Comparison of a real-time reverse transcriptase PCR assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus.
J Clin Microbiol
; 43(5): 2356-62, 2005 May.
Article
em En
| MEDLINE
| ID: mdl-15872266
ABSTRACT
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory infection of children. Understanding RSV pathogenesis and evaluating interventions requires quantitative RSV testing. Previous studies have used the plaque assay technique. Real-time reverse transcriptase PCR (RTrtPCR) offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multicenter studies. We developed RTrtPCR assays based upon the RSV N and F genes. The N-gene assay detected greater RSV quantity and was further evaluated. Standard curves utilized both extractions from RSV culture supernatants of known quantity and cloned purified copies of the target DNA. In vitro, the ratio of RSV subgroup A (RSV-A) genome copies to PFU was 1531. A total of 462 samples collected quantitatively from 259 children were analyzed in duplicate by RTrtPCR. Results were compared with those of RSV plaque assays performed on fresh aliquots from the same children. Duplicate RTrtPCR results were highly correlated (r2 = 0.9964). The mean viral load from nasal washes obtained on the first study day was 5.75 +/- standard error of the mean 0.09 log PFU equivalents (PFUe)/ml. Viral load by RTrtPCR correlated with plaque assay results (r2 = 0.158; P < 0.0001). Within individuals, upper and lower respiratory tract secretions contained similar viral concentrations. RSV-A-infected children had 1.17 log PFUe higher viral loads than did those with RSV-B (P < 0.0001). RSV quantification by RTrtPCR of the N gene is precise and has significant, though limited, correlation with quantitative culture. The utility of the RTrtPCR quantification technique for clinical studies would be solidified after its correlation with RSV disease severity is established.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Vírus Sinciciais Respiratórios
/
Infecções por Vírus Respiratório Sincicial
/
Carga Viral
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Tipo de estudo:
Clinical_trials
/
Diagnostic_studies
Limite:
Child
/
Humans
Idioma:
En
Revista:
J Clin Microbiol
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos