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 astrov irus infection in children hospitalized with acute gastroenteritis, a Northern hybridization method was used to screen stool samples for ast rovirus RNA. Positive results were confirmed using reverse transcripta se-polymerase chain reaction (RT-PCR), which showed surprisingly low s ensitivity. The low sensitivity of the RT-PCR method was considered li kely 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-RCR. Stu dy design: Stool samples from children hospitalized with acute gastroe nteritis were screened for astrovirus using Northern hybridization. Th e presence of astrovirus RNA was then confirmed using an astrovirus-sp ecific RT-PCR. Hybridization positive samples that failed to generate an RT-PCR product were cultured in CaCO-2 cells for 48 h. RNA was isol ated from cultures and re-tested using the same RT-PCR method. Results : Using Northern hybridization, human astroviruses were detected in th e stools of 31 patients and confirmed by RT-PCR in 16 samples. RNA ext racted 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 me thod for astrovirus positive faecal specimens improved the sensitivity of astrovirus-specific RT-PCR from 52 to 100%. The technique should b e of value as a confirmatory test in surveys of human astrovirus infec tion. (C) 1998 Elsevier Science B.V. All rights reserved.