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Seroprevalce, risk factors and zoonotic potential of HEV were studied.
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HEV antibodies occurs at high prevalence (30.9%) overall in Jordan.
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HEV infection associates with age and eating undercooked meat.
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Owning camels increased the odds of HEV seropositivity.
Abstract
Objectives
Hepatitis E virus (HEV) is hyperendemic in many countries, but data on this virus are not available in Jordan. This study determined the seroprevalence, risk factors and zoonotic potential of HEV in a Jordanian population.
Methods
A total of 450 sera samples from 8 different governorates were tested for HEV-IgG. A pre-tested and validated questionnaire was used to collect risk factor data including animal interaction and environmental exposures.
Results
The overall seroprevalence was 30.9%. Eating undercooked meat was significantly associated with HEV seropositivity (OR = 2.06, 95%CI 1.04–4.06) after controlling for age, gender, travel history and source of water. Age was also associated with HEV seropositivity; the youngest (≤14 years of age) and oldest age groups (60 and 80 years of age) had the highest prevalence (45.5% and 53.2%, respectively), compared to those between 20 to 29 years of age and 30 to 39 years of age (20.2 and 15.2%, respectively), although the small sample size among the youngest group tempers this association. There was evidence of a marginal association between owning camels and an increased odds of HEV seropositivity. Place of residence and source of drinking water were not associated with infection.
Conclusion
This is the first study to report HEV seroprevalence in Jordan and shows that HEV exposure is high in Jordan. Surveillance for acute and chronic Hepatitis E is needed to estimate the frequency of the actual disease.
Hepatitis E virus (HEV) is a single-strand RNA virus classified in the genus Hepevirus, family Hepeviridae and can lead to acute disease with symptoms ranging from subclinical (where patients clear the virus rapidly) to fulminant hepatitis (symptoms can include fever, anorexia, vomiting, nausea, myalgia and jaundice) (
). HEV-1 and HEV-2 primarily infect humans via the fecal-oral route and have been the main source of large waterborne HEV outbreaks in low- and middle-income countries (LMICs) (
). HEV-3 (in Europe and United States) and HEV-4 (in China, Japan and Southern Europe) outbreaks in humans have been associated with zoonotic exposure, including pork product consumption and handling (
). In addition, HEV has been detected in swine in the US, Japan, Madagascar, and several European countries such as Spain, Germany and the Netherlands (
Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States.
). Although the virus has not been isolated from ruminants, serological studies in the United States, Egypt, India, China and Spain have detected anti-HEV antibodies in these animals (
Sporadic cases and clusters of hepatitis E have been reported with increasing frequency in several European countries including the United Kingdom, Denmark, Spain, France, the Netherlands, Hungary, Germany and Norway (
). In MENA, hepatitis E accounts for a variable proportion (20–60%) of acute hepatitis cases, and seroprevalence of anti-HEV antibodies has been up to 20%, with Egypt reporting up to 80% (
). Despite the growing global threat of HEV, its existence in neighboring countries and the possible link to animal and environmental exposures, the epidemiology of HEV and risk factors remain unknown in Jordan. Thus, this study aimed to explore the seroprevalence of anti-HEV antibodies and associated risk factors in Jordan.
Materials and methods
Study design and population
Jordan is an upper middle-income country located in the MENA region with a population of 7.6 million, and a life expectancy of 74 years (
). For this cross-sectional study we analyzed 450 blood samples collected during a cross-sectional zoonotic disease study conducted in Jordan between November 2015 to May 2016 (the parent study).
Setting
Between two to six governmental health centers per each of the eight governorates were included in the study. Centers were selected randomly from the Ministry of Health directory. All blood samples were collected by registered nurses and medical professionals. Sera were stored at the health care centers at −20 °C until shipment to the Food Safety and Zoonotic Diseases Laboratory at Jordan University of Science and Technology (JUST).
Participants
In Jordan, patients are often accompanied by their relatives to medical examinations and testing. Registered nurses or medical professionals approached every relative during their visit to the health centers’ laboratory for participation in the study. All participants were briefed about the study objectives and the voluntary nature of the study.
Risk factors data collection
A pre-tested and validated questionnaire written in Arabic was developed to collect demographic and risk factor data including food consumption habits, travel history and animal exposure. The questionnaire was pretested among 20 participating and 20 non-participating individuals and revisions were made on the final questionnaire. The questionnaire was self-administered.
Sample size
Since there were no data on the prevalence of HEV in Jordan, we calculated a sample size based on a prevalence rate of 50% (requiring the largest sample size) and with a precision of 5%. The minimum required sample size needed was 384, and we tested 450 samples. The tested samples were randomly selected from the serabank to avoid any bias.
Ethical considerations
The Institutional Research Bioethics Committee of JUST granted approval for this study (IRB policy # 7601). Samples collection was also approved by the IRB committee of the Jordanian Ministry of Health to allow access the governmental health center. Signed informed consent for all adult participants and parental consent for children were obtained before collecting data. No names or other identifiers were collected. Data were available to the research team only.
Laboratory tests
Sera were analyzed using HEV-IgG ELISA and the results were interpreted according to the manufacturer’s instructions. This ELISA kit (Fortress Diagnostics) employs a solid phase, indirect ELISA method and uses a long recombinant protein (PE2) of a Chinese strain that belongs to genotype 1. The sensitivity of this kit was 95% in samples from patients with PCR-confirmed HEV infection (
). The kit is identical to the Wantai assay, which is widely used in HEV seroprevalence studies (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, Beijing, China) (
Performance of hepatitis E virus (HEV)-antibody tests: a comparative analysis based on samples from individuals with direct contact to domestic pigs or wild boar in Germany.
