Seroprevalence of Hepatitis E Virus Infection among Blood Donors in Bulgaria

Hepatitis E virus (HEV) infection is widespread among domestic pigs, industrial swine, and wild boars in Bulgaria. The aim of the current research was to present the HEV seroprevalence among blood donors in Bulgaria. In the present study, 555 blood donors (479 males and 76 females) were enrolled from five districts in the country (Shumen, Pleven, Stara Zagora, Plovdiv, and Sofia districts). All blood samples were tested for anti-HEV IgG using the recomWell HEV IgG ELISA test (Mikrogen GmbH, Neuried, Germany). Each participating donor completed a short, structured, and specific questionnaire to document data on the current study. Anti-HEV IgG positive results were detected in 144 (25.9%) blood donors, including 129 (26.9%) males and 15 (19.7%) females. The established HEV seropositivity was 28.8% (23/80) in Shumen district, 23.2% (22/95) in Pleven district, 27.1% (38/140) in Stara Zagora district, 27.5% (44/160) in Plovdiv district, and 21.3% (17/80) in Sofia district. A high HEV seroprevalence was found for persons who declared that they were general hunters (48.7%; 19/39; p = 0.001) and hunters of wild boars (51.6%; 16/31; p = 0.001). We present the first seroprevalence rates of HEV infection in blood donors from Bulgaria. The results of our research showed high HEV seropositivity among blood donors.

According to the World Health Organization (WHO) database, every year, there are approximately 20 million human HEV infections worldwide, leading to approximately 3.3 million symptomatic cases of HEV [10]. In this regard, WHO estimates that HEV caused 44,000 cases of lethal outcome in 2015 (accounting for 3.3% of the mortality due to viral hepatitis) [10].
The first HEV human cases were described in Bulgaria in 1995 [11]. Then, Teoharov et al. reported acute HEV infection in four cases in 53 tested patients [11]. For a period of about 20 years, there was a lack of research on HEV infection in humans and animals in the country. Since 2014, different studies on HEV infection in animals and humans have begun. Several studies have focused on hospitalized patients [12][13][14], outpatients [15], the general population [16,17], and hemodialysis patients [18]. Bulgarian researchers, in collaboration with Italian scientists, performed phylogenetic analysis of HEV isolated from people from Bulgaria [19,20]. In the last few years, the spread of HEV infection in animals in Bulgaria has been studied. Several seroprevalence studies on industrial pigs, domestic pigs, and wild boars have shown a high prevalence of HEV infection among animals in Bulgaria [21][22][23][24][25]. The established HEV seropositivity among industrial pigs varies from 40.0% to 60.3% [21][22][23], in domestic pigs-60.05% [24], and among wild boars-from 12.5% to 40.8% [24,25]. The prevalence of HEV in the only aboriginal pig breed (East Balkan swine) in Bulgaria was evaluated, and a high HEV seropositivity of 82.5% was found [26].
There is no available information on the HEV seroprevalence in Bulgarian blood donors. In this regard, we aimed to assess the current HEV seroprevalence rate among blood donors in Bulgaria. The present research gives new insights intothe epidemiology of HEV in one country from Southeastern Europe.

