Improved sensitivity of astrovirus-specific RT-PCR following culture of stool samples in CaCo-2 cells.
BACKGROUND
During an epidemiological study on the incidence of astrovirus infection in children hospitalized with acute gastroenteritis, a Northern hybridization method was used to screen stool samples for astrovirus RNA. Positive results were confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR), which showed surprisingly low sensitivity. The low sensitivity of the RT-PCR method was considered likely to be due to the presence of non-specific inhibitors.


OBJECTIVE
To develop and use a simple culture method to improve the sensitivity of diagnosis of astrovirus in clinical stool samples using RT-PCR.


METHODS
Stool samples from children hospitalized with acute gastroenteritis were screened for astrovirus using Northern hybridization. The presence of astrovirus RNA was then confirmed using an astrovirus-specific RT-PCR. Hybridization positive samples that failed to generate an RT-PCR product were cultured in CaCO-2 cells for 48 h. RNA was isolated from cultures and re-tested using the same RT-PCR method.


RESULTS
Using Northern hybridization, human astroviruses were detected in the stools of 31 patients and confirmed by RT-PCR in 16 samples. RNA extracted directly from 15 faecal specimens could not be amplified by RT-PCR. After culture for 48 h in CaCO-2 cells, RNA extracted from these samples could be amplified and confirmed the presence of astrovirus in all 15 specimens.


CONCLUSIONS
Development of a simplified culture method for astrovirus positive faecal specimens improved the sensitivity of astrovirus-specific RT-PCR from 52 to 100%. The technique should be of value as a confirmatory test in surveys of human astrovirus infection.
Department of Gastroenterology and Clinical Nutrition, Royal Children's Hospital, Parkville, Vic., Australia. h.mustafa@pgrad.unimelb.edu.au
H
H
Mustafa
E A
EA
Palombo
R F
RF
Bishop
D019350:Astroviridae Infections / Q000821:virology*; D015152:Blotting, Northern; D018938:Caco-2 Cells; D002675:Child, Preschool; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D007223:Infant; D001257:Mamastrovirus / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D012367:RNA, Viral