One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children.

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection.

Comparison of HeLa-I, HEp-2 and NCI-H292 cell lines for the isolation of human respiratory syncytial virus (HRSV).

Generally, laboratory diagnosis of viral respiratory infections utilizes virus isolation in cell culture and immunofluorescence assays. In this study, three cell lines (HEp-2, NCI-H292 and HeLa-I) were used for HRSV isolation of strains obtained from patients admitted at HU-USP with respiratory tract disease. HRSV was isolated in 46% (37) of 80 specimens inoculated in HeLa-I, 48% (39) in HEp-2, and 36.3% (29) in NCI-H292. Immunofluorescence was considered the gold standard and yielded 53% positive (43). The results from both methods combined had better sensitivity (73.2%) compared to either method alone. Comparing results between the cell lines with HEp-2 cells as the benchmark, the greatest sensitivity (72.2%) was observed in HeLa-I. This data shows that HeLa-I is adequate for HRSV isolation, giving results similar to the HEp-2 cells. The combined use of the HEp-2, HeLa-I and NCI-H292 cells improve the detection of HRSV.