Comparison of directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syncytial virus.

An enzyme immunoassay membrane test (Directigen RSV) for the detection of respiratory syncytial virus in clinical specimens was compared prospectively with isolation in cell culture and direct immunofluorescence (IF). A total of 315 nasopharyngeal wash specimens from pediatric patients were examined. Directigen RSV was 86.1% sensitive and 91.3% specific for specimens positive by isolation in cell culture and/or IF, with 88.6% agreement. The false-positive rate was 16%; 2 of 20 specimens giving false-positive reactions by Directigen RSV were true-positives by blocking assay. Twenty-seven specimens (8.5%) whose results were initially uninterpretable by Directigen RSV due to filtration difficulties were diluted and upon retesting produced acceptable results. Sixty-three viral isolates and/or IF identifications of virus antigens representing seven virus groups other than respiratory syncytial virus were also found; cross-reactions between Directigen RSV and other viruses were not observed. Directigen RSV will be useful as an immediate procedure and in facilities lacking a comprehensive virology laboratory.     

Evaluation of the Binax NOW, BD Directigen, and BD Directigen EZ assays for detection of respiratory syncytial virus

The Binax NOW assay (Binax, Inc., Portland, Maine) and the BD Directigen EZ assay (Becton Dickinson and Company, Sparks, Md.), two new rapid immunoassays for detection of respiratory syncytial virus (RSV), as well as the BD Directigen RSV assay (DRSV) (Becton Dickinson and Company) and direct immunofluorescence staining (DFA) were compared with culture for detection of RSV in fresh specimens from both children and adults during the 2002-2003 respiratory virus season. The majority (95%) of specimens were nasal or nasopharyngeal washes or aspirates. A total of 47 (26%) were culture positive for RSV. The overall sensitivities of DFA (n = 149), NOW (n = 118), EZ (n = 88), and DRSV (n = 180) compared with culture (n = 180) were 93, 89, 59, and 77%, respectively. The specificities of DFA, NOW, EZ, and DRSV were 97, 100, 98, and 96%, respectively. However, when results were separated into those from children and those from adults, DFA was the only rapid test adequate for detection of RSV (sensitivity of 100% compared to 0, 0, and 25% for NOW, EZ, and DRSV, respectively) in adults. For children the sensitivities of DFA, NOW, EZ, and DRSV were 93, 94, 72, and 81%. The NOW assay was the most sensitive and specific and the easiest to perform of the kit tests for detecting RSV in children. None of these three rapid kit tests was sensitive for detecting RSV in specimens from adults. DFA remains the rapid method of choice for detecting RSV in the adult population.     

Performance characteristics of VIDAS and directigen respiratory syncytial virus (RSV) antigen detection assays and culture for the identification of RSV in respiratory specimens

In a comparison of the Directigen and VIDAS respiratory syncytial virus antigen detection assays with viral culture, the sensitivity, specificity, positive and negative predictive values, and testing efficiency were 86, 93.1, 82.7, 94.6, and 91.2% for Directigen; 96.1, 90.8, 80.3, 98.3, and 92.3% for VIDAS; and 88.2, 100, 100, 95.7, and 96.8% for viral culture, respectively.     

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.     

Comparison of VIDAS with direct immunofluorescence for the detection of respiratory syncytial virus in clinical specimens.

BACKGROUND: requirements for infection control measures and the decision for treatment with antiviral agents make the rapid detection of respiratory syncytial virus (RSV) essential for hospitalized, pediatric and immunocompromised patients. Immunofluorescence is considered to be the most rapid and sensitive method for direct detection of RSV in clinical specimens, but several enzyme-linked immunoassays have also been commercially available. OBJECTIVES: to compare the performance of VIDAS RSV assay (Vitek ImmunoDiagnostic Assay System, BioMerieux Vitek), which is an automated enzyme-linked fluorescent immunoassay (ELFA) to direct immunofluorescence (DFA), in rapid detection of RSV in respiratory specimens. STUDY DESIGN: respiratory specimens collected during the 'RSV season', between the months of November 1997 and February 1998, were tested by both methods. DFA was performed upon receipt of the sample and an aliquot of the original specimen was stored in -70 degrees C for batch testing with VIDAS. RESULTS: from 238 samples that were tested, 231 could be evaluated by both methods. The two assays were in agreement for 213 specimens (92%), or 32 positive and 181 negative results. Eighteen discrepant results were generated; seven specimens were VIDAS-/DFA+ and 11 specimens were VIDAS+/DFA-. In addition, seven specimens had an inadequate number of cells for evaluation with DFA. One of these samples tested positive with VIDAS. VIDAS relative sensitivity and specificity were 82% and 94%, respectively, when compared to DFA. CONCLUSIONS: VIDAS is simple to perform, does not require expertise in interpretation and appears to be an acceptable method for the rapid detection of RSV.     

Evaluation of five methods for respiratory syncytial virus detection.

