Quantitation of human astrovirus by real-time reverse-transcription-polymerase chain reaction to examine correlation with clinical illness

Human astroviruses (HAstVs) cause gastroenteritis. Real-time, reverse-transcription-polymerase chain reaction (RT2-PCR) was developed to quantitate HAstV RNA. An 88 bp amplicon from the conserved 3′ genomic region was detected by binding of SYBR Green. RT2-PCR was reproducible, with a correlation coefficient of 0.998–1.00 and PCR efficiency of 94.4–100% (mean 97%). The coefficient of variation was 0.6–2.5%, dynamic range with RNA standard up to 5 × 108 RNA copies (RNACN) and sensitivity 5 RNACN. Of 54 blinded, archived stool samples from children hospitalized because of gastroenteritis tested by RT-PCR, 49 (91%) agreed by RT2-PCR for HAstV-positivity (Cohen κ = 0.81, 95%CI 0.66–0.97). HAstV RNACN in stools ranged from 7.6 × 101 to 3.6 × 1014 copies/0.1 g. Children coinfected with rotavirus had lower RNACN (mean log 4.22/standard deviation = 2.26) than those without coinfection (7.57/3.06; p = .019). Children taking infant formula also had lower RNACN (5.96/2.98) than breast-fed or weaned children (8.73/2.92; p = .027). Higher RNACN tended to occur with longer duration of diarrhea for the episode (r = 0.49, p = .064), but was not associated with change in age, gender, illness day, severity or breast-feeding. RT2-PCR quantitated HAstV RNA and RNACN in stool correlates with features of clinical illness.