Classification of Early Stages of NAFLD Based on Dual Diagnostic Methods

High prevalence of non-alcoholic fatty liver disease (NAFLD) has made this domain of medical diagnostics one of high professional and public interest. The major problem of NAFLD diagnostics is that in its initial phase non-alcoholic fatty liver tends to be benign without tendency to progress, while in its second phase -- non-alcoholic steatohepatitis (NASH) can progress to cirrhosis, which subsequently may cause hepatocellular carcinoma. This fact explains the need for more sensitive classifications that would allow early diagnostics of NAFLD. NAFLD diagnostics in most cases is based on clinicopathological criteria -- decision rules expressed through ultarasound signs and laboratory data, annotated by hepatologist/gastroenterologist. In this article we describe the process of creation of a classification of NAFLD early stages based on a decisional reasoning, which combines two methods of medical diagnostics

Clinical Utilities of Transient Elastography

Chronic liver disease causes 1.75 million deaths globally and is within the top 10 leading causes of death in middle income countries. Chronic liver injury occurs via a process of inflammation and fibrosis formation. Patients often do not present to healthcare until advanced stages of disease and when there is already decompensated cirrhosis. Liver biopsy has been used to identify earlier stages of fibrosis, but it is poorly accepted by patients and has limitations. Transient Elastography (TE) using Fibroscan ® is a non-invasive tool for the diagnosis liver fibrosis. The clinical application of Fibroscan in non-alcoholic fatty disease (NAFLD), chronic hepatitis B (CHB), and methotrexate induced liver fibrosis were examined. Clinical Utility of Transient Elastography in non-alcoholic fatty liver disease: Chapters 2, 3 & 4 Non-alcoholic fatty liver disease affects 20-35% of the global population, but only a small subset develop the histological subtype of non-alcoholic steatohepatitis (NASH), which can lead to progressive liver disease by causing fibrosis and eventually cirrhosis. Fibroscan can potentially identify those patients who have fibrosis and are at increased risk of further progression. Patients with type 2 diabetes, who are at high risk of NASH, were assessed. A liver stiffness measurement (LSM) ≥9.8 kPa, used as a cut-off for advanced fibrosis (1), was found in 12% (10/77) of subjects. Higher LSM readings correlated with higher BMI and the use of insulin therapy. Patients on insulin had LSM ≥9.8 kPa with likelihood ratio (LR): 12.3, p=0.002 (Chapter 2). The study was limited by a small sample size, and a high failure rate as the medium (M) probe was only available. A systemic review evaluating all non-invasive methods for diagnosing NASH and NAFLD fibrosis was undertaken. This included a meta-analysis that focused on what was found to be the most widely studied markers of NASH and NAFLD fibrosis: cytokeratin-18 (CK-18) fragments and transient elastography respectively (Chapter 3). Not only was TE found to be the most extensively studied, it had also one of the highest diagnostic accuracies with pooled sensitivities and specificities to diagnose F≥2, 3 and 4 to be: 79% and 75%, 85% and 85%, and 92% and 92% respectively. We then proceeded to perform a much larger study in diabetic subjects using the latest generation of Fibroscan ® 502 touch model (Chapter 4). This included the extra-large (XL) probe for obese subjects and also featured the novel Controlled Attenuation Parameter (CAP), which assesses liver steatosis. A total of 1918 diabetes patients at Prince of Wales Hospital, Hong Kong were recruited. Each had a TE and CAP to assess liver stiffness and steatosis. Reliable scans were achieved in 98.2% of patients using the M or XL probes. The proportion of patients with increased CAP (suggestive of steatosis) and increased LSM (suggestive of advanced fibrosis) were 72.8% and 17.7% respectively. By multivariate analysis, female gender, higher body mass index, triglycerides, fasting plasma glucose and alanine aminotransferase, and non-insulin use were associated with increased CAP. Longer duration of diabetes, higher body mass index, alanine aminotransferase, spot urine albumin-creatinine ratio, and lower high-density lipoprotein-cholesterol were associated with increased LSM. The17.7% prevalence of advanced fibrosis suggests type 2 diabetic patients would benefit from routine screening for liver disease. Clinical Utility of Transient Elastography in chronic hepatitis B: Chapters 5 and 6 Transient elastography was initially applied for staging patients with chronic hepatitis C (CHC) with data