Evaluation of three rapid enzyme immunoassays and cell culture for detection of respiratory syncytial virus

Mendoza, Joaquín; Rojas, Andrés Romero; Navarro, José María; Plata, Cecilia de; Rosa, Manuel de la

doi:10.1007/bf01961862

Cited by 15 publications

(7 citation statements)

References 15 publications

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“…At present, there is no vaccine available for prevention of the viral infection, although antiviral therapy is known to be effective in some patients (1, 2, 11). For appropriate treatment of RSV infection, it is crucial to have an accurate and timely diagnostic method for detection of the virus.A number of techniques are available for detection and identification of RSV, including cell culture, enzyme immunoassay (EIA), immunofluorescence (IF), and conventional reverse transcription (RT)-PCR (6,7,8,9,12,16,17,21,22,25). The classical cell culture method requires prompt inoculation of the labile virus and is a time-consuming process.…”

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Simultaneous Detection, Subgrouping, and Quantitation of Respiratory Syncytial Virus A and B by Real-Time PCR

Hu¹,

et al. 2003

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Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan Respiratory syncytial virus (RSV) is one of the major respiratory pathogens causing lower respiratory tract diseases in children (10,18,23). Based on genetic and antigenic variations in the structural proteins, RSV is classified into two subgroups, A and B (3, 24). At present, there is no vaccine available for prevention of the viral infection, although antiviral therapy is known to be effective in some patients (1, 2, 11). For appropriate treatment of RSV infection, it is crucial to have an accurate and timely diagnostic method for detection of the virus.A number of techniques are available for detection and identification of RSV, including cell culture, enzyme immunoassay (EIA), immunofluorescence (IF), and conventional reverse transcription (RT)-PCR (6,7,8,9,12,16,17,21,22,25). The classical cell culture method requires prompt inoculation of the labile virus and is a time-consuming process. In addition, the test has low sensitivity, detecting only 50 to 60% of RSV infections (17). More rapid immunoassays for the detection of RSV, such as EIA and IF, also have limitations in sensitivity and specificity. The accuracy of EIA, for example, is in the range of 57 to 98%, and that of IF varies from 65 to 92% (12). This wide variability in assay sensitivity and specificity may lead to inaccurate diagnosis of RSV infection, and consequently, the assays may be of limited value in patient care. Furthermore, such rapid immunoassays are rarely capable of differentiating RSV subgroups A and B, which may be associated with different disease severities (28).Conventional RT-PCR, which has recently been developed for the detection of RSV, is a more sensitive and specific diagnostic method that also allows for the subgrouping of the virus in a single reaction (6,7,8,9,17,25). However, it still requires time-consuming post-PCR analysis. Real-time RT-PCR, on the other hand, eliminates post-PCR processing (13). This is achieved by combining conventional RT-PCR with advanced fluorescence detection technology, which allows the fluorescence signals to be analyzed and recorded during PCR cycling. The advanced method also performs automatic sample analysis with enhanced sensitivity and specificity. In this report, we describe the development and application of a TaqManbased RT-PCR assay for simultaneous detection, subgrouping, and quantification of RSV A and B in clinical respiratory specimens. MATERIALS AND METHODSVirus stocks, plaque titration, and virus particle counts. RSV A2 (subgroup A) and 2B (subgroup B) were propagated in Vero cells. About 100 ml each of subgroup A and B virus stocks were prepared, and the stocks were stored in 0.5-ml aliquots for future use. For plaque titration of RSV stocks, confluent Vero cells in 24-well plates were inoculated with 100 l of a serial dilution of each virus at 37°...

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“…A number of techniques are available for detection and identification of RSV, including cell culture, enzyme immunoassay (EIA), immunofluorescence (IF), and conventional reverse transcription (RT)-PCR (6,7,8,9,12,16,17,21,22,25). The classical cell culture method requires prompt inoculation of the labile virus and is a time-consuming process.…”

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confidence: 99%

Simultaneous Detection, Subgrouping, and Quantitation of Respiratory Syncytial Virus A and B by Real-Time PCR

Hu¹,

et al. 2003

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“…Laboratory diagnosis by cell culture and viral serology is usually necessary to identify the etiologic agent, although the final results of RSV isolation by tissue culture usually require several days. A more rapid diagnosis can be made by direct detection methods, such as enzyme immunoassay and immunochromatography (IC) testing with nasopharyngeal swabs or aspirates (4,6,9). Recently these test have been widely used; however, they are only moderately sensitive in detecting RSV in respiratory tract specimens.…”

