Updated S2k Clinical Practice Guideline on Non-alcoholic Fatty Liver Disease (NAFLD) issued by the German Society of Gastroenterology, Digestive and Metabolic Diseases (DGVS) – April 2022 – AWMF Registration No.: 021–025

List of Abbreviations

AASLD: American Association for the Study of Liver Diseases

AFP: Alpha-Fetoprotein

AHA: American Heart Association

AI: Artificial Intelligence

ALD: Alcohol-related Liver Disease

ALT: Alanineaminotransferase

API: Active pharmaceutical ingredient

APASL: Asian Pacific Association for the Study of the Liver

APRI: AST/platelet ratio

ASH: Alcoholic Steatohepatitis

AST: Aspartate aminotransferase

ATI: Attenuation Imaging

AUC: Area under the curve

AUROC: Area under the Receiver Operating Characteristic (ROC) Curve

BMI: Body-Mass-Index

CAP: Controlled Attenuation Parameter

CDT: Carbohydrate-Deficient Transferrin

CHE: Cholinesterase

CIL: Cilofexor

CMV: Cytomegalovirus

CT: Computed Tomography

CVC: Cenicriviroc

DECT: Dual-Energy CT

DM: Duodenal Mucosa

DPP4: Dipeptidyl Peptidase 4

%EWL: Percentage Excess Weight Loss

EASL: European Association for the Study of the Liver

EBV: Epstein-Barr Virus

ELF: Enhanced Liver Fibrosis

ELIVATE: Study of Efficacy, Safety and Tolerability of the Combination of Tropifexor & Licogliflozin and Each Monotherapy, Compared with Placebo in Adult Patients with NASH and Liver Fibrosis.

EPA: Ethyl Eicosapentaenic Acid

ERCP: Endoscopic Retrograde Cholangiopancreatography

ESG: Endoscopic sleeve gastroplasty

EtG: Ethylglucuronid

F1–F4: Stages of Liver Fibrosis 1–4

FASCINATE: Study of TVB-2640 in Subjects with Nonalcoholic Steatohepatitis (NASH)

FASN: Fatty Acid Synthase

FF: Fat Fraction

FGF: Fibroblast Growth Factor

FIR: Firsocostat

FLI: Fatty Liver Index

FLINT: The Farnesoid X Receptor (FXR) Ligand Obeticholic Acid in NASH Treatment Trial

FLIGHT-FXR: Study of Safety and Efficacy of Tropifexor (LJN452) in Patients with Non-alcoholic Steatohepatitis

FPG: Fasting Plasma Glucose

FXR: Farnesoid X Receptor

γ-GT: Gamma-Glutamyl Transferase

GI: Gastrointestinal

GLP-1: Glucagon-like Peptide 1

GOT: Glutamic-Oxaloacetic Transaminase

GPT: Glutamate-Pyruvate Transaminase

HA: Hyaluronic Acid

HAV: Hepatitis A Virus

HbA1c: Hemoglobin A1c

HBV: Hepatitis B Virus

HCC: Hepatocellular Carcinoma

HCV: Hepatitis C Virus

HDL: High-Density Lipoprotein

HDV: Hepatitis D Virus

HEV: Hepatitis E Virus

HIS: Hepatic Steatosis Index

Histo: Histology

HOMA: Homeostasis Model Assessment

HOMA-IR: Homeostasis Model Assessment – Insulin Resistance

HVPG: Hepatic-Venous Pressure Gradient

iCCa: Intrahepatic CholangiIocarcinoma

IDF: International Diabetes Federation

IQR: Interquartile Range

LDL: Low-Density Lipoprotein

LFS: Liver Fat Score

LSG: Laparoscopic Sleeve Gastrectomy

LT: Liver Transplantation

MAFLD: Metabolic Dysfunction-Associated Fatty Liver Disease

MDB: Mallory-Denk-Bodies

Met: Metformin

mo: Month

MRCP: Magnetic Resonance Cholangiopancreatography

MRE: Magnetic Resonance Elastography

MRI: Magnetic Resonance Imaging

MRS: Magnetic Resonance Spectroscopy

n3 PUFA: n-3 Polyunsaturated Fatty Acid

NAFLD: Non-Alcoholic Fatty Liver Disease

NAS: NAFLD Activity Score

NFS: NAFLD Fibrosis Score

NASH: Non-Alcoholic Steatohepatitis

NASH CRN: NASH Clinical Research Network

NHLBI: National Heart, Lung and Blood Institute

NPV: Negative Predictive Value​

OAGB: One-Anastomosis Gastric Bypass

OCA: Obeticholic Acid

OGTT: Oral Glucose Tolerance Test

OTC: Over the Counter

Pat.: Patient

PCOS: Polycystic ovary Syndrom

PDFF: Proton Density Fat-Fraction

PEth: Phosphatidylethanol

PIIINP: Procollagen III peptide

Pio: Pioglitazone

PMID: PubMed Identifier

p. o.: per os

PPAR: Peroxisome Proliferator-Activated Receptor

PPV: Positive Predictive Value

Prosp.: Prospective

PUFA: Polyunsaturated Fatty Acids

RYGB: Roux-Y Gastric Bypass

SCD-1: Stearoyl-CoA Desaturase 1

SG: Sleeve Gastrectomy

SGLT2: Sodium Dependent Glucose Co-transporter 2

SWE: Shear-Wave Elastography

%TWL: Percentage Total Weight Loss

T2DM: Type 2 Diabetes mellitus

TANDEM: Study of Safety, Tolerability, and Efficacy of a Combination Treatment of LJN452 and CVC in Adult Patients ith NASH and Liver Fibrosis

TBWL: Total Body Weight Loss

TE: Transient Elastography

TG: Triclyceride

THRß: Thyroid Hormone Receptor-Beta

TIMP-1: Tissue Inhibitor of Metalloproteinase-1

TZD: Thiazolidindione

UDCA: Ursodeoxycholic Acid

US: Ultrasound

VCTE: Vibration-Controlled Transient Elastography

Vit C: Vitamin C

Vit E: Vitamin E

List of Figures

[Fig. 1]: Screening algorithm	e749
[Fig. 2]: Diagnostic algorithm in NAFLD for individuals with suspected increased risk of progression (consensus)	e753
[Fig. 3]: Drug recommendations for NAFLD depending on comorbidities and fibrosis stages (consenus) Please note that the dosing for liraglutide and semaglutide differ dependent on the indication (i. e. obesity treatment or type 2 diabetes therapy).	e763
[Fig. 4]: Step-by-step diagnosis by obesity and persistently elevated transaminases: Basic diagnostic tests for persistently elevated transaminases	e780

List of Tables

[Table 1]: Steering Committee	e739
[Table 2]: Members of the Guideline Development Group	e740
[Table 3]: Grid for grading recommendations	e741
[Table 4]: Classifying the strength of consensus	e741
[Table 5]: Criteria for the clinical diagnosis of metabolic syndrome	e744
[Table 6]: Differential diagnosis of hepatic steatosis (strong consensus)	e758
[Table 7]: Histopathological criteria of infantile NAFLD	e782

List of Appendices

Appendix 1: Liver biopsy request form	SupMat
Appendix 2: Table 4b-1: Randomized controlled trials of off-label drugs and nutritional supplements (selection)	SupMat
Appendix 3: Table 4b-2: Randomized controlled trials of novel NAFLD compounds	SupMat
Appendix 4: Table 4c: Studies on endoscopic procedures and effect on metabolic parameters and NAFLD	SupMat

1. Information on the Guideline

Editors

Lead Medical Society

German Society of Gastroenterology, Digestive and Metabolic Diseases (Deutsche Gesellschaft für Gastroenterologie, Verdauungs- und Stoffwechselkrankrankheiten, DGVS)

Scope and purpose

According to the current guidelines of the DGVS (German Society of Gastroenterology, Digestive and Metabolic Diseases), EASL (European Association for the Study of the Liver, 2016), AASLD (American Association for the Study of Liver Diseases, 2018), APASL (Asian Pacific Association for the Study of the Liver, HCC Guideline, 2017) and the World Gastroenterology Organisation, 2012), the entity “non-alcoholic fatty liver disease” (NAFLD) includes the categories of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NASH fibrosis and NASH cirrhosis. New nomenclatures (e. g. metabolic dysfunction-associated fatty liver disease, MAFLD) have been proposed, but are not universally established to date.

The progression of NAFLD and particularly NASH is associated with liver cell stress, consecutive inflammation and fibrosis, potentially leading to liver cirrhosis, portal hypertension and end-stage liver disease. NASH is also a relevant risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis and natural course of NAFLD are becoming increasingly better understood. However, the heterogeneity of the patients and the diseaseʼs multifactorial genesis encumber the assessment of the precise prognosis of affected individuals. In the near future, patients with NASH-associated end-stage liver disease are expected to represent the highest proportion listed for liver transplantation. Despite being modified by genetic factors, the disease is believed to primarily result from hyperalimentation and a hepatic manifestation of metabolic syndrome. The clinical presentation of non-cirrhotic NAFLD is usually non-specific. With a global prevalence of around 25 %, NAFLD is now the leading cause of chronic liver disease worldwide and a growing public health challenge. Given the current obesity epidemic, a further increase in the prevalence of NAFLD is to be expected, especially among adolescents and younger patients. Changes in lifestyles, demographic shifts and the increasing complexity of pharmacological therapies are causes for this rise. Medical healthcare professionals and patient advocate organizations must deal with this collectively and individually. The previous German S2k Guidelines on NAFLD expired in February 2020.

The current revision was needed to incorporate all recent scientific evidence on disease management. The guideline is intended to provide practical guidance on diagnosis, therapy and surveillance of people living with NAFLD, including lifestyle modification and pharmacological treatment. Diagnostic and therapeutic algorithms based on metabolic comorbidities and fibrosis stage are provided to improve its general applicability. The present Guideline aims to offer a compilation on the qualified and effective diagnosis and management of NAFLD that reflects the current state of scientific knowledge, thereby improving the targeted care of NAFLD patients.

Overarching aim of these Clinical Practice Guidelines

This Clinical Practice Guideline is designed to provide easy practical applicability for primary care physicians, internists, clinical nutritionists, surgeons, radiologists, cardiologists, pediatricians and gastroenterologists. Above and beyond that goal, this Guideline intends to set a “corridor of action” for taking common decisions.

The patient target population comprises patients with NAFLD of all ages.

Health care settings

Inpatient and outpatient, primary care, general practice, clinical nutrition/nutritional therapy, surgery, radiology, pediatrics, internal medicine and gastroenterology.

Target users/target audience

These Guidelines target all professional groups involved in the diagnosis and management of NAFLD: Internists, gastroenterologists, endocrinologists, diabetologists, obesity specialists, surgeons, clinical nutritionists, radiologists, specialists in pediatrics and adolescent medicine, pathologists, cardiologists, transplant physicians, patient representatives/advocacy groups as well as affected parties, family members and serves as information for benefits providers (health insurers, pension insurance funds). The German College of General Practitioners and Family Physicians (DEGAM) was invited to collaborate but declined to participate. Nevertheless, we deem these Guidelines to be equally relevant for general practitioners and family physicians.

Constitution of the Guideline Development Group: Stakeholder involvement

This development of these Guidelines was led by the German Society of Gastroenterology (DGVS), which commissioned Professor Ali Canbay, Bochum, Professor Elke Roeb, Giessen, and Professor Frank Tacke, Berlin to be the coordinators. The following were responsible for methodology: Dr. Petra Lynen Jansen, University Lecturer, and Ms. Pia Lorenz, DGVS Administrative Offices, Berlin. Throughout the process, Dr. Nothacker, Association of the Scientific Medical Societies in Germany (AWMF), Berlin, provided methodological advice and support and moderated the consensus conference as a neutral guideline expert. Torsten Karge was available to support the guideline portal and technically supported the consensus conference.

The guideline project was disseminated in the journal “Zeitschrift für Gastroenterologie” and published on the AWMF website to enable additional scientific medical societies/representatives to offer their collaboration. Letters were sent to the scientific medical societies and patient groups relevant to this specialty asking them to nominate mandate holders.

Representativeness of the Guideline Development Group: Scientific medical societies and associations involved

• Obesity Working Group in Childhood and Adolescence (Arbeitsgemeinschaft Adipositas im Kindes- und Jugendalter, AGA)
  S. Wiegand (Berlin)

• German Obesity Society (Deutsche Adipositas-Gesellschaft e. V., DAG)
  S. Engeli (Greifswald), S. Wiegand (Berlin)

• German Diabetes Society (Deutsche Diabetes Gesellschaft e. V., DDG)
  M. Roden (Düsseldorf), N. Stefan (Tübingen)

• German Society for Ultrasound in Medicine (Deutsche Gesellschaft für Ultraschall in der Medizin e. V., DEGUM)
  T. Bernatik (Ebersberg), T. Karlas (Leipzig)

• German Society for General and Visceral Surgery (Deutsche Gesellschaft für Allgemein- und Viszeralchirurgie e. V., DGAV)
  B. Müller (Heidelberg), K. Rheinwalt (Cologne)

• German Society of Surgery (Deutsche Gesellschaft für Chirurgie e. V., DGCH)
  D. Seehofer (Leipzig)

• German Society of Endocrinology (Deutsche Gesellschaft für Endokrinologie e. V., DGE)
  J. Bojunga (Frankfurt am Main)

• German Society for Nutritional Medicine (Deutsche Gesellschaft für Ernährungsmedizin e. V., DGEM)
  S. Bischoff (Stuttgart), M. Plauth (Dessau)

• German Society for Combating Lipid Metabolism Disorders and their Consequential Diseases DGFF (Lipid League) (Deutsche Gesellschaft zur Bekämpfung von Fettstoffwechselstörungen und ihren Folgeerkrankungen, DGFF (Lipid-Liga) e. V.)
  J. Bojunga (Frankfurt am Main),

• German Society of Internal Medicine (Deutsche Gesellschaft für Innere Medizin e. V., DGIM)
  R. Günther (Kiel)

• German Cardiac Society (Deutsche Gesellschaft für Kardiologie, DGK)
  M. Lehrke (Aachen)

• German Society of Pathology (Deutsche Gesellschaft für Pathologie e. V., DGP)/Federal Association of German Pathologists (Bundesverband Deutscher Pathologen e. V., BDP)
  H. Baba (Essen), T. Longerich (Heidelberg), A. Tannapfel (Bochum)

• German Roentgen Society (Deutsche Röntgengesellschaft e. V., DRG)
  K. Ringe (Hannover), A. Schreyer (Brandenburg)

• German Transplantation Society (Deutsche Transplantationsgesellschaft e. V., DTG)
  M. Sterneck (Hamburg)

• German Society for Pediatric Gastroenterology and Nutrition (Gesellschaft für Pädiatrische Gastroenterologie und Ernährung e. V., GPGE)
  J. de Laffolie (Giessen), P. Gerner (Freiburg), C. Hudert (Berlin), D. Weghuber (Salzburg)

• German Society of Pediatrics and Adolescent Medicine (Deutsche Gesellschaft für Kinder- und Jugendmedizin e. V., DGKJ)
  J. de Laffolie (Giessen), R. Ganschow (Bonn), C. Hudert (Berlin)

The German College of General Practitioners and Family Physicians (DEGAM) was invited to collaborate on these Guidelines, but was unable to support the guideline project due to staff bottlenecks; the same applied to the German Hypertension League (Deutsche Gesellschaft für Hypertonie und Prävention – Deutsche Hochdruckliga e. V., DHL). The German Psychological Society (Deutsche Gesellschaft für Psychologie e. V., DGPs) was similarly invited to collaborate, but did not respond. The German Society for Interventional Radiology and Minimally Invasive Therapy (Deutsche Gesellschaft für Interventionelle Radiologie und minimal-invasive Therapie, DeGIR) applied to collaborate. However, their participation was declined in light of the previously planned guideline contents. Topics relating to interventional radiology had already been addressed in the guidelines “Complications of Liver Cirrhosis” and “Hepatocellular Carcinoma” and will therefore not be discussed in these Guidelines.

Representativeness of the Guideline Development Group: Patient participation

Mr. I. van Thiel (Cologne) of the German Liver Patients Association (Deutsche Leberhilfe)

Besides the Steering Committee ([Table 1]), eight working groups were constituted that were each headed by two leads ([Table 2]). Working group 4 – “Management” is divided into three subgroups. In the working groups, the proportion of university to non-university-based physicians, hospital-based clinicians to private practitioners was well balanced. Participants in the working groups included gastroenterologists, endocrinologists, diabetologists, obesity specialists, pediatricians, specialists in pediatrics and adolescent medicine, pathologists, cardiologists, transplant physicians, clinical nutritionists/nutritional therapists, radiologists and surgeons.

Table 1 Steering Committee.

Name	City	Responsibility
A. Canbay	Bochum	DGVS
E. Roeb	Gießen	DGVS
F. Tacke	Berlin	DGVS
H. Bantel	Hannover	DGVS
J. Bojunga	Frankfurt am Main	DGFF/DGE
M. Demir	Berlin	DGVS
A. Geier	Würzburg	DGVS
W. Hofmann	Berlin	DGVS
J. Schattenberg	Mainz	DGVS
A. Tannapfel	Bochum	DGPathologie/BDP

Table 2 Members of the Guideline Development Group.

Working Group 1: Definition	Working Group Lead	E. Roeb, Giessen (DGVS) A. Tannapfel, Bochum (DGPathologie/BDP)
	Working Group Members	M. Bahr, Lübeck (DGVS) S. Hohenester, Munich (DGVS) T. Longerich, Heidelberg (DGPathologie/BDP)
Working Group 2: Screening for NAFLD	Working Group Lead	M. Demir, Berlin (DGVS) M. Krawczyk, Homburg (DGVS)
	Working Group Members	T. Bernatik, Ebersberg (DEGUM) P. Buggisch, Hamburg (DGVS) C. F. Dietrich, Bern (DGVS) C. G. Dietrich, Wiesbaden (DGVS) M. Lehrke, Aachen (DGKardiologie) K. Ringe, Hannover (DRG)
Working Group 3: Diagnostic features	Working Group Lead	H. Bantel, Hannover (DGVS) A. Canbay, Bochum (DGVS)
	Working Group Members	H. Baba, Essen (DGPathologie/BDP) T. Karlas, Leipzig (DEGUM) S. Mueller, Heidelberg (DGVS) S. Rossol, Frankfurt (DGVS) A. Schreyer, Brandenburg (DRG)
Working Group 4a: Management – Non-pharmacological conservative therapy	Working Group Lead	J. Bojunga, Frankfurt am Main (DGFF/DGE) J. Schattenberg, Mainz (DGVS)
	Working Group Members	S. Bischoff, Stuttgart (DGEM) S. Engeli, Greifswald (DAG) H. Hinrichsen, Kiel (DGVS) M. Plauth, Dessau (DGEM) I. van Thiel, Cologne (German Liver Patients Association)
Working Group 4b: Management – Pharmacological therapy	Working Group Lead	A. Geier, Würzburg (DGVS) F. Tacke, Berlin (DGVS)
	Working Group Members	V. Keitel-Anselmino, Magdeburg (DGVS) A. Kremer, Erlangen (DGVS) M. Roden, Düsseldorf (DDG) N. Stefan, Tubingen (DDG)
Working Group 4c: Management – Interventional therapy (endoscopic procedures, bariatric surgery, liver transplantation)	Working Group Lead	U. Denzer, Marburg (DGVS) M. Sterneck, Hamburg (DTG)
	Working Group Members	J. Kluwe, Hamburg (DGVS) B. Müller, Heidelberg (DGAV) K. Rheinwalt, Cologne (DGAV) H. Schmidt, Essen (DGVS) D. Seehofer, Leipzig (DGCH)
Working Group 5: Monitoring and long-term management	Working Group Lead	W. Hofmann, Berlin (DGVS) T. Lüdde, Düsseldorf (DGVS)
	Working Group Members	R. Günther, Kiel (DGIM) A. Pathil-Warth, Frankfurt am Main (DGVS) M. Rau, Wurzburg (DGVS) K. Stein, Magdeburg (DGVS)
Working Group 6: NAFLD/NASH in children (pediatrics)	Working Group Lead	J. de Laffolie, Giessen (DGKJ/GPGE) C. Hudert, Berlin (DGKJ/GPGE)
	Working Group Members	R. Ganschow, Bonn (DGKJ) P. Gerner, Freiburg (GPGE) D. Weghuber, Salzburg (GPGE) S. Wiegand, Berlin (AGA/DAG)
Coordinators		A. Canbay, Bochum (DGVS) E. Roeb, Giessen (DGVS) F. Tacke, Berlin (DGVS)

2. Methodology

2.1 Methodological principles

Literature search

Each working group performed their literature searches individually. The search and selection details are presented in guideline report.

Scheme of grading of recommendations

The strength of recommendation is derived from the formulation used (Should/Ought to/May be considered) in line with the grading illustrated in [Table 3]. The strength of consensus was established according to [Table 4].

Table 3 Grid for grading recommendations.

Description	Syntax
Strong recommendation	Should
Recommendation	Should
Open	Can

Table 4 Classifying the strength of consensus.

Consensus	% Agreement
Strong consensus	≥ 95
Consensus	≥ 75–95
Majority consensus	≥ 50–75
No Consensus	< 50

Statements

Statements are defined as descriptions or explanations of specific facts or key questions without direct calls to action. As part of a formal consensus procedure, statements are adopted according to the procedures in the recommendations and can be based on either study results or expert opinions.

3. External review and adoption

Adoption by the chairpersons of the publishing scientific medical societies/organizations

The full and complete Guideline was reviewed by all participating scientific medical societies and agreed on by consensus after a consultation version was posted on the DGVS and the AWMF website to be commented on by the professional community for 4 weeks in February 2022 (02.02. bis 28.02.2022). Commentaries were requested through the DGVS newsletter. All proposed changes are presented in the guideline report.

Editorial independence and guideline funding

These Clinical Practice Guidelines were developed according to the principle of editorial independence. The DGVS provided the funding for the use of the guideline portal, the online kick-off meeting and the online consensus conference. No third parties were in involved in the funding. The work done by the mandate holders and experts was exclusively on an honorary basis.

Disclosing and managing conflicts of interest

In line with the AWMF manualʼs guidance on managing conflicts of interest, all participants submitted their declarations completed on the corresponding AWMF standard form (form sheet 2018). The conflicts of interest forms were reviewed by the guideline coordinators and Dr. Nothacker (AWMF), then categorized according to the AWMF criteria as low, moderate and high in relation to the individual recommendations. Afterwards, they were presented to the Guideline Development Group prior to commencement of the consensus conference which, in turn, performed a mutual appraisal of the declarations of conflicts of interest.

Financial connections to industrial companies, for whose products no recommendations are issued in these Guidelines, were not appraised as conflicts of interest; this relates, among other things, to drugs under development. Lectures for manufacturers of diagnostics or therapeutics for NAFLD were appraised as low-level direct conflicts of interest. Activities on a scientific advisory board/work as consult or expert for a company in the health industry with a thematic connection to the guideline recommendations and the corresponding stock ownership were ranked as moderate direct conflicts of interest. The companies Siemens Healthcare, Echosens and GE Healthcare are ranked as relevant in terms of diagnostic procedures as are TECOmedical as the manufacturer of the CK18/M30 ELISA and Novo Nordisk as the manufacturer of antidiabetics. Patents were ranked as high conflicts of interest. As a result, eight experts were appraised to have moderate conflicts of interest. Moderate conflicts of interest produced an abstention during the voting and/or duplicated votes (once without, once with the affected parties, anonymous voting). Furthermore, the interdisciplinary and representative composition of the Guideline Development Group as well as the structured consensus development led by a neutral moderator are factors that help protect against bias. The declarations of interests submitted by all experts are listed in the Guideline Report.

4. Dissemination and implementation

Concept for dissemination and implementation

In addition to the journal “Zeitschrift für Gastroenterologie”, these Guidelines will be disseminated on the AMBOSS knowledge platform and via the homepages of the DGVS (www.dgvs.de) and AWMF (www.awmf.de). An English short version of the Guideline will likewise be published in the “Zeitschrift für Gastroenterologie”.

Period of validity and update processes

The guideline shall remain valid for around five years (until 30 September 2026). The revision will be initiated by the guideline coordinators of the DGVS. The Steering Committee will review the need for updating the Guideline annually. Ms. Lorenz ([e-mail]) at the DGVS Administrative Offices is available as contact partner.

Editorial note

Gender neutrality

In order to improve legibility, gender-specific terminology has not been used in this document. All personal designations are therefore to be understood as gender neutral.

Participatory decision-making

All recommendations contained in this guideline are to be understood as recommendations intended to be discussed and implemented within a participatory decision-making process involving the physician and the patient and/or the patientʼs family members.

