Prevalence and genetic diversity of rotavirus in Bangladesh during pre-vaccination period, 1973-2023: a meta-analysis

Introduction Rotavirus infection is a major cause of mortality among children under 5 years in Bangladesh. There is lack of integrated studies on rotavirus prevalence and genetic diversity during 1973 to 2023 in Bangladesh. Methods This meta-analysis was conducted to determine the prevalence, genotypic diversity and seasonal distribution of rotavirus during pre-vaccination period in Bangladesh. This study included published articles on rotavirus A, rotavirus B and rotavirus C. We used Medline, Scopus and Google Scholar for published articles. Selected literatures were published between 1973 to 2023. Results This study detected 12431 research articles published on rotavirus. Based on the inclusion criteria, 29 of 75 (30.2%) studies were selected. Molecular epidemiological data was taken from 29 articles, prevalence data from 29 articles, and clinical symptoms from 19 articles. The pooled prevalence of rotavirus was 30.1% (95% CI: 22%-45%, p = 0.005). Rotavirus G1 (27.1%, 2228 of 8219) was the most prevalent followed by G2 (21.09%, 1733 of 8219), G4 (11.58%, 952 of 8219), G9 (9.37%, 770 of 8219), G12 (8.48%, 697 of 8219), and G3 (2.79%, 229 of 8219), respectively. Genotype P[8] (40.6%, 2548 of 6274) was the most prevalent followed by P[4] (12.4%, 777 of 6274) and P[6] (6.4%, 400 of 6274), respectively. Rotavirus G1P[8] (19%) was the most frequent followed by G2P [4] (9.4%), G12P[8] (7.2%), and G9P[8], respectively. Rotavirus infection had higher odds of occurrence during December and February (aOR: 2.86, 95% CI: 2.43-3.6, p = 0.001). Discussion This is the first meta-analysis including all the studies on prevalence, molecular epidemiology, and genetic diversity of rotavirus from 1973 to 2023, pre-vaccination period in Bangladesh. This study will provide overall scenario of rotavirus genetic diversity and seasonality during pre-vaccination period and aids in policy making for rotavirus vaccination program in Bangladesh. This work will add valuable knowledge for vaccination against rotavirus and compare the data after starting vaccination in Bangladesh.


Introduction
Group A rotavirus is the main (family Reoviridae) pathogen of gastroenteritis among children under five years in Bangladesh (1)(2)(3).About 300 000 deaths related to rotavirus infection are reported worldwide every year (1).Rotavirus associated death has been reduced in the developed countries due to the implementation of rotavirus vaccination program (4)(5)(6).However, the health burden of rotavirus associated morbidity and mortality are still high in developing countries like Bangladesh without vaccination against rotavirus (7)(8)(9).The estimated incidence of rotavirus is 10,000 cases per 100,000 children under five years in Bangladesh.According to WHO, severe health conditions are reported from 25-30% of rotavirus infected children (8,9).Every year about 2500-3000 children under five die of rotavirus infection in Bangladesh.The TRIVAC model by Pan American Health Organization indicates that vaccination against rotavirus can prevent about 3 million cases and 4000 fatalities among children under five during next 10 years in Bangladesh (7)(8)(9)(10).
Four rotavirus A vaccines are prequalified by WHO and available internationally namely, RotaTeq (bovine-human reassorted vaccine), Rotarix (human rotavirus), Rotavac (bovine-human reassorted vaccine of G9P11), and RotaSiil (bovine-human reassorted vaccine) Besides, several other vaccines are available and licensed for use in India (Rotasiil, pentavalent human bovine reassortant vaccine), Vietnam (Rotavin-M1, human genotyp G1P [8]) and China (Lanzhou lamb, genotype G10P [12]of lamb rotavirus) (10).Though rotavirus vaccination is not included in the national immunization program in Bangladesh, international rotavirus vaccines are available in Bangladesh (9,10,13).The efficacy of rotavirus vaccines namely, Rotarix and RotaTeq is high in developed countries (13).After the introduction of rotavirus vaccination, the cases and fatalities among children under five reduced significantly in the developed countries.However, in developing and under-developed countries the vaccines have lower efficacy.A recent Rotarix trial in Bangladesh has revealed that vaccine efficacy is decreasing and it is below 50%.This lower efficacy may be attributed to the circulation of heterotypic strains and high number of mutational and reassortment events of homotypic rotaviruses (9,10).However, integrated studies on the prevalence and epidemiology of rotavirus are lacking in Bangladesh.Knowing the epidemiology and genetic diversity of rotavirus can contribute in evaluation of effects of rotavirus vaccines in Bangladesh.The main aim of this study is to determine the prevalence and molecular epidemiology of rotavirus in Bangladesh during pre-vaccination period, from 1973 to 2023.

