World Health Organization International Standard to Harmonize Assays for Detection of Hepatitis E Virus RNA

Sally A. Baylis

, Johannes Blümel, Saeko Mizusawa, Keiji Matsubayashi, Hidekatsu Sakata, Yoshiaki Okada, C. Micha Nübling, Kay-Martin O. Hanschmann, and the HEV Collaborative Study Group

Author affiliations: Paul-Ehrlich-Institut, Langen, Germany (S.A. Baylis, J. Blümel, C.M. Nübling, K.-M.O. Hanschmann); National Institute of Infectious Diseases, Tokyo, Japan (S. Mizusawa, Y. Okada); Japanese Red Cross Hokkaido Block Blood Center, Sapporo, Japan (K. Matsubayashi, H. Sakata)

Abstract

Nucleic acid amplification technique–based assays are a primary method for the detection of acute hepatitis E virus (HEV) infection, but assay sensitivity can vary widely. To improve interlaboratory results for the detection and quantification of HEV RNA, a candidate World Health Organization (WHO) International Standard (IS) strain was evaluated in a collaborative study involving 23 laboratories from 10 countries. The IS, code number 6329/10, was formulated by using a genotype 3a HEV strain from a blood donation, diluted in pooled human plasma and lyophilized. A Japanese national standard, representing a genotype 3b HEV strain, was prepared and evaluated in parallel. The potencies of the standards were determined by qualitative and quantitative assays. Assay variability was substantially reduced when HEV RNA concentrations were expressed relative to the IS. Thus, WHO has established 6329/10 as the IS for HEV RNA, with a unitage of 250,000 International Units per milliliter.

Hepatitis E virus (HEV) is a nonenveloped, single-stranded RNA virus belonging to the family Hepeviridae (1,2). In developing countries, HEV is a major cause of acute hepatitis, transmitted by the fecal–oral route and associated with contamination of drinking water. In industrialized countries, reports of HEV infection have been uncommon but are being reported more frequently; some cases are imported after travel to HEV-endemic areas, but reports of autochthonous cases are also increasing, and infection with HEV appears to be more prevalent than originally believed (3). Prospects for control of HEV infection are encouraged by recent efforts in vaccine development (4,5).
Four main genotypes of HEV, representing a single serotype, infect humans. Genotype 1 viruses are found mainly in Africa and Asia and genotype 2 in Africa and Central America; it is in these areas that prevention of HEV infection by vaccination would be most beneficial. Genotypes 3 and 4 viruses are generally less pathogenic, although some exceptions have been reported, particularly for genotype 4; these genotypes infect not only humans but also animals such as swine, wild boar, and deer. Although genotype 4 strains have mainly been restricted to parts of Asia, genotype 3 viruses are found widely throughout the world. Zoonotic transmission of HEV genotypes 3 and 4 to humans can occur by consumption of contaminated meat or meat products or by contact with infected animals (6,7). Shellfish, such as bivalve mollusks, have also been shown to act as reservoirs for HEV (8).
An alternate route of transmission of HEV by transfusion of blood components has been reported in Japan (9,10), the United Kingdom (11), and France (12,13). Studies in Japan (14) and the People’s Republic of China (15) have identified acute HEV infections in blood donors, confirmed by the detection of HEV RNA. Analysis of blood and plasma donors in Europe has identified HEV-infected donors in Germany (16–20), Sweden (18), and England (21). Transmission of HEV by solid organ transplantation has also been reported (22). Rates of HEV infection may be underreported in some countries, and misdiagnosis of HEV infection also occurs. For example, in some cases of suspected drug-induced liver injury, HEV has been determined as the cause (23). In one such recent case, HEV was shown to have been transmitted by blood transfusion (13).
Infection with HEV may cause particularly severe illness in pregnant women and in persons who have preexisting liver disease. Chronic infection with HEV genotype 3 is an emerging problem among solid organ transplant recipients and may also occur in persons with HIV and certain hematologic disorders (24). In patients with chronic infection, viral loads are monitored to investigate the efficacy of antiviral treatment (25,26) and effects of reduction of immunosuppressive therapy (27).
HEV infection is diagnosed on the basis of detection of specific antibodies (IgM and IgG), but the sensitivity and specificity of these assays is not optimal (28–30). Analysis of HEV RNA by using nucleic acid amplification techniques (NATs) is also used for diagnosis; this method can identify active infection and help confirm serologic results (31). Several NAT assays have been reported for the detection of HEV RNA in serum and plasma or fecal samples: conventional reverse transcription PCR (RT-PCR) and nested protocols (32), real-time RT-PCR, and reverse transcription loop-mediated isothermal amplification (33). The NATs include generic assays designed for the detection of HEV genotypes 1–4 (34,35).
In 2009, the World Health Organization (WHO) Expert Committee on Biological Standardization endorsed a proposal by the Paul-Ehrlich-Institut (PEI) to prepare an International Standard (IS) for HEV RNA for use in NAT-based assays. PEI recently completed an initial study that investigated the performance of HEV NAT assays in detection of HEV infection (36). In that study, dilution panels of HEV genotype 3 and 4 strains underwent blinded testing in laboratories that had experience in detection of HEV RNA. Results demonstrated wide variations in assay sensitivity (in the order of 100- to 1,000-fold for most assays).
After the initial study, 2 virus strains included in the panel (36) were selected for further development of a candidate IS for the WHO, and a candidate Japanese national standard (done in collaboration with the National Institute of Infectious Diseases in Tokyo). These viruses belong to genotype 3, which is widely distributed, and were genotype 3a and 3b strains, which were equally well detected in the initial study. The strains were derived from plasma samples that had sufficient titers of HEV RNA to prepare standards of good potency. An international collaborative study was conducted to establish the respective standards, demonstrate suitability for use, evaluate potency, and assign an internationally agreed-upon unitage.