Seroprevalence and incidence of hepatitis A in Southeast Asia: A systematic review.

BACKGROUND: A previous review on hepatitis A virus (HAV) seroprevalence in 2005 categorized Southeast Asia as a low HAV endemicity region. In 2010, the World Health Organization modified this from low to low/medium endemicity, pointing out that these estimates were based on limited evidence. Since then, there has been no attempt to review HAV epidemiology from this region. We conducted a systematic review of literature to collect information on HAV incidence and seroprevalence in select countries in the Southeast Asian region, specifically, The Association of Southeast Asian Nations over the last 20 years.
METHODOLOGY: This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. From the relevant articles, we extracted data and conducted a risk of bias assessment of individual studies.
RESULTS: The search yielded 22 and 13 publications on HAV seroprevalence and incidence, respectively. Overall, our findings point to a very low HAV endemicity profile in Thailand and Singapore and evidence of a shift towards low HAV endemicity in Indonesia, Lao People's Democratic Republic, Malaysia, the Philippines, and Vietnam. Only Singapore, Thailand, Malaysia, and the Philippines have existing HAV disease surveillance and reported incidence rates below 1 per 100,000. Several outbreaks with varying magnitude documented in the region provide insights into the evolving epidemiology of HAV in the region. Risk of bias assessment of studies revealed that the individual studies were of low to medium risk.
CONCLUSIONS/SIGNIFICANCE: The available HAV endemicity profiles in Southeast Asian countries, aside from Thailand, are limited and outdated, but suggest an endemicity shift in the region that is not fully documented yet. These findings highlight the need to update information on HAV epidemiology through strengthening of disease surveillance mechanisms to confirm the shift in HAV endemicity in the region.

Introduction

Hepatitis A disease is caused by the hepatitis A virus (HAV) and is transmitted via the fecal-oral route either through ingestion of contaminated food or water, or through close contact with an infectious person [1, 2]. Symptoms are initially non-specific (nausea, vomiting, fever, malaise and abdominal pain) and followed by bilirubinuria, pale stools, jaundice (usually 2–4 weeks) and pruritus (unusual case) that can last up to 6 months [3]. The severity and clinical outcome of hepatitis A are highly correlated with age at infection, with HAV infection being usually asymptomatic in children (~70% of cases) but commonly symptomatic in adults (>70% of cases) [3]. The presence of IgG antibodies confirms either vaccination or the initiation of the convalescent phase of infection, providing lifelong immunity to the individual [3]. Endemicity level (i.e. circulation) of HAV within a given region or population is readily estimated through serological surveys (measurement of HAV antibodies in the blood) and are reliable estimates of the burden of disease in a population.

HAV endemicity is typically high in low- and middle-income countries (where individuals are exposed to virus infection in their childhood years) in comparison to the situation in high-income countries which typically have very low endemicity (i.e. most of the population is naïve to HAV infection during their life) [4]. In recent decades, low- and middle-income countries have seen rising incomes and rapid urbanization and nearly the entire population (especially in urban areas) now have access to clean water. Consequently, these countries have reported a transition from high to low endemicity levels of HAV due to a progressive decrease of exposure to HAV during their childhood [4]. Paradoxically, this improvement place countries at higher risk for HAV [5-7]. Lastly, international trade, travel, and migration may also add to the high risk of outbreaks in countries reporting low or intermediate HAV endemicity [8].

A review investigating the seroprevalence of HAV in children and adolescents in the Southeast Asian region was published in 1998 [9]. Since then, no published review investigating exposure to HAV has been identified for the region. In 2005, a systematic review of the global seroprevalence of HAV ranked the Southeast Asian region as one with low endemicity, mainly driven by a large number of publications coming from Thailand [10]. In 2010, the World Health Organization (WHO) updated the aforementioned ranking and modified the HAV endemicity status for this region to low/medium endemicity [5], but this estimation was based on limited evidence with an average of less than one publication per country. A recent systematic review reported HAV seroepidemiology in the Asia-Pacific region, including all the East and South Asian countries [11]. However, results from only 3 out of 11 countries in the Southeast Asia Region (Thailand, Indonesia, and Singapore) were reported.

In this systematic review we evaluate the existing literature regarding HAV epidemiology by focusing on disease incidence and trends of exposure to HAV in the Southeast Asia region over the last 20 years. Specifically, the 11 members of the Association of Southeast Asian Nations (ASEAN) are included in the review (Brunei, Cambodia, Indonesia, Lao People’s Democratic Republic [PDR], Malaysia, Myanmar, the Philippines, Singapore, Thailand, Timor-Leste and Vietnam).

Methods

Search sources and strategy

The search was conducted in 5 electronic databases. We searched MEDLINE (via PubMed), Embase, Google Scholar and 2 regional databases: the Health Research and Development Information Network (HERDIN) from The Philippines [12] and MyJurnal from Malaysia [13]. We also consulted grey literature sources such as the official websites from the Ministry of Health (MoH) or ministry of public health (MOPH) and the National Health Agency of each country (including the official national report of waterborne diseases if available). To intensify the grey literature search we sent an information request letter to universities and research centers, known for their research activity in infectious diseases, requesting unpublished reports on HAV epidemiology. Lastly, we conducted a snowball search utilizing the bibliographies of the identified publications to retrieve any further relevant studies.