Special note

Medicine is subject to a continuous development process, so that all information, in particular on diagnostic and therapeutic procedures, can only correspond to the state of knowledge at the time of printing of the guideline. The greatest possible care has been taken with regard to the recommendations given for therapy and the selection and dosage of medications. Nevertheless, users are urged to consult the manufacturersʼ package inserts and expert information for verification and, in case of doubt, to consult a specialist. Any discrepancies should be reported to the DGVS. The user himself remains responsible for any diagnostic and therapeutic application, medication and dosage. In this guideline, registered trademarks (protected trade names) are not specially marked. It can therefore not be concluded from the absence of a corresponding reference that it is a free trade name. The work is protected by copyright in all its parts. Any use outside the provisions of copyright law without the written consent of DGVS is prohibited and punishable by law. No part of the work may be reproduced in any form without written permission. This applies in particular to reproductions, translations, microfilming and the storage, use and exploitation in electronic systems, intranets and the Internet.

Preamble

According to the current guidelines issued by the DGVS (German Society for Gastroenterology), EASL (European Association for the Study of the Liver), AASLD (American Association for the Study of Liver Diseases), APASL (Asian Pacific Association for the Study of the Liver) and the World Gastroenterology Organisation, non-alcoholic fatty liver disease (NAFLD) includes the spectrum of NAFL (non-alcoholic fatty liver), NASH (non-alcoholic steatohepatitis), NASH fibrosis and NASH cirrhosis. New nomenclatures, such as metabolic dysfunction-associated fatty liver disease, have been proposed, but are not universally established to date. The progression of NAFLD and particularly NASH is associated with liver cell stress, consecutive inflammation and fibrosis, potentially leading to liver cirrhosis, portal hypertension and end-stage liver disease. NASH is also a relevant risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis and natural course of NAFLD are becoming increasingly better understood. However, the heterogeneity of the patients and the diseaseʼs multifactorial genesis encumber the assessment of the precise prognosis of affected individuals. In the near future, patients with NASH-associated end-stage liver disease are expected to represent the highest proportion listed for liver transplantation. Despite being modified by genetic factors, the disease is believed to primarily result from hyperalimentation and a hepatic manifestation of metabolic syndrome. The clinical presentation of non-cirrhotic NAFLD is usually non-specific. With a global prevalence of around 25 %, NAFLD is now the leading cause of chronic liver disease worldwide and a growing public health challenge. Given the current obesity epidemic, a further increase in the prevalence of NAFLD is to be expected, especially among adolescents and younger patients. Changes in lifestyles, demographic shifts and the increasing complexity of pharmacological therapies are causes for this rise. Medical healthcare professionals and patient advocate organizations must deal with this collectively and individually. The previous German S2k Guidelines on NAFLD expired in February 2020.

1. Definitions

NAFL, NASH, NAFLD, Steatosis

Commentary 

Hepatic steatosis (liver cell steatosis) is characterized by the storage of fat in hepatocytes. Steatohepatitis is present if inflammation and hepatocyte damage can be detected in conjunction with hepatic steatosis [1]. Although a diet-related and alcoholic pathogenesis of steatosis and steatohepatitis is the most common cause, their differential diagnosis, covers a broad spectrum of possible causes for steatosis-associated liver damage (see [Table 6] Chapter “Diagnostics”). These causes should be investigated in the medical history and taken into account in the final interpretation of the patientʼs liver damage. The acronym for non-alcoholic fatty liver disease is NAFLD.

Both alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) are characterized by steatosis and lobular inflammation with ballooning of hepatocytes, resulting in wire mesh fibrosis (which progresses if the disease persists). A reliable differential diagnosis of ASH vs. NASH cannot generally be based on histological criteria alone. The differences between ALD and NAFLD identified in cohort comparisons are of a gradual nature and therefore not sufficiently reliable to typify the individual case (cave: Lifestyle modification prior to liver biopsy). Steatosis and the formation of glycogenated nuclei are often more pronounced in NASH, while the inflammatory activity and the detection of Mallory-Denk bodies (MDB) and satellitosis (granulocytic demarcation of a hepatocyte with MDB) is observed more frequently in ASH [2]. The mere evidence of sclerosing hyaline necrosis, which can develop as a result of extensive perivenular hepatocellular necrosis, was not regarded to be a result of NASH, meaning that sclerosing hyaline necrosis excludes a sole non-alcoholic origin of liver damage [3].

Most patients with NAFLD have central obesity and other components of metabolic syndrome. However, NAFLD can also develop in non-obese patients (referred to as lean NAFLD, comprising approx. 20 % of cases). It is assumed that these patients show less inflammatory activity and therefore have a better prognosis [4] [5] Due to the frequent association with metabolic syndrome, a consensus panel suggested that NAFLD be referred to as metabolic dysfunction associated fatty liver disease (MAFLD) [6]. Indeed, this term excludes some entities. On the one hand, lean NAFLD is poorly defined; on the other, metabolic disorders (e. g. mitochondriopathies, glycogenosis) represent separate pathogenetic and therapeutic entities. Further analyses are required to assess the acceptance for and the positive and negative consequences of renaming NAFLD to MAFLD [7]. Based on current knowledge, the term MAFLD should therefore not be used synonymously given its terminological imprecision [8]. The panel has decided to stick to the established term NAFLD, for which the vast majority of scientific evidence exists. The international European and US hepatological associations will hold a consensus meeting on this issue in the near future.

When diagnosed, NASH is considered a precancerous condition/lesion, implying that hepatocellular cancer (HCC) and, or less often, intrahepatic cholangiocarcinoma (iCCA; ratio: 5–7 HCC/1 iCCA) can develop and that surveillance should take place according to the corresponding S3 guideline [9] [10] [11] [12]. The HCC incidence in NASH patients without liver cirrhosis is reported at 0.02 % per year and increases up to 1.5 % per year in the presence of liver cirrhosis [9]. The specific aspects of NAFLD/NASH in children are discussed in the Chapter “Pediatrics”.

Commentary 

Detection of hepatocellular steatosis of up to 5 % is considered normal [13]. A significantly increased accumulation of triglycerides in the liver cells is called hepatic steatosis. A rule of thirds has been established for grading steatosis in the context of NAFLD (low: up to 33 %, moderate: 33–66 %, severe: > 66 % macrovesicular steatosis) [14] [15]. Although the conventional scoring systems mentioned below put the percentage of steatosis in relation to the number of hepatocytes, this approach is so impractical that the parenchymal area affected by macrovesicular steatosis usually tends to be estimated instead [16]. The NAFLD Activity Score (NAS) according to Kleiner not only evaluates lobular inflammation and hepatocellular ballooning but also steatosis as a subcomponent of inflammatory activity (grading) [15].As shown for bland steatosis, liver cell steatosis alone is not an independent risk factor for progression to liver fibrosis and is therefore unsuitable as a surrogate marker for the inflammatory activity of NAFLD [15]. Evidence of liver cell steatosis typifies the disease (steato-), while inflammatory activity is characterized by the extent of lobular inflammation and hepatocellular ballooning [10] [17].

Commentary 

The most important factor predicting the prognosis of NAFLD patients is the stage of fibrosis. A meta-analysis of five studies on 1495 biopsy-proven NAFLD patients and a follow-up of 17,452 patient-years showed that, compared to NAFLD patients without fibrosis (F0), those with fibrosis had an increased risk for both overall and liver-specific mortality, which increased continuously with the fibrosis stage. There was an exponential increase in risk in terms of liver-specific mortality [18]. The greatest risk of liver-specific, but also overall morbidity and mortality in NAFLD was demonstrated for advanced fibrosis (F3) and liver cirrhosis (F4). The following event rates were registered over an average observation period of 5.5 years: 8 % all-cause mortality, 8 % liver transplants, 19 % first-time hepatic decompensation, 9 % HCC, 3 % vascular events and 7 % non-hepatic malignancies. The transplant-free 10-year survival was 94 % for F3 and 45.5 % for F4. In stage F3, there were higher cumulative incidences of vascular events (7 % vs. 2 %) and non-hepatic malignancies (14 % vs. 6 %). By contrast, the frequency of hepatic decompensation and the development of HCC were increased in patients with liver cirrhosis: 44 % vs. 6 % and 17 % vs. 2.3 % [19]. These data suggest that cardiovascular and non-hepatic morbidity and mortality are more common in non-cirrhotic NAFLD patients, while complications of advanced liver disease determine the further prognosis in patients with manifest liver cirrhosis.

Xiao et al. conducted a meta-analysis of over 13,000 subjects to determine the best method for diagnosing liver fibrosis in NAFLD. In their comparison of APRI, FIB-4, BARD score, NAFLD fibrosis score (NFS), vibration-controlled transient elastography (VCTE), shear-wave elastography (SWE) and magnetic resonance elastography (MRE), the methods MRE and SWE showed the highest diagnostic accuracy for staging fibrosis. NFS and FIB-4 showed the best performance in detecting advanced fibrosis among the four non-invasive simple indexes [20]. According to current meta-analyses, complex biomarker panels and elastography can identify NAFLD-related fibrosis with moderate accuracy in obese individuals, but these methods have not yet been well validated [21]; also see Chapter “Diagnostics”.

Histological Grading and Staging

Commentary 

Macrovesicular steatosis of > 5 % suggests fatty liver disease. Steatohepatitis is said to be present if (steatosis-associated) inflammatory foci and hepatocellular ballooning are found concomitant with steatosis [14] [15]. Ballooning is defined as swelling and rounding of the hepatocytes. It is caused by a change in the intermediate filaments of the cytoskeleton, often with associated small lipid droplet accumulation and a dilatation of the endoplasmic reticulum. Immunohistologically, ballooned hepatocytes show a loss of keratin 8/18 expression in the hepatocytes [23]. Cytoplasmic inclusions in the form of Mallory Denk Bodies can also be detected in ballooned liver cells. Further typical but diagnostically unnecessary histomorphological characteristics of NAFLD are the detection of glycogenated nuclei and lipogranulomas. The fibrosis that occurs as a result of steatohepatitis often begins (in adults) in the center of the lobule in the form of perivenular and perisinusoidal fiber deposits (chicken wire type). Over the further clinical course, portal fibrosis develops with formation of bridging (portoportal and portocentral) septa that can ultimately lead to liver cirrhosis as the disease progresses [14] [15].

Two scoring systems (NAS and SAF) have been established for assessing inflammatory activity. While the SAF score allows the diagnosis of NASH (S_≥ 1A_≥ 2F_every), NAS was initially developed for use as a semi-quantitative scoring system in clinical trials in ordeto map the spectrum of the natural course of the disease [14] [15]. Both scoring systems have advantages and disadvantages. As with all graded histological parameters, the grading of ballooning is subject to a certain intra- and interobserver variability [24] [25]. In order to achieve the most comparable assessment possible, when using the NAS or the SAF score for grading the inflammatory activity, it is important to ensure that the scoring systems are used as per definition. To evaluate lobular inflammation, the mean value of all inflammatory foci counted in the core biopsy specimen per 200x magnification field is calculated, rather than just assessing the field of view with the highest number of inflammatory foci.

Whereas the inflammatory activity gives a snapshot of the current liver damage in the core biopsy specimen, the extent of the fibrotic parenchymal remodeling (staging) is used to define the disease stage. The staging provides an indication of the potential for regression and, in the case of repeated liver biopsies, of the dynamics of the liver damage. The histopathological staging systems grade the extent of the fibrosis and require a defined scale value (0–4). Therefore, these results cannot always be equated with the metric, continuous values ​​of non-invasive fibrosis detection methods. A major advantage of histopathological staging is the detection of early stages of fibrosis, where there is a high chance of complete reversibility.

Metabolic Syndrome

Commentary 

According to the International Diabetes Federation (IDF), the components of metabolic syndrome consist of obesity, insulin resistance, dyslipidemia and hypertension. Even before the criteria for diagnosing diabetes mellitus have been met, episodes of hyperglycemia and associated changes in blood lipids (increase in triglycerides and decrease in HDL cholesterol) can increase the risk of cardiovascular damage. The more components of metabolic syndrome are present, the higher is the cardiovascular mortality rate [26]. NAFLD is viewed by gastroenterological and diabetological societies as a hepatic manifestation of metabolic syndrome. According to the IDF consensus, metabolic syndrome exists if central obesity (defined as an enlarged waist circumference) plus two other criteria listed in [Table 5] can be demonstrated (https://www.idf.org/e-library/consensus-statements/60-idfconsensus-worldwide-definitionof-the-metablic-syndrome.html:). At BMI > 30 kg/m², central obesity can be assumed and waist circumference does not need to be measured additionally [27].

Table 5 Criteria for the clinical diagnosis of metabolic syndrome.

Measurands	Cutoff points
Enlarged waist circumference[*]	Population and country specific definitions
Elevated triglycerides (or drug treatment for hypertriglyceridemia [†])	≥ 150 mg/dL (1.7 mmol/L)
Reduced HDL-C (or drug treatment for reduced HDL-C [†])	< 40 mg/dL (1.0 mmol/L) for men; < 50 mg/dL (1.3 mmol/L) for women
Elevated blood pressure (or medication to lower blood pressure and a history of hypertension)	Systolic > 130 mmHg and/or diastolic > 85 mm Hg
Increased fasting glucose [‡] (or drug treatment for hyperglycemia)	≥ 100 mg/dL or previously diagnosed type 2 diabetes mellitus (T2DM). If the level is above 5.6 mmol/L or 100 mg/dL, OGTT is urgently recommended but is not required to define the presence of the syndrome.

HDL-C = high-density lipoprotein cholesterol.

^* Until further data are available, it is recommended to use the IDF cutoffs (waist circumference male ≥ 94 cm, female ≥ 80 cm) also ​​for non-Europeans and either the IDF or the AHA/NHLBI cutoffs (male ≥ 102 cm, female ≥ 88 cm) for persons of European origin.

^† Fibrates and niacin are very frequently used drugs for hypertriglyceridemia and reduced HDL-C. A patient taking any of these drugs can be assumed to have high triglycerides and low HDL-C. High doses of omega-3 fatty acids assume high triglycerides.

^‡ According to these criteria, most patients with T2DM have metabolic syndrome.

Minimum requirements for liver biopsy (technique, evaluation)

Commentary 

Liver biopsy is an invasive diagnostic method and associated with – albeit very low – morbidity and mortality [28]. The choice of access route (e. g. percutaneous, transjugular, laparoscopic, transgastral) should take into account the locally available expertise and the biopsy should be performed under optical guidance. Given the direct relationship between biopsy size and number of portal fields recorded [29], the core specimen should not be < 15 mm in length. When a needle caliber ≥ 1.6 mm (≤ 16G, ≤ 16 gauge) is used, this core specimen length ensures the presence of > 10 assessable portal fields [30] [31] [32]. Thinner needle calibers (< 1.6 mm) reduce the diagnostic accuracy [33] [34] [35] [36] [37]. In prospective studies, the use of larger needles was not associated with a higher risk of bleeding [38] [39]. The recommendation to take a core biopsy measuring at least 20 mm in length using a ≤ 16G needle is based on international consensus [31] [40] [41].

When taking liver biopsies, sampling errors can reduce the diagnostic accuracy of a liver biopsy. In NAFLD, this applies to both percutaneous and intraoperative samples taken under direct vision [42] [43] [44] [45]. In this context, taking two biopsies was able to reduce the sampling error [42] [46]. Taking more than two biopsies is associated with an increased risk of complications [47] [48] [49].

Minimum criteria for inclusion in the histology report

Commentary 

The report on histopathological findings should include a statement on the assessability of the biopsy (good, sufficient, borderline, inadequate). Factors that are taken into account here are the size of the biopsy (length, diameter), the number of portal fields assessed and the degree of fragmentation. Heavily fragmented biopsies can indicate liver cirrhosis, but are exactly what make it more difficult to assess the extent of fibrosis. The liver biopsy report should systematically describe all morphologically detectable changes in the portal fields, liver lobule and vessels. This ensures traceability within the framework of quality assurance measures.

According to the current definition, hepatocellular ballooning and lobular inflammation lead to the diagnosis of NASH [14] [15] [22] and must therefore be described on the histology report. They can be combined using established scoring systems like NAFLD Activity Score (NAS)/NASH-CRN [15] or the SAF/FLIP algorithm [14] [22]. When diagnosing NASH, information on the stage of the fibrosis is essential for further clinical management. For this, the definition of the fibrosis stages according to NASH-CRN/NAS is available, which is similarly used in the SAF Score/FLIP algorithm. As the histopathological findings should always be interpreted in the context of the overall clinical situation and take differential diagnoses into account, a description of all other histological abnormalities is desirable, or, in the context of NASH, also the absence thereof (such as MDB, apoptotic bodies or microvacuolar steatosis).

Indication, timing and performance of liver biopsies

Registration and initial processing of the biopsy

Commentary 

In addition to the indication for liver biopsy (see also Chapter “Diagnostics”), further processing must be conducted scrupulously. Immediately after collection, the liver biopsy should be transferred to neutral-buffered formalin (6.25–10 % formalin, corresponding to 2.5–4 % formaldehyde in phosphate buffer, pH7) and fixed for a sufficiently long time (optimally overnight) [50] [51]. This prevents any decomposition processes from taking place (e. g. autolysis). All examinations necessary for cytological diagnostics of fine-needle aspirates can be carried out on the formalin-fixed paraffin-embedded biopsy tissue. In addition, this material can be used for reliable investigations at the nucleic acid level (DNA/RNA) (with appropriate differential diagnostic considerations) [51]. Inflammatory manifestations of NASH are not evenly distributed around the liver tissue, which is why liver biopsy carries the risk of sampling error [42]. In addition, any possible comorbid conditions (e. g. primary biliary cholangitis) can manifest heterogeneously and segmentally. Therefore, a sufficient number of slices (at least 8) should be examined histologically.

The liver has a limited range of responses to damage. Therefore, histologically similar presentations or similar damage patterns can have different causes (e. g. ALD vs. NAFLD). For the optimal interpretation of liver biopsies, knowledge about the relevant clinical history as well as the relevant serological parameters (in relation to the differential diagnostic question) is essential. Here, a structured form, accompanying the liver biopsies has proven useful (an example is shown in the Appendix 1). In some situations, it can be useful to collect an additional core biopsy specimen, fixed in glutaraldehyde (especially in hereditary/childhood liver diseases) for electron microscopic examination or as native dry preparation (e. g. quantitative copper determination).

See Appendix 1

Differentiation of NAFLD from ALD

Differentiation of NAFLD from other hepatic steatoses

Commentary 

Recommendations and statements on the amount of alcohol were taken from the NAFLD guideline from 2015 [52] and are confirmed by the National Institutes of Health NASH clinical research network and the Asia Pacific Working Party on NAFLD Guidelines 2017 [53]. ALD cannot be ruled out with certainty in the case of higher daily alcohol consumption. Ethyl glucuronide (EtG) in urine or in the hair and phosphatidylethanol (PEth) in the blood are mainly used to confirm alcohol abstinence. By measuring EtG in the hair, alcohol consumption can be estimated retrospectively over a period of several months. EtG in urine is a suitable parameter for alcohol withdrawal or drinking withdrawal programs as well as for abstinence tests before liver transplantation (LT) or for inclusion on the waiting list.

The biochemical parameters ALT, AST and γGT can be used to identify existing alcoholic liver damage. However, the specificity is comparatively low. A combination of different biomarkers is advisable because they differ in their underlying pathomechanisms. GOT/AST, γGT, Hb and ferritin can provide clues for differentiating between ALD and NAFLD [54] [55]. The definition of harmful alcohol consumption is not uniform. In clinical trials on NASH, the definition of an average of no more than 14 units of alcohol per week for women and 21 for men was used [56]. In their meta-analysis, Larsson et al. found that approx. 12 g of alcohol correspond to approx. one drink and that studies use the following categories: Light (< 1 drink/day), moderate (1–2 drinks/day) and high (> 2–4 drinks/day) alcohol consumption [57]. According to the Royal Medical Colleges, studies on alcohol-related harm in women indicate that the level of consumption at which the relative mortality risk increases is around 16 g alcohol/day or around 2 drinks/day [58]. Aberg et al. observed a J-shaped association between alcohol consumption and mortality with alcohol consumption of 0–9 g/day compared to lifelong abstainers. However, these benefits have only been seen in non-smokers. Alcohol consumption > 30 g/day resulted in an increased mortality risk compared to lifelong abstainers [59]. Data from the National Health and Nutrition Examination Survey III have associated alcohol consumption with increased mortality in participants with fatty liver and metabolic syndrome. These findings suggest an overlap between NAFLD and ALD [60].

2. Prognosis & Screening

Incidence

Commentary 

The incidence of NAFLD has so far only been investigated in a few population-based studies. A meta-analysis from Asia of 237 studies and 13,044,518 individuals found a pooled annual NAFLD incidence rate of 50.9 cases per 1000 person-years (95 % CI 44.8–57.4) [61]. According to a meta-analysis for western countries, the annual incidence rate is 28 per 1000 person-years (95 % CI, 19.34–40.57), with only data from Israel being included [62]. A population-based study from the USA with 3,869 NAFLD patients and 15,209 controls found that the NAFLD incidence increased five-fold from 62 to 329 in 100,000 person-years between 1997 and 2014. There was a 7-fold increase in the group of 18–39 year-olds [63]. To date, there is no accurate information on the incidence of NAFLD in the general population.

Prevalence

Commentary 

Studies on the point prevalence of NAFLD in the general population show great variability. This is due, among other things, to regional differences, the different diagnostic modalities and the underlying definition of NAFLD, since liver biopsy as the gold standard in diagnosis cannot be used in population-based studies. A meta-analysis of 86 studies from 22 countries (n = 8,515,431) showed a global prevalence of NAFLD of 25.24 % (95 % CI: 22,10–28,65). The published prevalence rates were lowest for Africa at 13.48 % (95 % CI, 5.69–28.69) and highest for the Middle East (31.79 %; 95 % CI, 13.48–58.23) and South America (30.45 %; 95 % CI, 22.74–39.44) [62]. For Germany, the NAFLD prevalence was around 23 % in 2016 and is mathematically projected to be around 26 % in 2030 [64].

There are no population-based studies on the prevalence of NASH, as this requires obtaining a liver histology. The pooled NASH prevalence in “clinically indicated” NAFLD biopsies was 59.10 % globally (95 % CI: 47.55–69.73). In patients “without a NAFLD-related indication” such as before a living liver donation, the NASH prevalence was between 6.67 % (95 % CI: 2.17–18.73) in Asia and 29.85 % (95 % CI: 22.72–38.12) in North America [62]. For Germany it is estimated at 4.1 %, with a model based projected increase to 6 % in 2030 [64].

There is little data on the prevalence of NASH cirrhosis in the general population. Analysis of a population-based cohort from the USA during the periods 1999–2002 and 2009–2012 using surrogate markers for fibrosis showed a significant increase in the prevalence of NASH cirrhosis of 0.178 % in the period 2009–2012 compared to 0.072 % in the period 1999–2002 (p < 0.05). The prevalence of NAFLD with advanced fibrosis (F3) increased from 0.84 to 1.75 % (p < 0.001) over the same period. This corresponds to a 2.5-fold increase in the prevalence for NASH cirrhosis and a doubling of NAFLD-associated advanced fibrosis [65]. An analysis of the FLAG cohort with 507 NAFLD patients mainly from secondary care settings in Germany, using the FIB-4 score (modified cut-off of > 2.67 for ≥F3), showed a 10 % prevalence of advanced fibrosis or cirrhosis [66]. In contrast, a mathematical model estimated the prevalence of advanced fibrosis or cirrhosis (≥F3) in NAFLD in Germany to be 3.3 %, which corresponds to the number of cases totaling 600,000 [64].

Risk factors and prognosis

Commentary 

Liver-related morbidity and mortality: Although histological NASH is generally considered the progressive form of NAFLD, it has repeatedly been shown that NAFLD can also take a progressive course [67] [68] [69] [70]. In a meta-analysis of 11 studies with paired biopsies, the fibrosis progression by one stage was 14.3 years for NAFLD and 7.1 years for NASH [67]. In another large study (n = 646), the mean time to developing end-stage liver cirrhosis was investigated using biopsy-confirmed NAFLD over an observation period of 20 years. This showed for F0: 33.4; F1: 34.1; F2: 22.7; F3: 11.8 and F4: 5.6 years, respectively. The presence of NASH had no significant influence on these estimates (likelihood ratio test > 0.05 for all fibrosis stages) [69]. Nevertheless, the most important factor for prognosis in NAFLD is the underlying stage of fibrosis [18]. The greatest risk of both liver-specific and overall morbidity and mortality of NAFLD was found to be advanced fibrosis (F3) and liver cirrhosis (F4). The following event rates were registered over an average observation period of 5.5 years: 8 % all-cause mortality, 8 % LT, 19 % first-time hepatic decompensations, 9 % HCC, 3 % vascular events and 7 % non-hepatic malignancies. The transplant-free 10-year survival was 94 % for F3 and 45.5 % for F4. In F3, there were higher cumulative incidences of vascular events (7 % vs. 2 %) and non-hepatic malignancies (14 % vs. 6 %). In contrast, the proportion of hepatic decompensations and HCC was increased in patients with liver cirrhosis: 44 % vs. 6 % and 17 % vs. 2.3 % [19].

