Hepatitis B and C Viruses

Hepatitis B and C viruses (HBV/HCV) are among the leading causes of liver disease. HBV is a partially double-stranded circular DNA virus whose genome is approximately 3200 bases with four overlapping open reading frames (ORFs) and belongs to Hepadnaviridae family. HBV prevalence varies worldwide, with high rates reported in low-income countries. Approximately 90% of HBV infections are acute, 10% progress to chronic infection among adult patients. Although HBV can be prevented by immunisation, there is no licenced HCV vaccine. HCV is a positive-sense single-stranded RNA (+ssRNA) virus belonging to Flaviviridae family. The HCV global epidemiology varies, with high prevalence rates reported in low-income countries. Approximately 80% of acutely HCV-infected individuals develop chronic hepatitis disease, while 20% resolve spontaneously. Both HBV and HCV infections can result in both acute and chronic hepatitis, ranging in severity from asymptomatic to a life-threatening disease. The HBV and HCV are transmitted through contact with contaminated blood or its products. As compared with mono-infection, HBV/HCV co-infection has higher risk of liver damage. Thus, individuals who have active HBV and HCV infections are likely to be HCV-dominant with a high HCV viral load and low or undetectable HBV DNA levels

Protection from HCV infection – Identification of mechanisms of resistance to HCV infection in exposed uninfected injection drug users.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease. In the developed world, injection drug use (IDU) through sharing of infected needles and other paraphernalia remains the principal risk factor for HCV transmission. Effective but expensive treatment is now possible but there remains a pressing need for a vaccine. A proportion of people who inject drugs (PWIDs) remain uninfected despite HCV exposure from a long history of sharing needles and other paraphernalia. These cases are termed exposed but uninfected (EU) and test negative for both HCV antibodies and RNA and exhibit a phenotype of resistance to HCV infection. Improved understanding of the mechanisms that confer resistance in the EUs has the potential to aid development of an effective vaccine and novel therapeutic strategies. This thesis reports on the findings from 3 different strategies to identify characteristics of HCV resistance. I used urinary metabolomics, serum lipidomics and the study of adaptive and innate immune responses. Each of these methods has demonstrated clear differences between EU cases and healthy controls and/or spontaneous resolvers of HCV infection. Urinary metabolomics suggest a potential role of the gut microbiome, the serum lipidomics showed marked differences in lipid profiles in EU cases pointing towards a perturbed lipid/virus interaction, and the immune studies confirmed previous work identifying low level T cell responses in many EU cases but has also identified a marked upregulation of interferon alpha production to low dose viral RNA in EU cases utilising ELISA assay. In conclusion, this thesis reports data that identifies a number of new findings that provide insight into mechanisms of resistance to HCV infection. My findings suggest that the complex interplay between the virus and lipids together with an upregulated innate immune response may together help determine the outcome following HCV exposure. In summary, studies performed in this thesis have demonstrated that there are different pathways that define the EU phenotype. Despite being a heterogenous subgroup of PWIDs, the EUs are clearly distinct from a healthy control population.University of Plymout

Hepatitis B virus seroprevalence among Malawian medical students: A cross-sectional study

Background: Hepatitis B virus (HBV) predominantly spreads through contact with infected blood or other body fluids and causes liver disease. HBV vaccination for students at the College of Medicine, University of Malawi, is done without screening for the virus. It is important to assess the prevalence of HBV antigens among foundation-year students in order to consolidate evidence in support of HBV screening before vaccination. The aim of this study was to determine the seroprevalence of HBV antigens among 2013-2014 foundation-year students at the University of Malawi College of Medicine.Methods: A prospective cross-sectional descriptive study was conducted among 2013-2014 foundation year students at the Malawi College of Medicine. Out of the 234 foundation-year students, written consent was obtained from 89 students. Venous blood samples were collected and tested for HBV surface antigen using SD Bioline immunochromatographic rapid assays.Results: Out of the 62 (69.7%) male students, none tested HBV-positive, and out of 27 (30.3%) female students, none were seropositive. This suggested the absence of HBsAg among students or presence of HBsAg levels below detectable limits.Conclusions: This study showed levels of HBsAg below detectable limits among healthy young adults in Malawi. HBV screening for medical students should further be assessed to ensure adequate protection before they are assigned clinical duties. These findings provide enough grounds to agitate for further surveys to support the establishment of a universal HBV immunisation programme in Malawi

