203 J I M D C 2 0 1 7 203 Open Access F u l l L e n g t h A r t i c l e Hepatocellular Carcinoma in Patients Suffering from Chronic Hepatitis B versus Co-Infection of Hepatitis B and D Virus Riaz Hussain Khokhar 1, Anwar Ali Jamali 2, Shohabudin Rind 3 1 Postgraduate Department, Gastrointestinal & liver Diseases, Isra University Hospital at Hyderabad 2 Assistant Professor, Department of Medicine, Peoples University of Medical and Health Sciences Nawabshah, Sind 3 Postgraduate Department, Gastrointestinal & liver Diseases of Isra University Hospital at Hyderabad A B S T R A C T Objective: To determine the frequency of hepatic cancer in subjects suffering from chronic liver disease due to hepatitis B versus hepatitis B with co-infection of viral hepatitis D. Patients and Methods: This cross-sectional study was carried out in the Department of Medicine (Hepatology & Gastroenterology) at Isra University Hospital, Hyderabad Sindh, from March 2015 to February 2016. Both male and female patients, from 20-70 years of age either suffering from viral Hepatitis B alone or co-infected with viral hepatitis D were included in our research. Individuals having co-infection of Human Immunodeficiency virus, Viral hepatitis C, Wilson's disease, Hepatitis due to auto-immune diseases, alcoholic fatty liver disease, haemochromatosis, pregnant women and those patients who refused to give consent were excluded from study. Results: Out of total 200 patients, 142(71%) were males while 58(29) were females. Mean age was 52.83+15.6 years. Total 173(86.5%) patients were suffering from viral hepatitis B alone, while 27(13.5%) were infected by co-infection of Viral hepatitis B and D. Total frequency of hepatocellular carcinoma was 45(22.5%) in all cases. HCC was more frequent (33.34%) in patients with co-infection of chronic hepatitis B and D, as compared to only chronic hepatitis B patients (20.80%) but the difference was non-significant (p-value 0.14). Conclusion: Hepatocellular carcinoma was more frequent amongst patients co-infected with viral hepatitis D as compared to alone infection of hepatitis B virus. Key words: Hepatocellular Carcinoma, Viral hepatitis B, Viral hepatitis D. Author`s Contribution 1, Conception, synthesis, planning of research and manuscript writing 2 Interpretation and discussion Data analysis, 3 Interpretation, manuscript writing and Active participation in data collection Address of Correspondence Anwar Ali Jamali Email: dr.saeedarain786@gmail.com. Article info. Received: 2017 Accepted: 2017 Cite this article. Khokhar RH, Jamali AA, Rind S. Hepatocellular Carcinoma in Patients Suffering from Chronic Hepatitis B Versus Co-Infection of Hepatitis B and D Virus. JIMDC.2017;6(4): Funding Source: Nil. Conflict of Interest: Nil I n t r o d u c t i o n Hepatocellular carcinoma (HCC) is the commonest primary hepatic malignancy. It is also the leading cause of malignancy related mortality throughout the world. In the United States it is the ninth leading cause of mortality.1 According to recent data, the rate of HCC is still rising globally, although it is variable throughout the world. Chronic viral hepatitis can lead to cirrhosis and HCC. Hepatitis B, C and D infections are the commonest reasons of chronic hepatitis worldwide. Hepatitis B virus is the double-stranded circular DNA and having 8 genotypes “A to H”. Many epidemiological studies showed that hepatitis B infection is responsible for significant O R I G I N A L A R T I C L E 204 J I M D C 2 0 1 7 204 hepatocarcinogenicity.1,2 Hepatitis B virus carriers have 10%–25% lifetime risk of HCC development. Unlike other reasons of hepatitis severity, hepatitis B virus is the unique and may develop HCC without cirrhotic evidence.3 Persistent replication of hepatitis D virus and inflammation of liver may cause cirrhosis and development of hepatocellular carcinoma, while active replications of the both hepatitis B virus and hepatitis D virus may be responsible for high progression in disease, which leads to early liver cirrhosis and hepatic malignancy.4 Several epidemiological studies controversially showed different role of hepatitis D virus infection in the rising HCC risk. Many previously published studies did not found elevated rates of HCC in cases having hepatitis D co-infection, but in recent literature, it is mentioned that co-infection may increase the tumor incidence.4 Risk of the hepatocellular carcinoma should be reassessed according to the alteration of the natural history of hepatitis D chronic disease. However, burden of hepatitis D virus has decreased in several countries of the Western world, on other hand it is still prevalent still other countries, particularly in Asia Pacific Region.5 Association of HCC with co-infection of hepatitis D virus and hepatitis B virus or with hepatitis B viral infection alone, is still