Use of serological assays for diagnosis of hepatitis E virus genotype 1 and 3 infections in a setting of low endemicity

Abstract

Because of the occurrence of genotype 3 hepatitis E virus (HEV) in regions of low endemicity, it is important to validate the currently used serological assays for diagnosing infections with viruses belonging to this lineage, since these assays only use antigens derived from genotype 1 and 2 viruses. We evaluated the Genelabs enzyme-linked immunosorbent assay (ELISA) and the RecomBlot from Mikrogen for the detection of HEV-specific immunoglobulin M (IgM) and IgG under conditions of low endemicity. We compared test results of 16 patients with locally acquired genotype 3 HEV, 8 genotype 1 patients, 167 healthy controls from the general population, and 101 cases with hepatitis due to other viral causes. The measured specificities of the ELISA (98%) and the RecomBlot (97%) were comparable to those given by the manufacturer for IgM but were significantly lower for IgG (93% by ELISA and 66% by immunoblotting, versus reported values of 98% for ELISA and 95% for blotting). Antibody levels detected following infections with genotype 3 were lower than those following genotype 1 infections except for those measured in the IgM ELISA. Reactivity to the four antigens used in the immunoblot assay were analyzed and showed differences in the IgM immunoblot reactions between genotype 1 patients and genotype 3 patients. The ORF3 antigen was the most specific antigen. The specificity could be improved by a combined testing regimen with confirmation by immunoblotting of all positive ELISA results and by raising the cutoff of the IgG immunoblot assay without loss of sensitivity. We conclude that a combination of ELISA and immunoblotting is needed for acceptable specificity and sensitivity of HEV assays under conditions of low endemicity.

FIG. 1.

HEV-specific IgM and IgG antibodies measured by ELISA in the general population and in genotype 1- and genotype 3-infected HEV patients. Serum samples were from controls (n = 167) from the general population of The Netherlands, 8 genotype 1-infected patients (including 2 follow-up samples), and 16 genotype 3-infected patients (including 9 follow-up samples).

FIG. 2.

HEV-specific IgM and IgG antibodies measured by immunoblot assay in the general population and in genotype 1- and genotype 3-infected HEV patients. Serum samples were from controls (n = 65) from the general population of The Netherlands, 8 genotype 1-infected patients (including 2 follow-up samples), and 16 genotype 3-infected patients (including 9 follow-up samples).

FIG. 3.

Distribution of the incidence of strongly reactive HEV-specific bands in an immunoblot assay in the general population and in HEV genotype 1- and genotype 3-infected patients. Total number of serum samples tested: 65 controls, 10 genotype 1, and 25 genotype 3. Only immunoblot scores of individual bands of 2 to 3 were considered reactive, following the manufacturer's instructions.

FIG. 4.

Distribution of reactive HEV-specific bands in the IgM immunoblot assay of HEV genotype 1- and genotype 3-infected patients. Acute-phase serum samples from 8 genotype 1 and 16 genotype 3 patients were analyzed.

FIG. 5.

Distribution of reactive HEV-specific bands in the IgM and IgG immunoblot assay of HBV- and HCV-infected patients. (A) Detected IgM responses in serum samples from reactive HCV patiens (n = 4) and HEV patients (n = 2). (B) Detected IgG responses in serum samples from reactive HBV (n = 4) and HCV (n = 4) patients and HEV patients (n = 4).