Assessment of the accuracy of dried blood spot (DBS) sample in HIV-1 viral load as compared to plasma sample using Abbot assay

Background: HIV has claimed millions of lives with the Sub-Saharan Africa being the most affected. There is a significant increase in access to antiretroviral drugs which also demands frequent monitoring to determine the drug effectiveness and efficacy. Thus there is a great need to evaluate simplified methods to monitor treatment with such antiretroviral drugs. Use of dried blood spots (DBS) can be ideal if evaluated in resource limited countries such as Malawi since they are easy to collect, store and convenient. The main objective of this study was to evaluate the accuracy of a dry blood spot sample in the quantification of viral particles in HIV reactive patients using the Abbott m2000rt assay.Methods: 87 participants were recruited from the ART clinic at Queen Elizabeth Central Hospital using convenience sampling method. 29 were on antiretroviral therapy and 58 had not started the therapy. HIV-1 RNA extraction and quantification was performed from DBS and plasma using Abbott m2000sp and m2000rt systems respectively. The results were statistically analyzed by Bland-Altman method using medcalc software version 12.6.1.Results: 66 paired samples with detectable viral loads were analysed. These gave a correlation of 0.98. The mean difference was 0.05 log10 copies/ml with a standard deviation of 0.17 at 95% confidence interval.The Bland-Altman plots showed limits of agreement which ranged from -0.38 to 0.28 log10 copies/ml at 95% confidence interval.Conclusion: Results showed strong agreement between the plasma and DBS samples. A slight and clinically insignificant difference was observed between the two methods. A larger sample size can give support to the study findings. Since samples were less than a week old, it is not known if the results would be different if they were to be stored for a longer period

Infection with the hepatitis C virus causes viral genotype-specific differences in cholesterol metabolism and hepatic steatosis

Lipids play essential roles in the hepatitis C virus (HCV) life cycle and patients with chronic HCV infection display disordered lipid metabolism which resolves following successful anti-viral therapy. It has been proposed that HCV genotype 3 (HCV-G3) infection is an independent risk factor for hepatocellular carcinoma and evidence suggests lipogenic proteins are involved in hepatocarcinogenesis. We aimed to characterise variation in host lipid metabolism between participants chronically infected with HCV genotype 1 (HCV-G1) and HCV-G3 to identify likely genotype-specific differences in lipid metabolism. We combined several lipidomic approaches: analysis was performed between participants infected with HCV-G1 and HCV-G3, both in the fasting and non-fasting states, and after sustained virological response (SVR) to treatment. Sera were obtained from 112 fasting patients (25% with cirrhosis). Serum lipids were measured using standard enzymatic methods. Lathosterol and desmosterol were measured by gas-chromatography mass spectrometry (MS). For further metabolic insight on lipid metabolism, ultra-performance liquid chromatography MS was performed on all samples. A subgroup of 13 participants had whole body fat distribution determined using in vivo magnetic resonance imaging and spectroscopy. A second cohort of (non-fasting) sera were obtained from HCV Research UK for comparative analyses: 150 treatment naïve patients and 100 non-viraemic patients post-SVR. HCV-G3 patients had significantly decreased serum apoB, non-HDL cholesterol concentrations, and more hepatic steatosis than those with HCV-G1. HCV-G3 patients also had significantly decreased serum levels of lathosterol, without significant reductions in desmosterol. Lipidomic analysis showed lipid species associated with reverse cholesterol transport pathway in HCV-G3. We demonstrated that compared to HCV-G1, HCV-G3 infection is characterised by low LDL cholesterol levels, with preferential suppression of cholesterol synthesis via lathosterol, associated with increasing hepatic steatosis. The genotype-specific lipid disturbances may shed light on genotypic variations in liver disease progression and promotion of hepatocellular cancer in HCV-G3

