Assuntos
Infecções por Cardiovirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sistema Respiratório/virologia , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Theilovirus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Primers do DNA/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Adulto JovemRESUMO
The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.