These data suggest that cardiovascular and non-hepatic morbidity and mortality are more common in non-cirrhotic NAFLD patients, while complications of advanced liver disease determine the further prognosis in cases of established liver cirrhosis. The latter includes the risk of developing HCC. Depending on the region and study population, the prevalence rates range between 0.8 % and 34 % [19] [71] [72] [73] [74] [75]. The major challenge is that, even in the non-cirrhotic liver, the risk of NAFLD developing into HCC is 20–50 % [72] [74] [76] [77]. NAFLD is increasingly becoming an indication for LT. In the USA, it is currently the second most common LT indication with an increase of 167 % in the period 2003–2014; in Germany, this trajectory is constantly upwards [78] [79].

Cardiovascular and non-hepatic morbidity and mortality: Depending on the stage of fibrosis, patients with NAFLD have an increased liver-related mortality and all-cause mortality compared to healthy controls [18] [19] [62]. The main causes of death are cardiovascular related [62] [80]. In a retrospective analysis of 619 NAFLD patients over the period 1975–2005 and a median follow-up of 12.6 years, cardiovascular disease was the most common cause of death (38 %), followed by non-hepatic cancer (19 %) and complications of liver cirrhosis (8 %) [71]. Similar data were reported in two prospective studies from Sweden with a follow-up of up to 33 years: cardiovascular causes of death 43 % and 48 %, non-hepatic tumors 23 % and 22 % and liver-related mortality 9 % and 10 %, respectively [81] [82].

Extrahepatic tumors: A meta-analysis of 6,263 patients showed that NAFLD is associated with colorectal adenomas (OR 1.74; 95 % CI: 1.53–1.97) [83]. In one historical cohort study on 25,497 participants observed over 7.5 years, patients with NAFLD, particularly those with advanced fibrosis, showed an increased incidence of colorectal cancer in men and breast cancer in women, in addition to the known risk of HCC [84].

Screening

Commentary 

NAFLD usually has an asymptomatic course and is often diagnosed incidentally [85]. Despite an NAFLD prevalence of 20–30 %, the progression to NASH and NASH fibrosis is not high enough to recommend a screening for NAFLD in the general population [86] [87]. This makes screening all the more important in the group of patients with an increased risk. Elevated liver enzyme levels ​​alone are not a sufficient screening criterion, as NAFLD can also be present at normal transaminase levels. T2DM and obesity are independent risk factors for developing NASH-related fibrosis [88] [89]. Several studies show a clear association with factors related to metabolic syndrome [90] [91] [92]. If these risk factors are present, the NAFLD prevalence increases to 60–75 % and thus justifies screening [86]. In German cohorts, the above-mentioned risk factors were observed in patients with NAFLD plus age > 50 years [66] [93]. When several other diseases are present, NAFLD should be diagnostically clarified. There is a reciprocal correlation with coronary artery disease. NAFLD should also be considered in diseases such as polycystic ovarian syndrome (PCO), sleep apnea, hypothyroidism, depression and renal insufficiency [32] [94]. A general screening of relatives does not appear to be justified. Screening in the presence of the aforementioned factors has proven cost-effective, at least in the USA, by preventing liver-specific diseases and endpoints [95]. A decision to implement screening is determined more by the care structures of a particular health care system than by the availability of particular screening procedures. Nevertheless, the design of a screening algorithm must be aligned with the capabilities of those performing the screening (see Recommendation below) [96] [97] [98]. In Germany, almost all patients are primarily cared for by general practitioners. A proportion of the patients defined in the risk population (see previous Recommendation) are assigned to specialists (diabetologists/endocrinologists, cardiologists). However, large numbers of patients with T2DM, obesity and arterial hypertension are treated exclusively by general practitioners (e. g. as part of disease management programs).

Due to access to patients, comprehensive risk population screening in Germany can only be in the hands of primary care physicians, possibly supported by diabetologists and cardiologists. This group of physicians is particularly suited to broadly identify the major risk diseases for NAFLD and thus to determine the individual NAFLD risk in these patients [99]. This assessment is also in line with existing EASL recommendations [100] [101] and consistent with a recently developed algorithm for general practitioners and diabetologists [102]. Since screening should mainly be carried out by general practitioners see previous recommendation), screening tools must be widely available, inexpensive and non-invasive in order to increase acceptance [94] [98] [99] [101]. A two-step design with verification of steatosis and fibrosis risk improves the specificity (and in some cases even the sensitivity) of the screening [97] [103]. Positively screened patients must be transferred to a gastroenterologist/hepatologist for further evaluation. Patients with prolonged or repeated elevations in GPT/ALT should be referred for further evaluation as they are generally at increased risk for liver disease [104] [105] [106]. Assuming that at least moderate steatosis is relevant, the fatty liver index (FLI) and NAFLD liver fat score (LFS) perform best, with the highest AUROC values at a positive predictive value (PPV) of 99 %, but without reliable exclusion of steatosis below the respective cutoff [107] [108] [109]. FLI can be determined from routine parameters ​​in general practice and should therefore be used if, for example, an ultrasound cannot be performed [100]. Scores using readily available routine parameters for the fibrosis risk include the NAFLD Fibrosis Score (NFS), the FIB-4 Score, the APRI Score, the Forns Score and the BARD Score. The first two (NFS, FIB-4) are superior to the last three (APRI, Forns, BARD) in screening fibrosis in the NAFLD cohort [20] [110] and have also been investigated in Germany [93] [111]. In a recent systematic review, this was confirmed especially for the “hardest” endpoint (mortality) [112]. FIB-4 and NFS are also suitable for screening patients with normal ALT [113] and can be easily determined using internet-based calculators.

In population screening, all scores have noticeable weaknesses [114]. The discriminatory performance of all tests is markedly better in high-risk collectives [114]. Both FIB-4 and NFS have lower specificity in patients > 65 years of age [115], which may increase the referral rate to specialists due to a higher proportion of false-positive screened patients. Data from a screening study on type 2 diabetes patients show that the use of age-adjusted cutoffs on FIB-4 (in delineating negative vs. intermediate) reduces the number of patients tested as intermediate (from 38.3 % to 15.4 % [97].

The screening strategy proposed in [Fig. 1] is based on recent proposals and takes into account the aforementioned prerequisites for risk screening by general practitioners or primary care practitioners, but may not currently be evidence-based in several areas. This specifically applies to the handling of the intermediate-risk group, the screening interval in low-risk patients and the cost-effectiveness of the entire algorithm. Moreover, this stated screening recommendation requires appropriate training of the general practitioners.

Fig. 1 Screening algorithm [rerif].

Potential screening algorithm that contains the two main elements (detection of steatosis and fibrosis risk), can be modified according to availability and performed in the general practitionerʼs office. The algorithm corresponds well with the European algorithm of the EASL Clinical Practice Guidelines [101] [116] and a recently proposed approach for general practitioners and diabetologists [102] but is easier to apply. The sequence of fatty liver index and FIB-4 has been specifically studied for screening in a high-risk population of T2DM patients [97]. The application of age-adjusted cutoff points (in brackets) may be useful in order to reduce the high proportion of individuals tested as persons with intermediate risk. How to manage patients with intermediate risk is the subject of discussion and can be structured in various ways (re-screening or direct referral to a hepatologist).

Value of transabdominal sonography of the liver in NAFL

Commentary 

Reference is made to the National S3 Guidelines on Hepatocellular [11] [12]. US is a widely available, cost-effective, radiation-free method that allows assessment of hepatic steatosis. Hepatic steatosis results in an increase in the echogenicity of the liver parenchyma. With increasing steatosis, there is a dorsal weakening of the parenchymal signal. US is thus suitable as a screening method for hepatic steatosis: In moderate and severe hepatic steatosis, good sensitivity (approx. 85 %) is achieved with a specificity up to 98 % [117]. The best results are recorded above a liver fat content of 12.5 %. Above this threshold, there was no significant difference in the AUROC values ​​compared to 1H magnetic resonance spectroscopy (MRS) [118]. Nevertheless, sensitivity is markedly worse in mild steatosis and especially in microvesicular steatosis (sensitivity 69 %) [119]. It is therefore not possible to exclude hepatic steatosis using ultrasound. With regard to possible liver fibrosis, US allows neither a definite diagnosis nor reliable staging. US-based shear-wave elastography techniques are useful to rule out advanced liver fibrosis and liver cirrhosis in NASH. See also Chapter “Diagnostics”.

Value of MRI (magnetic resonance imaging) and computed tomography (CT) in NAFLD diagnosis and screening

Commentary 

Because of its radiation exposure, CT should not be used as a screening method for the detection of NAFLD. A differentiation between NAFL and NASH is not possible by CT. In terms of methodology, however, CT is a highly reproducible and objective imaging method for visualizing the fat content of the liver. If the density ratio of the liver and spleen on the native CT has a cutoff > 1.1, a diagnosis of at least moderate hepatic steatosis can be rendered [120]. Dual-energy CT (DECT) is a newer imaging technique based on data acquisition at two different energy settings. It can be used to draw conclusions about the composition of tissues. Individual studies on smaller cohorts have produced promising results for quantifying fat content in the liver, even in comparison with magnetic resonance imaging (MRI) [121].

MRI is a radiologic imaging technology without any radiation exposure. In principle, the proportion of water and fat in the liver can be separated using various magnetic resonance techniques. Fundamentally, the sequences are based on fat suppression techniques such as selective fat suppression, selective fat stimulation or the “short-tau inversion recovery” (STIR) sequence. Another approach is “in-phase” and “out of phase” imaging, where signal intensity alterations of fat and water in the tissue are used in the sense of “chemical shift imaging”. Sufficiently large studies are still lacking; individual studies on relatively small populations with histopathological correlation appear promising with regard to an exact fat assessment [122] [123].

According to the literature, magnetic resonance spectroscopy (MRS) has the highest accuracy for fat assessment but is currently limited to research centers due to a lack of standardization of the methodology and high demands on hardware and software. The same is currently true for MR elastography regarding the detection and staging of liver fibrosis [124] [125]. The MR-based quantification of liver fat content using the “proton density fat fraction” (PDFF) is increasingly recognized as the best method by virtue of its high accuracy, easy post-processing and better availability [126]. Compared to histology as the reference standard, PDFF-based determinations deliver high diagnostic accuracy for the detection of steatosis (histological grade 1–3) with an AUROC of 0.99 (05 % CI 0.98–1.00), a sensitivity of 96 % and a specificity of 100 % with a threshold of 3.7 % [127].

The MR-based differentiation of NAFL and NASH using liver-specific contrast media and T1 mapping is presented in publications as very promising. However, it is a method that has not yet become part of routine clinical practice given the small numbers of cases to date [128] [129].

The value of biomarkers in NAFLD screening

Commentary 

A wide range of tests and non-invasive algorithms have been developed to diagnose hepatic steatosis and liver fibrosis. The NAFLD Fibrosis Score (NFS) can be easily calculated from standard laboratory parameters using an online input screen (https://nafldscore.com). The following parameters are entered for the calculation: Age, BMI, IGF/diabetes yes/no, AST, ALT, platelets and albumin. A cutoff score below –1.455 excludes advanced fibrosis with 90 % sensitivity. An NFS > 0.676 confirms a diagnosis of advanced fibrosis with 97 % specificity and 67 % sensitivity. FIB-4 is another common algorithm used for non-invasive fibrosis prediction. It is straightforward to calculate from the parameters for AST, ALT, platelets and age of the patient. The analysis is based on two cutoff points: Patients with a cutoff point < 1.3 have a low risk of fibrosis, while patients with a cutoff point ≥ 2.67 have a high risk of advanced fibrosis [130]. Both scores are well suited for use in screening as they are mainly based on routine parameters. Other non-invasive fibrosis scores such as the ratio of AST to platelets (AST/platelet ratio, APRI) or the BARD score show good negative predictive values ​​(NPV) and are therefore suitable for excluding advanced fibrosis. A current meta-analysis of 16 studies showed that the Enhanced Liver Fibrosis (ELF) test is suitable for diagnosing advanced liver fibrosis in NAFLD patients [131]. The test showed a high NPV, especially in populations with a low NAFLD prevalence (e. g. when used in primary care). In contrast to NFS and FIB-4, the ELF test is comprised of a combination of three values that are not measured routinely: Type III procollagen peptide (PIIINP), hyaluronic acid (HA) and tissue inhibitor of metalloproteinase-1 (TIMP1). Therefore, further studies are needed to determine the use of this marker panel in primary care. The fatty liver index (FLI) was developed in 2006 by Bedogni et al. [132]. The calculation is based on BMI, waist circumference, gamma-glutamyl transferase (γGT) and triglycerides. In studies, the FLI has shown a diagnostic value (AUROC) of 0.813 (95 % CI, 0.797–0.830) for the detection of fatty liver [133]. Measuring cytokeratin 18 (K18, neoepitope K18Asp396-NE) ​​in serum is useful in distinguishing between NAFL and NASH: Higher concentrations of K18 fragments were detected in the blood of patients with NASH. K18 in serum can be measured with an M30 ELISA. More recent meta-analyses show a diagnostic accuracy (AUROC) of 0.82 (0.76–0.88) for the detection of NASH patients [134]. To date, a number of different cutoff points ​​for K18 serum concentrations have been published, which makes the use of this biomarker difficult.

Genetic risks (e. g. PNPLA3): It is assumed that about 20 % of the total NAFLD risk is caused by individual predisposition and 80 % by environmental factors [135]. In particular, carriers of the PNPLA3 p.I148 M risk allele have an increased risk of developing a fatty liver. The risk allele also increases the risk of developing liver damage: Carriers are more likely to develop liver fibrosis, cirrhosis and HCC [136] [137]. The PNPLA3 p.I148 M risk allele is present as a homozygous or heterozygous genotype in about 50 % of all Caucasians and is therefore a common risk factor for hepatic steatosis and fibrosis. Other, rarer variants, e. g. in the MBOAT7 and TM6SF2 genes, have been described as causal pathogenetic factors in NAFLD. More recent studies have shown protective effects from gene variants in MARC1 and HSD17B13. A current analysis [138] of patient cohorts from Italy, Germany and the UK Biobank showed that polygenic risk assessments based on the existence of risk variants in the above-mentioned four genes allow a stratification of NAFLD patients with regard to their liver cancer risk. The now low costs for genetic analyses enable genotyping to be used in routine clinical practice. However, routine genotyping of patients with NAFLD cannot yet be justified. A recently published biomarker combination of K18 fragments, C-terminal pro-collagen type III N-terminal peptide (Pro-C3), PNPLA3 p.I148 M genotype and acetyl-high mobility group box 1 significantly improved the diagnostic accuracy for NASH in patients with NAFLD (AUROC: = 0.87, sensitivity 0.71, specificity 0.87) [139].

New developments (microbiota, specific imaging, new biomarkers, AI-based algorithms, etc.)

Commentary 

Multiple studies indicate that the intestinal microbiome is involved in both the development and progression of NAFLD [140] [141] [142]. However, no specific gut microbiota composition can currently be phenotyped for NAFLD [143]. Therefore, stool diagnostics are currently not suitable for screening or diagnosing NAFLD [144].

3. Diagnostics

Initial diagnostics

Commentary 

Reference is made here to the National [11] [12], the EASL-EASD-EASO Clinical Practice Guideline NAFLD [116] and current reviews [145]. Ultrasound (US) is a widely available, cost-effective and radiation-free method that allows the diagnosis of hepatic steatosis when the liver parenchyma shows increased echogenicity. With increasing steatosis, there is additionally a dorsal weakening of the parenchymal signal. US is therefore suitable as a screening method for the detection of hepatic steatosis [146]. For the safe use of US diagnostics, knowledge of sound physics and device configuration is required. The examination should therefore be carried out under the guidance of or by experienced examiners. Under these conditions, US has an excellent specificity (> 95 %) for the detection of advanced hepatic steatosis [147], although its sensitivity is insufficient for minor changes (e. g. for grade S1 steatosis 65 %) [119]. It is therefore not possible to exclude hepatic steatosis using ultrasound.

Controlled Attenuation Parameter

Commentary 

Analysis of the ultrasound signal attenuation is a quantitative parameter for assessing the extent of steatosis. Controlled Attenuation Parameter (CAP) technology is a Vibration-Controlled Transient Elastography (VCTE), using software that evaluates the signal attenuation of ultrasound impulses in liver stiffness [148]. CAP was initially only available for the M-probe of the VCTE and has, in numerous histology-controlled studies, shown good diagnostic accuracy for the detection and grading of steatosis in liver diseases of various etiologies [149]. The cut-off for the detection of steatosis was 248 dB/m [149]. However, in patients with NAFLD, who are often obese, an XL probe is needed in many cases. To date, histology-controlled data available on using a XL-CAP probe show a potentially high diagnostic accuracy for the detection of steatosis in suspected NAFLD (sensitivity 80 % and specificity 83 % with a cut-off of 302 dB/m²) [150]. In NAFLD, even with an adequate combination of M and XL tubes, CAP did not show sufficient accuracy to differentiate the individual degrees of steatosis [151]. In a meta-analysis of individual patient data, various quality indicators [152] did not lead to any improvement in the accuracy of CAP [151].

In pilot studies, CAP technology was suitable for assessing steatosis progression, e. g. after bariatric intervention [153]. The prognostic significance of a CAP reduction has not been proven to date [154]. Initial data are available in the comparison of attenuation imaging (ATI) with histology and MRI [155] [156]. The diagnostic accuracy of ATI for detecting steatosis tended to be higher than that of CAP (AUC 0.90 vs. 0.85). The routine clinical use of CAP in NAFLD diagnostics cannot currently be recommended due to the limited amount of data.

Value of magnetic resonance imaging and computed tomography in the diagnosis of NAFLD

Commentary 

Reference is made to the EASL-EASD-EASO Clinical Practice Guideline NAFLD [116]. Because of its radiation exposure, CT should not be used as the primary diagnostic method for detecting NAFLD. In terms of methodology, however, CT is a highly reproducible and objective imaging method for visualizing the fat content of the liver. Hepatic steatosis can be diagnosed by multiparametric comparisons of parenchymal signal attenuation on native CT. For example, the attenuation of the parenchymal signal of more than 10 Hounsfield units compared to the spleen is a suitable diagnostic criterion. However, the sensitivity for steatosis of mild severity is low [157].

In principle, the proportion of water and fat in the liver can be separated using various magnetic resonance techniques. The Proton Density Fat Fraction (PDFF) method determines the signal ratio of triglyceride protons compared to the total amount of protons (triglycerides and water). The data is given in percent [158]. In several comparative studies, MR-PDFF showed the highest sensitivity and specificity of all non-invasive methods for the detection of hepatic steatosis [145] [159] and is currently the only method that can non-invasively grade the extent of steatosis in NAFLD with reliability. It should be noted that the information is based on the relative triglyceride content, but cannot provide any information about the histological distribution [160]. Due to the diagnostic precision, MR-based methods appear to be suitable as a reference standard for diagnostic and Interventional studies [158] [161]. However, clinical use is currently limited to centers due to low availability and hard- and software requirements. The predictive importance of the steatosis dynamics characterized by means of MR techniques in therapy studies has not yet been finally defined [124] [154].

Diagnostic algorithm

Commentary 

Non-invasive fibrosis scores such as FIB-4 or NFS are suitable for risk assessment during the primary diagnostic workup of high-risk patients (e. g. with obesity, T2DM or metabolic syndrome) in whom diagnostic imaging (e. g. by US) confirms hepatic steatosis or where elevated liver enzymes ​​(GOT, GPT and/or γGT) were found and NAFLD is suspected, once other causes have been excluded [32] [116].

Because of the high negative predictive value (NPV) of FIB-4 or NFS (≥ 90 %) [162] [163], advanced fibrosis can be excluded with a high degree of probability, taking into account the lower cutoff point (FIB-4 ≤ 1.3 or NFS <-1.455). In patients with a low risk of fibrosis, the monitoring, e. g. of FIB-4 or NFS and transaminases, can be carried out at regular intervals. For patients with FIB-4 or NFS in the intermediate group (between the two cutoff points) or high group (FIB-4 ≥ 2.67 or NFS> 0.676), an elastography using VCTE is recommended as a further test method after taking relevant comorbidities into account [32] [116] [145]. Alternatively, shear-wave-based elastography methods can be used, taking the manufacturer-specific cut-offs into account [162]. Shear-wave elastography (SWE) technologies are available as software components on many modern ultrasound devices and can therefore be used easily when performing abdominal ultrasound (see Recommendation below). However, compared to VCTE, the SWE procedures are not well evaluated for NAFLD risk stratification and have not yet been considered in the current recommendations from international specialist societies for clarifying NAFLD (EASL Guideline 2016, AASLD Practice Guidance NAFLD 2018) [32] [116] [145].

Elastography can be invoiced with the OPS Code 3–034 Version 2021 (Complex differential diagnostic sonography using tissue Doppler imaging and tissue deformation analysis). In several studies, the VCTE LSM showed a high NPV for the exclusion of advanced fibrosis, with cutoff values differing only slightly [164] [165]. For this reason, an LSM value of 8 kPa can be regarded as a sensitive threshold value in practice. LSM values ​​in the 12 kPa show a high sensitivity for the presence of liver cirrhosis. If the M and XL probes are used correctly, no adjustments of the cutoffs are necessary [150] [166]. Patients with FIB-4 > 1.3 or NFS ≥ –1455 and an LSM below the low cutoff point (< 8 kPa) can undergo monitoring of, for example, transaminases, FIB-4/NFS and VCTE, at regular intervals. Patients in whom the LSM is between the low and high cutoff value (8–12 kPa) have a medium risk of an underlying advanced fibrosis and require further clarification. Liver biopsy should be considered for these patients. A liver biopsy should also be considered in patients with a high LSM of > 12 kPa, unless there is clear evidence of liver cirrhosis from laboratory parameters, clinical symptoms or diagnostic imaging ([Fig. 2]). Patients with clear evidence of liver cirrhosis, diagnosed with non-invasive procedures, or confirmed advanced fibrosis/cirrhosis, diagnosed with liver biopsy, require regular monitoring for the development of liver-associated complications [11] [12].

Value of elastography and biopsy

Commentary 

Depending on the risk profile, the use of combined multi-stage methods or repeated elastography can improve the diagnostic accuracy. Parameters of classic B-scan and duplex sonography cannot reliably differentiate between simple NAFL and NASH with fibrosis [145]. In addition, even when using high-frequency probes, B-mode sonography has limited sensitivity for the detection of compensated advanced chronic liver disease (cACLD) [167]. Elastographic methods can better capture the extent of fibrotic changes in the liver parenchyma by measuring the elasticity of the liver tissue and also provide a quantitative assessment. Transient elastography (TE), particularly vibration-controlled TE (VCTE) in a stand-alone device, as well as shear-wave-based methods (SWE) such as point SWE (pSWE) and 2D-SWE, are available [145] [168]. The SWE methods are integrated into ultrasound devices and marketed by manufacturers under different names, e. g. Acoustic Radiation Force Impulse Imaging (ARFI, Siemens), Elast-PQ (Philipps) and Supersonic Shear-Wave Elastography. Strain elastography, which is often used for other organs, has no merits in clinical practice [145] [168] [169].

VCTE and SWE have been evaluated in histology-controlled studies mainly in patients with viral hepatitis [170] [171] [172] and showed a high NPV for the exclusion of advanced fibrosis and/or cirrhosis. Quality indicators of liver stiffness measurement using VCTE and pSWE are well established over a longer time [168] [171]. The following VCTE cutoffs have been proposed for the diagnosis of advanced fibrosis in NAFLD: 7.9 kPa with a sensitivity of 91 %; 9.6 kPa with a specificity of 92 %. VCTE using the M-probe is limited in obese patients (BMI> 30 kg/m²) and associated with false-positive results [173]. Meanwhile, the applicability of VCTE has been optimized by the XL probe [164] and its importance in NAFLD has been proven in large studies [150] [174] and meta-analyses [175] [176]. In morbid obesity, valid measurements ​​can only be obtained in approx. 60 % of cases [177]. In the largest prospective multicenter European study with 373 evaluated patients, these cutoffs were established: 8.2 kPa for F ≥ 2, sensitivity 71 %, spec. 70 %; 9.7 kPa for F ≥ 3, sensitivity 71 %, spec. 75 %; 13.6 kPa for F4, sensitivity 85 %, spec. 79 %; F4 Cutoff for 90 % sensitivity 10.9 kPa [150], independent of the probe used (M or XL) [150] [166]. Serial examinations could increase the positive predictive value if parameters were abnormal at both measurement times [178].

