HEV prevalence and potential risk factors in a large multi-ethnic youth cohort in China

This cohort study was designed to investigate the prevalence of and potential risk factors of HEV infection in a large multi-ethnic youth cohort in China. Blood samples were collected from participants (n = 6269) and serum was isolated. All serum samples were tested for anti-HEV IgG, anti-HEV IgM antibodies using commercial enzyme immunoassay kits (Wantai Biological Pharmacy Enterprise, Beijing, China). The overall rate of anti-HEV IgG and anti-HEV IgM prevalence was 4.78% and 0.14%, 0.03% were positive for both anti-HEV IgG and anti-HEV IgM antibodies. Anti-HEV IgG positivity is significantly higher in females (5.27%) compared to males (4.14%) (P = 0.028). Anti-HEV IgG prevalence is significantly (P = 0.0001) higher in Dong (17.57%), Miao (12.23%), Yi (11.04%), Gelao (9.76%), and Bai (10.00%) compared to other ethnic groups. It is significantly higher in Guizhou (11.4%), Sichuan (10.1%), Yunnan (9.3%), and Guangxi (6.9%) than that other province. We found that ethnicity and provincial background are significantly associated with HEV infection in this cohort. This study provides comprehensive information on HEV prevalence in multi-ethnic populations in China. However, our study only focused on a youth population from different provinces of China. Future studies are recommended to investigate HEV prevalence in other age groups of the ethnic populations.


Background
Hepatitis E, caused by the Hepatitis E virus (HEV) has emerged as an important health concern in both developed and developing countries [1]. In general, HEV infection is asymptomatic and self-limiting [2,3], but it can also cause severe diseases in specific populations, including pregnant women, immunocompromised patients, and patients with underlying liver diseases [4][5][6]. Globally, it is responsible for approximately 20 million new cases, over 3 million acute hepatitis cases, and 70,000 fatalities annually [7,8]. The HEV outbreaks were mainly common in developing countries compared to developed countries and its prevalence significantly varies based on ethnicity, socioeconomic conditions, food habits, water quality, sanitation and, geographic origin [9,10].
The People's Republic of China is the leading populated country in the world, is comprised of 56 different ethnic groups with different dietary habits and living lifestyle. Therefore, different Chinese populations are prone to many food-borne infectious diseases, such as HEV infection. According to recent studies, the overall sero-prevalence of anti-HEV immunoglobulin G (IgG) among the general Chinese people's ranges from 11-72% [7,9,11], and that of IgM is 1.8% [9]. However, HEV prevalence among different ethnic populations at the national level Open Access *Correspondence: znbalooch@yahoo.com † Huixia Li, Yinxia Zhang, and Zhongren Ma have contributed equally and should be consider as first authors 2 Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China Full list of author information is available at the end of the article has not been comprehensively studied. The majority of the ethnic populations are living in less developed areas in isolated communities with relatively poor sanitation, unhygienic food practices, socioeconomic condition, and lack of health facilities for prevention, diagnosis, and treatment of infectious diseases. Therefore, we designed this cohort study to investigate the sero-prevalence of IgG and IgM in large multi-ethnic Chinese youth and to understand potential risk factors.

Study design
We conducted this large multi-ethnic youth cohort study among the freshly enrolled students of Northwest Minzu University, a university dedicating to high education for ethnic populations in China. 5 ml of blood was taken in sterile syringes from 6269 students and was immediately transported to the laboratory for further processing. In addition to sample collection, we used structured questionnaire forms to document participants' demographic characteristics such as age, sex, ethnicity, area, political status, major subject, college, City, province. The serum samples were stored at −80 °C, until further testing. In this study, all participants were above 16 > year's age and never get HEV vaccination before.