The full electronic search strategy is described in the . Searches were limited to publications from January 01, 1999 to February 15, 2021 covering the last two decades of publications and conducted in both English and the local language of the country of interest.

Screening and selection

The identified publications were screened using the inclusion and exclusion criteria provided in . Original research from non-interventional studies was included if they reported the incidence of hepatitis A (usually defined as patients registered in clinical records or surveillance systems with the code B15 according to the International Classification of Diseases, Tenth Revision [ICD-10]) or the age-specific seroprevalence of HAV (defined as previous exposure to HAV confirmed by laboratory detection of HAV IgM/IgG in blood samples). Case reports and other publications such as commentaries, editorials and letters were excluded from this review. Both screening and selection of publications was performed by one author (GHS).

Risk of bias

The quality of individual studies included in this review from peer-reviewed sources was assessed using the tool for critical appraisal of prevalence studies in health research literature described by Hoy et al. [14]. The risk of bias assessment for each paper was performed independently by two reviewers (first reviewers KL, PB, ST, AS; second reviewers: DS, SB). Any disagreement was resolved by a third reviewer (GHS).

Risk of bias was not assessed for reports or publications from official MoH websites and outbreak specific studies. This was done because insufficient data and potentially biased disease reporting levels are known to exist in such reports as seen in other countries (such as United States) [15]. Thus, conducting a risk of bias assessment for these publications was considered out of the scope for this review.

Data extraction, statistical analysis and reporting

The information extracted from selected studies included study characteristics (year of publication, study design, main objective of the study and sample size), age group of the study population and case definition (laboratory confirmation methods and clinical definition of hepatitis A in the case of an outbreak study). Incidence cases were reported in numbers when available. Crude incidence rates and age specific seroprevalence were extracted and reported as such. If only incident cases were reported, the crude incidence rates were estimated for comparative purposes using the official population estimate from the corresponding National Statistics Bureau of each country. Annual incidence rates and age specific seroprevalence (by country or subregion) were plotted to describe the observed trend. HAV country endemicity was classified according to WHO criteria as follows: high (for countries reporting a seroprevalence above 90% by the time individuals were 10 years of age); intermediate (seroprevalence ≥50% by 15 years of age but <90% by 10 years of age); low (seroprevalence ≥50% by 30 years of age, but <50% by 15 of age); and very low (<50% by 30 years of age) [16]. A descriptive analysis of the extracted data and risk of bias assessment were performed to summarize the main outcomes. As heterogeneity of results was expected in the study populations due to differences in socioeconomic development and surveillance systems, no meta-analysis was planned for this review.

Results

Search results

Databases and snowball search yielded 448 publications. After removing 201 duplicates, and exclusions, a total of 35 publications were included in the final review (

Study characteristics

Among the 35 included publications, the largest number were for Thailand (n = 20) [23–38, 44–46, 51] followed by Malaysia (n = 5) [20, 41–43, 47]. While most countries contributed with a single study to this review, two studies provided data for immigrants coming from Vietnam, Philippines, Cambodia, Lao PDR and Myanmar [18, 21], no publications were found from Timor-Leste and Brunei. Twenty-two cross-sectional studies reported seroprevalence data while 13 publications reported only incidence data. Eight publications provided data from outbreak investigations and 5 publications were from official surveillance reports. Considering the study settings we found most reports community based (n = 10), followed by hospital based (n = 7) laboratory population ().

HAV seroprevalence

Among the 22 publications 15 reported results from Thailand and were mostly conducted before 2010 (). Seroprevalence surveys had sample sizes ranging between 100 and 4,260 individuals.

Thailand

At least 3 surveys conducted in Thailand in the study period [23, 29, 37] spanning the population of 12 different provinces may be representative of the national level (). These surveys independently showed a sharp increase in HAV exposure with age: from 20% or less in population <2 years of age to more than 90% in those ≥60 years of age. When comparing across surveys over time, we observed that exposure to HAV in individuals 15–20 years of age halved in the study period: from nearly 20% (1999) to less than 10% in 2014. It was also documented that the 50% exposure to HAV threshold was reached at 42 years of age in 2014 compared to 36 years of age in 2004, thus confirming the HAV epidemiological transition of Thailand into a very low endemicity country. During the same period, data from Bangkok focusing on chronic liver disease patients [33], intravenous drug users [27] and men having sex with men [26] showed a similar age pattern ().

Age Specific HAV SeroprevalenceƔ in Thailand in Different Regions and Years A) Surveys with National Representativeness 1999–2014 B) Surveys from Bangkok 1998–2007 C) Surveys across Different Provinces 2004 (Except Tak Province Ran in 2010). ƔNote: Seroprevalence measured by either detection of total HAV antibodies or specific IgG HAV in blood. Abbreviations: HAV, hepatitis A virus; IgG, immunoglobulin G.

The latest publication from Thailand analyzed exposure to HAV in older adults (≥60 years of age) and found it to be close to 100% [24]. In Thailand, seroprevalence surveys also depicted regional variability. In the exposure to HAV threshold of 50% was reached by the age of 15 years [38], while in the province of Chiang Rai, this threshold was reached by the age 50 years [29]. These results were in high contrast with the results from other regions which were closer to the nationwide average (). Results from 5 other studies from Thailand provided estimates of HAV exposure among medical students and medical cadets [28, 36, 52] and at specific age groups [30, 35].

