Rapid detection of respiratory syncytial virus with a monoclonal antibody.

We developed an indirect fluorescent-antibody test employing a mouse monoclonal antibody directed against the nucleoprotein of RSV for rapid detection of respiratory syncytial virus (RSV). This test demonstrated distinctive fluorescent inclusions in HEp-2 cells infected with 24 RSV isolates collected during 6 previous years but not in cells infected with 13 other respiratory viruses. Examination of nasal cells of 100 infants with acute respiratory illness showed that the indirect fluorescent-antibody test employing the monoclonal antibody was 79% sensitive and 100% specific, as compared with the combination of both culturing and a similar indirect fluorescent-antibody test with commercial anti-RSV serum. This monoclonal antibody is an easily produced, well-characterized, sensitive, and specific reagent for the rapid detection of RSV antigen.     

Serodiagnosis of respiratory syncytial virus (RSV) infection in children as measured by detection of RSV-specific immunoglobulins G, M, and A with enzyme-linked immunosorbent assay.

The diagnostic value of an enzyme-linked immunosorbent assay for detection of respiratory syncytial virus (RSV)-specific immunoglobulin G (IgG), IgM, and IgA in sera from infants and children with proven RSV infection, from a control group, and from patients with symptoms of viral respiratory disease was analyzed. Compared to virus isolation and RSV antigen detection methods, the sensitivity of this assay was 87% and the specificity was 79%. For IgG alone, these were 45 and 92%, for IgM alone they were 48 and 92%, and for IgA alone they were 74 and 95%, respectively.     

Comparison of nonspecific reactivity in indirect and reverse immunoassays for measles and mumps immunoglobulin M antibodies.

Serum specimens collected from patients convalescing from acute measles or mumps infections, other viral infections, or rheumatoid arthritis and from blood donors were tested in indirect and reverse assays for measles and mumps immunoglobulin M (IgM) antibodies. All the samples from patients convalescing from acute mumps and measles infections gave positive IgM results in both tests. However, 6% of sera from patients recovering from other viral infections, 68.4% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive results by the indirect measles IgM enzyme immunoassay (EIA). By the indirect mumps IgM EIA, 9% of sera from other viral infections, 70.1% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive reactions. The reverse test system for measles IgM gave false-positive results in 1.5% of sera from the group with other viral infections, and the reverse mumps EIA gave false-positive results in 0.9% of the patients. Other sera groups did not react in either measles or mumps reverse IgM assays. The results indicated that although nonspecific reactions are frequent in indirect IgM tests for viral antibodies, such reactions are rarely encountered when reverse IgM EIA tests are employed.     

Detection of antibody to bovine syncytial virus and respiratory syncytial virus in bovine fetal serum.

Batches of commercial fetal bovine serum, described by the suppliers as antibody-free, all contained antibody to bovine syncytial virus (BSV) when tested by indirect immunofluorescence. Antibody to bovine respiratory syncytial virus (RSV) was not detected in these sera. Twenty-four percent of individual fetal bovine sera contained antibody to BSV, and 14% contained antibody to RSV when tested by indirect immunofluorescence. BSV antibody titers in fetal sera from dams with high BSV antibody levels were variable but always higher than RSV antibody titers. Radial immunodiffusion studies with BSV-positive sera revealed the presence of immunoglobulin M (IgM), IgG, and IgA, but the quantity of these immunoglobulins was not directly related to the BSV antibody titers. The evidence suggests that the antibody present in fetal sera arose as the result of infection rather than from maternal transfer across the placenta.     

Detection of antibody to respiratory syncytial virus by membrane fluorescence.

An indirect membrane fluorescent antibody technique was established to study HEp 2 cells infected with respiratory syncytial (RS) virus. It was possible to detect IgG and IgM antibody to RS virus in the sera of patients with respiratory infections using this technique. The technique was further applied to the detection of IgA antibody to the same virus in colostrum.     

Immunoglobulin M antibody titers in the diagnosis of Legionnaires disease.

