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Highlights • Current nucleotide sequences ensure the reliability of rRT-PCR assays. • Development of a duplex rRT-PCR assay for the distinction of HRSV subgroups A and B. • Assay validation used an established pan-HRSV rRT-PCR as a reference test. • HRSV investigations and surveillance studies as duplex assay potential applications., Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region of the nucleoprotein gene combined in a single duplex reaction using all genome sequence data currently available in GenBank. The assay was validated for analytical sensitivity, specificity, reproducibility, and clinical performance with a geographically diverse collection of viral isolates and respiratory specimens in direct comparison with an established pan-HRSV rRT-PCR reference test. The assay was sensitive, reproducibly detecting as few as 5–10 copies/reaction of target RNA. The assay was specific, showing no amplification with a panel of 16 other common respiratory pathogens or predicted by in silico primer/probe analysis. The duplex rRT-PCR assay based on the most current available genome sequence data permits rapid, sensitive and specific detection and subgroup identification of HRSV.

An improved staphylococcal coagglutination test was developed for rapid detection, in a single assay, of rotavirus and adenovirus in stool samples (COARA). Suspensions of Staphylococcus aureus coated respectively with anti-rotavirus and anti-adenovirus sera were used to identify these viruses in 327 stool samples of children. The samples were also tested by an enzyme immunoassay. The data analysis has demonstrated a high degree of correlation between the two assays.