Prevalence and Associated Risk Factors Analysis of Hepatitis B & C Infections in the Low Socioeconomic Communities; A Cross-Sectional Study from Pakistan

Background: Pakistan is bearing the second highest global burden of hepatitis C and B virus, infecting 3-4 % of its overall 22 million population. These infections remain unchecked in most of the cases and such incidences become a continuous source of infection to the healthy population. Maximum efforts for screening, prevalence and surveillance of these viral infections is needed to stem the devastating impact on the underprivileged communities living in the outskirt of major cities. Methods: This study was designed to determine the prevalence of hepatitis B & C and the corresponding risk factors among the low socioeconomic communities of Islamabad. Participants (aged 10-70 years) were recruited from six localities inhabited with people living in underprivileged conditions. Relationship between hepatitis B/C incidence, demographics and risk factors was measured using Pearson's Chi-square test, univariate and multivariate regression analysis. A total of 1004 individuals were enrolled in this study. Results: Out of 1004 individuals, almost 4% were found positive for hepatitis C and 1% for hepatitis B after screening with PCR. Pearson’s Chi-square test showed a strong relationship of hepatitis B/C infection with marital status (p= 0.000), hepatitis B vaccination status (p= 0.000), blood or blood product recipient (p= 0.000), having a tattoo, family income (p= 0.026) and participant age (p= 0.000). Multivariable analysis showed hepatitis B vaccination odds ratio (OR) =5.309 (95% condence interval [CI] 2.812-10.025), population exposed to therapeutic injections four-times/past 6 months OR=4.328 (95% CI 1.319-13.617) dental visit four-time/past six months OR=11.9 (95% CI 3.350-40.098) people having exposure to HCV patients ≥ 6 times/past six months OR=3.095 (95% CI 1.577-6.074) and age of the participants OR=1.049 (95% CI 1.026-1.072) were independently associated with hepatitis C infection. Conclusions: These ndings show that the risk of hepatitis B/C is multifactorial. However, on multivariate analysis, no association was found between hepatitis C incidence and blood donation, blood recipient, nose/ear piercing, barber visit, tattooing, drug abuse, marital status, family income and education status.


Introduction
According to WHO estimation, every 1 in 3 people in the world have been infected with either hepatitis B or C. This accounts for about 3.9% (291.9 million) infections due to hepatitis B virus (HBV) and around 2.5% (177.5 million) infections due to hepatitis C virus (HCV) [1,2]. Every year, 1.4 million people die due to viral hepatitis, of whom 90% of deaths are due to hepatitis B or C [3,4]. Pakistan is bearing 2nd highest burden of global hepatitis C prevalence with 7.44% population carrying HCV [5]. Around 1.98% of the Pakistani population is having HBV antigen in their blood [6]. The population with chronic HBV or HCV infection is at high risk of developing liver cirrhosis, leading to hepatocellular carcinoma and metabolic disorders and in severe case, it can lead to a life-threatening situation [7].
Several risk factors such as ear/body piercing, intravenous drug abuse, tattoo, blood transfusion, needle stick injury, reuse of syringes, barber visits and dental checkups are reported to be associated with hepatitis B or C infection [8]. Moreover low socioeconomic status and lack of awareness about disease transmission routes also contribute signi cantly towards the increase in disease incidence [9]. To achieve the WHO target of eliminating 80% of hepatitis C incidence by 2030, each year about 36 million screenings need to be done and 660,000 cases need to be treated annually. Moreover, it would be ensured that 90% of diagnosed cases get treatment [10]. Therefore, ndings of the current study proposed to screen the general population of Pakistan at a large scale and ensure the HBV/HCV positive cases get proper treatment. In addition to that, we aim to qualify high-risk factors posing serious threats to a speci c population. Furthermore, this study intends to increase the knowledge about disease transmission in the general population, so that new cases could be prevented.

Study design, site and population
This cross-sectional study was conducted in Islamabad, the capital territory of Pakistan. The study population consisted of people belonging to lower socioeconomic status and was less educated in terms of formal education. Six regions consisting of such population were selected at random which included Police line H-11, IGFG church area F-7, G7/1, Iqbal Town, Abpara police station and Sohan near Faizabad. This study was conducted by setting up free awareness and screening camps from August 2018 to April 2019.

Sample size
The sample size was calculated using an online tool (https://www.surveysystem.com/sscalc.htm). Choosing a 95% con dence level and con dence interval of 3, the sample size for this study in Islamabad with the population of 1,095,000 people was calculated as 1066 individuals.

Inclusion and exclusion criteria
Participants having less than 10 years of age and greater than 70 years of age were excluded from the study. Only participants from 10 years to 70 years of age included in the study. Participants having total family income greater than 90,000 PKR were excluded from the study. Only male and female gender were included in the study and participant belonging to the third gender were excluded.

