Molecular and serodetection of hepatitis E virus among organ donors in the Czech Republic

Abstract

Introduction

The prevalence of hepatitis E virus (HEV) infection in the Czech Republic is high, and HEV represents a potential risk for immunosuppressed organ transplant recipients.

Aim

The aim of this study was to investigate the prevalence of anti-HEV IgG and IgM antibodies and the presence of HEV RNA in organ donors in the Czech Republic in order to assess the potential risk of HEV transmission.

Methods

In this study, 73 organ donors sampled between 2022 and 2024 were examined. IgG and IgM antibodies against HEV were detected using an electrochemiluminescence immunoassay, and HEV RNA was assessed by molecular testing.

Results

A high prevalence of anti-HEV IgG antibodies (49.3%) and a low prevalence of anti-HEV IgM antibodies (2.7%) were observed. HEV RNA was not detected in any of the samples, indicating the absence of active infection. Higher donor age was significantly associated with anti-HEV IgG positivity (p = 0.046). The observed IgG prevalence was significantly higher than previously reported data from the Czech Republic based on samples collected in 2001.

Conclusion

Despite the absence of detectable HEV RNA, the high prevalence of anti-HEV IgG antibodies indicates substantial HEV exposure among organ donors. These findings may inform discussion on HEV screening strategies for organ donors, particularly in settings with high background prevalence, with PCR testing serving as a confirmatory tool following serological screening. However, the relatively limited sample size and the absence of recipient follow-up data represent important limitations of this study.

Clinical trial

ClinicalTrials.gov ID: NCT06457438. The study used residual serum material from organ donors, which would have otherwise been disposed of. No pressure was exerted on the donors or their family members to agree to organ donation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-12567-5.

Introduction

Hepatitis E virus (HEV) has emerged as an important transfusion- and transplant-related pathogen, posing a particular risk to immunosuppressed recipients who may develop chronic infection following exposure to contaminated grafts or blood products [1, 2]. The increasing number of reported donor-derived HEV transmissions has led several countries to introduce routine screening of organ donors, including the United Kingdom (since 2017) and France (since January 2024) [3]. Despite this, screening policies remain inconsistent across Europe, and country-specific data are needed to better assess the risk and inform national strategies.

Globally, HEV is the second most common cause of viral hepatitis and is the leading cause of acute viral hepatitis in several European countries [4]. HEV belongs to the family Hepeviridae, subfamily Orthohepevirinae, and comprises eight genotypes (HEV1–HEV8). HEV1 and HEV2 cause large waterborne outbreaks in endemic regions, whereas HEV3 and HEV4 are zoonotic, widely distributed in pigs and wild mammals, and represent the major cause of autochthonous food-borne infections in Europe and East Asia [5, 6]. Transmission to humans occurs through consumption of undercooked meat, direct or indirect contact with infected animals, and, importantly, via blood transfusion or organ transplantation [1].

Most infections are asymptomatic, but 5%–30% of patients develop acute viral hepatitis, which is usually self-limiting in immunocompetent individuals. In contrast, immunosuppressed patients, particularly solid-organ transplant recipients, are at risk of developing chronic HEV infection, defined as detectable HEV RNA for more than three months [2].

The Czech Republic shows a high natural prevalence of HEV genotype 3. Anti-HEV IgG seroprevalence was 8.6% in a 2017 study using samples from 2001 [7], and the incidence of acute HEV reached 6.3 per 100,000 inhabitants in 2023—the highest rate since national reporting began in 1996 [8]. Acute HEV surveillance in the Czech Republic is based on the detection of HEV RNA and/or HEV IgM positivity within the national reporting system. Given this high background prevalence and the vulnerability of transplant recipients, data on HEV markers in organ donors are urgently needed.

Therefore, the aim of this study was to determine the prevalence of anti-HEV IgG, anti-HEV IgM, and HEV RNA in samples collected from organ donors in the Czech Republic between 2022 and 2024. Similar studies from Turkey, Denmark, India and Canada have reported variable results [9–13], underscoring the importance of regional surveillance to guide screening policies.

Methods

Study population and sample size

Serum samples obtained from organ donors between February 2022 and April 2024 were examined for IgG and IgM antibodies against HEV and for the presence of HEV RNA at the Department of Laboratory Medicine, University Hospital Ostrava. A total of 73 organ donors were included in the study. No formal sample size or power calculation was performed, as all available donors meeting the inclusion criteria during the study period were enrolled, and therefore the sample represents a complete cohort for the defined timeframe.

