Improved sensitivity of astrovirus-specific RT-PCR following culture of stool samples in CaCo-2 cells.

Abstract

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