Journal of Medical Virology 19:l23-134 (1986)

Detection of Respiratory Syncytial Virus in Nasopharyngeal Secretions by Enzyme- Linked Immunosorbent Assay, Indirect Immunofluorescence, and Virus Isolation: A Comparative Study Therese Popow-Kraupp, Gabriele Kern, Christa Binder, Wolfgang Tuma, Michael Kundi, and Christian Kunz

Institutes of Virology (TP.-K., G.K., W T , C.K.) and Environmental Hygiene (M.K.), University of Vienna, and St. Anna Childrens Hospital (C. B.), Vienna, Austria

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.

Key words: RSV, ELISA, IF, TC

INTRODUCTION

Respiratory syncytial virus (RSV) is one of the most important respiratory pathogens of infants and young children [Chanock and Parrott, 1965; Belshe et al, 19831. The morbidity is considerable, and RSV infections in this age group often require hospitalization [Kim et al, 19731. Methods for the rapid detection of RSV are therefore required to enable the accurate management of the patients. The indirect immunofluorescence technique (IF) described by Gardner and McQuillin [ 19681 was introduced in our laboratory in 1978 and has been used since then for rapid diagnosis of RSV infections in children. Nevertheless, besides the many advantages of IF, the

Accepted for publication January 16, 1986.

Address reprint requests to Therese Popow-Kraupp, Institute of Virology, University of Vienna, Kinder- spitalgasse 15, A-1095 Vienna, Austria.

0 1986 Alan R. Liss, Inc.

124 Popow-Kraupp et a1

need for intact cells containing viral antigens has caused some limitations to the use of this technique, since respiratory epithelial cells are rapidly destroyed, during specimen transportation. Only the acetone-fixed slide preparations of the epithelial cells can be sent over long distances without influence on the test result [Downham et al, 19741. Furthermore, screening by microscopy requires a skilled microscopist, and is time-consuming, especially if large numbers of specimens are to be examined.

More recently, an enzyme-linked immunosorbent assay (ELISA) for the detec- tion of viral antigens in nasopharyngeal secretions (NPS) of children with respiratory tract infections was described by several groups [Chao et al, 1979; Berg et al, 1980; Sarkkinen et al, 1981; Harmon and Pawlik, 1982; McIntosh et al, 1982; Hornsleth et al, 1982; Evans and Olson, 19821. This assay does not require pretreatment of the specimens for transportation, and the test system can be easily automated, thus allowing rapid screening of a large number of specimens per day.

We decided to introduce the ELISA for the diagnosis of RSV infections in our laboratory in order to overcome the problem of specimen transportation and to provide a rapid viral diagnostic service to hospitals and practitioners at a long distance from a virus laboratory. This should enable us to study the epidemiological pattern of RSV over a large geographical area. The aim of this work was to develop a highly sensitive ELISA for the detection of RSV antigens in NPS and to define its sensitivity and specificity by comparing this test to the two standard methods for RSV screening, IF and tissue culture virus isolation (TC).

MATERIALS AND METHODS Patients

Four hundred four nasopharyngeal aspirates from 384 patients were available in sufficient amounts for RSV screening by at least two different methods. These were obtained during the period January, 1983, to March, 1985. The patients were 3 weeks to 14 years old with 81% of them under 1 year of age (median 6 months). Most of them (94.9%) were hospital in-patients admitted with any form of respiratory illness; 26.3% of the children had an upper and 69.5% a lower respiratory tract infection. Specimens were taken on the day of admission. Most of the nasopharyngeal aspirates (71.6%) were obtained within the first week after the onset of symptoms (median third day).

Specimen Collection and Processing

Nasopharyngeal aspirates were obtained by suction from the nasopharynx as described previously [Popow-Kraupp et al, 19841 using a tracheal suction kit (Nunc, Kamstrup, Denmark). Tubes containing the NPS were transported in melting ice to the laboratory and processed immediately. Specimens were screened for RSV by ELISA and by at least one of the two other test systems employed. Of the 404 NPS, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC. A sufficient amount was available for parallel screening by all three methods from 89 specimens. NPS were further analyzed by ELISA and/or IF and TC for the presence of the following viral pathogens: adenoviruses (AD), influenza virus type A (I-A) and type B (1-B), parainfluenza virus type I, 11, I11 (PI, PII, PIII), and rhinoviruses (RH). NPS tested in addition to ELISA by IF were divided as far as possible into two equal parts prior to dilution. One of the two portions was further processed for immunofluores-

