Significant compartment-specific impact of different RNA extraction methods and PCR assays on the sensitivity of hepatitis E virus detection.

Abstract

RNA detection in plasma/stool is the gold-standard for diagnosis of hepatitis E virus (HEV) infection. The impact of viral extraction methods on HEV RNA detection is poorly investigated. We determined the limit of detection of the RealStar HEV RT-PCR V2.0 Kit (altona Diagnostics, RS) utilizing 3 RNA extraction methods (COBAS® AmpliPrep Total Nucleic Acid Isolation Kit, TNAi Roche; MagNA Pure 96 DNA, Viral NA SV Kit, MgP; QIAamp Viral RNA mini Kit Qiagen; VRK) in plasma and stool. The most sensitive method was evaluated in a total of 307 longitudinal samples of patients with HEV infection (acute=18/chronic=36) and compared to results with the former diagnostic standard of our centre (TNAi/FastTrack Diagnostic; FTD). The plasma-LOD was 49, 94 and 329IU/mL for extraction with MgP, VRK and TNAi respectively. In stool, the LOD was 21IU/mL, 528IU/mL and indefinable for extraction with TNAi, VRK and MgP respectively. Utilizing longitudinal patient plasma samples, MgP/RS revealed 56 HEV RNA-positive samples in 158 negative samples as determined by TNAi/FTD. In stool, from 37 HEV negative samples (TNAi/FTD), 15 were positive with TNAi/RS. At end of treatment, 8 out of 27 chronically infected patients were RNA positive with MgP/RS, while classified negative with TNAi/FTD. A relapse occurred in 3 of these patients. Different methods for RNA extraction and quantification have a significant, compartment-specific impact on the sensitivity of HEV detection. Knowledge about the favourable combinations of extraction and quantification has important implications for diagnosis and patients receiving antiviral therapy.