Choice of reference assay for the detection of rotavirus in fecal specimens: electron microscopy versus enzyme immunoassay

Abstract

Two previously demonstrated sensitive and specific enzyme immunoassays (EIAs) for rotavirus, one using polyclonal and monoclonal antisera (TestPack Rotavirus [TPK]; Abbott Laboratories) and the other using only monoclonal anti-rotavirus antibodies (Rotaclone [RTC]; Cambridge BioScience Corporation), were evaluated as potential reference assays for rotavirus testing in comparison with direct negative-staining electron microscopy (EM), the current laboratory standard. Two hundred and seven stool samples collected consecutively during the winter of 1989 from children with acute diarrhea admitted to a ward for infants from 0 to 2 years of age were tested by the EIAs and by EM. TPK specimens were read visually; RTC results were read spectrophotometrically. Specimens with discordant EIA and EM results were further evaluated by a fluorescent focus assay. Specimens positive by EM and those negative by EM but positive by fluorescent focus assay were considered to be positive for rotavirus. Of the 207 stools tested, 35 (17%) were positive for rotavirus by these criteria. EM had a sensitivity of only 80%. Specificities were 100% for RTC and EM and 89% for TPK. These findings indicate that EM, although very specific, is relatively insensitive compared with a highly sensitive monoclonal antibody-based EIA. An EIA with high sensitivity and specificity, such as RTC, is a more appropriate reference standard for rotavirus testing.

Determining the effectiveness of the pentavalent rotavirus vaccine against rotavirus hospitalizations and emergency department visits using two study designs

“…A potential bias with this method is that AGE controls may be misclassified if there are issues with the sensitivity or specificity of the rotavirus EIA results. In our study misclassification is unlikely to have substantially impacted our results since the rotavirus EIA is reported to be >95% sensitive, and >95% of bulk stool specimens were collected within 7 days [51]. Lastly, non-enrolled children were less likely to have insurance compared with enrolled children, so our analysis may under-represent the non-insured, which may be a less well-vaccinated population.…”

Evaluation of the ImmunoCardSTAT! Rotavirus Assay for Detection of Group A Rotavirus in Fecal Specimens

“…Rotavirus is as sensitive and specific as two widely used commercial EIAs for detection of group A rotavirus. Rotaclone has been found to be sensitive and specific compared with electron microscopy and with other commercially available immunoassays in several prior studies (4,5).…”

“…In all cases, electron microscopy technologists were unaware of laboratory results. Electron microscopy was carried out as described by Dennehy et al (4). Grids were examined for virus at a magnification of ϫ52,000 for approximately 5 min.…”

Viruses causing gastroenteritis

“…EIA has proven to be more sensitive than direct visualization by electron microscopy [191,192], and also has a high specificity in the detection of group A rotavirus, especially when monoclonal antibodies are used [193,194]. The LA technique has lower sensitivity than EIA [6,194].…”