Study instrument and variables
To obtain demographics and information about exposure to risk factors, using previously reported literature [11], a structured questionnaire was developed in the Urdu language. Demographics included age, gender, education, total family income/month, family members living in the same house and marital status.
Information about exposure to risk factors such as tattoo, history of drug abuse, history of blood donation or receiving and having a hepatitis B/C positive family member was recorded as yes or no. History of nose/ear piercing was recorded as none, one time, two times and three or more. Response to barber visit, exposure to injection, exposure to hepatitis B/C positive patient and dental checkup were recorded as none, one time, two times, three times, four times and six or more times per last six months.
The questionnaires were distributed among the camp participants. Those participants who could not read and write recorded their response by dictating it to camp volunteers. We got n = 1040 questionnaires back, out of which n = 33 was incomplete and hence excluded from the study. Those cases n = 3 which were positive for HCV antibodies but did not have detectable HCV RNA were also excluded from the study. Hence total n = 1004 cases were included in the current study.

Screening of HCV antibodies and HBV antigens
One-step rapid test kits (HEALGEN) were used for the detection of anti HCV antibodies and hepatitis B virus surface antigen (HBsAg) using whole blood.
Positive samples were run in duplicate and sample showing a positive result for the second time were reported as positive samples.

Con rmation of positive samples
Serum of people reported positive with one step rapid test kit was subjected to qualitative PCR using SaCycler-96 (Sacace-Italy) for the con rmation of HBV DNA and HCV RNA.

Data Analysis
The data of the current study was analyzed using SPSS 22. The demographics and exposure to risk factors are presented as frequencies and percentages.
Comparison of the study variables was assessed by using Pearson's Chi-square test. For potential risk factors, by using univariate binomial logistic regression, crude odds ratios (OR) and 95% con dence intervals were determined to take HCV status as a dependent variable. All variables showing p-value < 0.05 in univariate binomial logistic regression were subjected to multivariable binomial logistic regression analysis. This model further assessed for the presence of multicollinearity and interaction between independent variables. Hosmer-Lemeshow goodness-of-t test was used to examine the model t.

Results
In total n = 1004 samples were collected from six different areas of Islamabad and area-wise distribution of these samples is given in Fig. 1.
Out of the total, n = 727 (73%) population was having ≤ 10 years of education. A signi cant relationship was observed between education status and gender (χ2 = 22.93, p = 0.000). Similarly, there was a signi cant relation present in gender and history of drug abuse (χ2 = 15.09, p = 0.000) given that 39/42 (93%) drug abusers were male. Moreover, a signi cant relationship was observed between gender and nose/ear piercing (χ2 = 690.19, p = 0.000) given that 20/315 (6%) participants having at least one nose or ear piercing were male. In addition to that gender and presence of tattoo also have a signi cant relationship (χ2 = 5.42, p = 0.013) as 14/15 (93%) participants having tattoo were male. The detailed breakdown of the questionnaire is given in Table. 1.
Pearson's Chi-square test was calculated among the status of hepatitis and other study variables. This test shows a strong relationship between hepatitis B or C infection and marital status, hepatitis B vaccination status, blood or blood product recipient, having a tattoo, total family income and age of the participant. However, there was no signi cant association was observed between the status of hepatitis and nose/ear piercing, education status, barber visit, blood donation and drug abuse. Signi cant relationship of the status of hepatitis and other study variables are given in Table 2. Univariate binomial logistic regression test was performed to assess the risk factors for hepatitis C. Five variables having p value less than 0.05 overall or at least one of their categories were subjected to multivariable analysis. This analysis shows hepatitis B vaccination p = 0.000 odd ratio (OR) = 5.309 (95% con dence interval [CI] 2.812-10.025) as a strong predictor for hepatitis C infection. Compared with those without injection exposure population exposed to four-time to injection in the past 6 months were most likely to have HCV infection p = 0.015 OR = 4.328 (95% CI 1.319-13.617). Similarly, the population is more prone towards HCV infection who have visited a dentist four times in past six months p = 0.000 OR = 11.9 (95% CI 3.350-40.098) than those who have not visited a dentist at all. Moreover, people having frequent exposure to HCV patients ≥ 6 times during past six months were more susceptible towards HCV infection p = .001OR = 3.095 (95% CI 1.577-6.074) in comparison to those who have not exposed to HCV patients. Age of the participants proved to be a strong predictor of HCV infection p = .000 OR = 1.049 (95% CI 1.026-1.072). For multilinguistic binomial logistic regression analysis χ2 = 73.635, p = .000 and Hosmer-Lemeshow goodness-of-t test was recorded as p = 0.6. Table 3 shows a detailed analysis of risk factors for hepatitis C infection.