rapidly growing on its utility for the assessment in patients with CHB infection. Our study contributes to this by further evaluating the diagnostic accuracy and usefulness of TE, and also comparing its performance against the FIB4 index, Aspartate Platelet Ratio Index (APRI), Aspartate Alanine Aminotransferase Ratio (AAR), Age Platelet Index (API), Fibrosis Index (FI) and Caffeine Breath Test (CBT) (Chapter 5). In 71 CHB patients, the diagnostic performance of the LSM for Metavir fibrosis stage F≥1, 2, 3 and 4 were: Area under Receiver Operator Characteristic (AUROC) = 0.825, 0.792, 0.874 and 0.945 respectively. Patients with high ALT required higher LSM cut-offs. Dual cut-offs are needed to “rule in” and to “rule out” stage of fibrosis with a high level of certainty. Using normal vs high ALT specific cut-offs, F≥2 and F≥3 can be “ruled in” or “ruled out” with certainty in 49.3% and 57.7% of CHB patients respectively. TE was the superior non-invasive test when compared with FIB4-I, APRI, API, AAR and FI. Caffeine breath test compared well against TE in a small cohort, but is not as practical. Liver histology is limited by interobserver variability, with 44% of liver biopsies being classified a different stage on second evaluation, and the intraclass correlation coefficient showing moderate agreement (K =0.457). Although routinely compared, this highlights the limitations of assessing the accuracy of TE and other non-invasive tests against a reference standard that has such a degree of variation. The use of TE in the longitudinal monitoring of fibrosis is important in the follow up of patients with CHB (Chapter 6). Current literature was conflicting and seemed to suggest that decline in LSM was influenced more by the fall in ALT with decline in necroinflammatory activity, rather than fibrosis regression. We sought to evaluate the factors that affected LSM change and assess which clinical subgroups experienced an LSM decline. In 124 CHB patients who were followed for 31.2 months (SD 13.1), LSM decline was greatest in those who had active disease and were subsequently treated with antivirals. This is associated with ALT normalization, HBeAg seroconversion and viral suppression. In CHB patients with quiescent disease - ie did not require antiviral treatment, or who had persistently normal ALT irrespective of treatment - only a small or non-significant decline in LSM was observed. The change in LSM was strongly correlated with length of time and may suggest fibrosis regression. Further studies are required, as our findings are limited by a lack of correlation with liver biopsy, and the low baseline levels of liver stiffness in those with inactive CHB. Clinical Utility of Transient Elastography in methotrexate induced liver fibrosis: Chapter 7 Long term use of methotrexate has been associated with risk of liver fibrosis and the role of TE in this cohort was evaluated. The relationship between liver fibrosis and methotrexate dose, and other factors associated with moderate fibrosis (F2) using an LSM cut-off of ≥7.1 kPa were examined. In 39 patients with a mean intake dose of 5.3g of methotrexate, no correlation was found between the LSM and the cumulative dose or duration of treatment. Of the 7/39 cases of LSM≥7.1 kPa (17.9%), BMI≥30 was the only risk factor with a likelihood ratio (LR) of 4.442, p=0.029. One patient had cirrhosis (2.6%). This is much lower than rates reported from early studies [26% (2, 3)], and more in line with recent data [around 2% (4)], and lends support to the suggestion that early studies overestimated the risk of methotrexate induced fibrosis due to lack of controls for pre-existing liver disease (5). There was also no difference in the LSM of methotrexate subjects and matched population controls. Conclusion Our studies lend further support to the utility of LSM on identifying those at increased risk of liver fibrosis progression, which will continue to remain a significant clinical challenge in both individuals and as a public health burden. In particular we feel that major contributions have been made on the subject of screening for advanced fibrosis in a high-risk population of type II diabetic patients. Our longitudinal studies on the role of using TE in follow up and comparing its performance in CHB patients are also significant. Despite the small cohort of methotrexate users, this further supports the utility of TE in a wide range of liver diseases that manifest with progressive fibrosis. The next area of further development in the clinical use of TE is as a stand-alone marker that has prognostic significance

Emerging Nano-Theranostic Strategies against Non-Alcoholic Fatty Liver Disease: a review

