Time-resolved fluoroimmunoassay compared with virus isolation for rapid detection of respiratory syncytial virus in nasopharyngeal aspirates.

Two monoclonal antibodies against two distinct conserved epitopes of the respiratory syncytial virus (RSV) nucleocapsid protein were used in a direct time-resolved fluoroimmunoassay (TR-FIA) for the detection of RSV antigens in nasopharyngeal aspirates. The capture antibody was adsorbed to the solid phase of microdilution strip wells, and the indicator antibody was labeled with a europium chelate. Specimens and label were incubated simultaneously for 1 h at 37 degrees C in the coated wells. After the test samples were washed, fluorescence enhancement solution was added, strips were shaken, and the time-resolved fluorescence was measured. The test procedure took only 75 min, and the total time for 20 specimens, with pretreatment by sonication, was 2 to 3 h. We prospectively evaluated the detection of RSV in nasopharyngeal aspirates of pediatric patients by TR-FIA and by virus isolation in human diploid fibroblasts. TR-FIA detected 40 of 42 isolation-positive specimens. Nine additional isolation-negative specimens were positive by TR-FIA; all proved to be true positives by a blocking-type confirmatory assay. The sensitivity, specificity, positive predictive value, and negative predictive value for TR-FIA were 95, 96, 82, and 99%, respectively, of the values obtained by virus isolation and 96, 100, 100, and 99%, respectively, of the values obtained by virus isolation and the confirmatory assay.     

Monoclonal antibodies against respiratory syncytial virus and their use for rapid detection of virus in nasopharyngeal secretions.

We developed five monoclonal antibodies against respiratory syncytial virus. Three of these (23A3, 12A4, and 18B2) were used in an indirect fluorescent antibody test, and the results were compared with those of a similar indirect fluorescent test with commercial anti-respiratory syncytial virus serum. The results obtained with antibody 18B2 and commercial anti-respiratory syncytial virus serum were identical, whereas with antibodies 23A3 and 12A4 the incidence of positive identifications was around 50%.     

Rapid detection of respiratory syncytial virus antigens in nasopharyngeal secretions

The combined use of fluorescein-labelled monoclonal antibody and a cytocentrifuge for preparation of cell spots greatly reduced the time for rapid diagnosis, and improved the sensitivity and ease of detection of respiratory syncytial (RS) virus antigen in specimens of nasopharyngeal secretions.     

Rapid immunoperoxidase assay for detection of respiratory syncytial virus in nasopharyngeal secretions.

Samples of nasopharyngeal secretions obtained from 70 infants and young children with acute respiratory disease were examined for the presence of respiratory syncytial virus by immunoperoxidase assay (IPA). The IPA was compared with the immunofluorescence assay and with cell culture isolation. Respiratory syncytial virus antigen-positive cells were detected by both IPA and immunofluorescence assay in 28 specimens; 25 samples were positive in cell culture. The agreement between virus isolation and IPA and IFA was 89%. The applicability of IPA to rapid viral diagnosis of respiratory syncytial virus infection is discussed.     

Evaluation of the Becton Dickinson Directigen test for respiratory syncytial virus in nasopharyngeal aspirates.

A premarket trial of the Becton Dickinson Directigen respiratory syncytial virus membrane-based enzyme immunoassay compared the test with virus isolation for the detection of respiratory syncytial virus in 583 nasopharyngeal aspirates. After modification, the Directigen test showed a sensitivity of 83% and a specificity of 90%. It offers the potential for an efficient bedside test--without the need for any equipment--for the diagnosis of respiratory syncytial virus infection and requires only a 0.25-ml sample volume. However, for optimum reliability, freezing-thawing of samples and access to a confirmatory test were shown to be necessary.     

Commercial monoclonal antibodies for rapid detection of respiratory syncytial virus by direct immunofluorescence

A commercially available direct immunofluorescence (IF) test using a reagent consisting of a pool of two monoclonal antibodies against selected surface and internal proteins of respiratory syncytial virus (RSV) was evaluated in comparison with the indirect IF technique using a commercial bovine anti-RSV hyperimmune serum for the rapid detection of RSV in nasopharyngeal aspirates from 228 hospitalized children. Overall agreement between the two IF methods was 95%. The direct IF test was quicker to perform and easier to interpret than the indirect IF test.     

