Evidence of false-positive results in a commercially available rotavirus assay in the vaccine era, Australia, 2011 to 2012.

Concerns were raised about specificity of the VIKIA Rota-Adeno immunochromatographic kit. Only 28-37% of samples positive with the VIKIA kit could be confirmed using two real-time RT-PCR assays and three ELISA kits. On re-analysis of a subset of the positive samples, 86% remained positive with the VIKIA kit, however, 90% remained negative in the other assays. In a highly vaccinated population we found a high number of false-positive rotavirus tests with a widely-used commercial kit. We recently became concerned about the specificity of the VIKIA Rota-Adeno assay (bioMérieux, France) following an unexplained increase in positive results and feedback from clinicians. Accurate detection of rotavirus is essential for prevention and control of rotavirus outbreaks and disease monitoring. There are two common methods used for routine diagnosis: immunochromatographic (ICT) assays and enzyme-linked immunosorbent assays (ELISA). ICT assays are relatively inexpensive, easy to use, rapid (results within 20 min) and with reportedly good sensitivity (96.6%) and specificity (92.9%) [1]. Many diagnostic laboratories in Australia use the VIKIA Rota-Adeno assay for detection of rotavirus in faecal specimens. We therefore reexamined samples initially testing positive in the VIKIA Rota-Adeno ICT with other commercially available ELISA rotavirus assays and, for a subset of specimens, by RT-PCR. We obtained a convenience sample set of 133 faecal specimens submitted for diagnostic rotavirus testing and collected between July 2011 and August 2012 from patients with symptoms of acute gastroenteritis. Specimens were from two laboratories in Queensland (n=113: Pathology Queensland, a publically funded laboratory, and Sullivan Nicolaides Pathology, a private laboratory) and from a private laboratory network in Victoria (n=20: Melbourne Pathology). The latter were submitted to the National Rotavirus Reference Centre (NRRC) in Melbourne, Victoria, for genotyping. All samples had been tested initially for rotavirus using the VIKIA ICT method according to the manufacturer's instructions (Queensland: 81 positive, 32 negative; Victoria: 20 positive). Real-time RT-PCR, Queensland samples only All 113 Queensland specimens were tested initially in Queensland employing two real-time RT-PCR assays, using primers and TaqMan probe sequences described previously: ACAACTGCAGCTTCAAAAGAAGWGT) [4]. RNA extraction was performed by homogenising ca. 25 µl of stool specimen with 225 µl of phosphate buffered saline to provide a concentration of ca. 10%. Then 200 µl of this suspension were extracted into a volume of 50 µl using the Roche High Pure Nucleic Acid extraction kit as per kit instructions (Roche Diagnostics, Australia). As described previously, specimens were