A systematic review of the epidemiology of Hepatitis E virus infection in South – Eastern Asia

ABSTRACT Hepatitis E virus (HEV) infection is an emerging zoonotic viral disease, with an increasingly international public health challenge. Despite the concerns that the global disease burden may be underestimated. Therefore, evaluation of the disease epidemiology in South – eastern Asia through a systematic review will assist in unraveling the burden of the disease in the subregion. A priori protocol was prepared for the systematic review and followed by a literature search involving five electronic databases. Identified publications were screened for high quality studies and the elimination of bias and relevant data extracted. A total of 4157 citations were captured, and only 35 were included in the review. A wide range of HEV seroprevalence was recorded from 2% (urban blood donors in Malaysia) to 77.7% (lowland communities in Lao PDR). Sporadic HEV infection and epidemics were also detected in the subregion. Indicating hyperendemicity of the disease in South – eastern Asia.


Introduction
Hepatitis E virus (HEV) is the viral agent that causes an infectious disease known as hepatitis E. Hepatitis E to a large extent, manifests as acute icteric hepatitis, otherwise known as acute viral hepatitis (AVH) [1]. AVH is usually asymptomatic or self -limiting, but it may progress to acute liver failure (ALF) in some individuals. ALF is often associated with increased disease severity with extrahepatic manifestations and mortality [2]. Additionally, HEV infection may persist, leading to chronicity in immunocompromised persons. The disease severity and mortality are often higher in chronic infection and pregnant women with AVH. Hepatitis E mortality in the general population is usually between 0.2% -4%. On the other hand, mortality ranges between 15% -100% in pregnant women [3,4] and patients with chronic liver disease [5].
Although the actual burden of hepatitis E is not known, yet it is considered an emerging disease with an increasing public health threat globally [6]. Despite underestimating, the global burden of HEV infection is estimated at 2.3 billion, with an annual global incidence of 20 million [7,8]. HEV is responsible for both sporadic and epidemic infections. HEV is regarded as the commonest cause of acute viral hepatitis in the world [8]. HEV epidemics have been reported only in the developing countries of Asia, Africa, and in Mexico [9].
HEV is a small spherical virus with a diameter of 27-34 nanometers [10]. It is a non -enveloped positive -sense, single -stranded RNA virus with an approximate 7.2Kb genome [11,12]. The genome of HEV has three open reading frames (ORF); ORF1, ORF2, and ORF3. HEV belongs to the Hepeviridae family and has two genera; the Orthohepevirus (strains infect mammals and birds) and Piscihepevirus (strains infect fish) [10]. The genus Orthohepevirus has four distinct species (A, B, C, and D) while the second genus has one species; Piscihepevirus A. The Orthohepevirus A has eight known genotypes named HEV -1 to HEV -8 [13]. HEV -1 and HEV -2 are exclusively human viruses and enterically transmitted. Thus, responsible for most of the infections in developing countries. HEV -1 and HEV -2 are also associated with epidemics and severe infection in pregnancy [2]. Thus, often referred to as the "epidemic genotypes". On the other hand, HEV -3 and HEV -4 are swine genotypes that cause zoonotic infection in humans [9]. HEV -3 and HEV -4 are mostly responsible for infections in the developed countries [14] and for chronic HEV infection [2]. Also, HEV -7 [15] and Orthohepevirus C [16] have been reported as causes of chronic HEV infection in humans.
Systematic reviews (SR) are essential tools in human medical research, thus crucial for investigating HEV seroprevalence. SR of HEV epidemiology have been conducted in Africa [17,18], Europe as well as in some selected high -income countries [19]. However, there is no comprehensive SR of HEV infection in South -eastern Asia (SEA). SEA is a unique region; the region is multicultural, multiracial, multireligious, and comprises of countries that have both low and high income. These factors affect the distribution pattern of HEV; from outbreaks, sporadic infections, prevalent genotypes, mode of transmission to at risk population. Thus, a better understanding of hepatitis E epidemiology will provide more details on the pattern of the disease distribution in this region. It will also assist in implementing informed policy decisions and evidence based control measures for hepatitis E and associated healthcare challenges.
Therefore, the purpose of this study was to evaluate the epidemiology of HEV infection in SEA by reviewing and summarizing pertinent peer -reviewed publications. Sub -objectives were to determine the hepatitis E disease rates (seroprevalence, sporadic infection, outbreaks) in the subregion. The study also assessed the mode of disease transmission and identified circulating HEV genotypes. Additionally, the authors identified knowledge gaps and made recommendations for improved HEV studies. Authors also made recommendations for governments to implement measures to prevent HEV infection in at -risk populations and in the general population in the SEA subregion.

