Prevalence of Hepatitis D Among Patients with Hepatitis B Viral Infection Attending a Tertiary Care Centre of Nepal

Introduction: Worldwide there is variation in prevalence of Hepatitis D viral infection. Superinfection and co infection with hepatitis B viral infection is known to occur in 15-20 million people. Methods: This was a descriptive cross-sectional hospital based study carried out in NAMS, Bir hospital, Kathmandu, Nepal from period of January 2017 to June 2017. Consecutive patients of chronic hepatitis B viral infection of HBsAg positive with more than two-time upper normal limit of ALT were enrolled and tested for HDV IgG. Results: Forty patients were enrolled during study period. Mean age was 30.9±12.2 years. Males were 28 (70%) and females 12 (30%). Most of the patients were asymptomatic for HBV infection 32 (80%). HBeAg negative chronic hepatitis was most commonly present in 31 (77.5%). Family history of Hepatitis B viral infection was seen in 7 (17.5%) and sexual promiscuity in 5 (12.5%) as the mode of acquisition of hepatitis B viral infection. HBcIgM was positive in three patients with mean HBV DNA of 4.97x105±4.5x105 IU/ml in HBeAg positive group. HDV IgG was negative in all patients. Conclusions: Coinfection and superinfection of hepatitis D virus were found to be uncommon at Bir hospital, Nepal. _______________________________________________________________________________________


INTRODUCTION
Hepatitis D virus (HDV) was first discovered in 1977.It is a defective virus which requires Hepatitis B virus (HBV) for replication and expression. 1 Global burden of HDV is 15-20 million cases. 2 Coinfection or superinfection of HDV with HBV is considered to be a severe form of viral hepatitis, leading to rapid development of cirrhosis. 3Markers of HDV infection are antibody against HDV (IgM and IgG), detection of HDV antigen (HDV Ag), and HDV RNA quantification by polymerase chain reaction (PCR).Sensitivity and specificity of HDV IgG by enzyme linked imunosorbent assay (ELISA) are 98% respectively. 4 aimed to see the current prevalence of HDV in Hepatitis B infection in our setup.

METHODS
This is a cross sectional descriptive hospital based study carried out in National Academy of Medical sciences (NAMS), Bir hospital, Kathmandu.Informed written consent was taken from all patients.The study was approved by Institutional Review Board (IRB) of NAMS.Data was collected in structured proforma in period of March 2017 to August 2017.Any patient who presented to Bir hospital, Liver unit (OPD and indoor) with diagnosis of hepatitis B (HBsAg positive by ELISA) with two times upper normal limit of alanine aminotransferase (ALT) (Normal Upper limit of ALT 45 IU/ml) were included in study.Complete history was taken and clinical examination was done.Patients were specifically asked about history of blood transfusion, surgeries, hemodialysis, organ transplant, sexual promiscuity, intra-venous drug abuse (IVDA) and tattoo.All patients underwent two dimensional ultrasound of hepatobiliary system and selected patients underwent upper gastrointestinal endoscopy and their findings were recorded.Coexisting other diseases like Hepatitis C viral infection, Human immunodefienciency virus (HIV) infection, autoimmune hepatitis, wilson disease, hemochromatosis, viral hepatitis A and E, diabetes or alcohol abuse were excluded.Other tests like total serum billirubin, direct serum billirubin, aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), total protein, serum albumin, blood urea, serum creatinine and random blood glucose (RBG), prothrombin time (PT), International normalize ratio (INR) were sent.Patients were further evaluated for other markers of hepatitis B: hepatitis B envelope antigen (HBeAg), hepatitis B envelope antibody (HBeAb), Antibody against core antigen of Hepatitis B virus (Anti HBcIgM) and HBV DNA quantification by polymerase chain reaction (PCR).Immunoglobulin G against hepatitis D virus (Anti HDV IgG) was sent as a marker of HDV infection.If the sample tested positive for HDV IgG, we planned to send for HDV RNA quantification by PCR.During study period serum was collected and stored at -80 0 F before proceeding for HDV IgG by ELISA analysis (HDV Ab, Dia.Pro bioprobes srl).

Sample size was calculated as;
Sample size (n) = P (100-P) Assuming the prevalence of HDV (P) as 2% with Confidence Interval (CI) 95% (Z) and standard error (e) of 5%.
The calculated sample size was 31.
Data entry was done in Microsoft excel 2007 and analysis was done in SPSS version 23 and graph pad software.Variables were given in the form of ratio (%) and t-test was used for categorical variables.

RESULTS
A total of 46 patients were screened.Six patients were excluded among them two had drug induced liver injury, two were alcohol abuser, one had nonalcoholic steatohepatitis, and one had coexisting HIV infection.Prevalence of HDV among hepatitis B viral infection was zero.Most patients were males; M =28 (70%), F=12 (30%).Mean age of patients was 30.98±12.79 years with minimum age 15 years and maximum age 72 years (Table 1).When associated with the co-infection, it can lead to acute hepatic failure and when associated with superinfection, to chronic liver disease. 5HBsAg positive patients with HDV infection may develop hepatic failure and develop hepatocellular carcinoma as delta infection in HBV carriers is associated with more active and progressive disease, as suggested by clinical and histological evidence of high liver enzymes and a faster rate of developing cirrhosis. 6At local level there was a single study which was carried out in 1991 by SM Shrestha and Tusuda F. Their results suggest no patients were found to be positive with hepatitis D. 7 However prevalence of HDV in India is variable.Various studies suggest that its prevalence ranged from zero to 37.4% in patients with hepatitis B viral infection.Highest reported prevalence was from Bombay 37.4% in year 1992. 8In Ludhiana, its prevalence was 33% and most patients were children. 9In New Delhi prevalence was 8.1% in 1996. 10In a similar study conducted in 1998, prevalence of hepatitis D in Kolkata was 3.3 %. 11 Another study was conducted by Lal JS and colleagues in India in year 2014 involving large number of patients (n= 318) with Hepatitis B viral infection.These patients underwent three different modalities of tests (involving twice HDV IgG with two different manufacturers and HDV RNA by PCR).Surprisingly, no HDV infection was seen in this study. 12They concluded that low rate of HDV infection was possibly due to recent decline in HDV transmission secondary to improved healthcare practices which includes universal precautions and of use of disposable syringes. 12In a study from Bangladesh (n=180), 21.8% of asymptomatic HBsAg carriers and 25.6% of symptomatic hepatitis B patients were anti-HDV positive. 13milar to the above-mentioned study by Las JS and Shrestha SM, our study also showed a zero prevalence of HDV infection.However, our study had a lower sample size as compared to their study.We did not perform HDV RNA quantification by PCR as none of our participants were ELISA HDV IgG positive.In various studies the prevalence of HDV infection was high in IV abusers. 14Similarly high prevalence was recorded in hemophiliacs. 15This also may be one of the reasons for non-detection of HDV infection in our study as none of our patients were IVDA or hemophiliacs.
This study was aimed to find out prevalence of superinfection or coinfection of hepatitis D in patients attending to one of the tertiary care centre of Nepal.
Though it was small study, prevalence of HDV infection was zero.Our study has a limitation where patients might have acute hepatitis D viral infection however HDV IgM was not done.

CONCLUSIONS
The HDV infection in patients with HBV are not found in our setup.More epidemiological study is required whether to use HDV IgG in patients with hepatitis B viral infection in Nepal.