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 x 10(8) 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 kappa=0.81, 95%CI 0.66-0.97). HAstV RNACN in stools ranged from 7.6 x 10(1) to 3.6 x 10(14)copies/0.1g. 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.
Zhang
Zhian
Z
Center for Pediatric Research, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510, USA.
Mitchell
Douglas K
DK
Afflerbach
Carol
C
Jakab
Ferenc
F
Walter
Jolan
J
Zhang
Yan-Jin
YJ
Staat
Mary A
MA
Azimi
Parvin
P
Matson
David O
DO
D019350:Astroviridae Infections / Q000150:complications / Q000175:diagnosis / Q000821:virology*; D052160:Benzothiazoles; D018938:Caco-2 Cells; D002648:Child; D015169:Colony Count, Microbial / Q000379:methods*; D003959:Diamines; D006801:Humans; D001257:Mamastrovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012400:Rotavirus Infections / Q000150:complications
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I