). The specificity for the Wantai ELISA was evaluated in 9000 individuals and was 98.6% according to the package insert. The test results of each plate were validated separately by verifying the quality control criteria as described by the manufacturer.
Statistical analysis
The test results and questionnaire data were entered into Microsoft Excel and analyzed using Stata version 12.1 (StataCorp LLC, College Station, TX, USA). Frequency distributions were examined to assess the distribution of the data. Simple descriptive statistics, χ2 and t-test statistics were performed where appropriate. Bivariate analyses were conducted to assess associations between HEV seropositivity and independent variables collected (gender, age, education, occupation, rural or urban residence, type of house, animal exposure, travel history, consumption of raw meats or milk). Variables associated with the outcome at a p-value of 0.05 or less and variables reported in the literature to be important were included in the final model.
Results
Table 1 shows the distribution of hepatitis E virus (HEV) seropositivity across the main independent variables. Overall 30.9% of individuals sampled were positive for HEV antibodies and this did not differ by region (Figure 1). HEV seroprevalence was higher among males at 35.5% compared to females but this was not statistically significant. Seroprevalence varied by age group; the oldest age groups (those between 60 and 80 years of age) had the highest prevalence (53.2%), while those between 20 to 29 years of age and 30 to 39 years of age had significantly lower HEV seropositivity in comparison (20.2 and 15.2%, respectively). Although the sample size was small (n = 11), the youngest participants had a significantly higher seroprevelance at 45.5% compared to those 20 to 39 years of age. Table 1 also shows unadjusted (univariate analysis) and adjusted odds ratios (multivariate analysis) for being HEV seropositive. In the univariate analysis individuals with any education had a significantly lower odds of HEV seroprevalence compared with those who had no education (UOR = 0.51, p = 0.003), while those who had ever lived abroad had a significantly greater odds of being HEV seropositive (UOR = 1.79, p = 0.04) but these associations were not significant in the multivariate analysis. Eating undercooked meat was significantly associated with HEV seropositivity in both the univariate and multivariate analysis (AOR = 2.06, 95%CI 1.04–4.06 for the latter). Water source was not significantly associated with the outcome in both the univariate and multivariate analyses.
Table 1Prevalence of anti-Hepatitis E IgG, unadjusted and adjusted odds ratios (OR) for HEV in Jordan, 2015–2016.
Table 2 presents the unadjusted odds ratios between ownership of animals and growing vegetables and other related variables. Owning camels was associated with a greater odds of HEV seropositivity (UOR = 3.04, p = 0.15) although this was not significant. None of the other variables were associated with HEV seropositivity.
Table 2Zoonotic and environmental risk factors for anti-Hepatitis E IgG seropositivity and unadjusted odds ratios, Jordan, 2015–2016.
This study of HEV seroprevalence in Jordan found that 30.9% of those sampled were positive. High seroprevalence was reported in Nepal (47.1%), Bangladesh (49.8%) and Southwest France (34.0%) (
). High seroprevalence was also reported in the general population of France (31.9%), the Netherlands (27.0%), Switzerland (21.2%), Germany (29.5%) and Denmark (19.8%), meanwhile less than 15% seroprevalence was reported in Austria, UK, Italy and Czech Republic (
). All of these reported seroprevalence studies used the Wantai test. In addition to being the first such study in Jordan, this study has several other important characteristics; it includes data on animal and environmental exposures and sampled individuals throughout Jordan.
In the univariate analysis, males had greater odds of HEV seropositivity but this was not significant in the multivariate analysis. Several recent studies reported no differences in infection rates by gender (
). Statistically significant differences were observed between HEV seropositivity and age in both the univariate and multivariate analyses. When including age as a continuous variable in the model, the odds of HEV seropositivity are significantly greater. However, categorizing age unmasks that the youngest (those 6–14 years of age inclusive) and older age groups (50–80 years inclusive) had significantly greater odds of HEV seropositivity compared to those 15–19 years old. Several studies reported an increased odds of HEV seropositivity with increasing age (
), however most do not include children. The studies that have included children found lower HEV seroprevalence, with few exceptions in India and Egypt, that increased with age (
). We found a high prevalence of HEV seroprevalence among children between 6 to 14 years of age (the youngest positive child was 10 years of age). However, our results should be interpreted with caution since there were only 11 participants in the youngest age group. The steady increase in seroprevalence among participants between 15 to 80 years of age is generally consistent with previous research (
). More research is needed to better understand the implications of HEV seropositivity over the lifespan and its relationship to multiple exposures and infection events.
We report that eating undercooked meat appears to be significantly associated with an increased odds of HEV seropositivity (OR = 2.06, 95% CI 1.04–4.06). Previous studies have found a similar association (
). Future studies that capture detailed dietary history are recommended to identify food-borne risks. We also examined the association between animal ownership and HEV seropositivity and found that owning camels is associated with an increased odds of HEV seropositivity. Although this was not statistically significant and the number of participants who owned camels was small (n = 7), it does warrant further investigation in light of the recent discovery of HEV-7 (
Additionally, the cross-sectional nature of this data limits the interpretation of the results since only associations are reported. Despite these, this study is useful for providing a first look into this epidemiology of HEV in Jordan. Genotyping studies are important to better understand the epidemiology of HEV in Jordan, and a detailed history of environmental and animal exposures are also needed.
Conflict of interest
We declare no competing interests.
Acknowledgments
This research was supported by the Deanship of Research at Jordan University of Science and Technology. We acknowledge Alaa E. Bani Salman for her support in implementing this study.
References
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The global prevalence of hepatitis E virus infection and susceptibility: a systematic review.
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Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States.
Performance of hepatitis E virus (HEV)-antibody tests: a comparative analysis based on samples from individuals with direct contact to domestic pigs or wild boar in Germany.
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