Comprehensive investigation of the prevalence and risk factors of viral hepatitis B and C in PERSIAN Guilan Cohort Study CURRENT

Background Hepatitis B (HBV) and C (HCV) viruses are two serious infectious diseases with high global health impact. The aim of this study was to evaluate the prevalence of HBV and HCV in the Prospective Epidemiological Research Studies of the Iranian Adults (PERSIAN) Guilan Cohort Study through immunological and molecular methods. Methods The blood samples were obtained from 10520 enrolled participant. Complete biochemical and hematological assessments plus urine analysis were done. The presence of HBsAg, anti-HBs, anti-HBc and anti-HCV antibodies for all participants and HBeAg and anti-HBe antibody for HBV positive patients were evaluated. HBV genomic DNA and HCV genomic RNA were extracted from positive serum samples. Real time PCR assay was done to quantify HBV and HCV genomes. HCV genotyping was also performed. Results female


Abstract
Background Hepatitis B (HBV) and C (HCV) viruses are two serious infectious diseases with high global health impact. The aim of this study was to evaluate the prevalence of HBV and HCV in the Prospective Epidemiological Research Studies of the Iranian Adults (PERSIAN) Guilan Cohort Study through immunological and molecular methods.
Methods The blood samples were obtained from 10520 enrolled participant. Complete biochemical and hematological assessments plus urine analysis were done. The presence of HBsAg, anti-HBs, anti-HBc and anti-HCV antibodies for all participants and HBeAg and anti-HBe antibody for HBV positive patients were evaluated. HBV genomic DNA and HCV genomic RNA were extracted from positive serum samples. Real time PCR assay was done to quantify HBV and HCV genomes. HCV genotyping was also performed.
Conclusion Our detected HBV and HCV prevalence were lower than other cities/provinces of Iran, which may be due to the lifestyle or other unknown reasons.

Background
The hepatitis B virus (HBV) is a viral agent whose target tissue is liver and can cause both acute and chronic illnesses [1]. According to the 2016 World Health Organization (WHO) statistics, 240 million people who are positive for at least 6 months of HBsAg are reported as HBV positive individuals [2]. Meaningly, more than 686,000 people die each year due to the effects of the virus, including cirrhosis and cancer [3]. Finally, the highest rates of hepatitis B are found in Africa and East Asia [4][5][6][7].
The hepatitis C virus (HCV) is the main cause of chronic liver disease, which can lead to chronic hepatocellular carcinoma with high economic burden [8][9][10][11]. It has silent epidemiology and at the same time is a major blood-borne infection worldwide [12].
According to the latest global health statistics, 130-150 million people are infected with HCV [13] and 700,000 people die each year [14]. HCV has seven genotypes and 70 subtypes. HCV RNA assays are the most sensitive test for HCV infection and are a gold standard for proving hepatitis C infection [15]. Although certain population sub-groups such as hemophiliacs and hemodialysis are more susceptible to HBV and HCV [20-25], but evaluation of the prevalence of these two viruses in general population also is very important. Considering the importance of hepatitis B and C in this study, the prevalence of these viruses in people referring to the cohort of Guilan province will be discussed.

Participants
This study is nested in the Guilan center of Prospective Epidemiological Research Studies of the Iranian Adults (PERSIAN) cohort study [26,27], which named PERSIAN Guilan Cohort Study (PGCS). The PGCS was started at September 2014 in Some'e Sara (GPS coordinator Latitude: 37.308003 & Longitude: 49.315022), Guilan, Northern of Iran. All residents between 35 to 70 years were enrolled. These 10520 peoples will be followed at least for 10 years to understand new diseases and identify the underlying genetic susceptibility factors for chronic diseases. Moreover, participants were followed up yearly by telephone or medias and they were encouraged to participate in the study. Peoples who were unable to attend the clinic for examination or refusal by a person to participate in the study were excluded [28].

