A new assay for quantitative detection of hepatitis A virus

Sofia Persson,Erik Alm,Måns Karlsson,Theresa Enkirch,Heléne Norder,Ronnie Eriksson,Magnus Simonsson,Patrik Ellström

doi:10.1016/j.jviromet.2020.114010

Abstract

Hepatitis A virus (HAV) is mainly transmitted via contaminated food or water or through person-to-person contact. Here, we describe development and evaluation of a reverse transcription droplet digital PCR (RT-ddPCR) and reverse transcription real-time PCR (RT-qPCR) assay for detection of HAV in food and clinical specimens. The assay was evaluated by assessing limit of detection, precision, matrix effects, sensitivity and quantitative agreement. The 95 % limit of detection (LOD95 %) was 10 % higher for RT-ddPCR than for RT-qPCR. A Bayesian model was used to estimate precision on different target concentrations. From this, we found that RT-ddPCR had somewhat greater precision than RT-qPCR within runs and markedly greater precision between runs. By analysing serum from naturally infected persons and a naturally contaminated food sample, we found that the two methods agreed well in quantification and had comparable sensitivities. Tests with artificially contaminated food samples revealed that neither RT-ddPCR nor RT-qPCR was severely inhibited by presence of oysters, raspberries, blueberries or leafy-green vegetables. For this assay, we conclude that RT-qPCR should be considered if rapid, qualitative detection is the main interest and that RT-ddPCR should be considered if precise quantification is the main interest. The high precision of RT-ddPCR allows for detection of small changes in viral concentration over time, which has direct implications for both food control and clinical studies.

Highlights

Hepatitis A virus (HAV) is a major cause of acute viral hepatitis (Murray et al, 2015)
The virus belongs to the Hepatovirus genus of the Picornaviridae family and the genome consists of a positive-sense, sin gle-stranded RNA molecule of 7.5 kilobases with a single open reading frame (ORF) flanked by 3′ and 5′ untranslated regions (UTRs)
To overcome the limitation with the assay recommended in the ISO method (ISO, 2017; Persson et al, 2019), we developed a new assay for detection of HAV in food and clinical samples, designed for onestep RT-ddPCR and one-step RT-qPCR

Summary

Introduction

Hepatitis A virus (HAV) is a major cause of acute viral hepatitis (Murray et al, 2015). The human HAV strains are classified in three genotypes (I, II and III) and seven subtypes (IA, IB, IC, IIA, IIB, IIIA and IIIB) (Perez-Sautu et al, 2011; Pintoet al., 2012). Trans mission of HAV occurs mainly via the faecal-oral route through ingestion of contaminated food or water, or directly from person-to-person (Martin and Lemon, 2006; Mast and Alter, 1993; Murray et al, 2015). Foods previously associated with HAV outbreaks include oysters and clams (Costafreda et al, 2006; Sanchez et al, 2002), strawberries (Enkirch et al, 2018; Nordic, 2013; Scavia et al, 2017), raspberries (Hutin et al, 1999; Reid and Robinson, 1987), blueberries (Calder et al, 2003), dates (Rajiuddin et al, 2018), leafy green vegetables (Callejon et al, 2015; Herman et al, 2015; Rosenblum et al, 1990) and semi-dried tomatoes (Carvalho et al, 2012; Petrignani et al, 2010)

Methods

Results

Conclusion