JOURNAL OF CLINICAL MICROBIOLOGY, May 1990, p. 1021-1025 0095-1137/90/051021-05$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 28, No. 5

Evaluation of Five Methods for Respiratory Syncytial Virus Detection DIANE C. HALSTEAD,* SANDRA TODD, AND GALE FRITCH HealthEast Laboratories, The Allentown Hospital-Lehigh Valley Hospital Center, 1200 South Cedar Crest Boulevard, Allentown, Pennsylvania 18103 Received 27 October 1989/Accepted 5 February 1990

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 ELA (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.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in children less than 2 years old. Complications of RSV infection, including apnea, respiratory failure, or death, frequently occur in premature infants and children with congenital heart disease, pulmonary dysplasia, and immunosuppression (9). With the advent of therapy with antiviral agents such as ribavirin and with the emphasis on reducing nosocomial infections and costs for inappropriate therapy and hospital stays, it is imperative that laboratory results be available in a timely fashion. Virus isolation in cell culture permits the amplification of small amounts of virus that are present in a specimen and the recovery of several additional agents. RSV grows very slowly in cell culture, frequently taking 5 or more days before a cytopathic effect is detected. It is a labile virus; thus, it is mandatory that stringent adherence to transport guidelines and immediate inoculation of specimens into cell culture be performed for optimal recovery of the virus. In order to circumvent these problems, we evaluated four commercially available methods that are designed to rapidly detect RSV antigen and that do not rely on virus viability. We also report on the recovery of viruses in addition to RSV by the cell culture method when testing RSV antigen-

negative specimens. MATERIALS AND METHODS A total of 117 nasal aspirates (NAs) from pediatric patients with apparent lower respiratory tract disease were collected by using a sterile, infant suction set (catalog no. 3450; Becton Dickinson Acute Care, Franklin Lakes, N.J.), transported to the laboratory on wet ice, and processed immedi-

monoclonal RSV direct fluorescent-antibody (DFA) test reagents according to the directions of the manufacturer (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.). Slides were interpreted with an epifluorescence microscope (HBO 50; Optiphot; Nikon), an exciter filter (420 to 490 nm), and a barrier filter (515 W; Optical Apparatus Co., Inc., Ardmore, Pa.). At least three columnar epithelial cells per field at x 400 magnification were required for reporting a negative result, and the presence of one fluorescent large mononuclear or ciliated epithelial cell with cytoplasmic inclusionlike or particulate staining was required to consider a specimen positive for RSV antigen. Virus culture. Each NA was placed in a tube of viral transport medium (Bartels Immunodiagnostic Supplies, Inc.) and vortexed. Portions of 0.2 ml from a freshly collected specimen in viral transport medium were inoculated into a primary monkey kidney (PMK) shell vial (SV) and tube, an A549 SV, and human embryonic kidney, HEp-2, and MRC-5 cell culture tubes (Bartels Immunodiagnostic Supplies, Inc., or ViroMed Laboratory, Louisville, Ky.). Specimens that were frozen at -70°C (48 RSV antigen-positive specimens) were inoculated only into HEp-2 cell culture tubes. Specimens were allowed to absorb for 1 h at room temperature before 1.5 ml of refeeding medium (Bartels Immunodiagnostic Supplies, Inc.) was added to the tubes. Specimens were centrifuged in SV at 2,000 rpm for 45 min at 30°C in an

Omnifuge (American Scientific Products, Edison, N.J.), followed by the addition of 1.0 ml of refeeding medium. All cultures were refed the following day and were monitored daily for 14 days or until virus was isolated. Cell culture confirmation. At 48 h the PMK cells in SV were washed twice, i.e., two 5-min washes with 1.0 ml of phosphate-buffered saline, and were then scraped from the cover slip into approximately 0.3 ml of phosphate-buffered saline with a 1.0-ml disposable pipette (catalog no. 7520; Becton Dickinson Labware, Oxnard, Calif.). The cell suspensions were used to prepare one two-well slide and one

ately. RSV direct fluorescent-antibody test. Two two-well slides were prepared by using unwashed NAs. The slides were air dried, fixed with acetone at 4°C for 10 min, and stained with *