A global action agenda for turning the tide on fatty liver disease

Background and Aims: Fatty liver disease is a major public health threat due to its very high prevalence and related morbidity and mortality. Focused and dedicated interventions are urgently needed to target disease prevention, treatment, and care. Approach and Results: We developed an aligned, prioritized action agenda for the global fatty liver disease community of practice. Following a Delphi methodology over 2 rounds, a large panel (R1 n = 344, R2 n = 288) reviewed the action priorities using Qualtrics XM, indicating agreement using a 4-point Likert-scale and providing written feedback. Priorities were revised between rounds, and in R2, panelists also ranked the priorities within 6 domains: epidemiology, treatment and care, models of care, education and awareness, patient and community perspectives, and leadership and public health policy. The consensus fatty liver disease action agenda encompasses 29 priorities. In R2, the mean percentage of “agree” responses was 82.4%, with all individual priorities having at least a super-majority of agreement (> 66.7% “agree”). The highest-ranked action priorities included collaboration between liver specialists and primary care doctors on early diagnosis, action to address the needs of people living with multiple morbidities, and the incorporation of fatty liver disease into relevant non-communicable disease strategies and guidance. Conclusions: This consensus-driven multidisciplinary fatty liver disease action agenda developed by care providers, clinical researchers, and public health and policy experts provides a path to reduce the prevalence of fatty liver disease and improve health outcomes. To implement this agenda, concerted efforts will be needed at the global, regional, and national levels.publishedVersio

Diagnostic performance evaluation of hepatitis B e antigen rapid diagnostic tests in Malawi

Background The World Health Organization (WHO) has targeted a reduction in viral hepatitis-related mortality by 65% and incidence by 90% by 2030, necessitating enhanced hepatitis B treatment and prevention programmes in low- and middle-income countries. Hepatitis B e antigen (HBeAg) status is used in the assessment of eligibility for antiviral treatment and for prevention of mother-to-child transmission (PMTCT). Accordingly, the WHO has classified HBeAg rapid diagnostic tests (RDTs) as essential medical devices. Methods We assessed the performance characteristics of three commercially available HBeAg RDTs (SD Bioline, Alere, South Africa; Creative Diagnostics, USA; and Biopanda Reagents, UK) in two hepatitis B surface antigen-positive cohorts in Blantyre, Malawi: participants of a community study (n = 100) and hospitalised patients with cirrhosis or hepatocellular carcinoma (n = 94). Two investigators, blinded to the reference test result, independently assessed each assay. We used an enzyme-linked immunoassay (Monolisa HBeAg, Bio-Rad, France) as a reference test and quantified HBeAg concentration using dilutions of the WHO HBeAg standard. We related the findings to HBV DNA levels, and evaluated treatment eligibility using the TREAT-B score. Results Among 194 HBsAg positive patients, median age was 37 years, 42% were femaleand 26% were HIV co-infected. HBeAg prevalence was 47/194 (24%). The three RDTs showed diagnostic sensitivity of 28% (95% CI 16–43), 53% (38–68) and 72% (57–84) and specificity of 96–100% for detection of HBeAg. Overall inter-rater agreement κ statistic was high at 0.9–1.0. Sensitivity for identifying patients at the threshold where antiviral treatment is recommended for PMTCT, with HBV DNA > 200,000 IU/ml (39/194; 20%), was 22, 49 and 54% respectively. Using the RDTs in place of the reference HBeAg assay resulted in 3/43 (9%), 5/43 (12%) and 8/43 (19%) of patients meeting the TREAT-B treatment criteria being misclassified as ineligible for treatment. A relationship between HBeAg concentration and HBeAg detection by RDT was observed. A minimum HBeAg concentration of 2.2–3.1 log10IU/ml was required to yield a reactive RDT. Conclusions Commercially available HBeAg RDTs lack sufficient sensitivity to accurately classify hepatitis B patients in Malawi. This has implications for hepatitis B public health programs in sub-Saharan Africa. Alternative diagnostic assays are recommended

