Molecular characterization and genotyping of hepatitis B virus (HBV) from sudanese end stage renal disease (ESRD) patients with overt and occult HBV infection

Abstract

Hepatitis B virus (HBV), which causes infection globally, is endemic in sub-Saharan Africa. Currently, nine genotypes (A to I), distributed geographically, are recognised. In Sudan, located in north-east Africa, HBV prevalence ranges from 4% to 26%, and genotypes D and E predominate. Patients suffering from end-stage renal disease (ESRD) exhibit poor kidney function and require regular haemodialysis. HBV exposure in haemodialysis patients in Sudan is high, as the immunosuppressive nature of renal disease increases risk from HBV infection. The aim of this study was to molecularly characterise HBV from ESRD patients in Sudan undergoing haemodialysis and also to use phylogenetic analysis to determine if HBV infection was occurring in the four haemodialysis centres from which HBV DNA was successfully amplified. We undertook polymerase chain reaction (PCR) amplification and DNA sequencing of the basic core promoter/pre-core (BCP/PC) and the complete S (preS1/preS2/S) regions of 68 HBV DNA positive ESRD patients undergoing haemodialysis in four different treatment centres (Salma, Ibn Sena, Elturki and Alnaw) in Khartoum. In Hepatitis B surface antigen (HBsAg) positive (overt) HBV-mono-infected samples, PCR amplicons from the BCP/PC region were obtained from 28/46 (60.9%) of samples, with DNA sequences obtained from 24/46 (52.2%). Results for the S region were 23/46 (50%) and 15/46 (32.6%), for PCR amplification and sequencing, respectively. BCP/PC PCR amplification from 12 occult HBV-mono-infected samples resulted in 2/12(16.7%) amplifications of low density bands. Hence no DNA was obtained for sequencing. However, 4/12 (33.3%) of these samples yielded S region amplicons, with DNA obtained from 3/12 (25%). Ten HBV/HCV co-infected samples yielded 4/10 (40%) positive samples in the BCP/PC, with 3/10 sequenced (30%) in the complete S, 2/10 (20%) amplified with low DNA quality. Neighbour-joining phylogenetic analysis using 1000 bootstrap indicated that genotype D predominated at 11/15 (73.3%), followed by genotypes E and A, at 3/15 (20%) and 1/15 vi (6.7%), respectively. Genotype D was found in all centres and mostly detected at Salma 6/15 (40%), whereas genotype A was only detected at Elturki. Moreover, Genotype E was only detected in Salma and Ibn Sena. We identified a six nucleotide insertion occurring in the core region of HBV from four individuals from Salma centre. This was a novel finding in genotype D isolates as this insertion was previously found in genotype A. The preS1 mutation, ps1I48V, which occurs mostly in genotype A isolates, was found in one genotype A isolate from Sudan. We further identified a preS2 deletion in one genotype E isolate. The deletions in the pre S contribute to the development of hepatocellular carcinoma (HCC). The HBV reactivation marker, S174N was found in one HBsAg negative (occult) individual. We further identified HBV drug resistance mutations, rtS213 and rtQ215H occurring in two different individuals from Ibn Sena centre. Both of these individuals were not vaccinated. In conclusion, in Sudanese ESRD patients, genotype distribution varies between treatment centres, although genotype D predominates. In addition, drug resistance and vaccine escape similar to the ones found in South Africa exist in Sudan. The small sample size did not allow us to compare the mutations found in HBV isolates from individuals with overt or occult infection. Hence, further studies to identify distinguishing mutations between occult and overt samples, as well as in HBV/HCV co-infected individuals in Sudan are still needed.