Validation of a Laboratory-Developed Triplex Molecular Assay for Simultaneous Detection of Gastrointestinal Adenovirus and Rotavirus in Stool Specimens.

Enteric viral pathogens causing gastroenteritis include adenovirus and rotavirus, among others. Rotavirus is the leading cause of severe diarrhea in infants and young children worldwide. Among the adenoviruses known to cause gastroenteritis are those of species F (serotypes 40, 41). Here, we describe the development and validation of a laboratory-developed gastrointestinal triplex rRT-PCR (triplex) assay that targets adenovirus and rotavirus. Stool specimens were tested from patients across Ontario. Specimens were previously tested for adenovirus and/or rotavirus by electron microscopy (EM) or immunochromatographic test (ICT). Triplex sensitivity, specificity, positive and negative predictive values compared to Seegene assay (a commercial assay used here as the standard reference method) were 100%, 97.8%, 86.0%, 100% for adenovirus, and 99.1%, 98.4%, 96.3%. 99.6% for rotavirus, respectively. The triplex assay had a 95.2% and 97.3% overall percent agreements (OPAs) when compared to EM for adenovirus or rotavirus detection, respectively, and an OPA of 90.9% when compared to rotavirus ICT for rotavirus detection. Triplex assay exhibited similar performance to the Seegene assay for both adenovirus and rotavirus and detected more adenovirus and rotavirus than traditional testing methods. The high performance along with lower cost and reduced turnaround time makes the triplex assay a desirable testing method for a clinical microbiology laboratory.

Evaluation and verification of the Seeplex Diarrhea-V ACE assay for simultaneous detection of adenovirus, rotavirus, and norovirus genogroups I and II in clinical stool specimens.

Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.