Discussion
Hepatitis c is a global health problem and a major cause of liver transplant around the globe [12] while hepatitis B is still a life-threatening disease in developing countries associated with high morbidity and mortality rate [13]. Thus, it is very important to nd the risk factors associated with disease transmission, vaccination status, early diagnosis and treatment [14].
To measure the incidence rate and assessing the risk factors associated with hepatitis B/C infection, the current study was conducted. The Present study reports 4% HCV prevalence and 1% HBV prevalence in less educated population with the lower socioeconomic status of the federal capital Islamabad. Satti et al and Khokhar et al reported a similar result regarding HCV prevalence [15,16]. This study reports a quite low prevalence of hepatitis B than overall national HBV prevalence (2.5%) as reported by Mehmood et al. [6]. Due to less exposure to risk factors, the current study population may have a relatively low prevalence of HBV than the overall prevalence in Pakistan. This study reports a relatively high prevalence of hepatitis C in male (5%) than female (3%).
This study is in line with the study of Arshad, Arshad et al who reported 4.5% prevalence in male and 2% in female [17]. ul Huda et al and Akhtar et al Higher also reported higher HCV prevalence in male than female ul Huda, Jameel [18], [19]. High prevalence in male is attributed to social mobility and freedom, exposing male to risk factors more than female. In current study, strong gender biases were observed in nose ear piercing (χ2 = 690.19, p = 0.000), tattooing (χ2 = 5.42, p = 0.013) and history of drug abuse (χ2 = 15.09, p = 0.000). Related observations were previously reported by Yee et al. They reported 64.8% male and 23.3% female were found to be drug abuser, 20.5% male and 91.7% female underwent body/ear piercing, 20.5% male and11.7% were having tattoo [20]. Such discrepancies in the two studies are due to cultural norms and social trends as both studies are conducted in two different regions.
In terms of Pearson's Chi-square test, the present study reports a signi cant association of hepatitis B/C infection with marital status and low socioeconomic status both having p = .000. Results closer to the current study were previously reported by Akhtar et al [21] given that HCV infection and marital status had p = .007 and socioeconomic status p = .000. These ndings were also supported by Qureshi et al [22]. The married population is more vulnerable to risk factors such as sexual transmission and low socioeconomic status is associated with a high risk of exposure, less awareness about transmission routes and more liable to quack visit and unhealthy practices such as sharing of personal items.
This study reports a high association between hepatitis B/C infection and recipients of blood or blood products p = .000 which is in accordance with the results of Ejiofor et al, Adeyemi et al and Chakrabarty et al [23][24][25]. This association attributes to negligence or poor practices including the use of screening devices with low sensitivity in blood banks. Moreover, we observed a strong association between hepatitis B, C infection and tattooing in Chi-Square p = .015 but having tattoo was not proven to be a strong predictor of hepatitis C infection in univariate analysis. Thome & Homeberg, Jafri et al and Carney et al reported similar result p < .001 [13] but the only difference is that in there study having a tattoo is a strong predictor for HCV and HBV infection [26][27][28].
Most of the literature is equivocal regarding tattooing and hepatitis B/C infection. It is due to variability in the study population. Moreover, in the current study overall prevalence of population with a tattoo was quite low. Chi square analysis shows signi cant association of hepatitis B, C infection p = .000 with age Ziaee et al and Gacche & Al-Mohani reported likewise results p = .015 and p < .001 respectively [29,30]. This association depicts that with age, exposure to risk factor increases resulting in more infections in elderly age than in young age.

Conclusions
In this study, the hepatitis C prevalence was found to be 4% while hepatitis B was about 1%. It is thought that people belonging to low socioeconomic status are more prone to develop these infections given that less access to better health care facilities. This study reports that people belonging to the low socioeconomic class in the region of Islamabad have less prevalence of hepatitis C and B give that low exposure rate to risk factors. Exposure to dental clinics, therapeutic injections and hepatitis B/C patients along with age were found to be the strong predictor of hepatitis C. Overall the study population was not aware of the mode of transmission of hepatitis B/C. A targeted screening and vaccination approach could help to reduce the incidence rate and educating the public about transmission routes will further help to control the spread of such infections. -Studies involving animals must include a statement on ethics approval. Not applicable -Consent to publication Participants were well informed about the research work and the information to be published later.

Abbreviations
-Availability of data and material The data available is already shared in this manuscript. We don't have any other data to share.
-Competing interests Not applicable -Funding This project was funded by the student research fund of the National University of Sciences and Technology (NUST) Islamabad.

-Authors' contributions
This work was designed, conducted, and written by AN and FA. NA, ZS, AA, and FS helped in virus sequence analysis via various web tools and evaluated the manuscript. SN, SJK and TA helped in statistical analysis and nal editing of the manuscript.

-Acknowledgements
We are very thankful to the Islamabad lab and research center at Islamabad for helping us in testing the samples. Age-wise comparison of hepatitis B and C positive cases