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Comparison of an Immunochromatography Test with Multiplex Reverse Transcription-PCR for Rapid Diagnosis of Respiratory Syncytial Virus Infections

et al. 2004

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Human respiratory syncytial virus (RSV) is the most important viral pathogen causing lower respiratory tract infections in infants, immunocompromised hosts, and the elderly (1, 2, 10). Epidemics of RSV infection occur every winter in temperate climates, during rainy seasons, or year-round in tropical regions (11). RSV can infect the same individual repeatedly as well as infants under 6 months of age who still possess maternal antibodies against the virus (7). Many of these infections are difficult to distinguish clinically from other respiratory viral infections and some bacterial infections. Laboratory diagnosis by cell culture and viral serology is usually necessary to identify the etiologic agent, although the final results of RSV isolation by tissue culture usually require several days. A more rapid diagnosis can be made by direct detection methods, such as enzyme immunoassay and immunochromatography (IC) testing with nasopharyngeal swabs or aspirates (4,6,9). Recently these test have been widely used; however, they are only moderately sensitive in detecting RSV in respiratory tract specimens. On the other hand, immunofluorescent-antibody tests are highly sensitive for detecting RSV; however, they need special equipment and take at least 1 h to complete (4).We evaluated a new IC test, the SAS RSV test (SA Scientific, San Antonio, Tex.), for the first time to assess its clinical usefulness in detecting RSV antigens in nasopharyngeal swabs from subjects with RSV respiratory tract infections. This test takes 10 min to perform and relies on two specific monoclonal antibodies against RSV antigen. The sensitivity, specificity, and convenience of the test were assessed and compared to those of an existing IC test, Directigen EZ RSV (BD Biosciences, San Jose, Calif.). As a "gold standard " for detecting RSV in clinical samples, we used a multiplex reverse transcription-PCR (multiplex RT-PCR) method that was developed by Stockton et al. (12) for detecting and subtyping RSV (groups A and B). Recent studies suggest that RT-PCR is more sensitive than viral culture for detecting respiratory viruses in clinical specimens (13).One hundred two patients from 8 days to 9 years old (median ϭ 11 months) were included; 9, 1, 52, 23, and 17 subjects were diagnosed as having upper respiratory tract infection, laryngitis, tracheobronchitis, bronchiolitis, and pneumonia, respectively. The patients were seen during 2003 to 2004 at three institutions. Nasopharyngeal swabs obtained from each subject were suspended in 2 ml of 2% fetal calf serum-minimal essential medium and were used for two IC tests. The other was stored at Ϫ20°C until further analysis.For RNA extraction, we used 280-l samples according to the spin protocol of the QIAamp viral RNA minikit (Qiagen, Valencia, Calif.). For cDNA synthesis, 22.2 l of RNA solution was added to a reaction mixture (17.8 l) containing random hexamer (Takara, Otsu, Japan) and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, Calif.). The reaction mixture was incubated at...

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“…Direct antigen detection by indirect immunofluorescent assay (IFA) or commercial colorimetric enzyme immunoassay (EIA) has been used successfully in children. In this age group, using nasal washes or aspirates for sample collection, these tests appear to be both relatively sensitive and specific 6 . However, nasal washes are difficult to perform in older persons, particularly those with diminished capacity for cooperation.…”

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Evaluation of Four Methods for the Diagnosis of Respiratory Syncytial Virus Infection in Older Adults

Falsey

McCann

Hall

et al. 1996

J American Geriatrics Society

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Evaluation of three rapid enzyme immunoassays and cell culture for detection of respiratory syncytial virus

Cited by 15 publications

References 15 publications

Simultaneous Detection, Subgrouping, and Quantitation of Respiratory Syncytial Virus A and B by Real-Time PCR

Simultaneous Detection, Subgrouping, and Quantitation of Respiratory Syncytial Virus A and B by Real-Time PCR

Comparison of an Immunochromatography Test with Multiplex Reverse Transcription-PCR for Rapid Diagnosis of Respiratory Syncytial Virus Infections

Evaluation of Four Methods for the Diagnosis of Respiratory Syncytial Virus Infection in Older Adults

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