unclear. As in many studies it is reported that the development hepatocellular carcinoma itself is the complex process concerning cumulative gain and the loss of functions and mutations affecting tumor suppressor and oncogenic products.6,7 It has been reported that hepatitis B virus is more responsible for development of hepatocellular carcinoma, and hepatitis D virus seems to exert epigenetic control over HBV transcription and replication. A possible explanation may be that p24 and p27 both repress HBV enhancers, pIIE1 and PIIE2 inhibit replication, thus accounting for the low serum levels of HBV DNA in co-infected patients.8 On other hand, Pakistan have recognized a huge difference in the occurrence and sharing of HCC between population, who are suffering from viral Hepatitis B virus alone and viral hepatitis B co-infected with viral hepatitis D in different areas.9 The purpose of our study was to determine the frequency of HCC in subjects suffering from viral hepatitis B alone versus viral hepatitis B co-infected with viral hepatitis D virus. P a t i e n t s a n d M e t h o d s This cross-sectional research study was carried out in the Department of Medicine (Hepatology & Gastroenterology) Isra University Hospital, Hyderabad Sindh from March 2015 to February 2016. Informed written consent was taken from patients or their relatives. Both male and female patients of chronic liver disease, either suffering from viral Hepatitis B alone or co-infected with viral hepatitis D, from 20-70 years of age were included in our research. Individuals having age less than 20 years or more than 70 years, Co-infection with Human Immunodeficiency Virus, viral hepatitis C, Wilson's disease, hepatitis due to auto-immune diseases, alcoholic fatty liver disease, haemochromatosis, pregnant women and those patients who refused to give consent were excluded from study. Complete clinical examination of all patients was carried out. All the patients were divided in two groups. Group 1 included chronic HBV mono-infection patients and group 2 comprised of patients having co- infection of chronic HBV and HDV. Viral hepatitis B was diagnosed on the basis of HBsAg positive on enzymatic assays analysis through ELISA. Viral hepatitis D was diagnosed in HBsAg positive cases on the basis of hepatitis D Virus antibodies on ELISA. The diagnosis of hepatocellular carcinoma was suggested in those patients who had the clinical features suggestive of hepatocellular carcinoma which was further confirmed by tri-phasic contrast enhanced CT scan / magnetic resonance imaging abdomen, typically showing hyper vascular solid hepatic mass with support of high levels (>100 ng/ml) of alpha-fetoprotein. Socioeconomic status according to monthly income was defined as upper >50,000 rupees/month, Middle 15,000- 49,000 rupees/month and lower <15,000 rupees/month. All data was recorded in the pre design performa. Data were entered and analyzed in SPSS 16. Mean and standard deviation (SD) were calculated for quantitative variables. Frequency and percentage were calculated for qualitative variables. Chi- square test was applied to compare the frequency of HCC in HBV mono infection versus HBV+HDV co-infection. p- value less the 0.05 was considered as statistically significant. 205 J I M D C 2 0 1 7 205 R e s u l t s Out of total 200 patients, 142(71%) were male while 58 (29%) were females. Mean age of subjects was 52.83+15.6 years (range was 20-70 years). Most of our patients (67%) belonged to middle age group. Regarding the education level, out of 200 subjects 115(57.5%) were un-educated. Majority of subjects (56.5%) in our study belonged to lower economic class (Table1). Among these subjects, 173 were suffering from viral hepatitis B alone while 27 subjects were diagnosed to have viral hepatitis B and D co-infection (Figure 1). Figure 1: Patient’s distribution according to HBV and HBV+HDV Co-infection (n=200). Out of all study participants, 45 were diagnosed with HCC and 155 were without hepatocellular carcinoma. Thus, total HCC frequency was 22.5% (Figure 2). Figure 2: Patient’s distribution according to frequency of HCC (n=200). Ultrasound abdomen revealed that 127(63.5%) subjects had evidence of chronic liver disease without decompensation and remaining 73(36.5%) subjects were present with decompensation indicated by ascites, dilated portal vein, splenomegaly and malignant change / growth /mass. These suspected cases of HCC were stepped up for alpha-feto protein level, CT-scan abdomen. After performing these investigations, 45(22.5%) individuals were diagnosed as having hepatic cancer. All the suspected cases of HCC were also confirmed on biopsy. Mean alpha fetoprotein level in our study was 5322.44 +2779.82 ng/ml. HCC was more frequent (33.34%) in patients with co-infection of chronic hepatitis B and D, as compared to patients