Diagnosing rotavirus A associated IID: Using ELISA to identify a cut-off for real time RT-PCR

J Clin Virol. 2009 Mar;44(3):242-5. doi: 10.1016/j.jcv.2008.12.001. Epub 2009 Jan 29.


Background: The use of RT-PCR for diagnosis of group A rotaviruses is increasing, but up to 14% of healthy individuals may be positive by RT-PCR. If RT-PCR is not well correlated with disease, rotavirus A may not always be the cause of illness in RT-PCR positive patients with infectious intestinal disease (IID).

Objectives: To describe the differences in faecal viral load between ELISA positive IID cases, RT-PCR positive cases and healthy controls. To develop a cut-off in faecal viral load for attributing illness to rotavirus A in RT-PCR positive IID cases.

Study design: Faecal viral load was measured, using real time RT-PCR, in 118 community IID cases and 65 healthy controls, previously tested by ELISA. Cycle threshold (Ct) values from the real-time RT-PCR were used as a proxy measure of viral load. A cut-off for attributing illness to rotavirus A was selected, using ROC analysis.

Results: There was little overlap in viral load between ELISA positive IID cases (median Ct 17) and healthy controls (median Ct 37), but ELISA negative, RT-PCR positive IID cases (median Ct 37) had viral loads similar to healthy controls, indicating that RT-PCR is not detecting extra cases of group A rotavirus associated IID, only sub-clinical infections. The optimal cut-off in the real time RT-PCR was at Ct value 24-27.

Conclusion: ELISA is the best method for the laboratory diagnosis of rotavirus A associated IID. If RT-PCR is used, it is advisable to use a real time platform and to use a viral load cut-off equivalent to the detection limit of ELISA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Child
  • Child, Preschool
  • Diarrhea / virology*
  • Enzyme-Linked Immunosorbent Assay
  • Feces / virology*
  • Female
  • Gastroenteritis / virology*
  • Humans
  • Infant
  • Infant, Newborn
  • Male
  • Middle Aged
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Rotavirus / classification
  • Rotavirus / isolation & purification*
  • Rotavirus Infections / diagnosis*
  • Rotavirus Infections / virology
  • Young Adult