Sehrish Jabeen  ( Department of Microbiology, Federal Urdu University, Karachi, Pakistan )
Asif Rasheed  ( Center for Non communicable Diseases )
Nusrat Jabeen  ( Department of Microbiology, Federal Urdu University, Karachi, Pakistan )
Sehar Afshan Naz  ( Department of Microbiology, Federal Urdu University, Karachi, Pakistan )
Akhter Raza  ( Department of Computer Science, Federal Urdu University, Karachi, Pakistan )

Objective: To investigate the possible associations of hepatitis B and C virus infection with cardiovascular disease risk factors inperi-urban population.

Methods: The cross-sectional study was conducted from February to December 2016 in the periurban low-resource locality of Bin Qasim Town in Karachi. Serum samples were screened for hepatitis B surface antigen and hepatitis C virus antibodies. Anthropometric measurements were taken and markers related to cardiovascular disease were examined. Association of the two hepatitis virus infections with cardiovascular diseasewere investigated by anaylsing the data using SPSS 16

Results: There were 691 subjects. Serum triglyceride levels were significantly low in patients with hepatitis B virus (p<0.05). Those with hepatitis C virus had markedly low total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglyceride levels (p<0.05 each), whereas random blood sugar and body mass index values were significantly high (p<0.05 each. Hepatitis C virus infection was positively associated with body mass index and random blood glucose, and inversely associated with total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol(p< 0.05 each).

Conclusion: Hepatitis B virus infection showed a significant inverse association with triglyceride levels. However, hepatitis C virus infection was positively associated with body mass index and random blood sugar, and inversely associated with total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels.

Keywords: Hepatitis B, Hepatitis C, Coronary heart disease, Lipoproteins, Cholesterol. (JPMA 70: 58; 2020). https://doi.org/10.5455/JPMA.1542

Introduction

Hepatitis is a major global health issue, especially in the developing countries such as Pakistan,¹ where it is found mostly in the rural population. ² Hepatitis is an inflammation of the liver, principally caused by hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, and is responsible for minute disorders to liver cirrhosis or  hepatocellular carcinoma(HCC). ³ The prevalence of HBVand HCV worldwide is 10-15%.² Pakistan is the world's sixth largest country in terms of HBV and HCV prevalence at around 2.5% and 4.8%, respectively. ³ Chronic infection and associated inflammation may be Involved in the initiation and progression of atherosclerosis, ⁴ leading to cardiovascular diseases (CVDs). According to the World Health Organisation (WHO), till 2017 approximately 17.7 million people had died because of CVDs, representing 31% of global deaths, and, among those deaths, 7.4 million people died because of coronary heart disease (CHD).5 Main factors related with pathophysiology are age, male gender, family history, smoking, hypertension (HTN), diabetes mellitus (DM), obesity, high total cholesterol (TC), lowdensity lipoprotein cholesterol(LDL-C) and triglyceride (TG), as well as low high-density lipoprote in cholesterol(HDL-C) and less physical activity. ⁵ HBV may lead to chronic liver disease by dyslipidaemia. ⁶ Su et al. ⁷ investigated the association of a lower level of HDL-C and TC with asymptomatic chronic HBV infection. HCV is an independent risk factor for CVDs owing to its core protein nature. HCV can also play a crucial role in promoting insulin resistance (IR), oxidative stress (OS), and liver steatosis. Moreover, several direct and indirect mechanisms are assumed to be involved in the development of atherogenes is by HCV, including replication and colonisation in the arterial walls, fibrosis, increased levels of inflammatory markers and metabolic syndrome (MS). ⁷Association of HBV and HCV infection with CVD among the Pakistani population are not clear. The current study was planned to discern not only the prevalence of HBV and HCV infection and related determinants, but also to investigate the possible association of HBV and HCV infection with CVD risk factors in the target population.