Ethical considerations

The study was conducted in accordance with the guidelines set forth by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use and the principles outlined in the Declaration of Helsinki. Ethical approval for using pseudonymised donor samples was obtained from the Ethics Committee of the University Hospital Ostrava (project number: 823/2023). The trial is registered in the ClinicalTrials.gov database under the identifier NCT06457438.

Laboratory methodology

The examined samples were residual serum material collected from organ donors and sourced from the Transplantation Centre of the University Hospital Ostrava. IgG and IgM antibodies against HEV were detected using the ECLIA (electrochemiluminescence immunoassay) method, with Elecsys^® Anti-HEV IgG and Elecsys^® Anti-HEV IgM diagnostic kits (Roche Diagnostics), on a cobas^® Pro automatic analyser (Roche Diagnostics). Positive findings of IgG and IgM antibodies against HEV were confirmed by western blot using the HEV IgG/IgM recomLine diagnostic kit (Mikrogen Diagnostik). RNA was isolated from donor 400 µL serum samples using the MagPurix^® Viral Nucleic Acid Extraction Kit (Zinexts) on a MagPurix^® isolator (Zinexts), and HEV RNA was amplified by qRT-PCR using an ampliCube HEV 2.0 Quant kit (Mikrogen Diagnostik) on a CFX96 instrument (Bio-Rad). According to the manufacturer’s specifications, the limit of detection (LOD) of the qRT-PCR assay is < 50 IU/mL.

Statistical analysis

Numerical variables are expressed as median and interquartile range and categorical variables as absolute and relative frequencies (%). Between-group differences were assessed using the Mann–Whitney test or the Chi-square test of independence for contingency tables. The significance level was set at p ≤ 0.05, and statistical analysis was performed using R software (version 4.4.1, www.r-project.org).

Results

Study subjects

The present study comprised 73 organ donors (all brain-dead status), with a median age of 51 years (interquartile range: 41–58 years; mean ± SD: 50.9 ± 12.4). Of this group, 55 donors (75.3%) were male and 18 (24.7%) were female. The distribution of age and sex was assessed in relation to seropositivity for anti-HEV IgG and IgM antibodies. Supplementary Table S1 provides an overview of the donor cohort, including sex, age, and the types of organs retrieved (kidneys, liver, heart, lungs, and pancreas). It also specifies whether heart valves or pancreatic islets were included and lists any contraindications to donation. Together, these data illustrate the structure of the studied population with respect to organ donation profiles.

Prevalence of HEV antibodies and HEV RNA

Of the 73 organ donors, 37 (50.7%) were negative for anti-HEV IgG and 36 (49.3%) were positive. A total of 71 (97.3%) were negative for anti-HEV IgM, and only 2 (2.7%) were positive. All initially positive serological results were confirmed by Western blot. No discrepancies were observed and no samples were excluded. All 73 samples were negative for HEV RNA, as demonstrated by the absence of amplification curves in real-time PCR (Fig. 1).

Fig. 1.

Real-time PCR amplification plot for HEV RNA. Amplification curves of positive controls (blue) show typical exponential growth, whereas all donor samples (pink) remain at baseline fluorescence throughout all 45 cycles, indicating no detectable HEV RNA. The negative control (dark blue) shows no amplification. All 73 donor samples were tested and were therefore interpreted as HEV RNA-negative

The median age was significantly higher among those who tested positive for anti-HEV IgG compared to those who tested negative (Mann-Whitney test, p = 0.046; Fig. 2). This finding indicates a statistically significant association between increasing donor age and anti-HEV IgG seropositivity. Table 1 gives the prevalences of anti-HEV IgG and anti-HEV IgM among organ donors according to age group. Anti-HEV IgG positivity was not significantly associated with sex (Chi-square test of independence, p = 0.455; Fig. 2).

Fig. 2.

Anti-HEV IgG positivity in relation to age (Panel A) and sex (Panel B) among organ donors from February 2022 to April 2024

Table 1.