having only chronic hepatitis B (20.80%) but difference was not significant p-value 0.14 (Table.2). D i s c u s s i o n Hepatic carcinoma is the fifth most frequent cancer and 3rd common reason of mortality. It is documented that the Table 1: Demographic characteristics of participants (n=200) Characteristics n(%) Age groups (years) 20-40 44(22) 41-60 134(67) >60 22(11) Gender Male 142(71) Female 58(29) Educational status Un-educated 114(57) Primary 57(28.5) Middle-matric 13(6.5) Intermediate 10(05) Graduate Socioeconomic status 06(03) lower 113(56.5) Middle 70(35) Upper 17(08.5) Table 2: Association of HCC with HBV & HBV+HDV co-infection (n=200) Parameter HCC Total p-value With HCC n (%) Without HCC n (%) HBV 36(20.80) 137(88.20) 173 0.14 HBV+HDV 09(33.34) 18(66.66) 27 206 J I M D C 2 0 1 7 206 hepatitis B virus is one of the commonest oncogenic virus in human.10 Prevalence of HCC is high in population infected by hepatitis B virus. Additionally, increased risk of HCC is reported in HDV infected cases.10 Present study has been carried out to determine the proportional frequency of HCC in patients affected by chronic hepatitis B or chronic co-infection of hepatitis B and hepatitis D. The mean age of subjects was 52.83+15.6 years with minimum of 20 years and maximum 70 years. Similarly, study of Abbas Z et al10 reported that patient’s mean age was 54.6 ± 11.1 years. Another study of Kim HS et al11 also indicated that mean age was 48 years, with range of 18 years-94 years and male predominance (64.5%). These gender findings are also comparable to our study, as out of 200 patients, 142(71%) were male, while 58(29%) were females. In this study, 27(12.5%) subjects were suffering from viral hepatitis B and D co-infection. Similarly, a national study of Shaikh MA et al12 reported that anti-HDV was positive in (23.6%) patients with HBV positive. In some other international studies, the sero-prevalence of anti-HDV in HBV was reported as 11.5% in Iran and 10.6% in India.13,14 In Turkey, the prevalence of anti-HDV serological markers were observed as 27.5% in HBV related chronic hepatitis.15 In this current analysis, HCC was more frequent in patients with co-infection of chronic hepatitis B and D as 09(33.34%) out of 27 cases, as compared to only chronic hepatitis B patients as 20.80% out of 173 cases. Amougou MA et al16 reported that in HCC-cases found, hepatitis Delta antibody (Anti-HDV) co-infection was present in 41.4 %. Studies had also shown that extravagant reproduction of virus particles by HBV in combination with HDV leading to massive necro- inflammation results in HCC.17 Cases infected by combination of HBV and HDV also develop HCC earlier as compared to those having HBV infection only.18,19 Saravanan S et al20 researches showed the subjects of chronic hepatitis group, suffering from viral hepatitis B and are chronically ill, antibodies against hepatitis D Virus were present in approximately 5.7% of diseased persons. This generally leads to chronic liver disease (Cirrhosis) in about 5.9% patients. Co-infection including hepatitis D virus is linked to diverse patterns of the reciprocal inhibitions of the replication of virus.21 In literature it has been reported that HDV suppresses HBV replication with most cases being HBeAg -ve and with decreased HBV DNA level as compared to cases having HBV monoinfection.22-24 Potential mechanisms of virology of HBV suppression by HDV as inhibitions of HBV enhancers may include proteins of HDV (p24 and p27).25 Consistently previous studies reported that HBV/HDV co- infection has higher level of ALT and AST and decreased PLT and PTA levels.23,26 As well as patients infected by HBV/HDV co-infection are estimated, 1.43 times more likely to progress to ESLD (end stage liver disease) as compared to those cases having HBV infection only. It is suggested that HBV/HDV co-infection leads to more rapid progression in hepatic disease as compared to those only infected by mono-infection of HBV.16 It is suggested that studies should also be conducted in other centers, to find out association of HCC with chronic HBV mono-infection and chronic co-infection of hepatitis B virus and hepatitis D virus and to find out the mechanisms of their oncognesis. C o n c l u s i o n This study accomplished that HCC was more frequent amongst patients having co-infection with viral hepatitis D as compared to infection of hepatitis B virus alone. Mono infected viral hepatitis B or all HBV infected patients should be screened for HDV, because early recognition and treatment of co-infected HBV and HDV, may reduce the burden of associated morbidity and mortality. R e f e r e n c e 1. Balogh J, David Victor III EH, Burroughs SG, Boktour M, Saharia A, Li X, Ghobrial RM, Monsour Jr HP. Hepatocellular carcinoma: a review. 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