Improved detection of respiratory syncytial virus in nasal aspirates by seminested RT‐PCR

Abstract

A seminested RT-PCR for amplification of Respiratory syncytial virus (RSV)-RNA in nasal aspirates has been developed and used to test nasopharyngeal aspirates (NPAs) from 132 infants hospitalized with acute respiratory tract infections during winter epidemics. The results were compared with those obtained by virus isolation in tissue culture and antigen detection with an enzyme-linked immunosorbent assay (Ag-ELISA). RSV-RNA was detected by seminested RT-PCR in 57 of the 59 samples that were positive by virus isolation and/or ELISA, as well as in 25 of 73 samples negative by virus isolation and ELISA. Eighteen of these 25 samples were obtained from children older than one year of age, 17 of whom were experiencing reinfection, as indicated by the presence of preexisting serum RSV-IgG antibodies. These results indicate that seminested RT-PCR is more sensitive than conventional methods for the detection of RSV in patients experiencing reinfections and suggest that this assay might also be useful for rapid diagnosis of RSV infections in older people.

Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children

“…The prevalence of RSV was only slightly higher by PCR than by FA. Others have reported the prevalence of RSV to be 2-19% higher by PCR than by conventional methods, including FA (Gueudin et al, 2003;Hu et al, 2003;Ong et al, 2001;Whiley et al, 2002) and culture and enzyme immunoassay (Henkel et al, 1997;Kehl et al, 2001). Discrepant results in our study were likely due to the higher sensitivity of the RT-PCR assay.…”

GeneScan Reverse Transcription-PCR Assay for Detection of Six Common Respiratory Viruses in Young Children Hospitalized with Acute Respiratory Illness

“…We suspect that swab specimens yield less virus-often below the threshold for detection by virus isolation (2,22), but still above the threshold for detection by RT-PCR. In older children and adults, who often have low-titer HRSV in respiratory secretions, RT-PCR has also been markedly more sensitive than virus isolation (15,31). Given the increased sensitivity of our assay, we were concerned that we were unable to detect viral RNA in several culture-positive specimens.…”

Quantitation of respiratory syncytial virus RNA in nasal aspirates of children by real-time RT-PCR assay

“…Several groups have developed RT-PCR techniques to detect RSV RNA in respiratory samples from children with lower respiratory tract infections (Cubie et al, 1992;Freymuth et al, 1995Freymuth et al, , 1997Henkel et al, 1997;Paton et al, 1992).These methods usually detect about 5 Á/10% more RSV-positive samples than conventional methods for RSV detection. In this paper, we have shown that quantitative real-time RT-PCR is more sensitive than qualitative RT-PCR in infected cell cultures, and than isolation of respiratory samples in cell culture, identifying 6% more cases as being RSVpositive.…”