Prevalence of anti-HEV IgG and IgM antibodies by age group among organ donors during February 2022 to April 2024

Age range, years	Anti-HEV IgG-positive			Anti-HEV IgM-positive
	Total	Male	Female	Total	Male	Female
0–20	0/1 (0.0)	0/0 (0.0)	0/1 (0.0)	0/1 (0.0)	0/0 (0.0)	0/1 (0.0)
21–30	1/4 (25.0)	1/2 (50.0)	0/2 (0.0)	0/4 (0.0)	0/2 (0.0)	0/2 (0.0)
31–40	3/12 (25.0)	3/11 (27.3)	0/1 (0.0)	0/12 (0.0)	0/11 (0.0)	0/1 (0.0)
41–50	8/19 (42.1)	6/15 (40.0)	2/4 (50.0)	2/19 (10.5)	2/15 (13.3)	0/4 (0.0)
51–60	20/26 (76.9)	16/20 (80.0)	4/6 (66.7)	0/26 (0.0)	0/20 (0.0)	0/6 (0.0)
61–70	1/7 (14.3)	1/5 (20.0)	0/2 (0.0)	0/7 (0.0)	0/5 (0.0)	0/2 (0.0)
71–80	3/4 (75.0)	2/2 (100.0)	1/2 (50.0)	0/4 (0.0)	0/2 (0.0)	0/2 (0.0)

The values represent the numbers of anti-HEV IgG or IgM-positive organ donors relative to the number of organ donors in the age category, with the relative frequency (%) in parentheses. IgG positivity increased with age group

Discussion

The current results confirm the high prevalence of IgG antibodies against HEV among organ donors in the Czech Republic. The identified prevalence of 49.3% was significantly higher than the previously reported prevalence of 8.6% that was based on samples taken in 2001 [7]. Direct temporal comparisons of HEV IgG seroprevalence should be interpreted with caution, as different serological assays with varying sensitivity were used in earlier studies. Therefore, the observed difference likely reflects a combination of increased cumulative exposure over time and improved assay performance rather than a precise epidemiological trend. Importantly, while that study investigated the general population, our study focuses specifically on organ donors—a group at direct risk for transmitting HEV to immunocompromised recipients. This increase may be related to the increasing incidence of acute HEV infection sin the Czech Republic, which in 2023 reached the highest level since 1996 (6.3 cases per 100,000 people) [8]. The current data show a significant correlation between positive serology and increased donor age (p = 0.046), which is consistent with previous studies [14], as is the lack of a significant association between sex and HEV seropositivity (p = 0.455) [2].

An important finding is the absence of HEV RNA in all samples, indicating that none of the donors had an active infection. This result is important because the presence of viral RNA poses the highest risk of infection for the transplant recipient [1]. For example, a study in the UK found that HEV RNA helped identify donors with active infection, which led to the introduction in 2017 of mandatory tests for organ donors [15]. France followed this approach in 2024 [3], indicating the growing importance of such a measure.

The current study represents the first systematic evaluation of HEV antibody prevalence and HEV RNA presence among organ donors in the Czech Republic. Previous seroepidemiological studies have assessed antibody prevalence in the general population [7], but this work specifically focused on organ donors, as the risk of HEV transmission is particularly relevant in clinical practice in this context. As noted, there was a significantly higher prevalence of IgG antibodies (49.3%) compared to historical data from the general population (8.6%), suggesting increasing HEV exposure over recent decades. The findings support the need for routine HEV screening among organ donors in the Czech Republic, aligning with established practices in the UK and France [16]. Additionally, the results highlight the advantage of combining serological and molecular methods to ensure more accurate detection of potential HEV transmission risks for transplant recipients.

When interpreting HEV seroprevalence over time, differences in assay sensitivity must be considered. The 2001 prevalence survey used the recomWell HEV IgG/IgM ELISA (Mikrogen) [7], while our current study applied the Elecsys anti-HEV IgG/IgM assay (Roche) as a more sensitive method. In our recently published evaluation of 80 donor samples [17], Elecsys demonstrated higher sensitivity, with the recomLine HEV IgG/IgM immunoblot showing 78.7% concordance for anti-HEV IgG, whereas recomWell reached only 48.7%. For anti-HEV IgM, Elecsys identified three positive samples, all confirmed by immunoblot, while all ELISA-based methods remained negative, indicating lower IgM sensitivity and a potential underestimation of past prevalence in earlier ELISA-based studies.

A diagnosis of HEV mainly involves detection of specific IgG and IgM antibodies and direct detection of viral RNA using molecular methods. In this work, ECLIA was applied for detection of IgG and IgM antibodies, followed by western blot confirmation. ECLIA is a newer method compared to the more commonly used ELISA and offers higher sensitivity, making it more suitable for screening purposes [18]. Given its high sensitivity and reliability, ECLIA could be appropriate as a primary screening method for detecting HEV infection in organ donors.

International comparisons are instructive: HEV seroprevalence among blood and organ donors ranges from 5.5% in Canada [12], to 17.6% among transplant recipients in Turkey [9], and up to 20.6% in Denmark [10]. Such differences reflect geographic, dietary, and methodological variations, reinforcing the value of localized surveillance and policy development.