Other countries

Data from other countries included in the systematic review suggest high heterogeneity on the endemicity level of HAV across the region ().

Age specific trends in HAV Seroprevalence from ASEAN countries (1999–2020) (n = 10).

Note: The dotted line represents the 50% threshold to visually categorize HAV endemicity. Only latest information from Thailand is depicted intentionally. Abbreviations: ASEAN, Association of Southeast Asian nations; HAV, hepatitis A virus.

In Singapore, a population-based survey conducted in 1993 showed a HAV exposure level of 50% by 35 years of age, suggesting a very low endemicity profile [22]. In 1993 a survey ran in Yogyakarta, Indonesia [19] also showed an increase in exposure to HAV with increasing age, reaching the threshold of 50% at around 25 years of age, indicating low HAV endemicity. On the contrary, a survey from Vietnam [39], conducted in 1994, showed an almost complete seroconversion by 10 years of age. Twenty years later, a survey conducted between 2011 and 2017 among immigrant women in Korea [21] from countries including Cambodia (n = 58), the Philippines (n = 85) and Vietnam (n = 422), suggested an HAV endemicity age shift pattern in Vietnam (60% by 20–29 years of age and 80% by 30–40 years of age) and the Philippines (78% by 20–29 years of age and 72% by 30–40 years of age), but not in Cambodia (100% HAV seropositivity by 20–29 years of age).

A recent study from Lao PDR suggests a shift taking place in that country towards a low HAV endemicity profile, suggesting exposure to HAV of 50% among individuals around 20 years of age [17].

Incidence of hepatitis A

Reports on the incidence of hepatitis A were found for four countries in Southeast Asia including Malaysia [47], Singapore [51, 52], Thailand [51] and the Philippines [48], where hepatitis A is a notifiable waterborne disease. Please refer to for a geographical overview of this review.

Malaysia

Hepatitis A has been a notifiable disease in Malaysia since the late 1980s [53] and since then there has been a sharp and sustained decreasing trend of the number of HAV cases, from around 9.0 per 100,000 individuals in 1991, to 2.2 in 2004, to the lowest level thus seen of 0.14 per 100,000 individuals in 2017. The latest report from 2018 showed a slightly increased incidence rate of 0.30 per 100,000 individuals [47].

Singapore

Official reports from the national health statistics, dating back to 1999 reported a stable range of notifiable cases (range: 48 [2016]– 146 [2006]) [49]. The latest report from 2018 and 2019 [50] recorded 75 and 66 new cases (crude annual incidence rates estimate approximately 1.0 per 100,000 population), respectively [50].

Since 2003 National Health Statistics depicted stable incidence rates below 1.0 per 100,000 except in years 2005 and 2012 when incidence rates of 4 and 3 per 100,000 were estimated, respectively [51]. The latest report from 2019 stated the occurrence of 432 cases corresponding to an incidence rate of 0.65 per 100,000 [51].

The Philippines

Official reports from the national health statistics dating back to 2010 [48] reported a steady increase in annual cases from 440 to 563 in 2015. The two latest available reports from 2016 and 2017 showed 443 and 462 cases per year, respectively. Nearly 50% of the affected people were between 16–30 years of age. Estimated Crude annual incidence rates based on this information ranged between 0.4 and 0.6 cases per 100,000 population.

Outbreak reports

Outbreak information was reported for Indonesia [40], Malaysia [20, 41–43], Singapore [22] and Thailand [23, 44, 45]. No information on outbreaks occurring in the Philippines was found in the search ( The outbreak in Indonesia (n = 59) occurred by the end of 2015, in two very close junior high schools in the urban area of Surabaya. In Malaysia at least 3 outbreaks from different regions were documented: The first happened in 2000 [41] where 334 cases of hepatitis A were reported and the outbreak source was postulated to be of mixed sources and spread from person to person. The second outbreak was reported in 2002 [42] with 51 cases of hepatitis A and the outbreak source was postulated to be contamination of rivers used for recreational purposes and human sewage. The last outbreak was reported in 2012 [43] with 78 cases, and the outbreak source was postulated to be contaminated “Toddy”, an alcoholic beverage. In Singapore one outbreak attributed to consumption of raw shellfish resulted in 159 cases reported in 2002 [22]. The epidemiological report showed no specific food center to be implicated. In the other hand we found several reports from Thailand [23, 25, 44, 45]. The largest outbreak was reported in Buengkan province which affected more than 1,600 patients who attended the provincial hospital [25] and the latest outbreak report was from 2019, which reported 61 confirmed cases in a prison located in Bangkok [46].

Risk of bias assessment

The result of the risk of bias assessment revealed that the studies included in the review showed high internal validity (). Assessment scores ranked between low and medium risk according to the Hoy et al. scale [14], mainly affecting external validity. These studies showed flaws in sampling methodologies such as a lack of randomization or census procedures and applying convenience sampling strategies. Nine publications failed to provide confident representation of the targeted or the national population and six publications failed in the attempt to control the non-response bias.

