Molecular Epidemiology Of Hepatitis E In Jiangsu Province And Trend Analysis Of Global Research

IntroductionGlobally,hepatitis E virus(HEV)is the most common cause of acute viral hepatitis.While both outbreak and sporadic cases of HEV are reported in resource-limited countries,zoonotic transmission is considered the main source of the HEV infection in industrialized countries.HEV,a small(approximately 27-34 nm in diameter)non-enveloped positive-sense single-stranded-ribonucleic acid(ssRNA)virus,belongs to species Orthohepevirus A and can be divided in to eight distinct genotypes(HEV-1-8)infecting both humans and animals.Domestic pigs and wild boars are the main reservoir of animal HEV The spread of HEV in to the environment can pollute surface waters,which then can be the HEV infection source for humans and animals.The HEV transmission usually occurs through the fecal-oral route,and public health implications of its water-borne and food-borne transmissions along with environmental contamination raise high concerns regarding its zoonosis.Acute or chronic clinical manifestations are though linked to hepatitis but can involve extra-hepatic systems producing neurological,renal,or hematological abnormalities where the asymptomatic cases remain normal.Every year,nearly 20 million cases of HEV occur worldwide,of which 3.3 million infections are asymptomatic hepatitis.In 2015,according to the World Health Organization(WHO)estimates,approximately 44,000 deaths due to HEV have been reported worldwide.Rationale of the studyThe available literature shows that,the prevalence of HEV is high in developing countries.In developed countries,in the recent years the foodborne and zoonotic transmission of HEV has become a serious public health issue.Several studies reported a range of hosts responsible for spreading of HEV infection in humans.Therefore,the current study focused on the distribution of HEV in the context of one health approach.Justification of the studyIn developed countries,the revolution in the health industry has resulted in better health services for communities and reduced the financial burdens on individuals.However,developing countries are still lacking in the health facilities;therefore,they have to spend more on health treatments especially those,which involve the molecular testing.By understanding the distribution of HEV in selected cities of Jiangsu province,China this study is of worth importance for understanding the disease burden,and to improve the control measures and prevention strategies.Overall aimThe aim of the current study was to use serological and molecular approaches to determine the prevalence of hepatitis E in selected cities of Jiangsu province,China.Furthermore,to systematically assess the pooled prevalence of HEV infection in a high-risk population and to understand the global trends in hepatitis E research.Main objectives1.To find out the frequency distribution of hepatitis E in humans and pigs.2.To identify the existing genotypes of HEV.3.To systematically assess the published literature and determine the global pooled prevalence of hepatitis E infection in pregnant women.4.To characterize the global research trends and visualization mapping in hepatitis E research.Materials and Methods[A]Molecular epidemiologyEthical approvalThis study was approved by the Ethical Committee of "Jiangsu Provincial Centre for Disease Control and Prevention,China".All the experiments were performed according to the standard protocols and following the legal ethical procedure "The Declaration of Helsinki(1964)".Study designA molecular epidemiological cross-sectional study was conducted.Study subjectsThe study subjects were the human population,farmed pigs and the drinking and sewage water collected from the Yancheng and Zhenjiang cities of Jiangsu province.Samples collectionFrom each patient a sample of approximately 5 ml of venous blood sample was collected and kept it at room temperature for overnight to allow clotting and centrifuge.Serum was separate and stored at-80℃ before used.From new born to adult pigs’ bile and fecal samples were collected.The bile samples were collected from slaughter pigs in a 50-ml sterile centrifuge tube.The collected bile samples were suspended in 10%in 10%(w/v)diethyl pyrocarbonate-treated water,and further processed for HEV RNA detection.SerologyThe human serum was tested for the presence of anti-HEV antibodies using Enzyme-linked immunosorbent assay(ELISA)according to manufacturing instructions(Wantai Biologic Pharmacy Enterprise,Beijing,China).HEV RNA detectionThe samples were further tested for detection of HEV RNA using real-time fluorescence quantitative polymerase chain reaction(qPCR).The HEV RNA was extracted from 200 μL human serum using Trizol Reagent(Invitrogen,Carlsbad,USA).The cDNA was synthesized using a reverse transcriptase kit according to company instructions(TaKaRa,Tokyo,Japan).For the ORF2 fragment amplification a pair of primers was used(forward"5’-ATCCCGCATG ACATCGAC-3’",reverse "5’-GGTCGGT CCTGCTCATGTT-3’",and probe"FAM5’-CGGGGAATCTCGTGTAGTTATTC-AG-3’-TAMRA"),and the PCR conditions were as follows;"1 cycle of initial denaturation 94℃ for 5 min","35 cycles at 94℃ for 30s","55℃ for 30s",and "72℃ for 1 min",and a final extension at "72℃ for 5 min".The amplicon was separated through agarose gel electrophoresis(1.5%)and then visualized by ethidium bromide fluorescence under the ultraviolet(UV)rays.Statistical analysisThe obtained data was exported into Microsoft Excel 2019,and the result values were presented in frequencies and percentages.The data were presented in tables,figures,graphs,and pie charts.The required graphs and pie charts were generated using OriginPro 2018.For data management and statistical analysis,the relevant statistical tools and packages were used i.e.