A novel hepatitis B virus recombinant genotype D4/E identified in a South African population.

BACKGROUND: Genetic diversity is a characteristic trait of the hepatitis B virus (HBV) and has been associated with different clinical outcomes. In South Africa, HBV infection is a major public health concern. Most HBV infections are caused by genotype A strains. However rare cases of infection with HBV genotype D have been reported. The purpose of this study was to investigate the molecular characteristics of a rare HBV subgenotype D4 isolate. METHODS: The full-length genome of isolate ZADGM6964 was amplified in a one-step polymerase chain reaction. The amplified product was purified and cloned into a pGEM®-T Easy Vector System to investigate the genetic diversity of the viral quasi-populations. The primary isolate and clones were then directly sequenced and analysed using an array of bioinformatics software. RESULTS: Phylogenetic analysis showed that the primary isolate and cloned sequences formed a monophyletic cluster away from subgenotype D4 reference strains. Further recombination analysis revealed that isolate ZADGM6964 was in fact a D4/E recombinant strain with breakpoints identified within the X and overlapping pre-Core/Core open reading frames with a >70% bootstrap confidence level. The recombinant genotype D4/E was found to be unique from other D/E strains archived in the genetic database, GenBank. CONCLUSION: This study represents the first ever report on the isolation and molecular characterization of an HBV D4/E recombinant strain in South Africa. The findings provide evidence of further HBV genetic diversity in South Africa than has been previously reported.

Evidence of susceptibility to lamivudine-based HAART and genetic stability of hepatitis B virus (HBV) in HIV co-infected patients: A South African longitudinal HBV whole genome study.

BACKGROUND: Reports on the concomitant impact of HIV co-infection and long term highly active anti-retroviral therapy (HAART) on the genetic stability and molecular evolution of HBV are limited in sub-Saharan Africa. MATERIALS AND METHODS: This retrospective study investigated the molecular evolution of chronic HBV in HIV co-infected patients on lamivudine (3TC)-based HAART over a 5year period. Four HIV co-infected patients, consecutively recruited and followed-up, were screened for hepatitis B serological markers, and their viral loads determined. The HBV genome was amplified from longitudinal samples and characterized by Bayesian inference, mutational analysis, and identification of immune selection pressure. RESULTS: All patients exhibited persistent chronic HBV infection at baseline, as well as over the course of follow-up despite exposure to 3TC-based HAART. The polymerase gene in all isolates was relatively variable prior to HAART initiation at baseline and during the course of follow-up, although primary drug resistance mutations were not detected. All but one patient were infected with HBV subgenotype A1. The divergence rates between baseline and the last follow-up sequences ranged from 0 to 2.0×10(-3) substitutions per site per year (s/s/y). Positive selection pressure was evident within the surface and core genes. CONCLUSION: Despite persistent HBV infection in the HIV co-infected patients exposed to long term 3TC-based HAART, the molecular evolution of HBV over a 5year period was unremarkable. In addition, HBV exhibited minimal genetic variability overtime.

High prevalence of active and occult hepatitis B virus infections in healthcare workers from two provinces of South Africa.

BACKGROUND: Hepatitis B (HB) is a vaccine-preventable liver disease caused by infection with the blood-borne hepatitis B virus (HBV). South African healthcare workers (HCWs) may be at high risk of occupational exposure to HBV infection, since previous studies have found suboptimal levels of protection against HBV in HCWs. METHODS: A descriptive prevalence study based on self-administered questionnaires with data on demographics and HB vaccination status, and stored serum samples collected from 2009 to 2012, from 333 HCWs working or studying in Gauteng and Mpumalanga province hospitals or nursing colleges, was conducted. Samples were tested for HB surface antigen (HBsAg), antibodies to HBsAg (anti-HBs), antibodies to HB core antigen (anti-HBc), and HBV deoxyribonucleic acid (DNA). RESULTS: The majority of HCWs from whom the serum samples were drawn were black (91.4% [298/326]), female (82.6% [275/333]) and had received at least one dose of HB vaccine (70.9% [236/333]). The average age was 38.8years (range: 19-62). Of the HCWs, 23.2% (73/314) were susceptible (negative for all markers); 9.6% (30/314) were infected (HBsAg and/or DNA positive); 29.0% (91/314) were exposed (positive for either HBsAg, anti-HBc, or DNA); 18.8% (59/314) were immune due to natural infection (anti-HBs and anti-HBc positive only); while 47.8% (150/314) were immune due to vaccination (anti-HBs positive only). Furthermore, HBV DNA was detected in 8.6% (27/314) and occult HBV infection (OBI) (HBV DNA positive but HBsAg negative) was found in 6.7% (21/314) of samples. DISCUSSION AND CONCLUSION: This study, which is the first to report OBI in South African HCWs, found high rates of active HBV infection and sub-optimal protection against HBV in HCWs. There is a need to strengthen vaccination programmes through a policy that ensures protection for all HCWs and their patients.

