Lack of Anti-HDV Antibodies in HBsAg-Positive Children Two Decades after Compulsory HBV Vaccination: A Single Centre Study

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1 Med. J. Cairo Univ., Vol. 84, No. 1, June: , Lack of Anti-HDV Antibodies in HBsAg-Positive Children Two Decades after Compulsory HBV Vaccination: A Single Centre Study HANAN M. FOUAD, M.D.* and EHAB M. REYAD, M.D.** The Departments of Pediatrics* and Clinical & Chemical Pathology**, National Hepatology & Tropical Medicine Research Institute ''NHTMRI'', Cairo, Egypt Abstract Background: Hepatitis D Virus (HDV) infection is present worldwide and affects all age groups. About 5% of patients with chronic Hepatitis B Virus (HBV) infection are estimated to have HDV coinfection. HDV/HBV coinfection in children is associated with aggressive liver disease. Aim: To assess the prevalence of anti-hdv antibodies among a cohort of HBsAg-positive Egyptian children. Methods: This cross-sectional study included children who are HBsAg-positive, of both sexes, aged 1-18 years and were treatment-naïve. Laboratory tests including: Alanine aminotransferase, aspartate aminotransferase, HBsAg, HBcIgG, HBeAg, anti-hbe antibodies, anti-hdv antibodies and HBV DNA quantitation. Results: Sixty-five children were included. Their median age was 5 years, (range 1-16 years) and 64.6% were males. The commonest possible risk of HBV acquisition was HBsAgpositive mothers in 97%. Among the studied children; 49.2% were immune tolerant, 9.2% were immune active, 24.6% were inactive carriers. Anti-HDV antibodies were negative in all cases. Conclusions: There is lack of evidence of HDV infection in our group of Egyptian HBsAg-positive children who were HBV-infected mostly perinatally. Key Words: Anti-HDV antibodies Children Egypt HBV HDV. Introduction HEPATITIS D Virus (HDV) infection is present worldwide and affects all age groups. However, it does not have uniform distribution and its general pattern is parallel to that of Hepatitis B Virus (HBV) [1]. Among the million individuals harboring chronic HBV, approximately 15 million (~5%) are estimated to have HDV coinfection [2-4]. HDV is endemic in the Amazon Basin, the Mediterranean Basin, some parts of Asia and Central Africa [5,6]. Correspondence to: Dr. Hanan M. Fouad hananminaped@gmail.com HDV is a small RNA viral spherical particle approximately 36nm in diameter; it is also known as hepatitis delta virus [7,8]. It is a defective virus that requires Hepatitis B Surface Antigen (HBsAg) as its envelope protein [4]. HDV can occur as either a superinfection of chronic HBV infection or as simultaneous acute coinfection [7]. About 70-90% of patients coinfected with HBV/HDV show early HBeAg seroconversion and low serum levels of HBV DNA, as HDV infection can suppress HBV replication [9]. Control of HBV has distinctly diminished the epidemiologic and clinical impact of HDV in the developed world. The implementation of vaccine programs against HBV was associated with a decline in HDV infection [10]. HDV/HBV coinfection in children is associated with more aggressive liver disease than HBV monoinfection irrespective of HBeAg status [11,12]. They have rapid progression to cirrhosis and are more prone to hepatic decompensation, Hepatocellular Carcinoma (HCC) and death [3,5,13]. They show a high mortality rate of 2-20% in 5-10 years which is ten times higher than for those with HBV monoinfection [14]. The aim of this study is to assess the prevalence of anti-hdv antibodies among HBsAg-positive children, two decades after implementation of compulsory HBV vaccination program for infants in Egypt. Material and Methods This cross sectional study was carried out in the Pediatric Hepatology Clinic at the National Hepatology and Tropical Medicine Research Institute NHTMRI". The study was conducted from May to August The study included HBsAgpositive treatment-naive children, of both sexes, aged 1-18 years. Infants below 1 year of age were excluded to avoid misleading of antibodies trans- 777