Risk of bias assessment (n = 22¥).

¥Risk of bias was not assessed for reports or publications from official Ministries of Health websites and outbreak specific studies. Color coding: Per item result: green = Yes; red = No. Overall result: green, and amber dots correspond to low and medium risk of bias, respectively.

Discussion

This systematic review found evidences that suggest a full spectrum of HAV endemicity in Southeast Asia, where, aside from Thailand and the first recent study published from Lao PDR, none of the other countries have updated information on HAV seroprevalence at the national or regional level in the last 20 years.

Such lack of information might have failed to document the ongoing and expected HAV endemicity shift in these countries that have experienced major socioeconomic improvements in the past two decades [54].

The striking contrast in the number and size of HAV outbreaks in Thailand and Singapore, both countries with very low HAV endemicity profiles, illustrates the impact of a HAV endemicity transition with different pace over time. By 1993 and probably as a result of an earlier, faster, and greater socioeconomic development [55], Singapore already featured a very low HAV endemicity profile, while Thailand was just reaching intermediate levels (reaching the HAV exposure threshold of 50% among individuals 12 years of age) [23]. This example adds to the evidence that countries with an ongoing HAV endemicity shift, even those reaching low HAV endemicity levels, cannot rely solely on improvements in water hygiene and sanitation to contain the burden of hepatitis A infection [56, 57].

Although we only found one original outbreak report from Indonesia, there is evidence of several outbreaks across its territory. At least 47 episodes of hepatitis A outbreaks were documented between 1998 and 2018, mainly from the Isle of Java. Outbreaks were reported at schools and nearby areas (73%) and primarily involved people between 15–35 years of age [58]. Additionally, our review did not capture a recent outbreak report from July 2019 with over 950 cases in the Isle of Java [59] because it was in a press release format. These large numbers of outbreaks are underpinned by an ongoing transition of HAV endemicity that has not been documented yet. Lack of routine surveillance of hepatitis disease and low confidence in the existing epidemiological data in Indonesia has been previously reported [60]. The only HAV seroprevalence survey during our study period came from a single city (Yogyakarta) in 1996, showing a low HAV endemicity profile. As such, this data cannot be generalized to the entire Indonesian population [19].

This review did not find any published information on outbreaks in the Philippines, and very limited information on seroprevalence for the last two decades, although periodic surveillance reports of acute diseases have been published since 2010 showing incidence rates below 1 per 100000, similar to those reported from very low endemic countries in the region. The case of the Philippines shows that reporting up to date incidence data needs to be complemented with periodic seroprevalence surveys to fully capture the burden of HAV disease.

Among the 4 countries in Southeast Asia where hepatitis A is a notifiable disease, only Thailand provides updated information of HAV seroprevalence at a national level. This evidence confirms that there are clear gaps in the HAV epidemiology surveillance in the region. There is a clear need for establishing and improving nationwide HAV surveillance in most of the countries in Southeast Asia.

Seroprevalence studies captured from Malaysia [20] and Vietnam [21, 39] suggest that these countries might be at an intermediate endemicity level but fall short in determining the age at which a 50% threshold is reached (). In other words, the existing evidence is limited in assessing the age at midpoint of population immunity, defined as the youngest age at which half of the population has serologic evidence of prior exposure to HAV.

HAV Seroprevalence age trends reports from Lao PDR, suggesting a low endemicity profile in both rural and urban areas, contrast with those observed in Cambodia and Myanmar despite sharing the lower Human Development Indices in the region [54]. This could be attributed to the improvements in water sanitation achieved by Lao PDR in the last few decades [61, 62].

The burden of hepatitis A among high-risk groups, merits further discussion. Information, mostly from Thailand, related to specific populations such as healthcare professionals, intravenous drug users and men who have sex with men shows results consistent with the general population [23]. These results emphasize the identical baseline susceptibility of this group to the general population, with a higher risk of acquiring HAV due to the inherent nature of their profession or behaviors [63]. Similar information was lacking from the other countries in the region.

HAV seroprevalence surveys including individuals from early childhood (around 5 years or even less, when possible) to late adulthood remains the most relevant data for HAV epidemiological analysis and disease modeling given the strong cohort effect shown by the HAV endemicity transition [64, 65]. It is likely that the young birth cohorts in Southeast Asian countries will remain at high risk of HAV infection, although with regional and temporal variations.

It is fully acknowledged that breaking HAV transmission and, in this case, ensuring improvement of water sanitation and hygiene (WASH) remains the cornerstone of HAV containment in a country, but to accomplish it country and region wide is not an easy task in Southeast Asia [66]. Thus, depending on the epidemiological pattern, countries may consider targeted vaccination against the disease to stop its spread, protect the individual with life-long immunity and induce herd immunity [67].

This systematic review addressed some of the limitations of previous publications reporting seroprevalence and incidence data from Southeast Asia. It reported a risk assessment bias and included both a literature search in the local language and grey literature search to rule out the existence of potential data being excluded from the mainstream search.