Lipidomics analysis of fasting serum identifies novel lipid biomarkers specific for HCV genotype 3 and genotype 1 chronic hepatitis C virus infection

Background and Aims: HCV genotype 3 (G3) is now considered the most difficult to treat genotype in the era of new oral direct acting antiviral (DAAs) drugs, particularly in those with advanced liver disease. HCV-G3 is also associated with more rapid progression to cirrhosis. Evidence suggests that HCV-G3 has different effects on lipid metabolism compared to HCV-G1, associated with hepatic steatosis, low LDL cholesterol and distinct interactions in the formation of lipoviral particles. A lipidomic analysis was performed to identify lipid species differentially regulated in HCV-G3 compared to HCV-G1, to gain further insight into genotype-specific metabolic pathways. Methods: Lipidomic analysis was performed on fasting sera [N = 112 (73 G1, 39 G3); 25% cirrhosis] using a UPLC system coupled to a QToF mass spectrometer. Profiles were acquired in positive and negative ion modes. System suitability and stability was confirmed by injection of a quality control (QC) sample at regular intervals throughout the analytical run, prepared by combining equal aliquots of all the study samples. Data was pre-processed (alignment, noise filtering, normalisation) using XCMS and inhouse developed scripts, and subjected to multivariate statistical analysis using Simca-P. Principal component analysis (PCA) and orthogonal projection on latent structures-discriminant analysis (OPLS-DA) were performed on all data after pareto scaling. Results: Spectra were explored by PCA for initial visualisation to detect inherent trends and outliers. Samples clustered based on the differences between the HCV-G1 and HCV-G3. Pairwise analysis using OPLS-DA allowed establishing the lipids with the strongest contribution to the genotype separation in positive ion mode. Preliminary assignment based on mass, fragmentation pattern and retention time of lipid species upregulated in HCV-G3 included Cholesteryl linoleate [M+NH4] 666.621 m/z @ 15.47 min. Other lipid species were specifically increased in HCV genotype 1 [M+H] 784.588 m/z @ 6.29 min PC (36:30). In negative ion mode, additional novel lipid species were found to be differentially unregulated in HCV-G1. The novel lipid species are currently being identified and evaluated in an independent validation cohort. Conclusions: UPLC MS lipidomics methods identify novel lipid species differentially regulated in HCV-G3 compared to HCV-G1. Understanding of the lipidome in HCV-G3 may help identify factors associated with DAA relapse

The global NAFLD policy review and preparedness index: Are countries ready to address this silent public health challenge?

Background & aims: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, yet largely underappreciated liver condition which is closely associated with obesity and metabolic disease. Despite affecting an estimated 1 in 4 adults globally, NAFLD is largely absent on national and global health agendas. Methods: We collected data from 102 countries, accounting for 86% of the world population, on NAFLD policies, guidelines, civil society engagement, clinical management, and epidemiologic data. A preparedness index was developed by coding questions into 6 domains (policies, guidelines, civil awareness, epidemiology and data, NAFLD detection, and NAFLD care management) and categorising the responses as high, medium, and low; a multiple correspondence analysis was then applied. Results: The highest scoring countries were India (42.7) and the United Kingdom (40.0), with 32 countries (31%) scoring zero out of 100. For 5 of the domains a minority of countries were categorised as high-level while the majority were categorised as low-level. No country had a national or sub-national strategy for NAFLD and <2% of the different strategies for related conditions included any mention of NAFLD. National NAFLD clinical guidelines were present in only 32 countries. Conclusions: Although NAFLD is a pressing public health problem, no country was found to be well prepared to address it. There is a pressing need for strategies to address NAFLD at national and global levels. Lay summary: Around a third of the countries scored a zero on the NAFLD policy preparedness index, with no country scoring over 50/100. Although NAFLD is a pressing public health problem, a comprehensive public health response is lacking in all 102 countries. Policies and strategies to address NAFLD at the national and global levels are urgently needed