Hepatitis B virus infection in post-vaccination South Africa: occult HBV infection and circulating surface gene variants.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the prevalence of occult hepatitis B virus (HBV) infection and the HBV surface (S) gene variants circulating in the South African population after nearly two decades of universal hepatitis B vaccination. STUDY DESIGN: From a previous serosurvey, 201 serum samples with serological evidence of exposure to HBV were identified and these were stratified into post- and pre-vaccine introduction populations. For all samples, HBV DNA was screened and quantified using a real-time PCR assay and results analysed together with HBV serological markers. Where HIV results were available, subset analysis was performed. The HBV S gene was PCR-amplified and sequences analysed for a total of 37 isolates. RESULTS: The prevalence of occult HBV infection reduced from 70.4% in the pre-vaccine introduction era to 66.0% post-vaccine introduction. There was an association between HIV infection and an increase in prevalence of occult HBV infection within the post-vaccine introduction population, although this was not statistically significant. Furthermore, sequence analysis revealed the following HBV subgenotypes; A1 (n=34), A2 (n=2) and a rare D4 isolate. HBV S gene variants, including diagnostic escape mutants were isolated. CONCLUSION: There was a decline in the prevalence of occult HBV infection in post-vaccination South Africa, although the disease burden remains significant in the HIV co-infected population. After nearly two decades of a universal hepatitis B vaccination programme, no positive selection of vaccine escape mutants were observed.

Evidence for a change in the epidemiology of hepatitis B virus infection after nearly two decades of universal hepatitis B vaccination in South Africa.

The hepatitis B vaccine has been part of the South African Expanded Program on Immunization since April 1995 but its long-term impact remains unknown. This study tested 1,206 sera collected from patients aged 1-25 years from various health facilities across the country for HBV serological markers and HBV DNA. Based on the year the vaccine was introduced, samples were stratified by age into pre- and post-vaccine introduction populations, which were then compared for evidence of immunity and chronic carriage using the Chi-square test. Where HIV status was known, subset analyses were performed. Immunity to HBV infection increased from 13.0% in the pre- to 57.0% in the post-vaccine introduction population (P < 0.001). This decreased with increasing age within the post-vaccine introduction population (76.1% for 1-5 years, 50.0% for 6-10 years, and 46.3% for 11-16 years). In addition, HBV chronic carriage was significantly (P = 0.003) reduced in the post- (1.4%) compared to the pre-vaccine introduction population (4.2%). The difference in prevalence of active HBV infection in the serologically exposed pre- and post-vaccine introduction populations was not statistically significant. Subset analyses showed that evidence of immunity was significantly (P < 0.001) higher in the HIV negative compared to the HIV positive subset in both populations. Universal hepatitis B vaccination has been a remarkable success, with a significant increase in immunity to HBV infection. The observation that HBV chronic carriage increases as immunity wanes over time calls into question whether the time has come to consider a pre-adolescence vaccine booster dose policy.

Introduction of new subtypes and variants of hepatitis C virus genotype 4 in South Africa.

Hepatitis C virus (HCV) genotype is an important predictor of disease progression and treatment response. This descriptive study investigated the sequence diversity and genotypes of HCV in South Africa based on comparative analysis of the 5' untranslated region (UTR), C/E1, and NS5B regions of 60 sequences from 52 patients. Genotype distribution in the studied population was as follows: 54% (28/52) were genotype 5, 19% (10/52) were genotype 1, 19% (10/52) were genotype 4, and 2% (1/52) were genotype 3. Three of 52 (6%) individuals were infected with multiple genotypes based on the 5'UTR. Phylogenetic analysis of the 5'UTR was accurate in determining genotypes, while the C/E1 and NS5B coding region was able to differentiate both genotypes and subtypes, including an outlier group. Furthermore, this study observed the existence of distinct variants of HCV which were divergent from confirmed genotype 4 subtypes. For the first time in South Africa, this analysis has shown the presence of HCV subtypes 4k, 4q, and 4r, as well as evidence of intragenotypic recombinant 4l/4q within NS5B. In conclusion, while genotype 5a remains the predominant genotype in South Africa, the current study indicates the introduction of new subtypes and existence of variants of genotype 4 in South Africa.