As a major global cause of liver damage, non-alcoholic fatty liver disease (NAFLD) is associated with excessive hepatocellular accumulation of lipids in the liver, elevated levels of hepatic enzymes,and the fibrotic evidence. The primary therapies for NAFLD are changing lifestyle or managing comorbid-associated diseases. Lately, nanotechnology has revolutionized the art of nanostructure synthesis for disease imaging, diagnosis, and treatment. Loading drugs into nanocarriers hasbeen established as a promising strategy to extend their circulating time, particularly in treating NAFLD. In addition, considering a master modulator of adipogenesis and lysosomal biogenesis and function, designing novel nanostructures for biomedical applications requires using biodegradable materials. Various nanostructures, including inorganic nanoparticles (NPs), organic-based NPs, metallic nanocarriers, biodegradable polymeric nanocarriers, polymer-hybrid nanocarriers, and lipid-based nanocarriers have been designed for NAFLD treatment, which significantly affected serum glucose/lipid levels and liver function indices. NPs modified with polymers, bimetallic NPs, and superparamagnetic NPs have been used to design sensitive nanosensors to measure NAFLD-related biomarkers. However, certain limitations are associated with their use as diagnostic agents. The purpose of this review article is to shed light on the recent advancements in the field of nanomedicine for the early diagnosis, treatment, and prognosis of this progressive liver disease

Treatment options and combined therapies for metabolic associated fatty liver disease

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.Com o aumento da prevalência de obesidade, especialmente em países desenvolvidos, surge uma série de consequências metabólicas nas quais se inclui o fígado gordo não alcoólico. O fígado gordo não alcoólico caracteriza-se por uma acumulação de gordura intra-hepática excessiva (superior a 5% do volume do fígado) frequentemente associada a resistência à insulina. O fígado gordo não alcoólico é atualmente considerado a doença hepática com maior prevalência a nível mundial. Porém, até o momento, não existe nenhuma terapêutica farmacológica aprovada pelas autoridades reguladoras do medicamento. Com o intuito de facilitar o diagnóstico e assim aumentar a procura de novos fármacos, foi recentemente sugerido a alteração na nomenclatura da doença, de fígado gordo não alcoólico, para fígado gordo metabólico. Com esta monografia pretende-se sintetizar a informação existente relativamente à heterogeneidade da doença e, em especial, à sua abordagem farmacológica. A abordagem terapêutica inicial consiste na melhoria e controlo dos distúrbios metabólicos associados à síndrome metabólica, com alteração do estilo de vida, que envolve modificação da dieta, prática regular de exercício físico e perda de peso. No entanto, quando estes objetivos não são atingidos ou revelam ser insuficientes será necessário conjugá-los com terapêutica farmacológica que contrarie os mecanismos fisiopatológicos da doença. Considerando a sua prevalência e os fatores associados à progressão da doença torna-se imperativo que se investigue com maior detalhe a fisiopatologia do fígado gordo não alcoólico, realizando também a estratificação de risco de cada doente de modo a ser possível direcionar eficazmente a terapêutica, descobrir novos alvos terapêuticos e adaptar o tratamento a cada indivíduo.With the increasing prevalence of obesity, especially in developed countries, a series of metabolic consequences arise, including non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by excessive intrahepatic fat accumulation (greater than 5% of the liver volume) often associated with insulin resistance and that can progress to non-alcoholic steatohepatitis. NAFLD is currently considered the most prevalent liver disease worldwide. However, until now, there is no pharmacological therapy approved by the National Authority of Medicines. To facilitate the diagnosis and thus increase the research of new drugs, it was recently suggested a change in the disease nomenclature, from NAFLD to metabolic fatty liver disease (MAFLD). This monograph is intended to synthesize the existing information regarding the heterogeneity of the disease and, in particular, its pharmacological approach. The initial therapeutic approach, based on improvement and control of metabolic disorders associated with the metabolic syndrome, consists in lifestyle changes, involving dietary modification, regular physical exercise and weight loss. However, when these goals are not achieved or prove to be insufficient, it may be necessary to combine them with pharmacological therapy that counteracts the pathophysiological mechanisms of the disease. Considering the prevalence of NAFLD and the factors associated with the disease progression, it is imperative to investigate the pathophysiology of the disease in greater detail, making sure to perform risk stratification on each patient, so that it is possible to effectively target the therapy, discover new therapeutic targets and adapt the treatment to each individual