Scores of all seroprevalence papers included in this review ranked between low and medium risk, highlighting the reliability of the results included in the analysis. Most of the studies performed well in the internal validity items. The shortcomings mainly occurred over the external validity. This is not surprising in cross-sectional studies [68] as the generalization of a study outcome to a broader population is limited by the high variability across populations. Therefore, future studies should join efforts to reach local, regional, or national representatives to maximize the usefulness of HAV seroprevalence surveys.

Nevertheless, these results should be interpreted with caution owing to the limitations of the studies retrieved. Aside from Thailand, the low number of studies per country, their limited national representativeness and the heterogeneity of study population limit their external validity. In addition, the notable difference in the sampling methodology in the seroprevalence studies and differences in reporting and completeness of incidence data across countries in Southeast Asia hinder their comparability.

In summary (), this review documents the contrasting HAV endemicity and outbreak profiles of the countries in the Southeast Asian Region. It highlights the fact that ASEAN countries, aside from Thailand, have a substantial gap of up-to-date and reliable information on HAV epidemiology. This lack of information makes it difficult to fully document the epidemiology shifts, if any, in HAV endemicity. It is expected that several of these countries transit to low HAV endemicity in the next decades, which could consequently be associated with a higher risk and burden of disease in older age groups. Robust evidence on the HAV incidence and seroprevalence status of the population through the strengthening of surveillance systems will be useful for decision-makers to define targeted and comprehensive strategies to reduce the current and future burden of HAV disease [69] and potentially help in achieving the ultimate goal of eliminating hepatitis A in Southeast Asia by 2030 [70].

(DOC)

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Geographic representation of countries included in the review.

(TIFF)

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Search strategy.

(DOCX)

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Inclusion and exclusion criteria.

*References cited by screened articles were manually reviewed for relevance (i.e. snowballing). **References of included articles in these systematic reviews/meta-analyses were manually screened for additional relevant original articles (as deemed necessary by the reviewer).Abbreviations: HAV, hepatitis A virus; n.a., not applicable.

(DOCX)

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31 May 2021

PONE-D-21-06795

Seroprevalence and incidence of hepatitis A in South East Asia: a systematic review

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Reviewer #1: Yes

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Reviewer #1: The authors conducted a conducted a systematic review to evaluate the existing literature regarding HAV epidemiology, focusing on its incidence and seroprevalence trends in the countries of the Southeast Asian region. This is an important study.

Major comments

1) Authors describe five database were used in Method section, however Only Medline is used in Flowchart. Please correct.

2) The authors descripted different publications period criteria between the Methods (January 1st, 1999 to February 15th, 2021) and the Table 1 (January 1999 to December 2019).

3) The objective of the study was focusing on the incidence and prevalence of HAV. The case-control study design was in included in the inclusion criteria. What was authors plan to do with the data from case-control study?

4) In Table 2, additional information on the characteristics of included studies is needed to better understand the results. Authors should consider including information such as study setting (rural, urban, nationwide?), studied population, reported or calculated seroprevalence. The discussion of the results should also consider these characteristics.

5) In Table 3, Reporting HAV endemicity solely based on the most recent survey may be misleading in classifying the country as low or high endemicity. The prevalence depends on the study subjects, setting and period of data collection.

6) The manuscript is unnecessarily long and does not allow for grasping the main points of the research. The authors should write concisely and precisely. Also, Figure 4 does not provide more information for this research. Authors should consider deleting it, or adding information related to the aim of the study.

Minor comments

1) In abstract, as authors have divided into different paragraph (Background, Methodology, Conclusion), it is better to mention results in an individual paragraph

2) In the methods: the authors should have screened the biography of all included publication as well. This procedure might help finding more relative publications.

3) The quality of figure 2 and figure 5 are too low to read.

4) In different paragraph of Study characteristics, they have put abbreviations in some cases in some not. It is better to put all the abbreviation together in beginning or end of the article.

5) “Prevalence of HAV” is very confusing. More suitable word, such as “HAV exposure” should be used.

**********

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Reviewer #1: No

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17 Aug 2021

Dr. Emily Chenette

Editor-in-Chief

PLOS One

Wavre, June 24, 2021

Subject: Revised submission of manuscript entitled: Seroprevalence and incidence of hepatitis A in Southeast Asia: a systematic review

Dear Dr. Chenette,

On behalf of my co-authors, I would like to resubmit to your attention the following manuscript for publication in PLOS One as a systematic literature review. This systematic review aims to collect information on hepatitis A virus (HAV) incidence and seroprevalence in select countries in the Southeast Asian region. I acknowledge that all comments received from the peer-review process have been adequately revised as requested by the reviewers. We have provided each of these comments, along with their respective revisions including line and page numbers alongside the revised manuscript.

I declare that all authors fulfil the criteria for authorship, have read and approved the current manuscript and agree with its submission to PLOS One. I confirm that this manuscript has not been published elsewhere and is not under consideration by any other journal.

On behalf of the authors, I look forward to hearing from you with your decision at your earliest convenience.

Kindest regards,

Gustavo Hernandez-Suarez

To the editors:

1. We would like to confirm that the manuscript meets PLOS ONE’s style requirements.

2. Both the financial disclosure and competing interests state and describe the funding organization and the authors’ affiliation with this organization. These statements can be found in the “additional information” file.

