, Abdulrahman Y. Al-Haifi2
, Najlaa Abdullah Mohammed Al-Mutaa
2, Hassan Abdulwahab Al-Shamahy3,4
IMMUNOLOGICAL STATUS OF HEPATITIS B VIRUS INFECTION AMONG FRESHMEN UNIVERSITY STUDENTS IN YEMEN
Abdul Salam Mohamed Al Makdad1, Abdulrahman Y. Al-Haifi2, Najlaa Abdullah Mohammed Al-Mutaa2, Hassan Abdulwahab Al-Shamahy3,4
1Department of Medicine, Faculty of Medicine, Dhamar University, Dhamar, Yemen.
2Department of Microbiology, Faculty of Medicine, Dhamar University, Dhamar, Yemen.
3Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University, Republic of Yemen.
4Medical Microbiology department, Faculty of Medicine, Genius University for Sciences and Technology, Dhamar city.
Background: Hepatitis B virus (HBV) is a most important health problem, it's a global pandemic. Previous surveys showed a high prevalence of hepatitis B infection in Yemen. Hepatitis B vaccination is the most efficient way to prevent hepatitis B virus infection and its outcomes (such as hepatocellular carcinoma, cirrhosis, and liver failure).
Aim: To measure the immune status of hepatitis B virus amongst first-year students at the faculties of Thamar University during the academic year 2021-2022.
Methods: A total 196 of first-year students participated in this cross-sectional study and tested for anti-HBsAg and the antibody to HBV core antigen (anti-HBc) and HBsAg by using ELISA during the period from January to May 2022 at Thamar University. An anti-HBs antibody titer ≥10 mIU/mL was regarded as being protective against HBV infection.
Results: In this study, 22.4% of the students had protective levels against hepatitis B, while 77.6% had a non-protective level. HBV markers showed that 19.90%, 2.55%, and 75.51% of the students had been vaccinated and immunized due to a previous infection and exposed to HBV infection respectively and only 2.04% of them were non-obvious cases. The prevalence of anti-HBc was 4.6%. Risk factors that showed statistical significance were found between positive anti-HBc and family history of HBV (p=0.01).
Conclusion: The low prevalence of hepatitis B protection levels in Thamar University medical students needs further research and necessitates implementation of a screening and vaccination program for all non-immunized healthcare students.
Keywords: Antibodies, Hepatitis B virus, Immunization in Yemen, Low immunity.
INTRODUCTION
Of the global public health problems, hepatitis B infection is one of the most important. HBV belongs to the family Hepadnaviridae with partially double-stranded DNA1-3. Approximately 2 billion people worldwide have evidence of past or current infection with hepatitis B virus, 358 million or more people suffer from chronic lifelong infection, and about 887,000 people die each year from hepatitis B outcomes1-3. Hepatitis B virus is endemic in Yemen, the prevalence of HBsAg-positive virus in the general population and HCWs ranges from 8% to 20%, among infants, it was 4.1%, and up to 50% of health workers and residents have previous serologic evidence of HBV infection and this is all in the old reports4-8. On the other hand, recent studies have revealed that the incidence of HBsAg, is 0.7-2% among the general population and 4% among high-risk groups such as HCWs and hemodialysis patients and HBV also decreased further among children9-15.
It is well known that HBV vaccine is the foundation of hepatitis B infection prevention. To reach this, the vaccine must activate an immune response which would create protective hepatitis B surface antibody (anti-HBs) at a concentration of ≥10 mIU/mL at least 1 month and at most 2 months after the 3 dose6, this happens in more than 95% of infants, children and young adults. But the persistence of anti-HBs and thus protection against infection and the carrier state depends on the peak concentration of anti-HBs reached after the initial vaccination. One problem with this is that anti-HBs decay exponentially with the length of time since vaccination and so a booster shot is required6,17. Factors connected with reduced immune response to HBV vaccine comprise increasing age, nutritional status, gender, obesity, genetic factors, and smoking. Poverty, socioeconomic status, low of education, and weak health systems in Yemen are interrelated factors that influence nutritional status of people, which in turn affect their immune system6.
