Hepatitis B in sub-Saharan Africa: strategies to achieve the 2030 elimination targets.

The WHO global health sector strategy on viral hepatitis, created in May, 2016, aims to achieve a 90% reduction in new cases of chronic hepatitis B and C and a 65% reduction in mortality due to hepatitis B and C by 2030. Hepatitis B virus (HBV) is endemic in sub-Saharan Africa, and despite the introduction of universal hepatitis B vaccination and effective antiviral therapy, the estimated overall seroprevalence of hepatitis B surface antigen remains high at 6·1% (95% uncertainty interval 4·6-8·5). In this Series paper, we have reviewed the literature to examine the epidemiology, burden of liver disease, and elimination strategies of hepatitis B in sub-Saharan Africa. This paper reflects a supranational perspective of sub-Saharan Africa, and recommends several priority elimination strategies that address the need both to prevent new infections and to diagnose and treat chronic infections. The key to achieving these elimination goals in sub-Saharan Africa is the effective prevention of new infections via universal implementation of the HBV birth-dose vaccine, full vaccine coverage, access to affordable diagnostics to identify HBV-infected individuals, and to enable linkage to care and antiviral therapy.

Obesity as a cause of hepatocellular carcinoma.

During recent years the incidence of obesity has increased significantly, and in some instances rapidly, in many resource-rich countries. Paralleling this increase has been an increase in the incidence of hepatocellular carcinoma. It has been estimated that as many as 90% of obese adults will develop the metabolic syndrome. The worldwide incidence of this syndrome in adults at this time ranges from 9 to 34%. Furthermore, obesity in childhood increases the risk of obesity in adulthood, and hence the development of the metabolic syndrome and hepatocellular carcinoma. Ten to 20% of patients with non-alcoholic fatty liver disease progress to non-alcoholic steatohepatitis, and 8.3% of the latter develop cirrhosis. Up to 50% of these patients with cirrhosis, and a significant proportion of those without cirrhosis, progress to hepatocellular carcinoma. Much remains to be learnt about the mechanisms by which obesity and the metabolic syndrome cause hepatocellular carcinoma, although insulin resistance, increased tissue necrosis factor activity, alterations in serum lipids, non-alcoholic fatty liver disease and non-alcoholic steatosis play important roles. There is also increasing evidence that gut microbiota play a role in the development of the metabolic syndrome and hence of hepatocellular carcinoma.

Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. METHODS: We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence. RESULTS: Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. CONCLUSIONS: Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets.

Hepatic iron overload and hepatocellular carcinoma.

In recent years it has become increasingly evident that excess body iron may be complicated by the supervention of hepatocellular carcinoma (HCC). Hereditary hemochromatosis (HH) was the first condition in which hepatic iron overload was shown to predispose to the development of HCC. The inherited predisposition to excessive absorption of dietary iron in HH is almost always the result of homozygosity of the C282Y mutation of the HFE gene, which causes inappropriately low secretion of hepcidin. HCC develops in 8-10% of patients with HH and is responsible for approximately 45% of deaths in the HCC patients. Cirrhosis is almost always present when HCC is diagnosed. Dietary iron overload is a condition which occurs in rural-dwelling Black Africans in southern Africa as a result of the consumption, over time, of large volumes of alcohol home-brewed in iron containers and having, as a consequence, a high iron content. Iron loading of the liver results and may be complicated by malignant transformation of the liver (relative risk of approximately 10.0). Accompanying cirrhosis does occur but is less common than that in HH. The development of HCC as a consequence of increased dietary iron, and the fact that it may develop in the absence of cirrhosis, has been confirmed in an animal model. Drinking water with a high iron content might contribute to the high incidence of HCC in parts of Taiwan. The metabolic syndrome [obesity, insulin resistance type 2 (or diabetes mellitus type 2), non-alcoholic fatty liver or non-alcoholic steatohepatitis] has in recent years become a major public health problem in some resource-rich countries. A link between excess body iron and insulin resistance or the metabolic syndrome has become apparent. The metabolic syndrome may be complicated by the supervention of HCC, and recent evidence suggests that increased body iron may contribute to this complication.