Chronic liver disease detection and quantification

Chronic liver disease (CLD) is a major cause of death in the UK. The major contributors are alcohol, fat and viral hepatitis. The common pathway towards cirrhosis is progressive liver fibrosis. The utility of the traditional method of evaluating fibrosis, liver biopsy, is limited by procedural risk, sampling error and variability in histological analysis. This has driven exploration of non-invasive markers of liver fibrosis. I evaluated the performance of the Enhanced Liver Fibrosis (ELF) test to detect liver fibrosis in chronic hepatitis B and compared it to an alternative modality, transient elastography (TE), demonstrating good diagnostic performance in fibrosis assessment, with comparable performance to TE. Further, liver biopsy is not feasible in community settings, and although the role of non-invasive markers of fibrosis is expanding, they have not been widely evaluated in community settings. I estimated the incidence of CLD in a large cohort of community-based postmenopausal women and investigated the contribution of alcohol and overweight / obesity to risk of CLD, observing more clinical events attributable to cirrhosis among those who were overweight or obese, with the highest risk in those who were overweight or obese consuming the most alcohol. I estimated the association between skirt size, as a surrogate for overweight / obesity, and CLD, finding significantly increased risk in those with larger or increasing skirt size. I demonstrated that the ELF test predicts CLD in women with risk factors comprising alcohol excess and / or overweight or obesity. In addition to contributing to the epidemiological data in postmenopausal women, an important but under-evaluated group in terms of liver disease, I have provided data that could be used to design pathways for the early detection and stratification of CLD in the community

Cost-effectiveness of non-invasive methods for assessment and monitoring of liver fibrosis and cirrhosis in patients with chronic liver disease: systematic review and economic evaluation

BACKGROUND: Liver biopsy is the reference standard for diagnosing the extent of fibrosis in chronic liver disease; however, it is invasive, with the potential for serious complications. Alternatives to biopsy include non-invasive liver tests (NILTs); however, the cost-effectiveness of these needs to be established. OBJECTIVE: To assess the diagnostic accuracy and cost-effectiveness of NILTs in patients with chronic liver disease. DATA SOURCES: We searched various databases from 1998 to April 2012, recent conference proceedings and reference lists. METHODS: We included studies that assessed the diagnostic accuracy of NILTs using liver biopsy as the reference standard. Diagnostic studies were assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. Meta-analysis was conducted using the bivariate random-effects model with correlation between sensitivity and specificity (whenever possible). Decision models were used to evaluate the cost-effectiveness of the NILTs. Expected costs were estimated using a NHS perspective and health outcomes were measured as quality-adjusted life-years (QALYs). Markov models were developed to estimate long-term costs and QALYs following testing, and antiviral treatment where indicated, for chronic hepatitis B (HBV) and chronic hepatitis C (HCV). NILTs were compared with each other, sequential testing strategies, biopsy and strategies including no testing. For alcoholic liver disease (ALD), we assessed the cost-effectiveness of NILTs in the context of potentially increasing abstinence from alcohol. Owing to a lack of data and treatments specifically for fibrosis in patients with non-alcoholic fatty liver disease (NAFLD), the analysis was limited to an incremental cost per correct diagnosis. An analysis of NILTs to identify patients with cirrhosis for increased monitoring was also conducted. RESULTS: Given a cost-effectiveness threshold of £20,000 per QALY, treating everyone with HCV without prior testing was cost-effective with an incremental cost-effectiveness ratio (ICER) of £9204. This was robust in most sensitivity analyses but sensitive to the extent of treatment benefit for patients with mild fibrosis. For HBV [hepatitis B e antigen (HBeAg)-negative)] this strategy had an ICER of £28,137, which was cost-effective only if the upper bound of the standard UK cost-effectiveness threshold range (£30,000) is acceptable. For HBeAg-positive disease, two NILTs applied sequentially (hyaluronic acid and magnetic resonance elastography) were cost-effective at a £20,000 threshold (ICER: £19,612); however, the results were highly uncertain, with several test strategies having similar expected outcomes and costs. For patients with ALD, liver biopsy was the cost-effective strategy, with an ICER of £822. LIMITATIONS: A substantial number of tests had only one study from which diagnostic accuracy was derived; therefore, there is a high risk of bias. Most NILTs did not have validated cut-offs for diagnosis of specific fibrosis stages. The findings of the ALD model were dependent on assuptions about abstinence rates assumptions and the modelling approach for NAFLD was hindered by the lack of evidence on clinically effective treatments. CONCLUSIONS: Treating everyone without NILTs is cost-effective for patients with HCV, but only for HBeAg-negative if the higher cost-effectiveness threshold is appropriate. For HBeAg-positive, two NILTs applied sequentially were cost-effective but highly uncertain. Further evidence for treatment effectiveness is required for ALD and NAFLD. STUDY REGISTRATION: This study is registered as PROSPERO CRD42011001561. FUNDING: The National Institute for Health Research Health Technology Assessment programme

Current considerations for clinical management and care of non-alcoholic fatty liver disease: Insights from the 1st International Workshop of the Canadian NASH Network (CanNASH).

