European guideline on indications, performance and clinical impact of 13Cbreath tests in adult and pediatric patients: An EAGEN, ESNM, and ESPGHAN consensus, supported by EPC

INTRODUCTION 13 C-breath tests are valuable, noninvasive diagnostic tests that can be widely applied for the assessment of gastroenterological symptoms and diseases. Currently, the potential of these tests is compromised by a lack of standardization regarding performance and interpretation among expert centers. METHODS This consensus-based clinical practice guideline defines the clinical indications, performance, and interpretation of 13 C-breath tests in adult and pediatric patients. A balance between scientific evidence and clinical experience was achieved by a Delphi consensus that involved 43 experts from 18 European countries. Consensus on individual statements and recommendations was established if 80% of reviewers agreed and <10% disagreed. RESULTS The guideline gives an overview over general methodology of 13 C-breath testing and provides recommendations for the use of 13 C-breath tests to diagnose Helicobacter pylori infection, measure gastric emptying time, and monitor pancreatic exocrine and liver function in adult and pediatric patients. Other potential applications of 13 C-breath testing are summarized briefly. The recommendations specifically detail when and how individual 13 C-breath tests should be performed including examples for well-established test protocols, patient preparation, and reporting of test results. CONCLUSION This clinical practice guideline should improve pan-European harmonization of diagnostic approaches to symptoms and disorders, which are very common in specialist and primary care gastroenterology practice, both in adult and pediatric patients. In addition, this guideline identifies areas of future clinical research involving the use of 13 C-breath tests.This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.AbstractIntroduction: 13 C-breath tests are valuable, noninvasive diagnostic tests that can be widely applied for the assessment of gastroenterological symptoms and diseases.Currently, the potential of these tests is compromised by a lack of standardization regarding performance and interpretation among expert centers.Methods: This consensus-based clinical practice guideline defines the clinical indications, performance, and interpretation of 13 C-breath tests in adult and pediatric patients. A balance between scientific evidence and clinical experience was achieved by a Delphi consensus that involved 43 experts from 18 European countries. Consensus on individual statements and recommendations was established if  80% of reviewers agreed and <10% disagreed. Results: The guideline gives an overview over general methodology of 13 C-breath testing and provides recommendations for the use of 13 C-breath tests to diagnose Helicobacter pylori infection, measure gastric emptying time, and monitor pancreatic exocrine and liver function in adult and pediatric patients. Other potential applications of 13 C-breath testing are summarized briefly. The recommendations specifically detail when and how individual 13 C-breath tests should be performed including examples for well-established test protocols, patient preparation, and reporting of test results.Conclusion:This clinical practice guideline should improve pan-European harmonization of diagnostic approaches to symptoms and disorders, which are very common in specialist and primary care gastroenterology practice, both in adult and pediatric patients. In addition, this guideline identifies areas of future clinical research involving the use of 13 C-breath tests.

# Introduction
Breath tests are valuable, noninvasive diagnostic tests that are widely applied for the assessment of gastroenterological symptoms and diseases. C-breath tests provide the opportunity to diagnose Currently, the potential of breath testing is compromised by a lack of standardization regarding performance and interpretation among expert centers. This is highly relevant because modifications of the volume and/or composition of the test meal, of test performance and of the evaluation of data may markedly influence test results, diagnosis and thus, clinical usefulness of the investigation.
This consensus-based clinical practice guideline is needed within the gastrointestinal (GI) community to enhance pan-European harmonization of diagnostic approaches to symptoms and disorders, which are very common in specialist and primary care gastroenterology practice, both in adult and in pediatric patients. The guideline can add significantly to quality of investigation and, thus, the welfare of gastroenterological patients because it will allow a more rational approach to diagnostic evaluation and treatment. The guideline also aims to minimize disparities between health care systems across Europe, to facilitate cooperation between expert groups and the performance of multicenter clinical trials.

# Methods
The structured procedure, which was developed for the creation of this consensus-based clinical practice guideline, has previously been published.Briefly, this procedure was initiated by three representatives of the contributing societies (heads of guideline, JK, HH, MF) and started with formation of a representative core group of experts nominated from all participating societies and associations. This core group developed statements and recommendations, which were then submitted to reviewers in a three-stage Delphi voting process. The KELLER ET AL.