Immunofluorescence and Virus Isolation 125

cence according to Gardner and McQuillin [1980]. Briefly, NPS were dispersed in approximately 2 ml phosphate-buffered saline (PBS), pH 7.2. Thick fragments of mucus that would not break up were discarded. The cells were separated by centrifu- gation at 350g for 10 min at 4°C. The supernatant was removed and the cellular pellet (cell fraction) washed with PBS and resuspended in a sufficient amount of PBS (usually 0.5-1 ml) to obtain a satisfactory amount of cells on the slide. NPS smears were air-dried and fixed in acetone for 10 min.

The other portion of NPS was used for the screening by ELISA and, when all three methods were employed, also for virus isolation. For screening by ELISA, samples were prepared as described by Sarkkinen et a1 [1981]. Briefly, NPS were diluted approximately 1:3 in PBS, pH 7.2, containing 20% heat-inactivated fetal calf serum (FCS) and 2% Tween 20 and were sonicated for 5-10 sec (high power, amplitude 2) with an MSE sonifier to homogenize the mucouid nasopharyngeal aspirates and to release intracellular viral antigens. Fifty-five specimens tested by ELISA and IF could not be divided into two equal parts prior to dilution because of insufficient amounts of material. These secretions were diluted approximately 1 :3 in PBS, pH 7.2, and the cell fractions (CF), separated by centrifugation, were used for IF. The supernatant (mucus fraction; MF) containing the extracellular viral antigens was mixed with FCS and Tween 20 to final concentrations of 20% and 2 % , respec- tively, sonicated as described above, and tested by ELISA.

NPS to be tested in parallel by ELISA and TC were dispersed in minimal essential medium (MEM) containing 2% heat-inactivated FCS, 0.03 M MgCl2 X 6 H20, 200 pg/ml neomycin, and 1% (v/v) tryptose phosphate broth (maintainance medium); 0.5 ml was used for TC, and the rest, mixed with FCS and Tween 20 as described above, was sonicated and used for the screening by ELISA.

Viral Culture

For virus isolation, rhinovirus-sensitive HeLa cells, strain “Ohio” [Stott and Tyrrell, 19681, kindly provided by Prof. Tyrrell (Clinical Research Centre, Common Cold Research Unit, Salisbury, Wiltshire, England), were used. Cells grown in roller- tube cultures were inoculated before they were confluent, 24 hr after seeding. The growth medium (MEM containing 10% heat-inactivated FCS, 100 pg/ml neomycin, and 2 mM glutamin) was discarded and 250 pl of NPS in maintainance medium was inoculated into each of two roller tubes. After an incubation period of 2 hr at 32- 34°C on a roller drum, tubes were washed twice with Hank’s balanced salt solution, and 1.5 ml of maintainance medium was added to each of them. Cultures were further incubated at 32-34°C on a roller drum for a period of 10 days. All tubes were examined daily for cytopathic effect. Classification of viruses was based on acid and chloroform sensitivity [Grist et al, 19791. RSV and parainfluenza, influenza, and adenoviruses were further identified by IF or by ELISA, enteroviruses by neutraliza- tion with typing antisera.

Indirect lrnrnunofluorescence Method

The immunofluorescence staining procedure was carried out according to Gard- ner and McQuillin [ 19801. Calf immune sera against RSV and I-A were obtained from Wellcome Reagents Ltd. (Temple Hill, Dartford, England) rabbit immune sera against PI and AD (group specific were kindly provided by Prof. M. Grandien (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) and PI11 antiserum by

126 Popow-Kraupp et a1

Prof. P.S. Gardner (Dept. of Virology, University of Newcastle Upon Tyne, En- gland). The fluorescein-isothiocyanate (F1TC)-conjugated antibovine and antirabbit immunoglobulin from Wellcome Reagents, Ltd, (Temple Hill, Dartford, England) was used. Slides were read independently by two observers using a Leitz SM-Lux fluorescence microscope fitted with an HBO 50 W mercury vapour lamp, a BP 450- 490 exciter filter, and an LP 515 barrier filter. The number of virus-containing cells in 4 x 10 microscopic fields (diameter of 1 microscopic field 0.288 mm) was counted by the two observers, and the mean value was calculated.