Scope
A priori protocol (S1 File) was prepared based on the Preferred Reporting Items for Systematic Reviews and Meta -Analysis (PRISMA) guidelines [20] using the PRISMA assessment checklist (S2 File), before conducting this SR.
For this SR, the eligibility (inclusion and exclusion) criteria were defined using the acronym PICOT: • Population: Inclusion; studies conducted in the population of the South -eastern Asia countries (study location) as outlined by the United Nations [21] were included in the study. Exclusion; studies published in other languages other than English, studies that did not separate the prevalence of HEV from other viral diseases, studies covering topics other than HEV epidemiology (laboratory studies on the pathogenesis of diseases, molecular biology), and animal studies only were excluded. Also excluded were studies on case reports, letters, books, dissertations, review articles, unpublished reports, and conference papers.
• Outcome: Patients with serological evidence of HEV exposure (from seroprevalence, outbreaks, sporadic cases), risk factors, mode of transmission, and circulating genotypes. • Time frame: No limitations were placed on the year of publication.

Search strategy
The electronic search was conducted on 22 March 2020. The strategy embraced the assessment of all relevant literature citations captured by applying the search algorithm in five electronic bibliographic databases (Scopus, Science Direct, PubMed, MEDLINE, and ASEAN Citation Index). Also, a gray literature search was conducted via hand searching references of selected (review) articles and conference proceedings. Additionally, a related internet search was done in Google Scholar and Google on 10 June 2020. Details of specific algorithms used for searching each of the databases are outlined in the study protocol (S1 File). However, a sample search algorithm is given as follows; ("Hepatitis E Virus" OR "HEV infection" OR "HEV" OR "Viral Hepatitis E" OR "Hepatitis E" OR "Hepatitis E virus infection" OR "Hepatitis E antibodies") AND (Seroepidem* OR "Prevalence" OR Epidem* OR "Survey" OR "Seroprevalence") AND ("Indonesia" OR "Vietnam" OR "Thailand" OR "Singapore" OR "Malaysia" OR "Philippines" OR "Cambodia" OR "Myanmar" OR "Burma" OR "Laos" OR "Brunei" OR "Timor-Leste").

Data management
The obtained searched articles were compiled and deduplicated in an MS Excel spreadsheet. All steps of the SR, from screening to data extraction, were carried out on an Excel spreadsheet. The final dataset on the MS Excel spreadsheet was then subjected to further analysis. Selection process; the study selection was conducted by two independent reviewers, and a third reviewer decided about uncertainties based on discussion and consensus.
Data collection process; extraction of data was conducted simultaneously with the full text searching. Relevant information were extracted from each article included and recorded immediately in the respective data extraction files. Two independent reviewers carried out this process, and two others checked the information for verification.

Quality assessment
Each article included in this SR was deemed relevant after meeting the inclusion and exclusion criteria. The quality of each article was then evaluated based on the prevalence critical appraisal instrument developed by [22] for seroprevalence studies and the critical appraisal checklist by [23] for case series and outbreak studies. The ten questions used in each of the critical appraisal instrument was answered either with Yes, No, Unclear, or not applicable. Articles with ≤60% score, or ≥3 U, were considered to have failed the quality assessment test and were not included in the study. The details of the quality assessment and the articles assessed are given in the S1 Table.

Results
The systematic search conducted on the electronic databases captured 4151 citations, and additional six citations were found by manual searching. After a series of screenings, as shown in Figure 1 [20], 41 articles met the eligibility criteria, and a list of the articles is given in the S4 file. After quality assessment, 35 articles from 9 out of the 11 SEA countries were included. The distribution of included publication according to country is given in Figure 2. At the same time, the descriptive characteristics of the included studies are given in Table 1.

