Assessment of assays for the serodiagnosis of Venezuelan equine encephalitis.

An enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a plaque-reduction neutralization (PRN) assay and an immunoblot assay, all by means of an antigen prepared from the attenuated Venezuelan equine encephalitis (VEE) vaccine strain of virus, were compared with the conventional haemagglutination-inhibition (HAI) assay for the serodiagnosis of VEE. The HAI assay, which includes the use of wild type virus antigen, was less sensitive than the other assays when known-positive samples of serum from an epidemic of VEE were tested. The superior sensitivity of the IgG ELISA was confirmed by assaying both VEE epidemic samples and a bank of samples from VEE vaccinees. Samples with antibody specific for other Alphaviruses, however, cross reacted weakly in this assay. The PRN, immunoblot and HAI assays, although less sensitive than the ELISA, proved more specific. Experimental infection of guinea-pigs demonstrated the value of the IgM ELISA in the early detection of VEE virus infection. Immunoglobulin M was first found at 4 days post-inoculation (p.i.) during the viraemic phase of infection. Immunoglobulin G was detected by ELISA, PRN assay and IFA at 6 days p.i. Immunoblot and HAI assays, however, did not give positive results until 10 days p.i. The results support the diagnostic use of ELISA for detecting VEE virus-specific IgM and IgG, and the use of the specific PRN assay for confirming the diagnosis.     

病毒性脑炎脑脊液病毒特异性抗体ELISA检测结果分析

目的探讨儿童病毒性脑炎脑脊液病毒病原学特点及临床特征。方法应用酶联免疫吸附试验（ELISA）疗法埘121例临床诊断为病毒性脑炎患儿的脑脊液进行肠道病毒（EV）、单纯疱疹病毒（HSV）I型和Ⅱ型、EB病毒（EBV）、腺病毒（ADV）及流感病毒（FLuV）特异性IgM抗体检测。结果脑脊液病毒特异性IgM抗体检测阳性75例,阳性率61．98％。肠道病毒阳性35例,居第1位,其次是单纯疱疹病毒I型和Ⅱ型,共17例。肠道病毒感染多见于婴幼儿（6个月～3岁）,单纯疱疹病毒感染多见于学龄儿童;肠道病毒感染多见于夏秋季,单纯疱疹病毒感染多见于冬备季,EB病毒、腺痫毒及流感病毒各季节散发。结论肠道病毒是本地区儿童病毒性牺炎感染的主要病原;晒脊液病毒特异性IgM抗体检测町作为早期病原诊断的指标之一。     

A prospective assessment of the accuracy of commercial IgM ELISAs in diagnosis of Japanese encephalitis virus infections in patients with suspected central nervous system infections in Laos

Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia. We estimated the diagnostic accuracy of two anti-JEV immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) (Panbio and XCyton JEVCheX) compared with a reference standard (AFRIMS JEV MAC ELISA) in a prospective study of the causes of central nervous system infections in Laos. Cerebrospinal fluid (CSF; 515 patients) and serum samples (182 patients) from those admitted to Mahosot Hospital, Vientiane, were tested. The CSF from 14.5% of acute encephalitis syndrome (AES) patients and 10.1% from those with AES and meningitis were positive for anti-JEV IgM in the reference ELISA. The sensitivities for CSF were 65.4% (95% confidence interval [CI] = 51-78) (Xcyton), 69.2% (95% CI = 55-81) (Panbio), however 96.2% (95% CI = 87-100) with Panbio Ravi criteria. Specificities were 89-100%. For admission sera from AES patients, sensitivities and specificities of the Panbio ELISA were 85.7% (95% CI = 42-100%) and 92.9% (95% CI = 83-98%), respectively.     

Evaluation of three commercially available Japanese encephalitis virus IgM enzyme-linked immunosorbent assays

We evaluated performance of three commercial Japanese encephalitis virus (JEV) IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) kits with a panel of serological specimens collected during a surveillance project of acute encephalitis syndrome in India and acute meningitis and encephalitis syndrome in Bangladesh. The serum and cerebral spinal fluid specimens had been referred to the Centers for Disease Control and Prevention (CDC) for confirmatory testing. The CDC results and specimen classifications were considered the reference standard. All three commercial kits had high specificity (95-99.5%), but low sensitivities, ranging from 17-57%, with both serum and cerebrospinal fluid samples. Specific factors contributing to low sensitivity compared with the CDC ELISA could not be determined through further analysis of the limits and dilution end points of IgM detection.     