Histology-controlled data for use in NAFLD patients are also available for pSWE and 2D-SWE [179] [180]. The advantage of these techniques is the better applicability in obesity [175], whereby anthropometric factors have to be taken into account when interpreting the measurements ​​ [181]. Depending on the method, the cut-off points [182]​​ and the diagnostic accuracy [183] in NAFLD patients are in the range of VCTE. A diagnostic superiority compared to VCTE has not yet been proven [184]. VCTE and the SWE-based methods should be carried out by experienced users, whereby technical and patient-related influencing factors must be taken into account [185]. SWE-based methods are more complex to perform than VCTE and should be used by an experienced doctor. Liver stiffness should be determined at least three hours after the last meal in a standardized position, avoiding extreme breathing maneuvers. As acute hepatitis as well as extrahepatic diseases such as right heart failure and obstruction of the biliary tract lead to changes in the elasticity of the liver, these confounders must be recorded and evaluated with appropriate measures. In contrast, the influence of severity of steatosis on liver stiffness measurement is low [186]. For detailed instructions on how to use the individual elastographic methods, please refer to the recommendations of the specialist societies [168] [169] [187]. In particular, manufacturer-specific recommendations and technology-adapted cutoffs must be observed [188] [189].

Analogous to steatosis quantification, MR techniques can also be used to assess liver fibrosis. Compared to the ultrasound methods listed above, MR elastography (MRE) had a somewhat higher diagnostic accuracy [176] [184] and, in combination with serum markers, showed a high positive predictive value [190].

Fibrosis scores

Commentary 

The diagnostic value of the NFS and FIB-4 index to exclude advanced liver fibrosis is comparable, whereby the FIB-4 is based on fewer parameters. The FIB-4 index is therefore easier to determine, less expensive and should be prioritized.

The NFS includes age, BMI, glucose intolerance/diabetes mellitus, platelet count, albumin and AST/ALT ratio, which can be calculated free of charge using http://nafldscore.com. In a meta-analysis of 64 studies with 13 046 patients, the diagnostic accuracy (AUC) of the NFS for the diagnosis of advanced fibrosis (F ≥ 3) was 0.84 [20]. The NFS takes into account a low and high cutoff value of < –1455 and> 0.676. With an NFS of < –1455, advanced fibrosis could be excluded with a sensitivity of 82 % (exploration cohort, n = 480) or 77 % (validation cohort, n = 253) and NPV of 93 % or 88 % [162].

The FIB-4 Index is a cost-free test that can be calculated via http://gihep.com/calculators/hepatology/fibrosis-4-score/ and consists of age, platelet count, AST and ALT. For the FIB-4 index in NAFLD, a low and high cutoff value of 1.3 and 2.67 was identified for the assessment of advanced fibrosis [163]. In a study of 541 NAFLD patients, a NPV of 90 % for excluding advanced fibrosis was found for FIB-4 with a cutoff value of ≤ 1.3 [163]. In the meta-analysis of 64 studies with 13 046 patients, the diagnostic accuracy of the FIB-4 score was comparable to the NFS (AUC = 0.84) [20]. As the specificity of FIB-4 and NFS markedly decreases from ≥ 65 years, an age-adapted cutoff value (from ≥ 65 years) was determined for both. This is 2.0 for FIB-4 and 0.12 for NFS for the exclusion of advanced fibrosis (F3/4). With these cutoff points, the specificity in older patients could be improved to 70 % without reducing sensitivity (FIB-4: Sensitivity 77 % at cutoff 2.0; NFS Sensitivity 80 % with cutoff 0.12). The power of both tests is also markedly limited in young patients < 35 years of age [115]. How far the age-adapted cut-off points ​​for FIB-4 and NFS need to be taken into account must be further evaluated, as their use in individuals ≥ 65 years increases specificity but clearly reduces sensitivity [191].

NFS and FIB-4 were compared to other scoring systems such as APRI (AST/platelet ratio index) and BARD score, BMI, AST/ALT ratio and diabetes mellitus, and for TE using VCTE and evaluated for diagnosing advanced fibrosis in NAFLD. In a multi-center and single-center study of 741 and 323 NAFLD patients, FIB-4 and NFS were superior to the BARD score [111] [192] and APRI [192]. In the multi-center study, a comparison with TE using VCTE (cutoff points: < 7.9 kPa and ≥ 9.6 kPa) was also carried out. TE was shown to be superior to non-invasive scores, including NFS and FIB-4, in predicting advanced fibrosis (AUC: TE = 0.86, NFS = 0.77 and FIB-4 = 0.79; NPV: TE = 94 %, NFS = 87 % and FIB-4 = 85 %). The rate of false positive results was higher for TE and the rate of false negative results was higher for NFS and FIB-4 [192]. In another comparative study with 245 NAFLD patients, the TE (cutoff points were: < 7.9 kPa and ≥ 9.6 kPa) significantly better for the diagnosis or exclusion of advanced liver fibrosis (AUC 0.93) compared to the AST/ALT ratio (AUC: 0.66), APRI (AUC 0.74), FIB-4 (AUC: 0.80), NFS (AUC: 0.75) and BARD score (AUC 0.69) [165]. A multicenter study of 452 NAFLD patients from France also demonstrated a higher diagnostic accuracy of TE (0.83) for the detection of advanced fibrosis compared to BARD (0.69), APRI (0.75), FIB-4 (0.78) and NFS (0.73) [110]. The superiority of TE using VCTE (n = 126; cutoff values: 8 kPa) compared to NFS (n = 233), FIB-4 (n = 243) and APRI (n = 243) to diagnose advanced fibrosis was further confirmed in a single-center NAFLD study from Germany (NPV: 97 % versus 92 %, 91 % and 90 %; sensitivity: 91 % versus 69 %, 69 % and 77 %) [93]. Recently, the Hepamet Fibrosis Score (HFS), which takes age, gender, HOMA-IR, diabetes mellitus, GOT, albumin and platelets into account, was evaluated in a multicenter cohort of 2452 NAFLD patients and compared to NFS and FIB-4. This score takes into account a low and high cutoff (0.12 and 0.47) for the exclusion or detection of advanced fibrosis (sensitivity 74 %, specificity 97 %, NPV 92 %, PPV 76 %) [193]. The HFS is freely available online (https://www.hepamet-fibrosis-score.eu) and does not require age-adjusted cutoffs. The HFS also showed a high diagnostic value in NAFLD patients with normal weight or normal transaminases and was superior to the NFS or FIB-4.

A blood-based marker panel (NIS4), consisting of HBA1c, alpha2-macroglobulin, YKL-40 and miR-34a-5 p, using a low and high cutoff point (< 0.36 and > 0.63), excluded NASH with significant NAFLD activity (NAS ≥ 4) and fibrosis (≥ F2) with a sensitivity of 81.5 % and an NPV of 77.9 % and confirmed it with a specificity of 87.1 % and a PPV of 79.2 % [194].

Non-invasive diagnostics of inflammatory activity

Commentary 

In pilot studies, MR-based technologies as well as a liver stiffness score, attenuation measurement and laboratory parameters showed good diagnostic characteristics for the non-invasive prediction of NASH. Measuring inflammatory activity remains a challenge for imaging techniques. Conventional US diagnostics do not offer any reliable diagnostic criteria for the detection of NASH [145]. Liver stiffness is modulated not only by fibrotic changes but also by inflammatory activity, although the inflammatory component is moderate in most patients. Therefore, this technology alone does not offer any possibility of further differentiating between fibrosis and inflammatory activity [145]. The additional determination of tissue viscosity during elastographic analysis (dispersion slope) is a new method that shows a clear correlation with lobular inflammation [195] [196].

An algorithm consisting of liver stiffness (surrogate of fibrosis), attenuation measurement (surrogate of steatosis) and AST was, in a multicenter, histology-controlled study, predictive of underlying NASH with significant NAFLD activity (NAS ≥ 4) and fibrosis (≥F2) [197]. This FAST score showed an NPV of 85 % (sensitivity 90 %) with a cutoff of ≤ 0.35 and a PPV of 83 % (specificity 90 %) with a cutoff of ≥ 0.67 %) for the exclusion or confirmation of NASH with NAS ≥ 4 and ≥F2. In bariatric patients, the FAST score correlated with the decrease in inflammatory activity [198].

In addition to the US-based methods, MR-based methods appear to be suitable for differentiating NASH and NAFL [145]. In particular, the determination of the “iron-corrected T1” (cT1) is a promising parameter [199] which, in combination with a liver function test, showed promising results [200].

Individual serum markers

Commentary 

Cytokeratin-18 (K18) fragments are released from apoptotic hepatocytes and can be detected in the blood (M30 ELISA) [201] [202] [203]. This biomarker for cell death has been evaluated in numerous international studies to assess disease activity in NAFLD [201] [203] [204] [205] [206] [207]. In a meta-analysis of 11 studies with 822 patients, the overall sensitivity and specificity of K18 fragments were 66 % and 82 % for the diagnosis of NASH [134]. Another meta-analysis of 9 studies and a total of 856 patients reported an overall sensitivity of 78 % (0.64–0.92) and specificity of 87 % (0.77–0.98) as well as a diagnostic value (AUC) of 0.82 (0.78–0.88) [208]. The detection of K18 fragments showed a close correlation to histological inflammation and hepatocellular ballooning and thus reflected the inflammatory liver damage in NASH [207] [209]. Blood levels for K18 fragments also correlated with fibrosis In NAFLD [207] [209] [210]. Here, an AUC of 0.82 [201], 0.86 [207], 0.93 [209] and 0.88 [205] was determined for the K18 marker. The high diagnostic value of K18 determined in these studies could not be proven in a study on predominantly Latin American NAFLD patients. This showed a sensitivity/specificity of 58 %/68 % and 54 %/85 % with a correspondingly lower AUC of 0.65 and 0.68 for the diagnosis of NASH and fibrosis [210]. A recently published multicenter study of NAFLD patients from Germany showed that when the K18 marker detected false-positive NASH, the majority of patients had an inflammatory activity of at least 1 in the NAS and vice versa; the majority of patients with a false-negative result for NASH showed little or no fibrosis when the K18 marker was used [211]. One limitation of the K18 marker is the lack of a uniform cut-off for the detection of NASH in patients with suspected NAFLD. In several studies, a cutoff value for K18 of around 200 U/L was determined, which enabled a distinction between NASH and NAFL with the best possible sensitivity/specificity [201] [206] [209] [210].

The consideration of the K18 marker in scores such as the CHek-Score [212] or MACK-3-Score [213] is less well evaluated in contrast to its use as an individual parameter for assessing NAFLD activity. The MACK-3 score takes into account K18, GOT and an insulin resistance determined by HOMA (Homeostasis Model Assessment). A MACK-3 score ≤ 0.134 or ≥ 0.550 showed a 90 % sensitivity and 94 % specificity for the diagnosis of fibrotic NASH (NAS ≥ 4 and fibrosis F ≥ 2) [213]. Since the scores mentioned were mainly evaluated in patients with pronounced obesity or metabolic syndrome, their diagnostic value in NAFLD patients without obesity or metabolic syndrome remains unclear at present. Due to the lack of widely available elastography methods in extended primary diagnostics, e. g. in intermediate NFS/FIB-4 (FIB-4: 1.3–2.67 or NFS: -1,455–0,675), the subsequent determination of K18 or K18-based scores can be helpful in identifying patients with possible fibrotic NASH [211]. As an alternative to K18, the above-mentioned NIS4 for detecting fibrotic NASH could be considered in extended primary diagnostics or in prescreening for therapy studies. The determination of this marker panel in everyday clinical practice cannot currently be recommended.

Evaluation of risk factors and comorbidities in NAFLD

Commentary 

Because of the increased risk of cardiovascular morbidity and mortality in NAFLD, cardiovascular risk stratification should be undertaken. Initially, this should be done using risk scores (e. g. HEART score) and detailed recording of risk factors. Existing diseases should be identified at an early stage and preventive and therapeutic measures initiated. Metabolic risk factors such as arterial hypertension, dyslipidemia and particularly (visceral) obesity and T2DM are associated with a higher prevalence of NAFLD [214]. The NAFLD prevalence increases with increasing BMI and was over 95 % in patients undergoing bariatric surgery [215] [216]. Conversely, in a large meta-analysis, the global obesity prevalence in NAFLD was 51.34 % (95 % CI: 41.38–61.20) and with NASH in 81.83 % (95 % CI: 55.16–94.28) [62]. The association between T2DM and NAFLD is bidirectional. On the one hand, the prevalence of NAFLD in T2DM is twice that of the general population. According to a meta-analysis of 80 studies with 49,419 people from 20 countries, it was 55.5 % globally (95 % CI: 47.3–63.7), with the highest reported prevalence of 68 % (95 % CI: 62: 1–73) in Europe [217]. On the other hand, T2DM favors the advanced forms of NAFLD. The global prevalence of NASH in T2DM was 37.3 % (95 % CI: 24.7–50.0 %) and 17 % (95 % CI 7.2–34.8) of patients with NAFLD and T2DM have advanced fibrosis (≥ F3) [217]. The prevalence of NAFLD is also increased in underlying arterial hypertension. A cross-sectional study from Brazil that included 5362 people with normal blood pressure, prehypertension (untreated systolic blood pressure 120–139 mmHg or diastolic blood pressure 80–89 mmHg) and hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg or antihypertensive medication) showed significantly different NAFLD prevalence rates of 16.5, 37.5 and 59.3 % (p < 0.001) [218]. On the other hand, the global prevalence of hypertension in patients with NAFLD and NASH was 42.54 % (95 % CI: 30.06–56.05) and 70.65 % (95 % CI: 54.64–82.79) [62]. Dyslipidemia, defined as an increase in serum triglycerides (TG) and LDL cholesterol with low HDL cholesterol, is another metabolic risk factor for an increased NAFLD prevalence. A large cross-sectional study from Taiwan with 44 767 individuals showed that those with the highest total cholesterol to HDL and TG to HDL ratios showed a NAFLD prevalence of 78 %, while the NAFLD prevalence for the lowest ratios was 33 % [219]. The global prevalence of dyslipidemia in NAFLD and NASH is 40.74 % (95 % CI: 30.80–51.50) and 83.33 % (95 % CI: 36.87–97.72) [62].

In addition, age, gender and ethnicity influence the prevalence of NAFLD. A population-based study with 2811 participants from the Netherlands, mean age of 76.4 years (65.3–98.7 years) showed a prevalence of NAFLD of 35.1 %, which decreased with increasing age [220]. Being male also seems to be a risk factor for NAFLD [221] [222]. However, there are also data that show a higher NAFLD prevalence in females [223]. Ethnicity and related genetic differences are also likely to influence the prevalence of NAFLD. A meta-analysis from the United States showed that Latin Americans had the highest, while African Americans had the lowest NAFLD prevalence. White Americans are placed in between [224]. Familial accumulation of NAFLD due to genetic factors has been observed, e. g. occurring more frequently in monozygous compared to bizygous twins [225] and in family members of overweight children [226]. A possible genetic factor is a polymorphism in the PNPLA3 gene (adiponutrin). Meanwhile, the association of PNPLA3 SNP rs738 409 (Ile148Met) with steatosis and the progression to NASH fibrosis, cirrhosis and HCC has been confirmed repeatedly [136].

NAFLD is associated with extrahepatic diseases; the odds ratios (OR) obtained from meta-analyses are given below: Chronic renal failure (OR 1.37; (95 % CI: 1.20–1.53)), obstructive sleep apnea (OR 2.37; (95 % CI: 1.59–3.51)), hypothyroidism (OR 1.42; (95 % CI: 1.15–1.77)) and psoriasis (OR 2.15; (95 % CI: 1.57–2.94)) [227] [228] [229] [230]. Further associations exist with osteoporosis (prevalence 3.6 % with NAFLD vs. 1.5 % without; p < 0.005) and polycystic ovary syndrome (PCOS) with an almost four-fold increase in NAFLD prevalence [231] [232].

Estimating prognosis and risk stratification

Commentary 

The majority of NAFLD cases show a strong association with obesity and T2DM [233] [234] [235] [236] [237] [238] [239] [240], with some studies also reporting a NAFLD prevalence in the normal weight population of 7 %–16 % [4] [241] [242] [243]. Corresponding to the obesity-related occurrence of NAFLD, there are further associations with various metabolic dysregulations, which are summarized in [Table 5] (Definition) and which together define the metabolic syndrome. The presence of NAFLD is an independent risk factor for cardiovascular disease. This association is further strengthened by the occurrence of NASH. However, both disease entities do not form a cardiovascular risk equivalent, so that an individual risk assessment should be carried out taking into account previous cardiovascular diseases, age, gender, cholesterol, blood pressure and lifestyle using validated risk scores (e. g. Heart-SCORE). Additional risk modifiers ([Table 5]) must be considered. In addition, family history of cardiovascular disease, the presence of subclinical atherosclerosis, and socioeconomic status should be considered in the risk assessment.

Differentiation of NAFLD from other liver diseases with steatosis

Table 6 Differential diagnosis of hepatic steatosis (strong consensus).

Differential diagnosis of hepatic steatosis
Genetic diseases including lipid metabolism disorders	Abetalipoproteinemia Hypobetalipoproteinemia Familiar hyperlipidemia Lipodystrophy Hereditary fructose intolerance LAL deficiency (cholesterol ester storage disease (CESD), Wolmannʼs disease) Wilsonʼs disease Glycogen storage disease See also Table 2 (Liebe et al., 2021) [249]
Nutrition-related causes	Hyperalimentation acquired lipid metabolism disorders Fatty liver as part of the metabolic syndrome Total parenteral nutrition Malnutrition Acute weight loss (bariatric surgery, fasting) Pancreatectomy
Pregnancy	Acute fatty liver of pregnancy (AFLP)
Medications	Microvesicular steatosis:  Including, but not limited to valproic acid, tetracyclines, nucleoside analogs, acetylsalicylic acid, didanosine, stavudine, MDMA (amphetamines) Macrovesicular steatosis (+/– steatohepatitis):  Including, but not limited to amiodarone, tamoxifen, methotrexate, corticosteroids, anti-retroviral therapy, irinotecan, spironolactone, sulfasalazine
Endocrine causes	Type 2 diabetes Hypothyroidism Growth hormone deficiency Pituitary insufficiency Adrenocortical tumors Polycystic ovary syndrome (PCOS)/hyperandrogenism Estrogen deficiency/menopause Male hypogonadism
Chronic hepatitis C virus (HCV) infection	Specifically HCV genotype 3
Small intestinal disease	Celiac disease Bacterial overgrowth of the small intestine Short bowel syndrome (anatomical, functional)
Environmental factors, noxae, toxins	See also Table 1 (Liebe et al. 2021) [249]
Idiopathic diseases	Weber-Christian syndrome

Commentary 

NAFLD is associated with various comorbidities. These include cardiometabolic diseases, polycystic ovarian syndrome (PCOS) and sleep apnea syndrome. It has also been shown that subclinical hypothyroidism or low normal thyroid function is associated with progressive NAFLD [244]. Low-normal thyroid function was associated with increased all-cause and cardiovascular-related mortality [245]. Hypothyroidism contributed to triglyceride synthesis and insulin resistance and thus enhanced the development of NAFLD [246] [247]. In PCOS patients it could be shown that the apoptotic cell death detected by K18 fragments was associated with insulin resistance and a high NASH prevalence [248].

Differentiation between NAFLD and ALD

Commentary 

ALD is characterized by an increased GOT/GPT ratio (as long as there is no cirrhosis) and γGT as well as increased MCV and ferritin, which can be considered in the differential diagnosis to NAFLD However, ALD cannot be proven by an increase of these laboratory parameters. ALD cannot be excluded with certainty using individual methods. The clinical history and structured questionnaires such as the CAGE test or AUDIT (-C) questionnaire on alcohol consumption are important [250] [251]. The threshold dose for liver-toxicity is different for each individual and statistically defined and threshold values ​​do not guarantee that a patient would not sustain liver damage from very low alcohol consumption. Alcohol sensitivity is influenced, among other things, by genetics, ethnicity and gender. According to the European and American guidelines, the upper limit of 20 and 30 g alcohol per day is set for women and men, respectively [169] [252]. Both the Asian and German S2k Guidelines stipulate stricter upper limits of 10 and 20 g alcohol per day for women and men, respectively, also taking into account the carcinogenic effects of alcohol [52] [253].

Although there are slight histological differences between NAFLD and ALD, histology does not allow a reliable differentiation between the two diseases [254]. This also applies to hepatic iron overload that can occur in both diseases [54]. There are typical changes in the transaminases, depending on the etiology and the stage of fibrosis [255]. In a direct comparison of 30 patients with ALD and NAFLD (each matched for gender, fibrosis stage and age), there were differences found between the two diseases [55]. In this study, the γGT, GOT, ferritin and MCV were significantly higher in ALD, as expected, but the Hb was lower. In NAFLD, glucose and BMI were higher but not significantly. For differentiating between NAFLD and ALD, the NAFLD/ALD index was developed, which takes MCV, AST/ALT ratio, BMI and gender into account [256]. This score also requires prospective validation in larger cohorts in order to check the diagnostic value for its use in everyday clinical practice. Carbohydrate-deficient transferrin (CDT) can be detected in higher alcohol consumption (at least 50 g/day over a period of 1–2 weeks), but is not specific for the diagnosis of ALD and can be false negative in liver cirrhosis. The sensitivity and specificity for the detection of CDT can vary considerably, depending on the assay used 250]. Ethyl glucuronide (EtG), on the other hand, has a high specificity as a direct marker for detecting alcohol and can be found in the urine for up to 80 hours after alcohol consumption. With a cutoff for uEtG of 0.1 mg/L, substantial alcohol consumption can be excluded. EtG can be detected in the hair for even longer, with the detection reflecting alcohol consumption of one month per 1 cm of hair. With a cutoff of < 7 pg/mg, general alcohol abstinence can be assumed [250]. Measuring EtG is costly and is reserved for specific situations, such as the evaluation for listing patients for a liver transplant, which requires long-term abstinence from alcohol.

4. Treatment

4a Non-pharmaceutical therapy

Commentary 

Weight loss in overweight or obese NAFLD patients leads to a regression of steatosis [116] [257] [258] [259]. The reduction in steatosis and ALT is proportional to the weight loss; there is a clear dose-effect relationship [260] [261] [262]. In this context, it is irrelevant how the weight loss was achieved [32] [116] [257] [258].

The evaluation of paired liver biopsies from NASH patients before and after weight reduction show that a weight loss of at least 10 % should be achieved in order to accomplish regression of fibrosis and complete regression of NASH [263] [264] [265] [266] [267] [268] [269] [270] [271] [272] [273] [274]. Similar results were also found by systematic reviews [275] and guidelines [32] [116] [258] [276]. They also show that a minor weight loss primarily leads to an improvement in steatosis and transaminase levels [269] [272] [275] [276] [277] [278] [279] [280]. A controlled trial showed that 50 % of normal-weight NAFLD patients achieved a remission of steatosis with a weight reduction by 3–5 % [281].

In overweight and obese patients with liver cirrhosis, a 16-week lifestyle intervention with a low-calorie diet and aerobic exercise led to a significant decrease in body weight and portal hypertension; here, a weight reduction of at least 10 % was associated with a 23 % decrease in the hepatic-venous pressure gradient (HVPG) [282]. Regarding the regression of an already existing NASH cirrhosis or the prevention of disease progression with the development of HCC, no results from studies on lifestyle intervention are available to date. All in all, a weight reduction of at least 10 % is extremely effective in the treatment of NASH (90 % cure rate), but this target was only achieved by 10 % of patients in clinical practice [275]. Concepts such as web-based training [283] [284], text messaging [285] or increasing motivation through donations for charitable purposes [286] are new approaches to solving this dilemma.

Physical exercise should be carried out to reduce hepatic steatosis and to potentiate the effect of weight loss on the inflammation. Improvement in the necroinflammatory response has not yet been proven. Measuring liver fat using 1H-MRS shows that aerobic exercise without body weight changes led to a decrease in the hepatic fat content [287] [288] [289] [290]. Meta-analyses showed that aerobic training and/or isometric training in NAFLD patients also improved transaminase levels and hepatic fat content independently of weight loss [261] [291] [292] [293] [294]. Both training concepts are apparently equally effective [261] [292] [294].