Non-alcoholic fatty liver disease (NAFLD) affects approximately 8 million Canadians. NAFLD refers to a disease spectrum ranging from bland steatosis to non-alcoholic steatohepatitis (NASH). Nearly 25% of patients with NAFLD develop NASH, which can progress to liver cirrhosis and related end-stage complications. Type 2 diabetes and obesity represent the main risk factors for the disease. The Canadian NASH Network is a national collaborative organization of health care professionals and researchers with a primary interest in enhancing understanding, care, education, and research around NAFLD, with a vision of best practices for this disease state. At the 1st International Workshop of the CanNASH network in April 2021, a joint event with the single topic conference of the Canadian Association for the Study of the Liver (CASL), clinicians, epidemiologists, basic scientists, and community members came together to share their work under the theme of NASH. This symposium also marked the initiation of collaborations between Canadian and other key opinion leaders in the field representative of international liver associations. The main objective is to develop a policy framework that outlines specific targets, suggested activities, and evidence-based best practices to guide provincial, territorial, and federal organizations in developing multidisciplinary models of care and strategies to address this epidemic

Some epidemiological aspects of liver cirrhosis and hepatocellular carcinoma in Sweden

Background: Contemporary epidemiological studies examining incidence rates (IR) of cirrhosis and hepatocellular carcinoma (HCC) in Swedish populations are scarce. Cirrhosis and HCC are associated with a significant burden of health inequity and stigma. The importance of socioeconomic status (SES) in cirrhosis survival has scarcely been studied in Sweden. The impact of SES on HCC incidence and prognosis had never been investigated in Sweden. Aim: The overall aim of this thesis was to describe the contemporary epidemiology of cirrhosis and HCC in Swedish settings. We also aimed to improve the understanding of the importance of sociodemographic and clinical characteristics for the clinical course and early identification of cirrhosis and HCC. Methods: We used population-based medical registries to identify adult patients diagnosed with cirrhosis in the region of Halland between 2011 and 2018. Annual crude IR of cirrhosis were calculated (Paper I). Patients were followed-up until date of liver transplantation, death, moving from Halland, or until December 31st, 2019; whichever occurred first. Cox regression models were employed to estimate unadjusted and adjusted hazard ratios (HR and aHR) for several clinical and sociodemograhic variables (Paper II). The nationwide quality register for liver cancer was used to identify all adult patients diagnosed with HCC in Sweden between 2012 and 2018. Poisson regression was used to estimate IRs of HCC across several populations of interest (Paper III). Data extracted from the quality register were cross-linked to data from other nationwide registers. Multivariable logistic regression models were employed to identify factors associated with an increased likelihood for having unrecognized cirrhosis, or late-stage HCC at diagnosis. Patients were followed-up until the date of death, emigration from Sweden, or until December 31st, 2020; whichever occurred first. Cox regression modelling was used for the estimation of HRs and aHRs for several clinical variables (Paper IV). IRs of HCC were estimated for the whole adult population of Sweden and stratified by HCC etiologies (Paper V). Patients were stratified into those with non-alcoholic fatty liver disease (NAFLD) associated HCC and those with non-NAFLD-HCC. Furthermore, those with NAFLD-HCC were divided into those with and without underlying cirrhosis. Results: We identified a total of 598 patients with cirrhosis. The IR of cirrhosis in adults in Halland was estimated at 30 per 100,000 person-years, 39 for men, and 22 for women (Paper I). Patients with a low SES, defined as a low occupational skill level, had more advanced cirrhosis at diagnosis, lower mean survival, and higher mortality risk when compared to patients with high SES (Paper II). A total of 3,473 adult patients with HCC were identified and 68% were diagnosed with a late-stage HCC. Sex, country of birth, and individual- and contextual level SES were associated with the IRs of HCC. Men with a low household income and/or living in the most deprived neighborhoods had the highest IR of HCC (Paper III). Among patients with HCC, 2670 (77%) had underlying cirrhosis. Cirrhosis was unrecognized in 39% of all patients with underlying cirrhosis. Unrecognized cirrhosis was associated with more advanced HCC at diagnosis and worse survival (Paper IV). Among the 3,473 patients with HCC, 21% had underlying NAFLD, which also was the second-leading cause of HCC and the fastest- increasing cause of HCC (Paper V). Conclusions: The IRs of cirrhosis may be higher than previously estimated. Low SES was associated with a worse prognosis in cirrhosis, higher IRs of HCC, and increased risk of unrecognized cirrhosis in HCC. NAFLD is an increasing cause of cirrhosis and has become a leading cause of HCC. NAFLD is also associated with an increased risk of cirrhosis unrecognition in HCC