Detection of Japanese encephalitis (JE) virus-specific IgM in cerebrospinal fluid and serum samples from JE patients

Detection of Japanese encephalitis virus (JEV)-specific IgM by IgM-capture enzymed-linked immunosorbent assay (IgM-capture ELISA) has been accepted as the standard for serological diagnosis. In the present study, we analyzed the time course of the positive rate of JEV-specific IgM in serum and cerebrospinal fluid (CSF) specimens from confirmed JE patients. Serum and CSF samples were obtained from 155 JE cases for diagnostic purposes at hospitals in Thailand from 2002 to 2004. The levels of specific IgM were assessed by IgM-capture ELISA in the 171 serum and 156 CSF samples. Anti-JEV IgM was detected in 26 of 44 serum samples collected on days 1-4 of the disease period, in 31 of 44 samples collected on days 5-8, in 23 of 26 samples collected on days 9-12, and in all the samples collected on day 13 or later. Specific IgM was detected in 60 of 66 CSF samples collected on days 1-4 of illness, and in all the CSF samples but one collected on day 7 or later. The results indicate that the detection of JEV-specific IgM in CSF by IgM-capture ELISA is a reliable laboratory diagnostic method for confirmation of JE throughout the disease period, while the detection of IgM in serum samples is a reliable method on day 9 or later.     

Pseudotyped viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus.

Virus envelope proteins are the primary targets of neutralizing antibody responses. The epitopes recognized differ sufficiently between virus subtypes and species to distinguish viruses and provide an important basis for disease diagnosis. Venezuelan equine encephalitis virus (VEEV) causes acute febrile illness in humans and has high mortality in equines. The most specific detection methods for serum antibodies use live virus in neutralization assays or in blocking enzyme linked immunosorbent assays. However, work with Venezuelan equine encephalitis virus requires biosafety level 3 containment and select agent security in the United States. We report two new assays for detection of Venezuelan equine encephalitis virus neutralizing antibody responses, based on virus pseudotypes. The first provides detection by marker gene expression after 20 hours and is particularly suited for high-throughput screening; the second uses a new, rapid virus entry assay to give readouts within 1 hour. Both assays are safe, sensitive, and in general recapitulate neutralizing antibody titers obtained by conventional plaque reduction assays. Each is suitable as a rapid primary screen for detection of neutralizing antibodies against Venezuelan equine encephalitis virus.     

Enterovirus-specific IgM in the diagnosis of meningitis

Samples of serum from 557 patients with a clinical diagnosis of meningitis or encephalitis and referred to the Epsom Public Health Laboratory during a period of 3 years were tested for enterovirus-specific IgM in a mu capture enzyme-linked immunosorbent assay (ELISA). Enterovirus-specific IgM was detected in 45% samples from all age groups. In the 3-5-year age group, 67% specimens were positive. A notable male predominance (73%) was seen in the age group 0-15 years. As predicted, a seasonal increase in incidence was found in the summer and autumn months. Data from a questionnaire sent to the referring laboratories showed only a 5% enterovirus isolation rate from cerebrospinal fluids when isolation of a virus was attempted. The enterovirus IgM ELISA is a sensitive economical and rapid method for use in the diagnosis of viral meningitis.     

酶联免疫斑点法检测抗1型单纯疱疹病毒IgM分泌细胞在单纯疱疹病毒性脑炎早期诊断中的作用

目的 观察酶联免疫斑点法(ELISPOT)检测抗1型单纯疱疹病毒IgM(抗-HSV-1 IgM)分泌细胞在单纯疱疹病毒性脑炎(HSV-1脑炎)早期诊断中的作用并探讨其价值.方法 采用ELISPOT法和ELISA法分别检测患者脑脊液中的抗-HSV-1 IgM分泌细胞和抗-HSV-1 IgM,并对HSV-1腩炎23例、临床对照组40例进行回顾性分析,统计学处理采用Fisher精确检验法.结果 ELISPOT法检测HSV-1脑炎组9例患者2周内脑脊液中抗-HSV-1 IgM分泌细胞,8例阳性.敏感度为88.9%;临床对照组检测16例,15例阴性,特异度为93.8%.ELISA法榆测HSV-1脑炎组12例患者脑脊液2周内抗-HSV-1 IgM,2例阳件,敏感度为16.6%;临床对照组检测17例,15例阴性,特异度为88.2%.两种方法敏感度比较差异有统汁学意义(P<0.01).结论 应用ELISPOT法检测HSV-1脑炎患者脑脊液中抗-HSV-1 IgM分泌细胞优于 ELISA方法检测抗-HSV-1 IgM,具有更好的早期诊断意义.     

