Comparison of directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syncytial virus.

An enzyme immunoassay membrane test (Directigen RSV) for the detection of respiratory syncytial virus in clinical specimens was compared prospectively with isolation in cell culture and direct immunofluorescence (IF). A total of 315 nasopharyngeal wash specimens from pediatric patients were examined. Directigen RSV was 86.1% sensitive and 91.3% specific for specimens positive by isolation in cell culture and/or IF, with 88.6% agreement. The false-positive rate was 16%; 2 of 20 specimens giving false-positive reactions by Directigen RSV were true-positives by blocking assay. Twenty-seven specimens (8.5%) whose results were initially uninterpretable by Directigen RSV due to filtration difficulties were diluted and upon retesting produced acceptable results. Sixty-three viral isolates and/or IF identifications of virus antigens representing seven virus groups other than respiratory syncytial virus were also found; cross-reactions between Directigen RSV and other viruses were not observed. Directigen RSV will be useful as an immediate procedure and in facilities lacking a comprehensive virology laboratory.     

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

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

Peroxidase-antiperoxidase assay for rapid detection of respiratory syncytial virus in nasal epithelial specimens from infants and children.

A peroxidase-antiperoxidase (PAP) assay for the rapid detection of respiratory syncytial virus was compared with the indirect immunofluorescence method and with viral culture. Nasal epithelial specimens from 147 infants and children with acute respiratory infections were obtained and evaluated for the presence of respiratory syncytial virus antigens. Sensitivity, specificity, and accuracy by PAP were 91.7, 84.8, and 87.1%, respectively, and 87.0, 88.5, and 88.0%, respectively, by immunofluorescence compared with viral culture. The PAP assay was found to be as accurate as the indirect immunofluorescence method and more convenient to perform, since the color reaction and cell morphology were more easily observable by light microscopy. A new specimen collection method is reported; gentle scraping of the superficial nasal mucosa by the Rhino-probe method provided sufficient numbers of epithelial cells to perform multiple assays.     

Comparison of direct and indirect immunofluorescence staining of clinical specimens for detection of respiratory syncytial virus antigen.

Immunofluorescence staining methods for respiratory syncytial virus antigen detection were compared. Of 50 specimens originally positive for respiratory syncytial virus by direct immunofluorescence and culture, 49 were positive by repeat direct immunofluorescence and 32 were positive by indirect immunofluorescence. Additional results obtained on specimens originally negative for respiratory syncytial virus by direct immunofluorescence, culture, or both indicate that direct immunofluorescence staining for respiratory syncytial virus antigen was more sensitive than was indirect immunofluorescence.     

Comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques.

Staining of clinical respiratory specimens obtained by nasopharyngeal and throat swabs with direct fluorescein isothiocyanate-conjugated antisera to para-influenza virus types 1, 2, and 3, influenza A virus, respiratory syncytial virus, adenovirus, mumps virus, and measles virus was compared with isolation procedures in routine tissue culture systems. Direct staining of clinical specimens with the commercially available conjugated offered a rapid means of diagnosing respiratory infections on a routine clinical basis when used in conjunction with isolation in tissue culture systems. Of 292 patients who were culture positive for these viruses, 259 were diagnosed by detection of the viral antigen in clinical specimens by direct immunofluorescence. No specimens that subsequently yielded a different respiratory virus by culture were positive by the direct immunofluorescence method. Use of selected antisera based on clinical history of the patient reduced the cost of rapid viral diagnosis.     

Respiratory syncytial virus detection by immunofluorescence in nasal secretions with monoclonal antibodies against selected surface and internal proteins

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.     

Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study

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.     

Comparison of ortho respiratory syncytial virus enzyme-linked immunosorbent assay and HEp-2 cell culture.

Two hundred seventy nasopharyngeal aspirates were tested in duplicate with the Ortho Diagnostics, Inc. (Raritan, N.J.), respiratory syncytial virus antigen enzyme-linked immunosorbent assay. The test was sensitive (80 to 82%) and specific (96%) when compared with cell culture. The enzyme-linked immunosorbent assay detected seven antigen-positive specimens not among the 71 specimens that were positive for respiratory syncytial virus in cell culture. A blocking test confirmed those specimens as true positives (specificity, 100%).     

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.     

Surveillance for respiratory syncytial virus in infants hospitalized for acute lower respiratory infection in Chile (1989 to 2000)

Hospitalized infants (4,618) were studied for lower respiratory infections from 1989 through 2000 by routine immunofluorescence assay and viral isolation. The hospitalization rate for respiratory syncytial virus (RSV) averaged 2% per year. The fatality rate was 0.1%. Monthly RSV detection varied from 14 to 88%, and epidemics lasted 3.5 to 6 months. From 1994 high-early versus low-late epidemic patterns alternately were observed, the first influenced by a group B strain.     

Routine viral culture for pediatric respiratory specimens submitted for direct immunofluorescence testing.

From 1986 to 1987, 69 (25%) of 274 specimens from children with lower respiratory tract syndromes were positive for respiratory syncytial virus antigen by direct immunofluorescence assay (DFA). Comprehensive viral culture was performed on all 205 DFA-negative specimens, and 72 specimens yielded viruses; 5 specimens yielded multiple agents. Thus, 52% of specimens yielded a specific virus, supporting the routine use of viral culture. Isolates from the DFA-negative specimens included respiratory syncytial virus (n = 7), rhinovirus (n = 34), hemadsorbing viruses (n = 13), cytomegalovirus (n = 11), adenovirus (n = 8), enteroviruses (n = 3), and herpes simplex virus (n = 2). Although serologic confirmation is needed, cytomegalovirus may be an underappreciated cause of acute lower respiratory tract infection in normal children. Further studies must be conducted to document this possibility.     

