Serological tests are a suitable tool for describing the epidemiology of an infection independently of the acute infection represented by NAT. HEV gained attention, due to the observed high seroprevalence long before studies on acute infections via NAT, for example among blood donors, were published. Nevertheless, little is known about the long-term titers of immunoglobulins against HEV although these are necessary to assess a correlation between incidence and seroprevalence. The data presented here supplements these findings with the factor of long-term assessment. By tracking the detection of anti-HEV IgM and IgG antibodies over a period of up to 5 years in 370 blood donors, we systematically monitored long-term serology. Surprisingly, a proportion of 7.3% positive anti-HEV IgM (long-term IgM-positive) and 9.1% with negative anti-HEV IgG (seroreversion) were observed five years after index donation. Furthermore, differences were found in the frequency of these phenomena as well as in the quantification of titers between the sexes.The proportion of anti-HEV IgM positive donors was highest shortly after infection with HEV (three months follow-up), and the average IgM S/CO value decreased over the years and fell below the cut-off for a positive value in most donors. These kinetics were to be expected for IgM response [21]. Meanwhile, it should be noted that the serostatus IgM+/IgG− was only rarely detectable, which, however, fits in with an observation already published that anti-HEV IgM and IgG can be detected relatively simultaneously, but have their maximum with a delay [8, 23, 24]. In addition, it was also evident after three months that a large proportion (42.9%) had already seroconverted to IgM−/IgG+ status, which is consistent with a previously published observation in which a median time to disappearance of anti-HEV IgM of 85 days was determined [8].

Nevertheless, contrary to the usual kinetics of disappearing IgM detection after a few weeks, a proportion of 9.1% of anti-HEV IgM-positive donors were found even after five years, if the Euroimmun assay is used as a basis, and 7.3% if the 2:1 rule is applied. Riveiro-Barciela et al. published a similar observation over a period of 36 months in a group of 12 HEV-infected patients with acute hepatitis, 42% of whom still had an anti-HEV IgM-positive result when analyzed with the Mikrogen assay and 17% when using the Wantai assay. In our case, a positive result with the Euroimmun assay was consistent with the Mikrogen assay in 94.7%, while the Wantai assay gave a confirming positive result in 51.6% of cases. Taken together, the Mikrogen assay has a higher detection rate of IgM-positive samples compared to the Wantai assay. Whether this is due to a lower specificity or a higher sensitivity is intensively discussed elsewhere and cannot be conclusively clarified here [25,26,27,28]. In addition, our data suggest that the Euroimmun assay sensitivity and specificity is intermediate between these two assays. As the focus of our study was not on calculating the assay sensitivity but on the basic long-term course of the serology, we did not perform deeper comparative studies.

Similar to anti-HEV IgM, the number of positive anti-HEV IgG donations also increases rapidly after the index donation and reaches its maximum after three months. These observations are also consistent with the expected general kinetics of IgG antibodie response and also with descriptions in HEV infected individuals [8, 29]. A decrease in the IgG value is generally to be expected, but on average it did not fall below the detection limits, so that IgG may contribute to immunity against HEV over several years for most donors.

Noteworthy, 36.4% of the donors were IgG-negative five years after their index donation using the Euroimmun assay. Using the 2:1 rule, this only applied to 9.1%. Seroconversion post HEV RNA-positivity was detectable in all returning donors, although in 13 cases this seroconversion was not detectable with the assays provided by Euroimmun but with the other two assays.