Evaluation of the QuickLab RSV test, a new rapid lateral-flow immunoassay for detection of respiratory syncytial virus antigen

Rapid respiratory syncytial virus (RSV) diagnosis is vital to the prevention of nosocomial RSV infections. We evaluated a new rapid lateral-flow RSV immunoassay, the QuickLab RSV test, that requires use of only one reagent. We compared QuickLab to the Directigen RSV (DIR) assay, which requires six reagents, and direct fluorescent antibody (DFA) testing. DFA results were considered the "gold standard." For 133 nasopharyngeal aspirates tested, DFA results were 77 (57.8%) positive, 47 (35.3%) negative, and 9 (6.8%) indeterminate. The sensitivities, specificities, positive predictive values, and negative predictive values of QuickLab and DIR tests were 93.3% (70 of 75) and 80.8% (59 of 73), 95.6% (43 of 45) and 100.0% (46 of 46), 97.2% (70 of 72) and 100.0% (59 of 59), and 89.6% (43 of 48) and 76.7% (46 of 60), respectively. QuickLab was significantly (P = 0.02) more sensitive than DIR; the difference in specificities was not significant. DFA was more sensitive than DIR (P < 0.001) but not more sensitive than QuickLab (P = 0.45). The results of DIR testing were initially uninterpretable and required retesting with 15% of the specimens compared to 3% of QL results (P < 0.001). We conclude that the QuickLab RSV test has sensitivity similar to that of the DFA assay and better than that of the DIR assay. QuickLab testing is also simpler to perform and interpret than both DFA and DIR testing.     

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.     

Comparison of a new commercial enzyme immunoassay for rapid detection of respiratory syncytial virus

Two rapid methods for detection of respiratory syncytial virus in respiratory specimens were compared: direct immunofluorescence assay (DFA) with monoclonal antibody and an enzyme immunoassay (EIA) (Test-Pack RSV). Ninety-five nasopharyngeal washings and aspirates from 51 children were examined; the patients were hospitalized during a winter outbreak of RSV infection in the first trimester of 1990. A total of 41.0 % and 56.8 % of these samples were positive by EIA and DFA respectively. Considering only the 51 specimens collected at the onset of illness, EIA detected 72.5 % positive samples and DFA detected 78.4 %. In comparison with DFA, EIA was 92.5 % sensitive and 100 % specific for the acute phase of illness. When all the samples were taken into account, specificity was maintained but sensitivity fell to 72.2 %. The results show that both methods are useful during the acute phase of the illness, when the viral load is important. However, later on in the course of the infection DFA appears to be more sensitive than EIA.     

Diagnostic efficacy of two rapid tests for detection of respiratory syncytial virus antigen.

With the availability of ribavirin therapy for serious respiratory syncytial virus (RSV) infections, rapid diagnostic tests for the detection of RSV antigen are increasingly important. Efficacies of a commercially available enzyme immunoassay (EIA) (Abbott Laboratories, North Chicago, Ill.) and a fluorescent-antibody assay (FA) were evaluated in a study involving 135 specimens from children with respiratory symptoms. A nasal wash specimen was cultured immediately on RSV-sensitive A549 cells; the nasal wash was also used for EIA. FA was performed on a nasopharyngeal swab specimen with bovine anti-RSV and anti-bovine immunoglobulin G antisera (Burroughs Wellcome Co., Research Triangle Park, N.C.). A total of 39 specimens (28%) were tissue culture positive, including 35 EIA-positive and 37 FA-positive samples (sensitivities, 90 and 95%, respectively). All 96 tissue culture-negative specimens were EIA negative (specificity, 100%); 94 of these 96 specimens were FA negative (specificity, 98%). Positive and negative predictive values for the tests were as follows: 100 and 96% for EIA, respectively, and 95 and 98% for FA, respectively. Other viruses, including influenza A virus, adenovirus, enterovirus, and herpes simplex virus, were isolated in nine cases. One adenovirus-positive specimen had a false-positive RSV FA result; all nine specimens were RSV EIA negative. Both tests performed well in our study and provide cost-effective alternatives to tissue culture. The RSV EIA, in particular, uses standard serologic techniques and equipment and does not require expertise in virology. More widespread availability of rapid diagnostic tests for RSV will hopefully result in early and appropriate use of antiviral therapy in patients at risk for serious RSV infections.     