Commentary 

The rationale for weight loss is an improvement in the risk of comorbidity, transaminase activity and liver histology (necroinflammation); a Mediterranean diet can improve steatosis and insulin sensitivity. Overweight or obese NAFLD patients should be advised about a low-calorie diet in accordance with the guidelines for obesity management (S3 Guideline “Prevention and Therapy of Obesity” AWMF Register No. 050–001) [258] [295] [296]. The caloric target is 1200 kcal/day for women and 1400–1500 kcal/day for men, corresponding to a reduction by –500 to –1000 kcal/day [276]. The combination of a low-calorie diet with aerobic or isometric exercise has a synergistic effect and increases effectiveness in terms of improving steatosis and necroinflammatory activity [268] [269] [279] [297] [298]. When the energy balance was changed to the same extent by either a low-calorie diet alone or by a combination of less restrictive diet and physical activity, the participants in a systematic study achieved the same weight loss (-10 %) and the same improvement in transaminase activity, liver fat and insulin sensitivity [299]. Both interventions are effective on their own if the other variable – weight or physical activity – is kept constant. Aerobic training exercise without changing body weight led to a decrease in hepatic fat content [287] [288] [289] [290], as did weight reduction with a low-calorie low-carb or low-fat diet while maintaining an inactive, sedentary lifestyle [280]. NAFLD patients exhibit a low level of physical activity, in diabetic NAFLD patients, physical activity ranks in the lowest quartile [300]. Overweight or obese NAFLD patients show a reluctance to make lifestyle changes; only 10 % actively deal with the subject [301].

The study shows that a specific fat or carbohydrate composition of a low-calorie diet had no advantages with regard to weight reduction or improvement in transaminase activity or histological changes in NAFLD [32] [258] [276]. This also applies to formula diets, referred to as very low-energy diets (VLED), as meal replacement [302] [303]. Consuming a VLED (800 kcal/d), more than 80 % of a Munich cohort achieved a weight loss of at least 10 % in 52 weeks, accompanied by significant improvements in transaminase activity, FLI and NAFLD Fibrosis Score [304]. A high protein diet may be beneficial. In obese patients with T2DM, an isocaloric protein-rich diet led to an improvement in steatosis, insulin sensitivity and BMI after 6 weeks [305].

The rapidly increasing prevalence of obesity over the past few decades has been associated with the increasing consumption of fructose and fructose-containing corn syrup in processed foods and beverages [306] [307] [308] [309]. However, meta-analyses did not show that fructose consumption in the context of a normocaloric diet promotes the development or progression of NAFLD [310] [311] [312]. In a double-blind study on overweight people, excessive caloric intake, but not fructose compared to isocaloric amounts of glucose, was associated with an increase in the hepatic fat content and transaminases [313].

The results of seven interventional [272] [314] [315] [316] [317] [318] [319] and four observational studies [320] [321] [322] [323] suggest that a Mediterranean diet (MD) has beneficial effects on body weight, insulin sensitivity and hepatic steatosis. However, the data on the preventive effectiveness of MD with regard to the development of NAFLD is less clear [116] [275] [324] [325]. Data from the Framingham study show a reduced risk of new NAFLD disease in people with high adherence to MD {Ma, 2018 #332. In this context, a high-quality diet like MD was particularly effective when genetic risk factors were present. A Greek study found no association between MD adherence and the presence of NAFLD, but a negative correlation between MD adherence on the one hand and insulin resistance, transaminases, liver stiffness and histologically diagnosed steatosis and fibrosis on the other [321]. MD lowers the risk of cardiovascular disease and new onset T2DM, where obesity and insulin resistance play roles as etiological factors [326]. Compared with general dietary recommendations, an MD improves insulin sensitivity and steatosis even without weight loss [316]. In the CENTRAL study, MD was superior to a low-fat diet in terms of fat mobilization from the liver, heart and pancreas determined by whole-body MRI [327].

Compared with metabolically healthy individuals, the risk of mortality and cardiovascular events is more than threefold in normal weight patients with an underlying metabolic disease – a condition that affects around 20 % of the normal weight population [328] [329]. A controlled study of normal weight (BMI 22.7 kg/m²) NAFLD patients in Hong Kong showed that a low-calorie diet, achieving a weight reduction of 3–5 %, leads to remission of NAFLD in 50 % (measured by the hepatic fat content using ¹H-MRS) [281]. Other types of diet have yet to be evaluated [330] [331].

Aerobic or isometric exercise can reduce hepatic fat content and insulin resistance [287] [288] [289] [290] [332] [333]. It therefore seems logical to recommend these types of exercise to normal-weight NAFLD patients in order to improve steatosis and insulin sensitivity. A meta-analysis concluded that both types of exercise are equally effective with regard to hepatological endpoints, while isometric exercise proves less stressful for people with poor cardiorespiratory fitness [294]. The median effective aerobic exercise level was 4.8 MET (metabolic equivalent) provided in three 40-minute training units per week and 3.5 MET in three 45-minute units per week for isometric exercise [294].

According to WHO exercise guidelines of November 25th, 2020, published at https://www.who.int/publications/i/item/9789240015128, patients with NAFLD and a BMI > 20 and < 25 kg/m² should practice a minimum of 150 to 300 minutes of moderate-intensity aerobic physical activity or at least 75–150 minutes of high-intensity aerobic physical activity per week. Alternatively, an equivalent combination of medium and high intensity activity during the week can also be used.

Stimulant foods (alcohol, tobacco, coffee)

Commentary 

Retrospective studies that showed a favorable effect of moderate alcohol consumption on health must be assessed critically, as they only examined associations and not causalities [334] [335] [336]. In addition, prospective data from animal experiments clearly showed a negative effect of alcohol on, for example, diet-induced fatty liver [337] [338] [339] [340]. This could also be observed in NAFLD patients who, because of alcohol consumption, showed accelerated fibrosis progression [341]. Finally, a retrospective study showed that patients with NASH cirrhosis who also consume alcohol in small quantities have a significantly higher risk of developing HCC [342]. Alcohol consumption is a meaningful risk factor for the development of liver cirrhosis [343] and, especially in advanced stages of the disease, therefore, social alcohol consumption should be abstained from completely. In these instances, absolute abstinence should be recommended. In a small cohort analysis, patients with moderate, regular alcohol consumption (< 140 g/week) were more likely to have an advanced stage of fibrosis, especially those with T2DM [344].

Commentary 

Systematic reviews and meta-analyses suggest that drinking coffee leads to a reduction in the risk of HCC. Consuming larger amounts of coffee resulted in a higher risk reduction [345] [346]. However, increased coffee consumption is not associated with a reduced risk of hepatobiliary carcinoma [347]. The protective agents from coffee and the molecular mechanisms of HCC prevention have so far remained unclear.

Positive effects with regard to coffee consumption can be derived from epidemiological studies [348]. These showed a protective effect of coffee consumption in relation to the risk of developing NAFLD and also in relation to the fibrosis stage [349], but no controlled studies are available on this topic. In a pooled meta-analysis with a total of 11 studies, people who drank coffee had a relative risk of 0.77 (95 % CI 0.60–0.98) to develop NAFLD. In addition, there is also a significantly reduced risk of advanced liver fibrosis compared to patients who do not drink coffee (RR 0.68; 95 % CI 0.68–0.79) [349].

Commentary 

Patients with chronic liver disease fall into a risk group. According to the STIKO recommendations, all patients with chronic liver disease should be vaccinated against hepatitis A, hepatitis B, influenza and COVID-19. Patients who are awaiting organ transplantation or immunosuppressive therapy and immunosuppressed patients, e. g. those with liver cirrhosis, should be vaccinated against pneumococci. Vaccination against varicella is recommended for seronegative patients prior to any planned immunosuppressive therapy or organ transplantation. Vaccination with live vaccines is contraindicated after liver transplantation. Vaccinations should be carried out in accordance with the latest STIKO guidelines (www.rki.de/epidbull).

4b Drug therapy

Drug therapy for NAFLD regardless of comorbidities

Commentary 

The use of antioxidants such as vitamin E in patients with NAFLD fibrosis (≥F2) at a dose of 800 IU/day resulted in a histological reduction in steatosis and inflammation for two years without improving fibrosis [350]. Supplementation with vitamin E cannot be recommended, as some meta-analyses reported an increased all-cause mortality on long-term vitamin E treatment, especially at doses > 400 IU/day [351] [352] and an increased rate of prostate cancer in men [353]. Other dietary supplements such as omega-3 fatty acids, silymarin, polyphenols or drugs such as ursodeoxycholic acid (UDCA) and pentoxifylline did not produce any significant histopathological improvements in patients with NAFLD and can therefore not be recommended for treatment (see Appendix Table 4b-1; randomized controlled trials of off-label drugs and nutritional supplements). Pioglitazone was also evaluated in NAFLD patients without T2DM, for example in the PIVENS study [350]. Pioglitazone improved liver histology, steatosis, ballooning and lobular inflammation, but not fibrosis. In addition, pioglitazone cannot be recommended due to its side effect profile including significant weight gain, increased risk of bone fractures and, rarely, heart failure.

Regarding dietary supplements, most evidence available is on the use of omega-3 fatty acids and other polyunsaturated fatty acids. The WELCOME trial showed no effect on fibrosis stage [354] and a decrease in liver fat content was only found in subgroups [355]. Even smaller studies on omega-3 fatty acids [356] or omega-3 polyunsaturated fatty acids (3 PUFAs) [357] could not find any effect on the liver histology. Meta-analyses showed an improvement in transaminases only after 12 months alongside a decrease in liver fat content [358] [359]. In one study on overweight men, those in the subgroup with increased liver fat content showed no reduction of hepatic steatosis measured by MRI after 12 weeks [360]. Data from randomized trials on supplementation with trace elements in NASH are not available. The NHANES III study showed a lower mortality rate and lower non-invasive markers of fibrosis in NAFLD patients who had elevated serum selenium levels [361]. Controlled data on phytotherapy or “hepatoprotective substances” are scarce. A trial from Hong Kong compared Phyllanthus urinaria with placebo over a period of 24 weeks and found no effect on histological inflammation, obesity or fibrosis [362]. In a randomized controlled trial with histological endpoints, silymarin showed no effects on NAFLD [363]. Few clinical trials are available on vitamin D supplementation. Reduced liver values ​​were found over a period of 48 weeks [364]. These effects could not be observed in patients with T2DM [365]. A randomized trial on the supplementation with probiotics combined with prebiotics for one year showed no effect on non-invasive markers in NAFLD. Short studies have observed an effect on the liver enzymes ​​after 12 weeks [366]. Studies on the use of prebiotics, synbiotics and probiotics are limited to small case numbers and mostly only examined liver function tests and ultrasound over a limited period of time [367]. In a randomized trial, combined probiotics and prebiotics supplementation showed a change in the microbiome, but no benefit on liver fat content or liver stiffness as a surrogate for liver fibrosis [368]. In lean NAFLD patients (n = 50), synbiotics showed a benefit in terms of improving non-invasive surrogate markers of hepatic steatosis and fibrosis over 28 weeks [369]. Data on fecal microbiota transplant are not available [370].

See Appendix Table 4b-1

Drug therapy for NAFLD patients with diabetes

Commentary 

GLP1 analogues are only approved in combination with metformin (or as monotherapy in the case of metformin intolerance) for the treatment of T2DM. The 2020 national care guideline for T2DM suggests a combination therapy of metformin + SGLT2 inhibitors or GLP-1-RA for type 2 diabetes with cardiovascular risk factors and metformin monotherapy for type 2 diabetes without risk factors. The National Disease Management Guideline Type 2 Diabetes (long version, 2nd edition) is available: https://www.awmf.org/uploads/tx_szleitlinien/nvl-001l_S3_Typ_2_Diabetes_2021-03.pdf.

A placebo-controlled study with 52 NASH patients, 33 % of whom had T2DM, showed more resolution of NASH and less fibrosis progression after one year of liraglutide therapy [371]. Treatment with semaglutide for NASH and NASH fibrosis stage F1–F3 (62 % of the patients had T2DM) was associated with a significantly more frequent resolution of NASH, but without significant improvement in fibrosis [372]. The daily injections tested in this phase 2 study, however, correspond to a higher dose than is currently approved in Germany for the treatment of T2DM (in combination with metformin). In addition, GLP-1 analogues showed positive effects in cardiovascular endpoint studies (3P-MACE, cardiovascular death, non-fatal stroke, hospitalization due to heart failure and overall mortality) and have comparatively few contraindications, e. g. underlying or increased risk of pancreatitis, pregnancy or breast feeding. The European Medicines Agency (EMA) has approved semaglutide 2.4 mg/day for the management of obesity (BMI > 30 kg/m² or > 27 kg/m² with serious comorbidities), in conjunction with hypocaloric diet and physical activity, in January 2022.

Therapy with sodium dependent glucose transporter 2 (SGLT2) inhibitors showed a significant improvement in the liver fat content in patients with NAFLD and type 2 diabetes [373] [374] [375] [376]. Data from randomized controlled studies on the effect of SGLT2 inhibitors on liver histology are currently not available. SGLT2 inhibitors also show positive effects in cardiovascular and renal endpoint studies. The main side effects are genitourinary infection, dehydration and masking symptoms and clinical findings of diabetic ketoacidosis. The lack of histological data explains the overall lower strength of recommendation for SGLT2 inhibitors.

There are also a number of previous studies on the use of pioglitazone in patients with NASH who have either impaired glucose tolerance or T2DM. In a 6-month placebo-controlled study with a reduced-calorie diet, pioglitazone achieved a greater reduction in liver fat content and a significant improvement in NASH (hepatocellular ballooning and lobular inflammation) but not in fibrosis [377]. In an 18-month placebo-controlled study, based on a low-calorie diet of patients with NASH and prediabetes or T2DM, with a subsequent 18-month open-label follow-up, therapy with pioglitazone showed a greater reduction in liver fat content, a more frequent resolution of NASH as well as a greater improvement in fibrosis [378]. However, therapy with pioglitazone is contraindicated in several conditions, particularly in heart failure (NYHA I-IV) and in bladder cancer. Caution is advised in individuals with increased bone fracture risk and severe obesity, as pioglitazone promotes weight gain. These safety concerns justify the overall lower recommendation strength for pioglitazone.

There is currently insufficient experience on the possible use of GLP-1 receptor agonists, SGLT2 inhibitors or pioglitazone in patients with NASH-associated liver cirrhosis. SGLT2 inhibitors may require dose adjustment or discontinuation in case of impaired kidney function.

Other antidiabetic agents such as metformin, dipeptidyl peptidase IV inhibitors or insulin have so far not shown any specific advantages for the treatment of NAFLD. However, large retrospective studies have reported that metformin reduced the risk of developing hepatocellular carcinoma (HCC) in NAFLD patients [379]. Even in patients with NASH-associated Child A stage compensated cirrhosis, the use of metformin for treating diabetes is associated with a reduced risk of hepatic decompensation and HCC; it can therefore be used in compensated liver cirrhosis in a dose of up to 2 g/day, if the renal function is normal {Vilar-Gomez, 2021 #388} [380]. Metformin is contraindicated if the glomerular filtration rate (GFR) is below 30 ml/min. However, there are no prospective controlled studies on the use of metformin in liver cirrhosis.

A placebo-controlled study of patients with NASH and T2DM showed a greater reduction in liver fat content for vitamin E (800 IU/day) and a more frequent reduction in NASH without improvement in fibrosis [381]. The increased mortality and morbidity risk in vitamin E supplementation (see above) limits its use, particularly in patients with diabetes mellitus. In contrast, large studies in patients with T2DM show cardiovascular benefits with pioglitazone [382], liraglutide [383], semaglutide [384] and the SGLT2 inhibitors, such as empagliflozin [385] and dapagliflozin [386], so that these drugs should preferably be used for patients with T2DM.

Drug therapy for lipid metabolism disorders

Commentary 

There are no high-level studies that have investigated the treatment of NAFLD in lipid metabolism disorders. In underlying NAFLD, lipid metabolism disorders such as familial hypercholesterolemia, hypertriglyceridemia, lipoprotein (a) elevation or isolated HDL cholesterol reduction should be treated effectively, as they present a substantially increased risk for cardiovascular diseases; also, NAFLD increases the risk of cardiovascular diseases, independent of lipid metabolism disorders [233] [387]. There are no controlled studies showing the effectiveness of lipid-lowering agents on liver histology in NAFLD. In large cohorts, the use of statins in NAFLD was associated with a lower risk of liver disease progression [388] [389] [390]. Hepatotoxic side effects seem to occur very rarely, even when statins are used in patients with decompensated cirrhosis [391]. Statins also appear to have other benefits in cirrhotic patients. Clinical observations have shown a reduced risk of HCC [392] as well as a reduction in portal hypertension, improved endothelial dysfunction and reduced fibrogenesis [393].

Drug therapy for obesity

Commentary 

If GLP-1 receptor agonists (e. g., liraglutide, semaglutide) or orlistat use is indicated, the treatment can also have a beneficial effect on NAFLD or histologically confirmed NASH. Such data are not available for other approved weight-loss drugs. Although there are no approved drugs for the treatment of NAFLD in obesity, clinical trials in patients with NASH show a beneficial effect of treatment with glucagon-like peptide 1 (GLP-1) receptor agonists, of which semaglutide and liraglutide are approved in the EU for treating obesity [394]. However, there are no studies for GLP-1 agonists that exclusively included NAFLD patients with obesity. In a placebo-controlled study (n = 52) in patients with NASH, with a mean BMI of ⁓35 kg/m², one-year treatment with liraglutide of 1.8 mg/day s. c. more frequently produced a resolution of NASH and at the same time a less frequent progression of the fibrosis [371]. A current study of NASH examined the use of semaglutide, which has been approved in Germany for the treatment of T2DM as well as obesity. Semaglutide was used in patients with NASH and stage F1–F3 fibrosis, who had a mean BMI of ⁓36 kg/m². Therapy with semaglutide was associated with more frequent resolution of NASH without significant improvement in fibrosis [372]. The daily injections tested in this phase 2 study, however, correspond to a higher dose than for the treatment of T2DM (in combination with metformin). In addition, GLP-1 analogues showed beneficial effects in cardiovascular endpoint studies and have comparatively few contraindications (see above).

The drug orlistat, which is approved for the treatment of obesity, also showed positive effects on the course of NASH. In a placebo-controlled study (N = 50) over 36 weeks in patients with NASH and a BMI ≥ 27 kg/m² (mean BMI 36 kg/m²), who were all on a reduced-calorie diet and received 800 IU of vitamin E/day, orlistat therapy improved steatosis, ballooning and inflammation (but not fibrosis), particularly if the weight loss was ≥ 9 % [268]. Such data (favorable influence on NAFLD) are not available for other approved weight-loss drugs.

In addition to these treatment options, the fixed-dose combination of bupropion and naltrexone is also approved for weight reduction in Germany. A recently published post-hoc analysis of the approval data, which was financed by the approval holder, shows minor, clinically irrelevant, improvements in the ALT and the FIB-4 index. It should be noted that a heterogeneous patient group was included with the aim of weight reduction. The study did not aim to include patients with liver changes [395]. The validity of this analysis is therefore low. In the USA, the fixed-dose combination of phentermine and topiramate as well as lorcaserin are also approved for the treatment of weight loss. There is no data for either on the effect of NAFL or NASH [396].

To what extent does liver dysfunction in NAFLD influence therapy with statins, antihypertensives, antidiabetic drugs, anticoagulants and platelet aggregation inhibitors that are, or must be administered, for other indications?

Commentary 

A variety of enzymes and drug transporters are active in the liver metabolism. Their specific interaction has a decisive influence on the pharmacokinetics of drugs with hepatic metabolism and/or high biliary elimination. Animal and human studies on the changes in gene expression, protein expression and enzyme and transporter activity in NAFLD/NASH have been carried out for the most relevant enzymes of the cytochrome P450 family, the glucuronyl-, sulfo- and glutathione transferases, the influx transporter of the OCT (organic cation transport), OAT (organic anion transport) and OATP (organic anion transporting polypeptides) and the efflux transporter P-glycoprotein, BCRP (breast cancer resistance protein) and the multidrug resistance-associated protein (MRP) [397]. The changes appear to be more pronounced in NASH than in NAFLD. These data are not conclusive, mainly due to the small and heterogeneous database. The dose adjustment of a specific drug, however, cannot be derived from these data. The attempt to use physiologically based pharmacokinetic modeling (PBPK) to predict the kinetics of an individual drug on the basis of pharmacokinetic data in a specific patient population is possible in individual cases, but this also does not allow any general recommendations for clinical practice [398]. In these studies, as well as in those where the change in the area under the plasma drug concentration–time curve was determined for patients with NAFLD or NASH, deviations of ± 30–50 % or less were found [399] [400] [401]. Ultimately, the decision to adjust the dose of a drug, undergoing hepatic metabolic elimination and/or biliary elimination, remains an individual decision. Therapeutic drug monitoring is useful for decision-making, but is not established for many drugs or is not available in routine clinical practice. The focus should therefore be on drugs with a narrow therapeutic window and/or life-saving treatment. In this case, drug monitoring should also be carried out even the information for healthcare professionals does not explicitly recommend it.

Future pharmacological interventions

Commentary 

At present, the use of novel NASH-specific drugs in fibrosis stage F1 or F2 cannot be assessed conclusively. The benefits and risks of using newly approved NASH-specific drugs in fibrosis stage F4 are currently unclear. New therapeutic approaches that are currently being investigated in clinical trials are very promising and oftentimes focus on advanced fibrosis (F3). So far, however, there has been no scientific evidence that these substances improve long-term outcomes (survival, cardiovascular events, cancer, liver-related complications). As a surrogate for such long-term outcome data, the European Medicines Agency as well as the US Food and Drug Administration accept a significant improvement in liver histology as a result of the intervention, compared to a comparative treatment (currently placebo) for a “conditional approval”. It is required that in the follow-up biopsy either the histological features of NASH such as ballooning and inflammation have resolved without worsening of the fibrosis (NASH resolution) and/or the liver fibrosis has improved by at least one severity stage without worsening of the NASH characteristics (fibrosis improvement) [402] [403]. The most important aspect is the significant reduction in the (prognostically relevant) hepatic fibrosis resulting from NASH-specific drug treatment. Since these endpoints are clinically plausible and scientifically accepted, patients with an appropriate risk constellation, i. e. in particular with advanced bridging fibrosis (F3) and/or high disease activity and/or severe cardiometabolic risk factors, should preferably be included in clinical trials that investigate these endpoints. Even if the patients only receive placebo, they generally benefit from the close monitoring and lifestyle advice, as can be derived from the “placebo response rates” for the histological endpoints of 15–35 % [404].

A number of substances are currently being investigated in clinical phase 3 and phase 2 studies [405] [406] [407] [408] that act on the pathophysiological processes of glucose metabolism, inhibition of de novo lipogenesis, inflammation or fibrogenesis. The substance classes include agonists of the nuclear receptors FXR (or its downstream mediator fibroblast growth factor/FGF19) and PPAR, chemokine receptor (CCR) inhibitors, thyroid hormone receptor-ß (THR-ß) agonists, inhibitors of lipogenic key enzymes such as FASN and SCD-1 Enterohepatic hormones and their agonists such as glucagon-like-peptide-1 (GLP-1), FGF19 or FGF21. Medicines with a primarily antidiabetic effect such as the group of SGLT2 inhibitors should also be mentioned here.

Obeticholic acid (FXR agonist), resmetirom (THR-ß agonist), lanifibranor (PPAR agonist), semaglutide (GLP-1 receptor agonist) and aramchol (SCD-1 inhibitor) are currently being tested in phase 3 studies) (Appendix, Table 4b-2). An interim analysis showed positive data for obeticholic acid with regard to fibrosis improvement as a co-primary endpoint (REGENERATE Study) [409]. Several substances such as elafibranor (PPARα/δ agonist), cenicriviroc (CCR2/5 inhibitor) and selonsertib (ASK1 inhibitor) did not demonstrate positive efficacy results in phase 3 and are therefore no longer being developed for this indication. Further FXR agonists (tropifexor, cilofexor), recombinant FGF19 (aldafermin), different variants of FGF21 (pegbelfermin, efruxifermin), GLP-1 analogues (liraglutide, semaglutide), pan-PPAR agonists (lanifibranor) showed promising results in phase 2 studies (Appendix, Table 4b-2). Based on the current phase 3 interim analysis, obeticholic acid is the only drug with a significant benefit on fibrosis improvement and is the primary candidate for the first conditional approval; however, this was not granted at the time the guidelines were published [410].

In the future, NASH therapies will possibly consist of a combination of two or more drug classes with complementary effects in order to achieve an optimal therapy response. Such combination treatments are already being investigated in clinical trials (e. g. tropifexor plus cenicriviroc, semaglutide plus FXR agonist); most combinations contain at least an FXR agonist (Appendix, Table 4b-2). No statement can currently be made about additive or synergistic pharmacological effects due to the small number of cases available in phase 2 data [407] [408].

It is currently unclear, which patients will be the target population of future NAFLD treatment. Some of the recently conducted phase 3 studies included patients with stage F1 fibrosis and underlying risk factors, while others defined at least F2 or even exclusively F3 as the target population. Primarily, patients with more advanced fibrosis should be considered with a high degree of urgency because liver-associated as well as extrahepatic mortality are significantly increased [411]. It remains to be clarified to what extent patients with earlier stages of fibrosis should receive specific drug therapy or only those with the immediate highest risk of progression in F3. Presently, there are only very few study data (phase 2 or 3) available for pharmacological therapies in NASH cirrhosis [402].