Construction of replication competent plasmids of hepatitis B virus subgenotypes A1, A2 and D3 with authentic endogenous promoters.

Hepatitis B virus (HBV) is hyperendemic to southern Africa, with genotype A of HBV being the predominant genotype, and subgenotype A1 prevailing. Infection with this subgenotype is associated with rapid disease progression, and high frequency of hepatocellular carcinoma development. The objectives of our study was to construct recombinant 1.28 mer replication competent HBV DNA plasmids of subgenotypes A1, A2 and D3 containing authentic endogenous HBV promoters and to follow their replication in vitro after transfection of Huh7 cells. We found that subgenotype D3 replicated at a lower level, as measured by HBsAg and HBV DNA levels, when compared to cells transfected with genotype A. There was no difference in the intracellular and extracellular HBsAg between cells transfected with subgenotypes A1 or A2. Cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. In conclusion, the generation of these replication competent clones is an important step in the functional characterization of subgenotypes of HBV circulating in Africa and their comparison to strains circulating in other geographical regions of the world.

The role of cirrhosis in the etiology of hepatocellular carcinoma.

Abundant evidence supports the belief of a causal relationship between cirrhosis and hepatocellular carcinoma, but one that differs between high- and low-incidence regions of the tumor. In high-incidence regions, the cirrhosis is of the macronodular variety, is typically asymptomatic, and is caused predominantly by chronic hepatitis B virus infection, whereas in low-incidence regions, the cirrhosis, although usually macronodular, may be micronodular, is commonly symptomatic and of long-standing, and is caused by chronic hepatitis C virus infection, alcohol abuse over many years, the metabolic syndrome, or hereditary hemochromatosis. In a minority of patients, hepatocellular carcinoma develops in the absence of cirrhosis, supporting a direct hepatocarcinogenic effect of some of the causal agents. Cirrhosis is the major risk factor for tumor formation in patients with chronic hepatitis C virus infection. This virus does not integrate into cellular DNA, and malignant transformation results from increased liver cell turnover induced by recurring injury and regeneration of cells in the context of persisting inflammation, oxidative DNA damage, fibrosis, cirrhosis, and changes induced by the virus at a DNA level that have yet to be fully defined. Hepatitis B virus causes malignant transformation by both direct and indirect routes. The direct route results, in part, from integration of the viral DNA into host cellular DNA; transcriptional activation of host growth regulatory genes by hepatitis B virus-encoded proteins; and effects on apoptosis, cell signaling, and DNA repair. The direct route may share some similarities with that of hepatitis C virus infection. The metabolic syndrome may cause malignant transformation by production of oxidative stress and the induction of a variety of mutations, including some in the p53 gene.

Hepatocellular carcinoma: epidemiology and risk factors.

Hepatocellular carcinoma is one of the major malignant tumors in the world today. The number of new cases of the tumor increases year by year, and hepatocellular carcinoma almost always runs a fulminant course and carries an especially grave prognosis. It has a low resectability rate and a high recurrence rate after surgical intervention, and responds poorly to anticancer drugs and radiotherapy. Hepatocellular carcinoma does not have a uniform geographical distribution: rather, very high incidences occur in Eastern and Southeastern Asia and in sub-Saharan Black Africans. In these regions and populations, the tumor shows a distinct shift in age distribution toward the younger ages, seen to greatest extent in sub-Saharan Black Africans. In all populations, males are more commonly affected. The most common risk factors for hepatocellular carcinoma in resource-poor populations with a high incidence of the tumor are chronic hepatitis B virus infection and dietary exposure to the fungal hepatocarcinogen aflatoxin B1. These two causative agents act either singly or synergistically. Both the viral infection and exposure to the fungus occur from early childhood, and the tumor typically presents at an early age. Chronic hepatitis C virus infection is an important cause of hepatocellular carcinoma in resource-rich countries with a low incidence of the tumor. The infection is acquired in adulthood and hepatocellular carcinoma occurs later than it does with hepatitis B virus-induced tumors. In recent years, obesity and the metabolic syndrome have increased markedly in incidence and importance as a cause of hepatocellular carcinoma in some resource-rich regions. Chronic alcohol abuse remains an important risk factor for malignant transformation of hepatocytes, frequently in association with alcohol-induced cirrhosis. Excessive iron accumulation in hereditary hemochromatosis and dietary iron overload in the Black African population and membranous obstruction of the inferior cava cause the tumor in a few countries.