2 778 Lack ofanti-hdv Antibodies in HBsAg-Positive Children Two Decades mitted from their HBV-positive mothers. The study protocol was approved by the NHTMRI institutional review board. Enrollment was done after signing an informed consent by the parents/guardian. All children were subjected to: Thorough history taking, with special emphasis on possible risk factors for HBV acquisition and clinical examination. Laboratory tests including: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), HBsAg, HBcIgG, HBeAg, anti-hbe antibodies, anti-hcv antibodies, total anti-hdv antibodies and HBV DNA quantitation in IU/ml. A blood sample of 5ml was drawn from all children by venipuncture and added on EDTA. Samples were centrifuged and sera were divided into 2 aliquots, one was stored at 80ºC for HBV DNA assay by PCR and the other was stored at 20ºC for HBV, HCV and HDV serology profile assay. HBV serology was done by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Abbott Murex was used for HBsAg and Diasorin was used for HBeAg, anti-hbe and anti-hbc total (DiaSorin S.p.A., SALUGGIA (VERCELLI)-Italy). Anti-HCV antibody was also done using ELISA technique (Dia. Pro diagnostic Bioprobes Srl via Columella nº Milano-Italy). Serum total anti-hdv antibodies: Serum total anti-hdv antibodies are assayed in all enrolled subjects using ELISA technique (DiaSorin S.p.A., SALUGGIA (VERCELLI) -Italy). The assay is based on a double-antibody sandwich ELISA technique for the qualitative assay of anti-hdv in samples. In this technique, anti- HDV present in the sample and labeled anti-hdv antibodies compete for a fixed quantity of anti- HDV antigen bound to the solid phase. The quantity of enzyme tracer bound to the solid phase and consequently the enzyme activity are inversely proportional to the anti-hdv concentration present in samples or kit-controls. Enzyme activity is measured by adding a colorless chromogen/substrate solution. The enzyme action on chromogen/ substrate produces a color which is measured with a photometer. Statistical analysis of results: Data were collected and tabulated. Statistical analysis was performed to describe the group characteristics using Statistical Package for Social Science (SPSS) program version Mean and Standard Deviation (SD) or median and Interquartile Range (IQR) were estimates of quantitative data while frequency and percentage were estimates of qualitative data. Results Sixty-five HBsAg-positive children were included. Their median age was 5 years, (range 1-16 years) and 64.6% were males. The most common possible risk of HBV acquisition was HbsAgpositive mother in 96.9%. The median ALT was 31IU/ml (ranged IU/ml) and median AST was 28 (ranged IU/ml). HBsAg and HBcIgG were positive in all cases, HBeAg was positive in 70.8% and anti-hbe antibodies were positive in 29.2%. DNA was Below Detection Level (BDL) in 9.2%. Children with positive DNA were 59 (90.8%), they had a median level of 5.3 X 10 6 ranged between 41 and 260 X Among the studied children, 49.2% were immune tolerant, 9.2% were immune active, 24.6% were inactive carriers and 16.9% need longer follow-up for better classification in term of chronic hepatitis definitions. None of them had positive HCV antibodies. Data are shown in Table (1). All studied children tested negative for anti- HDV antibodies. Table (1): Characteristics of the studied HBsAg-positive infants and children. HBsAg-positive children N=65 Sex; N (%): Male Female Median age (IQR) in years [Min-max] Values 42 (64.6) 23 (35.4) 5 (5.5) [1-16] Risk; N (%): Maternal HBV 63 (96.9) Other family member; uncle 1 (1.5) Blood transfusion 1 (1.5) Median ALT (IQR) [Min-max] 31 (29) [11-307] Median AST (IQR) [Min-max] 28 (30) [15-280] Positive HBeAg; N (%) 46 (70.8) Positive anti-hbe Ab 19 (29.2) Positive HCV Ab; N (%) 0 HBV DNA IU/m l: BDL; N (%) 6 (9.2) Positive; N (%) 59 (90.8) Median (IQR) [Min-max] of 5.3 X 10 6 (44.9 X 10 6 ) positive cases [ X 10 6 ] Levels of viremia: Low (<20000) 15 (23.1) Moderate ( ) 5 (7.7) High (>200000) 45 (69.2) HBV status; N (%): Immune tolerant 32 (49.2) Immune active 6 (9.2) Inactive carrier 16 (24.6) Need more follow-up 11 (16.9) IQR : Interquartile Range. HBV : Hepatitis B Virus. ALT : Alanine Aminotransferase. AST : Aspartate Aminotransferase. HCV : Hepatitis C Virus. BDL : Below Detection Level.

