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


OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Phillips
Gemma
G
Department of Gastrointestinal, Emerging and Zoonotic Infections, Health Protection Agency Centre for Infections, London, UK. gemma.phillips@hpa.org.uk
Lopman
Ben
B
Tam
Clarence C
CC
Iturriza-Gomara
Miren
M
Brown
David
D
Gray
Jim
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D003967:Diarrhea / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000821:virology*; D005260:Female; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology; D055815:Young Adult