-599 heads of guideline and the core group members are listed as authors; the reviewers are listed as members of the European C-breath test group.
The following key questions were addressed in the guideline:
1. What is the role of [fig_ref] TABLE 1: Descriptors of grading 1 [/fig_ref].Of note, we aimed to develop recommendations on the specific indications for 13 C-breath tests, whereas general indications for H. pylori testing, gastric emptying testing and monitoring of pancreatic exocrine or liver function were cited from current national and international guidelines.
Statements and recommendations were distributed among all reviewers via email for voting and commenting. All reviewers voted on all statements and recommendations according to the 6-point Likert scale given in [fig_ref] TABLE 2: Six-point Likert-scale 1 [/fig_ref] and gave comments in case of disagreement. "Agreement" was established if  80% of reviewers voted A+/A AND < 10% D+/D. Statements and recommendations, which did not receive agreement during the first Delphi round, were modified according to comments as previously described,and all modified statements and recommendations underwent further rounds of the Delphi process.
Statements that achieved "agreement" after three Delphi rounds were accepted. Percentages for agreement and disagreement are given for all recommendations and statements. Recommendations additionally include quality of evidence (Q) and strength of recommendation (S).The guideline is organized in a way that information, which will enable the reader to perform clinical tests, is summarized in statements, recommendations and respective tables. Background information detailed in the comments explains the choice of test protocols and further methodological aspects.

## General 13 c-breath test methodology
Since their introduction into clinical practice in the 1970s, [bib_ref] Clinical diagnosis with the stable isotope 13 C in CO2 breath tests:..., Schoeller [/bib_ref] stable isotope C-breath tests have gained considerable importance and have been recommended for distinct diagnostic purposes by national and international guidelines and expert consensus papers. [bib_ref] Management of Helicobacter pylori infection-the Maastricht V/Florence consensus report, Malfertheiner [/bib_ref] [bib_ref] Joint ESPGHAN/NASPGHAN guidelines for the management of Helicobacter pylori in children and..., Jones [/bib_ref] [bib_ref] Expert consensus document: advances in the diagnosis and classification of gastric and..., Keller [/bib_ref] [bib_ref] United European Gastroenterology evidencebased guidelines for the diagnosis and therapy of chronic..., Lohr [/bib_ref] This is due to the fact that they are not only reliable, but also noninvasive, relatively simple and safe diagnostic tools. C is a stable, nonradioactive carbon isotope with a natural abundance of about 1% of carbon isotopes. The use of non-radioactive stable-isotope tracers in biomedical experiments and diagnosis is generally considered ethically acceptable in humans at all ages. Toxicity of 13 C has been examined in animals given amounts far in excess of those employed in the clinic. Up to sixty percent enrichment with C was achieved over prolonged periods of time without negative effects in adult animals and without signs of teratogenicity or embryotoxicity. [bib_ref] Stable isotopes in clinical research: safety reaffirmed, Jones [/bib_ref] [bib_ref] Stable isotopes: origins and safety, Klein [/bib_ref] [bib_ref] Substantial replacement of mammalian body carbon with carbon-13, Gregg [/bib_ref] Thus, the small requirements of C as a tracer in most clinical studies, particularly relative to its naturally high abundance, precludes any discernible risk of toxicity. Accordingly, C-exhalation from marker substances depends not only on the process under investigation (e.g., gastric emptying), but also on absorption of the marker substance and/or its metabolites, further (mostly hepatic) metabolism leading to production of 13 CO 2 , its transport to the lung and pulmonary excretion and distribution to other body compartments leading to a relevant loss of label, for example, into muscles or bone. [bib_ref] 13 C]bicarbonate kinetics in humans: intra-vs. interindividual variations, Irving [/bib_ref] All of these functions as well as the process under investigation can be influenced by demographic and physiological parameters, concomitant diseases and drug intake. [bib_ref] Influence of age on 13 C-urea breath test results in children, Kindermann [/bib_ref] [bib_ref] [ 