Rapid detection of respiratory syncytial virus in nasopharyngeal specimens obtained with the rhinoprobe scraper.

We compared the Rhinoprobe scraping technique for collection of superficial nasal mucosa epithelial cells and rapid detection of respiratory syncytial virus by immunofluorescence with paired, swab-collected specimens for virus culture from 1,257 infants and children with acute respiratory infections. Compared with viral culture as the reference test, the sensitivity, specificity, and accuracy of the immunofluorescence test were 83.6, 93.6, and 91.3%, respectively. We found the Rhinoprobe method safe, easy to use, and helpful in obtaining large quantities of epithelial cells for detection of respiratory syncytial virus and other respiratory viruses.     

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.     

Detection of respiratory virus antigens in nasopharyngeal secretions from patients with acute respiratory disease by radio-immunoassay and tissue culture isolation

An investigation was made of the sensitivity and specificity of four-layer radio-immunoassays (RIA) in the detection of adenovirus, respiratory syncytial virus, influenza virus types A and B, as well as para-influenza virus types 1, 2 and 3 from nasopharyngeal aspirates of 146 patients with acute respiratory disease. The sensivity of RIA was comparable with that of tissue culture isolation if the total number of positives is considered. The difference may have been caused both by a higher efficiency of the RIA for detection of inactivated or non-cultivable agents and by a higher efficiency of tissue culture methods if the samples contained only small amounts of antigen. Differences between the two antigen detection systems were found in particular with respiratory syncytial virus and influenza B virus. At present, the use of tissue culture isolation together with RIA is the optimal routine laboratory procedure for the diagnosis of respiratory infections.     

Evaluation of a commercial enzyme immunomembrane filter assay for detection of respiratory syncytial virus in clinical specimens

A commercial enzyme immunomembrane filter assay (EIFA) for respiratory syncytial virus (RSV) was compared prospectively with isolation in cell culture and an enzyme immunoassay. A total of 595 respiratory specimens, mostly from pediatric patients, were examined. The EIFA was 70.96 % sensitive and 72.40 % specific in comparison with cell culture. Results for 40 specimens (6.72 %) were uninterpretable, mainly due to filtration difficulties. Twenty-one (25 %) of 84 specimens whose results were initially considered false-positive were subsequently confirmed positive after a blocking test with bovine anti-RSV serum. On the basis of the total number of confirmed positive results, the sensitivity and the specificity of the test were 87.90 % and 75.77 %, respectively.     

Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus antigens was applied to the rapid diagnosis of acute infections in children and was compared with viral culture and immunofluorescence tests. The ELISA test employed commercially available reagents and was run on a day-to-day basis as specimens were received in the laboratory. Sensitivity and specificity by ELISA were 82 and 95%, respectively, compared with culture. In the same specimens, the sensitivity and specificity by immunofluorescence were 86 and 96%, respectively. Nasopharyngeal aspirates were proven to be a better source of viral antigen than were nasopharyngeal swabs. ELISA-positive samples remained positive even when left unrefrigerated for a week or mailed to the laboratory in plastic containers. Respiratory syncytial virus ELISA, like culture, became negative as the disease progressed and showed no superiority over culture for diagnosis late in the illness.     

Rapid and highly sensitive qualitative real-time assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology

The performance of a sensitive and specific qualitative respiratory syncytial virus (RSV) assay based on NASBA technology and real-time molecular beacon detection is presented. Very low detection limits for both RSV A and RSV B were determined: 95% detection hit-rate of 95 and 47 copies/input in isolation for RSV A and RSV B, respectively. RSV was detected in a wide variety of clinical samples including respiratory swabs, nasopharyngeal aspirates (NPA), bronchoalveolar lavages (BAL), endotracheal secretions, and sputum samples. In total 779 clinical samples were tested and a valid result was obtained for 765 (RSV NASBA assay), 765 (cell culture), and 529 (rapid direct immunofluorescence testing (IF)) samples. Of these samples, 229 (RSV NASBA assay), 61 (cell culture), and 122 (IF) samples were positive for RSV. In addition, 106 samples were reported as RSV negative using the NOW RSV assay (Binax). Subsequent testing using the RSV NASBA assay demonstrated that 32 (30%) of these samples were RSV positive. The RSV NASBA assay includes a homologous internal control, which offers a high degree of standardization and quality control. When the RSV NASBA assay was performed on the NucliSens EasyQ platform (bioMérieux), test results of 48 sample extracts were obtained in less than 2h.     

Comparative study of respiratory syncytial virus in nasopharyngeal aspirates using conventional cell culture, shell viral centrifugation culture, immunofluorescence and biotin-avidin enzyme linked immunosorbent assays.

133 nasopharyngeal aspirates (NPA) were simultaneously tested for the presence of respiratory syncytial virus (RSV) by conventional cell culture (CCC), shell vial centrifugation culture (SVC), immunofluorescence assay (IFA) and biotin-avidin enzyme linked immunosorbent assay (B-A ELISA). These yielded positive results in 32(24%), 45(33.8%), 36(27%) and 40(30%) of specimens, respectively. Specimens positive by IFA and B-A ELISA were all also positive by SVC. The sensitivity of CCC, IFA, and B-A ELISA comparing to SVC was 71%, 80%, and 88.9%, respectively. For rapid detection of RSV, we recommend the SVC method where a cell culture laboratory is available and the B-A ELISA method where a cell culture laboratory is not available.