Anti-HEV antibodies seroprevalence
Data on HEV antibodies seroprevalence was extracted from 33 studies in seven SEA countries. A summary of the major characteristics of the studies, including country, year of sampling, sample demographics, and assay used, is presented in Table 2. The distribution of the studies for each country; Cambodia (n = 3), Indonesia (n = 8), Lao PDR (n = 3), Malaysia (n = 3), Singapore (n = 2), Thailand (n = 7) and Vietnam (n = 6). Included studies used a wide range of assays for the anti-HEV antibody seroprevalence. However, the predominant assay employed is the kit from MP Biomedical, previously called Genelabs (n = 9). MP Biomedical is followed by the Wantai Bio-Pharm (n = 6). The variation seroprevalence across the countries is summarized in Figure 3.

Acute sporadic HEV infection
Data on sporadic HEV infection was captured from acute (sporadic) hepatitis cases in eight studies from six countries (Cambodia: n = 1, Indonesia: n = 2, Philippines: n = 1, Singapore: n = 1, Thailand; n = 2, and Vietnam: n = 1). Table 3 presents the characteristics of the studies, including country, year of sampling, method of diagnosis, case demographics, total sampled patients, number of cases, and percentage seropositivity.

HEV outbreaks
HEV outbreaks were recorded in two out of the 11 SEA countries studied. The outbreaks were reported in two studies, and details are summarized in Table 4. NR; not reported,

Risk factors and Mode of transmission
Risk factors, at risk groups, and the mode of HEV transmission was assessed and summarized in Table 5.
a; outbreak investigation study, DNS; data not statistically significant, NR; not reported, PDR; peoples' democratic republic.

Circulating HEV genotypes in SEA
Data on prevalent HEV genotypes in SEA is summarized in Table 6. Studies on HEV genotypes are reported from six countries. These countries are; Cambodia, Indonesia, Malaysia, Singapore, Thailand, and Vietnam.

Discussion
According to the United Nations (UN) geoscheme as well as the UN statistical division (UNSD) department, the subregion of South -eastern Asia is made up of 11 countries [47]. The list and characteristics of the countries that made up the SEA subregion are given in S3 File. There are two (Brunei and Singapore) developed (high -income) countries, and the remaining are developing (upper -middle, lower -middle and low -income) countries in the subregion. Of all the 35 articles included in the SR, there was no captured research in two (Brunei and Timor -Leste) out of the 11 countries. However, data was recorded in the Record identified through systematic searching on electronic databases. n = 4,151 Manual searching n = 6 Records after duplicates removed n = 3,368 Records screened on title n = 101 Full-text articles assessed for eligibility n = 41

Total records analysed n = 35
Identification Screening Eligibility

Duplicates n = 789
Excluded after title screening n = 3,267 Excluded after abstract screening n = 47

Excluded after full text screening (n = 13)
Outside population of interest Full articles not available Study not in English Case report Records screened on abstract n = 54