3. Figure 4 (now supplementary Figure 1) has been created for the purpose of this manuscript and does not contain images that may be copyrighted.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors conducted a conducted a systematic review to evaluate the existing literature regarding HAV epidemiology, focusing on its incidence and seroprevalence trends in the countries of the Southeast Asian region. This is an important study.

Major comments

1) Authors describe five database were used in Method section, however Only Medline is used in Flowchart. Please correct.

Response: Thank you for highlighting this point. Indeed, we conducted a search of five databases, MEDLINE (via PubMed), Embase, Google Scholar, the Health Research and Development Information Network (HERDIN) from The Philippines and MyJurnal from Malaysia. Among the studies included in this review, all studies were identified in Medline or through a grey literature search. We identified only duplicates among the studies found through searching other databases named above, we have edited the PRISMA flow chart (Fig 1) to describe the search results found on the remaining databases.

Line 172, page 10, Fig 1

2) The authors descripted different publications period criteria between the Methods (January 1st, 1999 to February 15th, 2021) and the Table 1 (January 1999 to December 2019).

Response: Thank you for your comment, the dates in table 1 (now supplementary Table 1) have been updated accordingly. This was incorrectly mentioned in table 1 as we had updated the search after 2019.

3) The objective of the study was focusing on the incidence and prevalence of HAV. The case-control study design was in included in the inclusion criteria. What was authors plan to do with the data from case-control study?

Response: Thank you for your comment. We included case control studies in the eligibility criteria because of two reasons which are as follows: Case control study design is often use for outbreaks investigation and controls might be population based and can provide information on the frequency of past exposure of HAV in the general. One case control was included in this review to describe an outbreak in Indonesia.

4) In Table 2, additional information on the characteristics of included studies is needed to better understand the results. Authors should consider including information such as study setting (rural, urban, nationwide?), studied population, reported or calculated seroprevalence. The discussion of the results should also consider these characteristics.

Response: Thank you for your comment. We have edited Table 2 (now Table 1) to include further information on study setting, population and seroprevalence as recommended by the reviewer. These characteristics have also been described in the results section of the text.

5) In Table 3, Reporting HAV endemicity solely based on the most recent survey may be misleading in classifying the country as low or high endemicity. The prevalence depends on the study subjects, setting and period of data collection.

Response: Thank you for your comment. We agree with the reviewer that the use of the most recent survey may not accurately describe the hepatitis A virus endemicity level of a country. As a result, we have decided to merge tables 2 and 3 together into one table (now Table 1) and removed the endemicity column from the new table entirely. Furthermore, we have edited the text in the discussion section to reflect the changes to the table 2 (now Table 1) by using words like ‘suggest’ rather than ‘show’ to interpret these results.

6) The manuscript is unnecessarily long and does not allow for grasping the main points of the research. The authors should write concisely and precisely. Also, Figure 4 does not provide more information for this research. Authors should consider deleting it, or adding information related to the aim of the study.

Response: Thank you for your comment, we have edited the discussion section by removing certain text regarding a discussion of the risk of bias related to the included publications. This has been adequately described in the ‘risk of bias assessment’ sub-section in the results section. We also strived to reduce any other text throughout the manuscript which we deemed as repetitive or contrary to the point being discussed. Lastly, while PLOS One does not have any word limits for review submissions, the word count for this manuscript after revisions comes to approximately 4000 words excluding tables, figures and references, following the PRISMA guidelines that recommends 5000 word-limit for Systematic Reviews.

We acknowledge that the figure 4 (now supplementary Figure 1) was created from scratch by our team and that no copyright conflicts were identified upon its completion. The open source map of Southeast Asia was utilized to highlight the geographical context of the study. The authors found it important to include this figure as it would provide the unspecialized reader with an overview of the region. We have moved this figure to Supplementary Fig 1 and leave this matter to the discretion of the reviewers and journal editors on whether to include this figure or delete it from the manuscript.

Minor comments

1) In abstract, as authors have divided into different paragraph (Background, Methodology, Conclusion), it is better to mention results in an individual paragraph

Response: Thank you for your suggestion. We have edited the abstract to include a paragraph which reports only the results of this review. This paragraph is now named ‘Results’ as recommended by the reviewer.

Line 39, page 3

2) In the methods: the authors should have screened the biography of all included publication as well. This procedure might help finding more relative publications.

Response: Thank you for your suggestion. The authors did screen the bibliographies of the publications included in this review, but it was not done systematically. This was because many countries had no other publications besides the ones already included in this review. Only Thailand had additional publications for which a bibliography screening was conducted. Manuscript text was edited in the methods and results sections, respectively.

3) The quality of figure 2 and figure 5 are too low to read.

Response: Thank you for highlighting this point. Upon checking the figures for quality standards described in the journal guidelines we can confirm that the figures adhered to the stipulated pixel limits. We have once again tried to provide the figures in a similar format.

4) In different paragraph of Study characteristics, they have put abbreviations in some cases in some not. It is better to put all the abbreviation together in beginning or end of the article.

Response: Thank you for your comment, we have revised the complete manuscript text in adherence to the journal guidelines. The guidelines stated that abbreviations must be mentioned in full at first use and only if used more than 3 times in the entire text.

5) “Prevalence of HAV” is very confusing. More suitable word, such as “HAV exposure” should be used.