Kinetics of IgM and IgG responses to Japanese encephalitis virus in human serum and cerebrospinal fluid

We measured levels of antibodies to Japanese encephalitis virus (JEV) in serum and in cerebrospinal fluid (CSF) specimens obtained from 32 patients with acute encephalitis by using "antibody-capture" solid-phase enzyme-linked immunoassays specific for IgM or IgG to JEV. The proportions of confirmed cases with IgM to JEV detectable in CSF were 68% (obtained on day 1), 100% (day 7), 96% (day 30), and 72% (day 180). For IgG in CSF the proportions were 47% (day 1), 89% (day 7), 100% (day 30), and 100% (day 180). Twenty-five CSF samples were obtained from control patients with other diseases with possible nervous system involvement (but none with a clinical diagnosis of viral encephalitis); none had detectable IgM to JEV. Five JEV-infected but asymptomatic siblings of patients with encephalitis were also examined; all had high levels of IgM to JEV in serum, but none had detectable IgM to JEV in CSF.     

High diagnostic yield by CSF-PCR for entero- and herpes simplex viruses and TBEV serology in adults with acute aseptic meningitis in Stockholm

Acute aseptic meningitis (AAM) affects 10-20/100,000 inhabitants per years in Sweden. Up to the beginning of the 1980s the diagnoses were made by virus isolation and/or determination of viral antibodies in serum. The development of PCR for detection of viruses in CSF samples has increased the sensitivity and diagnostic efficiency considerably. We investigated the aetiology of AAM and the diagnostic efficiency in an adult population in Stockholm, using a limited first-line combination of microbiological assays. CSF and serum samples, consecutively collected in 419 patients with clinical symptoms of AAM in northern Stockholm during 1999-2004, were included. PCR assays for herpes simplex virus (HSV) DNA and enterovirus (EV) RNA in the CSF as well as ELISA for IgM in serum to tick-borne encephalitis virus (TBEV) were performed routinely. A viral diagnosis was obtained in 255 of the 419 cases (62%) with these routinely performed assays. Clinical findings in combination with additional diagnostic tests resulted in an overall aetiological yield of 72%. EV was the major causative agent (27%) followed by TBEV (21%) and HSV-2 (19%). We conclude that consistent use of CSF-PCR for EV and HSV and TBEV serology established a diagnosis in the majority of AAM patients.     

寨卡病毒病研究进展

寨卡病毒病是由寨卡病毒引起的一种自限性急性传染病。寨卡病毒主要通过埃及伊蚊和白纹伊蚊叮咬传播,有证据表明也可通过性传播和母婴传播。临床表现主要为皮疹、发热、关节痛或结膜炎等非特异性症状,但寨卡病毒感染与新生儿小头畸形、格林-巴利综合征等存在密切关系。实验室检测方法包括实时荧光定量PCR检测病毒核酸和ELISA检测Ig M抗体。该疾病目前无有效的抗病毒药物和疫苗。预防措施主要为预防蚊虫叮咬和采取虫媒控制措施。     

Fatal outcome in Japanese encephalitis

Forty-nine consecutive patients with laboratory-confirmed acute Japanese encephalitis were studied to identify risk factors present at hospital admission which were associated with a fatal outcome. Sixteen patients (33%) died. The following constellation of findings correlated with a fatal outcome: infectious virus in cerebrospinal fluid (CSF), low levels of Japanese encephalitis virus-specific IgG and IgM in both CSF and serum, and a severely depressed sensorium. Age, sex, days ill before admission, distance from home to the hospital, past medical history, CSF protein content, and CSF leukocyte count were not significant risk factors. Among patients hospitalized for acute Japanese encephalitis, a vigorous virus-specific immunoglobulin response, both systemically and locally within the central nervous system, is a good marker for survival, and may be an inherently important factor in recovery from illness.     

Case Report: West Nile Virus Encephalitis: The First Human Case Recorded in Brazil

A Brazilian ranch worker with encephalitis and flaccid paralysis was evaluated in the regional Acute Encephalitis Syndromic Surveillance Program. This was the first Brazilian patient who met the Centers for Disease Control and Prevention (CDC) confirmation criteria for West Nile virus disease. Owing to the overlapping of neurological manifestations attributable to several viral infections of the central nervous system, this report exemplifies the importance of human acute encephalitis surveillance. The syndromic approach to human encephalitis cases may enable early detection of the introduction of unusual virus or endemic occurrence of potentially alarming diseases within a region.     

The humoral immune response to chlamydial infection in humans

The serodiagnostic methods commonly used for the detection of antibodies in various chlamydial infections, the value of serodiagnosis, and the role of humoral responses in human immunity are reviewed. The methods most commonly used for serodiagnosis of chlamydial infections are indirect immunofluorescence tests and enzyme-linked immunosorbent assays. These tests are highly sensitive and have been extensively used in epidemiologic and clinical studies of chlamydial infections. The evidence suggests that these tests are cost effective for population screening and that the presence of a high level of specific IgG or IgM in blood or of IgG or IgA in local discharges is a useful indicator for the provisional diagnosis of chlamydial infection.     

