Anti-HEV IgG Avidity Testing: Utility for Diagnosing Acute and Resolved Genotype 3 Infections

Abstract

European Association of the Study of the Liver (EASL) guidelines specify HEV RNA, as well as anti-HEV IgG and IgM as positive markers for acute HEV infection. HEV RNA assay sensitivity limitations may lead to false negative test results in patients with low levels of viremia. Moreover, anti-HEV IgM positivity is not a reliable indicator for distinguishing between acute and resolved infections given the ability of this antibody to persist several months after a resolved infection. Our study aims were to assess HEV IgG avidity for diagnosing acute and resolved infections, regardless of the anti-HEV IgM serostatus, and examine assay reliability when evaluating different genotype 3 (GT3) HEV subtypes. Patient serum samples (n = 104) were tested for HEV IgG avidity by utilizing the DIA.PRO kit on a DSX automated instrument. Among patients identified with acute HEV infections, 32 were infected with GT3: GT3c (n = 5), GT3e (n = 8), 3f (n = 17) and GT3-unsubtyped (n = 2). Avidity sensitivity was 91.2% and specificity was 100%. For patients with long-lasting anti-HEV IgM persistence, an Avidity Index >70% was observed. Thus, the DIA.PRO avidity assay may be utilized to distinguish between recently acquired and resolved HEV GT3 infections. However, for equivocal results (Avidity Index > 40-70%), HEV RNA molecular testing will be required to confirm a recent infection.

Keywords: HEV RNA; HEV acute hepatitis; HEV genotype 3; IgG avidity test; IgM; hepatitis E virus.

Figure 1

Subtyping of HEV GT3 patient-derived samples by phylogenetic analysis. Twenty-seven ORF2 patient-derived sequence fragments (shown in red) and reference sequences ([4], GenBank accession numbers shown) were used for the construction of a maximum-likelihood tree. Bootstrap values were based on 500 replicates. Bootstrap values > 60% are indicated on respective branches. The scale bar represents nucleotide substitutions per site. GenBank accession number of patient sequences are reported in Table S1 (see Supplemental Material).

Figure 2

Phylogenetic analysis based on partial open reading frame 1 (ORF1) (125 nt, nt78–202 of the HE-JA04–1911 isolate, GenBank accession number AB248521) sequences from patient-derived samples with a negative ORF2 RT-PCR result. The phylogenetic tree includes the prototype strains as indicated by Smith et al. [4] The maximum-likelihood tree was constructed with a bootstrap of 500 replicates. Bootstrap values > 60% are indicated on respective branches. The scale bar represents nucleotide substitutions per site. Supplementary data on sequence accession number of patients are provided in Table S1 (see Supplemental Material).

Figure 3

Correlation between HEV GT3 subtypes (GT3c, n = 5; GT3e, n = 8; GT3f, n = 17) and the mean Avidity Index, which was calculated for Group A GT3 patients. Boxes represent the first and third quartiles and the black line represents the median value. p-values were calculated using the Mann–Whitney test.

Figure 4

Group C patient samples with persistent (>24 months) anti-HEV IgM positivity were tested using the anti-HEV IgM rapid test. C+, positive control (HEV RNA positive, anti-HEV IgM positive, acute infection); C-, negative control (anti-HEV IgG positive, anti-HEV IgM negative, resolved HEV infection).

Figure 5

Power of the IgG avidity test in prediction of recent and prior (resolved) infections when considering HEV RNA positivity and clinical manifestations. Neg, patients who are HEV RNA negative and anti-HEV IgM negative. The kappa statistic was employed to measure the agreement between the Avidity Index (AI), HEV RNA, serological markers and clinical symptoms (see Section 3).