Adaptation and validation of a gastrointestinal panel to detect diarrheal virus pathogens on a high-throughput qPCR system.

With around 2 billion cases each year, infectious gastroenteritis remains a worldwide health problem. A major cause of acute gastroenteritis is infection with enteric viruses, which often leads to hospitalization in children and immunocompromised people. We adapted and validated a gastrointestinal qPCR panel, which simultaneously detects the most common enteric viruses: Norovirus GI and GII, Rotavirus, Adenovirus, Sapovirus, Astrovirus and Enterovirus in stool samples on a fully automated, high-throughput system (Roche cobas5800/6800/8800). Limits of detection (LOD), linear range and precision were determined using dilutions of clinical stool samples, which were quantified by digital droplet PCR. Specificity and sensitivity were evaluated using clinical stool samples from patients with diarrhoea and results were compared with commercial CE-IVD qPCR assays. LODs were below 100 for all targets except for Norovirus GI (3,180 copies/ml), Norovirus GII (299 copies/ml) and Rotavirus (851 copies/ml). The assay showed excellent linearity over 5-6 log steps for all pathogens (r²: 0.992-0.998). For inclusivity External Quality Assessment samples were correctly identified, and no false positives occurred in exclusivity panels containing 26 bacterial isolates and 12 clinical virus samples. Specificity and sensitivity determined by using 243 patient samples ranged between 98.2 and 100.0% and 85.7-100.0%, respectively. In this study we validated a lab-developed syndromic qPCR assay that reliably detects the seven most common enteric viruses in clinical stool samples. Our assay provides a fast, fully automated and easily scalable high-throughput solution for gastrointestinal routine virus testing and screening in high-risk patient groups and outbreaks.