Response: Thank you for your comment. We have edited the manuscript text to ‘’HAV exposure’’ instead of ‘’seroprevalence’’ based on the context of the sentence.

4 Oct 2021

Seroprevalence and incidence of hepatitis A in Southeast Asia: a systematic review

PONE-D-21-06795R1

Dear Dr. Hernandez-Suarez,

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Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

19 Nov 2021

PONE-D-21-06795R1

Seroprevalence and incidence of hepatitis A in Southeast Asia: a systematic review

Dear Dr. Hernandez-Suarez:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Yury E Khudyakov

Academic Editor

PLOS ONE

Table 1

Study characteristics of included studies (n = 35).

Author	Country	Publication year	Study year	Study design	Study population		Study setting	Age group (years)	Outcome	Clinical measurement	Platform manufacturer	Total number of samples tested	Seroprevalence
Khounvisith et al. [17]	Lao PDR	2020	2013–2018	Cross-Sectional	Population based/ Multiple cities		Urban/semi-urban (Peak district) or rural (Phasay district)	all ages	Seroprevalence	Anti-HAV IgG	Diasorin	1,195	51.05%
Poovorawan et al. [18]	Cambodia, Lao PDR, Myanmar	2009	2008	Cross-Sectional	Legal Immigrant workers in Thailand/ Single city		Hospital based, Bangkok	16–60	Seroprevalence	Anti-HAV IgG antibody	Murex Biotech	1,183	95.01%
Juffrie et al. [19]	Indonesia	2000	1995–1996	Cross-Sectional	Population based/ Single city		Regional (Gondokusuman) community based	4–32	Seroprevalence	Anti-HAV IgG antibody	Abbott	1,103	28.65%
Ahmad et al. [20]	Malaysia	2011	2009	Cross-Sectional	CLD patients/ Single City		Hospital based (Gastroenterology Clinic of Universiti Sains Malaysia, Kelantan)	≥21	Seroprevalence	Anti-HAV IgG antibody	Abbott	119	88.24%
Kwon et al. [21]	Philippines, Vietnam	2018	2011–2017	Cross-Sectional	Immigrant females to Korea/ Nationwide		Regional screening programs from different provinces in Korea	20–40	Seroprevalence	Anti-HAV IgG antibody	bioMérieux	575	73.91%
Lee et al. [22]	Singapore	2011	1993	Cross-Sectional	n.a.		n.a.	all ages	Seroprevalence	Anti-HAV IgG antibody	Abbott	930	25.91%
Sa-nguanmoo et al. [23]	Thailand	2016	2014	Cross-Sectional	Pediatric health check-up or outpatient clinic at hospitals/ Nationwide		Hospital based/ Seven provinces representing four regions of Thailand	>71	Seroprevalence	Anti-HAV IgG antibody	Abbott	4,260	34.53%
Posuwan et al. [24]	Thailand	2019	2017	Cross-Sectional	Single city (no further details provided)		Participants were from the Chum Phae district of Khon Kaen province in northeastern Thailand, no more details provided	60–85	Seroprevalence	Anti-HAV IgG antibody	Abbott	93	98.92%
Poovorawan et al. [25]	Thailand	2013	2012	Outbreak Study	Community-wide HAV outbreak		Hospital based/ Buengkan province	2–70	Confirmed case	Anti-HAV IgM antibody	Abbott	205*	82.93%
Linkins et al. [26]	Thailand	2013	2006–2008	Cross-Sectional	Men who have sex with men/ Single city		Men participating in the Bangkok Men who have sex with men Cohort Study	18–45	Seroprevalence	Anti-HAV total antibody	Murex Biotech	1,291	27.03%
Sunthornchart et al. [27]	Thailand	2008	2003–2005	Cross-Sectional	Intravenous Drug Users/ Single city		Hospital based Bangkok	≥20	Seroprevalence	Anti-HAV total antibody	Diasorin	1,107	60.16%
Samakoses et al. [28]	Thailand	2007	2001	Cross-Sectional	University students/ Single city		Students College of Medicine and the Nursing College of the Royal Thai Army	17–26	Seroprevalence	Anti-HAV total antibody	Abbott	432	15.28%
Chatproedprai et al. [29]	Thailand	2007	2004	Cross-Sectional	Healthy Children, 6 months of age/ Nationwide		Hospital passed 4 provinces from north, north–east, center and south of Thailand	0–85	Seroprevalence	Anti-HAV total antibody	Abbott	3,997	27.37%
Ratanasuwan et al. [30]	Thailand	2004	2000–2002	Cross-Sectional	Population based/Nationwide		Six provinces in the Central Region of Thailand	all ages	Seroprevalence	Anti-HAV total antibody	Abbott	1,514	52.25%
Jutavijittum et al. [31]	Thailand	2002	1998–2000	Cross-Sectional	Children, 4–16 years of age	Urban and rural schools School based/Single province		4–16	Seroprevalence	Anti-HAV total antibody	Sanofi Diagnostic Pasteur	1,145	9.61%