Serological tests
Serum samples were tested for the presence of anti-HEV IgG and IgM antibodies using commercial-available enzyme immunoassay kits (Wantai Biological Pharmacy Enterprise, Beijing, China) according to the manufacturer's instructions. Samples were tested in duplicate with cutoff values for IgG and IgM assays set at 0.22 and 0.357, respectively, which were determined based on the mean optical density 450 values from the negative controls (6 standard deviations). Samples with OD greater than or equal to the cutoff value were considered as positive.

Statistical analysis
The data was analyzed using SPSS version 20.0 (Chicago, IL, USA). Proportions were estimated with the 95% confidence interval (CI). χ 2 test or Fisher's exact test and Mantel-extension test for trend were performed to evaluate the difference in the prevalence of viral markers among sex, age, residential, and occupational groups. Univariate analysis using the χ 2 test or Fisher's exact test and multivariate logistic regression analysis was performed to identify potential risk factors for HEV infection by calculating odds ratios (ORs) and 95% CI. For all analyses, P-value < 0.05 was considered statistically significant.

Potential risk factors for HEV infection
To investigate the potential risk factors, only the samples positive for anti-HEV IgG were analyzed, which indicates past infection. Samples positive for IgM or both IgG and IgM, which indicates recent/ongoing infection, were not included because of the limited number. Univariate analysis revealed that sex, ethnic, and provincial backgrounds were significantly associated with a high rate of anti-HEV IgG antibody positivity. Interestingly, the multivariate analysis also indicated that ethnic and provincial background remained independent risk factors for the high rate of anti-HEV IgG positivity. The odds ratios and P-values of potential risk factors for anti-HEV IgG in the multivariate logistic regression model are shown in Table 3. In multivariate analysis, anti-HEV IgG positivity in females was higher compared to males but the difference was not significant (P = 0.058). Additionally, we found that there was a strong statistical association between geographic location and HEV prevalence (Table 3). Interestingly, in multivariate logistic ethnicity was also significantly (P = 0.017) associated with anti-HEV IgG positivity. The rate of anti-HEV IgG positivity was much higher in Dong, Miao, Yi, Gelao, Bai, and Yao compared to other ethnic groups (Table 3).
When we analyzed different potential risk factors data acquired from the self-reported questionnaires using univariate and multivariate logistic regression analysis. Our analysis revealed that ethnicity and provincial background are significantly associated with anti-HEV IgG (Table 3). We found that anti-HEV IgG sero-prevalence was higher in females as compared to males but the difference was not significant. Similar results have been reported [19][20][21] but others observed the opposite [22,23]. However, there is to date no evidence-based explanation on the sex disparity to HEV susceptibility, requiring further investigation. It is well accepted that HEV infection occurs at all ages, and thus the anti-HEV IgG rate increases with the growing of age. In this study, anti-HEV IgG prevalence ranges from 4.72-5.87% without significant differences among different age groups. This can be explained that we enrolled in a relatively homogeneous youth group. 94.6% of participants are less than 20 years old in this study, and therefore, young age might be the main contributor to the overall low HEV prevalence.
This study has limitations that must be considered when interpreting our findings. First, HEV RNA was not detected and therefore we could not confirm the circulating genotype in our population. Secondly, the information of several known risk factors, including dietary habits, living standards, travel history, family history, and profession were not available in this study. Finally, we were only able to recruit a youth cohort for practical reasons. Thus, the epidemiology of HEV infection in other age groups of Chinese ethnic populations remains to be investigated.

Conclusion
This study provides comprehensive epidemiological information on HEV prevalence in the multi-ethnic populations residing in different provinces of China. Funders has no role in the design of the study and collection, analysis, and interpretation of data, writing and publishing the manuscript.

Availability of data and materials
The aggregate data supporting findings contained within this manuscript will be shared upon request submitted to the corresponding author. Identifying patient data will not be shared.

Ethics approval and consent to participate
The protocol used in this study was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee at Northwest Minzu University Lanzhou, China. Written consent was individually obtained from the participants.

Competing interests
All authors declare they have no actual or potential competing interests.