Improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time PCR using nasopharyngeal flocked swabs

Detection of respiratory viruses by real-time multiplexed PCR (M-PCR) and of respiratory syncytial virus (RSV) by M-PCR and immunofluorescence (IF) was evaluated using specimens collected by nasopharyngeal flocked swabbing (NFS) and nasal washes (NW). In children with mild respiratory illness, NFS collection was superior to NW collection for detection of viruses by M-PCR (sensitivity, 89.6% versus 79.2%; P = 0.0043). NFS collection was noninferior to NW collection in the detection of RSV by IF.     

Comparison of nasopharyngeal aspirate with flocked swab for PCR‐detection of respiratory viruses in children

Fast‐ and high‐throughput molecular workflows require sample matrices to be suitable for automation. Respiratory swabs are better suited for this purpose compared to the more viscous nasopharyngeal aspirates. Samples collected by nasopharyngeal aspiration and nasopharyngeal flocked swab from 81 children were compared for detection and recovery of respiratory viruses. Using real‐time RT‐PCR, no statistically significant differences in virus detection between the two sample types were found, supporting the use of flocked swabs in children aged one month to two years.     

Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses

To determine the usefulness of nasal swabs as a simple method for detection of respiratory viruses, we compared nasal swabs and nasopharyngeal aspirates obtained at the same time from the opposite nostrils of 230 children with upper respiratory infection. The sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except respiratory syncytial virus.     

Comparison between pernasal flocked swabs and nasopharyngeal aspirates for detection of common respiratory viruses in samples from children

In this prospective study we compared the use of pernasal flocked swab samples with the use of nasopharyngeal aspirate (NPA) samples for the detection of respiratory viruses from 455 children less than 5 years of age. Overall, the sensitivity and the specificity of the pernasal flocked swab samples were 98.5% and 100%, respectively. The excellent sensitivity of the flocked swab samples in combination with the rapid means by which they may be collected makes them an alternative to NPA samples, whose collection is more invasive.     

Detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction

In this study, we present the multiple detection of respiratory viruses in infants during primary respiratory illness, investigate the sensitivity of nasal swabs and nasopharyngeal aspirates, and assess whether patient characteristics and viral load played a role in the sensitivity. Healthy infants were included at signs of first respiratory tract infection. Paired nasopharyngeal aspirates and nasal swabs were collected. Real-time polymerase chain reaction (PCR) was carried out for 11 respiratory pathogens. Paired nasopharyngeal aspirates and nasal swabs were collected in 98 infants. Rhinovirus (n = 67) and respiratory syncytial virus (n = 39) were the most frequently detected. Co-infection occurred in 48% (n = 45) of the infants. The sensitivity of the nasal swab was lower than the nasopharyngeal aspirate, in particular, for respiratory syncytial virus (51% vs. 100%) and rhinovirus (75% vs. 97%). The sensitivity of the nasal swab was strongly determined by the cycle threshold (CT) value (p < 0.001). The sensitivity of the swab for respiratory syncytial virus, but not rhinovirus, was 100% in children with severe symptoms (score ≥11). It is concluded that, for community-based studies and surveillance purposes, the nasal swab can be used, though the sensitivity is lower than the aspirate, in particular, for the detection of mild cases of respiratory syncytial virus (RSV) infection.     

Swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs

The purpose of this study was to compare the sampling efficacy of rayon swabs and nylon flocked swabs, and of oropharyngeal and nasopharyngeal specimens for the detection of respiratory viruses in elderly patients. Samples were obtained from patients 60 years of age or above who were newly admitted to Sorlandet Hospital Arendal, Norway. The patients were interviewed for current symptoms of a respiratory tract infection. Using rayon swabs and nylon flocked swabs, comparable sets of mucosal samples were harvested from the nasopharynx and the oropharynx. The samples were analysed using real-time polymerase chain reaction (PCR) methods. A total of 223 patients (mean age 74.9 years, standard deviation [SD] 9.0 years) were swabbed and a virus was recovered from 11% of the symptomatic patients. Regardless of the sampling site, a calculated 4.8 times higher viral load (95% confidence interval [CI] 1.3–17, p = 0.017) was obtained using the nylon flocked swabs as compared to the rayon swabs. Also, regardless of the type of swab, a calculated 19 times higher viral load was found in the samples from the nasopharynx as compared to the oropharynx (95% CI 5.4–67.4, p < 0.001). When swabbing for respiratory viruses in elderly patients, nasopharyngeal rather than oropharyngeal samples should be obtained. Nylon flocked swabs appear to be more efficient than rayon swabs.     