Methods Definitions
Epidemiology of rotavirus is defined as the distribution and determinants of the outbreaks in different population and strategies taken to minimize the health effect among them.The clinical feature was defined including both the signs and symptoms during and after the rotavirus infection.Transmission of rotavirus is defined as the transfer of the virus from source to human and carrier to human.This study included epidemiological, clinical and virological research published in Bangladesh on rotavirus.The rotavirus positive case was determined by ELISA-based method before the invention of RT-PCR laboratory-confirmed RT-PCR test.The standards of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement and Cochrane Collaboration were followed to conduct the study (18).

Study design
The study was performed by following different steps including, identification of precise objectives and search strategies, appropriate research articles, inclusion of manuscripts, collection of data, analysis, and summarization and inclusion of the findings, exclusion of irrelevant studies.We included previous original research articles focusing epidemiological studies, case studies, surveillance work, outbreak investigation, hospital-based surveillance and online databases.No previous study on the topic with strict parameters for the quality assessment is available.As a result, we relied on the quality reports of the selected articles by the authors.

Search strategy and selection criteria
We performed searches for collecting relevant articles in MEDLINE (through PubMed), Web of Science, EMBASE, Scopus, The New England Journal of Medicine (NEJM) and The Lancet with no restriction on language.The place of the search strategy was fixed for Bangladesh.All published manuscripts and scientific writings till December 31, 2022 were included in this study.The notable search term included Rotavirus, RVA, RVB, RVC, Epidemiology of rotavirus, Epidemiology of rotavirus in Bangladesh, Clinical features of rotavirus, Sign and Symptoms of rotavirus, Clinical characteristics of rotavirus, Cases of rotavirus, Transmission of rotavirus, Molecular epidemiology of rotavirus, Genetic diversity, Bangladesh, Children under five, and combination of these search terms.Distinguish searches were made for each term in every website and database.
Further, we performed search on the grey literature and Google Scholar database.These sources included data and articles from CDC (Centers for Disease Control and Prevention, USA), ECDC (European Centers for Disease Control and Prevention) WHO (the World Health Organization), Epicentre, ProMed, IEDCR of Bangladesh, ICDDR, B in Bangladesh.We searched for the yearly update of the data in these websites and databases.Further, articles and data were searched and included from different preprint databases including SSRN, bioRxiv, medRxiv, and AAS Open Research.Additionally, we manually analyzed the first twenty pages on the Google Scholar search system for each search term.We tried to determine the epidemiology of rotavirus by including prevalence over the years, genetic diversity, incidence, case reports, clinical history, case fatality rate, seasonality and vaccination.
Two authors, NS and NS conducted the evaluation of eligible scientific studies.After completion of searching of all the databases and websites, the potential articles were selected by removing the duplicates and screening out the eligible ones by NS and AKP.Articles with specific and relevant topics were selected for all cities and location in Bangladesh, all ethnicity, age group, sex and clinical features for full-text analyzing.We also included the articles focusing on the molecular epidemiology of rotavirus involving animals and human or only animal or only human published during 1973 and 2022.We excluded modelling and prediction studies, correlation of environmental factors studies, review articles and non-relevant studies to our objectives.Further, critical evaluation on the quality of the selected articles, screening out duplicated articles, and removing correspondence or comment of duplicated data were performed by NS, SKD and AK, separately.The seasonal exclusion criteria were implemented only on the studies on seasonality of rotavirus not on environmental impact.
The risk of bias was measured using The Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) assessment tool (19).The SYRCLE is consisted of 10 parameters to assess different biases in this study.The parameter included attrition bias, detection bias, reporting bias, performance bias, selection bias and other biases.The bias for each parameter was measured by using the possible outcomes as yes, no, and unclear, representing low, high, and unclear bias, respectively (19).Data on environmental factors were collected from different websites including, the official website of Bangladesh Meteorological Department (http://live4.bmd.gov.bd/satelite/v/sat_infrared/), meteoblue (www.meteoblue.com),AccuWeather (www.accuweather.com)and WeatherOnline (www.weatheronline.co.uk).Data on sociodemographic factors were included from the analyzed studies and database of national surveillance system on rotavirus in Bangladesh.

Case definition
A confirmed case of rotavirus was defined by following the WHO case definition and final classification guideline, the presence of rotavirus is confirmed by an enzyme immunoassay (EIA) or reverse transcriptase polymerase chain reaction (PCR)-based methods.The type of the used PCR assay will be determined on the basis of local guidelines and availability of the test method.

