1. Introduction

Hepatitis E Virus (HEV) is one of the leading causes of acute hepatitis in the world. HEV is a small icosahedral virus of the family Hepeviridae, which includes two genera Orthohepevirus and Piscihepevirus. The genus Orthohepevirus contains four species designated as Orthohepevirus A through D. Within Orthohepevirus A, 8 distinct mammalian genotypes (gt) have been identified to date (HEV 1-8) [1]. The gt 1 and 2 infect only humans and cause epidemics in developing countries in Africa and Asia, as well as in Mexico. They are primarily transmitted via the fecal-oral route due to fecal contamination of drinking water. Thus, it is related to the absence or failure of a safe water supply [2-3]. In contrast, gt 3 and gt 4 viruses have proven zoonotic potential and infect humans and several other animal species, such as pigs (Sus scrofa domesticus), wild boar (Sus scrofa scrofa), and deer (Cervidae) [4]. Zoonotic transmission of gt 3 or gt 4 occurs through consumption of undercooked meat or contact with infected animals [5]. Gt 5 and gt 6 infect wild boar, while gt 7 infects dromedaries (Camelus dromedarius). A recent study showed zoonotic transmission of genotype 7 related to consumption of camel meat. Gt 8 infects the Bactrian camel (Camelus bactrianus) [6]. HEV Infection is generally self-limiting in healthy human patients. Fecal-oral transmission is its primary route of transmission [7]. But recent work has reported that transmission of the virus is becoming increasingly zoonotic with different animal species (rats, deer, monkeys, and suids) as source of HEV [8]. The animal reservoir, primarily swine, plays a major role in this transmission. Food transmission by ingestion of raw or undercooked meat has been demonstrated by strain homology between the incriminating food and the infected subject and by case-control studies [2]. Occupational exposure is a risk factor; veterinarians, slaughterers/butchers, and forestry workers have shown a prevalence of HEV antibodies compared to individuals in other occupations [9-10]. Transmission of HEV within a herd occurs naturally through direct contact with HEV-infected animals or the feces of diseased animals, or through contaminated food or water sources [11]. Animals can acquire HEV virus at different times during their growing season. Most studies show that pigs become infected between 8 and 15 weeks of age, and some remain positive at slaughter [12]. In addition, 2-15% of pigs have been shown to be infected with HEV at slaughter [10]. Pork butchers may also be a confounding factor; it is likely that butchers live in less than optimal hygienic conditions, making them frequently exposed to HEV [13]. These data raise many concerns about the health of populations.

In Togo, to date, there is no information about HEV seroprevalence among pork butchers and in pigs. The objective of this study was to determine the seroprevalence of HEV in the pork butcher population of two regions of Northern Togo, Central and Kara, where pork production and consumption by the population are important, to identify risk factors associated with the infection that would contribute to its persistence in the population, and to determine the seroprevalence in slaughtered pigs intended for human consumption. This study provided the first mapping of HEV prevalence in the pork butchers and pig population in Togo.

2. Material and Methods

2.1 Study design and study site

From November 2021 to February 2022, a cross-sectional study was conducted and included 89 asymptomatic individuals in contact with pigs or pork meat (5 women and 84 men) and 289 individuals in general population (not in contact with pig) from Sokodé and 176 pigs slaughtered for consumption. The data from the general population (not in contact with pig) cohort analyzed in this study come from a previous serological survey conducted in Sokodé, Togo [14]. The study was approved by the Bioethics Committee for Health Research under No: 040/2019/CBRS, and furthermore the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as evidenced by the approval of the Ministry of Health and Public Hygiene (Togo) under N^o: 027/2020/MSHP/CAB/SG/DGAS/DPML/CBRS. An administrative authorization was also obtained from the Polyclinic and the Regional Blood Transfusion Center of Sokodé.

2.2 Study site, data collection and sampling

The central region with the capital city of Sokodé has a population of approximately 240 360 (INSEED, 2023) and is located in the center of Togo. The Kara region, located just above the central region, has the city of Kara as its capital, with a population of about 283 738 (INSEED, 2023). Located 320 km and 423 km from Lomé, respectively, the central region and Kara experience a single rainy season from April to October per year with a peak between July and September with a tropical climate. Participants (butchers) included in the research were face to face interviewed and their sera collected on site. Figure 1 shows the number of butchers and pigs tested by sample site. Demographic and background characteristics included were age, gender, area of residence, number of years in occupation, religion. We collected data regarding the practice of animal husbandry, wearing of work gloves, source of water use, times of hand washing, knowledge of hepatitis, having ever been screened or vaccinated against hepatitis (Table I). Five milliliters of blood were collected in a non-heparinized tube from each participant. The blood samples were processed according to the requirements of the Blood Bank Management Committee of the Regional Blood Transfusion Center (RBTC). The sample was centrifuged at 3000×g for 10 minutes at 25°C. Sera were collected in cryotubes and stored at -20 ± 5°C until serological analysis.

