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.
Center for Pediatric Research, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510, USA.
Zhian
Z
Zhang
Douglas K
DK
Mitchell
Carol
C
Afflerbach
Ferenc
F
Jakab
Jolan
J
Walter
Yan-Jin
YJ
Zhang
Mary A
MA
Staat
Parvin
P
Azimi
David O
DO
Matson
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