Performance of an antigen assay for diagnosing acute hepatitis E virus genotype 3 infection

BackgroundHepatitis E virus (HEV) is a major cause of hepatitis worldwide. Its diagnosis is based on the detection of anti-HEV IgM and/or HEV-RNA.ObjectiveTo evaluate the performance of the Wantaï HEV-antigen (Ag) ELISA^Plus assay for diagnosing acute HEV infections.Study designSpecificity was assessed using 100 blood samples containing no anti-HEV IgM, anti-HEV IgG, or HEV-RNA. Cross reactivity was assessed using samples positive for hepatitis C virus RNA (n = 10), Epstein-Barr virus DNA (n = 10) and cytomegalovirus DNA (n = 10). Serial dilutions of 4 HEV RNA positive samples were used to estimate the corresponding viremia detected with the Ag assay. Blood samples from 33 immunocompetent and 31 immunocompromised patients with an acute HEV genotype 3 infection, HEV-RNA positive, were tested to assess diagnostic sensitivity.ResultsThe HEV-Ag assay was 100% specific, with no cross-reactivity. The lower viremias detected ranged from 10³ copies/ml to 10⁵ copies/ml (800–80,000 UI/ml). Diagnostic sensitivity for an acute HEV infection was 91%, with no significant difference between immunocompetent (88%) and immunocompromised (94%) patients. The HEV-Ag assay was more frequently positive in immunocompromised patients at the acute phase (94%) than was the anti-HEV IgM test (71%; p = 0.04). The HEV-Ag assay ratio was correlated with HEV-RNA viral load (ρ = 0.54; p < 0.0001).ConclusionThe HEV-Ag assay performed well and could be suitable for laboratories with no molecular diagnosic facilities.     

Differences in capabilities of different enzyme immunoassays to detect anti-hepatitis E virus immunoglobulin G in pigs infected experimentally with hepatitis E virus genotype 3 or 4 and in pigs with unknown exposure

Hepatitis E virus (HEV), a major cause of acute viral hepatitis in humans in many developing countries, is highly prevalent in the pig population worldwide. The objective of this study was to assess the capability of three porcine prototypes of a human enzyme-linked immunosorbent assay (ELISA), an in-house ELISA and a line-immunoassay (LIA) to detect anti-HEV antibodies in pigs infected experimentally with HEV (n = 57), known to be negative for HEV infection (n = 27), or with unknown exposure to HEV infection (field samples, n = 90). All 27 samples from non-infected pigs were negative with all five assays. The earliest detection of anti-HEV antibodies occurred at 14 days post-inoculation (dpi) with four of five assays. From 42 dpi, all samples from infected pigs were detected correctly as anti-HEV positive. Kappa analysis demonstrated substantial agreement among tests (0.62-1.00) at 14 dpi and complete agreement (1.00) at 56 dpi. The overall area under the curve for all quantitative tests as determined by receiver operator characteristic analysis ranged from 0.794 to 0.831 indicating moderate accuracy. The results showed that all five assays can detect anti-HEV IgG antibodies accurately in pigs infected experimentally with HEV. In field samples, a higher prevalence of anti-HEV IgG was found in breeding herds than in growing pigs (100% versus 66.7-93.9%). These serological assays should be very useful in veterinary diagnostic labs for HEV diagnosis in swine.     

一株新型戊型肝炎病毒的部分克隆及分析

目的　对得自北京地区一例急性散发性戊型肝炎病人病毒 (ChinaT)作基因克隆分析。方法　用逆转录套式聚合酶链反应方法从患者粪便中克隆病毒并做核苷酸序列分析。结果　ChinaT株与 4株典型中国株HEV(ChinaA、ChinaB、ChinaC、ChinaD)、缅甸株、墨西哥株、美国株、非洲的乍得株核苷酸 /氨基酸同源性分别为 78% /92 %、78% /92 %、79% /90 %、78% /94%、76 % /92 %。结论　ChinaT株有较高的基因异质性 ,与其它HEV株有很大的不同 ,是一新型HEV。     

Coinfection of hepatitis A virus genotype IA and IIIA complicated with autoimmune hemolytic anemia,prolonged cholestasis,and false-positive immunoglobulin M anti-hepatitis E virus: a case report

A 37-year-old male presented with fever and jaundice was diagnosed as hepatitis A complicated with progressive cholestasis and severe autoimmune hemolytic anemia. He was treated with high-dose prednisolone (1.5 mg/kg), and eventually recovered. His initial serum contained genotype IA hepatitis A virus (HAV), which was subsequently replaced by genotype IIIA HAV. Moreover, at the time of development of hemolytic anemia, he became positive for immunoglobulin M (IgM) anti-hepatitis E virus (HEV). We detected HAV antigens in the liver biopsy specimen, while we detected neither HEV antigen in the liver nor HEV RNA in his serum. This is the first report of hepatitis A coinfected with two different genotypes manifesting with autoimmune hemolytic anemia, prolonged cholestasis, and false-positive IgM anti-HEV.     