13 C]aminopyrine and [ 13 C]caffeine breath test: influence of gender,..., Caubet [/bib_ref] [bib_ref] Gastric emptying time of fluids and solids in healthy subjects determined by..., Hellmig [/bib_ref] [bib_ref] Effects of postgastric 13 C-acetate processing on measurement of gastric emptying: a..., Goetze [/bib_ref] [bib_ref] Dose-response relationship between fat ingestion and oxidation: quantitative estimation using whole-body calorimetry..., Sonko [/bib_ref] [bib_ref] The magnitude of the naturally occurring isotopic enrichment of 13 C in..., Mccue [/bib_ref] [bib_ref] Physical activity alters normal values of the "European standard" 13 C-octanoic acid..., Keller [/bib_ref] [bib_ref] 13 Cbreath tests in peritoneal dialysis patients: influence of dialysis fluids, Bammens [/bib_ref] [bib_ref] Factors affecting 13 C-natural abundance measurement of breath carbon dioxide during surgery:..., Eaton [/bib_ref] [bib_ref] Influence of clinical parameters on the results of 13 C-octanoic acid breath..., Keller [/bib_ref] [bib_ref] Clinical utility of 13 C-liver-function breath tests for assessment of hepatic function, Afolabi [/bib_ref] A study investigating the influence of clinical parameters on the results of 13 C-OABT in more than 1200 patients has shown that 13 Cexhalation was increased in women and correlated with age. Diabetes mellitus and inflammatory bowel disease were associated with decreased, and bacterial overgrowth and malignant disease with increased 13 C-exhalation. 39 summarizes the general principle of 13 C-breath testing.
What are the general prerequisites for performance of 13 C-breath tests for clinical or research purposes? -603 production. Therefore, physical activity has to be avoided during C-breath testing (100%, 0%; Q:C S:A).
Even moderate physical activity such as walking roughly doubles energy expenditure compared with sedentary subjects and has corresponding effects on endogenous CO 2 production. [bib_ref] Physical activity and human energy expenditure, Westerterp [/bib_ref] [bib_ref] America's obesity epidemic: measuring physical activity to promote an active lifestyle, Keim [/bib_ref] In addition, exercise leads to a shift toward oxidation of nonlipid components. This increases 13 C-exhalation because the lipid molecules in the body contain substantially lower concentrations of 13 C than the nonlipid molecules, due to fractionation processes during lipid synthesis. [bib_ref] The magnitude of the naturally occurring isotopic enrichment of 13 C in..., Mccue [/bib_ref] Moreover, 13 C-breath tests using orally applied marker substances depend on GI transit which is accelerated by moderate exercise, while strenuous exercise has opposite effects. [bib_ref] Systematic review: exercise-induced gastrointestinal syndrome-implications for health and intestinal disease, Costa [/bib_ref] Accordingly, it has been shown that physical activity during 13 C-OABT markedly alters 13 CO 2exhalation in healthy volunteers as well as respective normal values. [bib_ref] Physical activity alters normal values of the "European standard" 13 C-octanoic acid..., Keller [/bib_ref] In summary, physical activity has profound and complex effects

## Concomitant diseases affecting digestive and metabolic
processes which ultimately lead to exhalation of 13 C-enriched breath may confound breath test results. In particular, major disturbances of GI transit and absorption, hepatic and lung function have to be considered. However, the influence of concomitant diseases on many 13 C-breath tests appears to be small:
Even in patients with nonalcoholic steatohepatitis or liver cirrhosis (50% Child-Pugh score C), 13 C-octanoic acid metabolism was found to be normal [bib_ref] Oxidative breakdown of octanoic acid is maintained in patients with cirrhosis despite..., Van De Casteele [/bib_ref] [bib_ref] The continuous real-time 13 C-octanoate breath test for patients with nonalcoholic steatohepatitis..., Mawatari [/bib_ref] [bib_ref] Total body metabolism of 13 C-octanoic acid is preserved in patients with..., Schneider [/bib_ref] and the 13 C-OABT correlated well with scintigraphy in patients who were critically ill. [bib_ref] Gastric emptying of a liquid nutrient meal in the critically ill: relationship..., Chapman [/bib_ref] Disturbances of 13 CO 2 -exhalation in patients with lung diseases are only expected in patients with very severe disease and reduced CO 2 -diffusion capacity. Data on potential impairment of 13 C-breath tests in such cases are not available in the literature.
