Hepatitis B and hepatitis D virus infections in the Central African Republic, twenty-five years after a fulminant hepatitis outbreak, indicate continuing spread in asymptomatic young adults

Abstract

Hepatitis delta virus (HDV) increases morbidity in Hepatitis B virus (HBV)-infected patients. In the mid-eighties, an outbreak of HDV fulminant hepatitis (FH) in the Central African Republic (CAR) killed 88% of patients hospitalized in Bangui. We evaluated infections with HBV and HDV among students and pregnant women, 25 years after the fulminant hepatitis (FH) outbreak to determine (i) the prevalence of HBV and HDV infection in this population, (ii) the clinical risk factors for HBV and/or HDV infections, and (iii) to characterize and compare the strains from the FH outbreak in the 1980s to the 2010 HBV-HDV strains. We performed a cross sectional study with historical comparison on FH-stored samples (n = 179) from 159 patients and dried blood-spots from volunteer students and pregnant women groups (n = 2172). We analyzed risk factors potentially associated with HBV and HDV. Previous HBV infection (presence of anti-HBc) occurred in 345/1290 students (26.7%) and 186/870 pregnant women (21.4%)(p = 0.005), including 110 students (8.8%) and 71 pregnant women (8.2%), who were also HBsAg-positive (p = 0.824). HDV infection occurred more frequently in pregnant women (n = 13; 18.8%) than students (n = 6; 5.4%) (p = 0.010). Infection in childhood was probably the main HBV risk factor. The risk factors for HDV infection were age (p = 0.040), transfusion (p = 0.039), and a tendency for tattooing (p = 0.055) and absence of condom use (p = 0.049). HBV-E and HDV-1 were highly prevalent during both the FH outbreak and the 2010 screening project. For historical samples, due to storage conditions and despite several attempts, we could only obtain partial HDV amplification representing 25% of the full-length genome. The HDV-1 mid-eighties FH-strains did not form a specific clade and were affiliated to two different HDV-1 African subgenotypes, one of which also includes the 2010 HDV-1 strains. In the Central African Republic, these findings indicate a high prevalence of previous and current HBV-E and HDV-1 infections both in the mid-eighties fulminant hepatitis outbreak and among asymptomatic young adults in 2010, and reinforce the need for universal HBV vaccination and the prevention of HDV transmission among HBsAg-positive patients through blood or sexual routes.

Fig 1. Phylogenetic analysis of HDV partial genome (400 bp) comparing mid-eighties delta fulminant hepatitis clones (FH1985) to HDV direct sequences (CAR2010) sampled in 2010 from serum (s) and dry blood spot (d) among asymptomatic young adults in Bangui.

We aligned 45 cloned sequences from the 12 FH strains obtained from the historical cohort (labelled ‘1985’ in green) and the 6 strains obtained from asymptomatic students in 2010 represented in duplicate from serum (s) and dried blood (d) (labelled ‘2010’ in blue). We also included 1 strain from a hospitalized case of acute HDV hepatitis in Bangui in 2010 (sd525-CAR2010) and HDV-1 sequences from 9 African samples characterized in Bobigny (dFr) or Lyon (dLy), France, in addition to CAR HDV sequences published by Andernach and coworkers [22], sampled in 2009. Further comparison included HDV strains from various parts of the world and genotype-reference prototypes (labelled in red). Bayesian analyses (10M generations) gave the consensus tree represented in Fig 1, after discarding 25% of trees from early topology exploration. Branch values indicate posterior probabilities >0.9. Interestingly, the fulminant 1985 and asymptomatic student 2010 strains are all affiliated to HDV-1 with a 100% posterior probability value (thick branch) and the clade topology do not distinguish the mid-eighty strains from the 2010 strains.

Fig 2. Alignment of the L-HDAg amino acid deduced sequences from fulminant-associated isolates from the historical cohort (1985) and from young asymptomatic HDV-infected students (2010).

The alignment is compared together with L-HDAg sequences from HDV-1 prototype (Italy, Accession Number:X04451) and HDV-3 prototype (Peru-1, Accession Number:L22063), representing the prototypes of two HDV genotypes associated with fulminant hepatitis outbreaks. Dots represent the same amino acid as in the Italy prototype and question mark ambiguities. A: Full-length coding sequences (214 codons) were obtained for 3 FH isolates (FH27, FH88, FH123). B: COOH terminal part of L-HDAg of the corresponding HDV 1985 (45 FH clones) associated with fulminant hepatitis and 2010 (seven) sequences associated to asymptomatic infections. Note that all the African sequences have the A202S mutation and that FH27 clone 4 has a frameshift mutation of the carboxy-terminal end of L-HDAg (ORF-K), leading to the disappearance of the farnesylation CXXX box.