E L S A for RSV Antigen Detection Propagation of the virus and purification of the nucleocapsid (NCS). Hep-2

cells (Flow Laboratories, Rockville, MD) grown in Roux-bottles and infected with the Long strain of RSV (100 TCIDSO/ml, 10 ml per Roux-bottle) were maintained in MEM (Earle's) supplemented with 2 % heat-inactivated FCS, 2 mM glutamine, penicillin (200 U/ml), streptomycin (200 pglml), neomycin (100 pg/mI), and fungizon (5 ,ug/ml) at 35°C until an extensive cytopathic effect was observed (5 days postinfec- tion). RSV NCS antigen was prepared according to the instructions of Ilonen et a1 [ 19801. Briefly, RSV-infected Hep-2 cells, dislodged into the culture fluid by vigorous shaking with glass beads, were pelletted by centrifugation at 1,400g for 30 min at 4°C. The supernatant was removed, and the virus-cell pellet was washed once with PBS, pH 7.2, and resuspended in 1/150 of the original volume in PBS, pH 7.2. RSV- infected Hep-2 cells in PBS were disrupted by approximately 30 strokes with a glass homogenizer at 4"C, and trypsin (2.5% in PBS; Seromed, Biochrom KG, Berlin, Federal Republic of Germany) was added to a final concentration of 0.25% (v/v). The virus-cell suspension was incubated at room temperature for 1 hr and once more homogenized with 10-20 strokes in the glass homogenizer. Large debris was removed by centrifugation at 2,OOOg for 20 min, at 4°C and aliquots of the supernatant were then layered on top of CsCl gradients [2 ml40% (w/v), 2 ml 30%, 2 ml 25% CsCl in 10% (v/v) PBS in distilled water] and centrifuged at 75,OOOg for 3 hr at 4°C. NCS were collected after fractionation with an ISCO model 640 fractionator (Instrumenta- tion Specialities Company, Lincoln, NE) with continous monitoring at 254 nm. Fractions containing RSV NCS were pooled, dialyzed over night against 10% PBS in distilled water, and examined by electron microscopy after negative staining with 1 % uranyl acetate at pH 4.0. Aliquots of NCS preparations were stored at -80°C until immunization. The protein concentration of the RSV-NCS preparations was deter- mined according to the method of Schaffner and Weissmann [ 19731.

Immunization protocol. Rabbits were immunized subcutaneously with 400 pg of RSV NCS per animal in complete Freund's adjuvant. Booster injections, 400 pg of antigen in incomplete Freund's adjuvant per animal, were given three times with an interval of 4 weeks between each inoculation. The same immunization schedule was used for guinea pigs, except that 200 pg of antigen per animal was administered. Animals were bled 3 weeks after the final injection, and the RSV-specific antibody concentration was determined by an ELISA using Hep-2 cell lysates as antigen and uninfected Hep-2 cell lysates as control antigen.

ELISA for the determination of RSV antibodies in the immunesera. RSV antigen and control antigen were prepared according to Br&kovh et a1 [ 19811. The tests for RSV antibodies were performed as described earlier for mumps virus antibodies [Popow-Kraupp, 19811. Horseradish peroxidase-conjugated goat antibodies against rabbit or guinea pig immunoglobulins were purchased from Nordic Immuno-

Irnrnunofluorescence and Virus Isolation 127

logical Laboratories (Lausanne, Switzerland). The absorbance at 492 nm was mea- sured by the use of a multichannel photometer (Multiscan, Flow Laboratories, Bonn, Federal Republic of Germany), and the specific absorbance (difference between absorbance obtained with antigen and controlantigen) was calculated for each serum dilution. The titer of an immune serum represented that serum dilution yielding a specific absorbance of 2 0.1. Rabbit and guinea pig immune sera had titers of lo6-

Preparation of the immunoreagents for the RSV antigen ELISA. Immuno- globulin fractions from the RSV immune sera were obtained by precipitation of the sera with a saturated solution of ammonium sulphate [final concentration 50% (w/v)] followed by extensive dialysis against PBS. The RSV immunoglobulin preparations were further absorbed with a 2% Hep-2 cell suspension [final concentration 1 % (v/ v)] according to Gardner and McQuillin [1980] and tested by ELISA against Hep-2 cell lysate antigen. No reaction of the absorbed RSV immunoglobulin preparations with the Hep-2 cell lysate antigen (absorbance < 0.1) could be observed. The protein concentrations of the absorbed RSV immunoglobulin preparations were determined according to Schaffner and Weissmann [1973], and aliquots were stored at -20°C.