Lack of Marseillevirus DNA in immunocompetent and immunocompromised Italian patients

Marseilleviridae is a family of viruses which have only been propagated in acanthamoeba. Marseillevirus sequences have been recently detected in different human matrices by viral metagenomics. Single-center studies worldwide have estimated a low prevalence of marseillevirus both in symptomatic patients and in healthy donors but, to date, no informations are available on the prevalence of this giant virus in Italy. By a polymerase chain reaction targeting the ORF152 viral sequence, we tested sera from 197 immunosuppressed patients and 285 healthy donors, and 63 and 30 respiratory and cerebrospinal fluid samples, respectively, of patients with various clinical conditions and referring the Virology Division for diagnostic purposes. We observed no evidence of Marseillevirus DNA in all 575 samples tested. Marseillevirus probably does not cause infection in human.     

Human cytomegalovirus (HCMV)-specific immunoglobulin E as a serologic marker for HCMV infection in immunocompromised patients

Summary An antibody capture assay using an enzyme-linked human cytomegalovirus (HCMV) antigen for the detection of specific immunoglobulin E (IgE) was established. IgG, M, and E responses to HCMV were studied in 497 sera obtained from 44 renal transplant recipients and 51 acquired immunodeficiency syndrome (AIDS) patients. The results were compared with those obtained from 58 HCMV-seropositive healthy individuals. HCMV-specific IgE was detected in 11 (91.7%) renal transplant recipients with primary HCMV infection. In contrast, antibodies of the IgG and IgM classes were detected in only 6 (50.0%) of these patients. Specific IgE was detected in 10 (90.9%) out of 11 renal allograft recipients suffering from secondary HCMV infection. Significant IgG titer rises and IgM were detected in 2 (18.2%) and 6 (54.6%) of these patients, respectively. IgG titer rises and IgM and IgE antibodies were seen in 5 (12.2%), 1 (2.4%) and 18 (43.9%) AIDS patients respectively. All healthy immunocompetent HCMV-seropositive individuals were tested IgE negative. The results obtained in our study indicate that IgE against HCMV is a more reliable serologic marker for primary and secondary HCMV infection than IgM in immunocompromised individuals, especially in organ transplant recipients, since it is not affected by the prophylactic application of HCMV hyperimmune globulin preparations.Abbreviations AIDS acquired immunodeficiency syndrome - BAL bronchoalveolar lavage - CDC Centers for Disease Control, Atlanta, USA - ELISA enzyme-linked immunosorbent assay - HCMV human cytomegalovirus - HIV human immunodeficiency virus - Ig immunoglobulin - PBS phosphate buffered saline - RTR renal transplant recipients     

戊型肝炎病毒结构区基因的克隆表达及其在诊断中的初步应用

目的研制戊型肝炎病毒诊断试剂。方法利用融合蛋白表达载体ｐＧＥＸ－３Ｘ表达了戊型肝炎病毒第二读码框区（ＯＲＦ２４０２～６６０）优势抗原表位。表达抗原溶于水，可利用商品化的谷胱甘肽Ｓｅｐｈａｒｏｓｅ－４Ｂ亲合层析柱得到纯化的抗原。结果经应用发现，此基因重组抗原作为酶联免疫试剂的抗原，用于检测血清中抗戊型肝炎病毒抗体，与新加坡进口试剂盒有高度的一致性，和血清中抗体反应性更强。结论预示该基因抗原可能具有较好的反应谱，应用前景乐观     

Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay

Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [³H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher (P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.     

亚洲戊型肝炎病毒结构蛋白的变异

目的了解戊型肝炎病毒的变异。方法用双脱氧法对亚洲主要戊型肝炎流行国家的戊型肝炎病毒结构基因区段进行了分析，并根据ｃＤＮＡ序列确定了其氨基酸的序列。结果证明亚洲国家包括印度、缅甸、中国、巴基斯坦、吉尔吉斯坦流行的戊型肝炎病毒在基因水平和氨基酸水平上均有变化，核苷酸变化幅度在５％以内。氨基酸在测定的区域内未发现变异，与美洲发现的墨西哥株相比，亚洲株拟来自同一个祖先，基因和氨基酸水平的变化仅属基因漂移的范围。结论以亚洲戊型肝炎病毒基因为基础的疫苗将有广泛的保护作用     

Hepatitis E virus infection in developed countries

Hepatitis E was considered to be endemic infectious disease in developing countries in tropical or subtropical regions with poor sanitary conditions. Large, previously reported outbreaks were mainly due to contaminated water or heavy flooding. Prototype hepatitis E viruses of genotypes I and II were obtained from such endemic cases. In developed countries, in contrast, hepatitis E was rare and diagnosed only in travelers or imported cases. However, the development of accurate diagnostic tests, mainly PCR detection elucidated that autochthonous hepatitis E in developed countries is far more common than previously thought. Although the main route of transmission is food-borne, other routes including blood-borne have been suggested. Recent developments of gene-based diagnostic assays and molecular epidemiology have disclosed the significance of hepatitis E virus infection in developed countries.     