Excluded full text (n = 6)
Failed quality assessment using critical appraisal tools.  remaining nine countries, indicating that HEV infection is prevalent in the region. The endemicity of HEV infection in SEA is further confirmed by the seroprevalence data extracted from 32 publications with 33 studies in seven countries of the region ( Table 2). Judging from the earliest recorded study, it is evident that HEV infection has been present in the subregion for at least two decades and a half [48]. HEV seroprevalence showed wide variations between countries, within the countries, and from one population to another. The highest recorded seroprevalence was 77.7% among the healthy population of lowland communities in Lao PDR [49]. On the other hand, the lowest rate was reported among urban blood donors in Malaysia, with 2% [50]. The wide variations seen in the HEV seroprevalence between countries in SEA is like the results obtained from Africa. In a similar SR conducted by Kim and colleagues in Africa, HEV seroprevalence varied by country from 0% (in Gabon) to 84.3% (in Egypt) [17]. Studies in Europe have also shown HEV seroprevalence variations between countries in that region [51,52]. However, the variations in Europe are not as wide as those seen in this SR study and those observed in Africa [51,52]. The reason for the seroprevalence variation by country could be due to several factors. These factors include, among others, the assay method employed, and publication year. The assay used for the seroprevalence study could explain the observed variation in seroprevalence across countries. These assays vary in their performances (sensitivity and specificity) thus may give results that differ from one study to another. There seems to be no consistency in the assay type used across the countries and even within countries. Different assays are employed within and across countries for determining the HEV seroprevalence. Several studies investigating performance of assays used in HEV seroprevalence studies have established that employed assay type is a predictor of seroprevalence [53][54][55][56]. Implying that type of assay used in a study can influence seroprevalence estimations. In one of the studies, a broad range (42%-96%) of sensitivity was reported for anti -HEV detection among the five assay types studied [53]. Another essential predictor for variation in HEV seroprevalence is the year of publication. As shown in the results of this SR, chronologic time could show either increasing or decreasing  [57]. However, in Singapore, the situation was the reverse of what is observed in Cambodia. Here, IgG seroprevalence increased from 14% in 2007 to 35% in 2016 [58]. In addition to factors enumerated as influencing variation of seroprevalence between countries, cultural practices, eating habits, and whether a country is developed or developing [59] will affect seroprevalence within a particular country. The high rate HEV seroprevalence observed in this SR study indicates that HEV is hyperendemic in the subregion. The majority of countries in this study are developing with either low -income, lower -middle income, or upper -middle income economies. This could explain the high rate of HEV seroprevalence observed.
However, even in Singapore, a developed country with a high -income economy, the seroprevalence (of 10.5% -22.3%) appeared relatively high [58,60]. This scenario goes to prove that the endemicity of HEV is no longer restricted to only developing countries, but even developed countries are no exception [51]. Other factors that may lead to HEV seroprevalence variation in the subregion include study type, sample size or frame [18], migration, tourism, and proportion of specific ethnic groups in a country [19]. Studies were conducted   [61][62][63]. The seroprevalence rate of the general population in Indonesia ranges from 5.9% to 18%, while the susceptible group (those living in previous outbreak areas) have a range of 18% to 59%. In Vietnam, the seroprevalence of the at risk group of individuals exposed to pigs is 53% and 31% among healthy populations. The results are similar in Lao PDR, Malaysia, and Singapore. This pattern of seroprevalence has also been reported in an SR of HEV epidemiology conducted in Africa [17]. Furthermore, evidence of HEV infection endemicity in the SEA is not limited to seroprevalence studies alone. Reports on sporadic cases also exist to buttress further the fact that hepatitis E is endemic in the subregion. Seven hundred and fifteen confirmed acute hepatitis E cases from 1996 to 2019 were reported in the subregion out of 7079 sampled patients. The seropositivity rate ranges from 1.1% [61] to 88% [64] amongst different age groups (Table 3). Acute hepatitis E cases in most countries are low despite the high rate of HEV seroprevalence in the respective countries. However, the clinical HEV infection of 88% in Indonesia is in tune with the high seroprevalence rate reported in the country [48,64].
Nonetheless, the low rate disparity indicates either a high rate of asymptomatic HEV infection cases or misdiagnoses/missed diagnoses in the subregion. The results obtained in this SR study is not dissimilar with what was observed elsewhere in an SR conducted in Africa and Europe [17,51]. These results are also in line with reports from several primary studies showing high asymptomatic HEV infection [65][66][67]. There are also suggestions that certain HEV genotypes may be responsible for more symptomatic (HEV -1 and HEV -2) and asymptomatic (HEV -3) infections [68]. Therefore, a low rate of acute HEV infections within a particular region or country, if due to asymptomatic cases, may imply the prevalence of HEV genotype with a less virulent course. They are thereby resulting in more asymptomatic cases as opposed to presentation with symptoms.