Sampling and biochemical assessments
The aseptic blood samples were collected from the cubical vein into vacutainers. Total number of WBC, RBC, platelet, lymphocyte, monocyte, and granulocytes were counted.
The serum sample was harvested and stored at -20 °C until use for complete biochemical assessment. The Hb concentration and level of Hct, MCV, MCH, MCHC, RDWCV, RDWSD, plateletcrit, MPV, PDW were also evaluated. Urine sample was collected and used immediately for measuring of specific gravity (SG), pH, and creatinine level.

Virological assessments
The presence of HBsAg, anti-HBs, anti-HBc and anti-HCV antibodies were determined by Electrochemiluminescence (Cobas e 411, Roche, Germany). For positive patients, these four tests plus presence of HBeAg and anti-HBe antibody were measured again. HBV genomic DNA was extracted from positive serum samples by viral DNA extraction kit (QIAGEN, Germany). HCV genomic RNA was extracted from positive serum samples by viral RNA extraction kit (Roche, Germany). To quantify HBV and HCV genomes, qPCR assay was carried out using TaqMan-based commercial available kit (QIAGEN, Germany) based on the manufacturer's instructions. HCV genotyping was done using HCV Genotype Plus Real-TM kit (Sacase Biotechnologies, Italy).

Ethical consideration
This study was confirmed by the Ethics Committee of Guilan University of Medical Sciences (Ethics code: IR.GUMS.REC. 1396.254).

Statistical analysis
Qualitative data were expressed as frequency and percentage and their association with HBV and HCV statuses were analyzed using Chi square test. Quantitative data were presented as mean ± SD and between HCV/HBV positive and negative groups differences were analyzed using two independent sample t test. All statistical analysis were performed using SPSS version 23. The P value lesser than 0.05 was considered as significant difference.

Results
Demographic characteristics of our patients and statuses of HBV and HCV infection are presented in Table 1. Most of our participants were female (53.5%), rural (56.1%), married (97.2%) with primary education (< 12 years) (72.1%) without smoking (75.2%) or alcohol consumption (85.3%). In addition, most of them had history of surgery (63.3%) and hospitalization (80.6%) and had no transfusion (89.5%) or genital aphthous (98.8%). The HBV prevalence was 0.24% (95% CI, 0.16% to 0.36%) and the HCV prevalence was 0.11% (95% CI, 0.06 % to 0.19 %). The geographic distribution of HBV positive and HCV positive patients based on gender are presented in Figure 1. Rural participants were significantly more HBV positive than urban peoples (P=0.045) while male individuals were significantly more HCV positive than female participants (P=0.013). No other significant associations were detected between other evaluated demographic variables with HBV and HCV prevalence.
The complete blood and urine analysis of our participants are presented in Table 2 Table 3.
Most HBV positive patients (52%) had lesser than 300 copies of HBV DNA per ml. While, most HCV positive patients (58.4%) had 10 5 -10 6 copies of HCV RNA per ml. Most detected HCV genotype was 2a ( Figure 2). First-degree relatives of all HCV positive patients were also checked for HCV infection by qPCR. Just child of one patients had HCV infection with genotype similar to her mother as 1a.

Discussion
In the present study, the prevalence of HBV and HCV in the Guilan site of PERSIAN cohort were reported. We found the prevalence of 0.2 and 0.1 for HBV and HCV, respectively.   positive, as we found in this study, also reported previously [62]. It can be said that, both HBV and HCV influenced the liver tissue and the changes in biochemical and hematological parameters can be related to these changes in the hepatic functions.

Conclusions
In summary, we found lower HBV and HCV prevalence compared to other regions of Iran.
Also, compared to previous reports from our province, Guilan, the HBV and HCV prevalence also decreased. This may be due to the preventive strategy or increase of the medical knowledge of peoples, which must be evaluated in the further studies.

Availability of data and materials
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Ethical approval of the study was obtained from Guilan University of Medical Sciences.
(Grant number: 1397.163). Written consent was taken after informing the purpose and importance of the study to each participant. To ensure confidentiality of participant's information, codes were used where by the name of the participant and any identifier of participants was not written on the questionnaire.

Consent for publication
Not applicable.