Development of a Fully Automated, High-Throughput Molecular Assay for Detection of Rat Hepatitis E Virus in Routine Diagnostics.

Recently, cases of human infection with rat hepatitis E virus (ratHEV, Rocahepevirus ratti) have been reported worldwide. Due to the significant genetic differences between ratHEV and human HEV genotypes 1-4 (Paslahepevirus balayani), current HEV diagnostic assays are unable to detect ratHEV. The aim was to establish and validate a laboratory-developed ratHEV RT-qPCR assay for use with human plasma and stool samples on a fully automated, high-throughput platform. Published primers and probes were optimized for use on cobas 5800/6800/8800 systems using European Union In Vitro Diagnostics Regulation (IVDR)-grade reagents, including an RNA full-process inhibition control. Analytical sensitivity (21 repeats), linear range (five repeats) and precision (three repeats over 3 days) were evaluated using viral particles from cell culture (MN450851.1). The inclusivity was verified using DNA oligonucleotides and known positive samples (rat liver and human serum). The limits of detection were 98.9 copies/ml in plasma and 60.3 copies/ml in stool, and the assay showed excellent linearity over at least 5 log (r2: 0.991 in plasma and 0.9989 in stool) and high precision (< 0.62 ct). The assay reliably detected different ratHEV C1 subgenotypes, returning positive results for all 11 rat liver samples and one known ratHEV RNA-positive human plasma sample, while no false positives were detected in the broad cross-reactivity set (n = 41). In the pilot ratHEV surveillance cohort, 1.1% of plasma samples (n = 1999) were positive for HEV RNA, but none were positive for ratHEV RNA. Our new, fully automated, lab-developed ratHEV assay can be used in compliance with the IVDR for routine human diagnostics. Further studies are needed to determine the clinical relevance in different human cohorts.