The purpose of this study was to determine whether measurement of immunoglobulin M (IgM) antibodies against Legionella pneumophila serogroup 1 can aid in the diagnosis of Legionnaires disease. On the basis of measurements of antibody levels in 1,942 control sera, we used an IgM titer of 1:256, observed in 2.3% of the controls, as presumptive evidence of Legionnaires disease. Measurement of IgM titers permitted us to presumptively or definitively diagnose Legionnaires disease in 13 of 34 patients earlier than we would have if only IgG titers had been measured. Of the 13 patients, 5 were diagnosed serologically only by IgM antibody determination. IgM titers were presumptively diagnostic in week 1 of clinical symptoms in 4 of the 13 patients. We conclude that conjugates used for antilegionella indirect fluorescent-antibody tests should be capable of detecting IgM antibodies so that the value of serological results in diagnosing and managing Legionnaires disease will be maximized.     

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population.     

Immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay

Immunoglobulin class-specific enzyme immunoassay (EIA) was used for determination of antibody responses in sera collected from 26 children with acute primary respiratory syncytial virus (RSV) infections. All 26 patients had IgG antibody responses with a significant titer increase in 24 (92%); an IgM antibody response was detected in 19 of the 26 (73%). From patients aged 6 months or less only 5 of 8 produced detectable IgM antibodies, whereas all patients aged 1–2 years did so. IgM antibodies appeared within 1 week after onset of illness and persisted from 20 days to 2–3 months. An IgA antibody response was observed in 20 of 26 (77%) patients with a significant titer increase detected in 17 of 26 (65%) patients. In some patients the persistence of IgA antibodies followed that of the IgM antibodies, but in others the IgA antibody titers remained stable up to the end of the follow-up. The most sensitive assay system for serological diagnosis of acute RSV infection in children was the determination of titer increases by IgG antibody.     

Clinical and epidemiologic characteristics of respiratory syncytial virus subgroups A and B infections in Santa Fe, Argentina

Respiratory Syncytial Virus (RSV) has two major antigenic groups, A and B. The implications of these variants in the epidemiology and pathogenesis of RSV infection are not well defined. This study was undertaken to compare the two RSV subgroups in patients admitted to hospital. Clinical and epidemiologic features of RSV subgroups in children under 30 months of age with proven RSV acute lower respiratory infections were examined during 4 winters from 1993 to 1996 in Santa Fe, Argentina. RSV typing was carried out with monoclonal antibodies in nasopharyngeal cells by indirect immunofluorescence. Of the 177 RSV positive nasopharyngeal aspirates obtained from 1993 to 1996, 85 (48%) were available for typing. Seventy-three (85.9%) specimens were identified as Subgroup A and 12 (14.1%) as Subgroup B. Except in 1993, in which only Subgroup A was detected, both variants circulated throughout the epidemic season. Subgroup A infections produced more severe disease than Subgroup B infections, as assessed by the length of the hospital stay and the use of respiratory support. This difference was age related, being evident in infants 0-6 months old. Patients with Subgroup B infections were also significantly less frequently breast-fed (95% vs. 75% for A and B subgroups, respectively; P = 0.04). It is concluded that the severity of disease in Argentinian patients admitted with acute RSV infections may be associated with Subgroup A strains as determined by a serogrouping method.     

An epidemic of respiratory syncytial virus in elderly people: clinical and serological findings

In 1984-1985, an outbreak of respiratory syncytial virus (RSV) infection occurred in two geriatric wards. Among 68 patients (mean age +/- SD = 82.5 +/- 12.5 with respiratory signs, 52 had signs caused by RSV infection. Among all patients, the clinical and serological attack rates were 61.2% and 75.0%, respectively. The most frequent clinical presentation was intensive coughing (96.1%) and fever (96.1%) associated with expectorate (63.5%). The duration of the respiratory symptoms was 5 to 7 days. The disease gradually resolved, although in eight (15.4%) patients complications occurred. For periods of up to 1 year after infection, 172 sera were obtained and tested by complement fixation test (CFT), fluorescent assays for titrating specific IgG, IgA, and IgM, and Western blotting. Specific IgM appeared in six (11.5%) of the infected patients and peaked 2 to 6 months after infection, and there was no significant correlation with severity of clinical symptoms. However, higher peak G and A antibody responses were observed in persons with rales (CFT: P = 0.008; IgG: P = 0.042; IgA: P = 0.020), cough (IgG: P = 0.034), sputum (IgG: P = 0.030), dyspnea (CFT: P = 0.024), conjunctivitis (CFT: P = 0.025), and bronchitis (CFT: P = 0.018). The temporal patterns of IgA and CFT results were found to be similar, whereas IgG peaked later, i.e., between 2 and 6 months. The patients with the most severe symptoms had the highest antibody titers obtained by conventional tests and by Western blots. Thus, RSV can be an epidemic pathogen among elderly persons, although this illness is usually mild.     