“Statistical Package for the Social Sciences"(SPSS)version 25.0 software for windows.The results of the descriptive statistics are presented in mean,frequencies or percentages.Chi-square and Fisher’s exact tests were used to find the association of HEV antibodies with demographic and clinical data.Simple and multiple logistic regression analyses were performed to determine the relationships of gender,age,profession,blood transfusion,and HEV antibodies.Mann Whitney u test was used to compare the median age and weight between positive and negative qPCR in pig samples.Fisher’s exact test was used to compare the proportion of positive qPCR among collection areas.Simple logistic regression analysis was used to determine the relationship of age,weight and sample collection areas with PCR findings in pig samples.Odds ratio(OR)and 95%confidence interval(CI)were calculated and a P-value less than 0.05 was recorded statistically significant.[B]Meta-analysis sectionMethodologyData sourcesThe ScienceDirect and PubMed databases were utilized to retrieve relevant studies on HEV published in English language between 2015 to 2018.Data analysisThe extracted data were analyzed by using Stata,version 12.0 and R software,version 3.6.0 for windows.The pooled prevalence was determined by applying the random effects model at 95%CI.[C]Bibliometric analysisMethodologyA bibliometric study was conducted to identify and characterize the global trends in HEV research.A comprehensive search was performed in the Web of Science Core Collection(WoSCC)database without any search limitations.The keyword "Hepatitis E" was used in the title field.The retrieved dataset was assessed for a number of attributes including author,journal,publication year,document type,institution,category,funding source,and country.The obtained dataset was exported in to Microsoft Excel 2019 for data arrangement and descriptive analysis.Furthermore,the data were exported into HistCiteTM and VOSviewer software’s for windows for citations analysis and visualization mapping respectively.Results[A]From July 2018 to November 2019,a total of 430 human serum samples were examined.The mean age of study participants was 48.1 years.Of which,59.1%was male and 40.9%was female.The prevalence of anti-HEV-IgG antibody was 50.9%(219/430),with high prevalence in male 57.5%(126/219),while the anti-HEV-IgM antibody was detected in 6.3%(27/430)samples.Age wise distribution shows that,individuals between 41 to 60 years were more affected than other age groups(P=<0.001).The individuals with farming profession had high prevalence of HEV 65.3%(143/219)(P=0.001).The anti-HEV-IgG antibody prevalence was high in Dongtai location 86.8%(190/219)(P=<0.001).HEV RNA was detected in 18.3%(11/60)samples,of which HEV sub-genotype 4d was detected in 72.7%(8/11)samples.A total of 344 pig samples(bile samples)were examined.The mean age of pigs was 5.6 months with mean weight 103.8kg.In total,HEV RNA was detected in 10.5%(36/344)samples.The Changgang COFCO Breeding Department(45%),Tang Yang(26.2%),and Xinjie Jinyang(20%)areas had higher prevalence(P=<0.001).HEV genotype 4 was detected in 69.4%(25/36)samples.[B]In pregnant women,the pooled prevalence of HEV was 16.51%(95%CI:0.10-0.23).The highest prevalence of HEV was in Sudan(61.29%)and the lowest was recorded in Italy(3.41%).The heterogeneity level was I2=98%;p<0.01.[C]A total of 4,642 publications were analyzed.These publications were written by 13,818 authors published in 714 journals.The top leading author in hepatitis E research was Izopet J(n=172,3.7%).The "Journal of Hepatology" was the top leading journal(n=210,4.5%).The most frequent year of publication was 2018(n=390,8.4%).Network visualization mapping shows that United States of America(USA)was the top ranked country with highest total link strength.Conclusions[A]The infection rate of hepatitis E is high in Jiangsu Province,especially in men ages 41 to 60 years old.Hepatitis E infection still exists in pigs.The most common HEV subtype was 4d.[B]In pregnant women,the global pooled prevalence of HEV was high,particularly in African countries.[C]In HEV research,the highly contributing country was USA followed by China and France.PreventionConsidering the importance and wide circulation of HEV in humans and animals,global public health priority needs to be given to enhancing monitoring and surveillance as well as adopting adequate mitigation strategies for prevention and control of HEV and associated zoonotic significance of the virus.There is a need to increase awareness about precautionary measures of safe drinking water and food practices,strengthen diagnosis and monitoring,health care and treatment options,drugs and therapeutic regimens,adapt large-scale vaccination along with mobilizing resources on a priority basis.RecommendationsA better understanding of the viral dynamics and disease process can be elucidated by developing models that are amicable to natural host or disease pathology to support future research and minimize risks.Special attention should be paid to vulnerable and high-risk populations.In the absence of specific knowledge and lack of interventions,general preventive measures can be helpful.The best practices to prevent the HEV in endemic areas are improve safe drinking water and food supply,following appropriate standards of personal hygiene and sanitation,preventive measures in blood safety and organ transplantation procedures are strongly needed.The HEV zoonotic transmission is a significant public and environmental health challenge.Consumption of uncooked pork meat and meat products constitute the high-risk factors associated with HEV.These need to be checked adequately to prevent food-borne transmission of this virus to humans.Prevention and control strategies need to be directed particularly for the protection of immunocompromised individuals,by limiting the sources of HEV Special attention should be paid to vulnerable and high-risk populations.Safety measures and disease awareness are of great importance while traveling in HEV endemic areas.Advances in research with regards to the development of effective diagnostics,vaccines,drugs and therapeutics need to be exploited to their full potential to counter this important virus,which has high public health concerns.There is a dire need for effective treatments and a commercially available vaccine to prevent and control the HEV infection with particular emphasis on low-income countries.