Study carried out in Saudi Arabia for students of Taibah University elucidated that the hepatitis B markers showed that only 15.2% of students had protective levels against the disease, while the rest showed negative markers17. Similar study conducted in Iran for first year medical students was demonstrated that 36.2% showed a non-protective anti-HBs response (anti-HBs < 10 mIU/mL) and 164/257 individuals (63.8%) showed a protective anti-HBs response (anti-HBs≥ 10 mIU/mL)18.
To our knowledge, there are no published studies on the immunological status of hepatitis B virus among university students in Yemen. Therefore, the aim of the current study is to assess the immunological status of hepatitis B virus among first-year students at Thamar University, during the academic year 2021-2022.
SUBJECTS AND METHODS
Study area: This study was carried out at Thamar University located in Dhamar city; Dhamar governorate (15°40’N 43°56’E) is located at the central area of the western highlands region of Yemen1600–3200 meters above sea level.
Study design and sample: A cross-sectional study was conducted from January to May 2022 (the time allowed for fieldwork for a master's thesis) at Thamar University, Dhamar Governorate, Yemen. The study targeted the first-year students at all faculties of Thamar University. The total number of students enrolled at the time of the study at Thamar University in their first-year was 1724 students and the study conducted on 196 respondent students for the assessment of their immune status by conducting Anti-HBs, Anti-HBc and HBsAg tests.
Inclusion Criteria: First year students from all faculties of Thamar University who were present during the sample collection and who signed consent form to participate in the study.
Exclusion Criteria: First year students who are not available at the time of data collection due to different reasons (absence, sick leave, maternity leave etc) and students who declined to offer consent to the study.
Sample Size Determination: Sample size was calculated by EPi Info 7™ using the STATCALC utility based on a 15.2% predicted frequency17, a 95% confidence level, 5% confidence limits, and a 90% response rate. Therefore, the sample size was planned to be 196 students among all first-year students in the faculties of Thamar University (total=1724).
Sampling Method: The choosing for them by systematic random sample method among all Thamar University first year students from all faculties (196 students out of 1724) and according to gender was included within two groups [50% females and 50% males].
Data Collection: Data was collected by pretested structured questionnaire. The study variables include Socio economic factors (monthly income, Fathers and mothers education level etc.), demographic factors (residence, age, sex etc.) and potential risk factors of HBV infections (Previous history of surgical operations, a history of infected family, share of personal objects and blood transfusion etc.). Results of HBV markers that was detected, they was added to questionnaire chart. The potential risk factors were used as the independent variables, while the positive results of HBV markers was considered as the study outcome (dependent variables).
Collection of Blood sample: From each student; five mL of whole blood aseptically by venipuncture was collected. After clotting of the blood serum was separated by centrifugation. At –20°C sera specimens were kept until tested for the HBV markers.
Laboratory test: All samples were tested for Anti-HBs, Anti-HBc and HBsAg. Serological assays for of the HBsAg, Anti-HBsAg and total Anti-HBcAg serological markers were performed on the ELISA System (Roche Cobas e 411 analyzer) using the electrochemiluminescence immunoassay “ECLIA”.
Statistical analysis: The data were analyses performing Epi Info statistical program version 6 (CDC, Atlanta, USA). Conveying the quantitative data like mean values, standard deviation (SD), as the data were normally distributed. The qualitative data were expressed as percentages; for comparison of two variables to determine the p value, the Chi square test was used. Odd ratio (OR) was used with 99% confidence interval. p value <0.05 was regarded as statistically significant.
Ethical consideration: From all students consents were taken and students notified that participation is voluntary and that they can refuse without giving any reason.
