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Published on: January 28, 2019
Rotavirus shedding in symptomatic and asymptomatic children using reverse transcription-quantitative PCR
Indrani Mukhopadhya1, Rajiv Sarkar, Vipin Kumar Menon
1Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India.
Insights
Reverse transcription-real-time polymerase chain reaction (RT-qPCR) detected group A rotavirus shedding in children. Symptomatic children shed more virus for longer durations than asymptomatic children, highlighting RT-qPCR
Area of Science:
- Virology
- Pediatrics
- Infectious Diseases
Background:
- Group A rotavirus is a significant cause of diarrheal disease in children worldwide.
- Understanding rotavirus shedding patterns is crucial for infection control and transmission studies.
- Previous methods for quantifying rotavirus shedding have limitations in sensitivity and reproducibility.
Purpose of the Study:
- To investigate and compare rotavirus shedding patterns in children with symptomatic (diarrhea) versus asymptomatic infections.
- To evaluate the utility of reverse transcription-real-time polymerase chain reaction (RT-qPCR) for characterizing rotavirus shedding.
Main Methods:
- Collected sequential stool samples from 10 children with rotavirus diarrhea and 5 asymptomatic children over 2 months.
- Utilized a validated RT-qPCR assay targeting the rotavirus VP6 gene for viral quantification.
- Assessed assay reproducibility using inter-assay and intra-assay coefficients of variation (CV).
Main Results:
- The RT-qPCR assay demonstrated high reproducibility (inter-assay CV: 1.40-2.97%; intra-assay CV: 0.03-3.03%).
- Median shedding duration was longer in symptomatic children (24 days) compared to asymptomatic children (18 days).
- Symptomatic children presented with significantly higher viral loads (median C(q) 17.21 vs. 30.98) and exhibited a distinct shedding pattern with secondary shedding.
Conclusions:
- RT-qPCR is a reliable and effective method for characterizing rotavirus shedding patterns in children.
- Symptomatic rotavirus infection is associated with prolonged and higher-quantity viral shedding compared to asymptomatic infection.
- Findings provide valuable insights into rotavirus transmission dynamics and the clinical course of infection.
Abstract:
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.
