Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver

Eva Trojnar,Sabine Burkhardt,Matthias Contzen,Anja Carl,Dietrich Mäde,Jochen Kilwinski,Ulrich Schotte,Sascha Mormann,Reimar Johne,Dominik Moor,Anne Kontek,Kornelia Berghof-Jäger,Nadine Althof

doi:10.3390/microorganisms8101460

Abstract

Background: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. Methods: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. Results: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. Conclusions: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

Highlights

Infection of humans with the hepatitis E virus (HEV) genotypes 1 to 4 can result in hepatitis E, which is usually characterized as an acute self-limiting inflammation of the liver
A major part of these infections is considered to be a result from zoonotic virus transmission via the consumption of raw or undercooked meat, liver and meat products from infected pork or wild boar
Pig livers were obtained from food business operators in Germany and pre-tested by real-time RT-PCR for the presence of HEV-RNA as described [31]

Summary

Introduction

Infection of humans with the hepatitis E virus (HEV) genotypes 1 to 4 can result in hepatitis E, which is usually characterized as an acute self-limiting inflammation of the liver. A major part of these infections is considered to be a result from zoonotic virus transmission via the consumption of raw or undercooked meat, liver and meat products from infected pork or wild boar. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. Conclusions: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, be applied to routine food control as well as in monitoring studies

Methods

Results

Conclusion