3 Hanan M. Fouad & Ehab M. Reyad 779 Table (2): Rates of HDV in the pediatric age group. Prevalence of HBsAg/HBV DNA % Studied pediatric population Prevalence of anti- HDV antibodies/ HDV RNA % Reference Africa: Egypt 4 HBsAg-positive 0 Current study Chronic liver disease/cirrhosis 8.9 Morcos et al., 2000 Burkina Faso HBsAg-positive 20.5 Central African Republic 44.3 HBsAg-positive 2.9 Andernach et al., 2014 Europe: Greece 0.95 HBsAg-positive 15.3 Manesis et al., 2013 Asia: Mongolia 9.8 HBsAg-positive 2 (RNA) Tsatsralt-Od et al., 2007 (88.7% immunized) America: Greenland 5-10 HBsAg-positive 51 Børresen et al., 2010 (no HBV vaccination) HBV: Hepatitis B Virus. HDV: Hepatitis D Virus. Discussion Egypt is considered moderately endemic for HBV, with 4% of the population having evidence of chronic HBV infection [15]. HBV vaccination was integrated in the expanded program of immunization for children in Egypt since late The program used a yeast recombinant DNA vaccine (10µg) to be given at 2, 4 and 6 months of age [16]. For the time being, there is no perinatal screening of pregnant women in Egypt. After 2 decades of compulsory HBV vaccination, our results showed lack of anti-hdv antibodies among studied HBsAg-positive children. There are scanty published data about the prevalence and characteristics of HDV infection in the pediatric population in Egypt. After initiating HBV vaccination program, Morcos et al., in 2000 [11] reported a prevalence of 8.9% among Egyptian children with chronic liver disease. Worldwide, studies reported wide range of HDV rates in HBsAgpositive children ranging between 2-51% [17-20]. Available published data in children are shown in (Table 2). The wide range of anti-hdv antibodies can be attributed to the introduction of HBV vaccine in the vaccination program in some countries and its lack in others. In Egypt, Darwish et al., [21] reported anti- HDV prevalence of 21.9% among HBsAg-positive healthy blood donors and 23.5% among adult patients with chronic HBV. More recently, Gomaa et al., [22] reported a prevalence of 4.7% among HBsAg-positive healthy blood donors. Worldwide, adult rates of anti-hdv antibodies ranged from 0% to 51% in different populations [20,23]. In the present study, anti-hdv antibodies were not detected in any of all the enrolled children. Among the studied children, the most common possible risk of acquisition was positive HBsAgmothers in about 97%. Despite that HBV transmission mainly occurs through perinatal transmission (from mother to child during birth) [24], HDV is not transmitted via perinatal route [25]. Recent studies have excluded the perinatal transmission as a main route of HDV infection. They confirmed horizontal rather than vertical route of transmission. They proposed that HBV/HDV superinfection is the predominant scenario in children with coinfection [18,20]. Family members are supposed to be the reservoirs for transmission of HDV infection [3,5]. Anti-HDV antibodies were used for screening based on prior recommendation of its use in testing HBsAg-positive patient [26]. The antibodies are detected within a few weeks of infection and may persist for years even when the patient has experienced HBsAg seroconversion [27] or has undergone liver transplantation [28]. HDV infection should be confirmed by the detection of serum HDV RNA [5]. HDV RNA is not recommended for screening of infection as there is no evidence that its direct testing in the absence of anti-hdv antibodies is of any use because anti-hdv antibodies develop in every infected individual [29]. An important trend in worldwide HDV infection is a global decline in the prevalence of both acute and chronic forms of the disease. This decreasing trend is the result of global HBV vaccination, increasing awareness, improved prevention strategies (e.g. screening of blood products) and socio-