Subjects and Methods

The cross-sectional study was conducted from February to December 2016 in the peri-urban low-resource locality of Bin Qasim Town in Karachi. Only those living in the selected community and willing to participate were included, and those who refused to participate were excluded. Convenient sampling was used and consent was obtained from each subject. The sample size was calculated considering mean cholesterol among Pakistani HCV patients to be 218.38±1.54 mg/dl, ⁸ and using 99% confidence inter val (CI) and 0.2 bound of error. Blood samples were drawn from each non-fasting participant, and the sample was centrifuged. Serum was isolated and store d at - 800C. A self-designed questionnaire was used to acquire demographic information, including age, gender, family history, medicati on history, and tobacco con sumption. Anthropometric measurements, including subject's body weight, height, body mass index (BMI), waist and hip circumferences, and systolic and diastolic blood pressures, were also taken. Data relevant to the coronary artery disease(CAD)prevalence was also collected. The advance quality single-step test strip (In-Tec Products, Incorporation, Xiamen, China) was used for the visual detection of hepatitis B surface antigen(HBsAg) and anti- HCV antibodies in serum samples. Biochemical analysis was performed for quantitative measurement of serum cholesterol, HDL-C, LDL-C, TG, and glucose for all participants. All biochemical tests were performed based on the routine enzymatic colorimetric method by automatic an alyser (H itachi 902, Hitachi H igh- Technologies Corporation, Tokyo Japan). According to the Centre for Disease Control (CDC) prevention, waist-to-hip ratio (WHR) >0.90 for males and >0.80 for females is considered an indicator of the risk of heart diseasein South Asian population. ⁹ BP measurements were performed in a standardized manner. ¹⁰ BMI was calculated using the standard formulaweight (kg)/height (m2). ⁹ TC200 mg/dL, LDL-C 130 mg/dL, reduced HDL-C 40mg/dL in males and 50 mg/dL in females, and TG150mg/dL were considered abnormal values as recommended by the American Heart Association (AHA). ¹¹ DM was defined as random blood glucose (RBG) 140 mg/dL and or with the treatment of hypoglycaemic agents as suggested by the American Diabetes Association (ADA). 12 Data was analysed using SPSS 16. To check the null hypothesis that there is no relationship between HBV or HCV infection and CVD risk factors, univariate (chi square test and t-test) and multivariate (linear regression model) analyses was done. T-test and Chi-square tests were applied for comparison between sero-positive and seronegative groups with p<0.05 being significant. To define the strength of association of HBV or HCV infection with CVD risk factors, linear regression model was used. In multivariable analysis, gender, DM, HTN, tobacco consumption and BMI were used for adjustment.

Results

Of the 3,000 individuals approached, 691(23%) agreed to participate, among those two were co-infected patients and excluded from the analysis and finally presenting the total of 689.The prevalence percentages of HBV and HCV infection were 75(10.9%) and 43(6.2%), respectively (Table 1).



The prevalence of HBV infection was predominant in males(p <0.05). Conversely, the prevalence of HCV infection was high in females though the difference was not significant (p>0.05). The prevalence of HBV infection was high in subjects aged 21-40 years 44 (14.3%) compared to those aged 20 years19(8.4%) and 41 years 12(7.6%). The highest prevalence of HCV infection was in those aged 41 years 30(19.1%) followed by 20 years 0.4% (1) and 21-40 years 3 .9% (12). Bo th HBV an d HC V infec ti ons were considerably high in married individuals and (p<0.05). HBV-infected subjects had a lower meanWHR, TC, LDLC, RBS and, to some extent, low HDL-Ccompared to those for HBV-negative subjects (p>0.05). In contrast, TG levels were significantly low in HBV-infected subjects than in HBV-negative subjects (p<0.05). Also 57.3% HBV-infected subjects were married compared to 45.2% among the non-infected with a low prevalence of CAD and family history of coronary heart disease. In addition, only tobacco consumption (64.0% vs.45.5%) was substantially high in HBV-infected subjects. HCV-infected subjects had markedly lower meanserum TC (162.2±64 vs. 197. 4 ± 63. 8mg/dL), LDL-C (90. 1± 48.6vs. 116.2±50.4mg/dL), HDL-C (47.7±18.7 vs. 53.1±16.0 mg/dL), and TG (122.0±68 vs. 140.0±90.2 mg/dL) than HCV-negative subjects. By contrast, RBS levels and BMI were significantly high in HCV-infected subjects (p<0.05 each). Furthermore, most HCV-infected subjects were married and the prevalence of DM (11.4% vs. 2.8%) was higher than in HCV-negative subjec ts. Tobacco consumption was comparably high but was not significant (p>0.05). Unadjusted coefficient of CVD risk factors showed that HBV infection was inversely associated with TG even after adjustment (p<0.05). Unadjusted coefficient of CVD determinants demonstrated that HCV infection was positively associated with BMI and RBS, but it was inversely associated with TC, HDL-C and LDL-C (p<0.05). Similarly, adjusted coefficients of CVD risk factors showed a significant positive link with BMI (p<0.05). Considerably inverse associations were observed among HCV infection and cholesterol level, TG, and LDL-C (p<0.05 each) (Tables 2-3).





