Comparison of Binax NOW and Directigen for rapid detection of influenza A and B.

Directigen Flu A + B and Binax NOW Flu A and Flu B tests detected 33 (55.9%) and 31 (52.5%) of 59 influenza-positive samples, respectively. In children under 2 years of age, sensitivity increased to 75% for both tests. Three samples tested falsely-positive for influenza B using Binax NOW.     

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.     

Performance of a rapid assay (Binax NOW) for detection of respiratory syncytial virus at a children's hospital over a 3-year period

A rapid assay, Binax NOW RSV, was compared to viral culture for 14,756 pediatric respiratory specimens obtained from 2003 to 2006. There were 794 viral culture-confirmed respiratory syncytial virus infections. Sensitivity was 81%, and specificity was 93.2%. Sensitivity was greatest for neonates (91.1% versus 80.7% [P < 0.01]).     

Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients. OBJECTIVES: To rapidly and accurately quantify and subtype RSV in respiratory samples, we developed and evaluated two real-time RT-PCR assays. STUDY DESIGN: A quantitative assay was designed using primers for a consensus region of the matrix protein gene and a subtype-specific assay for RSV-A and RSV-B detection was designed using primers for the polymerase gene. Quantitative RSV RT-PCR results of pediatric nasal wash samples submitted to the University of Washington Virology Laboratory from December 2002, through May 2003, were compared to those of an indirect fluorescent antibody RSV antigen detection assay (FA). RESULTS: Specificity of the RT-PCR assay was high, with no amplification of eleven common respiratory viruses and eight herpes viruses. Among 751 samples, RSV was detected in 267 (35.6%) by FA and in 286 (38.1%) by RT-PCR. Median RSV copy number in nasal wash samples that were positive by both FA and RT-PCR was 2.5 x 10(7) copies/mL versus a median of 3.0 x 10(4) copies/mL for samples positive by RT-PCR only (P < 0.001). The detection and quantity of RSV in respiratory specimens was associated with younger age, but not with gender or hospitalization. Among positive samples from this Seattle cohort, 52% were subtype A and 48% were subtype B. Both subtypes were detected with similar viral loads among all patient groups (stratified by age, gender, and hospitalization), and throughout the specimen collection period. CONCLUSIONS: These real-time RT-PCR assays provide a rapid, specific, and highly sensitive alternative for detecting, quantifying, and subtyping RSV in clinical specimens.     

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.     

Evaluation of an enzyme membrane immunoassay (Directigen RSV) for the rapid diagnosis of respiratory syncytial virus infections

A rapid enzyme immunoassay (EIA) membrane test, the Directigen respiratory syncytial virus (Becton Dickinson), was compared with cell culture, an indirect immunofluorescence (IF) test, the Monofluokit respiratory syncytial virus (Diagnostics Pasteur), and a conventional enzyme immunoassay antigen test, the Abbott respiratory syncytial virus enzyme immunoassay in nasal aspirates specimens from children with suspected respiratory syncytial virus (RSV) bronchiolitis. The sensibility and specificity of the Directigen, respiratory syncytial virus were 91% and 98% respectively, as compared with those of culture, and of 93% and 86% as compared with the Monofluokit respiratory syncytial virus. In the comparison of the two enzyme immunoassays, Directigen respiratory syncytial virus detected more positive specimens: 68/127 than the other: 46/127. Directigen respiratory syncytial virus is a very rapid test, (15-min), sensitive and specific, which can be used as an alternative technique for detection of respiratory syncytial virus in nasal samples when viral isolation or immunofluorescence direct are not available.     

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.     

Respiratory syncytial virus detection by Remel Xpect, Binax Now RSV, direct immunofluorescent staining, and tissue culture

The performance characteristics of Xpect RSV (XP) and Binax Now RSV (BN) were compared to those of direct fluorescent-antibody staining and/or tissue culture for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirate and wash samples from children (n = 110) and adults (n = 66). The sensitivity, specificity, positive predictive value, and negative predictive value of XP were 75%, 98%, 95%, and 90%, respectively; and those of BN were 74%, 100%, 100%, and 90%, respectively. The performances of the assays were similar within a given age group and specimen type (nasopharyngeal aspirate or wash specimen). XP and BN are useful for screening for RSV in respiratory specimens when large volumes are tested or low levels of staffing occur.     