Figure 1: A. Map of the Kara region and B. Map of the Central region. Sampling site were red spot between Sotouboua up to Kouméa / Number of butchers (B), pigs (P) and general population (GP) tested by sample site

Table I: Sociodemographic characteristics of butchers

2.3 Serological analyses

A total of 89 serum samples collected from butchers were tested for the presence of HEV antibodies (IgM and IgG) with enzyme-linked immunosorbent assays (ELISA): HEV ELISA kit for HEV immunoglobulin G (IgG) and HEV IgM ELISA 3.0 kit for HEV immunoglobulin M (IgM) (MP Biomedicals Asia Pacific Pte Ltd, Singapore; formerly Genelabs Diagnostics Pte). 176 swine serum samples were tested with the enzyme-linked immunosorbent assay (ELISA): VHE ELISA 4.0 kit (For veterinary use only) for total HEV antibodies (IgG, IgM, and IgA) (MP Biomedicals Asia Pacific Pte Ltd,). The tests were performed according to the manufacturer's instructions. MP Diagnostic HEV ELISA 4.0 uses a proprietary recombinant antigen, which is highly conserved between different strains (12-14) of HEV, to detect the presence of specific antibodies, including IgG, IgM and IgA, to HEV. The MPD HEV IgM ELISA 3.0 and MPD HEV IgM ELISA 3.0 are indirect immunoassays that utilize a highly conserved conformational epitope encoded by the hepatitis E virus open reading frame 2 (ORF 2). The MPD HEV IgM ELISA 3.0 and MPD HEV ELISA have a sensitivity of 98% and a specificity of 96.7%. All three ELISAs calculate a result based on the signal-to-cut ratio (CO). Specimens with absorbance values less than the CO value are considered non-reactive, and those with absorbance values greater than or equal to the CO value are considered initially reactive. The cut-off value (CO) is the average absorbance value of the three negative controls multiplied by 0.20 for the MP Diagnostic HEV ELISA 4.0, by 0.40 for the MPD HEV IgM ELISA 3.0 and by 0.50 for the MPD HEV IgM ELISA respectively. Human samples were tested with the Rapid Diagnostic Test Bioline^TM Malaria Ag Plasmodium falciparum Histidine 2 Rich (HRP2) which has a sensitivity of 99.7% and a specificity of 99.5%, to identify false positives or cross-reactions with HEV antigens.

2.4 Statistical analysis

Data analysis was performed with SPSS version 21 software. Descriptive statistical analysis was used to calculate the mean, median, percentages of sociodemographic variables, percentage of seropositivity to anti-IgM and anti-IgG antibodies at the 95% confidence interval (95%CI). Statistical analysis was performed using the Chi-square test (x²). The test was considered significant at a p value < 0.05.

3. Results

3.2 Socio-demographic characteristics of study participants

A total of 89 butchers participated in this study, of which 84 (94.4%) were men and 5 (5.6%) were women. The mean age of the butchers was (32.3 ± 9.9SD). For men, it was (32.3 ± 9.9SD) with an age range of 16 to 65 years and for women, it was (33.4 ± 8.7SD) with an age range of 18 to 51 years. As for the general population of the 289 individuals who participated in the previous study [14], 118 (40.8%) were female and 171 (59.2%) were male. The age range was 17 to 51 years, with a mean age of 26.16 years (± 6.53SD) and a median of 24 years. 46 (51.7%) of the butchers had less than 5 years of experience and 46 (51.7%) had a high school level compared to 163 (56.4%) in the general population. 272 (94.1%) of the general population lived in urban areas compared to 74 (83.1%) of butchers who lived in rural areas. 3 (1%) butchers (not in contact with pork), were observed in the general population. Indeed, before starting their activity, the future butchers underwent a certain number of tests, including those for viral hepatitis, in order to practice freely. Of the butchers surveyed, only 29 (32.6%) had been tested for hepatitis B and/or C and only 16 (18%) had received the hepatitis B vaccine. Other types of hepatitis viruses were unknown or ignored. A total of 176 pigs were included in this study, all of which were of slaughter age (3 months and older). Table I presents the socio-demographic characteristics of the butchers.

