Rockville, Marvland tract epithelial cells of pediatric patients with acute respiratory disease.

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1 JOURNAL OF CLINICAL MICROBIOLOGY. Dec p /83/ /0 Copyright t 1983, American Society for Microbiology Vol. 18. No. 6 Respiratory Syncytial Virus Detection by Immunofluorescence in Nasal Secretions with Monoclonal Antibodies Against Selected Surface and Internal Proteins HYUN W. KIM, * RICHARD G. WYATT,3 BRUCE F. FERNIE.4 CARL D. BRANDT.' JULITA 0. ARROBIO.' BARBARA C. JEFFRIES.' AND ROBERT H. PARROTT' 2 Research Foucndaition of Clhildren 's Hospital Nationtal Medical Center, antd Department of Clhild Healtlh anxd Delvelopment. Geor,ge Washington University Sc/hool of Medicine,2 Washington, D.C ; Laboratory of Infectious Diseases, National Institlute of Allergy aiind Infectious. Diseaises, Betlhesda., Maryland ;3c(i/ Division of Molecular Virology and Immunology, Deparl-tmelent of Microbiology, Georgetown Univ-ersity, Rockville, Marvland Received 8 July 1983/Accepted 19 September 1983 Specimens containing respiratory tract epithelial cells from infants and children with acute respiratory disease were evaluated by using an indirect immunofluorescence technique with two specific respiratory syncytial virus monoclonal antibodies. One (RS/HN 13-1) was directed against a cell surface viral antigen, and the other (RS/HN 25-2) was directed against viral antigen present in large cytoplasmic inclusions. The same results on presence or absence of respiratory syncytial virus were obtained by cell culture and immunofluorescence in 93% of 252 patients tested adequately by both methods. The sensitivity of indirect immunofluorescence was approximately equal to that of cell culture. A total of 84 specimens were positive for RSV by immunofluorescence; 82 of them were positive with both monoclones, and the remaining 2 were positive only with the monoclone directed against the internal protein. The fluorescence pattern of the latter monoclone was unique and easily recognized. Indirect immunofluorescence testing with monoclonal antibodies to respiratory syncytial virus proved to be a very useful diagnostic technique, and results could be obtained within 4 h of specimen collection. Respiratory syncytial virus (RSV) is the most important respiratory viral pathogen in pediatric age groups, particularly in early infancy (2). The diagnosis of RSV infection by cell culture is time consuming and generally requires 3 to 10 days for isolation and additional time for specific identification of the virus. In 1968, McQuillin and Gardner reported a sensitive and specific immunofluorescence technique for the detection of RSV antigens in the nasopharyngeal secretions collected during the acute phase of the disease (13). Our attempts to develop a rapid and useful diagnostic test for RSV infection have been hampered by a lack of specific antibody with high titer. We recently evaluated an indirect immunofluorescent antibody (IFA) technique which used two specific RSV monoclonal antibodies; one was directed against a cell surface viral antigen (3, 4) and the other was directed against viral antigen associated with large cytoplasmic inclusions (3). The goal was to enable us to assess, within 24 h of admission to the hospital, the presence or absence of RSV in upper respiratory tract epithelial cells of pediatric patients with acute respiratory disease. This paper describes the successful evaluation of specimens from 252 respiratory disease patients who were tested for RSV infection by IFA with RSV monoclonal antibodies and by cell culture. MATERIALS AND METHODS Patients. We studied 277 infants and children between 5 days and 70 months of age who were hospitalized with acute respiratory disease or who acquired respiratory infection nosocomially. Viral studies were requested by the patients' physicians. During the period January 1982 througb March 1982, 80 patients (group 1) were studied. and 197 patients (group 2) were studied during the period November through March 9, The majority of specimens were obtained within 24 h of admission to the hospital (2 to 8 days after the onset of illness). Specimens for IFA testing. Nasal wash specimens were collected with a De Lee suction catheter with mucus trap (Sherwood Medical Industries. St. Louis. Mo.) by gentle suction into 3 ml of phosphate-buffered saline (PBS). ph 7.4. Nasal swab specimens and combined nasal and oropharyngeal swab specimens 1399