Discussion

The continuous rise of HBV and HCV infection is an alarming situation in Pakistan. Several international reports have supported the association of HBV and HCV infection with CVD, but not a single report regarding this association from Pakistan is available. The current study included 691 individuals from a population belonging to the periphery of Karachi. The prevalence of HBV infection is notably high in males mostly middle-aged. Males are more prone to have chronic HBV infection compared to females. ¹³ HBV can be transferred through sexual activity; ¹⁴ and in males, HBV can be transferred through homo- as well as heterosexual activities, especially in the middle-age group. ¹⁵ In addition, a high carrier rate among people aged 40- 50 years was observed. ¹⁶ In the current study, HBV infection is highly prevalent in married persons, which is in line with the findings of a previous study. ¹⁷This study determined that the prevalence of HCV infection was prominently high in the old age group (41 years). The same trend has been described, ¹⁸ but slight variation was observed as the highest prevalence of HCV infection was detected in people aged 50-59 years. ¹⁹In our study, HCV infection was more prevalent in female participants, but was not statistically different, and comparable results have been reported in the past. ¹⁹ We found that HBV-infected subjects had low TC, TG, LDL, and RBG mean values compared with healthy participants. These outcomes are comparable with previous studies. ⁷ Current results indicated that HCVinfectedpatients had significantly low TC, LDL-C, and TG levels compared with HCV-negative individuals; similarly, low TC, LDL-C, and TG levels were determined in HCVinfected patients. ²⁰ The reduced levels of serum LDL-C, HDL-C, and TC were positively associated with the severity of liver disease. Significant reduction in TC and TG levels in cirrhotic patients has been confirmed, whichis because liver biosynthesis is reduced. ²¹ Studies have mentioned that lipids have a significant function in responding to any infection because lipoproteins can bind various viruses to diminish their toxic effects. ^22,23 We found that HBV infection was inversely associated with TG. Studies have supported the inverse association of HBsAg sero-positivity with hypertriglyceridemia and decreased HDL-C after adjustments. The inverse correlation between chronic hepatitis B(CHB) and MS may be accredited as favourable consequences to the lipid profiles of infected subjects. ²⁴In contrast, no significant association between the risk of CAD and HBV infection was found. ²⁵ The reason for the inverse association is unclear, but a low inflammatory burdenof CHB infection may be a possible reason. ²⁵ Another possibility is the low prevalence of traditional risk factors for CAD, such as DM, HTN and hyperlipidaemia. ²⁶ Possible association between HCV infection and CVD risk has been concluded with different results. ^20,27Our findings demonstrated that HCV infection was positively associated with BMI and RBG, but was inversely associated with TC, TG, HDL-C, and LDL-C. After adjustment for CVD risk factors, HCV infection was positively associated with BMI and inversely associated with TC and LDL-C. These results are comparable with a study that demonstrated that HCV infection seems to be an independent risk factor of CVD because of its association with steatosis, DM and IR. ²⁷ A study showed that chronic HCV infection appears to predispose the infected individuals to developing atherosclerosis, which directs them toward alterations in carotid plaques, ²⁸ although blood lipid levels and the prevalence of MS are low in HCV-infected subjects. A study²⁹ observed an alteration in the glucose breakdown in HCV-infected population. The core protein of HCV can degrade insulin receptor substrates (IRS) 1 and 2 by enhancing the expression of tumour necrosis factor-alpha (TNF-) and the inhibition of the cytokine signaling-3, leading to defective phosphorylation of PI3K and Akt.6 It might be possible that decreased TC, LDL and HDL levels in the serum ofcirrhotic patients may be related to the further development of cirrhosis. ²¹Therefore, establishing a mean range of lipid profile that can be used as a prognostic marker for patients at risk of liver cirrhosis is crucial. The outcomes of the current study are not very close to the past studies as our study has certain limitations. For example, we were unable to collect fasting samples. Alanine transaminase (ALT ) levels of the participants were also not measured. Besides, the time and logistics did not allow us to continue the clinical/collection unit for the required time. Longitudinal follow-up studies may help to better understand the association of HBV and HCV with CVD.