Future personalized therapy concepts are to be expected. This could consist of a targeted “correction” of the intestinal microbiota to reduce NAFLD and cardiometabolic comorbidities [412] or targeted therapies based on genetic risk stratification. The classic example of this would be the single nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, which codes for the missense mutation I148 M. Targeted drugs (e. g. antisense oligonucleotides, tyrosine kinase inhibitor momelotinib) could inhibit PNPLA3 levels in 148 M homozygous persons and thus modify a pathomechanism for progression [413] [414].

See Appendix, Table 4b-2

4c Interventional therapy approaches

Indications for bariatric surgery

Commentary 

Bariatric surgery has been proven to be the most effective therapy for morbid obesity. Furthermore, bariatric surgeries usually lead to an improvement and often to a complete remission of obesity-associated secondary diseases as well [415]. According to the current German S3 guideline of the DGAV from 2018, bariatric surgery is indicated in severe obesity with a BMI ≥ 40 kg/m² (even without concomitant diseases), if conservative weight reduction measures (diet change, exercise and possible behavioral therapy) have failed. Moreover, this procedure should be offered to patients with a BMI ≥ 35 kg/m² and at least one major obesity-related concomitant disease such as NAFLD and NASH, once conservative weight reduction measures alone have failed [415].

It should be noted that NAFLD and NASH are frequent comorbidities in obese patients [75]. Various studies and a current meta-analysis show that bariatric surgery led to high remission rates of NAFLD and NASH in these patients [271]. The highest-quality data come from the “Lille Bariatric Cohort”, which showed a high remission rate in histologically confirmed NASH over five years. The study moreover showed that bariatric surgery could lead to long-term improvement in existing liver fibrosis, even though fibrosis progression occurred in a small percentage of patients [416]. This observation is highly relevant since fibrosis is considered the most important risk factor for the progression of NAFLD and NASH to cirrhosis or HCC [18] [69]. Registry-based data suggest that bariatric surgery reduces the risk of HCC and progression to cirrhosis [417] [418]. Finally, a cost-benefit analysis showed an advantage for bariatric surgery, especially in NASH [419].

Although there are studies that demonstrate the positive effects of weight reduction even in normal weight NASH patients [420] and despite the excellent surgical results obtained in prospective and retrospective cohort studies on patients with BMI ≥ 35 kg/m², it cannot generally be recommended to offer metabolic surgery for NAFLD and NASH to patients with BMI < 35 kg/m² and failed conservative therapy. The reasons for this are that the data only stem from non-randomized trials and that no prospective data exist on the effects of metabolic surgery for patients with BMI < 35 kg/m² and NAFLD and/or NASH. However, a recently published network meta-analysis suggests that bariatric surgery is a more effective therapeutic option than drug therapies [421]. Future studies should compare metabolic surgery directly with the most effective drug therapy and in this way investigate the value of metabolic surgery in NAFLD and NASH patients with a BMI < 35 kg/m².

In patients with established liver cirrhosis, bariatric surgery should only be performed in the compensated cirrhosis stage. Mosko et al. have shown that mortality in decompensated cirrhosis and/or previous bleeding increases remarkably due to portal hypertension (compensated cirrhosis: 0.9 %; decompensated cirrhosis: 16.3 %) [422]. Overall, the risk of perioperative complications in patients with compensated liver cirrhosis is markedly increased, but still within an acceptable range [423] [424] [425]. Patients with liver disease and signs of portal hypertension should undergo extensive diagnostic evaluation preoperatively [426]. This includes at least one consultation with a gastroenterologist/hepatologist, a preoperative esophagogastroduodenoscopy (EGD) to assess potential esophageal varices and/or hypertensive gastropathy alongside a portal venous CT to evaluate bypass circuits. If necessary, invasive measurement of the portal venous pressure can also be considered. Furthermore, the indication for bariatric surgery in patients with portal hypertension can only be made within the framework of a careful, critical interdisciplinary risk-benefit assessment [427]. At centers with a wealth of experience and maximum care capacity, lowering the portal vein pressure may be considered prior to bariatric surgery [17] [427]. Bariatric surgery can also be performed on well-selected LT candidates at experienced centers highly specialized in liver transplantation and bariatric surgery [428].

Bariatric surgery in NAFLD

Commentary 

Various surgical procedures have been established, with laparoscopic sleeve gastrectomy (LSG) and laparoscopic Roux-Y gastric bypass surgery (RYGB) being used most frequently in Germany and worldwide. The use of laparoscopic one-anastomosis gastric bypass (OAGB) is also becoming increasingly popular. Thus far, there has not been a conclusive assessment with regard to the effectiveness and risk-benefit analysis of the various procedures. Randomized controlled trials with a 5-year follow-up comparing sleeve gastrectomy and RYGB produced overall equivalent outcomes. RYGB leads to a marginally better weight loss (approx. 1–2 BMI points after 5 years), while sleeve gastrectomy is associated with fewer complications and re-operations [429] [430]. Nevertheless, sleeve gastrectomy has been linked to a markedly increased risk of de novo gastroesophageal reflux with a subsequent risk of developing Barrettʼs esophagus and associated esophageal cancer [431] [432]. Corresponding long-term data are lacking, thereby making EGD-guided follow-up care in patients with sleeve gastrectomy indispensable. A randomized controlled trial comparing RYGB with 200 cm OAGB showed a comparable effect on weight loss and metabolic outcome after two years of follow-up. In the trial, however, OAGB was associated with a significantly higher rate of deficiency symptoms [431]. Their significance is currently still unclear, as they mainly led to anemia and only a few cases resulted in a relevant protein deficiency, which could be of importance for patients with impaired liver function.

Gastric banding is inferior to the other methods in terms of long-term weight loss and metabolic effects and should therefore only be used in exceptional cases [432]. Although biliopancreatic diversions (Scopinaro and duodenal switch) have the most significant metabolic effects, their use is limited due to side effects, particularly by the occurrence of malnutrition [432].

With regard to the effectiveness of the various surgical procedures in NAFLD and NASH, no concluding assessment is possible due to limited data. Studies using histology, taken during the initial surgery and in the follow-up, show contradicting results. The publications by Froylich et al. and Schönfels et al. compared RYGB with sleeve gastrectomy [433] [434]. While Froylich et al. found no difference between RYGB and sleeve gastrectomy, Schönfels et al. showed a more frequent normalization in liver histology findings after sleeve gastrectomy. When comparing RYGB with adjustable gastric banding, Caiazzo et al. showed the clear superiority of RYGB over adjustable gastric banding in terms of improved liver histology [274]. Further studies with long-term results and histological endpoints are urgently needed to determine the best practice for NAFLD and NASH.

The safety of the various surgical procedures should be considered carefully when used in NAFLD and NASH patients. Liver failure after bariatric surgery is a very rare but serious complication. An analysis of ten patients with liver failure after bariatric surgery showed that this only occurred after bypass procedures (RYGB and OAGB); to date, no such cases have been described in the literature after sleeve gastrectomy or gastric banding [435] [436]. Thus, in patients with severe NAFLD and liver dysfunction with the risk of postoperative liver failure, malabsorptive bypass procedures such as distal RYGB or an OAGB with> 200 cm biliopancreatic leg length should be avoided. Moreover, a recent meta-analysis showed that the perioperative complication risk after sleeve gastrectomy was reduced down to a third compared to RYGB in patients with liver cirrhosis [437]. Sleeve gastrectomy also has the advantage that changes to upper gastrointestinal tract anatomy are minimized and that endoscopic access to the biliary tract remains. This is particularly relevant for potential LT candidates. Thus, sleeve gastrectomy should be the surgical procedure of choice in patients with liver cirrhosis.

Endoscopic procedures in NAFLD (requirements, methods)

Commentary 

Endoscopic procedures are less effective and long-lasting than surgical procedures in terms of weight reduction but can be used if conservative therapy has failed and surgical bariatric procedure are contraindicated. The AWMF “Clinical Practice Guideline: Obesity Surgery and the Treatment of Metabolic Diseases” from 2018 states a “can be considered” recommendation for endoscopic procedures – mainly in favor of the intragastric balloon as based on the data available at the time that guideline was written [438] (AWMF Register No. 088–001).

The best studied endoscopic procedure is the intragastric balloon (IGB); it is indicated from a BMI of ≥ 30 to 40 kg/m² with an implantation period of 6 months. One recent meta-analysis examined 13 RCTs (endoscopic IGB vs. sham or lifestyle interventions) with 1523 patients and showed a significant advantage for IGB with regard to percentage excess weight loss (%EWL) and percentage total weight loss (%TWL), e. g., of –17.98 % and – 4.40 %, respectively [439]. An older meta-analysis from 2008 [440] of 3698 patients demonstrated a good safety profile for this method with serious complications below 1 % (small intestinal obstruction 0.8 %, gastric perforation 0.1 %) and the need for earlier removal due to pain/sense of pressure in 4.2 % of patients. IGB as bridging intervention in patients with a BMI > 50 kg/m² before bariatric surgery proved to not be significantly effective [441] in a recent meta-analysis with regard to weight loss. Therefore, there is no advantage for bridging with IGB in such severely overweight patients, at least with regard to weight loss.

IGB is also the best studied endoscopic procedure with regard to metabolic and hepatic parameters. Table 4c in the Appendix gives an overview of the studies and results. At 6 months after IGB there was not only a significant decrease in BMI but also a significant decrease in plasma glucose, insulin levels and triglycerides, in addition to a significant decrease in liver enzyme levels ​​(AST, ALT). Two papers examined the effect on liver histology, using the NAFLD activity score, and showed a decrease of 2–3 points [442] [443]. It was also shown in 4 studies that imaging procedures (MRI, CT, US) showed a significant decrease in liver volume [444], steatosis grade [445] [446] and liver fibrosis stage [443].

See Appendix Table 4c

Endoscopic sleeve gastroplasty (ESG) has been established over the last few years in parallel with the introduction of endoscopic suturing. A recent Danish meta-analysis analyzed 23 studies – mainly cohort studies and case series – including 3 non-prospective comparisons versus laparoscopic gastric sleeve (LSG), surgical endoluminal sleeve gastroplasty (primary obesity surgery endoluminal procedure, POSE) and endoscopic intragastric balloon (IGB) [447]. There was a mean weight loss over 12 months of –16.3 %. The intervention was superior to IGB and lifestyle modifications with regard to weight loss and inferior to the surgical procedures, albeit with a lower rate of adverse events than surgery. Two studies investigated ESG (OverStitch technique) with regard to metabolic and hepatic parameters (see Appendix Table 4c). In addition to weight loss, there was a significant decrease in HbA1c [448] [449] and a lowering of the NFS and hepatic steatosis index after 12 and 24 months [449]. At present, there are 2 ongoing prospective randomized studies on the procedure: ESG (OverStitch) + lifestyle modification vs. sham + lifestyle modification in patients with histologically confirmed NASH [NCT 03426111] and ESG versus LSG [NCT04060368].

The third endoscopic approach involves bypassing the proximal small intestine (duodenum), as this is where metabolic processes take place. This has a particularly favorable effect on T2DM and thus can also reduce lipogenesis and lipid storage in the liver. The endoscopic duodenal-jejunal bypass using EndoBarrier^® consists of a metal stent that is anchored in the duodenal bulb. A plastic tube is attached to it and bridges the duodenum into the jejunum. A meta-analysis of 15 heterogeneous studies, including 5 RCT, demonstrated a more effective weight loss for the EndoBarrier^® compared to lifestyle interventions [450]. The improvement in the metabolic parameters HBA1c and fasting glucose did not reach statistical significance. There were 27 adverse events, nausea, vomiting, mucosal laceration and ulceration in the duodenal bulb, 6 of which were severe. A retrospective study from Germany examining hepatic parameters (see Appendix Table 4c) showed improved diabetes control, decreased AST and ALT levels, and elastographically (VCTE) a decrease in liver stiffness and a decrease in hepatic steatosis [451]. The procedure is currently not approved in Germany.

The duodenal mucosa resurfacing (DMR) (Revita System) is based on circular thermal ablation of 10 cm duodenal mucosa using an endoscopic balloon catheter. Initial high-quality prospective data published on obese patients with T2DM showed an improvement in diabetic metabolism and a decrease in liver enzyme levels (see Appendix Table 4c) [452] [453]. Further prospective studies on effectiveness, side effects and the influence on steatosis and liver fibrosis in NAFLD are to be expected.

Another endoscopic procedure, the partial jejunal diversion, is a partial jejunoileal bypass, which is generated by the endoscopic insertion of 2 magnets (100 cm distal to the Treitz ligament and about 100 cm proximal to the ileocecal valve). Only small feasibility studies are currently available (see Appendix Table 4c) showing a positive influence on the metabolic situation [454]. Further data are expected.

What are the indications for liver transplantation in NAFLD?

Commentary 

Liver transplantation (LT) is an established treatment option for patients with decompensated liver cirrhosis or HCC developing from NASH cirrhosis. When rendering the indication, reference is also made to the S2k Guidelines on Liver Transplantation that is expected to be published in 2022.

Numerous studies have looked at the survival of patients with post-LT NASH compared to patients with other liver diseases. In most studies, the 1-year survival of NASH patients tends to be slightly worse [455] [456]. In a large study by the European Liver Transplant Registry, the 1 and 10-year survival in the 1667 patients with NASH cirrhosis without HCC compared to the 47 063 non-NASH patients without HCC was not significantly different (1 and 10 year -Patient survival: NASH 84.1 % and 62.1 % versus non-NASH 86.2 % and 62.9 %). The same also applied to patients with HCC, although the overall survival of the HCC patients was significantly worse. In the multivariate Cox regression analysis, the patient age > 61 years (HR 2.07) or > 65 years (HR 1.72) was associated with an increased mortality compared to the patient age < 45 years. Furthermore, there was an increased mortality for patients with BMI > 40 kg/m² (HR 1.96) but also with a low BMI < 18.5 kg/m² (HR 4.29) [457]. An analysis of the United Network for Organ Sharing (UNOS) database including patients between 2002 and 2016 showed a comparable patient survival rate between NASH patients and patients with cryptogenic, autoimmune or alcoholic liver cirrhosis [458]. On the other hand, a large meta-analysis that included 37 studies from 11 countries between 1996 and 2016 found a negative effect of obesity on transplant success. Patients with BMI > 30 kg/m² or BMI > 35 kg/m² had a significantly poorer survival rate compared to normal-weight patients (72.6 % and 69.8 % versus 84.2 %; p = 0.02 and p = 0.03, respectively) [459].

In the benefit/risk assessment, the indication for LT in patients with decompensated NASH cirrhosis is emphasized by the fact that waiting list mortality appears to be higher in patients with NASH or obesity than in other patient groups. An analysis of the UNOS database showed that obese patients benefited more from LT than patients of normal weight [460]. However, the BMI is only of limited significance in patients with NASH cirrhosis and hydropic decompensation. No influence of weight on survival was found when weight was corrected for ascites [461]. On the other hand, the BMI does not reflect the existing muscle mass. Sarcopenia, which is associated with poorer survival after transplantation, can also occur in obese patients and can be recorded, for example, by measuring the muscle mass index.

The risk of recurrence of NASH is not per se a contraindication to LT. Although 41–54 % of all NASH patients develop NAFLD again within 1 year after transplantation [462] [463] and 89 % in the long-term course [464], the development of fibrosis appears to be markedly slower than in patients. In a recently published single-center American cohort study with 226 patients, transplanted for NASH cirrhosis, histological NASH recurrence was found in 49 % 3 years after transplantation, but cirrhosis was seen in only 4 patients after 9 years [465]. Another single-center American study, which included 103 patients transplanted for NASH cirrhosis, found advanced fibrosis in 20.6 % (median) by histology after 47 months post-LT and in 26.8 % by transient elastography after 75 months (> F3) [464]. This recent study showed higher recurrence rates of NASH cirrhosis than older, larger studies, in which the cirrhosis recurrence rate was between 4–10 % after approx. 10 years. Nevertheless, the recurrence rate appears to be acceptable compared to other transplant indications and by no means a reason for the rejection of NASH cirrhosis as a transplant indication.

What are the specific risks of LT in NAFLD patients?

Commentary 

At the time of listing for LT, patients with NASH cirrhosis suffered more often from a metabolic syndrome than patients with any other cirrhosis pathogenesis. In addition, it could be shown that NASH patients tend to be older [466]. In LT candidates with NASH cirrhosis, the prevalence of coronary artery disease (CAD) is also markedly higher at 21 %–29 % than in patients with cirrhosis of other etiologies (5–11 %) [466] [467]. This corresponds to the higher complication rate post-LT, proven in numerous studies [468] [469] [470]. In the study by Vanwagner et al. [471], cardiovascular events occurred in 26 % of the 115 NASH patients in the 1st year post-LT, but only in 8 % of the 127 patients with ethyl toxic cirrhosis (p < 0.001). In the multivariate analysis, NASH was a significant risk factor for the occurrence of cardiovascular complications, regardless of age, T2DM, nicotine consumption and the presence of a metabolic syndrome. NASH cirrhosis was also associated with high cardiovascular mortality (50 %), with 70 % of events occurring perioperatively. According to a large cohort analysis of the UNOS database on over 32 800 liver transplant patients, the presence of NASH cirrhosis was a risk factor for the occurrence of serious cardiovascular events 30 and 90 days post-LT (OR 1.6) [472]. In the largest current meta-analysis by M. Barone et al. [473], which included 24 studies, there was a significantly increased mortality risk at 30 days, 1, 2 and 5 years after LT, especially for the subgroup of patients with BMI ≥ 40 kg/m². A BMI > 30 kg/m² already posed an increased risk of postoperative complications. The increased cardiovascular risk should be taken into account when evaluating patients for LT. Hogen et al. [474] therefore suggested that coronary angiography always be performed in patients with NASH cirrhosis, if more than 2 of the following risk factors exist: Age > 50 years, T2DM, hypertension, family history of cardiovascular disease, nicotine consumption or known cardiovascular disease. Patients with 1–2 risk factors can initially be examined using dobutamine stress echo; coronary angiography should only be performed if CAD is suspected.

Furthermore, infectious complications – in particular wound healing disorders – were observed more frequently in patients with NASH cirrhosis than in patients with other transplant indications. This could be explained by the higher prevalence of T2DM. Overall, in some studies, increased perioperative morbidity is also reflected in a longer intensive care and hospital stay after LT. In a study by Malik et al. infections were actually the most common cause of death (57.1 %) post-LT [475].

Due to the increased perioperative risk, patients with NASH cirrhosis should therefore be comprehensively and critically assessed in a multidisciplinary setting as part of the transplant evaluation, including not only gastroenterologists/hepatologists and transplant surgeons, but also cardiologists, anesthetists and diabetologists [476].

As shown above, patients transplanted for NASH cirrhosis are at high risk of recurrence of NAFLD and NASH. Risk factors are insulin-dependent diabetes mellitus before transplantation [463], older age in conjunction with a metabolic syndrome [477], female gender [478], genetic factors [479] alongside severe weight gain and obesity post-transplantation [480]. This should definitely be considered with regard to the management of patients post-LT and when choosing immunosuppressive therapy. Steroids are associated with the occurrence of metabolic syndrome, but both calcineurin inhibitors – tacrolimus and cyclosporine – also have a negative effect on the development of insulin resistance [481]. In contrast, there was no association between the use of everolimus and the occurrence of NAFLD post-LT.

When is liver transplantation contraindicated?

Commentary 

NAFLD is often associated with relevant extrahepatic comorbidities that can endanger the therapeutic outcome of LT [233]. Numerous studies associated obesity with a poorer clinical outcome post-LT [457] [459] [468] [473] [476] [482]. Particularly noteworthy is a current European registry study, evaluating over 66,000 liver transplants. The multivariate analysis and, specifically the subpopulation of patients with NASH without HCC, showed a significant association between morbid obesity (BMI ≥ 40 kg/m²) and poorer survival LT [457]. This association has already been documented in earlier registry studies that were, however, evaluated independently of the diagnosis [468] [482] [483]. The older practice guidelines of the American Association for the Study of Liver Diseases (AASLD) therefore defined a BMI ≥ 40 kg/m² (WHO class III) as a relative contraindication to LT [484].

However, it must be noted that there are also studies on larger patient populations that did not identify a higher BMI as an independent risk factor for post-LT mortality [461] [485] [486]. The falsification of BMI due to ascites was a relevant confounding factor for presumed associations between obesity and post-LT prognosis [461]. It is noteworthy that the European transplant registry study not only found an association for morbid obesity (and cachexia) with poorer survival in the NASH cohort, but also for a BMI in the normal weight range (18.5–25 kg/m²) [457]. A plausible explanation for this negative treatment outcome could be that a normal weight BMI in the usually overweight population of NAFLD/NASH patients may indicate a history of muscle wasting and established sarcopenia. Moreover, an older retrospective study of over 25,000 waiting list patients calculated that LT had a relevant survival benefit even in morbidly obese patients that was no less than across other BMI ranges [487].

All of this shows that the BMI is not a universally applicable, sufficiently accurate tool to define a contraindication to LT in NAFLD patients. Although a recent meta-analysis also described a significant association between obesity with BMI ≥ 30 kg/m² and poorer survival post-LT, it was opposed to recommending the use of BMI as an exclusion criterion due to the heterogeneity of the included studies [459]. With that in mind, BMI should be used as a guide to the risk of complications in overweight patients post-LT and not as a categorical exclusion criterion.

Future studies may show that, in patients with NASH cirrhosis, other parameters reflect the chances of success of LT better than BMI and are also associated with survival on the waiting list or post-LT, such as frailty [488], myosteatosis [489] or cardiopulmonary performance capacity [490].

Specific lifestyle interventions and drug therapy while waiting on the list pre-LT

Commentary 

A large proportion of patients with NASH cirrhosis are overweight or obese. In a recent European registry study, the mean BMI of patients who had to undergo LT due to NASH was 32.6 kg/m² [457]. As described above, there is abundant evidence that obesity is associated with a poorer post-LT prognosis [459]. In addition to obesity, NASH patients with advanced liver cirrhosis often have prognostically unfavorable disorders such as malnutrition, sarcopenia and myosteatosis. Many patients with liver cirrhosis also suffer from malnutrition and sarcopenia and, in conjunction with obesity, present as combined clinical picture of sarcopenic obesity [489] [491] [492] [493] [494]. These clinical conditions can be treated with targeted interventions. In addition to the nutritional recommendations for treating overweight patients with NAFLD, offering a Mediterranean diet with plenty of vegetables, fruit, grains, fish and olive oil as the main source of fat [325] [495], measures such as snacks and late meals, a protein-rich diet, and the addition of branched-chain amino acids [496] [497] can be used. A diet adapted to the energy requirement is likely to have a positive effect on malnutrition [498] [499] [500] [501]. A differentiated assessment of the nutritional status and body composition of NASH patients on the transplant waiting list is therefore indicated. A retrospective study indicated that nutritional interventions improve survival and quality of life in patients with liver cirrhosis [502]. Weight reduction should not be recommended for patients with advanced NASH cirrhosis, as this could worsen sarcopenia and malnutrition.

Obesity can be associated with mental illnesses such as eating disorders or depression [503] [504] and vice versa, it can also have psychosocial effects that negatively impact the patientʼs prognosis [505]. For this reason, and because psychological support can help implement the required lifestyle changes, current obesity guidelines recommend the psychological assessment of obese patients and the integration of psychological interventions into the obesity management strategy [503] [504] [506]. Accordingly, obese patients with NASH cirrhosis should also be given a targeted psychological assessment as part of the LT evaluation focusing on obesity-associated mental illnesses and the need for additional psychotherapeutic treatment.

While it is well established that weight reduction through lifestyle changes lead to a histological improvement in patients with non-cirrhotic NASH [269], little evidence is available on patients with advanced NASH cirrhosis. An uncontrolled pilot study examined the effect of an intensive lifestyle intervention with an individualized low-calorie diet and 60 minutes of physical training per week in 60 overweight or obese patients with compensated NASH cirrhosis and portal hypertension. Significant weight reduction was achieved after 16 weeks that was accompanied by an improvement in portal hypertension. Liver decompensation was not reported during the intervention [282]. Numerous smaller studies also showed that adapted programs of physical exercise did not have any adverse effects in patients with liver cirrhosis but suggested positive effects on aspects such as maximum oxygen capacity (VO2), muscle mass, mobility and quality of life [507] [508] [509] [510]. However, the large proportion of Child A cirrhosis patients, included in the studies, must be pointed. Whether lifestyle intervention can bring about clinical improvement in patients with decompensated Child C liver cirrhosis has not yet been investigated in large, controlled studies. However, adapted movement exercises to maintain mobility seems to be sensible.

