper quadrant pain. She had abnormal hepatic function and mild splenomegaly, and was diagnosed as having primary Epstein–Barr virus (EBV) infection. Coagulation profiles indicated low plasma activity of protein C (49%) and protein S (47%), which normalized 3 weeks later. Hypercoagulability in transient protein C and protein S deficiency might contribute to the development of splenic infarction in infectious mononucleosis.     

A controlled study of sleep related disordered breathing in obese children.

BACKGROUND: Unlike the adult sleep related disordered breathing (SDB) patients who are typically obese, the relation between obesity and childhood SDB is not clear. AIMS: To investigate whether obese children are more at risk of obstructive SDB when compared to normal population, and whether this risk is potentiated by the presence of pharyngeal lymphoid tissue. METHODS: Forty six obese children (age 10.8 (SD 2.3) years; BMI 27.4 (SD 5.1)), and 44 sex and age matched normal weight children (age 11.7 (SD 2.1) years; BMI 18 (SD 1.8)) were studied. All children underwent a set of physical examinations (including the upper airways) and sleep studies. RESULTS: The obese children were different from the normal weight children in terms of type (predominantly obstructive), frequency, and severity of respiratory disturbances. Depending on the criteria used, 26% or 32.6% of obese children had SDB; 2.3% of normal controls had OAI > or =1 and 4.5% had RDI > or =5. Presence of SDB was related to presence of tonsils (size >2; range 0-4) (OR 12.67, 95% CI 2.14 to 75.17) and BMI (OR 1.20, 95% CI 1.08 to 1.33). CONCLUSIONS: Results suggest that obese children are at increased risk of obstructive SDB; the presence of any pharyngeal lymphoid tissue enlargement in obese children should therefore be aggressively managed.     

PCR法检测140例儿童EB病毒DNA的临床分析

目的探讨应用聚合酶链反应技术(PCR)检测EBV-DNA的临床应用及140例EBV阳性病例的临床分析。方法应用PCR和荧光检测技术检测外周血EBV-DNA,并对140例阳性病例的临床特点进行回顾性分析。结果病例年龄1月～12岁,中位年龄3岁,<3岁84例,3～7岁45例,>7岁11例;140例患儿中传染性单核细胞增多症17例(12.1%),呼吸道感染20例(14.3%),另外还有原发性血小板减少性紫癜、肝炎、嗜血细胞综合征、过敏性紫癜等及隐性感染病例;结论PCR法检测EBV-DNA时间短,准确性好,灵敏度高,在EB病毒感染相关疾病诊断中很有价值。     

EB-virus-specific IgM and IgG antibodies in first-degree relatives of children with acute lymphoblastic leukaemia.

EB-virus-specific IgM and IgG antibodies (to virus capsid and soluble complement fixing antigens) were estimated in sera from mothers and sibs of children with acute lymphoblastic leukaemia, from patients with infectious mononucleosis, and from control induviduals. IgM antibodies were present in 12 of 16 mothers and 3 of 4 sibs of children with acute lymphoblastic leukaemia. They were also present in 14 of 16 patients with infectious mononucleosis, but in only 1 of 12 control individuals.     

A controlled study of sleep related disordered breathing in obese children

Background: Unlike the adult sleep related disordered breathing (SDB) patients who are typically obese, the relation between obesity and childhood SDB is not clear. Aims: To investigate whether obese children are more at risk of obstructive SDB when compared to normal population, and whether this risk is potentiated by the presence of pharyngeal lymphoid tissue. Methods: Forty six obese children (age 10.8 (SD 2.3) years; BMI 27.4 (SD 5.1)), and 44 sex and age matched normal weight children (age 11.7 (SD 2.1) years; BMI 18 (SD 1.8)) were studied. All children underwent a set of physical examinations (including the upper airways) and sleep studies. Results: The obese children were different from the normal weight children in terms of type (predominantly obstructive), frequency, and severity of respiratory disturbances. Depending on the criteria used, 26% or 32.6% of obese children had SDB; 2.3% of normal controls had OAI ⩾1 and 4.5% had RDI ⩾5. Presence of SDB was related to presence of tonsils (size >2; range 0–4) (OR 12.67, 95% CI 2.14 to 75.17) and BMI (OR 1.20, 95% CI 1.08 to 1.33). Conclusions: Results suggest that obese children are at increased risk of obstructive SDB; the presence of any pharyngeal lymphoid tissue enlargement in obese children should therefore be aggressively managed.     

Malignant B-cell lymphoma following and associated with infectious mononucleosis. A comparison of two cases

The present report describes two young males with clinically diagnosed infectious mononucleosis (IM) who subsequently were diagnosed as having malignant B-cell lymphoma (i.e., immunoblastic sarcoma of B-cells). Despite these apparent similarities, there were fundamental differences between the two cases. The first patient, who lymphoma was diagnosed 9 months after IM, was one of a well-described kindred with the X-linked lymphoproliferative syndrome (XLP) in which affected young males lack the ability to mount an effective immune response to primary infection with the Epstein-Barr virus (EBV) (i.e., infectious mononucleosis), and subsequently develop fatal lymphoproliferative disorders of the B-cell type. This was in contrast to a second patient, also a young male, who did not have the X-linked lymphoproliferative syndrome, who did develop specific antibodies to the Epstein-Barr virus and whose malignant lymphoma was closely associated in time (i.e., 5 weeks) with the clinical diagnosis of infectious mononucleosis. The comparative immunologic and virologic features are discussed as well as the importance of careful clinicopathologic correlation in young adults and children developing malignant lymphoma both following and in association with infectious mononucleosis.     