Statistical analysis
Total number of cases, circulating genotypes and seasonality were determined for each decade by summation of the reported cases per genotypes.Pooled statistical analyses were conducted for determining the overall prevalence of rotavirus and genotypic distribution by using SAS version 9.4.Logistic regression analysis was conducted to determine the impact of sociodemographic factors and environmental factors on the odds of rotavirus infection.

Studies analyzed and included
This study detected 12431 research articles on the prevalence and molecular epidemiology of rotavirus in Bangladesh (Figure 1).Firstly, among 12431 articles, 231 were screened and initially considered for analyzing abstract.Secondly, from 231 articles we selected 96 articles for full text investigation and excluded 135 articles.Thirdly, after analyzing the full texts, we excluded 67 studies and included 29 (30.2%)studies for further analysis.Among the selected manuscripts, we extracted molecular epidemiological data from 29 articles, prevalence data from 29 articles, clinical symptoms from 19 articles (Figure 1).Finally, 23 studies were included in the meta-analysis.

Pooled prevalence of rotavirus infection
Prevalence of rotavirus infection varied from the first report to the last report of rotavirus during the last 50 years in Bangladesh.The pooled prevalence of rotavirus infection was 30.1% (95% CI: 22%-45%, p = 0.005) in Bangladesh before the introduction of mass vaccination.Among these 29 studies during 1973-2023, rotavirus infection was reported from 25 (86.2%)studies (20-50).Rotavirus A (23 of 25, 92% studies) was the most predominant followed by rotavirus B (1 of 25, 4% study) and C (1 of 25, 4% study).Rotavirus A was reported from about 99.93% (45671 of 45679) of the cases (Table 1).Most of the cases (80%) were reported among children.Co-prevalence of pathogenic bacteria, virus and parasites were reported in 7 studies.Male was the most commonly affected group.In majority of the studies, ELISA and reverse-transcriptase polymerase chain reaction was used to detect the presence of rotavirus (Table 1).Before 2010, studies on rotavirus were conducted in only three cities including Dhaka, Mymensingh and Matlab.Procedure of study selection on rotavirus in Bangladesh.The standards of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement was followed to select the studies.

Temporal and spatial distribution of rotavirus infection
Rotavirus infection was detected across the country.The first report was from Matlab, Chandpur during 1977-1979 with lower prevalence and bacterial co-infection (20).Only few studies were conducted in selected cities in Bangladesh during 1975-1999 (20-25).Larger prevalence (>20%) of rotavirus among children was detected in Matlab during 1978-1979 and 1987-1989 followed by Dhaka and Mymensingh during 1988 and 1989 (Figure 2).However, cases of rotavirus were not reported from any other part of the country during 1975-1999.The number of studies  3) (26-49).Majority of the studies were reported from the three cities including Dhaka, Mymensingh and Matlab until 2010 in Bangladesh.Two surveillance studies during 2012-2015 and 2012-2017 included rotavirus cases from 8 divisional cities in Bangladesh and reported higher prevalence of about 64% (Figure 3).Besides these divisional cities studies on rotavirus have been conducted only in limited areas including Sirajganj, Savar and Tangail.Presence of other enteric viruses and bacterial co-infection have been reported from Dhaka, Chittagong, Savar, Tangail, and Sirajganj during 2010-2019 (43,44).
We also found that only limited studies were conducted in Dhaka, Mymensingh and Matlab before 2000 in Bangladesh.The number of studies and localities increased during 2000-2010.Majority of the studies reported rotavirus from Dhaka, Matlab, Mymensingh (91%) and extended to Sylhet, Chittagong, Rangpur, Khulna and Barisal during 2010-2023.This study reports that published documents represent mostly hospitalized patients in selected areas (20-50).In future, more studies including samples from both home staying and hospitalized children with diarrhea from all over the country should be conducted.Hospital oriented surveillance during 2012-2017 included divisional cities and large number of samples.Molecular surveillance is lacking significantly to represent actual diversity of rotavirus in Bangladesh.
Clinical symptoms of rotavirus positive people were similar with previous reports (40)(41)(42)(43)(44). Diarrhea was the most prevalent symptoms followed by dehydration and vomiting.We found fever and abdominal pain inconsistently reported from the infected children in Bangladesh.Studies reporting co-infection of other bacterial pathogens have found higher frequency of fever, abdominal pain and vomiting than only rotavirus infection.Hospitalization was required for majority of the rotavirus infected children (80-100%) in Bangladesh.These findings are in good agreement with previous studies in Bangladesh (40)(41)(42)(43)(44).
We found significant impact of environmental factors and seasonality on rotavirus infection in Bangladesh.Rotavirus infection was detected all around the year with a sharp increase of cases during the winter season (December to February) with average air temperature 19-21°C.Further, we found that risk of rotavirus infection was high at lower temperature, lower UV index and lower amount of rainfall.These findings reflect seasonality and association of increased rotavirus infection with environmental parameters in Bangladesh (21, 23,24,29,30,32,35,36,[40][41][42][43][44].In some of the previous studies two peaks of rotavirus, one in winter and another in summer have been reported (40)(41)(42)(43)(44).However, in this integrated study we found a significant frequency of cases (about 48%) were documented during winter.Our findings are completely supported by previous studies in the developing countries.Previous studies have reported abundance of rotavirus infection among male children aged <5 years.However, lack of statistical analysis to evaluate the relationship between socio-demographic factors and rotavirus infection were found.In this study, we found that male (aOR: 1.48, 95% CI: 1.14-1.92,p = 0.003) children aged <2 years (aOR: 1.76, 95% CI: 1.23-2.32,p = 0.001) living in rural areas had higher risk of rotavirus infection in Bangladesh.These findings will add knowledge on the impact of sociodemographic factors on rotavirus infection.Our findings are supported strongly by previous findings (40-44, 51, 53-68).
The main limitation of this study was not including any data on rotavirus vaccination due to lack of studies.Further, we could not include mutational data of Bangladeshi rotavirus strains due to lack of studies.This study could be improved if data from all the regions were available and included for longer periods.