Indications and contraindications for endoscopic intervention or bariatric surgery in NAFLD patients before, during or after LT

Commentary 

A BMI ≥ 40 kg/m² is viewed by many centers as a relative contraindication to LT due to the increased post-LT morbidity and mortality [511]. In addition, cirrhotic patients with a BMI ≥ 40 kg/m² on the waiting list show an increased risk of mortality or an increased risk of being removed from the waiting list [512]. The relationship between a BMI ≥ 40 kg/m² and a deterioration in post-LT outcomes cannot be demonstrated to the same extent in all analyses [513]. Therefore, in patients on the transplant list with severe obesity (BMI > 35 kg/m²) and other prognostically unfavorable factors (e. g. T2DM), obesity surgery should be discussed on an individual basis, after conservative measures have failed [514].

From the data available thus far, no general recommendations for the optimal timing of bariatric surgery (before, during or after LT) can be derived [514], because each time point is impacted by its own specific risks and contraindications [515]. Therapy planning for patients with a BMI > 35 kg/m² and a possible indication for LT should therefore be carried out individually after a careful risk-benefit assessment [516]. This should be done in centers with experience in bariatric surgery and transplant medicine, as hospital mortality in cirrhotic patients at centers, performing > 100 bariatric surgical interventions per year is markedly lower than at centers with lower case numbers (OR 0.3, p < 0.0001 compared to centers with < 50 operations per year) [422]. LT candidates can also undergo bariatric surgery relatively safely in highly specialized centers for LT and bariatric surgery that possess the corresponding experience [428].

Pre-LT bariatric surgery can be helpful to give patients with morbid obesity access to LT in the first place and at the same time this makes sense in terms of favorably influencing modifiable risk factors for survival post-LT [511]. However, bariatric surgery prior to LT can only be carried out in patients with a low MELD score and acceptable risk with no signs of clinical decompensation. Except in cases of underlying HCC as a transplant indication, patients with well-compensated cirrhosis usually do not qualify for LT. Thus, preoperative bariatric surgery does not play any major role in clinical practice. Furthermore, an increased percentage of patients suspended from the transplant list due to sarcopenia following bariatric surgery has been reported [517].

It could be shown in smaller case series that sleeve gastrectomy in patients with Child A stage compensated cirrhosis is associated with an increased but still low overall complication rate [424] [427] [437]. Decompensated cirrhosis or significant portal hypertension carry a high postoperative complication and mortality rate, meaning that the indication for pre-LT bariatric surgery should no longer be rendered that readily. An analysis of data from the US Nationwide Inpatient Sample based on 3888 obesity surgeries in patients with compensated cirrhosis and 62 with decompensated cirrhosis showed that even with compensated cirrhosis, a longer hospital stay and increased mortality compared to patients without cirrhosis (0.3 % vs 0.9 % OR 2.17). These figures only concern hospital mortality; later decompensation and readmissions were not taken into account. In contrast, the mortality in patients with decompensated cirrhosis was 16.3 % (OR 21.2, CI 5.39–82.9) [422]. With regard to operation type in cirrhotic patients, sleeve gastrectomy has markedly fewer complications than RYGB [437].

Reliable data for a better risk stratification of patients with morbid or severe obesity based on the MELD score, the HVPG or liver function tests are not yet available. In general, the 30-day mortality rate increases linearly with increasing MELD score, namely by 1 % for every MELD point between 8 and 20 and by a further 2 % for every MELD point above 20 [518] [519] [520].

It is not clear whether preoperative TIPS implantation in patients with cirrhosis and portal hypertension can reduce the postoperative complication rate for bariatric surgery. However, data on other abdominal interventions suggest that preoperative TIPS can reduce complications from portal hypertension [521] [522].

If transplantation is not an option in patients with decompensated cirrhosis due to morbid obesity alone, the indication for simultaneous obesity surgery and LT can also be considered on an individual basis. Acceptable outcomes for LT with simultaneous sleeve gastrectomy, were reported mainly from single-center case series. It could be shown that a simultaneous sleeve gastrectomy during an LT markedly reduces post-transplantation BMI [523] [524]. On the other hand, simultaneous sleeve gastrectomy increases perioperative morbidity and mortality post-LT In addition, malnutrition can occur in the early phase after transplantation, which can delay convalescence in patients often already suffering from catabolic symptoms (sarcopenic obesity) [525]. On the other hand, successful simultaneous LT with sleeve gastrectomy resulted in permanent weight reduction up to 3 years post-LT (weight loss 34.8 ± 17.3 % after 3 years) and additional longer term advantages with regard to arterial hypertension, insulin resistance and hyperlipidemia [524].

Regarding the choice of procedure, most experience is available for sleeve gastrectomy in the context of LT. Possible advantages of SG for (potential) liver transplant patients are the simpler technical feasibility, the preservation of the endoscopic access to the biliary tract system and the avoidance of malabsorption. The latter also plays a role in terms of the reliable absorption of immunosuppressants.

A third option is potential bariatric surgery after LT if conservative measures fail with persistent or worsening morbid obesity. Conservative treatment of severe obesity and metabolic complications post-LT can be particularly difficult, among other things because side effects of immunosuppression make metabolic control more difficult [526]. Successful case series have also been published on obesity surgery post-LT [527] [528] [529]. Overall, the complications are within acceptable limits [527] [530] [531] [532]. Because of expected adhesions and the level of immunosuppression, obesity surgery should, if possible, not be carried out before the end of the first year after transplantation. Isolated case reports exist on the use of the IGB in patients with liver cirrhosis or post-LT [533].

NAFLD patients as organ donors

Commentary 

The problem of accurately evaluating liver function in higher grades of hepatic steatosis in an organ donated after death using ex vivo machine perfusion was discussed in [534]. Patients with NAFLD should always be considered as organ donors. The acceptable degree of hepatic steatosis depends on the type of donation (living vs. cadaveric donors), the type of steatosis (macrovesicular vs. microvesicular) and other donor and recipient factors and must be determined in each individual donor/recipient case. With regard to the accurate evaluation of living liver donation many other factors must be taken into account, not just the steatosis grade alone.

In the context of liver donation after brain dead, macrovesicular steatosis> 30 % is an extended donation criterion. Microvesicular steatosis, on the other hand, is less relevant. A higher grade macrovesicular steatosis of the graft represents an independent risk factor for postoperative complications up to primary graft failure. One of the reasons for this is increased ischemia-reperfusion injury observed in steatotic liver grafts [535]. In addition to macrovesicular steatosis, other factors such as donor age and time of ischemia have a major influence on subsequent transplant function [536]. Recipient factors must also be taken into account in the individual organ acceptance in order to estimate the cumulative risk in the transplantation of organs with extended donor criteria. The procedure regarding organ acceptance of steatotic donor livers does not differ between recipients with and without NASH. There are no data that justify a different approach, especially since steatosis of the transplant is reversible in the early phase after transplantation [116] [515].

A better assessment of the liver function and conditioning of steatotic transplants may be used in the future for regenerating liver tissue ex vivo via machine perfusion. Results based on stronger evidence are mainly available for normothermic perfusion of donor livers. A prospective randomized trial showed that more transplants were used after normothermic machine perfusion and that these showed less graft damage postoperatively than the control group with cold preservation without machine perfusion [537]. In addition, by using normothermic machine perfusion, 22 of 31 (71 %) livers that were initially assessed as unsuitable for transplantation could be transplanted after machine perfusion and testing for functionality. A high proportion of the livers were rejected for transplantation due to underlying steatosis alone or for combined reasons [538]. There are also indications that the ischemia-reperfusion damage may be less pronounced after machine perfusion, which could be of particular advantage in steatotic transplants [539].

If living donation is planned, it is important to rule out relevant steatosis in the potential living donor, both for reasons of donor protection and with regard to a possible initial non-functioning of the liver in the recipient. Therefore, the limits for steatosis in living donation, especially adult recipients, are markedly lower than in post-mortem donation. Living donors with a macrovesicular steatosis of> 30 % are rejected in most centers. The exact extent of acceptable steatosis in living donors depends, among other factors, on the donorʼs age and the volume of the donorʼs residual liver after living donation (future liver remnant) [540] and therefore cannot be given in general terms. Potential donors with excessive or borderline hepatic steatosis can use conservative measures (diet, lifestyle change) to help reverse steatosis. This also applies to non-overweight donors with NAFLD [541].

To assess the liver parenchyma of the living donor, a sonography and a VCTE are performed to quantify steatosis using CAP and fibrosis using stiffness measurement. Additional biopsy-guided assessment of the degree of steatosis is necessary, primarily in living donors with an increased BMI, since the prevalence of steatosis in potential donors with a BMI > 28 kg/m2 is as high as 76 % [542]. The same applies if the CAP measurement is increased to > 248 dB/m or a VCTE measurement is increased to > 7 kPa.

5. Monitoring and long-term management

Surveillance (frequency)

Commentary 

The close association between NAFLD and metabolic diseases must be viewed bidirectionally, i. e. that NAFLD can already precede the development of T2DM by years. Conversely, according to a current multinational meta-analysis, 72 % of patients with NAFLD have a metabolic syndrome and 47 % have T2DM [32] [543] [544]. In addition, the vast majority of NAFLD patients are overweight and have arterial hypertension. The frequency of cardiovascular diseases is higher in NAFLD than in control groups and already lead to cardiovascular events such as myocardial infarction and stroke from the early stages of fibrosis [18] [545].

A recommendation of the follow-up extent and time interval must therefore be adjusted to the heterogeneity of NAFLD with its risk factors and comorbidities. The following factors play a decisive role: Cooperation between primary and secondary care, knowledge of and reference to other national health care guidelines (specifically on obesity, T2DM, CAD, gastrointestinal oncology and HCC) as well as reference to existing preventive screening examinations and cancer prevention in accordance with G-BA guidelines is required.

Surveillance of fibrosis progression

Commentary 

In general, NAFLD can be regarded as a rather slowly progressing liver disease [408]. As the only histologic feature, liver fibrosis is associated independently with long-term overall mortality, LT and liver-related events [82] [546]. A current meta-analysis, which included 13 studies with a total of 4428 patients and histologically confirmed NAFLD, showed a steadily increasing risk for all-cause mortality in F4 compared to F0 (RR, 3.42 (95 % CI, 2.63–4.46), liver-related mortality (RR, 11.13 (95 % CI,, 4.15–29.84), die LT (RR, 5.42 (95 % CI, 1.05–27.89) and liver-related events (RR, 12.78 (95 % CI, 6.85–23.85) [411].

Measured over a longer period of time, the initial stage of fibrosis correlates with the development of severe liver disease. One retrospective cohort study on 646 liver-biopsied NAFLD, patients were followed for an average of 20 years. Patients with F3 fibrosis developed liver decompensation over a period of 6 years whereas patients with F1 fibrosis took up to 35.6 years [69]. It therefore seems appropriate to also assess the progression of fibrosis over time during long-term monitoring of NAFLD patients in order to identify more equivocally those patients at high risk for clinical endpoints. As paired liver biopsies outside of clinical trials are not acceptable, it makes sense to repeatedly use non-invasive surrogate markers for liver fibrosis, such as the FIB-4 index, the AST/ALT quotient, the NFS or imaging methods (including transient elastography, shear-wave elastography, ARFI). Earlier data on chronic hepatitis C have shown that repeated measurement of liver stiffness and calculation of FIB-4 combined allow a better prediction of clinical endpoints than measuring each parameter alone [547]. The interventional FLINT study with paired liver biopsies showed that, for NAFLD, the non-invasive tests FIB-4 and NFS correlated with an improvement in liver histology [548]. Data from the Swedish population-based AMORIS study (n = 40,729), in which the FIB-4 index was determined at baseline value in the years 1985–1996, with a repeated measurement on average 2.4 years later, suggest that the increase in FIB-4 is associated with an increasing risk of advanced liver disease later on [549]. However, half of the liver-related events also occurred in the permanently “low” FIB-4 group, implying that the sensitivity for the repeated FIB-4 calculation with regard to liver endpoints must be classified as low (sensitivity 10–40 %). It should also be pointed out that increasing age might also be relevant when calculating FIB-4, because age is a parameter in the calculation determining the FIB-4 score [115].

Non-invasive determination of fibrosis progression: A pragmatic recommendation at this point in time may be the calculation of a low-cost score such as the AST/ALT quotient or the FIB-4 repeated every 2–3 years, especially in patients who have one or more risk factors for advanced fibrosis: Age 45–55 year; BMI > 30–32 kg/m²; T2DM; arterial hypertension. Alternatively, depending on availability, liver stiffness measurements can be repeated every 2–3 years. Future studies will clarify whether new surrogate markers (e. g. NIS4, FAST score) or imaging methods such as MR elastography or MR PDFF can also be used to assess the individual rate of fibrosis progression and the course of NASH [550].

Risk profiles

Commentary 

Basic diagnostics for long-term management 

• Physical examination with determination of:

  • BMI

  • Waist size

  • Blood pressure

• Clinical laboratory tests:

  • ALT, AST, γ-GT, bilirubin, AP

  • Blood count

  • Lipid status (triglycerides, cholesterol, HDL, LDL cholesterol)

  • Fasting blood sugar, HbA1c

In patients with incident NAFLD without advanced fibrosis and without typical concomitant diseases, these examinations can be carried out every 2–3 years, e. g. as part of the regular health check every 36 months. In patients with advanced fibrosis and/or NASH, baseline examinations should be performed annually.

Two large-scale meta-analyses in recent years have convincingly shown the increased incidence of liver and cardiovascular endpoints in NAFLD patients with an increasing stage of fibrosis [18] [411]. In the recently published meta-analysis by Taylor et al. the unadjusted relative risk for all-cause mortality was between 1.12 (95 % CI 0.91–1.38; for F0 versus F1) and 3.42 (95 % CI 2.63–4.46; F0 versus F4 fibrosis) [411]. Cardiovascular diseases such as myocardial infarction and stroke are the main causes of overall mortality. In a retrospective cohort study from the German Disease Analyzer Database (IQVIA), data from 22,048 NAFLD patients followed up at 1262 general practices between 2000–2015 were compared with a control group without NAFLD. The risk of newly emergent cardiovascular diseases was compared between the two groups that were adjusted for the incidence of arterial hypertension and diabetes mellitus, among others. The hazard ratio (HR, 95 % CI) was 1.34 (1.10–1.63) for the occurrence of myocardial infarction, 1.35 (1.25–1.45) for CAD, 1.15 (1.04–1.26) for atrial fibrillation and 1.09 (0.95–1.24) for stroke (n. s.) [545].

Furthermore, current studies have shown the higher incidence of cancers in patients with NAFLD compared to those without NAFLD [551]. One study conducted in the United States on 4722 patients with NAFLD demonstrated an almost doubling of the number of new cancers, especially gastrointestinal tumors, over an average follow-up of 8 years. Further analysis of the German Disease Analyzer Database (IQVIA, (see above) found that tumors of the urogenital system in men with an HR (Hazard Ratio) of 1.26; skin tumors (regardless of gender) with an HR of 1.20 and breast cancer in women with an HR of 1.22 were increased compared to patients without NAFLD [552]. In patients with NASH and/or fibrosis, annual checkups, including the basic examinations (see above), should be carried out as part of long-term management. It should also be ensured that, in addition to preventive measures and interventions (reference Chapter “Therapy”), subject-specific estimations of the individual risk profile (e. g. based on of the S3 Guideline “General practitioner risk advice on cardiovascular prevention”; ESC Pocket Guidelines of the European Society of Cardiology/German Society of Cardiology). Furthermore, patients should be encouraged to participate in the statutory cancer screening program in Germany (e. g. information on cancer screening from the German Federal Joint Committee (G-BA)).

HCC Surveillance in NAFLD cirrhosis

Commentary 

The association of NAFLD cirrhosis and HCC development has been well studied and documented over the past few years. Patients with confirmed NAFLD cirrhosis have a markedly higher annual risk of HCC. In two different American cohort studies, the HCC risk was found to be 1.56 % and 2.6 %, respectively; overall, it can be classified at over 1.5 % [342] [553]. This is slightly lower than in patients with HCV-related cirrhosis, in whom the annual incidence of HCC is approximately 4 %. In view of the fact that NAFLD-associated HCC often has a worse prognosis, as they occur more frequently in older and more severely ill patients and are often discovered late, regular surveillance is recommended [554] [555]. In patients with NAFLD cirrhosis, this is cost-effective, since the annual incidence of HCC is more than 1.5 % [556] [557] [558]. However, in a large matched case-control study conducted within the U. S. Veterans Affairs healthcare system, no improvement in cancer-associated mortality was obtained in patients with liver cirrhosis who underwent regular screening [559]. These results question the usefulness of the HCC screening in patients with liver cirrhosis and must therefore be examined further in follow-up studies.

Liver ultrasound is a widely available, inexpensive and effective screening method for HCC detection in risk groups [560] [561] [562]. Since screening quality depends largely on the examinerʼs experience and the equipment quality, an experienced examiner with a sufficient number of examinations per year and a device with a quality level analogous to DEGUM II are recommended for screening examinations (http://www.degum.de/) (https://www.leitlinienprogramm-onkologie.de/Leitlinien.7.0.html). If the liver cannot be adequately assessed by ultrasound, an additional MRI can be offered. Two studies on the role of MRI in HCC screening were evaluated in a meta-analysis on HCC screening [563] [564] [565]. This showed a pooled sensitivity for HCC detection of 83.1 % (95 % CI 72.0 % –90.5 %) and a specificity of 89.1 % (95 % CI 86.5 % –91.3 %) [565]. One of the two studies showed a significantly better sensitivity and specificity of MRI compared to ultrasound for the detection of HCC in cirrhosis patients [563]. Further studies are necessary to clarify the cost effectiveness of MRI examinations for HCC surveillance.

German and European HCC guidelines do no longer recommend mandatory testing of the tumor marker alpha-fetoprotein (AFP) for HCC surveillance [116] [566], as any unequivocal added benefit to ultrasound alone remained unclear. However, a current meta-analysis on HCC surveillance suggests that the addition of AFP to ultrasound increases sensitivity of HCC detection. In total, 32 studies with 13 367 patients were analyzed. Ultrasound alone had a lower sensitivity of 45 % for HCC detection than the combination with AFP of 63 % (relative risk 0.88; 95 % CI 0.83–0.93 for all stages, in the early stage RR 0.81; 95 % CI 0.71–0.93) [565]. Due to the widely discussed value of AFP alone, several alternative biomarkers or combinations of several biomarkers for HCC screening have been tested in studies in the last few years. In this context, the GALAD score has emerged as a promising screening method that determines the HCC risk using the patientʼs age, gender and the biomarkers α-fetoprotein (AFP), the AFP isoform L3 (AFP-L3) and the des-gamma-carboxy prothrombin (DCP) [567]. Patients with HCC showed an AUC of 0.96.

Various microRNAs were also investigated as biomarkers for HCC. In patients with chronic hepatitis B or C, markedly decreased levels of microRNA-139 were found in HCC [568] [569]. There are discordant results for mircoRNA-182. One study showed an increased risk of HCC due to the upregulation of miR-182, another showed a significant downregulation of the same miR-182 in HCC patients with chronic hepatitis C [570] [571]. MiR150, miR331–3 p or miR193 also seem to be interesting markers for diagnosing or predicting the course of the disease [106] [570] [572]. In total, there is not enough data to make clear recommendations for microRNA testing in the context of screening.

Surveillance in NAFLD without cirrhosis

Commentary 

NAFLD has meanwhile become one of the main risk factors for the development of HCC and will possibly replace viral hepatitis as the main cause in a few years [573]. Regular monitoring of patients with proven NAFLD cirrhosis with regard to the development of HCC is therefore established and recognized (see above). It is much more difficult to evaluate the extremely heterogeneous group of patients with NAFLD without the full clinical picture of cirrhosis. A general HCC surveillance of all NAFLD patients is neither cost effective nor practicable – especially given the high and steadily increasing number of cases – since the annual incidence of HCC in this group is well below 1.5 % [574]. Several studies in the last years have shown that up to 42 % of all cases of NAFLD-associated HCC occur in non-cirrhotic livers [575]. This fact requires the identification of patient groups with an increased risk profile. The following risk factors were identified:

Advanced hepatic fibrosis: A meta-analysis by Dulai et al. identified the NAFLD-associated liver fibrosis as the most important risk factor for mortality in NAFLD [18]. In particular, the risk of liver-specific mortality including HCC was exponentially increased in fibrosis patients and depended on the degree of fibrosis (maximum HR 42.30 (95 % CI 3.51–510.34) in F4. Therefore, a non-invasive determination of the degree of fibrosis should be carried out in all patients with NAFLD (e. g. as a combination of two different test modalities, e. g. fibrosis scores (FIB-4, NFS) and imaging methods (transient elastography, shear-wave elastography, ARFI, etc.) [9]. Further current studies have identified liver fibrosis in NAFLD patients as a risk factor for the development of HCC [76] [576]. An American retrospective cohort study on 296,707 NAFLD patients with the same number of controls found that alongside older age, male sex and T2DM there was an increased risk of HCC from constantly increased FIB-4 above 2.67 in the subgroup of patients without cirrhosis [76] [553]. In the case of NAFLD-associated fibrosis, it therefore makes sense to investigate further risk factors for the development of HCC.

Male sex has meanwhile emerged from many studies on NAFLD patients as an independent risk factor for HCC [76] [553] [577]. A European study investigated 100 patients with NAFLD-HCC and 275 control patients with NAFLD. In the group of HCC patients, 82 % were male, in the control group only 59 % [577]. A similar assessment came from Ioannou et al. on a cohort from within the U. S. Veterans Affairs healthcare system [553].

Several studies have identified elderly patient age as a risk factor for developing HCC [76] [106] [342] [553] [578] [579]. Two studies conducted within the U. S. Veterans Affairs healthcare system diagnosed HCC significantly more often in NAFLD patients who were older than 60 (aHR 2.09) and 65 (0.41 per 1000 PY) years of age [76] [553]. In a Taiwanese cohort, age > 55 years was already a relevant risk factor for HCC in NAFLD patients (HR 7.78 95 % CI 3.12–19.44) [106]. Also in a Japanese cohort of NAFLD patients, age> 60 was one of the risk factors for HCC (HR: 4.27; 95 % CI: 1.30–14.01) [578]. Altogether, it can be said that with increasing age of NAFLD patients, the risk of developing HCC also increases significantly. This leads to the well-known problem that patients with HCC in the context of NAFLD are often older and more seriously ill than patients with HCC of a different etiology and are therefore more difficult to treat.

T2DM A retrospective cohort study from Japan with 6508 NAFLD patients showed T2DM to be an independent risk factor for HCC (HR: 3.21; 95 % CI: 1.09–9.50) [578]. This observation could be confirmed in further recent studies, so that T2DM, frequently occurring in the context of metabolic syndrome in NAFLD patients, is to be regarded as a relevant risk factor for HCC [76] [579].

Elevation of transaminase levels: A persistent inflammatory reaction of the liver could also be identified as a risk factor for developing HCC in NAFLD patients. Several studies found an increase in AST alone (AST> 40 IU/l, HR 8.20; 95 % CI 2.56–26.26) or in ALT (HR 6.80; 95 % CI 3.00–15,42) or AST/ALT ratio or FIB-4 as a risk factor for HCC development [76] [106] [553] [578]. This is consistent with the fact that persistent inflammation in the liver promotes carcinogenesis.

Genetic risk factors: In the last few years several genetic risk factors for the development of NASH cirrhosis have been described. In particular for SNP in the genes PNPLA3, TM6FS2 and MBOAT7, a clear association with increased intrahepatic fat accumulation and fibrosis was shown [136] [580] [581] [582]. Additionally, PNPLA3 rs738 409 C>G in patients with F3 fibrosis or cirrhosis was determined to carry a significantly increased HCC risk (HR 2.66; 95 % CI, 1.02–7.13) [576]. Even for these SNP, heterozygous NAFLD patients had an increased risk of HCC and this risk was even 5 times higher in homozygosity for GG compared to CC [577]. For the MBOAT7 variant re641 738 C<T an increased risk of HCC in NAFLD patients was also demonstrated in a current study, especially in pre-existing fibrosis (OR 1.65, 95 % CI 1.08–2.55) [583]. A combination of risk alleles in PNPLA3, MBOAT7 and TM6FS2 led to an increasing risk of HCC for each additional risk allele.

Overall, the study situation is insufficient for a surveillance recommendation. There is no risk score on the basis of which a recommendation for or against HCC surveillance can be made. In summary, the subgroup of NAFLD patients with proven fibrosis and other risk factors (male gender, older age, T2DM, chronic hepatic inflammation, genetic risk factors) have a significantly increased risk of developing HCC. In the absence of an existing risk stratification score in the sense of an individualized treatment concept, HCC screening analogous to the procedure in NAFLD cirrhosis can be offered.