Reliability of two new test kits for rapid diagnosis of respiratory syncytial virus infection.

Two new rapid enzyme immunoassays (EIAs) for detecting respiratory syncytial virus (RSV), Directigen (Becton Dickinson Microbiology Systems) and TestPack (Abbott Diagnostics) were compared with virus isolation and direct immunofluorescence by using fresh specimens. The sensitivities of both EIAs were low (72 to 73%), but when initial specimens were used, TestPack had a high sensitivity (92%) in contrast to that of Directigen (76%). Because of its high sensitivity and specificity, TestPack can be used for diagnosis of RSV in acute disease.     

A reverse passive haemagglutination test for the detection of respiratory syncytial virus in nasal secretions from infants

A reverse passive haemagglutination (RPH) test has been developed for the detection of respiratory syncytial (RS) virus in nasal secretions, taken from infants with acute respiratory illness. In the final form of the procedure, RS virus was detected in 24 of 25 samples positive for RS virus by tissue culture and/or fluorescence antibody staining and in two samples negative for RS virus by these techniques. The simplicity of the technique and the rapidity with which it may be performed together with its apparently high degree of sensitivity should make RPH useful in the rapid diagnosis of RS virus.     

Antibiotic use in children is not influenced by the result of rapid antigen detection test for the respiratory syncytial virus.

BACKGROUND: Rapid antigen detection test (RADT) for respiratory syncytial virus (RSV) is widely used in children hospitalized with acute respiratory tract infection (ARTI), but its influence on antibiotic (AB) use is uncertain. OBJECTIVE: To evaluate if confirmation of RSV infection by RADT modified AB use and elucidate others factors associated with the continuation of antibiotics. STUDY DESIGN: Charts of children hospitalized with viral ARTI aged 0-35 months were reviewed. Modification of antibiotics according to RSV RADT results was compared using Kaplan-Meier estimates and multivariate Cox regression. RESULTS: Of children receiving antibiotics when the RSV RADT result was available, RSV RADT was positive in 144 and negative in 54. Positive RSV RADT results did not lead to modification of antibiotic use. Factors independently associated with cessation of intravenous antibiotics were age > or = 3 months (HR 2.44 [1.41-4.21]) and absence of pneumonia (HR 1.50 [1.03-2.19]). Absence of otitis was associated with cessation of oral antibiotics (HR 9.16 [95% CI, 2.35-35.76]). CONCLUSION: Confirmed presence of RSV by RADT did not influence antibiotic use in young children with ARTI. Except with pneumonia, the risk of bacterial superinfection of RSV infected children is minimal and confirmation of RSV infection should prompt treating physicians to interrupt antibiotics.     

Evaluation of three rapid enzyme immunoassays and cell culture for detection of respiratory syncytial virus

Three rapid enzyme immunoassay techniques for the detection of respiratory syncytial virus antigen (Becton Dickinson Directigen RSV, Abbott RSV Testpack and Abbott RSV EIA) and cell culture were evaluated in a total of 250 nasal washings. The sensitivity and specificity were 62 % and 76 % respectively for Directigen, 64 % and 86 % for RSV Testpack, and 76 % and 81 % for RSV EIA, taking cell culture as the reference method. Agreement between cell culture and EIA techniques was 79 % (70 positive and 128 negative results). All three EIA techniques gave positive results in 69 samples (52 positive and 17 negative in the cell culture). In 121 samples all three EIA techniques gave negative results (103 negative and 18 positive in the cell culture). Using the cell culture technique 46 strains other than respiratory syncytial virus were isolated.     

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay.