A total of 117 nasal aspirates were cultured for respiratory syncytial virus (RSV) and tested for RSV antigen by a direct fluorescent-antibody (DFA) test (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), the Directigen enzyme immunoassay (EIA; Becton Dickinson Microbiology Systems, Cockeysville, Md.), the TestPack EIA (Abbott Laboratories, North Chicago, Ill.), and RSV EIA (Abbott). Agreement of two of five methods or a positive RSV culture were required to validate a result. A total of 57 of 117 (48.7%) specimens were culture positive in HEp-2 cells, A549 cells, or both. A total of 5 of 117 (4.3%) additional specimens met the criteria of a positive specimen; i.e., 62 of 117 (53.0%) specimens were positive. Results obtained from 77 of 117 (65.8%) specimens were concordant for all five methods. The sensitivities, specificities, and positive and negative predictive values for the culture and DFA methods were 91.9, 100, 100, and 91.7% and 91.9, 96.4, 96.6, and 91.4%, respectively. The sensitivities, specificities, and positive and negative predictive values for the three EIA procedures, Directigen, TestPack, and RSV EIA, were 75.8, 80.0, 81.0, and 74.6%; 93.6, 100, 100, and 93.2%; and 71.0, 100, 100, and 75.3%, respectively. New self-contained EIA configurations and the DFA method offer attractive alternatives to the culture method. Technical simplicity, rapid turnaround time, performance, and cost must all be considered when selecting a system for RSV detection.     

Immunofluorescence with respiratory syncytial virus

The growth of respiratory syncytial (RS) virus in tissue cultures of HEp-2 cells at a low multiplicity of infection has been studied by means of several staining procedures including immunofluorescence, as well as by serial infectivity titrations.Specific fluorescent staining was first noted 10 hours after infection and was restricted entirely to the cytoplasm throughout the growth cycle.A significant increase in the amount of infectious virus present in the system was first detected 10 hours after inoculation, and peak values were achieved after 48 hours.The pattern of development of RS antigen has been compared with that of other medium-sized ether-sensitive agents and possible taxonomic implications are discussed.     

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.     

Comparison of the RSV respi-strip with direct fluorescent-antigen detection for diagnosis of respiratory syncytial virus infection in pediatric patients

The RSV Respi-Strip was compared to the SimulFluor respiratory screen for detecting respiratory syncytial virus in nasopharyngeal aspirates from pediatric patients. Of samples tested, 115/239 (49%) were positive by direct fluorescent-antigen detection. The sensitivity and specificity of the RSV Respi-Strip were 92% (95% confidence interval [CI], 86% to 96%) and 98% (95% CI, 94% to 100%), respectively, with a diagnostic efficiency of 95%.     

Comparison of three rapid diagnostic techniques for detection of respiratory syncytial virus from nasal wash specimens.

We report results of three rapid tests for respiratory syncytial virus antigen detection. An immunofluorescence assay using commercial antibody and two commercial enzyme immunoassays (Ortho Diagnostics, Inc., Raritan, N.J., and Abbott Laboratories, North Chicago, Ill.) were applied to 199 nasal wash specimens. The Abbott enzyme immunoassay was the most sensitive technique, with a sensitivity of 93.8%. The specificities of the three techniques were comparable and greater than 95%. The availability of reliable rapid diagnostic techniques will allow for better care of infants with severe respiratory syncytial virus infection.     

Serological studies with respiratory syncytial virus

Summary Cross-neutralization tests with 9 strains of RS virus and antisera prepared against them by intra-nasal infection of ferrets, showed that antigenic variation does occur among RS virus strains. One strain, 8/60, differed from all the other strains tested, and there were smaller differences between some other strains.     

A rapid test for detection of respiratory syncytial virus in nasopharyngeal secretion

A new rapid membrane enzyme immunoassay (MEIA; Directigen RSV) for detection of respiratory syncytial virus (RSV) was evaluated using samples of nasopharyngeal secretion from infants and children with acute respiratory disease. The MEIA was compared with an immunofluorescent antibody (IF) technique using a sensitive biotin-avidin (BA) EIA as reference. Of 242 samples tested, 108 were positive by the MEIA and 123 by the BA-EIA. Of 144 samples which were also tested by the IF technique, 57 were positive by the BA-EIA and 43 by the IF technique. These results give a sensitivity of 86 % and 72 % for the MEIA and IF technique respectively. Of 57 samples found to be positive by the BA-EIA, 41 were positive by the IF technique, but 48 were positive by the MEIA. The MEIA is thus more sensitive than the IF technique but less sensitive than the BA-EIA in detecting RSV in nasopharyngeal secretions.     

Practical recommendations for the detection of pediatric respiratory syncytial virus infections.

In our private clinic-hospital setting, respiratory syncytial virus (RSV) was isolated from infants more frequently and sooner from nasal washes (84%; 4.2 days) than from throat swabs (45%; 5.5 days) or nasopharyngeal swabs (39%; 5.7 days). Immunofluorescence of nasal wash cells identified 72% of the infants with virus isolations from nasal washes in less than one day. We therefore recommend the combination of isolation and immunofluorescence on nasal wash specimens for optimal detection of RSV-infected infants. Immunofluorescence of respiratory tract cells was also useful for monitoring the presence of RSV antigen in intubation secretions during ribavirin antiviral therapy. RSV infectivity was maintained in phosphate-buffered saline at room temperature for 6 h. Transport and inoculation of specimens in less than 6 h yielded RSV isolates from 50% of sampled infants during the two RSV seasons examined. For optimal RSV isolation, we recommend inoculation of HEp-2 tubes less than or equal to 4 days old. Replacing medium after 3 days as compared with 7 days did not increase recovery of RSV and provided little practical reduction in time to detection of cytopathology.     

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.     

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.