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.     

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.     

Culture vs direct antigen assays for detection of microbial pathogens from lower respiratory tract specimens suspected of containing the respiratory syncytial virus

Following the introduction of effective antiviral chemotherapy, rapid antigen assays have been utilized increasingly, instead of cell cultures, for detection of the respiratory syncytial virus from lower respiratory tract specimens. Because antigen assays, unlike cell culture, cannot amplify low levels of the virus to a detectable level, assay sensitivity is especially dependent on high-quality specimens. In addition, the assays are unable to detect other viruses or bacteria with which the patient may be infected. This review summarizes results from clinical studies of the performance of cell cultures and the more commonly used antigen assays, describes factors that may lead to false-positive or false-negative test results, and makes recommendations for the selection of procedures for the reliable detection of microbial pathogens from patients suspected of being infected with respiratory syncytial virus.     

Prospective evaluation of a dot-blot enzyme immunoassay (Directigen RSV) for the antigenic detection of respiratory syncytial virus from nasopharyngeal aspirates of paediatric patients

This study investigated the efficacy of a commercial enzyme immunoassay (Directigen RSV, ColorPAC) in comparison with the shell vial culture method (using Hep-2 cells) for the detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates from children with bronchiolitis. During the period 1995-2002, 4950 samples were examined. RSV was detected in 1660 (33.5%) samples, with a sensitivity of 80.9%, a specificity of 97.5%, a positive predictive value of 93.8%, a negative predictive value of 91.6%, and a testing efficiency value of 92.2% compared with shell vial culture. In 83 (5%) samples, the ColorPAC was positive and the shell vial assay was negative. Of these, 71 (85.6%) were false-negative by cell culture. The true false-positive results obtained by ColorPAC represented only 0.7% of all RSV-positive samples. In general, no statistically significant differences were detected between the different months and epidemic periods studied. Compared with ColorPAC, the shell vial culture method displayed a sensitivity of 95.8% and a specificity of 100%. Overall, the ColorPAC assay was an acceptable, simple and rapid method for the antigenic detection of RSV in paediatric respiratory samples.     

Comparison of three enzyme-linked immunosorbent assays and a direct fluorescent-antibody test for detection of respiratory syncytial virus antigen.

We prospectively evaluated three enzyme immunoassays (EIAs) and a direct fluorescent-antibody (DFA) test for respiratory syncytial virus detection. Of 90 specimens, 79% gave the same results in all four tests (30 positive and 41 negative) and 97% were in agreement in three of the four assays. The agreement between the direct fluorescent-antibody test and each enzyme immunoassay was greater than or equal to 86%.     

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.     

Evaluation of direct immunofluorescence, enzyme immunoassay, centrifugation culture, and conventional culture for the detection of respiratory syncytial virus.

Four methods of detecting respiratory syncytial virus (RSV) from clinical specimens were evaluated. A total of 410 specimens consisting of nasopharyngeal washes, aspirates, and swabs were simultaneously tested for the presence of RSV by direct immunofluorescence assay (DFA), enzyme immunoassay (EIA) (Kallestad Pathfinder), shell vial centrifugation culture (SVC), and conventional culture. DFA identified 146 (83%) of the 175 positive cases, EIA detected 153 (87%), SVC detected 127 (73%), and conventional culture detected 70 (40%). Conventional culture isolated an additional 19 respiratory viruses other than RSV. DFA and EIA were able to detect nonviable virus not isolated by a culture method, and SVC isolated low-titer virus not detected by conventional culture. DFA and EIA gave similar results; however, the EIA system was less dependent on technical expertise. The use of SVC enhanced the conventional culture system with 63 RSV isolates not recovered from the tube culture. We recommend complementary use of both culture and nonculture methods in the 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.     

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.