ELISA for the detection of RSV antigens in NPS. All tests were carried out in Immulon U-shaped removastrips (Dynatech, Plochingen, Federal Republic of Germany). Optimal dilutions of the reagents were determined by checkerboard titra- tions. For coating, guinea pig RSV immunoglobulin (capture antibody; CAB) was diluted in carbonate-bicarbonate buffer, pH 9.6, to a protein content of 8 pg/ml, and 50 pl of the solution was placed in each well and incubated over night at 4°C. Removastrips with the coating solution could be stored in a moist chamber at 4°C for several weeks without affecting the results. Immediately before the test was carried out, the coating solution was removed, and 50 p1 per well of the diluted and sonicated NPS was added to the wells. After an incubation period at 37°C over night, the wells were washed thoroughly with PBS, pH 7.2, and 50 pl per well of rabbit RSV immunoglobulin (detector antibody; DAB), diluted in PBS, pH 7.2, supplemented with 20% heat-inactivated FCS and 2% Tween 20 (dilution buffer) to a protein content of 16 pg/ml, was added, and the wells were incubated for 1 hr at 37°C. Thereafter, the DAB was washed out, and 50 pl per well of species-specific horserad- ish peroxidase-conjugated donkey antibodies against rabbit immunoglobulin (Amer- sham International plc., Amersham, England) diluted 1 : 1 ,OOO in dilution buffer was added. After another incubation period, 1 hr at 37"C, and a final washing procedure, 50 p1 per well of substrate (0-phenylenediamine, 1 mg/ml in 0.1 M phosphate-citrate buffer, pH 5.0, containing 1 p1 perhydrol/ml) was added and the reaction stopped after 30 min by the addition of 100 p1 per well of 2 N H2S04. Absorbance at 492 nm was measured by a multichannel photometer (Multiscan, Flow Laboratories, Bonn) against a reagent blank. A positive control consisting of RSV-infected cell culture supernatant fluid was included in each test.

Determination of the Cut-Off Level

Thirty-three nasopharyngeal aspirates, RSV-negative by IF and virus isolation, were tested by the RSV antigen ELISA. The mean absorbance value (OD) plus 2 SD was 0.36. Therefore an OD of 0.4 was taken as the cut-off for a positive reaction.

lo7.

128 Popow-Kraupp et a1

Confirmatory Test

To confirm the specificity of the positive reaction of certain specimens, espe- cially of those with an OD only slightly above the cut-off or of those negative by IF or TC, a blocking test was performed as follows: NPS were tested in duplicate as described above except that prior to the addition of the DAB the specimens were incubated with horse preimmune and RSV immune serum for neutralization (Flow Laboratories Inc., Bonn; lot 43631002; minimal neutralizing titer 1:320) for 1 hr at 37°C. RSV-specific binding was indicated by a 50% or greater decrease of the OD obtained with the well incubated with the horse RSV immune serum compared to the well incubated with the horse preimmune serum. An ELISA reading that failed to show a 50% or greater decrease in the blocking test was considered false-positive.

Immunoreagents for the adenovirus, parainfluenza viruses types I, 11, 111, influenza virus A and B virus antigen ELISAs were kindly provided by Prof. P. Halonen (Department of Virology, University of Turku, Finland).

A result of one of the three tests employed was considered false-negative if in the same specimen a reproducible positive result was obtained by at least one of the two other methods employed. The sensitivity of the test system investigated was calculated as the percentage of concordant positive results in both the assay investi- gated and the reference test in relation to the positive samples in the reference system. The specificity of the test system investigated was calculated as the percentage of concordant negative results in both the assay investigated and the reference test in relation to the negative samples in the reference system.

Statistical Methods

The x2 test and the x2 test of McNemar was used to test significant differences.

