Hepatitis B virus (HBV) infection and recombination between HBV genotypes D and E in asymptomatic blood donors from Khartoum, Sudan

Abstract

Sudan is a highly endemic area for hepatitis B virus (HBV), and >5% of blood donors are chronically infected. To examine potential strategies to improve HBV blood safety, 404 replacement donor samples previously screened for HBV surface antigen (HBsAg) were tested for antibody to HBV core (anti-HBc), anti-surface antigen (anti-HBs), and HBV DNA. Of 145 anti-HBc-containing samples (36%) identified, 16 retested were HBsAg positive (11%). Anti-HBs was detected in 43/77 (56%) anti-HBc-reactive samples. Six samples were HBsAg(-)/anti-HBc(+)/anti-HBs(+) and contained HBV DNA, meeting the definition of occult HBV infection (OBI). OBIs had low HBV DNA loads (<10 IU/ml) and were genotype B (n = 1) or genotype D (n = 5). Pre-S/S and/or whole genome sequences were obtained from 47 randomly selected HBsAg-positive donors added to the previous 16. Genotype E was identified in 27 strains (57.5%), genotype D in 19 strains (40.5%), and genotype A2 in 1 strain (2%). Two outlier strains within genotype D ultimately were identified as recombinants of genotypes D and E with identical recombination points, suggesting circulating, infectious, recombinant strains. Anti-HBc screening does not appear to be a sustainable blood safety strategy because of the cost and the negative impact on the Sudanese blood supply, even when reduced by anti-HBs testing. Being at the junction between two main African HBV genotypes, genetic recombination occurred and became part of the molecular epidemiology of HBV in Sudan.

FIG. 1.

HBV viral load distribution of 64 Sudanese HBsAg-positive blood donor samples. Black bars characterize the total population of donors, white bars characterize donors infected with HBV genotype D, and gray bars characterize donors infected with HBV genotype E.

FIG. 2.

Phylogenetic tree of Sudanese HBV strains based on Pre-S/S sequences. Phylogenetic analysis was performed with the neighbor-joining algorithm based on the Kimura two-parameter distance estimation method. Only bootstrap values of ≥75% are shown (1,000 replicates). Sudanese sequences are indicated in boldface, and HBV reference sequences of genotypes/subgenotypes A1 to A3, B, C, D1 to D8, E, F, G, and H are identified by their GenBank accession numbers (HQ385227 to HQ385272). For a more comprehensive analysis, genotype E sequences (a) and genotype D sequences (b) are shown in two distinct trees. Suspected recombinant strains are indicated by thick bars.

FIG. 2.

Phylogenetic tree of Sudanese HBV strains based on Pre-S/S sequences. Phylogenetic analysis was performed with the neighbor-joining algorithm based on the Kimura two-parameter distance estimation method. Only bootstrap values of ≥75% are shown (1,000 replicates). Sudanese sequences are indicated in boldface, and HBV reference sequences of genotypes/subgenotypes A1 to A3, B, C, D1 to D8, E, F, G, and H are identified by their GenBank accession numbers (HQ385227 to HQ385272). For a more comprehensive analysis, genotype E sequences (a) and genotype D sequences (b) are shown in two distinct trees. Suspected recombinant strains are indicated by thick bars.

FIG. 3.

Schematic representation of Sudanese HBV D/E recombinant strains. (a) Schematic representation of recombination points in Sudanese strain S81 and in three recombinant HBV-D/E (D8) strains from Niger (bne281 and bne367; GenBank accession numbers FN594769 and FN294770) and Ghana (GH16; GenBank accession number GQ161754), which were included for comparison. Numbers above the bars represent the exact genome positions (EcoRI cut) of the recombination points as indicated by SIMPLOT in an alignment of 3,257 nucleotides that includes all genotype-specific gaps. A representation of the three open reading frames of the HBV genome is shown above the figure. (b) Schematic representation of the clones isolated from the Sudanese S81 and S347 plasma samples in the Pre-S/S region. Numbers indicate the nucleotide positions of recombination as indicated by SIMPLOT.