Human cytomegalovirus-specific CD4 and CD8 T cell responses in primary infection of the immunocompetent and the immunocompromised host

The kinetics of primary human cytomegalovirus (HCMV) infection and specific T-cell responses were investigated in 16 immunocompetent pregnant women and 8 solid-organ transplant recipients (SOTR). T-cell responses to whole HCMV and to pp65 and IE-1 peptides were determined by flow cytometry evaluation of IFNγ production. HCMV-specific CD4⁺ and CD8⁺ T-cells appeared earlier and simultaneously in immunocompetent subjects, whereas specific CD8⁺ T-cells preceded CD4⁺ T-cells in half of the SOTR examined. The magnitude of the HCMV-specific T-cell pool was comparable. HCMV load reached peak levels 100–1000 times higher in SOTR than in immunocompetent women, while the virus persisted for months in blood of both groups. T-cells directed to pp65 and IE-1 were only detected in a portion of subjects developing a full T-cell response to the whole virus. Thus, the development of cell-mediated immune response in primary HCMV infection may be missed when looking at pp65 and IE-1 peptide-stimulated T-cells only.     

Serological diagnostics of hepatitis E virus infection

Development of accurate diagnostic assays for the detection of serological markers of hepatitis E virus (HEV) infection remains challenging. In the course of nearly 20 years after the discovery of HEV, significant progress has been made in characterizing the antigenic structure of HEV proteins, engineering highly immunoreactive diagnostic antigens, and devising efficient serological assays. However, many outstanding issues related to sensitivity and specificity of these assays in clinical and epidemiological settings remain to be resolved. Complexity of antigenic composition, viral genetic heterogeneity and varying epidemiological patterns of hepatitis E in different parts of the world present challenges to the refinement of HEV serological diagnostic assays. Development of antigens specially designed for the identification of serological markers specific to acute infection and of IgG anti-HEV specific to the convalescent phase of infection would greatly facilitate accurate identification of active, recent and past HEV infections.     

Prolonged jaundice attributed to super infection of hepatitis E virus in a case of resolving leptospirosis

India is endemic for both Leptospira and hepatitis E virus (HEV). The clinical presentations of these diseases have overlapping features. We report a case of superinfection of HEV in a patient with resolving leptospirosis with underlying Hodgkin lymphoma. The diagnosis of HEV in our case was established by HEV-RNA PCR as our patient was immunosuppressed. The present study highlights the need for molecular diagnosis in the case of HEV infection with strong clinical suspicion and negative serological results.     

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genusHepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs inWestern Europe and in North and South America and cause zoonotic infections inhumans. Several serological assays to detect HEV antibodies in pigs have beendeveloped, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop asensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 openreading frame-2 was produced and coated onto polystyrene ELISA plates. Afterincubation of porcine sera, bound HEV antibodies were detected with anti-porcineanti-IgG and anti-IgM conjugates. For primary estimation of sensitivity andspecificity of the assay, sets of sera were used from pigs experimentally infectedwith HEV Gt3. For further validation of the assay and to set the cutoff value, abatch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3assay and two other serologic assays based on HEV Gt1 antigens. Since there is nogold standard available for HEV antibody testing, further validation and a definitesetting of the cutoff of the developed HEV Gt3 assay were performed using astatistical approach based on Bayes'' theorem. The developed and validated HEVantibody assay showed effective detection of HEV-specific antibodies. This assay cancontribute to an improved detection of HEV antibodies and enable more reliableestimates of the prevalence of HEV Gt3 in swine in different regions.     

Human cytomegalovirus-specific CD4 and CD8 T-cell response determination: Comparison of short-term (24 h) assays vs long-term (7-day) infected dendritic cell assay in the immunocompetent and the immunocompromised host

Human cytomegalovirus (HCMV)-specific CD4⁺ and CD8⁺ T-cells were measured in the immunocompetent host as well as in 13 solid-organ transplant recipients (SOTR), and 12 young hematopoietic stem cell transplant recipients (HSCTR) by using a long-term (7-day) assay based on PBMC stimulation by HCMV-infected dendritic cells (iDC), and two short-term (24 h) assays, one for CD4⁺ stimulation by infected cell lysate (iCL), and the other for CD8⁺ stimulation by a pool of 34 epitopic peptides (pep-pool). In the immunocompetent, the number of T-cells activated by either iCL or the pep-pool was significantly reduced with respect to iDC. In both SOTR and HSCTR, the number of T-cells activated by iDC was comparable to that activated by iCL or the pep-pool. A significant correlation between iDC-activated T-cells and T-cells activated by either iCL or the pep-pool was observed. In conclusion, whenever a rapid result is needed, short-term assays may efficiently replace the iDC assay.     

