Journal of clinical microbiology 2017 02 22() pii 10.1128/JCM.02334-16
Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically-infected patients. A reverse transcription, real-time PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for simultaneous detection and quantification of HEV RNA in human serum was developed based on adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame (ORF) 2/3 nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the 1(st) World Health Organization (WHO) International Standard for HEV RNA, code 6329/10, and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. Analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% CI, 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO International Reference Panel for HEV Genotypes, code 8578/13, showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF 2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. Availability of secondary standards calibrated to the WHO HEV international standard can improve standardization and performance of assays for detection and quantification of HEV RNA.