Corresponding author. 1021




eight-well slide. After the two-well slide was air dried and fixed with acetone at 4°C, it was stained by an indirect fluorescent-antibody assay with a pool of monoclonal respiratory viral antibodies to RSV; adenovirus; influenzavirus types A and B; parainfluenza types 1, 2, and 3; and antimouse fluorescein isothiocyanate conjugate with Evans blue counterstain, as described in the package insert (Bartels Immunodiagnostic Supplies, Inc.). Nonimmune mouse antiserum (Bartels Immunodiagnostic Supplies, Inc.) was used as a negative control to rule out nonspecific fluorescence. A positive antigen control for each antiserum specimen tested was processed as outlined above. If the antibody pool was positive, the eight-well slide was stained and identified by using specific monoclonal antisera (Bartels Immunodiagnostic Supplies, Inc.) and fluorescent conjugate. If the PMK SV was negative at 48 h of incubation, the A549 SV was processed at 5 days, as outlined above. The protocol included testing of PMK tubes for guinea pig erythrocyte hemadsorption at 5 and 10 days if the test results described above were negative. If the hemadsorption test results were positive, smears were prepared and stained with the antibody pool and then with virus-specific antisera if the pool was positive. The 48 previously frozen RSV DFA-positive specimens that were inoculated only into HEp-2 cell cultures in order to confirm the presence of RSV were monitored for 14 days for a characteristic syncytial cytopathic effect. Virus isolates that were not identified by these procedures were subjected to further testing based on the type of cytopathic effect, the length of time to detection, and the cell line infected. Cytomegalovirus (CMV) isolates were confirmed with monoclonal anti-CMV indirect fluorescent-antibody reagents (Syva Co., Palo Alto, Calif.). Isolates other than RSV, CMV, and rhinovirus were confirmed by the Pennsylvania Bureau of Laboratories (Lionville) as part of our Community Respiratory Virus Surveillance Program. Enzyme immunoassay (EIA) procedures. NAs that were frozen at -70°C and thawed one time were diluted 1:3 in viral transport medium and tested according to the directions of each of the manufacturers, i.e., Directigen (DTG; Becton Dickinson Microbiology Systems, Cockeysville, Md.) and TestPack (TP) and RSV EIA (Abbott Laboratories, North


controller was removed from the ColorPAC, the membrane was washed. Four drops of enzyme-conjugated anti-RSV monoclonal antibodies were allowed to absorb completely for 2 min; this was followed by a washing step and 4 drops of reagents 4 and 5 (enzyme substrate) were added. After incubation for 5 min at room temperature, 4 drops of stop reagent were added. A purple triangle of any intensity or a "bull's-eye" effect (a white triangle with a purple background) on the membrane (ColorPAC) was interpreted as positive for RSV antigen. If a purple dot appeared, the test was interpreted as negative for the RSV antigen. If neither a purple dot nor a triangle appeared, the test was uninterpretable.

(ii) TP procedure. By the use of a transfer pipette that was supplied with the TP kit, 750 ,ul of specimen was added to the specimen treatment cup along with 3 drops of dithiothreitol buffer. Following gentle mixing and incubation for 5 min at


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VOL. 28, 1990

TABLE 2. Evaluation of five methods for RSV detection Method

Culture DFA (Bartels) DTG (Becton Dickinson) TP (Abbott) RSV EIA (Abbott)

Sensitivity (%)

Specificity (%)



91.9 91.9 75.8 93.6 71.0

100 96.4 80.0 100 100

100 96.6 81.0 100 100

91.7 91.4 74.6 93.2 75.3

Positive value (%)


value (%)

inserted into the specithe bottom. A total of 3 drops of anti-RSV-coated microparticles and 3 drops of anti-RSV biotin were added to the filtered specimen to form an antibody-antigen-antibody complex. This was mixed and incubated for 10 min. The entire contents of the filter tube were then poured into the center of the purple focuser, which was securely seated on the Reaction Disc containing inactivated RSV. After approximately 30 s, the focuser was discarded and 3 drops of alkaline phosphatase-labeled antibiotin antibody was added to the Reaction Disc, followed by incubation for another 3 min. After a wash, 3 drops of

room temperature, a filter tube was men treatment cup and depressed to

chromogen were added, followed by incubation for 2 min and an additional wash. As with the DTG test, results were read immediately by three laboratorians, and the strengths of the reactions were graded qualitatively. The specimens were coded to ensure that the tests were interpreted objectively. Plus and minus signs were considered positive and negative, respectively, for RSV antigen; without signs the tests were uninterpretable.