2 1400 KIM ET AL. were collected with a wet cotton-tipped applicator by gentle swabbing of the nasal mucosa alone or both the nasal and oropharyngeal mucosa. These swabs were agitated in 3 ml of PBS. Tracheal aspirates were collected by nurses who suctioned material through a plastic catheter into a few drops of normal saline. The one brush biopsy pulmonary specimen was collected by another physician. Specimens from group 1 were transported to the laboratory at room temperature. whereas those from group 2 were immediately transported in ice water. All specimens (from group 1 within 4 to 22 h of collection and from group 2 immediately or within I h of collection) were processed by centrifugation for 10 min at 1,000 to 1,200 rpm at 4 C. The resultant sediment was suspended in 3 ml of PBS and again centrifuged for 10 min at to 1,200 rpm at 4 C. The pellet was suspended in a few drops of PBS, mixed well with a capillary pipette, distributed evenly on each circle of a three-circle acetone-resistant microscope slide (Roboz Surgical Instrument Co.. Inc., Washington, D.C.), air dried, and fixed in acetone for 10 min at 4 C. One group of fixed nasal wash specimen slides from 80 patients (group 1) was held frozen at -70)C ftor 8 to 10 months before staining. A second group of fixed specimen slides from 197 patients (group 2) was held at 4 C before staining within 48 h. Specimens for virus isolation. For virus isolation, we also obtained combined nasal and oropharyngeal swab specimens in 6.0 ml of veal infusion broth containing 0.5% bovine serum albumin and antibiotics from the same patients at the time of specimen collection for IFA testing. To detect RSV and other viruses in cell cultures, generally within 5 h of collection, 0.2 ml of combined nasal and oropharyngeal swab specimens from all patients were inoculated (without prior freezing) into two tubes each of Hep-2, monkey kidney, and WI-38 diploid cell cultures (Flow Laboratories. McLean Va., and M.A. Bioproducts, Walkersville Md.), as previously described (10). Monoclonal antibodies. Two of seven monoclonal immunoglobulin G (lgg) antibodies recently prepared by Cote et al. were evaluated (3). One (RS/HN 13-1) is a monoclonal antibody directed agatinst surface protein (4) and neutralizes RSV, whereas the other (RS/HN 25-2) is a monoclonal antibody directed against internal protein and does not neutralize RSV. IFA technique. We used a standard indirect immunofluorescence technique (5). Undiluted culture fluids of RS/HN 13-I or RS/HN 25-2 (50 as VI). the first antibodies, or 50 p1 of hypoxanthine-aminopterinthymidine medium, as a control, were placed each on one of the three circles of each slide. The slide was then incubated for 30 min in a moist chamber at 37 C together with an RSV-infected Hep-2 cell control slide for each test. Each slide was washed for 30 min in three changes of PBS and drained, and 50 I of a 1:100 dilution of the fluorescein-conjugated sheep antimouse IgG (Cappel Laboratories. West Chester, Pa.) containing rhodamine contrast strain (1:100) was applied to all circles of each slide. The slides were then incubated for 1 h in a moist chamber at 37 C, washed for 30 min in three changes of PBS, given a final wash for 2 min in distilled water, and mounted with buffered-glycerol mounting medium. ph 8.0. The slides J. CLIN. MICROBIOL. were viewed with a Leitz DIALUX 20 UV microscope at a magnification of x250. Each specimen was examined for fluorescence by two persons, one of whom read all specimens under code. Only specific intracytoplasmic fluorescence with characteristic staining patterns for the indicated RSV antigens was recorded as positive. RESULTS Frequency of adequate specimens. Only 76% of the previously frozen slides from 80 nasal wash specimens were adequate for evaluation of immunofluorescence, i.e., they had ca..5 columnar epithelial cells per high-power field (Table 1). In attempts to obtain better IFA specimens, different specimens (including a nasal swab alone or combined nasal and oropharyngeal swabs) were obtained from a second group of study patients. A small number of tracheal aspirates and one brush biopsy pulmonary specimen were also evaluated. Nasal wash specimens were adequate, but even after careful washing they contained debris such as mucus, squamous cells, leukocytes, and erythrocytes. Also, the numbers of cells obtained by nasal wash were smaller than those obtained by nasal and oropharyngeal swabs combined. Specificity of monoclonal antibodies. Hep-2 cells infected with RSV and rhesus monkey kidney cells infected with parainfluenza virus types 1, 2, or 3 or influenza viruses A or B were tested with the two RSV monoclonal antibodies by indirect immunofluorescence. There was no cross-reactivity of the antibodies (or the conjugate when tested alone) with heterologous viral antigens. Yellow-green fluorescence was seen only with the RSV-infected cells. Furthermore, RSV-specific immunofluorescence was not detected in respiratory tract cells from 30 patients who yielded other respiratory virus isolates, including adenovirus (13 patients). parainfluenza virus (4 patients), influenza A virus (11 patients), influenza B virus (1 patient), and rhinovirus (1 patient). Comparison of IFA and cell culture. An RSV TABLE 1. Frequency of adequate specimens' Group 1 Group 2 Type of specimen (n = 80) (n = 197) frozen slides unfrozen slides Nasal wash 61/80 (76%)" 13/13 (1007)4 Nasal swab ' 76/77 (99%) Nasal and orophar- 82/83 (99%) yngeal swab Tracheal aspirate 19/23 (83%) Brush biopsy pulmo- 1/1 nary specimen " Approximately five or more cells per high-power field. "Poor quality of specimen with debris. Not available.

