Assessment of ISO Method 15216 to Quantify Hepatitis E Virus in Bottled Water

Abstract

Hepatitis E virus (HEV) is one of the causative agents of water-borne human viral hepatitis and considered in Europe an emerging zoonotic pathogen. Analysis of bottled water through a standard method validated for HEV can contribute towards the risk management of this hazard. Putting some recent reports by the European Food Safety Authority in place, this study aimed to assess the performance of the concentration and extraction procedures described in ISO 15216-1:2017 for norovirus and hepatitis A virus on HEV detection. Following the ISO recommendation, the bottled water samples were spiked using serially diluted HEV fecal suspensions together with mengovirus as process control and concentrated by filtration via positively charged nylon membranes. In order to extract viral RNA from the resulting concentrates, two different methods were compared in this study: The one recommended in the ISO norm, NucliSens® MiniMag® system (NS), and an alternative commercially available kit NucleoSpin®RNA virus kit (MN). Finally, three reverse transcription quantitative PCR (RT-qPCR) assays were used to quantify HEV titers. The evaluated procedures resulted in average HEV recoveries of 14.08 ± 4.90% and 3.58 ± 0.30% for the MN and NS methods, respectively. The limit of detection (LoD_95%) was 1.25 × 10⁴ IU/L for both extraction methods combined with the three RT-qPCR assays tested, with the exception of NS extraction coupled with RT-qPCR1 that showed a LoD_95% of 4.26 × 10³ IU/L. The method characteristics generated in this study support the limited suitability of the ISO 15216-1:2017 concentration procedure coupled with the evaluated RT-qPCR assays for detecting HEV in bottled water.

Keywords: RT-qPCR; bottled water; concentration method; hepatitis e virus (HEV).

Figure 1

Hepatitis E virus (HEV) recovery in bottled water samples using the ISO 15216-1:2017 procedure and comparing two extraction kits and three RT-qPCRs assays. MN: NucleoSpin^®RNA virus kit (Macherey-Nagel GmbH & Co.); NS: NucliSens^® MiniMag^® system (BioMerieux SA); RT-qPCR1: modified from [20]; RT-qPCR2: CeeramTools^® Hepatitis E Virus Detection KHEV kit (BioMérieux SA); and RT-qPCR3: Described in [21] and modified in [22]. Different letters denote significant differences among methods for each virus (p < 0.05).