A Study on the Age-Related Changes in Hepatitis B and C Virus Serology

Objective: There are a limited number of studies on the seroprevalence of Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infection in Turkey. This study aimed to compare HBV and HCV seroprevalence rates before and after the implementation of national hepatitis B vaccination program (NHPVP) and to explore age-related changes in the seroprevalences. Materials and Methods: Results of Hepatitis B surface antigen (HBsAg), Hepatitis B virus surface antibody (antiHBs) and antiHCV tests performed during 2009 and 2010 in a state hospital laboratory were reviewed retrospectively. As NHPVP was started in 1998, 12 years of age and younger children were considered postNHPVP period subjects. Results: A total of 3280 HBsAg, 2444 antiHBs and 3188 anti-HCV test results were reviewed, representing approximately 3% of the population of the city where the study was conducted. HBsAg, antiHBs and anti-HCV positivity were 3.2%, 36.12% and 0.97%, respectively. HBsAg and antiHBs positivity were higher among males (p<0.001). When assessed by age groups, HBsAg positivity was found to be the lowest in the age group 0-12 and highest in the age group 51-60. HBsAg positivity was lower in the postNHPVP period versus pre-NHPVP period (p=0.01). Anti-HCV positivity was similar for preand postNHPVP periods (p>0.05). Conclusion: Owing to the use of NHPVP, HBsAg seropositivity has decreased, and it is expected to decrease further in the next years with a wider implementation of the program. Efforts should be continued to increase the level of awareness in the community for HBV and the importance of immunization.


Introduction
Hepatitis B virus (HBV) and hepatitis C virus (HCV) cause liver disease, which leads to major health issues and socioeconomic problems.Nearly one-third of the global popula-tion has been exposed to HBV and approximately 2 -3% of the world population is currently infected by HCV [1,2].
Transmission in high endemic regions is horizontal either prenatally or during childhood [3].This type of transmission is associated with the increased likelihood for the develop-ment of chronic HBV infection [4].Turkey is considered as an intermediate endemic region with respect to HBV incidence (2-8%) and mostly, transmission occurs horizontally during childhood or adulthood [1,5].Persons living in high-risk communities should be screened for HBV and those with negative results should be given HBV vaccination in order to reduce the spreading of infection [6].Studies from Turkey have shown that 0.3-1.8% of the population is infected with HCV and it seems to be decreasing in the young population [7].This study aimed to investigate the rates of HBV and HCV seroprevalence before and after the vaccination program and explore the age-related changes in the seroprevalences in one of Turkey's eastern provinces.

Patients and Methods
This study was based on a retrospective review of medical files of the patients who admitted to Ardahan State Hospital between January 2009 and December 2010.Ardahan is located in the Eastern Anatolian region of Turkey, and a part of it lies in the Eastern Black Sea region.It has a border with the Republic of Georgia in the north east.Total population was 105.454 according to 2010 Turkish census [8].In the county of Ardahan, there is one city hospital where this study was conducted and one town hospital serving a limited number of patients due to physical and technical incapability.The majority of patients residing in the county are admitted to Ardahan State Hospital (study site) for diagnosis and treatment because of its improved capabilities in terms of physical and laboratory facilities and staff.
During the study, Hepatitis B virus surface antigen (HBsAg), Hepatitis B virus surface antibody (antiHBs) and anti-HCV tests performed by the microbiology laboratory were reviewed.Tests were conducted using a fully automated Roche Modular E-170 (Roche Diagnostics; Basel, Switzerland) ELISA instrument and kits.
Test results that were equal to or greater than the cut-off value were considered positive for HBsAg and anti-HCV and an antiHBs value ≥10 IU/mL indicated positivity.To avoid duplicate testing, initial test was taken into account in those patients with multiple tests.Repetitive control testing results for Chronic HBV patients have been excluded.Since the national hepatitis B vaccination program (NHPVP) was introduced in 1998 in Turkey, 12 years of age and younger children were considered post-NHPVP subjects.Subjects were divided into seven groups based on their age: ≤12 years, 13 -20 years, 21 -30 years, 31 -40 years, 41 -50 years, 51 -60 years and age over 60.