RESULTS A total of 62 of 117 (53%) NAs submitted from children with symptoms compatible with bronchiolitis, pneumonia, or other respiratory disease were positive for respiratory syncytial virus or antigen. In order to validate a positive result, a positive RSV culture or at least two of the five methods used to detect RSV were required. Of the 117 specimens, 57 (48.7%) were culture positive in HEp-2 cells, A549 cells, or both. Only five specimens (4.3%) were culture negative but were positive by at least two of four antigen

detection systems;


two specimens were positive by

DFA and DTG only; one specimen was positive by RSV EIA and DTG only; and two specimens were positive by DFA, DTG, TP, and RSV EIA. Results of all five methods were in agreement for 77 of 117 (65.8%) NAs. A total of 42 specimens were negative and 35 specimens were positive for RSV by the culture method and by all four antigen detection systems. Thirteen of the RSVnegative specimens were positive for the following viruses: influenza A virus (n = 6), influenza B virus (n = 2), adenovirus type 2 (n = 2), rhinovirus (n = 1), CMV (n = 1), and echovirus type 22 (n = 1). Additionally, two specimens that were positive by DTG only were also positive for rhinovirus and influenza B virus. The remaining 40 specimens (34.2%) gave nonconcordant results. TP and culture results were in agreement for 36 of the 40 specimens (21 positive and 15 negative); DFA and culture results were in agreement for 30 of 40 specimens (18 positive and 12 negative); RSV EIA and culture results were in agreement for 21 of 40 specimens (6 positive and 15 negative); and DTG and culture results were in agreement for 9 of 40 specimens (7 positive and 2 negative). Table 2 lists


the sensitivities, specificities, and positive and negative predictive values for culture, DFA, DTG, TP, and RSV EIA. DISCUSSION The significant morbidity and mortality associated with RSV infections, particularly in high-risk individuals, makes specific and rapid diagnosis imperative. Over the past years, several commercial vendors have addressed this need by introducing indirect fluorescent-antibody tests and enzymelinked immunosorbent assays which provide results in hours rather than in the days associated with traditional cell culture techniques (12). Particularly since the advent of the antiviral agent ribavirin, still more rapid tests have been introduced, including the DFA test, which can be performed in less than 1 h (6, 8; D. C. Halstead, Clin. Microbiol. Newsl. 9:181-185, 1987). More recently, Abbott Laboratories and Becton Dickinson have introduced the TP and DTG tests, respectively, which provide results within 15 to 21 min. Since our virology laboratory serves large pediatric, neonatal, and geriatric populations, which are target groups for infection with RSV, we were anxious to evaluate new methods that might decrease the turnaround time for results, so that isolation procedures to prevent nosocomial spread and protocols for appropriate therapy could be implemented. We previously used the RSV EIA procedure for identifying RSV antigen-positive patients and compared these results with the two newly marketed TP and DTG EIAs and with the methods we were using at that time, which were the DFA test and the cell culture isolation method. The culture, DFA, and TP test results compared favorably. The apparent differences in the sensitivity and negative predictive value of TP and the gold standard culture fell within the range of sampling error. Another possible explanation for the apparent differences is the fact that 48 of 117 specimens were frozen, which has been shown to compromise the recovery of RSV in cell culture (11). The sensitivities and specificities of monoclonal antibody DFA tests have been reported to range between 81 and 90% and 70 and 100%, respectively (10, 11). The excellent results obtained in our study with unselected specimens may be attributed to the quality of the NAs that were processed and the fact that direct NA smears were prepared without washing or centrifugation steps. In contrast to EIA procedures that require a certain concentration of antigen, the interpretation of our DFA procedure required only one cell containing RSV antigen to constitute a positive test result. The sensitivities and negative predictive values of the DTG test and RSV EIA, as well as the specificity and positive predictive value of the DTG test, were inferior to those values obtained by the other methods. However, the majority of false-positive DTG test results, as well as false-positive RSV EIA and TP results, based on the agreement of two of the five methods or a positive RSV culture, were weak to very weak reactions. Concordant were at least as strong as the positive DTG and TP reactions positive control reactions within each assay in 27 of 35 (77%) and 32 of 35 (91.4%) positive specimens, respectively. The RSV EIA absorbance values for 32 of 35 (91.4%) positive specimens were equal to or greater than 0.500; i.e., they were considerably higher than the cutoff values of 0.146 to 0.152. Because of the inordinately high number of specimens that were positive by the DTG test only, a blocking antibody test was performed as described by the Becton Dickinson & Company Research Center, which supplied the necessary reagents. Because of limited specimen volume, only 7 of 11