3 VOL. 18, 1983 isolate (83-122) was titrated in Hep-2 cells (Flow Laboratories) at serial 10-fold dilutions, as done previously in the study by McQuillin and Gardner (13). RSV could be detected by IFA as well as by observation of cytopathic effect (CPE) in tissue culture cells at a dilution of 10-1 but not at higher dilution 7 days after inoculation. We also reinoculated five of the unfrozen specimens which were previously positive by cell culture, but were negative by IFA, onto Hep-2 cells (four tubes for each specimen). We observed for CPE and tested one tube daily for 4 days by IFA. No CPE was observed, nor did IFA become positive during the observation period. Two of these five specimens were inoculated again and were positive by IFA as well as by development of CPE on day 5. Detection of RSV. The characteristic pattern of RS/HN 13-1 staining was one of brilliant yellowgreen fluorescence of coarse to fine intracytoplasmic granules as well as of diffuse cytoplasmic staining (Fig. 1A). In contrast, the characteristic pattern of RS/HN 25-2 was one of brilliant fluorescence of coarse-to-fine intracytoplasmic granules and prominent large round or oval inclusions (Fig. 1B). The latter pattern was especially unique and easily recognized. Infected cells stained with hypoxanthine-aminopterin-thymidine medium and conjugate alone were negative. Table 2 shows the results with the group of 61 slide preparations which had been frozen at -70 C (group 1). There were equivalent results for 57 (93%) of these patients who were tested both for RSV isolates and for immunofluorescence (V = 0.871, Kendall coefficient of association; x2 = 46.3, P < ). Four patients were positive by cell culture, but were negative by immunofluorescence. Table 3 shows the results with the subsequent prospective study in which unfrozen specimens were examined (group 2). There was again a 93% agreement between cell culture and immunofluorescence results (V = , Kendall coefficient of association; x2 = 123.4, P < ). Of 49 specimens, 9 (18%) were positive only by immunofluorescence, but the fluorescence pattern was characteristic. Specimens from these nine patients were obtained 3 to 13 days (median 7 days) after the onset of illness. These nine patients did not differ in age from other patients in the study who yielded RSV by cell culture and were positive for RSV by IFA. Five specimens were negative initially by immunofluorescence and positive by cell culture; on retesting of a duplicate slide, three of the five were subsequently read as positive. Thus, there were at least three false negative results by the initial immunofluorescence observation. Overall, in both groups tested, there was a 93% agreement after initial testing. RSV DETECTION 1401 All IFA specimens from both groups, whether previously frozen or unfrozen, were tested with both monoclonal antibody preparations. A total of 84 specimens were positive for RSV by immunofluorescence; 82 were positive with both monoclones, and the remaining 2 were positive only with the monoclone directed against the internal protein. The results of immunofluorescence of prospective unfrozen specimens were usually available to physicians on the day of specimen collection. With optimal timing of the collection and processing of specimens, results were available in approximately 4 h. DISCUSSION In 1968, McQuillin and Gardner reported a sensitive and specific immunofluorescence technique for the detection of RSV antigens in nasal secretions, using antisera prepared in rabbits (13). Detection of RSV antigens by immunofluorescence techniques, indirect or direct, has since been reported by several investigators (1, 8, 12, 14, 15). These investigators used antibodies derived by immunization of rabbits (8, 12, 14) or a gnotobiotic calf (15) or by monoclonal antibody techniques (1). Bell et al. (1) reported that an IFA test with a mouse monoclonal antibody was highly sensitive (79%) and specific (100%) for the detection of RSV in comparison with a combination of virus cultivation and an IFA test with a commercially available bovine anti-rsv serum. They observed more prominent inclusions and less background fluorescence