High frequency of hepatitis B virus infection in patients with beta-thalassemia receiving multiple transfusions

Abstract

Background and Objectives: Hepatitis B virus (HBV) may occasionally be transmitted through transfusion of blood units that are hepatitis B surface antigen (HBsAg) negative but HBV DNA positive. Children with β-thalassemia are particularly susceptible to HBV because they receive multiple blood transfusions. These children have high infection rates despite vaccination against HBV. Post-vaccination infections may be a result of viruses harbouring surface (S)-gene mutations (e.g. G587A) in a region critical for reactivity to antibody to hepatitis B surface antigen (anti-HBs). The true prevalence of HBV in individuals with β-thalassemia has not been studied previously. Patients and Methods: Seventy patients with β-thalassemia (median age 6 years; range 8 months to 22 years; 49 male), who had received seven to 623 (median 61) units of blood each and three doses (10/20 µg) of HBV vaccine (Engerix B) before presentation to us, were included in the study; 50 of the 70 patients had received transfusions prior to vaccination. Enzyme-linked immunoassay for serological markers [HBsAg, antibody to hepatitis B core antigen (anti-HBc) and quantitative anti-HBs] and polymerase chain reaction (PCR) followed by Southern hybridization for molecular detection of hepatitis B, was performed on all samples. The PCR-amplified product was cloned, sequenced and the nucleotide and deduced amino acid sequences for the HBV S and polymerase (P) genes were analysed for mutations. Results: Four of 70 (5·7%) individuals with β-thalassemia were HBsAg positive and 14 (20%) were anti-HBc positive. The prevalence of serological markers increased with number of transfusions (P < 0·01). Of 70 patients, 53 (75·7%) had an anti-HBs titre of > 10 IU/l following vaccination and 17 (24·3%) were non-responders (< 10 IU/l); 22 (31·4%) of the 70 were DNA positive. The frequency of HBV infection in β-thalassemia was similar in vaccine responders and non-responders. The virus was of subtype ayw (genotype D) in the five DNA-positive samples in which a 388-nucleotide region of the S gene was sequenced. Mutations occurred at 13 positions in the S gene and at 10 positions in the P gene. Hydrophobicity plots revealed differences in amino acid regions 117–165 and 195–211. Some of these amino acid substitutions coincided with the putative cytotoxic T-lymphocyte epitopes of both S and P proteins. Conclusions: A high frequency of HBV infection was seen using molecular methods in thalassemic patients. The frequency of infection was similar in vaccine responders and non-responders. A number of mutations were observed in the S gene, which could have implications for viral replication as well as virus–host cell interaction.