Performance of virus isolation and Directigen Flu A to detect influenza A virus in experimental human infection.

BACKGROUND: Few data exist to assess the sensitivity of different specimen types for viral detection during the course of influenza virus infection. OBJECTIVES: This study assessed the relationships between quantitative influenza A virus replication and antigen detectability by the enzyme immunosorbent assay (EIA) Directigen Flu A in different type of samples during experimental human infection. STUDY DESIGN: Fourteen volunteers were inoculated with influenza A virus A/Texas/36/91 (H1N1). Four specimens types were collected in sequence for quantitative isolation in cell culture and antigen testing from days 1 to 8 after inoculation. RESULTS: Seventy-one (63%) of nasopharyngeal wash specimens were culture positive, compared to 51 (46%) of throat gargles, 51 (46%) of nasal swabs, and 27 (24%) of throat swabs. All subjects shed virus in their nasopharyngeal wash at least one day and 86% of subjects had a positive nasopharyngeal wash culture on day 2 after inoculation. The mean viral titers were highest on day 2 post inoculation for all specimen types and averaged 3.6 log10 TCID50/ml for nasal washes, 1.2 log10 TCID50/ml for throat gargles, 1.8 log10 TCID50/ml for the nasopharyngeal swabs, and 0.6 log10 TCID50/ml for the throat swabs. Mean viral titers in the nasal washes were significantly different (P<0.05) compared to other specimen types. The peak of sensitivity of EIA (compared to culture) was the second day after inoculation. Nasopharyngeal and throat swab results were combined for this analysis and considered positive by culture if positive in either or both samples. Thus, on day 2 the number of EIA positive samples relative to the number culture positive was 9/12 (75%) for nasopharyngeal wash specimens, 2/9 (22%) for throat gargles, and 7/11 (64%) for the combined throat and nasal swabs specimens. CONCLUSIONS: Nasopharyngeal washes are the most sensitive sample type detecting influenza A virus in adults. For rapid diagnosis the Directigen Flu A is an alternative with a sensitivity compared to culture ranging between 64 and 78% if performed on nasopharyngeal specimens on day two or three after experimental infection in adults. However, if performed on other specimens or later in the course of infection the sensitivity is lower.     

Comparison of Midturbinate Flocked-Swab Specimens with Nasopharyngeal Aspirates for Detection of Respiratory Viruses in Children by the Direct Fluorescent Antibody Technique