Aflatoxins as a cause of hepatocellular carcinoma.

Aflatoxins, metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, are frequent contaminants of a number of staple foods, particularly maize and ground nuts, in subsistence farming communities in tropical and sub-tropical climates in sub-Saharan Africa, Eastern Asia and parts of South America. Contamination of foods occurs during growth and as a result of storage in deficient or inappropriate facilities. These toxins pose serious public health hazards, including the causation of hepatocellular carcinoma by aflatoxin B1. Exposure begins in utero and is life-long. The innocuous parent molecule of the fungus is converted by members of the cytochrome p450 family into mutagenic and carcinogenic intermediates. Aflatoxin-B1 is converted into aflatoxin B1-8,9 exo-epoxide, which is in turn converted into 8,9-dihydroxy-8-(N7) guanyl-9-hydroxy aflatoxin B1 adduct. This adduct is metabolized into aflatoxin B1 formaminopyrimidine adduct. These adducts are mutagenic and carcinogenic. In addition, an arginine to serine mutation at codon 249 of the p53 tumor suppressor gene is produced, abrogating the function of the tumor suppressor gene, and contributing to hepatocarcinogenesis. Aflatoxin B1 acts synergistically with hepatitis B virus in causing hepatocellular carcinoma. A number of interactions between the two carcinogens may be responsible for this action, including integration of hepatitis B virus x gene and its consequences, as well as interference with nucleotide excision repair, activation of p21waf1/cip1, generation of DNA mutations, and altered methylation of genes. But much remains to be learnt about the precise pathogenetic mechanisms responsible for aflatoxin B1-induced hepatocellular carcinoma as well as the interaction between the toxin and hepatitis B virus in causing the tumor.

Occult hepatitis B virus infection in chacma baboons, South Africa.

During previous studies of susceptibility to hepatitis B virus (HBV) infection, HBV DNA was detected in 2/6 wild-caught baboons. In the present study, HBV DNA was amplified from 15/69 wild-caught baboons. All animals were negative for HBV surface antigen and antibody against HBV core antigen. Liver tissue from 1 baboon was immunohistochemically negative for HBV surface antigen but positive for HBV core antigen. The complete HBV genome of an isolate from this liver clustered with subgenotype A2. Reverse transcription PCR of liver RNA amplified virus precore and surface protein genes, indicating replication of virus in baboon liver tissue. Four experimentally naive baboons were injected with serum from HBV DNA-positive baboons. These 4 baboons showed transient seroconversion, and HBV DNA was amplified from serum at various times after infection. The presence of HBV DNA at relatively low levels and in the absence of serologic markers in the baboon, a nonhuman primate, indicates an occult infection.

Epidemiology of hepatocellular carcinoma in sub-Saharan Africa.

Published incidences of hepatocellular carcinoma in the Black population of sub-Saharan Africa underestimate the true incidence of the tumor because of the many instances in which hepatocellular carcinoma is either not definitively diagnosed or is not recorded in a cancer registry. Despite this, it is manifestly evident that the tumor occurs commonly and is a major cause of cancer deaths in Black African peoples living in the sub-continent, particularly in those living in rural areas. 46,000 new cases of hepatocellular carcinoma have been recorded to be diagnosed in sub-Saharan Africa each year, and age-standardized incidences of the tumor as high as 41.2/100,000 persons/year have been documented. The highest incidence of hepatocellular carcinoma has been recorded in Mozambique. The tumor occurs at a young age in rural dwelling and, to a lesser extent, urban dwelling Black Africans. It is also more common in men than women, particularly in the younger patients. Cirrhosis co-exists with hepatocellular carcinoma in about 60% of patients and is equally common in the two sexes. The tumor is not only common in the Black African population, it also carries an especially grave prognosis, with about 93% of the patients dying within 12 months of the onset of symptoms. Caucasians living in the sub-continent have a low incidence of hepatocellular carcinoma and it occurs at an older age.