Conclusions
This is the first meta-analysis including all the studies on prevalence, molecular epidemiology, and genetic diversity of rotavirus from 1973 to 2023 in Bangladesh.This study created an integrated overview of rotavirus infection in Bangladesh during 1973 to 2023.Vaccine coverage of rotavirus is still low in Bangladesh as rotavirus vaccines are not included in the EPI program.As a result, high prevalence of rotavirus (30.1%, 95% CI: 22%-45%, p = 0.005) in Bangladesh contributing to majority of health problems and mortality among children aged <5 years till now.We also found great genetic diversity of rotaviruses in the study regions with high prevalence of G1P [8], G2P [4], G9P [8] and G12P [8].Introduction of existing rotavirus vaccines and development of more effective vaccines to prevent the health burden associated with rotavirus infection are immediately needed.This study will provide overall scenario of rotavirus infection during prevaccination period and aid in policy making for rotavirus vaccination program in Bangladesh.

FIGURE 2
FIGURE 2 Spatial and temporal distribution of rotavirus cases and prevalence in Bangladesh during 1973-1999 and B. 2000-2023.Blue color circle indicated cases, red color circle indicated prevalence yellow color box indicated population density in p/km 2 .

FIGURE 3
FIGURE 3 Spatial and temporal distribution of rotavirus cases and prevalence in Bangladesh during 2000-2023.Blue color circle indicated cases, red color circle indicated prevalence yellow color box indicated population density in p/km 2 .

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FIGURE 4 Year-wise genotypic distribution of rotavirus (A) G-type, data were represented from 1985 to 2019.Five years interval was used.The bar in the right side represented total diversity during 1985-2019, (B) P-type, data were represented from 1990 to 2019.Five years interval was used.The bar in the right side represented total diversity during 1990-2019, and (C) G[P]-types, data were represented from 1990 to 2019.Five years interval was used.The bar in the right side represented total diversity during 1990-2019.

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FIGURE 5 Adjusted odds ratio of having symptoms of (A) Diarrhea, (B) Severe dehydration, (C) Fever, (D) Watery stool, (E) Abdominal pain, (F) Hospitalization among people with rotavirus positive vs negative infection.Blue bar indicated rotavirus positive and brown bar indicated rotavirus negative cases.
FIGURE 6 (A) Seasonal distribution of rotavirus infection described in different studies during 1973-2023 in Bangladesh.According to the variation of average temperature, four seasonal groups were included, December to February, March to May, June to August and September to November.The last bar in the right side represented cumulative seasonality of rotavirus infection.(B) Seasonal distribution of rotavirus positive cases and trends of average temperature in Bangladesh during 1973-2023.

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FIGURE 7Adjusted odds ratio of rotavirus infection by (A) Seasonality, (B) UV index, (C) Average temperature and (D) Average rainfall.

TABLE 1 Continued
*non-human primates.#Birds increased during 2000-2023 in Bangladesh.About 22 studies were conducted on the prevalence and epidemiology of rotavirus on human during 2000-2023 (Figure TABLE 2A Spatial and temporal distribution of rotavirus G type in Bangladesh.

TABLE 2B
Spatial and temporal distribution of rotavirus P type in Bangladesh.
TABLE 2C Spatial and temporal distribution of rotavirus genotypes in Bangladesh during 1973-2023.

TABLE 3
Clinical symptoms of rotavirus infection among children in Bangladesh during 1973-2023.

TABLE 4
Month-wise proportionate distribution of rotavirus cases during 1973-2023 in Bangladesh.