in nasal cells from an RSV-infected infant by using an IFA test with a mouse monoclonal antibody than one with the bovine anti-rsv serum. For IFA testing, we employed two specific RSV monoclonal antibodies, one directed against surface protein (3, 4) and the other directed against internal protein (3). Overall, the immunofluorescence results with two groups of specimens with these two RSV monoclonal antibodies agreed in 93% of cases with cell culture results. The sensitivity of RSV detection by immunofluorescence and observation of CPE in Hep-2 cell cultures were the same. In an earlier study, McQuillin and Gardner reported a 100-fold greater sensitivity by their immunofluorescence technique, using Bristol HeLa and Hep-2 cells (13). We do not know the relative sensitivity of their cell lines to RSV. We used Hep-2 cells (Flow Laboratories), which are sensitive to RSV, for titration of the RSV isolate (83-122). Of our 49 unfrozen specimens, 9 (18%) were positive by immunofluorescence and not by cell culture, but the fluorescent staining pattern was characteristic; thus, we believe that these detected actual RSV infections. Bell et al. (1) reported that 9 (19%) of their 48 culture-negative

4 1402 KIM ET AL. J. Cl-IN. MICROBIOL. FIG. 1. Epithelial cells in a combined nasal and oropharyngeal swab specimen from a patient with RSV infection showing specific RSV fluorescence with fluorescein-conjugated IgG fraction sheep anti-mouse IgG after monoclonal antibody RS/HN 13-1 (A; note diffuse cytoplasmic staining) or monoclonal antibody RS/HN 25-2 (B; note the staining of prominent large round or oval inclusions [white arr-ows] and coarse-to-fine intracytoplasmic granules). x 8)0.

5 VOL. 18, 1983 TABLE 2. Immunofluorescence for RSV on slides frozen for 8 to 10 months as compared with cell culture performed within 5 h of specimen acquisition' Cell Immunofluorescence result culture result Positive Negative Positive 35 4 Negative 0 22 " All specimens were obtained by nasal wash. V 0.871, Kendall coefficient of association: X- = P < specimens were positive by both the IFA test with a mouse monoclonal antibody and the IFA test with a commercially available bovine anti- RSV serum. They concluded that both the IFAs were more sensitive than virus culturing. These observations may be explained by findings of Gardner et al. (6), who studied paired nasopharyngeal secretions and demonstrated that RSV antigen can be detected by immunofluorescence for several days after RSV can no longer be successfully isolated from patients. Further, they showed that eight single secretions collected from patients who had been ill for 5 to 14 days were positive by immunofluorescence in the absence of virus isolation. They suggested that the findings may be due to neutralization of virus by a patient's developing antibody, by antigen deterioration, or by faulty isolation technique. Kaul and colleagues (9) reported that RSVspecific secretory IgA as well as IgG and IgM antibody appear in respiratory tract secretions as early as the 3 days after the onset of illness caused by RSV infection. This antibody may neutralize RSV. The production of easily recognizable and characteristic staining patterns by these monoclonal antibodies. particularly the internal monoclonal antibody. make them valuable for the specific diagnosis of RSV infection. Adequate quality and quantity of cells present in the specimens were essential for IFA evaluation, TABLE 3. Immunofluorescence for RSV on unfrozen slides within 48 h of specimen acquisition as compared with cell culture performed within 5 h" Cell Immunofluorescence result culture resullt Positive Negative Positive 40 5" Negative " Specimens were from various sources. V = , Kendall coefficient of association: X P < h Of these five specimens, three were positive by immunofluorescence on retesting. RSV DETECTION 1403 and the use of combined nasal and oropharyngeal specimen was preferable to other specimens. In many laboratories, virus recovery in tissue culture has for years been the diagnostic method of choice for RSV diagnosis. Many other viruses, including RSV, can be recovered by this method. However, tissue