Since digestion of dietary lipids by pancreatic lipase cannot occur before the meal has entered the duodenum, results of the 13 C-MTGT are influenced by and correlate with gastric emptying parameters. 14 This predominantly applies to early postprandial 13 C-exhalation rates (1-3 h), which have insufficient specificity for detection of PEI.
However, this problem can be overcome by prolongation of the breath sampling period so that gastric emptying is of minor importance for 13 C-exhalation.
Vice versa, 13 CO 2 -excretion following administration of 

## C-urea breath test
H. pylori is a common bacterial pathogen responsible for substantial GI morbidity worldwide. In addition to causing inflammatory gastroduodenal alterations, H. pylori is the major risk factor for gastric cancer development and is associated with various other, partly non-GI diseases. [fig_ref] TABLE 3: Indications for H [/fig_ref] 

## Summarizes important indications for
H. pylori testing in adults and children recommended by current European guidelines. [bib_ref] Management of Helicobacter pylori infection-the Maastricht V/Florence consensus report, Malfertheiner [/bib_ref] [bib_ref] Joint ESPGHAN/NASPGHAN guidelines for the management of Helicobacter pylori in children and..., Jones [/bib_ref] There are differences in the approach to H. pylori infection between adults and children. Thus, the European Society of Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) explicitly recommends against a test and treat strategy in children, as well as against H. pylori testing as part of the initial investigation in children Meta-analyses confirm that 13 C-UBT achieves 95% sensitivity and specificity in both adults and children. [bib_ref] Non-invasive diagnostic tests for Helicobacter pylori infection, Best [/bib_ref] Theoretically, food in the stomach may dilute the marker substance and thereby impair the contact between the infected mucosa and the substrate leading to lower 13 CO 2 production and decreased sensitivity of the test. Although this aspect is still controversial, some studies suggest that fasting before the 13 C-UBT indeed improves accuracy of test results. [bib_ref] 13 C urea breath test with nondispersive isotope-selective infrared spectrometry: reproducibility and..., Mana [/bib_ref] [bib_ref] Review article: 13 C-urea breath test in the diagnosis of Helicobacter pylori..., Gisbert [/bib_ref] Accordingly, it appears to be prudent to perform the 13 C-UBT in fasting conditions. [bib_ref] Review article: 13 C-urea breath test in the diagnosis of Helicobacter pylori..., Gisbert [/bib_ref] In adults, fasting overnight appears reasonable. If this is not feasible, a fasting period of 4-6 h is deemed sufficient. In young children, long fasting periods may be problematic. However, sensitivity of the 13 C-UBT was markedly reduced to about 50% when children were fed a meal immediately before the test. [bib_ref] Carbon 13-labeled urea breath test for the diagnosis of Helicobacter pylori infection..., Rowland [/bib_ref] Therefore, a fasting period of 4 h is suggested, including in young children.

## Recommendation 2.7
Prior to the patients. [bib_ref] Expert consensus document: advances in the diagnosis and classification of gastric and..., Keller [/bib_ref] [bib_ref] Gastroparesis and functional dyspepsia: excerpts from the AGA/ANMS meeting, Parkman [/bib_ref] However, prior to gastric emptying testing, the exclusion of mucosal or structural disorders such as inflammatory or malignant diseases as the underlying cause of symptoms is required. [bib_ref] Expert consensus document: advances in the diagnosis and classification of gastric and..., Keller [/bib_ref] Specific indications for gastric emptying testing in adults as suggested by international guidelines and expert consensus papers are given in. [bib_ref] Expert consensus document: advances in the diagnosis and classification of gastric and..., Keller [/bib_ref] [bib_ref] Clinical guideline: management of gastroparesis, Camilleri [/bib_ref] [bib_ref] Management strategies for gastrointestinal, erectile, bladder, and sudomotor dysfunction in patients with..., Kempler [/bib_ref] For pediatric patients, there are no generally accepted guideline recommendations on gastric emptying testing. The recommendations given inare derived from a recent review of the literature, which shows that nausea, vomiting, and abdominal pain are the most common symptoms in children, while early satiety, postprandial fullness, bloating, and weight loss occur less frequently in pediatric gastroparesis. 72
When should 13 C-GEBT be utilized?.
