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Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses
Published on: May 2, 2019
Improved detection of rotavirus shedding by polymerase chain reaction
J Wilde1, R Yolken, R Willoughby
1Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Insights
A new reverse-transcriptase polymerase chain reaction (RT-PCR) method improves rotavirus detection in infants. RT-PCR identified rotavirus RNA longer and earlier than enzyme immunoassay, aiding in understanding infant rotavirus shedding.
Area of Science:
- Virology
- Molecular Biology
- Pediatrics
Background:
- Rotavirus is a leading cause of gastroenteritis in infants.
- Accurate and timely detection of rotavirus shedding is crucial for infection control in hospital settings.
- Current diagnostic methods like enzyme immunoassay may have limitations in detecting prolonged or early viral shedding.
Purpose of the Study:
- To develop and evaluate a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for improved identification of rotavirus RNA in infants.
- To compare the diagnostic performance of RT-PCR with a solid-phase enzyme immunoassay (EIA) for detecting rotavirus shedding in hospitalized infants.
- To assess the duration and onset of rotavirus shedding as detected by both RT-PCR and EIA.
Main Methods:
- A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for rotavirus RNA amplification.
- The RT-PCR assay was compared against a solid-phase enzyme immunoassay (EIA) for rotavirus detection.
- Faecal samples from 40 hospitalized infants were analyzed, with a focus on 9 infants with prolonged hospital stays and rotavirus detection.
Main Results:
- RT-PCR detected rotavirus RNA in 58% of faecal samples, compared to 36% detected by EIA (p < 0.002).
- The geometric mean duration of rotavirus shedding was significantly longer with RT-PCR (9.5 days) than with EIA (5.7 days) (p < 0.018).
- RT-PCR identified rotavirus shedding for 2-7 days longer in 5 infants and detected shedding 1 or more days earlier in 4 infants compared to EIA.
Conclusions:
- RT-PCR is a more sensitive method than EIA for detecting rotavirus RNA in infants, identifying shedding for longer periods and earlier.
- The enhanced sensitivity of RT-PCR may have implications for understanding rotavirus transmission dynamics in hospital environments.
- Further research is needed to determine if infants shedding rotavirus detectable only by RT-PCR can transmit the wild-type virus.
Abstract:
To improve identification of children excreting rotavirus a method for the amplification of rotavirus RNA by the polymerase chain reaction (PCR) was developed. The assay was compared with a solid-phase enzyme immunoassay in the detection of rotavirus shedding by infants in hospital during the winter peak of rotavirus infections. Forty children were studied in an intermediate care unit after transfer from intensive care units. Only two were admitted primarily because of diarrhoea; the other thirty-eight were admitted for management of various other disorders. Rotavirus shedding was detected by enzyme immunoassay in twenty of the infants, and nine of these (aged 1 week to 8 months) remained in hospital for more than 5 days after the initial detection of rotavirus and could be studied long term. Of 103 faecal samples from the nine infants, 60 (58%) contained rotavirus RNA detected by reverse-transcriptase (RT)/PCR, whereas only 37 (36%) were positive for rotavirus antigen by the immunoassay (chi 2 = 10.3, p less than 0.002). The geometric mean time of rotavirus shedding was 9.5 (range 1-19) days as detected by RT/PCR and 5.7 (range 1-17) days by the immunoassay (p less than 0.018). In five of the nine children, RT/PCR detected rotavirus shedding for 2-7 days longer than the immunoassay and in four children RT/PCR was positive 1 or more days before rotavirus antigen was detected. Further studies should attempt to find out whether infected infants are capable of spreading wild-type virus during periods when they are not shedding antigen as detectable by enzyme immunoassay.