Endoscopic surveillance in NAFLD cirrhosis

Commentary 

The current recommendations are based on the report of the “Baveno VI Consensus Workshop” on risk stratification and individual treatment of portal hypertension [585], which largely is in line with the AASLD recommendations on portal hypertension and bleeding in cirrhosis [586]. In the Baveno VI recommendations, criteria were also developed to select patients with compensated liver cirrhosis in a subgroup of patients with compensated cirrhosis using non-invasive markers (elastography value < 20 kPa and platelets > 150 000/µL) in whom there is no necessity for initial screening endoscopy, but who should only have the non-invasive tests repeated annually. These recommendations only applied to patients with viral liver disease. However, since a current study was able to validate the Baveno VI criteria in patients with metabolic liver disease [587], future studies could implement non-invasive screening examinations for varices status in patients with compensated NASH cirrhosis and thus reduce the number of unnecessary screening endoscopies in compensated NASH cirrhosis. In this context, recommendations to extend monitoring intervals are defined in the Baveno VI criteria based on the persistence or elimination of underlying liver-damaging diseases (e. g. recovery from viral hepatitis, complete abstinence from alcohol), while no clear recommendations can currently be made in this regard in the context of the NAFLD.

6. Pediatrics

Preamble

Children, adolescents and adults with obesity face an ever-present, persistent form of social stigma. They are often faced with discrimination in the workplace as well as in education and health care. Research shows that weight stigma can damage health, undermine human and social rights, and is unacceptable in modern societies. There is international consensus to consistently avoid stigmatizing language [588]. In this context, “people-first language” is a recognized linguistic standard that is also used in this guideline.

Prevalence and incidence

Commentary 

As part of the global obesity pandemic, the incidence and prevalence of NAFLD is increasing. The obesity rate has more than tripled since the 1960 s and is now 18.5 % in the USA and affects 13.7 million children and adolescents [589]. In Germany, the latest data from the 2nd wave of the KIGGS study found a prevalence of 15.4 % for overweight and 5.9 % for obesity [590] [591] [592].

The prevalence of NAFLD in children and adolescents varies significantly with the screening method used (transaminases, ultrasound, biopsy) and the patient population (regional-ethnic, genetic and environmental differences, gender, other risk factors). Accurate non-invasive biomarkers are missing for the precise recording of the NAFLD prevalence: Estimates range from 3–10 % of all children and adolescents in western industrialized nations [588] [589] [590]. In a pooled analysis of over 16,000 children with obesity the prevalence was 34.2 % (Confidence interval [CI] 95 % 27.8–41.2 %) compared to 7.6 % (CI 95 % 5.5–10.3 %) in the general pediatric population [593]. In one autopsy study conducted in San Diego County (USA), a histologically confirmed NAFLD was found in 9.6 % of all children and adolescents examined, with the prevalence in obesity being 38 % [594].

Classification of metabolic syndrome

Commentary 

In children and adolescents with obesity and other components of metabolic syndrome, the risk of developing NAFLD increases to 60–70 % [595] [596]. A multicenter study of 675 children with NAFLD showed a prevalence of T2DM of 30 %, whereby these children also had a higher risk of developing NASH [597]. Considering the strong metabolic influencing factor in the etiology of the disease, a renaming of the nomenclature to Metabolic (Dysfunction) Associated Fatty Liver Disease (MAFLD) was recently discussed among experts [597].

Natural course of NAFLD

Commentary 

Very little high-quality data are available on the long-term course of pediatric NAFLD. This includes the question of the progression of a simple NAFL to NASH, the development of fibrosis in children and adolescents with NASH as well as the rate of NASH-associated cirrhosis and the occurrence of HCC. In adult patients with NASH, mortality is largely determined by the degree of fibrosis [16]. One of a few published case descriptions in children with sequential liver biopsy reports a potentially rapid progression of NASH to cirrhosis within a few years [598]. In a histopathological study, Mann et al. describe portal inflammatory activity as an independent risk factor for progression to advanced fibrosis [599]. This is of particular importance since portal involvement primarily affects younger children (see Chapter “Histopathology”). Ultimately, the question of the capacity of the liver for regression of moderate or advanced fibrosis under adequate therapy remains, especially in childhood and adolescence.

The lifespan, and thus the imminent cumulative risk of progression or the occurrence of complications, is increased in pediatric NAFLD. The latency here can be significant. A large retrospective study examined the relationship between the BMI in 244 464 school children in Copenhagen (born between 1930 and 1989) and the incidence of NAFLD in adulthood. Weight gain in childhood was shown to be an independent risk factor for developing NAFLD and liver cirrhosis later in life. Another Danish study shows a clear association between BMI at the age of 18–20 years and severe liver-related morbidity and mortality 40 years later (increase 5 % per BMI unit over 11.5 kg/m² for cirrhosis, decompensation and liver-associated death) [600]. In a cohort of 66 adolescents (age 13.9 ± 3.9 years), observed over 20 years, the liver transplant-free interval in patients with NAFLD was significantly shorter than in the general US population (standardized mortality ratio 13.6 (95 %) Confidence interval, 3.8–34.8; p < 0.0001)), with 2 patients requiring LT due to decompensated liver cirrhosis. [601] Overall, there is concern about an earlier occurrence of serious hepatic or cardiovascular complications in adolescence or early adulthood [602].

Risk factors for developing fibrosis in NAFLD include the presence of other components of metabolic syndrome, such as insulin resistance or dyslipidemia [603]. According to current data, low or moderate alcohol consumption into young adulthood is not, as previously assumed, protective for the development of fibrosis [604] [605]. Important epigenetic influences are the maternal diet and behavior and consequent intrauterine deficiency or overnutrition (high fat diet in animal models [606], epidemiological data [607] [608] and a number of DNA methylation and histone modification profiles as well as microRNA profiles in the liver and blood circulation [609].

Genetics

Commentary 

The PNPLA3 148 M variant is the genetic influencing factor that has been best studied in children and adolescents. It is associated with an increased risk for NAFLD and also with the histological severity of steatosis, inflammation and fibrosis. The minor allele frequency of PNPLA3 is positively associated with the prevalence of NAFLD. Meanwhile, several pediatric studies with histologically proven NAFLD have confirmed the association of the PNPLA3 I148 M variant with a higher degree of steatosis, a higher NASH risk and an increased risk of fibrosis [610] [611] [612].

The fact that only a subset of patients with NAFLD develop progressive NASH initially pointed to a multifactorial background of the disease beyond purely lifestyle and environmental factors and it is proved that the individual genetic predisposition plays a decisive role in the phenotype of NAFLD. Numerous genetic risk factors for NAFLD have now been identified. These are clinically relevant, especially in children and adolescents with an early onset of the disease and thus long duration of the disease. In the aforementioned autopsy study by Schwimmer et al. ethnic origin was also examined. After correction for the BMI, children with a Hispanic family background had the highest risk (11.8 %), while children with an African American background were protected (1.5 %) [594]. The increased risk for children with a Hispanic-Mexican background could also be reproduced in population-based studies. It turns out that the minor allele frequency (MAF) of the pathogenic I148 M allele in the Mexican population is markedly elevated at 0.73 compared to Caucasians (0.26–0.32) and African-Americans (0.18) [613].

While the data on TM6SF2 in children does not currently permit a final assessment, a recent meta-analysis showed no effect of MBOAT7 in children and adolescents, in contrast to adults [614]. The study of 685 children and adolescents showed for the protective variant HSD17B13a lower degree of steatosis, lower transaminases and a lower fibrosis score [615]. The study of 685 children and adolescents showed for the protective variant HSD17B13a lower degree of steatosis, lower transaminases and a lower fibrosis score [615]. The interaction of the risk from PNPLA3 1148 M with fructose consumption in children would warrant further investigation into risk stratification. Currently, this does not result in changing advice on lifestyle changes or pharmacological therapy options [616]. In clinical trials, genotyping should be carried out for a more precise evaluation of outcomes.

Screening

Commentary 

Chronic liver disease must also be identified promptly in obese children and adolescents. NAFLD progresses in childhood and adolescence and, in exceptional cases, can also lead to liver cirrhosis. Diagnostics enables the exclusion of other chronic liver diseases and an early attempt at therapy [601] [617] [643] [659]. ALT levels should be compared to gender-specific reference ranges [618].

Algorithms for persistent transaminase elevation

Commentary 

Since other hepatopathies can present clinically, in laboratory tests, in imaging and histologically with very similarly pictures, the primary goal is not the diagnosis of NAFLD, but the reliable exclusion of other causes for the elevated transaminases (infections, autoimmune diseases, metabolic and endocrinological diseases).

Commentary 

Basic diagnostic tests for persistently elevated transaminases: 

Medical history and clinical findings: Blood pressure should be measured with the correct cuff size.

Screening:

• AST, ALT, γ-GT, AP, bilirubin (direct, indirect), CK, LDH, partial thromboplastin time (Quick-test)

• Ultrasound examination

When warning signs are present, i. e. clinical, laboratory or sonographic evidence of progressive liver disease or in the presence of cholestasis, immediate further diagnostics should be initiated, otherwise follow-up within the infection-free interval after 6–12 weeks.

Basic diagnostic tests (level 1):

If elevated liver values ​​persist for more than 6 months without “red flag signs” such as splenomegaly, sonographic or clinical signs of high-grade liver fibrosis/cirrhosis, cholestasis, abnormal family or travel history, blood count, Quick, PTT, autoantibodies (ANA, SMA, LKM, SLA), immunoglobulins (IgG, IgA, IgM), transglutaminase-IgA-Ab, serum ceruloplasmin, serum alpha-1-antitrypsin, cholesterol, HDL, LDL, T3, fT4, TSH, virus serology (CMV, EBV, HAV, HBV, HCV, HEV) should be measured.

Extended basic diagnostic tests (level 2):

Copper excretion in the 24-hour urine with or without administration of penicillamine, phenotyping of alpha-1-antitrypsin, if necessary, in borderline results.) LAL-D enzymes, hepatitis E serology.

In addition, special diagnostic procedures such as genetic and metabolic diagnostics such as amino acids in plasma, organic acids in urine, lactate and ammonia in serum, sweat test, echocardiography, etc. can also be helpful.

Ultrasound examination by elevated ALT provides the probably most sensitive method for the early detection of NAFLD. A disadvantage of sonography is the inability to quantify hepatic steatosis.

Indication for liver biopsy and extended diagnostics

Commentary 

An immediate liver biopsy as part of the initial diagnosis is recommended, if there is suspected progressive liver disease, e. g. with high IgG or positive liver-associated autoantibodies or suspected Wilsonʼs disease (low ceruloplasmin, increased copper excretion in the urine, Kaiser-Fleischer ring).

In cases of radiological evidence of hepatic steatosis, Wilsonʼs disease or other specific hepatopathies cannot be ruled out with certainty without liver biopsy. Patients with normal or only slightly elevated transaminases can already have significant fibrosis. In addition to the puncture-related risks (pain, bleeding, biliary leakage, injury to other organs, pneumothorax), liver biopsy carries a considerable sampling error, especially since the histological picture of NAFLD is not homogeneously distributed in the liver (1: 50 000 sample volume) [46]. Further uncertainty arises from the fact that the histological assessment is dependent on the examiner [619]. The importance of the non-invasive markers is shown in the Chapter “Monitoring”.

Commentary 

NAFLD covers a spectrum of diseases from the NAFL to NASH-associated cirrhosis. The term NAFL is also used in children and adolescents for describing non-alcoholic fatty liver or benign hepatic steatosis. Accordingly, the term NASH is used in pediatrics for the more aggressive form of liver cell steatosis involving hepatocytic degeneration and fibrosis. To diagnose NAFLD in childhood, it is required that at least 5 % of the hepatocytes have macrovesicular fat deposits. In analogy to the diagnosis of hepatic steatosis in adults, there are low-grade (mild) steatosis (less than a third of the hepatocytes affected), moderate (moderate) steatosis (two-thirds of the hepatocytes affected) and high-grade (severe) steatosis (more than two-thirds of the hepatocytes affected). If, during the diagnostic process, there is evidence of a different liver disease, the diagnosis should be sought immediately, if necessary with a liver biopsy.

Differential diagnostics

Commentary 

Differential diagnoses for NAFLD in childhood and adolescence:

• Nutritional disorders: Acute or chronic malnutrition, parenteral nutrition,

• Hepatopathies: Infectious hepatitis, autoimmune diseases (autoimmune hepatitis, primary sclerosing cholangitis PSC, inflammatory bowel disease, celiac disease), metabolic diseases (lysosomal acid lipase deficiency, Wilsonʼs disease, α1-antitrypsin deficiency, glycogen storage disease, familiar hyperlipoproteinemia, abetalipoproteinemia, oxidation- urea cycle disorders, hemochromatosis), endocrine diseases (after CNS surgery near the pituitary gland or chemotherapy, hypothyroidism, pituitary insufficiency),

• Disease syndromes: e. g. Bardet Biedl syndrome, Prader Willi syndrome, lipodystrophy,

• Hepatotoxic drugs: e. g. amiodarone, methotrexate, steroids, L-asparaginase, vitamin A, zidovudine and other “highly active antiretroviral therapy” (HAART) for HIV, valproate.

Ethical considerations for inclusion in clinical trials: 

Participation in a study is very desirable; non-participation has no effect on the therapy. Avoiding (repeated) liver biopsies in childhood using validated non-invasive markers is an important goal in order to be able to treat as many children as possible in studies. No non-invasive marker is currently sufficiently validated to adequately replace liver biopsy. Another goal of clinical trials is to develop new drug targets.

Histopathology of pediatric NAFLD

Commentary 

Schwimmer and coworkers have described two different forms of NASH in children [620]. Type 1 shows a picture comparable to that of NAFLD in adults. This form is characterized by steatosis in zone 3 (centrilobular) frequently associated with hepatocellular ballooning and the development of perisinusoidal fibrosis. It seems to be more common among adolescents.

Type 2 of childhood NAFLD is more common in younger children and shows a pronounced, partly panacinar steatosis in zone 1. The largest fat vacuoles are found periportally. Hepatocellular ballooning and perisinusoidal fiber deposits are usually absent, or these features are only mild. Although a milder lobular inflammation was initially described, the analysis of a larger cohort showed no statistically significant difference between the two variants of hepatic steatosis in children. Mallory bodies are very rarely found. However, children with periportal steatosis more pronounced in zone 1 have fibrosis emanating from the portal field. This occurs more often than in NASH with zone 3 steatosis already with septa present, and thus shows a potential for progression [620] [621]. Already in the original work by Schwimmer et al. as in other studies, an overlap between these two types of NAFLD in children could be demonstrated, which is why the classification does not seem practicable. In addition, the available data derive from cross-sectional studies, making it impossible to assess whether the two types of steatosis are prognostically relevant. It seems important that there are distinct steatosis phenotypes (in contrast to adults) and that NAFLD can also be progressive in children, although the formal criteria for diagnosing NASH (fat + ballooning + inflammatory foci) are not met.

In the differential diagnosis, Wilsonʼs disease, other, also rare, hepatic metabolic diseases or weight loss due to diarrhea in the context of chronic inflammatory bowel diseases with liver involvement must be considered. The diagnostic differentiation from viral hepatitis is particularly important.

Table 7 Histopathological criteria of infantile NAFLD.

Population	Children	Adolescents
Steatosis	Zone 1 up to panacinar	Zone 3 up to panacinar
Inflammation	More portal than intra-acinar inflammation, especially in the early stages	Intraacinar inflammation dominant
Extensive ballooning	Rare or absent, no Mallory-Denk bodies	In zone 3
Fibrosis	Incipient periportal fibrosis	Perisinusoidal fibrosis, in zone 3

Treatment: Prevention, lifestyle therapy/obesity therapy

Commentary 

With regard to lifestyle interventions and weight reduction, there is currently no systematic difference between children and adolescents with obesity and/or NAFLD The pillars of therapy include increasing exercise and modifying diet. The probability of weight loss and its maintenance increases with support from multidisciplinary teams [622] [623].

The multimodal lifestyle intervention is suitable for all children and adolescents for the primary treatment of NAFLD and has been shown to reduce the intrahepatic fat percentage [624] [625]. The combination of nutrition and exercise therapy is more effective than the respective individual intervention [626].

In a randomized intervention study with 40 children and adolescents with obesity and NAFLD, the dietary restriction of free sugars over 8 weeks led to a greater reduction in the hepatic fat content from 25 % to 17 % (measured by MR-PDFF) compared to the control group. In an 8-week randomized trial, the restriction of carbohydrates was superior to a low-fat diet when the daily caloric requirement was maintained, producing a significant reduction in hepatic fat content and an improvement in insulin resistance [627]. The prevention of metabolic syndrome in the sense of secondary prevention is achieved through early and long-term successful obesity therapy. Exercise is particularly effective in multimodal lifestyle therapy for improving insulin resistance [628]. Exercise therapy is probably also relevant for children of normal weight and NAFLD, as there is a connection between insulin resistance and skeletal muscle mass [629]. https://www.awmf.org/uploads/tx_szleitlinien/050-002l_S3_Therapie-Praevention-Adipositas-Kinder-%20Jugendliche_2019-11.pdf

Treatment: Bariatric procedure (surgery/endoscopy)

Commentary 

Long-term therapeutic success in children/adolescents with extreme obesity and a metabolic syndrome with NAFLD is rarely achieved. For this reason, bariatric procedures as a way of treating NAFLD in children/adolescents have been discussed controversially for years [630]. The basic indication for bariatric measures takes into account the characteristics of these age groups. Particularly in the case of irreversible bariatric surgical procedures (stomach reduction; gastric bypass), the lifelong consequences must be taken into account [631] [632]. On the other hand, pediatric patients with extreme obesity can undergo bariatric procedures to improve their metabolic situation and thus improve their NAFLD [633]. The greatest evidence is available for the methods of gastric reduction and gastric bypass [634]. For small numbers of cases, surrogate markers of NAFLD (transaminases, sonography, elastography, scores) are usually described over the course of the disease [635], and in individual studies also for bariatric endoscopic procedures such as gastric banding [636]. These show in each case a significant improvement in the NAFLD (based on the different markers), but there is a lack of uniform criteria and long-term observations. Only one pediatric study evaluates liver biopsies before and 1 year after stomach reduction in 20 adolescent patients and found significant improvements in both histology and adipokines [637] [638]. In this context, the usefulness of a routine intraoperative liver biopsy in pediatric patients is also discussed [639]. The small number and heterogeneity of pediatric studies on the topic of “bariatric procedures and NAFLD” currently do not allow any clear recommendations on indications or contraindications. On the contrary, there is an urgent need for a structured documentation and follow-up in this particular patient group. Recommendations for endoscopic bariatric procedures such as a gastric balloon are given in the S3 guideline on Obesity Surgery (AWMF Register No. 088–001) under certain conditions for adulthood. Due to insufficient studies, no recommendation can be made for adolescents.

Management: Pharmacological Therapy (Vitamin E & N-3)

Commentary 

Although in the Treatment of NAFLD in Children (TONIC) Trial [640], a multicenter, placebo-controlled study, neither vitamin E (800 IE/d) nor metformin (500 mg twice daily) could achieve the primary endpoint of a substantial and persistent ALT reduction in children and adolescents, there was a significantly reduction in NASH (58 % vs 28 %, p = 0.006) and a significant decrease in the histological activity index (–1.8 vs –0.7) in the vitamin E group. There was less ballooning in the metformin group. In adults, there are considerable safety concerns about treatment with vitamin E (increased all-cause mortality, stroke and prostate carcinoma) [353]. The long-term safety of high-dose vitamin E treatment in children is unknown [641].

Studies on the effectiveness of omega-3 fatty acids have yielded conflicting results. While a combination of eicosapentaenoic acid and docosahexaenoic acid showed no significant therapeutic benefit in one study [642], the administration of docosahexaenoic acid (250 mg/day) for 6 months led to a significant improvement in liver fat content and cardiometabolic risk factors in another study [643]. It also reduced ALT levels at follow-up more than a year later [644]. The response to DHA seems to correlate with PNPLA3 polymorphisms (I148 M variant less responsive) [645]. Choline combined with DHA and vitamin E showed a significant reduction in ALT and steatosis [646]. Administration of cysteamine bitartrate (CBDR) for 1 year led to a significant reduction in ALT and inflammation but did not improve histological scores [647].

Although the relationship between altered gut microbiome and NAFLD have been well documented, randomized trials on factors influencing the gut microbiome in NAFLD are rare. A randomized trial with 8 weeks of Lactobacillus rhamnosus strain GG (12 billion CFU/day) led to significantly lower ALT regardless of changes in BMI [648]; another study showed a significant decrease in steatosis under VSL # 3 treatment (Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum, Streptococcus salivarius subsp. thermophilus) lasting 4 months [649]. In view of the low risk of undesirable effects, further studies on the long-term course and cost-benefit analyses are required. Individual substances are currently being tested in phase 2 studies on pediatric populations, including the dual PPARα/δ agonist elafibranor and the AT1 antagonist losartan. Losartan inhibits the production of plasminogen activator inhibitor 1 and blocks the renin angiotensin system, thereby exerting an anti-inflammatory and anti-fibrotic effect. However, data on effectiveness are still pending (STOP NAFLD trial, NCT03467217).

Monitoring: Clinical management/connection to obesity/hepatology centers

Commentary 

The clinical management of pediatric NAFLD is a multidisciplinary challenge and includes care in a pediatric liver care center as well as a child obesity center. In addition to medical care, the integration of ecotrophology, physiotherapy and, if necessary, child and adolescent psychiatry as well as pediatric social care are decisive influencing factors for sustainable therapeutic success.

Monitoring: Non-invasive progress parameters (imaging/biomarkers)

Commentary 

Specific non-invasive biomarkers for assessing the progression of fibrosis are not sufficiently validated, but are urgently required for efficient risk stratification. The clinical assessment of the course of NAFLD in children and adolescents can relate in particular to the course of steatosis, the development of NASH and the quantification of fibrosis.

Anthropometric data (height, body weight, BMI, BMI percentile and SDS) including the physical status should always be collected. Any success of obesity therapy can also be documented within this context. As part of the laboratory workup, it is useful to regularly examine not only the liver parameters (ALT, AST, γ-GT) but also metabolic sequelae (fasting glucose, fasting insulin plasma levels, HOMA-IR, lipid profile).

An easy-to-use and inexpensive imaging technique is the ultrasound scan of the liver. However, the sensitivity is low and generally reliable proof of steatosis (device and operator-dependent) is only possible > 30 % fat accumulation in the liver [650]. Long-term monitoring can only provide a rough quantification of steatosis. The quality of the examination is further limited in extreme obesity. Detection of steatosis by MRI, e. g. using magnetic resonance proton density fat fraction MR-PDFF [651] [652] or measuring the hepatic fat fraction [653].

The prognosis of NAFLD in children is influenced by the development of NASH and progressive liver fibrosis. Non-invasive biomarkers (serum markers and imaging methods) for the detection of NASH and classification of the fibrosis degree were also increasingly being investigated in children and adolescents, but have not yet been adequately validated. Distinguished are clinical fibrosis scores (based on standard-of-care laboratory and clinical parameters), experimental serological biomarkers and imaging methods.

Clinical fibrosis scores were repeatedly examined in pediatric cohorts with histologically confirmed NAFLD. A South Korean study by Yang et al. [654] investigated 77 children and adolescents; it showed the highest test quality for the detection of moderate fibrosis (F ≥ 2) by determining the FIB-4 (AUROC 0.81). The validation of these data in a multicenter study conducted in the United States by Mansoor et al. [655] was unsuccessful and, with an AUROC of 0.69, markedly below the result described above. In fact, none of the examined fibrosis scores (AST/ALT ratio, FIB-4, NAFLD fibrosis score, APRI) showed sufficient test quality to detect any moderate or advanced fibrosis. A more recent study by Jackson et al. [656], who examined 146 children and adolescents with NAFLD, found AUROC values between 0.57 (NAFLD fibrosis score, PNFS [657]) and 0.67 (AST to platelet ratio index, APRI and pediatric NAFLD fibrosis index, PNFI) [658]. Interestingly, the determination of the ALT and the AST alone gave AUC-ROC values of 0.64 each. There is currently no superiority in the use of clinical fibrosis scores over measuring transaminase levels alone.

Another method for quantifying hepatic fibrosis is the determination of mechano-elastic tissue properties using elastography. For this purpose, both ultrasound-based modalities (transient elastography [659], Shear-Wave Elastography [660], Time-Harmonic Elastography [661]) and MR-based elastography [653] [662] are available. The diagnostic accuracy of these investigations is well above the results of clinical fibrosis scores (in particular AUROC > 0.87 for the detection of moderate fibrosis across all modalities), but further studies on independent cohorts are required to validate these methods. The diagnostic challenge lies particularly in patients with extreme obesity, since the penetration depth of the transient elastography is limited by the subcutaneous fatty tissue, and consequently leads to inaccurate measurements or are technically not feasible [663].

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