Pancakes marked with 13 C-octanoic acid are an acceptable and palatable solid test meal for children but cannot be used in case of allergy to egg, milk or wheat, in coeliac disease or very young children. [bib_ref] Gastric emptying of solids in children: reference values for the (13) C-octanoic..., Hauser [/bib_ref] [bib_ref] Variability of the 13 C-octanoic acid breath test for gastric emptying of..., Hauser [/bib_ref] The lack of standardization is of concern because it makes Scintigraphic equivalent values or breath test data corrected for scintigraphy according to [bib_ref] Measurement of gastric emptying rate of solids by means of a carbon-labeled..., Ghoos [/bib_ref]. [bib_ref] Measurement of gastric emptying rate of solids by means of a carbon-labeled..., Ghoos [/bib_ref] [Corrections added on June 28, 2021 after first online publication: Typos have been corrected in.] KELLER ET AL.
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patients. [bib_ref] A stable isotope breath test with a standard meal for abnormal gastric..., Szarka [/bib_ref] The test was approved by the FDA for evaluation of gastric emptying in 2015 (https://www.accessdata.fda.gov/scripts/ cdrh/cfdocs/cfpma/pma.cfm?id=P110015), and it is also CE marked according to the producer's information (https://sercon-instruments.
com/wp-content/uploads/2017/06/019-gebt.pdf). However, it has not been marketed in Europe, so far. C-acetate has been used as a marker for acaloric and caloric liquids such as formula diets. [bib_ref] Is the 13 C-acetate breath test a valid procedure to analyse gastric..., Gatti [/bib_ref] [bib_ref] Simultaneous 13 C/14C dual isotope breath test measurement of gastric emptying of..., Chew [/bib_ref] [bib_ref] Measuring gastric emptying of semisolids in children using the 13 C-acetate breath..., Braden [/bib_ref] [bib_ref] Reproducibility and reliability of the 13 C-acetate breath test to measure gastric..., Barbosa [/bib_ref] [bib_ref] Comparison between gastric scintigraphy and the [ 13 C]-acetate breath test with..., Sanaka [/bib_ref] In young children or children dependent on gastrostomy feeding, the test meal often consists of milk or milk-based formulas. [bib_ref] Variability of the 13 C-acetate breath test for gastric emptying of liquids..., Hauser [/bib_ref] Apple juice has also been used. [bib_ref] Gastric emptying time of fluids and solids in healthy subjects determined by..., Hellmig [/bib_ref] Results of the test closely correlate with those of scintigraphy in adults and children. [bib_ref] Is the 13 C-acetate breath test a valid procedure to analyse gastric..., Gatti [/bib_ref] The Wagner-Nelson method has been suggested for analysis of C-GEBT by Sanaka et al. [bib_ref] The Wagner-Nelson method can generate an accurate gastric emptying flow curve from..., Sanaka [/bib_ref] This method has been developed to describe the entrance of ingested drugs into the venous system based on its urinary excretion data. When applied on breath tests, it describes the manner in which 13 CO 2 appears in the venous system based on pulmonary 13 CO 2 excretion data. [bib_ref] The Wagner-Nelson method can generate an accurate gastric emptying flow curve from..., Sanaka [/bib_ref] Dietary and other restrictions, which generally apply before and during 13 C-breath testing, have been explained in General Methodology. Physical activity, in particular, has to be avoided during 13 C-GEBT, not only to standardize CO 2 -production but also because physical activity influences gastric emptying velocity. [bib_ref] Systematic review: exercise-induced gastrointestinal syndrome-implications for health and intestinal disease, Costa [/bib_ref] Indications for pancreatic function testing in adults and children as recommended by international guidelines are given in. [bib_ref] United European Gastroenterology evidencebased guidelines for the diagnosis and therapy of chronic..., Lohr [/bib_ref] [bib_ref] English language version of the S3-consensus guidelines on chronic pancreatitis: definition, aetiology,..., Hoffmeister [/bib_ref] [bib_ref] Guidelines for the investigation of chronic diarrhoea, Thomas [/bib_ref] [bib_ref] Nutritional considerations in pediatric