specimens that were false positive by the DTG test were retested. Blocking antibody, however, did not alter the test results ofany ofthe seven specimens. Four specimens tested by DTG filtered slowly, even after the specimens were diluted 1:3 as recommended by the manufacturer. All were interpreted as positive; one specimen was weakly positive by the DTG test and by the RSV EIA, and three specimens were positive by the DTG test only. Two specimens that were positive by the DTG test only were positive for rhinovirus and influenza B virus. It is unknown whether the results were due to cross-reactions. Further evaluation of this product is necessary to resolve this concern. In contrast to other published studies of RSV EIA reporting sensitivities and specificities of 96 and 96% (4) and 87.5 and 95% (13), respectively, the sensitivity as well as the negative predictive value of the RSV EIA in our study were considerably lower. Because of a limited specimen volume, blocking assays were not performed to resolve this apparent discrepancy. In general, the sensitivities of EIAs have varied between laboratories, e.g., 61 to 88% (1, 7). These differences may reflect variations in types, e.g., NA versus nasal swabs (1, 5), the volume required for the assay, and the quality of the specimens that were submitted. Both the DTG test and the RSV EIA require a smaller volume, 250 and 100 ,ul, respectively, compared with TP, which requires 750 ,ul. It is unknown whether the quality of the specimens influenced the outcome of EIAs in our study. The EIA procedures used were designed to test fresh or frozen and diluted nasopharyngeal washes and NAs (in addition to other respiratory specimens). Freezing of the NAs did not appear to influence TP results. It is only speculation whether freeze-thawing had an impact on the sensitivities of the DTG monoclonal antibody assay or the RSV EIA with polyclonal antibodies. M. D. Tolpin and M. A. Collins (Editorial, Clin. Microbiol. Newsl. 10:109-111, 1989) observed that polyclonal-based assays such as the RSV EIA tend to retain reactivity more than monoclonal antibody-based assays do when specimens are frozen for extended periods, e.g., 2.5 years at -20°C. The specimens used in our study were stored at -700C for less than 6 months, which is an acceptable alternative to processing fresh specimens, according to the recommendations of the manufacturer. Discrepancies related to the DTG method may be attributed to the fact that the assay is based on membrane filtration; i.e., the antigen is captured on a membrane and is then detected by anti-RSV antibody. In contrast, the TP captures antibody-antigen-antibody complexes on an RSVcoated membrane, which may enhance the sensitivity as well as the specificity. The high predictive values obtained in this study may have been due to the high prevalence of disease in this unselected sampling of pediatric patients. More than half of the specimens tested were positive by the culture method or RSV antigen detection. We attribute the positivity rate (53%) to strict adherence to our guidelines for specimen collection of NAs, transport on wet ice, and timely processing. Recovery of virus in cell culture is still considered the method of choice for establishing the presence of virus(es) in clinical specimens (10; E. M. Swierkosz, M. K. Nichols, T. Armstrong, L. Melvin, and R. R. Schmidt, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, C-51, p. 331). In the interest of cost-containment, we routinely culture only RSV antigen-negative specimens unless we are specifically requested to do otherwise. This appears to be consistent with thé practice in other laboratories as well (1-3). In support of