PCR testing for HEV RNA is considered the gold standard for the diagnosis of active infection, especially in immunosuppressed patients whose serological response may be attenuated [2]. In the current study, qRT-PCR was used with a commercial diagnostic kit that allowed accurate detection of viral RNA. HEV RNA can be present in the blood even when serological tests are negative [18], and PCR testing could be used as a confirmatory method after screening to ensure the most accurate assessment of the presence of active infection.

In recent years, the European Union has considered the introduction of a harmonised approach to the detection of HEV, particularly in the context of the safety of blood transfusion and transplantation [14]. Some countries already test all blood donors for HEV RNA, which reduces the risk of transmission [1]. The current study shows that a similar approach could be appropriate for organ donors in the Czech Republic, especially given the increasing prevalence of HEV [19].

These results support the introduction of routine screening of organ donors for HEV in the Czech Republic. Given the high prevalence of IgG antibodies, the increasing incidence of acute cases, and the importance of molecular diagnostics for detecting active infection, it is important to consider incorporating PCR testing into standard protocols to confirm positive serological screening. Further multicentre studies could better characterise the epidemiology of HEV among organ donors and determine the optimal approach to screening [3].

Although this study provides important evidence of a high HEV IgG prevalence among organ donors, several limitations should be considered when interpreting the results. First, the number of included donors was relatively small, which may reduce the statistical power, particularly in subgroup analyses. Second, the monocentric design limits the generalisability of the findings to the entire Czech donor population, as regional epidemiological differences in HEV exposure are likely. Third, no systematic long-term follow-up of transplant recipients was included. As a result, possible donor-derived HEV transmissions or post-transplant seroconversions could not be evaluated, which may lead to an underestimation of the true clinical impact. Liver tissue was not available for histological examination or intrahepatic HEV RNA testing, as liver biopsy is not routinely performed in organ donors in the absence of macroscopic abnormalities. Consequently, subclinical hepatic infection cannot be entirely excluded.

Despite these limitations, our findings have relevant public health implications. The high HEV IgG seroprevalence in donors highlights sustained exposure to HEV within the Czech population and suggests a potential risk for immunosuppressed recipients. These data may support national discussion on HEV screening strategies for organ donors. However, the cost-effectiveness of universal PCR screening remains to be assessed, especially in comparison with targeted screening approaches (e.g., high-risk donors or organs intended for highly immunosuppressed recipients). A formal economic evaluation would be valuable for informing future policy decisions and ensuring optimal allocation of healthcare resources.

Future research should aim to provide a more comprehensive national overview by conducting a multicentre study with a larger donor cohort. Such research should ideally include post-transplant follow-up to assess HEV seroconversion or viraemia among recipients, allowing a more accurate estimation of donor-derived transmission risk. The implementation of combined serological and molecular testing in a prospective model could further clarify the most effective screening algorithm for clinical practice.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ALT

Alanine aminotransferase

ECLIA

Electrochemiluminescence immunoassay

ELISA

Enzyme-linked immunosorbent assay

ESOT

European Society for Organ Transplantation

IgG

Antibody IgG

IgM

Antibody IgM

HEV

Hepatitis E virus

LOD

Limit of detection

qRT-PCR

Quantitative reverse transcription polymerase chain reaction

RNA

Ribonucleic acid

Author contributions

HS study preparation, sample processing, manuscript preparation, and project administration; TG sample and data collection and proofreading of the manuscript; AK statistical analysis preparation and proofreading of the manuscript; AH sample processing; PK research funding support.

Funding

This study was supported by the Ministry of Health, Czech Republic – Conceptual Development of Research Organization (FNOs/2024) Grant/Award Number: 05/FNO-OR/2024.

Data availability

Data supporting the study findings are available from the corresponding author.

Declarations

Ethic approval and consent to participates

The study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of the University Hospital Ostrava (project number: 823/2023). Written informed consent to participate was not required, as the study involved a retrospective analysis of samples obtained during the standard medical assessment of deceased organ donors. According to Czech legislation (Act No. 285/2002 Coll., the Transplantation Act), the Czech Republic operates under a presumed consent system, meaning that unless individuals have registered their objection during their lifetime, they are considered potential organ donors. Therefore, the requirement for informed consent was waived in accordance with national regulations.

Consent for publication

All authors have perused the manuscript and consent to its publication in the journal.

Competing interests

The authors declare no competing interests.