culture techniques are time consuming, requiring 3 to 10 days for isolation of virus and additional time for specific identification of the virus. Thus, tissue culture is not a rapid diagnostic test for RSV infection. From the standpoint of time, we prefer the monoclonal IFA test over cell culture. It also appears that the commercial antisera will be comparable to monoclonal antibodies in sensitivity. Rapid diagnosis by IFA can hasten the institution of infection control measures, such as among young infants with congenital heart disease who much more frequently experience severe illness or die with RSV infection than do those without such infection (11). Rapid diagnosis of RSV infection by immunofluorescence will be especially useful if ribavirin (7) or other antiviral drugs are routinely used in treating severe RSV disease. ACKNOWLEDGMENTS We thank J. L. Gerin Division of Molecular Virology and Immunology. Depairtment of Microbiology. Georgetown University. for supplying the monoclonal antibodies: R. M. Chanock. Laboratory of Infectious Diseases. National Institute of Allergy and Infectious Diseases. for critical aippraisatl of this work: and T. Popkin, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, for assistance with photography. LITERATURE CITED I. Bell, D.M., E. E. Walsh, J. F. Hruska, K. C. Schnabel, and C. B. Hall Rapid detection of respiratory syncytial virus with a monoclonal antibody. J. Clin. Microbiol. 17:1t) Chanock, R. M., H. W. Kim, C. D. Brandt, and R. H. Parrott Respiratory syncytial virus, p In A. S. Evans (ed.). Viral infections of humans. 2nd ed. Plenum Publishing Corp., New York. 3. Cote, P. J., Jr., B. F. Fernie, E. C. Ford, J. W.-K. Shih, and J. L. Gerin Monoclonal antibodies to respiratory svncvtial virus: detection of virus neutralization and other antigen-antibody systems using infected human and murine cells. J. Virol. Methods 3: Fernie, B. F., and J. L. Gerin Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion. Infect. Immun. 37: Gardner, P. S., and J. McQuillin Rapid virus diagnosis: application of immunofluorescence. 2nd ed., p Butterworth Inc., London. 6. Gardner, P. S., J. McQuillin, and R. McGuckin. 1970). The late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence. J. Hyg. 68: Hall, C. B., J. T. McBride, E. E. Walsh, D. M. Bell, C. L. Gala, S. Hildreth, L. G. Ten Eyck, and W. J. Hall Aerosolized ribavirin treatment of infants with respiratory svncvtial viral infection: a randomized double-blind study. New Engl. J. Med. 308: Hornsleth, A., B. Friis, P. Andersen, and E. Brenoe Detection of respiratory syncytial virus in nasopharyngeal secretions bv ELISA: comparison w\ith fluorescent anti-

6 1404 KIM ET AL. J. CLIN. MICROBIOL. body technique. J. Med. Virol. 10: Kaul, T. N., R. C. Welliver, D. T. Wong, R. A. Udwadia, K. Riddlesberger, and P. L. Ogra Secretory antibody response to respiratory syncytial virus infection. Am. J. Dis. Child 135: Kim, H. W., J. 0. Arrobio, C. D. Brandt, B. C. Jeifries, G. Pyles, J. L. Reid, R. M. Chanock, and R. H. Parrott Epidemiology of respiratory syncytial virus infection in Washington, D.C. I. Importance of the virus in different respiratory tract disease syndromes and temporal distribution of infection. Am. J. Epidemiol. 98: MacDonald, N. E., C. B. Hall, S. C. Suffin, C. Alexson, P. J. Harris, and J. A. Manning Respiratory syncytial viral infection in infants with congenital heart disease. New EngI. J. Med. 307: McIntosh, K., R. M. Hendry, M. L. Fahnestock, and L. T. Pierik Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples. J. Clin. Microbiol. 16: McQuillin, J. and P. S. Gardner Rapid diagnosis of respiratory syncytial virus infection by immunofluorescent antibody techniques. Brit. Med. J. i: Minnich, L., and C. G. Ray Comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques. J. Clin. Microbiol. 12: Sarkkinen, H. K., P. E. Halonen, P. P. Arstila, and A. A. Salmi Detection ot r-espiratory syncytial. parainfluenza type, 2. and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasophar-yngeal specimens from children with acute respiratory disease. J. Clin. Microbiol. 13: Downloaded from on February 26, 2019 by guest