Comparison of the VIDAS RSV assay and the Abbott Testpack RSV with direct immunofluorescence for detection of respiratory syncytial virus in nasopharyngeal aspirates.

The sensitivity and accuracy of the VIDAS RSV assay in testing fresh specimens were 82.7 and 87.1%, respectively, whereas specimens previously frozen at -70 degrees C gave a sensitivity of 96.2% and an accuracy of 95.4%. The sensitivity and accuracy of Abbott Testpack RSV were 92.6 and 91.3% for fresh specimens and 86.8 and 88.1% for frozen specimens. The advantages and drawbacks of the two assays are discussed.     

Comparison of two rapid methods for detection of respiratory syncytial virus (RSV) (Testpack RSV and ortho RSV ELISA) with direct immunofluorescence and virus isolation for the diagnosis of pediatric RSV infection.

The ability of two commercial immunoassays to detect respiratory syncytial virus (RSV) in respiratory specimens was evaluated as follows: 152 specimens were tested by TestPack RSV (Abbott), and 72 were tested by Ortho RSV ELISA (Ortho). Test outcomes were compared with those of virus isolation alone, direct immunofluorescence assay (DFA) alone, or virus isolation and/or DFA. TestPack RSV versus virus isolation showed 91% sensitivity, 96% specificity, 93% positive predictive value (PPV), and 95% negative predictive value (NPV). TestPack RSV versus DFA showed 89% sensitivity, 97% specificity, 96% PPV, and 93% NPV. When TestPack RSV performance was compared with that of virus isolation and DFA, the sensitivity was 87% and the specificity was 100%. Ortho RSV ELISA versus virus isolation showed 88% sensitivity, 87% specificity, 79% PPV, and 93% NPV. Ortho RSV ELISA versus DFA showed 91% sensitivity, 88% specificity, 81% PPV and 95% NPV. When Ortho RSV ELISA performance was compared with that of virus isolation and DFA, the sensitivity was 86%, the specificity was 89%, the PPV was 86%, and the NPV was 89%. The accuracy of the TestPack RSV in combination with ease of performance and no need for specialized equipment or special skills make it an attractive alternative to DFA for rapid direct detection of RSV.     

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 Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus.

Directigen FLU-A, an enzyme immunoassay membrane test, was compared prospectively to isolation in cell culture and direct immunofluorescence (IF) for the detection of influenza A virus. One hundred ninety specimens were evaluated by Directigen FLU-A and cell culture; 184 of these specimens were also tested by direct IF. The sensitivity of Directigen FLU-A compared to isolation in cell culture and direct IF was 100%. The specificities of Directigen FLU-A compared to isolation and direct IF were identical, 91.6%. Fourteen specimens that were positive by Directigen FLU-A did not yield virus in culture; two of the specimens, however, were positive by direct IF, and four other specimens were not specimens of choice for the test. A positive Directigen result had positive predictive values of 62.6 and 75.0% compared to isolation and direct IF, respectively; a positive Directigen result with an intensity reading of 2+ or greater, however, had positive predictive values of 85 and 100% compared to isolation and direct IF, respectively. In all comparisons, the negative predictive value was 100%. There was no evidence that cross-reactivity occurred with non-influenza A antigens. Directigen FLU-A should serve as a convenient screening test for influenza A and as a rapid test supported by isolation in cell culture during an influenza outbreak.     

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.     

Comparison of rapid immunofluorescence procedure with TestPack RSV and Directigen FLU-A for diagnosis of respiratory syncytial virus and influenza A virus.

A rapid immunofluorescence format requiring 20 min for completion was as effective as conventional indirect and direct immunofluorescence procedures for detecting respiratory syncytial virus and influenza A virus antigens in clinical specimens. Rapid immunofluorescence was more sensitive than TestPack RSV and comparable to Directigen FLU-A immunosorbent assays, which require 20 min for completion.     

A comparison of Thermo Electron RSV OIA to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

BACKGROUND: Rapid diagnostic methods for respiratory syncytial virus are useful tools available for the clinician. OBJECTIVES: The Thermo Electron RSV OIA (optical immunoassay kit) was prospectively compared with direct immunofluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah. STUDY DESIGN: Specimens from three hundred and thirty patients exhibiting respiratory symptoms were collected for testing by the three methods above. Several specimens were positive by both OIA and DFA with a negative culture result. These culture results were verified by RT-PCR analysis. RESULTS: Overall, 107 specimens were positive for RSV by the reference tests (culture or RT-PCR). DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests the sensitivity, specificity, positive, and negative predictive values of the OIA for detection of RSV were 87.9%, 99.6%, 98.9% and 94.5%, respectively. CONCLUSIONS: The rapid OIA assay format proved to be cost effective, and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.     

Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen.

Immunofluorescence staining methods for respiratory syncytial virus antigen detection were compared. Of 50 specimens originally positive for respiratory syncytial virus by direct immunofluorescence and culture, 49 were positive by repeat direct immunofluorescence and 32 were positive by indirect immunofluorescence. Additional results obtained on specimens originally negative for respiratory syncytial virus by direct immunofluorescence, culture, or both indicate that direct immunofluorescence staining for respiratory syncytial virus antigen was more sensitive than was indirect immunofluorescence.     

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%.     

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.     

Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay.