可溶性白细胞介素-2受体检测在儿童EB病毒感染相关疾病中的应用价值

目的探讨可溶性白细胞介素(白介素)-2受体检测在儿童EB病毒(EBV)感染相关疾病鉴别诊断中的应用价值。方法将72例患儿分为IM组、IM转化EBV相关的噬血细胞淋巴组织细胞增生症(EBV-HLH)组和EBV-HLH组;采用酶联免疫吸附试验(ELISA)分别检测患儿血清可溶性白介素-2受体和EBV抗体四项(EBV壳抗原VCA-IgM、VCA-IgG和EBV早期抗原EA-IgG、EBV核抗原-1 EBNA-1-IgG),荧光实时定量PCR检测患儿血浆EBV-DNA的表达,流式细胞术分析淋巴细胞亚群(CD3,CD4,CD8,CD19,CD56)。结果 72例患儿急性期可溶性白介素-2受体水平均超过2 400 U/ml;IM转化组在急性期可溶性白介素-2受体水平仅轻度增高(4 320 U/ml),与IM组(3 310 U/ml)无明显差异,治疗后却明显增高(8 970 U/ml),并接近EBV-HLH组水平(11 230U/ml);EBV抗体四项显示IM转化组和EBV-HLH组在治疗后VCA-IgG和EA-IgG仍然持续高滴度,同时EBV核抗原-1-IgG仍持续阴性;三组急性期都有EBV-DNA拷贝数从低拷贝至高拷贝的病例,治疗后IM转化组和EBV-HLH组仍可检测到EBV-DNA(3×103~4×105copies/ml);IM转化组和EBV-HLH组CD8+细胞在治疗后仍持续较高水平[(61.32±4.63)%,(68.36±4.32)%],并同时出现NK细胞(CD56+)比例下降[(9.23±3.28)%,(10.52.±3.34)%]。结论结合EBV抗体、EBV-DNA和淋巴细胞亚群检测,可溶性白介素-2受体检查有可能成为追踪观察EBV感染相关疾病的指标之一。     

Social and medical problems in children of heroin-addicted parents. A study of 75 patients

Between January 1985 and December 1987, seventy-five children of intravenous heroin-addicted parents (one or both) were studied. Their ages ranged from 4 days to 14 years. All patients had suffered from several pediatric diseases. Three major types of problems were found among the children studied: infectious diseases, nutritional diseases, and parental neglect and/or disinterest. The most common diagnoses at discharge were gastroenteritis (24%), pneumonia (21%), malnutrition (17%), upper airway infectious diseases (13%), septicemia (12%), child abuse (4%), acquired immunodeficiency syndrome (3%), and other infectious diseases (24%). Their parents reported hepatitis B virus infection, acquired immunodeficiency syndrome (AIDS), and alcoholism. The unemployment rate among the fathers was 37%. Sixteen percent of mothers were prostitutes. There was an imprisonment record of 19% for mothers and fathers combined. A multidisciplinary approach for this group of children would make prevention possible and care less expensive.     

Splenic infarction in a patient hereditary spherocytosis,protein C deficiency and acute infectious mononucleosis

Splenic infarction is a common cause of left upper quadrant pain and must be suspected in patients with hematologic or thromboembolic conditions and signs of localized or systemic inflammation. Although several mechanisms have been proposed for splenic infarction in patients with various hematologic disorders, hereditary spherocytosis (HS) is usually not associated with an increased risk for thromboembolic events. We report a 13-year-old male with HS who was referred to our hospital with a 4-day history of fever and left upper quadrant pain. Ultrasound scans and magnetic resonance imaging showed lesions suggestive of splenic infarction. Initially, antibiotic treatment was started because secondary infection was suspected. However, 1 week after admission the patient developed typical clinical signs of acute infectious mononucleosis. Further laboratory work up confirmed the diagnosis of acute Epstein–Barr virus infection and additionally revealed protein C deficiency. This association has not been reported previously and may have contributed to the development of splenic infarction. Since infectious mononucleosis is a common cause for clinical consultations in adolescence, physicians caring for children with hematologic disorders should be particularly aware of those possible complications. An erratum to this article can be found at     

Changes in lipid profile observed in children over the course of infectious disease

Lipid profile was evaluated prospectively in 23 consecutive children, aged 3.2–14.9 years, admitted to the hospital with a febrile illness (pneumonia, upper respiratory tract infection, diarrhea, pyelonephritis, mononucleosis, appendicitis). The degree of dyslipidemia associated with fever was assessed using each child as his/her own control and by comparison with 93 non-febrile children who had no evidence of fever during the past six months. Total cholesterol decreased during the symptomatic phase of the disease. The magnitude and duration of its decrease appeared to be related to the degree and duration of fever. Low HDL-cholesterol and hypertriglyceridemia were observed during the late stage of the febrile disease and were still detected in the convalescent phase. This study suggests that in children, transient and sometimes prolonged lipid changes may occur in association with an infectious febrile disease. This effect is important for defining the appropriate timing for screening for dyslipidemias.