Differentiation of Herpes simplex virus types 1 and 2 in sera of patients with HSV central nervous system infections by type-specific enzyme-linked immunosorbent assay.

OBJECTIVE: To differentiate by enzyme-linked immunosorbent assay (ELISA) using type-specific glycoprotein G herpes simplex virus (HSV) type 1 and type 2 in serum collected from patients with HSV central nervous system (CNS)infections. METHODS: HSV 1 and 2 typing in convalescent sera of 17 patients with HSV acute encephalitis, myelitis, or meningitis was determined by the type-specific IgG ELISA kit (Gull Laboratory, Inc.). HSV CNS infections were diagnosed by polymerase chain reaction (PCR) or conventional serologic tests from acute to convalescent stages. RESULTS: In 13 of 17 patients, HSV type 1 and HSV type 2 antibodies were positive; 11 patients with HSV type 1 and 2 patients with HSV type 2 were found. All 10 patients with encephalitis showed equivocal or positive results for HSVtype 1. In two of three cases of myelitis, HSV type 1 was demonstrated. Each case of myelitis and meningitis reacted to both types 1 and 2. CONCLUSION: These data suggest that the kit is useful for type differentiation of HSV CNS infections from convalescent sera, and can play a supplementary role in HSV typing by PCR or previous serologic tests.     

Comparison of performance of serum and plasma in panbio dengue and Japanese encephalitis virus enzyme-linked immunosorbent assays

Abstract. We examined the comparative performance of serum and plasma (in dipotassium EDTA) in Panbio Dengue enzyme-linked immunosorbent assays (ELISAs) for detection of non-structural protein 1 (NS1), IgM, and IgG, and a dengue/Japanese encephalitis virus (JEV) combination IgM ELISA in a prospective series of 201 patients with suspected dengue in Laos. Paired comparisons of medians from serum and plasma samples were not significantly different for Dengue IgM, and NS1 which had the highest number of discordant pairs (both 2%; P = 0.13 and P = 0.25, respectively). Comparison of qualitative final diagnostic interpretations for serum and plasma samples were not significantly different: only 1.5% (3 of 201 for Dengue/JEV IgM and Dengue IgG) and 2.0% (4 of 201; IgM and NS1) showed discordant pairs. These results demonstrate that plasma containing EDTA is suitable for use in these ELISAs.     

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.     

Evaluation of enzyme-linked immunosorbent assays with two synthetic peptides of Epstein-Barr virus for diagnosis of infectious mononucleosis

To diagnose infectious mononucleosis caused by Epstein-Barr virus (EBV), a peptide from the EBV nuclear antigen (EBNA) 1 (p107) and an EBNA 2 peptide (polyproline) were used as antigens in enzyme-linked immunosorbent assays for IgG and IgM. Well-characterized serum samples (360) from healthy individuals and patients with EBV or cytomegalovirus infections were examined. The p107 IgG and IgM assays were also tested with serum from 1000 patients with suspected EBV-related disorders. The p107 and polyproline IgG assays were 100% specific for EBV seropositivity. Low p107 IgG titers (less than 1000) were found in 98% of patients with EBV infectious mononucleosis but also in 18% of patients with other diseases. A p107-to-polyproline IgG ratio of less than 1 was 98% specific for EBV infectious mononucleosis; sensitivity was 86%. In EBV capsid antigen-IgG seropositive patients, a p107 IgG titer of less than 1000 together with a p107 IgG-to-IgM ratio of less than 1 was 98% sensitive and specific for EBV infectious mononucleosis. Thus, this ratio appears adequate to measure EBNA antibodies for diagnosis of EBV mononucleosis.     

West Nile virus meningomyeloencephalitis--value of interferon assays in primary encephalitis

A 68 year-old woman contracted West Nile fever after a stay of one month in Israel. Mild encephalitis and severe myelitis, resembling the "polio syndrome", developed, with important sequelae. Specific antibodies against West Nile fever virus progressively increased in the patient's serum. Epidemiological and clinical data about neurologic aspects of this infection are reviewed. Meningoencephalitis is not unusual, but only one previous case of acute anterior myelitis has been described in humans although this pathology is well known in experimental and veterinary diseases. Very few studies concern interferon and arbovirus infections in humans. Interferon assays in our patient's serum and cerebrospinal fluid showed its presence and persistence: this is in relation with the replication of the virus in the central nervous system, as it has been demonstrated in other primary viral encephalitis.