A commercial enzyme immunoassay (EIA) for the rapid detection of respiratory syncytial virus (RSV) in respiratory secretions was evaluated by comparison with both virus isolation in HEp-2 cells and indirect immunofluorescence (IFA) staining of exfoliated respiratory cells. Initial examination of 80 nasopharyngeal aspirates collected from infants with acute respiratory illness showed that the RSV EIA was positive for 21 of 24 specimens positive by virus isolation or IFA (87.5% sensitivity) and negative for 53 of 56 specimens negative by virus isolation and IFA (95% specificity). The EIA appears to be an acceptable and more rapid test than virus isolation for the detection of RSV, especially for laboratories in which prompt inoculation of specimens is not always possible. IFA staining with commercial bovine anti-RSV serum was found to be the most sensitive and rapid test for the detection of RSV. However, three of four specimens positive by IFA and negative by virus isolation were not cultured under optimal conditions. In addition, the IFA test requires a highly trained technologist to interpret the staining results.     

Rapid detection of respiratory syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper.

We compared the Rhinoprobe scraping technique for collection of superficial nasal mucosa epithelial cells and rapid detection of respiratory syncytial virus by immunofluorescence with paired, swab-collected specimens for virus culture from 1,257 infants and children with acute respiratory infections. Compared with viral culture as the reference test, the sensitivity, specificity, and accuracy of the immunofluorescence test were 83.6, 93.6, and 91.3%, respectively. We found the Rhinoprobe method safe, easy to use, and helpful in obtaining large quantities of epithelial cells for detection of respiratory syncytial virus and other respiratory viruses.     

A simple and rapid microassay for the titration of human respiratory syncytial virus

A microassay, using tissue culture microplates for the titration of human respiratory syncytial virus by syncytium formation, is described. Virus titers obtained agreed well with those obtained in a larger assay system; the microassay, however, is more rapid and economical. Large numbers of virus samples are easily and rapidly processed as the assay necessitates an incubation period of only three days.     

Comparison of an immunochromatography test with multiplex reverse transcription-PCR for rapid diagnosis of respiratory syncytial virus infections

A new commercial rapid 10-min one-step immunochromatography (IC) test, SAS RSV test, was compared to another IC test, Directigen EZ RSV, employing RT-PCR as the "gold standard" for detecting respiratory syncytial virus. Of 102 clinical samples, 79 were positive by RT-PCR, 66 (82.5%) were positive with the SAS RSV test, and 55 (69.6%) were positive with Directigen EZ RSV. The specificity of the new test was 91.3% (21 of 23), similar to that of Directigen EZ RSV (100% [23 of 23]). This test performs well enough to be used for patient care.     

Evaluation of the Abbott TESTPACK RSV enzyme immunoassay for detection of respiratory syncytial virus in nasopharyngeal swab specimens.

The Abbott TESTPACK RSV assay (Abbott Laboratories, North Chicago, Ill.), a rapid (20-min) enzyme immunoassay, was compared with culture and direct immunofluorescence (DFA) of nasopharyngeal cells for the detection of respiratory syncytial virus (RSV) in nasopharyngeal swab specimens. Nasopharyngeal swab specimens, collected from 234 infants, were placed in viral transport medium. Portions of specimen in transport medium were used for each test. Of 234 specimens, 70 (30%) were culture positive, 103 (44%) were DFA positive, 107 (46%) were culture or DFA positive, and 112 (48%) were TESTPACK RSV positive. Of 19 specimens positive by TESTPACK RSV but negative by culture or DFA, 15 were positive by the blocking assay. A total of 122 specimens were culture, DFA, or blocking assay positive; TESTPACK RSV detected 108 specimens (sensitivity, 89%). The specificity, positive predictive value, and negative predictive value of TESTPACK RSV as compared with those of culture, DFA, and the blocking assay were 96, 96, and 89%, respectively. By comparison, the sensitivity, specificity, positive predictive value, and negative predictive value of combined culture and DFA were 88, 100, 100, and 88%, respectively. TESTPACK RSV is a rapid and reliable enzyme immunoassay for the direct detection of RSV antigen in nasopharyngeal swab specimens.