Conclusion

Most HBV and HCV-infected patients were asymptomatic and had low awareness about the disease and the risk. Such individuals not only serve as carriers but may experience extremely serious consequences that are costly as well as untreatable. Moreover, the horizontal transmission was found to be an essential mode of transfer of HBV and HCV infection. Proper screening, vaccination, and treatment need to be addressed urgently.
Disclaimer: The text ispart of an M.Phil thesis submitted to the Graduate Research Management Council of the Federal Urdu University of Arts, Science and Technology, Karachi.

Conflict of Interest: None.

Source of Funding: The Centre for Non-Communicable Diseases (CNCD), Pakistan.

References

1. Averhoff FM, Glass N, Holtzman D. Global burden of hepatitis C: considerations for healthcare providers in the United States. Clin Infect Dis 2012; 55: S10-15.

2. Waheed Y, Shafi T, Safi SZ, Qadri I. Hepatitis C virus in Pakistan: a systematic review of prevalence, genotypes and risk factors. World J Gastroenterol 2009; 15: 5647-53.
3. Butt AS. Epidemiology of viral hepatitis and liver disease in Pakistan. Euroasian J Hepatogastroenterol 2015; 5: 43-8. 4. Kiechl S, Egger G, Mayr M, Wiedermann CJ, Bonora E, Oberhollenzer

F, et al. Chronic infections and the risk of carotid atherosclerosis prospective results from a large population study. Circulation 2001; 103: 1064-70.

5. World Health Organization. Cardiovascular diseases. Fact Sheet:
317. [Online] 2017 [cited 2017 May 17]. Available from: URL: http://www.who.int/mediacentre/factsheets/fs317/en/

6. Su TC, Lee YT, Cheng TJ, Chien HP, Wang JD. Chronic hepatitis B virus infection and dyslipidemia. J Formos Med Assoc 2004; 103: 286-91.

7. Adinolfi LE, Zampino R, Restivo L, Lonardo A, Guerrera B, Marrone A, et al. Chronic hepatitis C virus infection and atherosclerosis: clinical impact and mechanisms. World J Gastroenterol 2014; 20: 3410-7.

8. Bashir MF, Haider MS, Rashid N, Riaz S. Association of Biochemical Markers, Hepatitis C Virus and Diabetes Mellitus in Pakistani Males. Trop J Pharm Res 2013; 18: 845-50.

9. Centers for Disease Control and Prevention. Healthy weight. [Online] 2015 [cited 2015 May 15]. Available from : URL :

https://www.cdc.gov/healthyweight/assessing/index.html
10. American Heart Association. [Online] 2017 [cited 2017 Nov 30].