providing virus isolation capabilities, 62 of 178 antigennegative specimens (34.8%) processed over an 18-month period were culture positive. Isolates included RSV (n 22), influenza A virus (n = 8), influenza B virus (n = 3), parainfluenza virus type 1 (n = 1), parainfluenza virus type 3 (n = 7), adenovirus (n = 6), rhinovirus (n = 9), enterovirus (n = 4), and CMV (n = 2). Sensitive and specific RSV antigen detection EIAs may play a valuable role in the rapid detection of RSV for effective management and treatment of infected patients and in preventing or curtailing the spread of RSV infection. The choice of RSV kits should be based not only on the quality of the reagents but also on the specimen volume, the cost ofthe reagents, and the availability of necessary equipment and trained personnel and should relate to the specific needs of the laboratory to identify other viruses that may be present. Because our laboratory has an epifluorescence microscope and trained microscopists, we found that the DFA test fulfills the criteria as a rapid, cost-effective RSV screen with the additional benefit of direct visualization of specimens as a quality control measure. For those laboratories that can ensure virus viability during transport to the laboratory and that have cell culture capabilities, we also advocate testing of all RSV antigen-negative specimens in cell culture for the comprehensive recovery of viruses. =

ACKNOWLEDGMENTS We are indebted to Judy Aronson for editorial assistance in preparing the manuscript. We also acknowledge Abbott Laboratories for supplying a portion of the kits under evaluation and Becton Dickinson Microbiology Systems for supplying kits and RSV blocking antibody. LITERATURE CITED 1. Ahluwalia, G., J. Embree, P. McNicol, B. Law, and G. W. Hammond. 1987. Comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay. J. Clin. Microbiol.

25:763-767. 2. Arens, M. Q., E. M. Swierkosz, R. R. Schmidt, R. Armstrong, and K. A. Rivetna. 1986. Strategy for efficient detection of respiratory viruses in pediatric clinical specimens. Diagn. Microbiol. Infect. Dis. 5:307-312. 3. Blanding, J. G., M. G. Hoshiko, and H. R. Stutman. 1989. Routine viral culture for pediatric respiratory specimens submitted for direct immunofluorescence testing. J. Clin. Microbiol. 27:1438-1440. 4. Bromberg, K., G. Tannis, B. Daidone, L. Clarke, and M. Sierra. 1987. Comparison of HEp-2 cell culture and Abbott respiratory syncytial virus enzyme immunoassay. J. Clin. Microbiol. 25:

434-436. 5. Bruckova, M., M. Gradien, C. A. Pettersson, and L. Kunzova.

1989. Use of nasal and pharyngeal swabs for rapid detection of respiratory syncytial virus and adenovirus antigens by enzymelinked immunosorbent assay. J. Clin. Microbiol. 27:1867-1869. 6. Chao, R. K., M. Fishaut, J. D. Schwartzmann, and K. Mclntosh. 1979. Detection of respiratory syncytial virus in nasal secretions from infants by enzyme-linked immunosorbent assay. J. Infect. Dis. 139:483-486. 7. Freymuth, F., M. Quibriac, J. Petitjean, M. L. Amiel, P. Pothier, A. Denis, and J. F. Duhamel. 1986. Comparison of two new tests for rapid diagnosis of respiratory syncytial virus infections by enzyme-linked immunosorbent assay and immunofluorescence techniques. J. Clin. Microbiol. 24:1013-1016. 8. Gardner, P. S., and J. McQuillin. 1980. Rapid virus diagnosis, 2nd ed. Butterworth and Co., Ltd., London.

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9. Hall, C. B. 1981. Respiratory syncytial virus. In R. D. Feigin and J. D. Cherry (ed.), Textbook of pediatric infectious diseases. W. B. Saunders & Co., Philadelphia. 10. Hughes, J. H., D. R. Mann, and.V. V. Hamparian. 1988. Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay. J. Clin. Microbiol. 26:588-591. 11. Kadi, Z., S. Dali, S. Bakouri, and A. Bouguerniouh. 1986. Rapid diagnosis of respiratory syncytial virus infection by antigen immunofluorescence detection with monoclonal antibodies and


immunoglobulin M immunofluorescence test. J. Clin. Microbiol. 24:1038-1040. 12. Krilov, L. R., L. Marcoux, and H. D. Isenberg. 1988. Comparison of three enzyme-linked immunosorbent assays and a direct fluorescent-antibody test for detection of respiratory syncytial virus antigen. J. Clin. Microbiol. 26:377-379. 13. Swenson, P. D., and M. H. Kaplan. 1986. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay. J. Clin. Microbiol. 23:485488.

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) t...
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