Rotavirus shedding in symptomatic and asymptomatic children using reverse transcription-quantitative PCR

Reverse transcription-real-time polymerase chain reaction (RT-qPCR) for the VP6 gene was used to study group A rotavirus shedding in children with symptomatic and asymptomatic rotavirus infection. Sequential stool samples (n=345) from 10 children with rotavirus associated diarrhea and from five children (n=161) with asymptomatic rotavirus infection were collected over a period of 2 months. A RT-qPCR assay on the samples using a rotavirus VP6 plasmid standard demonstrated high reproducibility, with an inter-assay coefficient of variation (CV) of 1.40-2.97% and an intra-assay CV of 0.03-3.03%. The median duration of shedding was longer in children with diarrhea compared to asymptomatic children (24 days vs. 18 days; P=0.066). The median quantitation cycle (C-q) at presentation in symptomatic children was 17.21 compared to 30.98 in asymptomatic children (P=0.086). The temporal pattern in symptomatic children consisted of a high initial viral shedding coinciding with the duration of diarrhea, followed by a rapid fall, and then a small increase in secondary shedding 21 days later. Compared to children with rotavirus diarrhea, those with asymptomatic infection shed lower quantities of virus throughout the observation period. No difference was noted between the G and P genotypes of samples collected at onset of infection and during the shedding period. Shedding was intermittent in a subset of children in both groups. RT-qPCR is a useful method to characterize shedding patterns. J. Med. Virol. 85:1661-1668, 2013. (c) 2013 Wiley Periodicals, Inc.