Hemadsorption immunosorbent technique for determination of mumps immunoglobulin M antibody

We used hemadsorption immunosorbent technique (HIT) to detect mumps immunoglobulin M (IgM) antibody. IgM from human sera was adsorbed into anti-human IgM-coated wells in plates, and mumps-specific IgM was detected by adding mumps virus hemagglutinin and guinea pig erythrocytes consecutively. Specific IgM-positive sera showed hemadsorption, whereas negative sera showed hemagglutination. All 81 patients with current mumps infections tested showed mumps-specific IgM antibody, with titers ranging from 160 to 327,680. In most cases IgM antibody was already present on day 1 or 2 after the onset of illness. IgM antibody persisted for 6 to 10 weeks. Of 57 patients with acute respiratory illnesses caused by parainfluenza virus, 5 showed cross-reactions in the hemadsorption immunosorbent technique assay for mumps IgM. The hemadsorption immunosorbent technique assay is specific for the IgM class of antibody and avoids false-positive results due to rheumatoid factor. This test is an efficient and sensitive method for rapid and early diagnosis of mumps infections.     

Development and evaluation of serological assays for detection of human hantavirus infections caused by Sin Nombre virus.

BACKGROUND: The hantavirus cardiopulmonary syndrome (HCPS) was first recognized in 1993 after a cluster of acute respiratory distress syndrome deaths in the southwestern of the United States. The major causative agent of HCPS in North America is the Sin Nombre virus (SNV) carried by the deer mouse Peromyscus maniculatus. The first HCPS case imported to Europe was reported in 2002. OBJECTIVES: The objective of the study was to develop and evaluate ELISA and Western blot tests for the serological detection of human infections caused by SNV including those imported to Europe. STUDY DESIGN: A polyhistidine (His)-tagged recombinant nucleocapsid (rN) protein of SNV was expressed in Saccharomyces cerevisiae and purified by nickel chelation chromatography. On the basis of the purified SNV rN protein mu-capture and indirect IgM and IgG ELISAs and an IgG Western blot were developed. The evaluation of the tests was performed using a negative serum panel and a blinded serum panel from the US containing acute-phase sera from HCPS patients. RESULTS: Based upon the results obtained using a panel of negative control sera the specificity for SNV mu-capture and indirect IgM and IgG ELISAs were found to be 100%. All 33 sera from SNV-infected HCPS patients included in the blinded panel were detected by the SNV mu-capture and indirect IgM ELISAs. Twenty-nine out of the 33 SNV-IgM positive sera reacted also in the SNV-IgG ELISA. An SNV-IgG Western blot confirmed the data of the SNV-IgG ELISA. Although the majority of anti-SNV positive sera cross-reacted with rN proteins of Puumala virus and Dobrava virus, the lacking reactivity of a few sera with these heterologous rN antigens in the corresponding IgM and IgG ELISAs demonstrates the value of virus-specific test formats for acute-phase sera. CONCLUSIONS: The novel SNV ELISA and Western blot tests represent a useful tool for the serological detection of SNV infections.     

RSV molecular characterization and specific antibody response in young children with acute lower respiratory infection.

The presence of respiratory syncytial virus (RSV) in nasopharyngeal aspirates (NPA) were studied in 254 hospitalized Argentinean children with acute lower respiratory infection tract (ALRI). The specific humoral immune response and partial sequences of the G protein gene were studied in a subset of 22 children with RSV confirmed infection. The RSV IgM detection and the RSV IgG titration were made by immunofluorescence assay (IFA) in pairs of sera. The partial RSV G gene sequences were obtained by an RT-PCR amplification directly from de NPAs. RSV was present in 44.5% of the children. The RSV IgM was detected in 22.7 and 68.8% of the first and second sera, respectively. The IgG geometric mean titers of the acute and convalescent sera were 8 and 589. The RSV IgG titration was able to define 86.4% of the RSV confirmed cases. The percentage of coincidence between RSV IgM detection in the second sera and diagnosis by RSV IgG titration was 72.7% and no significant differences were observed. The nucleotide sequence of one group A and three group B viruses were identified. The first one was related with circulating viruses in Madrid, Montevideo and Mozambique during 1992, 1989 and 1999, respectively. The three sequences identified as group B viruses were closely related with circulating viruses in 1998 from South Africa and Canada during 1999 and 2000. The data obtained in our study provide the first approach at the molecular level (nucleotide) of the RSV circulating strains in Argentina and the lack of genotype patterns previously determined make necessary a continuous molecular surveillance in order to contribute to the understanding of the behavior of this virus in our community.     