Pancharoen et al. [32]	Thailand	2001	1998–1999	Cross-Sectional	Population based/ Single city		Nurseries, school and graduate schools in Bangkok	1–30	Seroprevalence	Anti-HAV IgG antibody	Murex Biotech	895	12.85%
Pramoolsinsap et al. [33]	Thailand	1999	1998	Cross-Sectional	Chronic Hepatitis B & C patients and healthy blood donors		Hospital based/ single city	16–60+	Seroprevalence	Anti-HAV IgG antibody	Abbott	195	Healthy donors 64.62% (126/195) Asymptomatic HBV carriers 68.6 (348/507) HBV-Related CLD (162/196) HCV- related CLD (106/117)
Pilakasiri et al. [34]	Thailand	2009	n.a.	Cross-Sectional	University students/ Single city		Nursing students at the Royal Thai Army Nursing College, Bangkok	16–41	Seroprevalence	Anti-HAV total antibody	Abbott	381	8.92%
Luksamijarulkul et al. [35]	Thailand	2003	1999–2000	Cross-Sectional	Population based/ Single city		Residents hill-tribe communities in the north of Thailand. (rural area)	15–24	Seroprevalence	Anti-HAV total antibody	General Biologicals Corp	190	87.89%
Chatchatee et al. [36]	Thailand	2002	1996–2001	Cross-Sectional	University students/ Single City		Students of medical college in Bangkok	20–22	Seroprevalence	Anti-HAV total antibody	Abbott	135	11.11%
Poovorawan et al. [37]	Thailand	2000	n.a.	Cross-Sectional	Population based/ Nationwide		Participants of vaccination trial against hepatitis B virus infection collected in 5 representative provinces in Thailand	1–18	Seroprevalence	Anti-HAV Total antibody	Abbott	961	7.91%
Rianthavorn et al. [38]	Thailand	2011	2009–2010	Cross-Sectional	Hospital based/ Single city		Tak province, border province between Thailand and Myanmar	all ages	Seroprevalence	Anti-HAV total antibody	Abbott	308	70.78%
Hau et al. [39]	Vietnam	1999	1999	Cross-Sectional	Population based		Laboratory based community study	0–87	Seroprevalence	Anti-HAV IgG antibody	Abbott	646	96.90%
Wahyuddin et al. [40]	Indonesia	2019	2015–2016	Case-Control	Healthy children		Regional, school based	12–14	Confirmed case	Anti-HAV IgM antibody	Chemux BioScience	72	75.00%
Mohd et al. [41]	Malaysia	2001	2000	Outbreak Study	Population based		Residents of Kuala District, Terengannu state	2–71	Confirmed case	Anti-HAV IgM antibody	n.a.	334	100.00%
Venugopalan et al. [42]	Malaysia	2004	2002	Outbreak Study	Population Based		Residents Hulu Langat District, Selangorstate	1–40	Confirmed case	Anti-HAV IgM antibody	n.a.	51	100.00%
Yusoff et al. [43]	Malaysia	2015	2012	Outbreak Study	Population Based		Residents of Manjung District, Perak State	13–72	Confirmed case	Anti-HAV IgM antibody	n.a.	78	100.00%
Poovorawan et al. [44]	Thailand	2005	2002–2003	Outbreak Study	Institution based/ Single city		Childcare center suburban area of Bangkok	1–6	Confirmed case	Anti-HAV IgG and anti-HAV IgM antibodies	Abbott	112	IgG: 66.07%; IgM: 62.50%
Phanwong et al. [45]	Thailand	2008	2005	Outbreak Study	Population based		Wiangpapao district, Chiang Rai Province	n.a.	Confirmed case	Anti-HAV IgM antibody	n.a.	1,308	n.a.
MOH, Thailand [46]	Thailand	2019	2017	Outbreak Study	Institution based/ Single city		Prisoners, Bangkok City	n.a.	Confirmed case	Anti-HAV IgM antibody	n.a.	141	43.26%
MOH, Malaysia [47]	Malaysia	2019	n.a.	Surveillance system	n.a.		n.a.	All ages	Confirmed case	Incidence HAV rate	n.a.	n.a.	n.a.
DOH, Republic of Philippines [48]	Philippines	2019	n.a.	Surveillance system	Population based		Nationwide surveillance report	All ages	Confirmed case	Incidence HAV rate	n.a.	n.a.	n.a.
MOH, Singapore [49]	Singapore	2018	n.a.	Surveillance system	Population based		Nationwide surveillance report	All ages	Confirmed case	Incidence HAV rate	n.a.	n.a.	n.a.
MOH, Singapore [50]	Singapore	2019	n.a.	Surveillance system	Population based		Nationwide surveillance report	All ages	Confirmed case	Incidence HAV rate	n.a.	n.a.	n.a.
MOH, Thailand [51]	Thailand	2019	n.a.	Surveillance system	Hospital based/ Nationwide		Nationwide surveillance report	All ages	Confirmed case	Incidence HAV rate	n.a.	n.a.	n.a.

*Out of a total of 1,619 patients with clinical symptoms of hepatitis A who visited the hospital during the outbreak.

Abbreviations: CLD, chronic liver disease; DOH, Department of Health; HAV, hepatitis A virus; HBV, hepatitis B virus; IgG, immunoglobulin G; IgM, immunoglobulin M; MOH, Ministry of Health; n.a., not applicable.