Materials and Methods
The current study was performed in accordance with the ethical principles of the Declaration of Helsinki (June 1964, last revision in October 2013). Blood samples were processed in accordance with the requirements of the Ministry of Health, Sofia, Bulgaria and complied with the good medical practices of the medical specialty "Transfusion Hematology" (part of the medical specialties in the Republic of Bulgaria). Informed consent was obtained from all blood donors included in the research. Information and approval documents were printed and signed by the blood donor and medical staff (physician or nurse). These documents were given to the medical staff and such documents are kept for 1 year. Medical staff checked that all the required documentation was complete and consent forms were signed before testing. Samples were discarded if the documents were incomplete or unsigned.
The Bulgarian territory is divided into 28 administrative districts. Five hundred and fifty-five blood samples were collected from non-remunerated blood donor voluntaries (n = 555) enrolled from five districts ( Figure 1) [27]. The blood samples were collected from 1 June 2020 to 31 October 2020. The collection of blood samples was carried out through blood donation campaigns in the five districts of Bulgaria mentioned above.  [27]. The blood samples were collected from 1 June 2020 to 31 October 2020. The collection of blood samples was carried out through blood donation campaigns in the five districts of Bulgaria mentioned above. Each participating donor completed a short, structured, and specific questionnaire to document data on the current study. The questionnaire contained information about demographics and baseline characteristics (sex, age, level of education, and area of residence), contact with animals (raising a domestic pig), the consumption of meat (pork meat, and meat from wild animals), derived meat products (pork sausage, salami, etc.), the consumption of seafood, hunting in nature (general hunting and hunting of wild boars). The questionnaire was validated by the Infectious Disease specialist and Transfusion Hematology specialist.
All samples were tested for anti-HEV IgG using recomWell HEV IgG ELISA kits (Mikrogen GmbH, Neuried, Germany). The recomWell HEV IgG is a qualitative and/or quantitative in vitro test employed for the detection and reliable identification of IgG antibodies to HEV in human serum or plasma. The recomWell HEV IgG is a screening test based on the principle of an indirect sandwich ELISA. In qualitative evaluation, the cutoff (limit) is the arithmetic mean that is calculated from the extinction values of both cutoff controls (at the beginning and end of the series). In quantitative evaluation, the corresponding antibody activity in units per mL is assigned to the extinction values using a formula. The measurement units U/mL are arbitrary units, which do not allow conclusions concerning (international) reference values. The recomWell HEV IgG ELISA test has a 98.9% sensitivity and 98.5% specificity.
All the data derived from the questionnaire were compared by the Chi-square test and T-test. The results on the seroprevalence of HEV infection in blood donors by district, part of the country, and region were compared by Fisher's exact test and the Chi-square test. Binary logistic regression was used to evaluate the risk of positive results according Each participating donor completed a short, structured, and specific questionnaire to document data on the current study. The questionnaire contained information about demographics and baseline characteristics (sex, age, level of education, and area of residence), contact with animals (raising a domestic pig), the consumption of meat (pork meat, and meat from wild animals), derived meat products (pork sausage, salami, etc.), the consumption of seafood, hunting in nature (general hunting and hunting of wild boars). The questionnaire was validated by the Infectious Disease specialist and Transfusion Hematology specialist.
All samples were tested for anti-HEV IgG using recomWell HEV IgG ELISA kits (Mikrogen GmbH, Neuried, Germany). The recomWell HEV IgG is a qualitative and/or quantitative in vitro test employed for the detection and reliable identification of IgG antibodies to HEV in human serum or plasma. The recomWell HEV IgG is a screening test based on the principle of an indirect sandwich ELISA. In qualitative evaluation, the cutoff (limit) is the arithmetic mean that is calculated from the extinction values of both cutoff controls (at the beginning and end of the series). In quantitative evaluation, the corresponding antibody activity in units per mL is assigned to the extinction values using a formula. The measurement units U/mL are arbitrary units, which do not allow conclusions concerning (international) reference values. The recomWell HEV IgG ELISA test has a 98.9% sensitivity and 98.5% specificity.
All the data derived from the questionnaire were compared by the Chi-square test and T-test. The results on the seroprevalence of HEV infection in blood donors by district, part of the country, and region were compared by Fisher's exact test and the Chi-square test. Binary logistic regression was used to evaluate the risk of positive results according to the part of the country and region. Statistical analysis was performed by MS Excel 2007 (Washington, DC, USA) and IBM SPSS Statistics version 21.0 (New York, NY, USA). A p-value < 0.05 was considered statistically significant.