Maternal antibody and respiratory syncytial virus infection in infancy

One hundred newborn infants were studied prospectively for 1 year for evidence of infection with respiratory syncytial virus (RSV). The indirect membrane fluorescence technique was used to determine specific antibody in sera. Infection was shown in 29 cases. In 31 infants exposed to an RSV epidemic season, there was no evidence of infection. Maternal antenatal sera were also tested, and a wide range of IgG antibody to RSV was found. Mean titre of maternal IgG antibody to RSV was significantly higher (P < 0.001) in those mothers whose babies remained uninfected than in those whose babies had proved RSV infection before 6 months of age. Babies born to mothers with high levels of IgG antibody to respiratory syncytial virus were protected against infection with this virus during the first months of life when the risk of severe disease was greatest.     

Evaluation of four commercial immunoglobulin G (IgG)- and IgM- specific enzyme immunoassays for diagnosis of Mycoplasma pneumoniae infections

The four following commercially available enzyme immunoassays (EIAs) were assessed and compared for their performance in detecting Mycoplasma pneumoniae specific IgG and IgM antibodies: EIA-Platelia, EIA-Bmd, EIA-Sorin and EIA-Biotest. Three groups of patients were investigated: 39 patients (27 children and 12 adults) with respiratory infections and a M. pneumoniae PCR-positive in respiratory specimens (group I; 52 sera), 61 healthy children and adults (group II; 61 sera) and 20 patients with rheumatoid factor, antinuclear antibodies or positive antiviral IgM (group III; 20 sera). In group III, the IgM specificity for the EIA-Platelia, EIA-Bmd, EIA-Biotest and EIA-Sorin was 100%, 90%, 65% and 25%, respectively. In the children from group I, the four EIAs had similar IgM sensitivity (89 to 92%) but a striking difference in IgM sensitivity was observed in adult patients: 16% EIA-Platelia and EIA-Bmd, 50% EIA-Biotest, 58% EIA-Sorin. The sensitivity for IgG was greater with EIA-Bmd and EIA-Biotest, especially in detection of IgG in acute-phase serum : 61% EIA-Bmd and EIA-Biotest, 15% EIA-Platelia and 31% EIA-Sorin. Discrepant and unexpected results were observed in IgM detection from control healthy patients using EIA-Sorin and EIA-Biotest, confirming the lack of specificity of these two EIA-tests and making them inaccurate for routine diagnosis. A high IgG seroprevalence were found in healthy adults by the four EIAs (43-70%). In healthy children, EIA-Bmd and EIA-Biotest gave a higher IgG seroprevalence than EIA-Sorin and EIA-Platelia (45% each for the former as compared to 17% and 20%, respectively, for the latter).These results confirm that the IgM EIA serology test is a valuable tool for the early diagnosis of M. pneumoniae infections in children, as long as the EIA test used is specific. In adults, the difficult interpretation of EIA tests suggests that paired sera, combined with PCR detection on respiratory tract specimens collected on admission of patient, should be required for accurate diagnosis.     

Practical implementation of a multiplex PCR for acute respiratory tract infections in children

Molecular testing for acute respiratory infections (ARIs) has documented value but limited implementation due to questions that typically slow the acceptance of new tests. This study sought to address these questions and achieve implementation. Rhinovirus was added to a nested multiplex PCR (M-PCR), increasing its diagnostic yield. Over one winter, three hospital pediatric departments used the M-PCR to complement their direct fluorescent-antibody assay (DFA) for respiratory syncytial virus (RSV). Clinicians recorded "pretest probability estimates" (using continuous scales for various pathogen groups) for comparison with test results; treatments and test turnaround times were also recorded. Transnasal and throat swabs, with or without nasopharyngeal aspirate (NPA), were M-PCR tested. NPA-containing sample sets found to be RSV positive by DFA were not further tested. Single PCR for human metapneumovirus (hMPV) was performed retrospectively. Of 178 ARI episodes representing 172 patients, NPA was included in 97 sample sets; 54 (56%) were determined to be RSV positive. The other NPA-containing sample sets (n = 43) yielded 27 findings (63%), and the swab-only sets (n = 81) yielded 47 findings (58%); rhinovirus was found most often. Testing for hMPV yielded seven positive results. M-PCR median turnaround times were 4 days in swab-only samples and 5 days with NPA. Antibiotics were prescribed in 50 episodes, at rates similar for RSV and rhinovirus. Pretest probability estimates of a viral cause were lower in episodes caused by rhinovirus than in episodes caused by RSV. The hospitals continued to use M-PCR for NPA-containing samples found to be RSV negative by DFA. Test implementation is more likely with higher diagnostic yield and a protocol that reflects day-to-day clinical and laboratory operations.     

