Methods
Study design
This study was an evaluation of frozen stool specimens from children < 5 years of age with watery diarrhea and moderate to severe dehydration collected and tested with the VIKIA® Rota-Adeno assay during rotavirus surveillance in Niger [3]. A random sub-sample of 734 specimens collected between July 2010 and May 2011 were tested at the French national reference laboratory for enteric viruses in Dijon, France, for enteric viruses with Seeplex® Diarrhea-V ACE assay (Seegene, Seoul, Korea). We randomly selected 140 rotavirus positive and 140 rotavirus negative specimens based on the reference method in order to estimate an expected sensitivity of 85% with an accuracy of ± 6% and an expected specificity of 90% with an accuracy of ± 5%. Unfortunately, 8 negative samples and 21 positive samples could not be retrieved. Thus, 119 positive and 132 negative samples by RT-PCR were included in the study. Samples for this analysis were similar to the overall study population in terms of demographic characteristics.
Selected samples were tested with VIKIA® Rota-Adeno and Premier™ Rotaclone® tests at CERMES, Niamey, Niger following the manufacturer’s recommendations. Two experienced laboratory technicians independently read the results of the RDT and EIA, with a third reading in case of discrepancy. They were all blind to the results of other tests and reference method.
Rapid diagnostic test (RDT)
VIKIA® Rota-Adeno is a rapid, qualitative, chromatographic immunoassay for the simultaneous detection of rotavirus and adenovirus. The test was performed according to the manufacturer’s recommendations and the results were read visually within 10 min and interpreted according to manufacturer’s recommendations, including reading as a positive result only lines that showed the expected color.
Enzyme immuno assay (EIA)
The Premier™ Rotaclone® kit is the only multi-well rotavirus EIA kit approved by the US Food and Drug Administration for in vitro diagnosis, it uses monoclonal antibodies raised against rotavirus structural protein VP6. It is suited for analyses of large numbers of samples and the results can be read after 1 h. The assay was performed according to manufacturer’s recommendations and results of the Premier™ Rotaclone® test were read both visually (VR) and using an optical density (OD) spectrophotometer (Emax microplate reader with Softmax®Pro5 S/N E13193; Molecular Devices; California) and interpreted according to manufacturer’s recommendations.
Reference method
The reference method for the detection of rotavirus was the Seeplex® Diarrhea-V ACE assay (Seegene, Seoul, Korea), which is a commercial multiplex PCR system for the detection of the following human diarrheal viruses: astrovirus, group A rotavirus, enteric adenovirus and norovirus.
Briefly, viral RNA was extracted from 20% faecal suspensions in phosphate-buffered saline (PBS) using the NucliSENS® EasyMAG™ platform (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Viral RNA was reverse transcribed using the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and multiplex PCR was performed with the Seeplex® Diarrhea-V ACE system (Seegene, Seoul, Korea) according to the manufacturer’s instructions.
Data management and analysis
Results of the index and reference tests were recorded in an Excel database (MS Corporation, Seattle, Washington, USA). Data analysis was conducted in Stata® 12.1 (College Station, Texas, USA). Sensitivity and specificity were estimated by comparing the results of the index test to those of the reference method using the diagt command, which displays summary statistics for diagnostic tests and provides exact binomial confidence intervals. Inter-reader reproducibility was assessed using Cohen’s kappa coefficient.