Results
The overall mean age, mean male age, and mean female age were 37.2 ± 8.0 years (95% CI: 35.5-38.9), 37.1 ± 7.9 years (95% CI: 35.3-38.9), and 37.8 ± 8.8 years (95% CI: 32.6-43.0), respectively. The male sex dominated among analyzed blood donors (sex ratio: male/female = 1/0.13). People who had a low/intermediate level of education were 22.8% more frequently reported than participants who had high level of education (Table 1). In the group with a high level of education, men represented 78.0% (167/214) and women represented 22.0% (47/214) of the group, whilst in the group with a low/intermediate level of education, men represented 91.4% (312/341) and women represented 8.6% (29/341) of the group. The number of blood donors from a city was nearly nine times higher than that of blood donors from a village. The highest levels of anti-HEV IgG positive results were found in Shumen district, and the lowest were recorded for Sofia county ( Table 2). The distribution of HEV positive data in different age groups in the studied districts varied in different ranges. The mean HEV seropositivity in those aged 18-29 years old was 18.2% (95% CI: 11.0-25.4), the highest was found in Shumen county (32.0%), and the lowest was recorded in Sofia district (6.2%). The highest HEV seroprevalence in the age group 30-39 years was estimated in Pleven district (27.7%), the lowest was recorded in Sofia county (14.8%), and the overall percentage for all districts was 23.4% (95% CI: 19.5-27.3). The mean HEV seropositivity in those aged 40-49 years old was 27.2% (95% CI: 22.5-31.9), the highest was in found in Plovdiv county (35.8%), and the lowest was recorded in Sofia district (20.6%). The highest HEV positive results in older people (≥50 years) were calculated in Sofia county (54.4%), the lowest were recorded in Pleven district (0.0%), and the overall mean for all districts was 32.2% (95% CI: 16.6-47.8). To estimate the risk for HEV seropositivity, the odds ratio (OR) in different parts of the country and regions was performed by binary logistic regression. The OR of anti-HEV antibody occurrence in Southern Bulgaria was determined in comparison to that in Northern Bulgaria (Table 3). We found that the odds of HEV infection were 1.018 times higher in Southern Bulgaria than in Northern Bulgaria. We estimated that the odds of HEV infection were 1.117 times higher in the North-Central region, 1.394 times higher in the South-Central region, and 1.495 times higher in the Northeastern region than in the Southwestern region. The review of the questionnaire showed different results (Table 4). We did not find significant interactions between anti-HEV IgG positive results and the consumption of pork meat (p = 0.213); pork sausage, salami, etc. (p = 0.303); and seafood (p = 0.598). The breeding of domestic pigs was not found to be a significant factor for HEV seropositive among blood donors. A significant interaction was estimated between people who declared the consumption of meat from wild animals and anti-HEV IgG antibodies (37.8%; 45/119; p = 0.001). The statistical analysis showed significant results between general hunting and HEV seropositivity, as 48.7% (19/39; p = 0.001) of the general hunters had anti-HEV IgG positive results. Among hunters who declared that they go wild boar hunting, 51.6% (16/31; p = 0.001) of them were anti-HEV IgG positive participants.
In other countries of the Mediterranean Basin, the prevalence of HEV infection was similar to that of the Balkan Peninsula (excluding France, where a much higher seropositivity was established). In Italy, a positivity of 8.7% (869/10,011; 95% CI: 8.14-9.25) [35], 9.1% (12/132) [36], and 9.8%/10.2%/17.4% (in various ELISA tests in one research) [37] [56,57], France (22.4%) [38], and Thailand (29.7%) [58]. The data from our study showed a high HEV seropositivity among blood donors from Bulgaria. We think that this could be influenced by various factors. One of the possible reasons for the result is the high HEV seropositivity among animals in the country. A high HEV prevalence has been observed among domestic pigs (60.05%) [24], industrial swine (40.0% and 60.3%) [21,23], wild boars (40.8%) [25], and East Balkan swine (aboriginal pig breed) (82.5%) [26] in Bulgaria. Consequently, meat and subproducts (sausage, salami, etc.) from these animals could be HEV-contaminated. The subsequent probable food-borne pathway may infect humans. This is one of the potentially possible explanations for our result. In this regard, we observed that a significant risk factor for HEV infection was the consumption of game meat (p = 0.001). Other authors have also reported that the consumption of raw and undercooked meat and meat products was a risk factor for HEV infection. Mansuy et al. found that HEV infection was more frequently observed in individuals eating pork meat, raw pork liver sausages (incl. figatelli), game meat, offal, and oysters [38]. Capai et al. reported that the highest risk for infection was the consumption of figatellu, pork meat, fittonu, sausages and pates, offal, liver, and wild berries [39]. Mooij et al. established that there was a direct connection between anti-HEV IgG seropositivity and eating traditional Dutch dry raw sausages (cervelaat, fijnkost, salametti, and salami), other dry sausages, smoked beef, steak, and raspberries among Dutch blood donors [53]. Among Nepalese blood donors, Shrestha et al. noted that pork consumption increased the risk for HEV infection [59].
Another possible reason for the high HEV seroprevalence among blood donors in Bulgaria (25.9%) could be the applied diagnostic ELISA test. Theoretically, it is possible, but we think that, in our study, this was unlikely, because the applied recomWell HEV IgG Mikrogen test a had high sensitivity (98.9%) and specificity (98.5%).
Regarding the HEV gender prevalence, we did not find a statistically significant sex difference in our study (p = 0.184). However, a review of the scientific literature showed that the HEV seropositivity was higher in males than in females [35,38,[60][61][62].
In the present research, we observed that the highest HEV seropositivity was estimated among adults (40-49 years) and the lowest was found in younger participants (18-29 years [66]. The present study has some limitations that need to be addressed. Only the ELISA test was used for the detection of HEV IgG antibodies, without a confirmation test, and a confirmatory immunoblot test was not performed (including HEV RNA testing). In this regard, the results and conclusions should be interpreted with caution. Furthermore, the current research included several counties out of a total of 28 districts in the country, so it was not a nationwide survey. Despite these limitations, this work represents the first research on the anti-HEV IgG prevalence among blood donors in Bulgaria, enriching the knowledge on this infection for one country from Southeastern Europe.

Conclusions
We have presented the first seroprevalence rates of HEV infection in blood donors from Bulgaria. The results of the current study and those from previous investigations among animals show a high seroprevalence of HEV in the country. We think that this is probably linked to the autochthonous spread of HEV in the country, associated with the zoonotic characteristic of the virus. This hypothesis must be verified by future research on the molecular analysis of HEV in animals, humans, and food. It would be helpful to develop an information campaign to enrich people's knowledge on HEV infection.