We evaluated prospectively the detection of respiratory syncytial virus (RSV) by culture and by direct antigen detection using an indirect immunofluorescence assay (IFA), a direct monoclonal immunofluorescence assay (DFA), and a monoclonal enzyme immunoassay (EIA). Of 221 specimens, 95 (43%) were culture positive for RSV, 4 (1.8%) contained more than one virus, and 17 (7.6%) contained a virus other than RSV. Overall, HEp-2 and Flow 6000 cells grew significantly more RSV isolates (82 and 72%, respectively) than A549 cells, which grew only 29% of the isolates. The mean time for RSV detection with HEp-2 cells was 2.9 days. This was significantly less than the mean time for RSV detection with either Flow 6000 cells (6.1 days) or A549 cells (6.4 days). Of 221 specimens, 129 were tested simultaneously by culture, IFA, and DFA. Of these 129 specimens, 62 (48%) were positive by culture, 69 (53%) were positive by IFA, and 70 (54%) were positive by DFA. For 92 specimens screened simultaneously by culture, IFA, and EIA, positive results were obtained for 33 (36%) of the specimens by both culture and IFA and for 29 (32%) of the specimens by EIA. Of 126 culture-negative specimens, 21 (17%) were positive for RSV when determined by IFA. Conversely, 14 (15%) of 95 RSV culture-positive specimens were negative by IFA, whereas DFA missed 19% of the culture-positive specimens. Compared with culture, the Kallestad EIA kit had a sensitivity and specificity of 73 and 92% respectively, but missed 9 (27%) of 33 culture-positive specimens. These data demonstrate that isolation by culture continues to be important for viral diagnosis of REV infections and that for valid comparative studies between viral isolations and rapid detection methods, both sensitive host cells and appropriate test conditions are required.     

Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion.

We identified by immunochemical methods 13 polypeptides associated with the infectious respiratory syncytial virus virion. Eight of these polypeptides (VP200, VP84, VP66, VP43, VP40, VP37, VP28, and VP19) were identified as virus specific. Two other polypeptides, (VP) 22 and (VP) 12, are provisionally considered to be of viral origin. Three nonviral proteins are also intimately associated with the infectious virion. These nonviral proteins were identified as cellular actin and two proteins with bovine serum albumin immunospecificity. VP40 was identified as the major ribonucleoprotein. Based on biochemical and biophysical similarities with paramyxovirus proteins, other respiratory syncytial virus proteins are believed to have these specific viral functions: VP84, "hemagglutinin"; VP66, undissociated fusion protein, F1,2; VP43, F1; and VP19, F2, VP66 contains a major determinant involved in viral infectivity since all neutralizing antibodies tested, including a monoclone, precipitated this protein.     

Comparison of viral isolation, direct immunofluorescence, and indirect immunoperoxidase techniques for detection of genital herpes simplex virus infection.

Seventy-six consecutive patients presenting to a genital herpes simplex virus (HSV) clinic were enrolled in a study comparing viral isolation (VI), indirect immunoperoxidase (indirect IP), and direct immunofluorescence (direct FA) techniques for the detection of HSV antigen. Of the 76 patients, 61 (80%) demonstrated HSV by VI, compared with 66% by indirect IP and 55% by direct FA (P less than 0.05). Genital lesions from nine patients demonstrated HSV antigen by direct FA or indirect IP but were VI negative; eight of nine patients had subsequent episodes of genital HSV confirmed by VI. During the vesicular-pustular stage of the disease, VI was positive in 90%, indirect IP was positive in 76%, and direct FA was positive in 71% of the lesions, whereas with ulcerative lesions, VI was positive in 72%, indirect IP was positive in 55%, and direct FA was positive in 38%. These commercially available rapid viral diagnostic techniques are specific and useful, if adequate specimens are obtained from early genital lesions.     

Comparison of immediate and delayed inoculation of HEp-2 cells for isolation of respiratory syncytial virus.

Immediate inoculation of HEp-2 cells is generally advocated for the isolation of respiratory syncytial virus. However, delayed laboratory inoculation of properly transported specimens obtained by aspiration of nasopharyngeal mucus provided an isolation rate similar to that obtained with immediate inoculation.     

Microwave-accelerated direct immunofluorescent staining for respiratory syncytial virus and influenza A virus.

A microwave-accelerated direct immunofluorescence staining method which requires only 20 min from specimen receipt to interpretation is as effective as conventional methods for detecting respiratory syncytial virus and influenza A virus antigens in clinical specimens. The time required compares favorably with that for the less sensitive Abbott Test Pack RSV.     

Separation and concentration of respiratory syncytial virus (RSV) antigens

Respiratory syncytial virus has been concentrated by precipitation with polyethlene glycol-6000 and partially separated from cellular debris by sucrose-gradient centrifugation. Treatment of the concentrated virus with 0.5% sodium deoxycholate results in a resolution of this material into two fractions with different (1.27 and 1.19 g/cm³) densities and distinct antigenic properties. The materials separated are of sufficient antigenic purity for use as reagents in radioimmunoassay procedures.     

Routine viral culture for pediatric respiratory specimens submitted for direct immunofluorescence testing.

From 1986 to 1987, 69 (25%) of 274 specimens from children with lower respiratory tract syndromes were positive for respiratory syncytial virus antigen by direct immunofluorescence assay (DFA). Comprehensive viral culture was performed on all 205 DFA-negative specimens, and 72 specimens yielded viruses; 5 specimens yielded multiple agents. Thus, 52% of specimens yielded a specific virus, supporting the routine use of viral culture. Isolates from the DFA-negative specimens included respiratory syncytial virus (n = 7), rhinovirus (n = 34), hemadsorbing viruses (n = 13), cytomegalovirus (n = 11), adenovirus (n = 8), enteroviruses (n = 3), and herpes simplex virus (n = 2). Although serologic confirmation is needed, cytomegalovirus may be an underappreciated cause of acute lower respiratory tract infection in normal children. Further studies must be conducted to document this possibility.     

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.