Additionally, disease outbreaks are apparent indications of the occurrence of such disease in the location. Accordingly, HEV discovery was traced back to the historic 1955 epidemic of acute hepatitis in Delhi, India [69]. Ever since the first recorded outbreak, several HEV infection outbreaks have been reported in the developing countries of Africa and Asia. Likewise, in this SR, two outbreaks were identified in Myanmar [70] and Indonesia [64]. Table 4 showed that the Myanmar outbreak occurred in 1989, with 108 confirmed cases. Jaundice was the most frequent presenting symptom. However, the clinical attack rate and case fatality rate were not reported. The Indonesian epidemic occurred between 1997-1998, involving 110 confirmed cases with no mortality [64]. More females were affected than males, and the clinical attack rate was 19% [64]. Both Myanmar and Indonesia are developing nations with low income economies. Thus, reports of hepatitis E outbreak in these countries agree with established results of restriction of hepatitis E outbreaks to low income nations [69]. The trend of HEV outbreaks occurring in low income countries was also the same in Africa, as reported by Kim et al. (2015). Although, in Kim's study, the frequency of the epidemics in Africa is more than what is reported in this study. In Kim's study, more outbreaks were reported in almost alternate years. Whereas, in this study, only two outbreaks were identified and in two countries. The possible explanation for this could be that outbreaks in SEA are seldom reported in peer review journals.
Moreover, in addition to evaluating seroprevalence, clinical HEV infection, and outbreaks investigations, this study also looked at the associated risk factors, at risk groups, and the route of HEV transmission. It is believed that the evaluation of risk factors will provide a clue for the observed country-to -country, and regional differences in the seroprevalence. Thus, in addition to other enumerated factors such as the assay method, assessing the disease risk factors that prevail in each country or region will offer more clarity as to why the variations in seroprevalence. From the results of most studies included in this SR, there are several risk factors for HEV infection or seropositivity. These factors can be categorized based on risk practices, gender, age, occupation, and place of residence. Certain practices were identified as predisposing factors to HEV seroprevalence. The risk practices or conditions range from close contact with animals or animal waste, eating of uncooked or undercooked swine meet to blood transfusion (Table 5). In one of the studies, the eating habit of the population was attributed to the high anti -HEV antibody seropositivity. People in that community are known for consuming uncooked pig intestines and fresh blood mixed with vegetables [71]. Thus, dietary preference for eating raw or undercooked animal  products, particularly pig liver, will predispose to a high rate of HEV seroprevalence in a region. Other recorded risk activities include poor hygienic practice, drinking fecal contaminated water, and human waste disposal into water bodies. The findings in this SR is in line with an SR conducted in some selected non -endemic countries. Wherein dietary preferences for uncooked liver and HEV contamination of food sources were identified as factors impacting HEV IgG seroprevalence [19]. The male gender and elderly age group were also identified as at -risk groups for high HEV seropositivity in most studies. However, some studies recorded no significant difference between male and female HEV seroprevalence.
Regarding sporadic HEV infection, male gender and advanced age were also reported to be more affected in most of the studies. Of the few studies that investigated the predisposition of places of residence to HEV seroprevalence all, identified rural residence as a risk factor. HEV seroprevalence was also noted to have a predilection to some individuals in certain occupations. Animal transporters, abattoir workers, slaughterhouse workers, swine farm workers, swine farmers, and livestock farmers were all acknowledged as at -risk occupations for HEV seropositivity. This study also identified several transmission routes for HEV infection from analyzed studies, as outlined in Table 5.
Consequently, a new categorization for the HEV transmission route based on the findings of this SR may not be out of place. So, transmission routes for HEV can be divided into three broad groups; nonzoonotic, zoonotic, and vertical transmission. The non -zoonotic transmission can be subdivided further into waterborne (due to fecal contamination), bloodborne, and person-to-person (direct contact) transmission. The zoonotic transmission can be either direct or indirect. Direct zoonotic transmission will entail transmission from animals to humans through; direct contact with animals, their fluids and secretions, or wastes. The indirect zoonotic transmission has three subdivisions. One, waterborne; from contamination of water with HEV infected animal waste. Two, foodborne; from consuming contaminated animal products of HEV infected animals. Three, bloodborne; the sources for bloodborne transmission for zoonotic and non -zoonotic could be any of these; -organ transplant, hemodialysis, blood transfusion, and intravenous drug administration/abuse. These bloodborne sources have already been established in several studies as avenues of HEV transmission [72,73].
