Detection of HEV

The commonly used tests for HEV infection in humans and animals include the detection of IgM and IgG anti-HEV antibodies, less frequently anti-HEV IgA, and the detection of HEV RNA. IgM anti-HEV antibodies can be detected during the first few months after HEV infection, whereas IgG anti-HEV antibodies represent either recent or past exposure. HEV infection is usually confirmed by (quantitative) RT-PCR. However, these assays should be standardized to increase reliability. The presence of HEV RNA indicates current infection, be it acute or chronic [297].

Laboratory diagnosis uses serum samples to detect anti-HEV antibodies with western blot assays, indirect enzyme-linked immunosorbent assays (ELISAs) or line assays. For serological tests in domestic pig, meat juice may be a feasible alternative to serum [298]. In humans, anti-HEV IgM levels peak around the time of the ALT peak and may persist for up to five months after the onset of disease [299]. Shortly after the appearance of IgM, IgG antibodies develop and persist throughout the acute and the convalescent phases, remaining high for at least one year after illness recovery. The presence of IgG antibodies is a marker of previous exposure to HEV, but the exact duration of immunity to HEV is not clarified, since reinfection with HEV has been documented [300]. IgA has a similar onset, but although detectable in serum, it is not screened for in most studies [301]. Commercially available immunoassays differ substantially in their sensitivity and specificity for the diagnosis of acute HEV infections [302], with a sensitivity of around 90% and false-positive results varying from 0.3% to 2.5% [302,303]. The variability in HEV genome leads to antigenic variations with important impact on the construction of specific, sensitive and reliable immunoassays [304]. HEVgt1 to gt4 is represented by one serotype, and HEV-specific antibodies appear to be detectable with antigens of all four genotypes. In various assays different antigens were used, even rat HEV antigen [154,305-307]. Broad performance variability among HEV strains with poor sensitivity were seen in assays focusing on pORF3. Conversely, all HEV isolates share some important cross-reactive antigens, especially within the pORF2 being an important key antigen that stimulates the host immune response [100]. HEV recombinant proteins have been used in different formats of diagnostic assays.

For the detection of antibodies against HEV in human serum specimens, a commercially available immunoblot test recomBlot HEV IgG/IgM using overlapping recombinant proteins

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covering the entire ORF2, and one recombinant protein covering ORF3 was recently developed (Mikrogen GmbH, Neuried, Germany). This test was adapted for detecting antibodies in swine and used to show HEV-specific seroprevalence of 49.8% among domestic pigs in Germany [114].

Different ELISAs for the detection of anti-HEV antibodies in serum specimens using E. coli-expressed HEV antigens and VLPs coli-expressed in insect cells have been developed [308-314].

Most diagnostic formats are based on the use of host-specific detection reagents, such as anti-human or anti-swine antibody. A host-independent detection is the double-antigen sandwich ELISA [299]. An assay based on this format was recently developed for the detection of anti-HEV in human and animal specimens [315]. Two of the most widespread commercial tests are the MP Diagnostics HEV ELISA kit (MP Biomedicals, Santa Ana, CA, USA; formerly Genelabs Diagnostics, Singapore), utilizing short C-termini of pORF2 and pORF3 of HEVgt1 and gt2, and the Abbott HEV EIA (Abbott Diagnostics, Lake Forest, IL, USA), using the complete pORF3 and a significant portion of pORF2 of gt1. In European surveys the HEV Ab-ELISA kit (Axiom, Buerstadt, Germany) is often used, which is a double-antigen sandwich ELISA based on pORF2 of gt1. The PrioCHECK HEV Ab porcine assay (Prionics, Schlieren-Zurich, Switzerland) is an indirect ELISA for the detection of anti-HEV IgG in domestic pig, and is based on pORF2 and pORF3 from both gt1 and gt3.

In addition, immunochromatographic methods for the detection of serological markers of HEV infections have been developed. A rapid immunochromatographic assay ASSURE™

has been developed by Genelabs Diagnostics, Singapore (nowadays MP Biomedicals, Santa Ana, CA, USA), and evaluated for the detection of IgM anti-HEV in serum specimens from patients with acute hepatitis E infection [316].

To date, detection of HEV RNA by molecular genetic methods is considered the “gold standard” [317]. Detection of RNA is performed by different RT-PCR methods, amplifying genomic fragments in one of the three ORFs [151,287,318-320]. RT-PCR assays published so far are in-house tests characterized by a high degree of performance variability [321]. HEV RNA can be found in blood and feces of patients during the late prodromal phase, and is detectable in the feces for another two weeks [92,322]. The time of viremia is very short;

however, undetectable HEV RNA does not exclude HEV infection. Sequencing of the PCR product allows further determination of the HEV strain.

In immunocompromised patients such as transplant recipients, HEV diagnosis is generally based on the detection of HEV RNA, as testing for antibodies may give false-negative results due to immunosuppression. In this setting, HEV RNA detection and quantification may also have a role in monitoring the clinical response to antiviral therapy [300,323]. In different tissue specimens negative sense HEV RNA, which is an indicator of active viral replication, can be demonstrated by in-situ-hybridization as well [270,273,324].

Recently, the proof of the presence of HEV antigen was introduced as an additional early diagnostic marker [317,325]. However, the application of HEV antigen screening is currently inferior for the early detection of HEV infection due to the decreased sensitivity compared to nucleic acid amplification technology methods [326]. The presence of HEV antigen in different tissues using immunohistochemistry was recently demonstrated in swine [268].

More recently, an immunohistochemical method for the detection of HEV antigen in liver tissue of hepatitis E patients was described representing a valuable tool for the detection of HEV infection in biopsy, autopsy and explant liver tissues [327].

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