3.1 HEV Seropositivity

The table II shows the seroprevalence of the different study populations. A total of 18 samples (20.22%) (95% CI: 19.33-21.10) of the 89 pork butchers were positive for IgM antibodies, indicating a recent HEV infection, and 5 samples (5.6%) (95% CI: 5.09-6.10) were positive for IgG antibodies, indicating a recovering or old infection. There were 2 double positive IgG+IgM+. Because the risk of IgG false positive associated to Malaria fever was found in much serological analysis. We also evaluate the Malaria status of the human population thus five samples (27.7%) (95% CI: 5.09-6.10) of the anti-IgM HEV positive were found to be positive for the Rapid Diagnostic Test Plasmodium falciparum versus none in the anti-IgG HEV positive lot.

Regarding detection in pigs 141/178 (80.1%) (95% CI: 79.66-80.55) were positive for total HEV antibodies.

Table II: Results of HEV total and M immunoglobulin detection in butchers / pigs

IC: Confidence interval, n: size, (-): Not calculated

Whatever was the geographical origin of the pigs, the HEV seroprevalence is the same (Table III), indicating that circulation of HEV is probably an old story but with some silence period as the youngest animals have already the very same prevalence as the oldest one. In Bohou there was a tendency to a prevalence increase with age that may signed a continuous virus circulation.

Table III: Seroprevalence of pigs (P) tested per sample site

3.3 Risk factors associated with HEV infection

Observation of the risk factor analysis shows that no factor was significantly associated with anti-HEV IgM in butchers (p ≥ 0.05). To avoid confounding factors with risk factors associated with infection, the 5 individuals positive for both anti-IgM HEV and plasmodium in butchers were excluded from the data. In the general population, 1% (3/289) of butchers who did not touch pigs or handle pig carcasses were observed and 37.0% (107/289) were involved in raising poultry, goats, cattle, dogs and cats. The ratio of anti-IgM VHE of butchers and the general population was (OR: 2) with p = 0.202. Tables IVa and IVb present successively the risk factors and behavioral risk factors associated with anti-IgM HEV infection in both the general population and butchers and those associated with anti-IgG HEV infection in butchers.

Table IVa: Anti-HEV immunoglobulins associated with risk factors

Table IVb: Anti-HEV immunoglobulins associated with comportemental risk factors

*Significant (p<0.005), n: size, (-): Not calculated

a: Comparison of butchers’ exposure to Anti-IgM VHE+ and the risk factor

b: Comparison of butchers’ exposure to Anti-IgG VHE+ and the risk factor

c: Comparison of general population exposure to Anti-IgM VHE+ and the risk factor

4. Discussion

In previous studies in Togo, it was established evidence of the diffusion of HEV in the general human [14]. Although contamination of drinking water is the main source of virus spread, numerous studies have shown that zoonotic transmission is also one of the sources of HEV spread. Pork meat, which is highly appreciated by consumers because of its taste, is increasingly becoming part of the eating habits of most Togolese, especially those living in the northern part of the country. Pork butchers find it a great opportunity to increase their income. Unfortunately, the activity is unstructured and poorly organized, so that pigs are slaughtered in makeshift slaughterhouses without any health regulations. We observed that the pork butchery attracts mostly males, 84 (94.4%) who reside in the outskirts of towns 74 (83.1%) with a secondary education level 46 (51.7%). This result was similar to that reported in Benin where 92.8% were male [15] and in Chad where 77% were educated [16]. These data suggest that, like any activity requiring a great deal of physical effort, men were the most solicited and the essential link in this economic activity.