Statistical Assessment
Epi Info 5.0 (CDC; Atlanta, USA) statistical software package was used for statistical analyses.Student's t-test was used for comparing quantitative variables and chi-square for comparing qualitative variables.The statistical significance level was accepted at a p value <0.05.

Results
Totally 3280 HBsAg (1983 females, 1297 males), 2444 antiHBs (1484 females, 960 males) and 3188 anti-HCV (1985 females, 1203 males) tests were included in the analysis.Rates of HBsAg, antiHBs and anti-HCV positivity were found to be 3.2%, 36.12% and 0.97%, respectively.Rates of HBsAg and antiHBs positivity were higher among males compared to females (p<0.001)(Table 1).There were no statistically significant differences between genders with respect to HBsAg and antiHBs positivity or anti-HCV positivity among post-NHPVP subjects (p>0.05).However, HBsAg positivity was more prevalent among pre-NHPVP subjects in males (p<0.001,OR (odds ratio): 3.06).On the other hand, no statistical difference was found between genders with respect to anti-HCV positivity during the pre-NHPVP period (p>0.05).Copositivity between HBsAg and anti-HCV was not detected.
Number of tests that performed among age groups in the study period can be seen in Table 2.When the age groups were assessed, the lowest rate of HBsAg positivity was observed for the age group 0 -12 and the highest for the age group 51 -60.Within-group assessment of only HBsAg positive subjects showed that the age group 31 -40 had the highest positivity rate (30.4%).HBsAg positivity was lower in post-NHPVP subjects versus pre-NHPVP subjects (p = 0.01).However, anti-HBs positivity was highest in the age group 0 -12, which constituted the post-NHPVP subjects.Among age groups, anti-HCV positivity was most commonly observed in subjects in the age group >61 years, whereas within-group assessment of anti-HCV positive subjects showed a clustering of cases in the age group 21 -30 (Table 3).HBsAg and anti-HCV positivity was observed to increase with advancing age (Figure 1).