Paired nasopharyngeal aspirate (NPA) and midturbinate flocked-swab specimens from 153 children with respiratory symptoms were examined by the direct fluorescent antibody (DFA) technique. Seventy-four infants (49%) had a viral infection documented by DFA. The flocked-swab specimens had 93% sensitivity and 96.7% agreement with the NPA specimens, with a kappa coefficient of 93.4% (95% confidence interval, 0.877, 0.991).The direct fluorescent antibody (DFA) technique revolutionized the rapid detection of respiratory viruses. Since its inception in 1968, it has been one of the mainstays in clinical virology laboratories throughout the world (4). The ability of DFA to detect respiratory viruses depends on many things, but it all begins with good specimen collection. The nasopharyngeal aspirate (NPA) has been considered the best specimen to detect respiratory viruses in infants (4). However, it is difficult to collect because it requires special equipment, such as a catheter, trap, and vacuum source, and specialized training. A traditional nasopharyngeal swab is the next best specimen, especially in older children or adults, because it utilizes common supplies; however, the collection end of the swab, comprised of wound Dacron fibers, has limited absorbent capacity to trap virus-infected exfoliated epithelial cells. A nylon nasopharyngeal flocked swab with enhanced absorptive properties introduced in 2006 compared favorably to the NPA for the detection of respiratory viruses by DFA (2). Recently, a midturbinate flocked swab developed by Smieja, et al. (7), and marketed by Copan, Inc., has offered a more intuitive approach for the collection of nasopharyngeal specimens (1). It has compared favorably to the NPA and the flocked nasopharyngeal swab in the diagnosis of respiratory viruses by culture, antigen detection, and PCR, none of which require intact exfoliated epithelial cells for visualization; there is no published experience of midturbinate flocked swabs with DFA in children (1, 5, 6). The midturbinate flocked swab differs from the nasopharyngeal swab. It has a sampling depth indication gauge and also has a larger absorptive capacity than the smaller nasopharyngeal swab.The present study was designed to compare the efficacy of the midturbinate flocked swab with the NPA in the detection of respiratory viruses by DFA.The study was conducted from 5 January 2010 through 11 March 2010. All children 2 years of age or less admitted to the infant''s floor of the hospital with respiratory symptoms were enrolled. The study was reviewed by the Children and Youth Institutional Review Board, who waived the need for a formal review because the study was deemed an evaluation comparing a new specimen collection device to the standard nasopharyngeal aspirate; parents were allowed to opt out of the use of the new specimen device. A nasopharyngeal aspirate specimen was collected through one nostril. A second specimen was collected through the other nostril with a midturbinate FLOQ swab (Copan Diagnostics, Inc., Murrieta, CA) designed for children 2 years of age or less; the swab was inserted up to the collar on the shaft. Both specimens were placed in 3 ml of Copan UTM transport medium, transported to the virus laboratory, and processed within 6 h. The suspension was centrifuged, and the cellular pellet washed. The cells were then spotted to glass slides. The cells were stained for DFA using a D3 Ultra respiratory screening identification kit (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The kit screened for respiratory syncytial virus (RSV), influenza viruses (IFV) A and B, parainfluenza viruses (PFV) 1, 2, and 3, and adenovirus (AdV). An additional stain for human metapneumovirus (hMPV) (DHI) was included. The DFA readers were not blinded to the specimen source. The degree of DFA agreement between specimens collected by NPA and midturbinate flocked swabs was calculated with Cohen''s kappa coefficient of agreement.One hundred fifty-three infants entered the study. Paired specimens were collected from every infant. Respiratory viruses were identified in 74 (48.6%). Respiratory syncytial virus was most frequent, found in 47 patients (30%), with hMPV in 25 (16.3%), PFV in 1 (0.7%), AdV in 1 (0.7%), and IFV in none (0.0%). The 2009 H1N1 influenza A virus had last been identified in the laboratory in November 2009, more than 1 month before the start of the study. DFA of NPA specimens identified all the viruses. DFA of the flocked-swab specimens failed to detect 4 RSV and 1 hMPV isolate that had been detected in the NPA specimens. The negative DFA test results on flocked-swab specimens agreed with the negative DFA test results on NPA specimens. Overall, the positive and negative DFA test results on flocked-swab specimens had 96.7% agreement with the DFA test results on NPA specimens, with a Kappa coefficient of 93.4% (95% confidence interval [CI], 0.877, 0.991; P < 0.00001). The sensitivity of the flocked swab was 93.2% (95% CI, 0.849, 0.978).The midturbinate flocked swab proved to be comparable to the NPA for the detection of common respiratory viruses, such as RSV and hMPV, in a DFA test in the present study. The absence of IFV and the low numbers of AdV and PFV isolates in specimens prevented an assessment of the swab''s utility in detecting these viruses; however, earlier studies with nasopharyngeal flocked swabs suggested that the midturbinate swab would give similar results (3). In an earlier study, the sensitivity of the NPA in detecting either IFV or RSV was greater than the sensitivity of flocked nasopharyngeal swabs, although the difference was not statistically significant; the differences may be attributed to the greater number of respiratory epithelial cells available for examination in NPA specimens (2). The advantage of the midturbinate collection over nasopharyngeal collection resides in the relative ease of collection and the resultant patient cooperation, especially among the very young; however, the observations made in the present study may not extend beyond the pediatric population.     

Pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults

A pooled sample of oropharyngeal swabs, nasopharyngeal swabs and nasopharyngeal washings, taken from each of 1,000 subjects, was compared to separate specimens from the same sampling. Multiplex real-time polymerase chain reaction (mqRT-PCR) was used to identify 12 respiratory viruses. Two hundred and forty-three (97%) of the 251 viruses identified in the separate samples were also identified in the mixed samples. The sensitivity rate was identical at 100% for all virus groups except coronaviruses. This sensitivity rate clearly justifies the use of pooled samples instead of separate samples for clinical and epidemiological purposes. The reduction in costs attained from the use of pooled samples may represent a critical advantage when considering its use in extensive clinical and epidemiological studies.     

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

BACKGROUND: The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.     

Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens.

Directigen FLU-A, a new enzyme immunoassay membrane test, rapidly detects influenza A virus antigen in specimens from patients. Nasopharyngeal washes and pharyngeal gargles were used to determine the effectiveness of the assay as applied to different types of routinely collected clinical samples. All specimens had been previously shown to contain influenza A virus by virus isolation in tissue culture. Directigen FLU-A was 90% sensitive (95% confidence interval, 56 to 99.7%) with nasopharyngeal washes but only 39% sensitive (95% confidence interval, 17 to 64%) with pharyngeal gargles (P = 0.018) when used with samples containing similar amounts of infectious virus (50% tissue culture infective dose, 1.0 to 4.5). The intensity of the positive reaction with Directigen FLU-A did not correlate with the amount of virus in the specimens. Directigen FLU-A was found to detect cell-associated antigen more readily than free virus; only 20 infected cells were required to identify cell-associated influenza A virus antigen, whereas the limit of detection for free virus was 1.63 x 10(3) infectious virus particles. These findings suggest that Directigen FLU-A detects the cell-associated antigen present in clinical specimens rather than free virus. In addition, Directigen FLU-A detected avian and swine influenza A viruses in both cloacal swabs (75% sensitivity) and swine lung homogenates (86% sensitivity), indicating its potential usefulness in the surveillance of nonhuman influenza A viruses.     

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.     

Dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time NASBA

This paper describes the molecular detection of influenza A, influenza B, respiratory syncytial virus and human metapneumovirus using real-time nucleic acid sequence based amplification (NASBA) from respiratory samples collected on simple dry cotton swabs, non-invasively and in the absence of transport medium. Viral RNA was detectable on dry cotton and flocked swabs for at least 2 weeks at room temperature and was readily extracted using magnetic silica extraction methods. Dry cotton respiratory swabs were matched with traditionally collected respiratory samples from the same patient, and results of traditional laboratory techniques and real-time NASBA were compared for all four viral targets. The results not only showed a significant increase in the detection rate of the viral targets over traditional laboratory methods of 46%, but also that dry swabs did not compromise their recovery. Over two subsequent winter seasons, 736 dry cotton respiratory swabs were collected from symptomatic patients and tested using real-time NASBA giving an overall detection rate for these respiratory virus targets of 38%. The simplicity of the method together with the increased detection rate observed in the study proves that transporting a dry respiratory swab to the laboratory for respiratory virus diagnosis using molecular methods is a suitable and robust alternative to traditional sample types.     

A comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time PCR

We tested the hypothesis that swabs from the nasopharynx carry a higher viral load than swabs from the oropharynx in patients with real-time polymerase chain reaction (PCR)-confirmed influenza infection. Using flocked swabs, oropharyngeal and nasopharyngeal samples were harvested from hospital-admitted influenza patients no later than 3 days after the initial detection of influenza virus. Comparison of cycle threshold (CT) values was performed to assess differences in viral load in the specimens. Seventeen patients were diagnosed with influenza B, 14 patients with influenza A(H1N1)pdm09, and one patient with influenza A(H3N2). Nasopharyngeal samples were positive at a lower CT value than the oropharyngeal samples [mean difference in CT 5.75, 95 % confidence interval (CI) 3.8–7.7, p?<?0.01], suggesting that, on average, the calculated viral load of the nasopharyngeal samples was 54 times higher (95 % CI 13.7–210.8) than those of the oropharyngeal samples. The corresponding difference in the calculated viral load for influenza A(H1N1)pdm09 virus was 23 times (95 % CI 3.8–136.2, p?<?0.01) and for influenza B virus, it was 80 times (95 % CI 9.3–694.6, p?<?0.01). In patients with acute influenza, nasopharyngeal swabbing was clearly superior to oropharyngeal swabbing in terms of diagnostic yield by real-time PCR.     

Evaluation of nasal and nasopharyngeal swab collection for the detection of Epstein‐Barr virus in nasopharyngeal carcinoma

Epstein‐Barr virus detection using nasopharyngeal swabs has been suggested as a potential screening test that could improve the specificity of current EBV‐based serological assays. However, application requires insertion of the swab deep into the nasopharynx, a procedure not amenable to non‐clinic screening. We reasoned that swabbing the more easily accessible nasal cavity might provide an appealing alternative for NPC detection. Patients > 18 years of age diagnosed with histologically confirmed NPC were recruited from the Otolaryngology Department at the National Taiwan University Hospital. ENT clinicians collected both nasal and nasopharyngeal swabs. EBV DNA and cellular beta‐globulin DNA were quantified using quantitative PCR targeting a highly‐conserved region of the BKRF1 gene. EBV DNA was detectable (non‐zero) in all 34 nasopharyngeal swabs and above the positivity threshold of 1666 EBV copies in 30 (88.2%) patients. EBV DNA was detectable in 50% of 34 nasal swabs and above the positivity threshold in four (11.8%) patients. Average EBV DNA levels were >3‐fold higher (P < 0.001) in nasopharyngeal compared to nasal swabs. Among the 17 NPC patients with detectable EBV DNA in both swab types, we observed correlation (P < 0.01) between EBV DNA measurements. Our data represent the first evaluation of EBV DNA collected from nasal swabs. Given current EBV DNA amplification techniques, nasopharyngeal swabs remain more sensitive than nasal swabs for NPC detection.     

Comparison of the FilmArray RP,Verigene RV+, and Prodesse ProFLU+/FAST+ Multiplex Platforms for Detection of Influenza Viruses in Clinical Samples from the 2011-2012 Influenza Season in Belgium

Respiratory tract infections (RTIs) are caused by a plethora of viral and bacterial pathogens. In particular, lower RTIs are a leading cause of hospitalization and mortality. Timely detection of the infecting respiratory pathogens is crucial to optimize treatment and care. In this study, three U.S. Food and Drug Administration-approved molecular multiplex platforms (Prodesse ProFLU+/FAST+, FilmArray RP, and Verigene RV+) were evaluated for influenza virus detection in 171 clinical samples collected during the Belgian 2011-2012 influenza season. Sampling was done using mid-turbinate flocked swabs, and the collected samples were stored in universal transport medium. The amount of viral RNA present in the swab samples ranged between 3.07 and 8.82 log₁₀ copies/ml. Sixty samples were concordant influenza A virus positive, and 8 samples were found to be concordant influenza B virus positive. Other respiratory viruses that were detected included human rhinovirus/enterovirus, respiratory syncytial virus, parainfluenza virus type 1, human metapneumovirus, and coronavirus NL63. Twenty-five samples yielded discordant results across the various assays which required further characterization by sequencing. The FilmArray RP and Prodesse ProFLU+/FAST+ assays were convenient to perform with regard to sensitivity, ease of use, and low percentages of invalid results. Although the limit of sensitivity is of utmost importance, many other factors should be taken into account in selecting the most convenient molecular diagnostic assay for the detection of respiratory pathogens in clinical samples.     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

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.