4 780 Lack ofanti-hdv Antibodies in HBsAg-Positive Children Two Decades economic conditions [24,25,30,31]. Increasing vaccination coverage for children will have a huge impact on HBV prevalence alone but the introduction of adult vaccination will have the greatest impact on both HBV and HDV prevalence [2,30, 31]. Clearly, the challenge that prevention of HBV with an effective vaccination program will eliminate the possibility of HDV infection [24]. Thus, HDV can be controlled using interventions directed towards HBV but not vice-versa. Therefore, HBV adult vaccination programs can play an important role in eradication of both viruses showing a primary effect on HBV prevalence and a secondary effect on HDV prevalence [25]. Our study limitation is that it is a single centre study with a limited number of patients. Despite the limited number of the studied HBsAg-positive children, the strength of the present study arises from the fact that previous information regarding anti-hdv antibodies in children is lacking worldwide and in Egypt as well. Also the HDV serostatus of the parents is not known but another study in Egypt reported an HDV prevalence of 4.7% among HBsAg-positive healthy blood donors in 2013 [22]. In conclusion, our study revealed lack of anti- HDV antibodies in HBsAg-positive Egyptian children 2 decades after implementation of obligatory HBV vaccination in infancy. References 1- World Health Organization (WHO): Hepatitis Delta fact sheet (WHO/CDS/CSR/NCS/2001.1). WHO, Geneva, Switzerland. HepatitisD_whocdscsrncs2001_ 1.pdf?u_ ABBAS Z., JAFRI W. and RAZA S.: Hepatitis D, Scenario in the Asia-Pacific region. World. J. Gastroenterol., 16: , BAHCECIOGLU I.H., AYGUN C., GOZEL N., et al.: Prevalence of hepatitis delta virus infection in chronic hepatitis B patients in eastern Turkey: still a serious problem to consider. J. Viral. Hepat, 18: , HUGHES S.A., WEDEMEYER H. and HARRISON P.M.: Hepatitis delta virus. Lancet, 378: 73-85, WEDEMEYER H. and MANNS M.P.: Epidemiology, pathogenesis and management of hepatitis D: Update and challenges ahead. Nat. Rev. Gastroenterol. Hepatol., 7: 31-40, RIZZETTO M. and CIANCIO A.: Epidemiology of hepatitis D. Semin. Liver Dis., 32: 211-9, TAYLOR J.M.: Hepatitis delta virus. Virology, 344: 71-6, KOYTAK E.S., YURDAYDIN C. and GLENN J. S.: Hepatitis D. Curr. Treat. Options. Gastroenterol., 10: , CROSS T.J., RIZZI P., HORNER M., et al.: The increasing prevalence of Hepatitis Delta Virus (HDV) infection in South London. J. Med. Virol., 80: , DEGERTEKIN H., YALCIN K., YAKUT M., et al.: Seropositivity for delta hepatitis in patients with chronic hepatitis B and liver cirrhosis in Turkey: A meta-analysis. Liver Int., 28: 494-8, MORCOS M.M., MIKHAIL T.H., HANNA W.M., et al.: The prevalence of delta virus infection in chronic liver disease in Egyptian children in comparison with some other countries. Panminerva. Med., 42: , ABBAS Z., SOOMRO G.B., HASSAN S.M., et al.: Clinical presentation of hepatitis D in Pakistani children. Eur. J. Gastroenterol. Hepatol., 26: , RIZZETTO M.: Hepatitis D: Thirty years after. J. Hepatol., 50: , BUTI M., HOMS M., RODRIGUEZ-FRIAS F., et al.: Clinical outcome of acute and chronic hepatitis delta over time: A long-term follow-up study. J. Viral. Hepat., 18: , World Health Organization (WHO): Hepatitis B, WHO fact sheet No. 204, Available from: who.int/mediacentre/factsheets/fs204/en/index.html. [Accessed 2015 December]. 16- MANSOUR E., ABDUL-RAHIM S., BATOUTY G., et al.: Integration of hepatitis B immunization in the Expanded Program on Immunization of the Child Survival Project. J. Egypt Public Health Assoc., 68: , TSATSRALT-OD B., TAKAHASHI M., ENDO K., et al.: Prevalence of hepatitis B, C, and delta virus infections among children in Mongolia: Progress in childhood immunization. J. Med. Virol., 79: , BØRRESEN M.L., OLSEN O.R., LADEFOGED K., et al.: Hepatitis D outbreak among children in a hepatitis B hyper-endemic settlement in Greenland. J. Viral. Hepat., 17: , MANESIS E.K., VOURLI G., DALEKOS G., et al.: Prevalence and clinical course of hepatitis delta infection in Greece: A 13-year prospective study. J. Hepatol., 59: , ANDERNACH I.E., LEISS L.V., TARNAGDA Z.S., et al.: Characterization of Hepatitis Delta Virus in Sub- Saharan Africa. J. Clin. Microbiol., 52: , DARWISH M.A., SHAKER M., RASLAN O.S., et al.: Delta virus infection in Egypt. J. Egypt Public Health Assoc., 67: , GOMAA N.I., METWALLY L.A., NEMR N., et al.: Seroprevalence of HDV infection in HBsAg positive population in Ismailia, Egypt. Egypt J. Immunol., 20: 23-8, ONYEKWERE C.A., AUDU R.A., DURO-EMMANUEL F., et al.: Hepatitis D infection in Nigeria. Indian J. Gastroenterol., 31: 34-5, THOMAS E., YONEDA M. and SCHIFF E.R.: Viral hepatitis: Past and future of HBV and HDV. Cold. Spring. Harb. Perspect. Med., 5: A021345, 2015.

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