Control of viral hepatitis infection in Africa: are we dreaming?

BACKGROUND: At least five different types of viral hepatitis cause problems of significant public health importance in Africa, where together they constitute a huge burden of disease. But until now, efforts to control the infections have been largely piecemeal. Analysis of the strategies needed to control each virus, however, reveals major overlaps. PROPOSAL: We propose that the control of these infections in the WHO African Region should start with the common strategies rather than with each disease. But this approach presents potentially huge problems to overcome, such as the difficulty of integrating multiple health service elements - the track record for successful integration of such services is not good. This is despite encouraging rhetoric from donors and national leaders alike. And to succeed, disparate programmes must work closely together. But we believe that the time is right to create new opportunities for prevention and treatment of hepatitis, including increasing education, and promoting screening and treatment for more than 500 million people already infected with hepatitis B and C viruses. IMPACT: The impact of these efforts on decreasing mortality and morbidity will be significant because of the high burden of disease from these infections, and also because the effect will spill over to benefit the control of other communicable diseases and health systems strengthening. Such a project will inevitably involve multiple strategies that will vary somewhat according to the epidemiology of the diseases and the location.

Hepatitis B virus / human immunodeficiency virus co-infection and its hepatocarcinogenic potential in sub-saharan black africans.

CONTEXT: Since the introduction of highly active anti-retroviral regimen for human immunodeficiency virus-1 infection, a significant increase in the incidence of hepatocellular carcinoma has been reported in patients already chronically infected with hepatitis B virus and then given this form of regimen for their retroviral infection. EVIDENCE ACQUISITION: This phenomenon was initially attributed to the far more prolonged survival of those patients who received this new regimen, which provided sufficient time, allowing hepatitis B virus-induced hepatocellular carcinoma to develop. RESULTS: The current belief is that the increased incidence of hepatocellular carcinoma is because of co-infection with the two viruses, one known to be hepatocarcinogenic and the other suspected to increase the carcinogenic potential of the other. Because both hepatitis B virus and human immunodeficiency virus -1 are endemic in the Black population of sub-Saharan Africa and are transmitted in similar ways, as many as 20% of this population are co-infected with the two viruses. In this way, the already high risk of Black African patients developing hepatitis B virus-induced hepatocellular carcinoma is further increased. CONCLUSIONS: The pathogenetic mechanism or mechanisms involved in the carcinogenic interaction between the hepatitis B virus and the human immunodeficiency virus-1 in sub-Saharan Black Africans and other populations co-infected with these viruses have yet to be determined.

Hepatocellular carcinoma in developing countries: Prevention, diagnosis and treatment.

Hepatocellular carcinoma (HCC) occurs commonly and with increasing frequency in developing countries, where it also carries an especially grave prognosis. The major risk factor for HCC in these regions is chronic hepatitis B virus (HBV) infection, although dietary exposure to aflatoxin B1 also plays an important etiological role. Prevention of HCC in developing regions is unlikely in the foreseeable future. Although an effective vaccine against HBV is available, the percentage of babies born in developing countries that receive the full course of immunization remains low. Moreover, the usually long interval between infection with HBV and the development of HCC means that 30 to 50 years will elapse before the full effect of the vaccine will be realized. Practical measures to prevent aflatoxin B1 exposure are not in place. Serum α-fetoprotein levels are a useful pointer to the diagnosis of HCC in low-income countries, but definitive diagnosis is hampered both by the lack of the sophisticated imaging equipment now available in developed countries and by obstacles to obtaining histological proof. In the majority of patients in low-income regions, the tumor is inoperable by the time the patient presents. Hepatic resection is seldom possible in sub-Saharan Africa, although the tumor is successfully resected in a larger number of patients in China. Liver transplantation for HCC is rarely performed in either region. Sophisticated new radiotherapy techniques are not available in developing countries. The beneficial effects of the multikinase inhibitor, sorafenib, are encouraging, although financial considerations may restrict its use in low-income countries.

The effect of the G1888A mutation of subgenotype A1 of hepatitis B virus on the translation of the core protein.