For a complete outlook of HEV epidemiology in the subregion, this study also assessed the prevailing HEV genotypes. The most prevalent genotype in the subregion is HEV -3, followed by HEV -4 (Table 6). HEV -3 being the most prevalent genotype, further justifies the observed inverse relationship between HEV seroprevalence and rate of clinical HEV infection detected in most countries. Also, interesting to note, is the prevalence of HEV -1 in Singapore, a developed country. HEV -1 is believed to be restricted to only developing countries, but here, in addition to HEV -3 and HEV -4, HEV -1 was also reported in Singapore ( Figure 4). However, the study alluded to the fact that HEV -1 might be imported into the country by travelers or non -residents [58]. Another striking observation also is that despite recorded HEV outbreak in Indonesia, HEV -1 and HEV -2 were not reported in the country. Instead, HEV -4 was the identified genotype and not the so -called epidemic genotypes (HEV -1 and HEV -2). Indonesia is a developing country with hygienic and sanitary challenges that favors HEV -1 and HEV -2 prevalence. However, other risk practices and conditions in favor of HEV -4 and HEV -3 also exist in the country. So, possibilities are that the epidemic genotypes are the most prevalent but yet to be identified. Another probability could be that HEV -1 is most prevalent in the outbreak areas and HEV -4 in non -epidemic areas. Also, there is the likelihood of a predominant genotype switch occurring in Indonesia from HEV -1 to HEV -4, since it is possible to have mixed genotype prevalence in a country. A similar situation also exists in China, another outbreak country. Previously, HEV -1 was believed to be the most abundant genotype in China. However, most recent studies have reported HEV -4 as the most prevalent [74].
This SR as well, identified knowledge gaps and limitations relating to HEV research and epidemiology. HEV study paucity was identified in some countries in the region. Of interest are the two countries (Brunei and Timor-Leste) that did not report any study. So, more studies are needed, particularly on sporadic infection and outbreak investigation in the subregion. Likewise, more seroprevalence studies on susceptible groups are required. Studies on susceptible groups such as pregnant women, immunocompromised, and hemodialysis patients are of a limited number in the subregion. To standardize HEV diagnostic method, more studies should be conducted examining the performance characteristics of different assay types. This type of investigation will allow for the adoption of assays with similar superior performance characteristics for future HEV diagnostic studies. Also, in conducting future investigations, global or regional standard generic protocols could be generated to be adopted for the research. This protocol will allow for harmonization and better comparison among different studies. A similar proposal for a standard protocol in HEV seroprevalence research has been made earlier [17]. The high rate of asymptomatic cases noted here may lead to underestimating the hepatitis E burden in the subregion. Thus, implementation of routine HEV screening in the hospitals, at least among the high risk groups, will be worthy. Consequent to the above enumerations and findings, there is a need for recommendations. Recommendations that will help shape policy formulation toward effective control and prevention of hepatitis E in the subregion. A joint sub-regional /multilateral collaboration among member countries for the control of hepatitis E is needed. This collaboration will allow for harmonizing immigration and migration policies across the subregion. It will also work on the effective livestock movement and animal husbandry practices within the member nations. Within countries, there is a need for robust HEV control and prevention programmes. Starting from improved government intervention in the area of HEV research. Improved research will unravel the actual burden of hepatitis E. It may also lead to the development of novel vaccine(s) to prevent the disease. Also, governments at all levels should focus on improving decent basic hygiene. Concerted efforts should be made to provide safe drinking water. Ensure adherence to standard guidelines for public water supply. Also, the adoption of eco -friendly sanitation measures regarding sewage disposal (for both animal and human) in communities will help prevent the disease.
Furthermore, health education on improved personal hygiene and safe dietary preference behaviors will help HEV infection prevention and control. Discouraging the consumption of uncooked or undercooked animal products and contaminated beverages will have a great impact on HEV prevention. Enhancing surveillance for early outbreak detection and upgraded blood screening strategy will reduce epidemic impact and improve control.

Strength and limitation of the study
This is the first SR study on the epidemiology of HEV infection in the SEA subregion. This SR is unique and robust in two aspects. One, the screening process was culminated with a vigorous critical appraisal to determine the included studies. This ensured that only quality studies are included for the SR and eliminated bias. Two, a comprehensive approach was adopted to include the most critical aspects of disease epidemiology as the study's outcomes. However, the study is not without some limitations. There is the possibility of unintentional omission of relevant publications since only five databases were searched for this SR. Also, only publications in English language were considered eligible for inclusion in the study.