Against this background, the Euroimmun assay seem to have a lower detection rate in late-stage post infection than the other two assays, whereby the result of the Mikrogen assay contradicted the result of the Wantai assay more often and yielded a positive result. Particularly in view of the fact that the Euroimmun assay yielded negative results in samples with average lower (semi-) quantitative values in the other two assays, we assume that this assay has a lower sensitivity, which has already been described in a study by Avellon et al. [26]. Contrary to data from other publications, which reported a sensitivity range of 74—97.1% for the Wantai assay and 65–95.3% for the Mikrogen assay, our data suggest that the Mikrogen assay is more sensitive than the Wantai assay [26,27,28]. In our study, 13 out of 370 donors (3.5%) showed no seroconversion in the Euroimmun assay. In comparison, in a Swiss study, only 1 out of 90 donors (1.1%) who were analyzed with the Wantai assay showed no seroconversion [30]. Therefore, when discussing the results below, a certain inaccuracy of the results depending on the assay used must always be taken into account. Comparative studies have shown that the assessment of the serostatus of HEV-infected persons is variable for different serological assays, which was basis for the decision of our testing strategy [22, 31].

Seroreversion (falling below the cut-off for a positive anti-HEV IgG titre) was observed for a certain proportion of donors, although it is a matter of sensitivity and the interpretation of cut-offs depending on the assay used. Seroreversion rates of 1.9% annually (among HEV seropositive individuals observed over 12 years; no male bias, Mikrogen assay), 15% annually (population-representative HEV serology positive cohort; 19% in females, Wantai assay) or 19% in follow-up samples (HIV- and HEV-coinfected individuals; Wantai assay) have been described in other studies [32,33,34]. The extent to which an HEV infection once experienced offers lifelong protection or protection against complications in the event of reinfection can only be evaluated through observation over decades demonstrably reinfected people. Whether reinfections occur at all among healthy individuals has not been clarified.

In addition to the question of reactivability at low-level IgG titers, an analysis of the avidity of IgG antibodies, cellular immunity and vaccine-induced immunity is a promising approach in future studies to investigate the immune response to HEV in more detail.

We observed several donations (n = 28) that were anti-HEV IgG positive at the index donation. However, it is not possible to differentiate whether the donors underwent reinfection or were already in a late phase of infection as the serostatus from before the donation was unknown. Nevertheless, it is interesting that in individual cases we were able to determine an IgM−/IgG+ serostatus at the index donation (n = 3) and a subsequent donation showed an IgM+/IgG+ status. The reason for this could be that (a) anti-HEV IgG can be detected before anti-HEV IgM if the titers increase at the same time, but the sensitivities of the assays differ, or that (b) these individuals have been reinfected, particularly since the index donation was already anti-HEV IgG positive.

Another striking feature of our data was the difference in long-term serology between the sexes. This was reflected in both, the qualitative observations and quantitative values. Compared to the general proportion of females in the subcohorts (between 21.3 and 25.9%), a higher proportion of those with long-term IgM (34.2%) and a lower proportion of IgG seroreverts (15.3%) were women. This is interesting against the following background: studies, for example, in German cohorts, have not found significant differences in seroprevalence between men and women, while a higher incidence among men was found when screening for HEV RNA [21, 32]. A study of Bulgarian blood donors shows that although the HEV seroprevalence was similar in men and women, the incidence was twice as high in men in the same cohort [35]. A male bias in immune response is known to occur in various viral infections and is associated with physiological, sex-specific factors such as immunomodulators encoded on the X chromosome or hormone-dependent gene expression regulators [36, 37]. Due to the lack of long-term studies, reference is usually made to the stronger humoral immune response in women shortly after infection. In our data, the long-term differences in antibody formation against HEV were more pronounced. A similar observation was made in the long-term observation of antibodies against SARS-CoV2, with a significantly greater decline in IgG antibody titers in men than in women after one year [38].

In conclusion, the evaluation of seroprevalence and incidence, for example in blood donors, offers interesting conclusions on the epidemiology of HEV. Long-term studies are needed to link these two parameters. Based on the data analyzed in this study with a unique cohort, it should be noted that.

1.

IgM positive results without NAT testing do not confirm recent infection (long-term IgM positivity).

2.

A negative anti-HEV IgG result does not exclude a prior HEV infection (seroreversion).

3.

Sex-dependent antibody response against HEV is contributing to the divergence between sex-based incidence and seroprevalence.