In the previous study, the anti-IgM and IgG seroprevalence of HEV in the general population in 2023 was 11.7% and 5.6% respectively. In the present study, the anti-IgM HEV seroprevalence was 20.22% with ratio (OR: 2; p= 0.202), and that of IgG was 5.6%. These data suggested that HEV circulates more actively in this population group of swine butchers and they had twice the chance of contracting the virus. These results could be explained by the lack of hygiene around this activity, handling meat, organs and feces of pigs without protection. The high IgM to IgG ratio in these studies may suggest that there are several foci of transmission with a higher frequency of new infections or that those tested had associated autoimmune diseases or had malaria at the time of testing with a possible interaction. The difference could be due to the high sensitivity of the anti IgM kits, the versions of the tests to detect low concentrations of IgG, but also to differences in the type of IgG antibodies present in the sera [17]. Five (5; 27.7%) were positive for antibodies to Plasmodium falciparum out of 18 positives for HEV anti-IgM. As some studies have shown, antibodies to Plasmodium Ag may interact nonspecifically with antigens of other agents such as HEV that could test positive, resulting in false positives to anti HEV. Thus, this false positive rate could abnormally increase the seroprevalence of HEV immunoglobulin. Certainly, a study with a higher number of samples could help to understand this difference. In Burkina Traoré et al. (2015) reported a similar seroprevalence which was 1% for anti-IgM and 76% for total anti-HEV antibodies in the population of pig butchers. This reflects that HEV is endemic and circulating in this population. The seropositivity for gender was 20.2% (95% CI: 19.4 - 21.0) in men and 20.0% (95% CI: 6.0 - 34.0) in women, this result translates that they were exposed to the same source of contamination and were unaware of the hygiene rules. Many studies suggest that zoonotic transmission of HEV may occur through frequent contact with biological samples (feces, blood) and organs of HEV-infected animals [18]. In agreement with many similar studies in the world, we have observed high seroprevalences in pork butchers who have direct contact with pigs or pork meat [19-21]. Comparing this study to other studies conducted in other countries of the sub-region, such as Burkina Faso [9], the seroprevalence observed among butchers was higher than that observed in the general population. The data from these studies suggested that zoonotic contamination could be one of the main sources of HEV infection; however, our data suggest that in Togo, in addition to the zoonotic source of contamination, there are other reservoirs of contamination that are totally unknown and that fuel the spread of infection. Although in this study no risk factors such as gender, age, education, area of residence, number of years in business, husbandry practices and hygiene were associated with anti-IgM and IgG HEV seroprevalences, studies have reported that they may constitute significant risks in the contamination and spread of HEV [22]. In addition, the seroprevalence of total anti-HEV antibodies observed in pigs at threshing age was 80.11%. This result suggests that HEV is endemic in pig populations in Togo. This result was similar to those observed in Ghana 62.4% [23] and Burkina 80% [9], which could be a source of contamination and spread of HEV for professionals in the sector [10,22]. In addition, the study revealed similar seroprevalences according to the age of the pigs which were 80%, 78% and 82% respectively in 3-6 months, 6-12 months and over 12 months of age. This suggests that, pigs at slaughter age could be at the same risk of HEV contamination.

Limitations of this study

This study has limitations. The study did not allow the detection of HEV RNA, and the characterization of HEV genotypes circulating in the pig and pork butcher population was not performed. The genotypic characterization of HEV to be determined in pigs and humans will provide a more accurate picture of the zoonotic transmission of this virus.

Conclusion

This study showed the presence of HEV antibodies in the serum samples from butchers and pigs slaughtered for consumption, and could reflect that they were in contact with HEV. The relatively low IgG seroprevalence in the butchers reflects a very low endemic status of the infection that is not different from the general population which is in contrast to the observed situation in Burkina. We did not know if this was might be due to a lack of sensitivity or the current test compared to the old test used in Burkina by our group. Whatever the case, however, the high seroprevalence of IgM might reflects a major risk of HEV transmission in this population and a largely changing status. In addition, the high seroprevalence of total antibodies observed in pigs confirms their HEV reservoir characteristics and their potential role as a source of contamination and virus propagation. Further studies are needed to characterize the circulating genotypes in pork butchers and pigs in order to establish the link between them and to identify other factors fueling the HEV status in the human population.

Consent to Publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.

Funding

This study was financed by the Directorate of Scholarships and Training of the Togolese State.

Author Contributions

KVMS, KAT, JBO, ET, PR, SDK, and NB participated in the study design. KVMV, BKN, JA, GK and KAT performed serological analyses, KVM, KAT, BLO, and PR performed the statistical analysis, and all authors participated in drafting and revising the manuscript. All authors read and approved the final version.

Acknowledgements

Not applicable.

Ethical Approval

The study was approved by the Bioethics Committee for Health Research of the Ministry of Health and Public Hygiene of Togo under No.: 040/2019/CBRS, and furthermore the study protocol complies with the ethical guidelines of the 1975 Declaration of Helsinki, as evidenced by the approval of the Ministry of Health and Public Hygiene (Togo) under No.: 027/2020/MSHP/CAB/SG/DGAS/DPML/CBRS. The administrative authorization was also obtained from the Polyclinic and the Regional Blood Transfusion Center of Sokodé. Informed consent was obtained from all study participants after they were informed of the project.

Availability of Data and Materials

Not applicable.

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