RESULTS
A total of 196 students participated in this study in their first academic year at Thamar University with the age range between 18 and 22 years and the mean age (±SD) age of 20.14 (±1.09) years. Half 50% of the participants were male and nearly two-thirds 76% of them lives in urban areas.
Distribution of Anti-HBs level by gender: 22.4% of students had protective level against HBV, the percentage of males and females with low immunity were the same 6.1%, while, the percentage of females who had adequate immunity and high immunity were 6.1 and 0.5 versus 2 and 1.5 of males respectively. There was no statistically significant variation between both sexes (Table 2).
Distribution of Anti-HBs level by Age: Table 3 shows that no correlation was found between age and anti-HBs level *=Kruskal Wallis test; χ2=3.48, p=0.32. About 77.6% of students had anti-HBs titer <10 mIU/mL, 12.2% had between 10-100 mIU/mL, 8.2% had between 100-1000 mIU/mL and only 2% above 1000 mIU/mL. There was no significant association between anti-HBc positive status and risk factors, except, family history of HBV infection χ2=9.74, (95% CI 1.74 –8.62); p=0.01. Being cupping showed a trend but not a statistically significant difference χ2=1.28 (95% CI 1.28–42.00) p=0.057 (Table 4).
DISCUSSION
The present study showed that a high proportion of students had a non-protective (anti-HBs <10mIU/mL) titer of 77.6% against hepatitis B virus, while only 22.4% of the students had a protective titer (anti-HBs ≥10 mIU/mL). The present findings of non-protective titer are consistent with those of Mosaad et al.,17 who reported that more than 84.80%, of medical students had no protection anti-HBs levels. In contrast to the present findings, two Iranian studies among dental hygienists and medical students showed that most of the students had a protective titer against hepatitis B virus of 93.6% and 95.1%, respectively19,20. The difference in the rate of protection against hepatitis B virus can result from the difference in coverage of HBV vaccine among the target groups in the studies, the difference in the economic level, the geographical and regional differences, and the consideration of the cold chain for vaccine storage, the vaccination periods, also from the number of vaccines injected, and the type of combination applied to assess the titer of HBs, the genetic variation of the participants, the sex, the greater obesity, the age, the place and method of injection as well as the nutritional status6,21.
According to sero-analysis of hepatitis B markers, the present study showed that 19.9%, 75.51% of the students were vaccinated and susceptible, respectively. The overall prevalence of anti-HBc in this study was 4.6%. Similar rates have been reported in Iran 4.9%22 and Jordan (2.0-4.1%)23. Higher rates of anti-HBc positivity have been reported in studies conducted in Syria (10.3%)24. Of the 4.9% of current tested students with positive anti-HBc, 2.04% had unexplained cases (positive anti-HBc and negative anti-HBs).
Pattern of infection with hepatitis B virus, complete recovery from acute and chronic hepatitis B correlated with loss of HBsAg and appearance of anti-HBs in serum. Thus, anti-HBc is usually accompanied by HBsAg or anti-HBs. However, the detection of “anti-HBc alone" is not an uncommon serological pattern. On the other hand, this pattern is one of the more confusing HBV results and can have several possible interpretations such as resolving acute HBV infection, i.e., in the period between HBsAg loss and detectable anti-HBs development, false-positive results, in chronic and past infections as well as anti-HBc alone is the most common seromarker in Occult hepatitis B virus infection (OBI) individuals. The incidence of OBI in anti-HBc (+) but anti-HBs (−) blood donors has been reported to be as high as 7–15% making this an important clinical issue, therefore, screening for anti-HBc can help to identify OBI25-27. As well, in this study family history of HBV infection was significantly associated with anti-HBc positive status. This finding is consistent with recent study conducted in Ethiopia28. However, Risk factors such as male gender, surgical operations, dental procedures, blood transfusion and sharing shaving instruments were not significantly associated with anti-HBc positivity (Table 4).