Discussion
Hepatitis B vaccination has been implemented since 1982.It is probably the major preventive measure against the development of HBV infection and its complications.The primary goal of Hepatitis B vaccination program is to immunize all new-borns in order to prevent the occurrence of HBV infection later in the early childhood [9].Vaccination coverage rates in the post-NHPVP era in Turkey were 66% in 2000, 82% in 2006 and 96% in 2010 [10].As a result of the mass vaccination program for HBV, Turkey was included in low risk countries for HBV infection by World Health Organization [11].In this hospital-based retrospective study, antiHBs positivity was found to be 85.6% among children born after the introduction of NHPVP.Consistent with our national data, antiHBs positivity, in other words, vaccination coverage was 100% among children born in 2009 and 2010.
According to the 2010 Technical Report published by The European Centre for Disease Prevention and Control (ECDC), HBsAg positivity is between 2 -8% in general population in  Turkey.Corresponding rates in western and central/eastern cities were reported to be 2 -4% and 6 -8% respectively [1].A community-based prevalence study conducted by the Turkish Association for the Study of the Liver (TASL) found a 4% of HBsAg positivity in Turkish population [12].In our study, HBsAg positivity rate was 3.2%.AntiHBs positivity was the highest in the post-NHPVP group with a rate of 85.56%, whereas lowest rate was observed for the age group 21 -30 (20.5%).Starting from this age group, antiHBs positivity showed a second rise with advancing age and exceeded 40% among subjects older than 50 years of age.This high rate observed in the post-NHPVP group is associated with the efficient implementation of the vaccination program.Similar finding was observed in Yılmaz et al. 's [13] study.They found that the anti-HBc positivity was decreased after the implementation of NHPVP.However, escalation in antiHBs rate after 30 years of age may be associated with an immune response elicited by exposure to HBV.This observation is consistent with the studies conducted in Turkey.A nation-wide research in Turkey reported that while the rate of HBV exposure was around 40% among subjects in their 30s, it increased with advancing age and exceeded 60% in subjects older than 64 years of age [14].The age group 21 -30, which had the lowest antiHBs rate, consisted of the subjects who were not given vaccination or exposed to HBV.We believe that a national policy should be developed for vaccinating this age group.
Another important finding of our study was a 3-fold increased rate of HBsAg and antiHBs positivity among males compared to females (p<0.05).This difference was observed in children born during the pre-NHPVP era and not in those who were born during the post-NHPVP era.This suggests that during the pre-NHPVP era, exposure to HBV was more prevalent among males versus females.It may be associated with risky procedures with no aseptic and antiseptic measures applied, such as blood brotherhood (two or more men swearing loyalty to each other in a ceremony known as a blood oath, where the blood of each man is mingled together), circumcision and the use of alum stone during shaving at the barber's shop.As a matter of fact, studies conducted in Nigeria and Yemen showed a higher HBsAg seroprevalence among traditionally circumcised females compared to females not going through this process [15,16].In recent years, advances in reducing exposure to HBV were achieved by strict implementation of childhood vaccination program and through raising awareness for routes of transmission of HBV and HCV and increased level of education.HBsAg and antiHBs levels in the post-NHPVP era observed in this study corroborate this theory.
While anti-HCV positivity fell to 0.49% in the post-NHPVP era, it was statistically similar to the values in the pre-NHPVP era (p>0.05).The primary reason for this finding is the lack of immunization using a vaccine, as in the case of hepatitis B. However, it was observed that anti-HCV positivity showed a decrease over the years in all age groups.Coskun et al. [7] showed that anti-HCV positivity decreased from 3.35% in years 1998-2000 to 0.74% in years 2000-2005.Increased level of awareness in the community for routes of transmission of HCV may have resulted in lower anti-HCV rates over the years.Continued efforts to educate the community should contribute to further decreases in exposure to both HBV and HCV in the coming years.Media, universities and industrial companies should take collaborative action to inform the community on these issues.However, illicit drug use, which is increasing globally, may also affect anti-HCV positivity in Turkey [17].Thus, particularly younger people should be informed about the possibility of transmitting viral hepatitis by sharing needles.
The major limitation of this study was its retrospective design, which lacked concurrent assessment of HBsAg, anti- HBs and anti-HCV tests for all patients.While all of the three tests were performed in some patients, only two (HBsAg and anti-HCV) tests were available for others.This resulted in a variable number of studied tests.Another limitation of our study was the absence of anti-HBcIgG and confirmation tests such as HCV RNA.Anti-HBcIgG is not routinely performed in hospitals due to economic concerns.Accordingly, it could not be ascertained whether the observed antiHBs results were associated with natural immunity or vaccine-induced immunity.Moreover, this study is conducted as hospital-based and only in one eastern province of Turkey.If this work had been done population-based in more than one city from different regions, the data would be more powerful.
In conclusion, the national vaccination program has changed the HBV epidemiology in Turkey and resulted in an apparent trend towards reduced illness levels.Hopefully, with vaccination, HBV incidence will be further decreased and the risk associated with the virus, representing a major cause of morbidity and mortality for the community will be minimized in the coming years.Additionally, reduction in the complications associated with HBV and HCV would make a substantial economical contribution to national budgets.

Table 2 . Number of tests among age groups in the study period
HBsAg: Hepatitis B surface Antigen; AntiHBs: Hepatitis B surface antibody; Anti-HCV: Hepatitis C virus antibody Figure 1.Changes in HBsAg and anti-HCV rates by age groups.

Table 3 . Hepatitis B and C seroprevalence distribution among age groups
HBsAg: Hepatitis B surface Antigen; Anti-HBs: Hepatitis B surface antibody; Anti-HCV: Hepatitis C virus antibody Eurasian J Med 2016; 48: 37-41