A distinctive characteristic of subgenotype A1 of hepatitis B virus is G1888A in the precore region. This transition introduces an out-of-frame AUG, creating an overlapping upstream open reading frame (uORF), terminating five nucleotides downstream from the core AUG. This uORF can potentially be translated into a seven amino acid peptide. In addition to stabilizing the encapsidation signal by forming a base pair with T1871, this mutation may affect translation of the core protein. The aim of this study was to use reporter constructs to determine whether G1888A had any modulating effect on core protein translation. The complete core gene with part of the precore of subgenotype A1 was cloned into the amino terminal of a green fluorescent protein (GFP) plasmid. Core/GFP fusion protein expression was measured using flow cytometry following transfection of Huh 7 cells. The introduction of uORF resulted in an 18.75% reduction of core gene expression. When the suboptimal Kozak sequence of the 1888 AUG was replaced with an optimal one, this reduction was enhanced (64.84%). By increasing the distance between the stop of the overlapping uORF and the core AUG, by a minimum of 15 nucleotides, core/GFP expression was almost doubled, indicating that stalling of ribosomes at the stop of the uORF may be interfering with initiation at the core AUG through steric hindrance. Our findings indicate that the G1888A mutation, may interfere with initiation at the downstream 1901 core AUG, decreasing core protein translation. This decrease may account for the relatively low viral loads seen in individuals infected with subgenotype A1.

Distinct mutant hepatitis B virus genomes, with alterations in all four open reading frames, in a single South African hepatocellular carcinoma patient.

Sequence variation of hepatitis B virus (HBV) can influence the replication, antigen expression and pathogenicity of the virus. We report on the mutational analysis of HBV performed in a 28-year-old Black South African female diagnosed with HBV-induced hepatocellular carcinoma. Full-genome amplification and DNA sequencing of HBV was carried out. Five distinct complete genomic clones were described with extensive genomic and intragenic variation. Phylogenetic analysis revealed that all five clones belonged to subgenotype A1 and that there were at least four virus populations with genomes of different lengths ranging from 3194 to 3253 base pairs. In this particular patient, four major characteristic features, not previously reported to occur simultaneously in HBV isolated from a single patient, were observed. Firstly, all the clones harboured a 13 base pair deletion and a 45 base pair insertion in the basic core promoter (BCP). Secondly, a 37 base pair insertion in the core gene with three adjacent single nucleotide deletions were observed. Thirdly, premature S gene stop codons were observed in some clones and lastly X gene initiation codon mutations were also observed. The complex nature of the mutations in the HBV isolated from this single patient may have contributed to the early onset of hepatocarcinogenesis.

Hepatitis B virus x protein in the pathogenesis of hepatitis B virus-induced hepatocellular carcinoma.

Currently available evidence supports a role for the hepatitis B virus (HBV) x gene and protein in the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). HBx gene is often included, and remains functionally active, in the HBV DNA that is frequently integrated into cellular DNA during hepatocellular carcinogenesis. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumour suppressor gene and other tumour suppressor genes and senescence-related factors. However, the molecular mechanisms responsible for HBx protein-induced HCC remain uncertain. Only some of the more fully documented or more recently recognised mechanisms are reviewed. During recent years evidence has accumulated that HBx protein modulates transcription of methyl transferases, causing regional hypermethylation of DNA that results in silencing of tumour suppressor genes, or global hypomethylation that results in chromosomal instability, thereby playing a role in hepatocarcinogenesis. HBx protein has both anti-apoptotic and pro-apoptotic actions, apparently contradictory effects that have yet to be explained. Particularly important among the anti-apoptotic properties is inhibition of p53. Recent experimental observations suggest that HBx protein may increase the expression of TERT and telomerase activity, prolonging the life-span of hepatocytes and contributing to malignant transformation. The protein also interferes with nucleotide excision repair through both p53-dependent and p53- independent mechanisms. Carboxy-terminal truncated HBx protein loses its inhibitory effects on cell proliferation and pro-apoptotic properties, and it may enhance the protein's ability to transform oncogenes. Dysregulation of IGF-II enhances proliferation and anti-apoptotic effects of oncogenes, resulting in uncontrolled cell growth.