散发性戊型肝炎病毒部分核苷酸序列分析

目的为阐明戊型肝炎病毒（ＨＥＶ）毒株的地理分布、流行特征以及研制血清学诊断试剂和基因工程疫苗提供资料。方法选择１例浙江仙居山区散发性ＨＥ患者，从其血清中分离ＨＥＶＲＮＡ，通过逆转录－套式聚合酶链反应法（ＲＴ－ｎＰＣＲ），扩增该散发性ＨＥＶ（Ｚ－３３株）ＯＲＦ２区部分ｃＤＮＡ片段（４９７ｂｐ），然后进行直接序列分析，并与ＨＥＶ各主要代表株序列作比较。结果Ｚ－３３株与新疆流行株ＣＨ１．１的核苷酸及氨基酸序列同源性分别为９６．７％及９８．２％；与缅甸流行株的同源性分别为９４．２％及９９．１％；与缅甸散发株的同源性分别为９５．２％及９９．１％，与墨西哥株的同源性分别为８１．８％及９４．５％。结论浙江仙居散发性ＨＥＶＺ－３３株和新疆流行株ＣＨ１．１一样，与ＨＥＶ缅甸株可能为同一亚型。     

Identifications of polyphyletic variants in acute hepatitis suggest an underdiagnosed circulation of hepatitis E virus in Argentina

Background

In recent years, an increasing number of infections with genotype 3 hepatitis E virus (HEV) have been reported in western countries. Data in South America, however, are still scarce. Swine and human variants previously described in Argentina are closely related to a human Austrian one.

Objective

To identify whether HEV is still circulating in Argentina.

Study design

Sera and stool samples from adults and children with unexplained acute liver disease referred to our center during the last six years were prospectively studied. Dual infection with hepatitis A was retrospectively studied in a group of children with fulminant hepatic failure.

Results

Fifteen new cases (13 adults and 2 children), seven of whom required hospitalization, were diagnosed. Nine had detectable HEV RNA, and one had imported genotype 1. Subgenotype 3i HEV-related variants are still circulating. Five autochthonous sequences, related to European, American and Japanese ones, grouped in subgenotype 3a. One case had a subgenotype 3b variant.

Discussion

The polyphyletic variants widespread in Argentina suggest multiple sources of infection. Whether or not their reservoir is swine merits further investigation. Since hepatitis E is still considered rare, differential laboratory testing in unexplained acute liver disease is not routinely performed in Argentina. Broadening awareness of this disease is important in light of the decrease in hepatitis A incidence since universal vaccination was implemented in 2005. The diagnosis of hepatitis E with a combination of serological and molecular tools is needed to better understand its epidemiology and impact on the clinical management of patients with unexplained increased transaminases.     

我国汉、回和藏族部分人群HEV感染的血清学调查

目的了解我国不同民族的健康人群戊型肝炎病毒感染情况。方法采用ELISA方法检测人群血清中戊型肝炎病毒（戊肝,HEV）IgG抗体。汉族人群血清分别来自于四川、北京、黑龙江和山东,回族和藏族人群血清来自于甘肃、宁夏和青海,总共10448份血清采集于2006—2008年。结果七省市人群HEV抗体总阳性率为17．97％（1878／10448）。汉、回和藏不同民族人群HEV抗体阳性率分别为24．32％（1794／7376）、3．59％（81／2258）和0．37％（3／814）。不同地区人群HEV抗体阳性率分布,四川、北京、黑龙江和山东汉族人群阳性率分别为27．45％、20．30％、22．89％和22．68％,甘肃汉和回族分别为24．63％（184／747）和6．12％（77／1258）,宁夏回族和青海藏族分别0．40％和0．37％。汉回藏不同民族各年龄组人群HEV感染分布,汉族各年龄组人群HEV抗体阳性率,在≤10岁年龄组为5．19％,11～20岁组为11．64％,21～30岁组为20．08％,31～40岁组为34．17％,41—50岁为41．75％,51～60岁组为48．58％,≥61岁组为57．43％。回族人群各年龄组人群HEV抗体阳性率依次为3．11％,3．96％,2．11％,3．98％,2．52％,4．57％和6．67％。藏族人群3份阳性者分布在21—30岁组、31—40岁组和51～60岁组各1份,阳性率为0．63％、0．58％和1．01％。结论汉族人群戊肝病毒感染明显高于回族和藏族,感染率随年龄增长而升高。回藏族人群HEV抗体阳性率低下,应加强对HEV感染的监测。