RESULTS Sensitivity of the Test System

To determine the lowest amount of RSV-specific nucleocapsids detectable by the immunoreagents prepared by us, a purified nucleocapsid preparation (Fig. 1A) was diluted to a protein content of l,OOO, 100, 10, 1, 0 ng/ml and tested by ELISA. As can be seen in Figure lB, the test system was capable of detecting RSV nucleocap- sids at a protein concentration of 10 ng/ml.

Application to Clinical Samples

Of the 404 nasopharyngeal aspirates, 278 were screened by ELISA and IF, 205 by ELISA and virus isolation, and 89 by all three methods. Results are summarized in Table I.

Comparison of ELISA to IF. Enough material was available from 223 of the 248 NPS screened by ELISA and IF to test their cell fractions (CF) by both methods; 55 specimens could not be divided in two equal parts prior to dilution because of an insufficient amount of material. The cell fractions of these secretions were used for IF, and the supernatants (mucus fraction; MF) were tested by ELISA. These speci- mens are further designated as mucus fractions. Of the 278 NPS, 223 CF and 55 MF, 62 (52 CF, ten MF) were RSV-positive and 189 (150 CF, 39 MF) were RSV-negative by both test systems. Discordant results were obtained with 27 specimens (Table I). Nine of these were positive only by ELISA and 18 (12 CF and six MF) only by IF.

nmunofluorescence and Virus Isolation 129

Fig. 1. Determination of the sensitivity of the respiratory syncytial virus (RSV) antigen ELISA by testing a purified RSV nucleocapsid preparation (A) in the test sytstem (B).

Of the nine ELISA RSV-positive and IF-negative secretions, eight showed a more than 50% reduction of their ODs in the blocking test. One with an OD slightly above the cut off-was unblockable and therefore represents a false-positive ELISA result. This specimen was also negative by culture. Four of the eight ELISA RSV- positive and IF-negative specimens were also tested by virus isolation, and RSV was isolated from all of them (Fig. 2c). One of these four additionally contained parainflu- enza virus type 111, confirmed by ELISA, IF, and virus isolation.

Of the 18 specimens positive only by IF, 12 were CF and six were MF. Virus isolation was carried out in four of the 12 cell fractions, and RSV was recovered from two (Fig. 2c). To analyze the reasons and factors leading to a false-negative ELISA result, NPS were classified into three groups according to the extent of their fluores- cence (Fig. 2a): group 1, NPS with 0 RSV-positive cells in 4 X 10 microscopic fields; group 2, NPS containing 1-15 RSV-positive cells; and group 3; NPS with more than 15 RSV positive cells. Specimens negative for RSV by ELISA and positive by IF were all in group 2.

Older children with reinfections or patients whose samples were drawn late in their illness excrete lower amounts of RSV. To determine whether they represent a group among whom false-negative ELISA results would be found more frequently, patients of group 1 and group 2 were analyzed with respect to their age and the number of days from the onset of symptoms to NPS sampling. No significant difference between the two groups could be found ( x 2 test).

The sensitivities of the ELISA in relation to IF were 77.5% and 86.7% when the mucus fractions were excluded. The specificity of the ELISA compared to IF was 95.7%.

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Comparison of ELISA with culture. Seventy-one of the 205 NPS tested by ELISA and virus isolation were RSV-positive and 118 were RSV-negative in both tests. Discordant results were obtained with 16 specimens (Table I, Fig. 2b). Of the eight specimens RSV-positive only by ELISA, one represents an unspecific reaction as described above. The remaining seven specimens showed a 50% reduction in the blocking test, and four of them were also RSV-positive by IF (Fig. 2c). One of the four specimens RSV-positive by ELISA and by IF and negative by culture additionally contained adenovirus as detected by ELISA, IF, and culture. Of the eight specimens RSV-positive by culture and negative by ELISA, six were also screened by IF, and two were RSV-positive (Fig. 2c). The sensitivity of the ELISA compared to culture was 89.9% and the specificity 94.4%.

