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Previous Article | Next Article 
Journal of Clinical Microbiology, May 2008, p. 1842-1843, Vol. 46, No. 5
0095-1137/08/$08.00+0 doi:10.1128/JCM.02329-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
National Center for Immunization and Respiratory Diseases, Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta,1 Atlanta Research and Education Foundation, Decatur, Georgia,2 Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio3
Received 4 December 2007/ Returned for modification 28 January 2008/ Accepted 3 March 2008
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(The information in this report was presented at a poster session at the 45th Annual Meeting of the Infectious Diseases Society of America, 4 to 7 October 2007, in San Diego, CA.)
We examined existing bulk stool and rectal swab samples collected from 1 March to 30 June 1999 as part of an active surveillance study conducted at the Cincinnati Children's Hospital Medical Center (CCHMC) (2). In brief, children below the age of 5 years with AGE presenting to the CCHMC emergency department were enrolled if they met study criteria (2). During this period, 165 children provided either a bulk stool specimen or a rectal swab. Sixty bulk specimens were randomly selected, and then each was matched with a swab specimen on the basis of the children's ages (±3 months) and the dates of collection (±2 weeks).
To evaluate the rate of rotavirus detection in healthy children, an existing set of bulk stool specimens from nonhospitalized children in the same community who were free of symptoms of diarrhea or vomiting for at least 14 days prior to specimen collection was tested. The specimens from healthy children were randomly selected at a 1:2 frequency to match those of the children with AGE on age (±3 months) and date of collection (±2 weeks).
Specimens were tested for rotavirus antigen by a Rotaclone commercial microplate EIA (Meridian Diagnostics) and for rotavirus RNA by conventional RT-PCR with VP6 primers at the Centers for Disease Control and Prevention (CDC). For RT-PCR, briefly, RNA was extracted from stool specimens by a guanidine-silica-based method and was analyzed by RT-PCR for the rotavirus VP6 gene by using a commercial one-step kit (Qiagen) (4, 6). Differences were evaluated for statistical significance (P < 0.05) by a chi-square test for the assay and specimen type and by a Wilcoxon rank-sum test for the severity score and age.
In total, 145 specimens were analyzed. Fifty-seven bulk stools and 60 rectal swabs were from children with AGE, and 28 bulk stools were from healthy children. For children with AGE from whom bulk samples were obtained, illness, determined by a modified 17-point Vesikari score (14), was similar to that of patients from whom rectal swabs were obtained (P = 0.60). The mean age of children classified with severe disease was 16 months (95% confidence interval, 10 to 22 months), with no age difference by specimen type (P = 0.57).
The results of RT-PCR and EIA testing are displayed in Table 1. The rate of detection of rotavirus in children with AGE was significantly greater with bulk stool specimens than with rectal swabs by EIA (49% versus 27%; P = 0.01). There was no significant difference by assay when bulk specimens were evaluated (49% versus 53% for EIA and RT-PCR, respectively; P = 0.71). No stools from healthy controls were positive by EIA, but five (18%) were positive by RT-PCR. Therefore, the difference in the rate of detection of rotavirus in bulk stool specimens between children with AGE and healthy controls was greater by the EIA (49 versus 0%, respectively; P < 0.01) than by RT-PCR (53 versus 18%, respectively; P < 0.01), although the rates were significantly different by both methods.
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Our results also suggest that a bulk or whole-stool specimen is superior to a rectal swab specimen for rotavirus detection, especially with the EIA. By EIA testing, the rate of detection of rotavirus in bulk stools was significantly higher than that in rectal swabs. The smaller amount of fecal material contained in swab specimens likely resulted in reduced sensitivity of detection by EIA. This hypothesis is supported by the observation that RT-PCR, which can detect smaller amounts of virus, increased the detection rate of rotavirus in rectal swab specimens by 15%, from 27% to 42%.
We did not obtain both a rectal swab and a bulk stool specimen from the same child for this evaluation, and while patients were matched, it is possible that children with bulk stool and rectal swab specimens were not directly comparable. The specimens used in this evaluation had been stored 8 years before the testing for this study was conducted. While rotavirus is durable, the freeze-thaw cycles after collection may have resulted in a reduction of the amount of detectable virus. The tests we compared in this study were all performed in 2007, but it remains possible that detection of antigen by EIA was affected more than detection of RNA by RT-PCR, because the amount of antigen necessary for detection by EIA is larger than the amount of RNA needed for detection by RT-PCR.
In conclusion, the findings of our study support the recommendations in the WHO generic protocol for hospital- and community-based surveillance of rotavirus disease (13) that bulk stool specimens should be obtained from children with AGE and should be tested by EIA for purposes of routine surveillance.
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ACKNOWLEDGMENTS |
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We acknowledge Tricia Busse, Michelle Roth, Cara Kohlrieser, Nancy Roberts, Marina Bischoff, Mary Sandquist, Marilyn Rice, Dick Ward, and Donna Sander of the CCHMC for valuable contributions to this study. We also thank all the children and their families for choosing to participate in the study.
Mary A. Staat received research funding from Wyeth Laboratories, Inc., and Merck & Company and serves on the Rotavirus Advisory Board for Merck & Company and GlaxoSmithKline. David I. Bernstein received research funding from GlaxoSmithKline, Merck & Company, and Wyeth Laboratories and is an author of the patent for the GlaxoSmithKline rotavirus vaccine.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
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FOOTNOTES |
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Published ahead of print on 19 March 2008.
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