Short CommunicationDiagnosing rotavirus A associated IID: Using ELISA to identify a cut-off for real time RT-PCR
Section snippets
Background
Enzyme linked immunosorbent assay (ELISA) has traditionally been the method of choice for laboratory diagnosis of group A rotavirus associated infectious intestinal disease (IID).1, 2 However, with the availability of reverse transcription-polymerase chain reaction (RT-PCR) assays for rotavirus A3, 4 and the move towards multiplexing in clinical virology,5, 6 the use of RT-PCR is increasing. Whilst RT-PCR does identify more rotavirus A infections than ELISA,7 up to 14% of healthy individuals
Objectives
Research objectives were to describe the differences in rotavirus A viral load detected in IID cases positive by ELISA, IID cases negative by ELISA but positive by RT-PCR and healthy controls; to develop a cut-off in faecal viral load for attributing illness to rotavirus A in IID cases.
Specimens
Faecal specimens were collected from IID cases and healthy controls during the Infectious Intestinal Disease Study for England (1993–1996).11 IID cases were recruited from a community cohort, or at consultation with their general practitioner. IID cases had acute diarrhoea or vomiting lasting less than 2 weeks, with no known non-infectious cause, preceded by a symptom-free period of 3 weeks.12 Healthy controls, with no history of IID for the preceding 3 weeks, were recruited concurrently to IID
Descriptive analysis
IID cases were aged up to 83 years and controls were aged up to 46 years; 60% of cases and 71% of controls were aged less than 5 years. The median Ct value in ELISA positive IID cases was substantially lower than in controls (Table 1) and there was very little overlap between the distributions of Ct values in these two groups (Fig. 1). There was no evidence of a difference in Ct value distribution between the ELISA negative, RT-PCR positive IID cases and the controls (Fig. 1, Table 1), in all
Discussion
We have used faecal viral load to demonstrate that ELISA diagnosis is highly correlated with disease in rotavirus A infection, in accordance with other community and hospital-based studies,13, 19, 20, 21 and that RT-PCR is probably only detecting additional infections in IID cases at levels not associated with illness. We have selected a cut-off in the real time RT-PCR assay to improve the specificity of diagnosing rotavirus A associated IID by this method.
A major strength of this study is the
Conclusion
RT-PCR does not provide sufficient specificity for attributing illness to rotavirus A in IID cases. As the use of multiplex PCR assays for enteric viruses increases in routine diagnosis of IID, there will be a need to interpret the results of these sensitive tests to determine disease aetiology.22, 23 We have shown that ELISA positivity remains a good correlate of disease in rotavirus A infection, supporting the use of ELISA in the WHO protocol for surveillance of rotavirus associated
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study was funded by the Food Standards Agency. We would like to thank Corrine Amar, Fenella Halstead, Dalia Choudhury and Mihaela Cirdei who completed the laboratory testing.
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