Evaluation of an indirect hemagglutination test for Legionella pneumophila serogroups 1 to 4

Parallel testing of 895 sera by indirect hemagglutination and indirect fluorescent-antibody techniques showed 97.3% agreement. Although the indirect hemagglutination technique usually showed more cross-reactivity among serogroups than the indirect fluorescent-antibody technique with Formalin-fixed antigens and a conjugate which detected primarily immunoglobulin G antibodies, heterologous serogroup reactions were significantly lower than homologous serogroup titers and the etiological serogroup could be easily defined. The indirect hemagglutination techniques showed no cross-reactivity with a crude extract of Escherichia coli O13:K92:H4. Since the indirect hemagglutination technique was shown to detect both immunoglobulin M and immunoglobulin G antibodies and was found to be rapid, simple, and inexpensive, it appears to be an excellent alternative to the indirect fluorescent-antibody test for serodiagnosis of legionellosis.     

The detection of antibodies to cytomegalovirus in the sera of renal transplant patients by an igm antibody capture assay

An IgM antibody capture assay for detection of cytomegalovirus (CMV) IgM antibody (MACRIA) was developed. It was shown to be of similar sensitivity to the indirect immunofluorescence test but has the advantage that rheumatoid factor does not react in it and pretest fractionation of serum is not required. It does, however, give false results with some Paul Bunnell-positive sera. The assay was used to measure the IgM response in 28 renal transplant patients followed prospectively. Seven patients (100%) with primary infections and six of 13 (46%) patients with secondary infections developed IgM by MACRIA. Nine of 13 (69%) patients with CMV IgM-positive sera had symptoms other than pyrexia associated with CMV infections, while only one of seven (14%) IgM-negative infections were symptomatic. Four of seven irreversible rejection episodes were associated with CMV IgM. The possible significance of CMV IgM production is discussed.     

3820例呼吸道感染患儿病原体的检测结果及流行病学特点分析

目的:了解垫江地区儿童呼吸道病原体流行情况,为疾病的预防和诊治提供实验室依据。方法:回顾性分析2018年1月至2020年10月本院收治的3820例呼吸道感染患儿入院时检测的血常规、血清淀粉样蛋白A(Serum amyloid A,SAA)、C反应蛋白(C-reactive protein,CRP)结果及采用间接免疫荧光法检测的11项呼吸道病原体IgM抗体结果。结果:病毒IgM抗体总检出率为51.65%(1973/3820),以流感病毒B(Influenza virus B,INFB)、流感病毒A(Influenza virus A,INFA)和肺炎支原体(Mycoplasma pneumoniae,MP)感染为主;在1973例呼吸道病原体阳性患者中有321例患者存在混合感染,混合感染率为16.27%;女性患儿阳性率高于男性患儿(χ²=9.67,P=0.002);学龄前组与学龄组呼吸道病原体阳性率较高,婴儿组最低,差异具有统计学意义(P<0.05)。夏季病原体阳性检出率明显低于其它三个季节(χ²=25.62,P=0.00);肺炎衣原体(Chlamydia pneumoniae,CP)组SAA水平最高,明显高于副流感病毒(Parainfluenza virus,PIV)组(P<0.05),INFA组和呼吸道合胞病毒(Respiratory syncytial virus,RSV)组白细胞(White blood cells,WBC)明显高于其它4组,其中INFA组以单核细胞(Monocytes,MONO#)增高为主,RSV组以淋巴细胞(Lymphocyte,LYMPH)增高为主。结论:INFB,INFA和MP是垫江地区儿童最主要的呼吸道感染病原体,不同性别、不同年龄段和不同季节病原体感染率存在显著差异,且感染不同类型病原体后实验室检测结果也有所差异,应针对性采取有效的措施预防感染和合理诊治。     

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.