Comparison of ELISA with TC and IF. Enough material for the screening by all the three methods was available from 89 NPS. Concordant results were obtained with 72 of 89 (80.9%) of the specimens. Of the 29 NPS RSV-positive by ELISA, 28 were also positive by culture and/or IF, and one gave a false-positive result by ELISA. A false-negative ELISA result was obtained with eight NPS, a false-negative IF result was also obtained with eight specimens. Six specimens were falsely classified as negative by culture. Paired serum samples from these six patients were tested for RSV-specific IgG and IgA antibodies by ELISA. Seroconversions were found in all the six paired sera.

Forty-seven (11.6%) of the 404 NPS contained other viruses diagnosed by IF, ELISA, and/or TC. Ten specimens were positive for parainfluenza virus type 111, 12 for adenoviruses, 21 for rhinoviruses, one for parainfluenza virus type I, one for influenza virus type B, one for Coxsackie virus B1, and one for Echo virus type 20. None gave a positive result by the RSV antigen ELISA (mean OD 0.124, SD 0.108).

DISCUSSION

Detection of RSV antigens in nasopharyngeal secretions by ELISA or radioim- munoassay (RIA) with a varying degree of test sensitivity has been reported by several groups. Immunoreagents employed in these tests either were commercially available [Chao et al, 19791 or were produced by immunization with purified RSV [Hendry and McIntosh, 19821, RSV polypeptides [Hornsleth et al, 19821 or RSV NCS [Sarklunen et al, 198 11. We decided to use NCS antisera as immunoreagents for our assay because RSV NCS can be purified in sufficient amounts and represent the predominant virus protein in infected cells.

The sensitivity of our ELISA as measured by testing a purified nucleocapsid preparation was 10 ng of protein/ml (Fig. l a and b). When the results obtained with ELISA were compared to those of the indirect IF (Table I, Fig. 2a), nearly the same proportion of RSV-positive cell fractions was false-negative by IF (eight of 72) and by ELISA (12 of 72). Of the 16 RSV-positive specimens whose cell fractions were screened by IF and whose mucus fractions were tested by ELISA, six (37.5%) were false-negative by ELISA, indicating that the supernatant of an unsonicated nasal secretion is unsuitable material for ELISA testing.

All the specimens with false-negative ELISA results were obtained from patients whose NPS contained a low concentration of RSV antigens (Fig. 2a). Hornsleth et a1 [1982] reported a higher percentage of NPS with a low RSV antigen concentration and a false-negative ELISA result in children older than 9 months of age. We were

Immunofluorescence and Virus Isolation 133

not able to define, however, a specific group according to age or number of days between onset of symptoms and NPS sampling among whom false-negative ELISA results would be more frequently encountered.

One reason for discrepant results obtained with specimens containing low amounts of RSV antigen is that, even with one cell containing viral antigens, a positive result can be obtained by IF, whereas a certain concentration of antigen is required for a positive ELISA result. Therefore, IF is somewhat more sensitive if specimens are carefully prepared and screened, which is extremely time-consuming . In the case of a large number of samples, however, it is likely that, because of a lack of time, the small number of RSV-positive cells in some samples is overlooked leading to a loss of IF sensitivity.

False-negative results were also obtained by virus isolation (Table I, Fig. 2b,c). Considering the fact that nearly the same proportion of specimens was false-negative by all the three test systems (Table I, Fig. 2a-c), the small but statistically not significant differences between the three methods are most probably due to difficulties in separating nonhomogeneous material into two or three portions containing equal amounts of antigen.

None of the 47 NPS containing other viruses gave a false-positive result in the RSV antigen ELISA.

Our data show that ELISA for the detection of RSV antigens in NPS from children with acute respiratory tract infections is as sensitive and specific as indirect IF and virus isolation. In contrast to these two tests ELISA can easily be automated, specimen handling is simple, and the results are not influenced by unfavorable conditions during the transport of specimens to the laboratory [McIntosh et al, 19821. This test therefore represents an excellent tool for a rapid viral diagnosis service for pediatric hospitals and practitioners a long distance from a virus laboratory. It also allows widescale screening over a large geographical area, which is of particular importance for collecting data on the prevalence of respiratory virus infections throughout the country.

ACKNOWLEDGMENTS

We wish to thank Mrs. I. Hartnett, Mrs. H. Dippe, and Mrs. C. Rabeck for their excellent technical assistance and Mag. U. Apfelthaler for typing the manuscript. This work was supported by the Dr. Josefine Frank Fund.

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