Available from: URL: https://www.heart.org/en/health-topics/highblood-pressure/understanding-blood-pressure-eadings#.WJHHVGER4dU 

11. American Heart Association. [Online] 2017 [cited 2017 Dec 01]. Available from: URL: https://www.disabled-world.com/calculatorscharts/cholesterol-chart.php
12. American Diabetes Association. Diabetes. [Online] 2018 [cited 2018

August 29]. Available from: URL: http://www.diabetes.org/livingwith-diabetes/treatment-and-care/blood-glucose-control/checking-your-blood-glucose.html 

13. Yang F, Yin Y, Wang F, Zhang L, Wang Y, Sun S. An altered pattern of liver apolipoprotein AI isoforms is implicated in male chronic hepatitis B progression. J Proteome Res 2009; 9: 134-43.
14. Kabir A, Tabatabaei SV, Khaleghi S, Agah S, Kashani AHF, Moghimi

M, et al. Knowledge, attitudes and practice of Iranian medical specialists regarding hepatitis B and C. Hepat Mon 2010; 10: 176-82.

15. Hou J, Liu Z, Gu F. Epidemiology and prevention of hepatitis B virus infection. Int J Med Sci 2005; 2: 50-7.

16. Kaygusuz S. The results of seropositivity of HAV, HBV and HCV in relation to age and sex in Kirikkale. Viral Hepatit Derg 2003; 3: 160-5.

17. Nurgalieva ZZ, Hollinger FB, Graham DY, Zhangabylova S, Zhangabylov A. Epidemiology and transmission of hepatitis B and C viruses in Kazakhstan. World J Gastroenterol 2007; 13: 1204-7.

18. Tolmane I, Rozentale B, Keiss J, Arsa F, Brigis G, Zvaigzne A. The prevalence of viral hepatitis C in Latvia: a population-based study. Medicina (Kaunas) 2011; 47: 532-5.
19. Niu Z, Zhang P, Tong Y. Age and gender distribution of Hepatitis C

virus prevalence and genotypes of individuals of physical examination in Wu Han, Central China. Springerplus 2016; 5: 1557-65.

20. Butt AA, Xiaoqiang W, Budoff M, Leaf D, Kuller LH, Justice AC. Hepatitis C virus infection and the risk of coronary disease. Clin Infect Dis 2009; 49: 225-32.

21. Ghadir MR, Riahin AA, Havaspour A, Nooranipour M, Habibinejad AA. The relationship between lipid profile and severity of liver damage in cirrhotic patients. Hepat Mon 2010; 10: 285-8.
22. Grunfeld C, Feingold KR. Tumor necrosis factor, interleukin, and interferon induced changes in lipid metabolism as part of host defense. Proc Soc Exp Biol Med 1992; 200: 224-7.

23. Leong JC, Kane JP, Oleszko O, Levy JA. Antigen-specific nonimmunoglobulin factor that neutralizes xenotropic virus is associated with mouse serum lipoproteins. Proc Natl Acad Sci 1977; 74: 276-80.
24. Huang CY, Lu CW, Liu YL, Chiang CH, Lee LT, Huang KC. Relationship

between chronic hepatitis B and metabolic syndrome: A structural equation modeling approach. Obesity 2016; 24: 483-9.

25. Wijarnpreecha K, Thongprayoon C, Panjawatanan P. Hepatitis B virus infection and risk of coronary artery disease: a meta-analysis. Ann Transl Med 2016; 4: 423- 8.

26. Ghotaslou R, Aslanabadi N. Ghojazadeh M. Hepatitis B virus infection and the risk of coronary atherosclerosis. Ann Acad Med Singapore 2008; 37: 913-5.

27. Negro F. Facts and fictions of HCV and comorbidities: steatosis, diabetes mellitus, and cardiovascular diseases. J Hepatol 2014; 61: S69-78.

28. Adinolfi LE, Restivo L, Zampino R. Chronic HCV infection is a risk of atherosclerosis. Role of HCV and HCV-related steatosis. Atherosclerosis 2012; 221: 496-502.

29. Ampuero J, Romero?Gómez M, Reddy KR. Review article: HCV genotype 3-the new treatment challenge. Aliment Pharmacol Ther 2014; 39: 686-98.