Serological tests for hepatitis B markers also showed that only 11/54 of the students who said they had received the HBV vaccine were vaccinated, whereas the majority of them were susceptible 39/54. A possible interpretation is that the antibody titer drastically decreased with time. In addition, 3–20% known vaccinated failure rate, which can be attributed to vaccine factors (e.g. type, dose, schedule and injection site) or host factors (e.g. male sex, smoking, and chronic illness)6,23. However, the cases where the subjects were vaccinated and had low or undetected titer cannot be interpreted as having vaccine failure for two main reasons: first, to indicate vaccine failure, post-vaccination testing must be performed within 1-2 months after the third dose of the vaccine has been administered; second, the current study did not explore their memory cells to show if they still had anti-HBs antibody-secreting cells29,30. The level of anti-HBs among the study subjects was not significantly affected by age, as the range was narrow enough, 18-22 years, not to show any statistical difference. This results agreement with results of AL-Shamahy et al.,6,7 and not agreement with results of many studies that showed had proven antibody levels decrease with over time and increasing age31,32. A decrease in the level of anti-HB in the blood mainly indicates a decrease in protection and the necessitate for a booster dose of the hepatitis B vaccine. As per WHO recommendations, booster immunization for hepatitis B virus is not suggested and protection lasts for at least 20 years, and probably lifelong. Nevertheless, numerous studies emphasize the importance of booster doses for stimulating the memory immune system and maintaining a higher protective rate of anti-HBs. Boosters will activate immune memory and provide reassurance of protective immunity against super-infection33,34.
The present study showed slightly higher protective rate of anti-HBs antibody in females (25%) compared to males (19.4%) but this variation was not significant (p=0.30). The same findings were reported in previous studies conducted in Yemen6, and other countries35,36; Gender differences might be due to the opposite effects of sex hormone androgen and estrogen. Moreover, there are numerous immunological genes appearing on the X chromosome while few ones are mapped on the Y chromosome. Estrogen activates monocyte to secrete IL-10, which induces Immunoglobulin G (IgG) and Immunoglobulin M (IgM) secretion through B-cells in turn, while testosterone damages the production of IgG and IgM from B-lymphocytes, as well as restrains producing IL-6 from monocyte37.
The results of this study showed that there is a statistical significance between the immunological status of the students and the education of the mother (p=0.04) (Table 1), and most of the mothers of unprotected students had a level of education less than secondary (82%). A study previously conducted in Yemen showed that the social and economic status and lack of education, in many regions of Yemen are interrelated factors that affect the growth of children, which in turn affects their immune systems6. In this study, we did not find a significant correlation between levels of Anti HBs and smoking. This result was in agreement with study conducted by Peces et al.,38 and it was not in agreement with a similar study conducted by Alavian et al.,20 which showed that there is a relationship between smoking and decreased immune response to HBV vaccine. Chronic diseases such as autoimmune hepatitis and kidney failure are risk factors for vaccine non-response and reduced body immunity39,40.
However, in the current study there is no significant relationship between the history of chronic diseases and the immunological status of the students. Perhaps this is because the subjects who took part in the study were young, and there were no common chronic diseases among them.
Limitation of the study
The main limitations of this study included small sample size, and potential self-reporting errors in the questionnaire. Nevertheless, this study could serve as a bridgehead for further studies with larger sample sizes to test the findings discussed.
CONCLUSION
The low prevalence of hepatitis B protection levels in Thamar University medical students needs further research and necessitates implementation of a screening and vaccination program for all non-immunized healthcare students.
ACKNOWLEDGEMENTS
The authors would like to thank the Thamar University, Dhamar city, Yemen for the support.
CONFLICT OF INTEREST
No conflict of interest associated with this work.
AUTHOR’S CONTRIBUTIONS
This research is part of a master's degree in the Biology Department, Faculty of Applied Sciences, Thamar University, second author Najlaa Abdullah Mohammed Al-Mutaa, who conducted field work, and who did laboratory work and other authors contributed to data analysis, drafting and review of the paper, and gave final approval to the research.
REFERENCES