Comparison of 2 Assays for Diagnosing Rotavirus and Evaluating Vaccine Effectiveness in Children with Gastroenteritis

Jacqueline E. Tate

, Slavica Mijatovic-Rustempasic, Ka Ian Tam, Freda C. Lyde, Daniel C. Payne, Peter Szilagyi, Kathryn Edwards, Mary Allen Staat, Geoffrey A. Weinberg, Caroline B. Hall, James Chappell, Monica McNeal, Jon R. Gentsch, Michael D. Bowen, and Umesh D. Parashar

Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (J.E. Tate, S. Mijatovic-Rustempasic, K.I. Tam, F.C. Lyde, D.C. Payne, J.R. Gentsch, M.D. Bowen, U.D. Parashar); University of Rochester School of Medicine and Dentistry, Rochester, New York, USA (P. Szilagyi, G.A. Weinberg, C.B. Hall); Vanderbilt University Medical Center, Nashville, Tennessee, USA (K. Edwards, J. Chappell); Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA (M.A. Staat, M. McNeal)

Abstract

We compared rotavirus detection rates in children with acute gastroenteritis (AGE) and in healthy controls using enzyme immunoassays (EIAs) and semiquantitative real-time reverse transcription PCR (qRT-PCR). We calculated rotavirus vaccine effectiveness using different laboratory-based case definitions to determine which best identified the proportion of disease that was vaccine preventable. Of 648 AGE patients, 158 (24%) were EIA positive, and 157 were also qRT-PCR positive. An additional 65 (10%) were qRT-PCR positive but EIA negative. Of 500 healthy controls, 1 was EIA positive and 24 (5%) were qRT-PCR positive. Rotavirus vaccine was highly effective (84% [95% CI 71%–91%]) in EIA-positive children but offered no significant protection (14% [95% CI −105% to 64%]) in EIA-negative children for whom virus was detected by qRT-PCR alone. Children with rotavirus detected by qRT-PCR but not by EIA were not protected by vaccination, suggesting that rotavirus detected by qRT-PCR alone might not be causally associated with AGE in all patients.

Commercially available enzyme immunoassays (EIAs) traditionally have been used to detect rotavirus in children who have acute gastroenteritis (AGE). The rate of rotavirus detection is higher with EIAs than with conventional and semiquantitative real-time reverse transcription PCRs (qRT-PCRs) (1–6), but some qRT-PCR–positive samples could represent low-level viral shedding from patients with asymptomatic infections or recently resolved rotavirus infections (6). qRT-PCR cycle threshold (C_t) values correlate inversely with the amount of viral RNA in a specimen. In a study from the United Kingdom, specimens from patients with AGE that tested positive for rotavirus by EIA had significantly lower qRT-PCR C_t values (higher viral loads) than did qRT-PCR–positive specimens from patients with AGE that tested negative by EIA and from healthy controls; C_t values for the latter 2 groups did not differ (7). Another study found that C_t values correlated inversely with severity of disease in patients with AGE and EIA-positive specimens (8).
Two rotavirus vaccines (RotaTeq [RV5], Merck, West Point, PA, USA, and Rotarix [RV1] GSK Biologicals, Rixensart, Belgium) are recommended for use worldwide (9,10). These vaccines have demonstrated high efficacy (> 85%) against severe rotavirus-associated AGE in the United States and other high-income countries (11–14). As vaccine use increases, monitoring vaccine impact is important and requires sensitive and specific detection of rotavirus-associated AGE. Several case–control studies of rotavirus vaccine effectiveness have used patients with AGE who test negative for rotavirus by EIA as a comparison group for patients with AGE who test positive by EIA, and concerns have been raised about whether the rotavirus EIA might fail to detect a proportion of true rotavirus cases and thus lead to bias from misclassification of some cases (11–20). We compared rates of rotavirus detection by EIA and qRT-PCR among children with and without AGE and examined rotavirus vaccine effectiveness against severe cases of rotavirus-associated AGE, as defined by using different combinations of the EIA and qRT-PCR results.