pancreatitis: a position paper from the NASPGHAN Pancreas..., Abu-El-Haija [/bib_ref] When should 13 C-PFBT be utilized? [bib_ref] triglyceride breath test: methodological problems and clinical applications, Mixed [/bib_ref] Currently, the original 127 or modified versions [bib_ref] 13 C-mixed triglyceride breath test to assess oral enzyme substitution therapy in..., Dominguez-Munoz [/bib_ref] [bib_ref] )C-mixed triglyceride breath test detects moderate pancreatic exocrine insufficiency, Keller [/bib_ref] [bib_ref] Sensitivity and specificity of an abbreviated (13)C-mixed triglyceride breath test for measurement..., Keller [/bib_ref] [bib_ref] 13 C-mixed triglyceride breath test for evaluation of pancreatic exocrine function in..., Keller [/bib_ref] of the with specificity ranging between 80% and 90% in adults. [bib_ref] English language version of the S3-consensus guidelines on chronic pancreatitis: definition, aetiology,..., Hoffmeister [/bib_ref] A modified test using comparably high lipid loads in subjects explicitly avoiding physical activity during testing reached high sensitivity and specificity rates even in mild to moderate PEI (100% and 92%, respectively). [bib_ref] )C-mixed triglyceride breath test detects moderate pancreatic exocrine insufficiency, Keller [/bib_ref] However, as evident from the test principle, the 13 C-MTGBT is a test of lipid digestion and absorption. Therefore, the 13 C-MTGBT is accepted as an appropriate alternative to the coefficient of fat absorption, both for the diagnosis of PEI and for evaluating the efficacy of pancreatic enzyme replacement therapy (PERT) in clinical practice. [bib_ref] United European Gastroenterology evidencebased guidelines for the diagnosis and therapy of chronic..., Lohr [/bib_ref] [bib_ref] 13 C-mixed triglyceride breath test to assess oral enzyme substitution therapy in..., Dominguez-Munoz [/bib_ref] [bib_ref] Development and diagnostic accuracy of a breath test for pancreatic exocrine insufficiency..., Dominguez-Munoz [/bib_ref] [bib_ref] 13 C-mixed triglyceride breath test for evaluation of pancreatic exocrine function in..., Keller [/bib_ref] [bib_ref] Comparison of 72-hour fecal fat quantification and the 13 C-mixed triglyceride breath..., Wejnarska [/bib_ref] On the other hand, 13 CO 2 exhalation is not only decreased by lipase deficiency but also by other causes of lipid malabsorption, for example, celiac disease, short bowel syndrome, or postcibal asynchrony following gastric resection. [bib_ref] Analysis of fat digestive and absorptive function after subtotal gastrectomy by a..., Nakamura [/bib_ref] Thus, the specificity of the test for the differential diagnosis of chronic diarrhea is limited.
Unfortunately, fecal elastase, the other noninvasive test which is predominantly used for pancreatic function testing in the clinic, also has limited specificity in these cases.Direct comparisons between C-MTGBT and fecal elastase favor the 13 C-MTGBT for diagnosis of steatorrhea, [bib_ref] Usefulness of a 13 C-labeled mixed triglyceride breath test for assessing pancreatic..., Nakamura [/bib_ref] and generally in patients with chronic pancreatitis after pancreatic resections. [bib_ref] Diagnosis of exocrine pancreatic insufficiency in chronic pancreatitis: (13)C-mixed triglyceride breath test..., Gonzalez-Sanchez [/bib_ref] In infants with cystic fibrosis, sensitivity of the 13 C-MTGBT for diagnosis of steatorrhea was high, but specificity was low.Compared to 13 C-GEBT, A mathematical correction for altered CO 2 -production rates in physically active subjects using heart rate recordings improves diagnostic accuracy of the 13 C-MTGBT in non-resting subjects. [bib_ref] Improving the specificity of the [ 13 C]mixed triacylglycerol breath test by..., Slater [/bib_ref] However, under clinical conditions, it is pragmatic to request that patients remain seated throughout the test. [Corrections added on June 28, 2021 after first online publication: Typos have been corrected in].

## -
UNITED EUROPEAN GASTROENTEROLOGY JOURNAL PERT. [bib_ref] 13 C-mixed triglyceride breath test to assess oral enzyme substitution therapy in..., Dominguez-Munoz [/bib_ref] [bib_ref] Development and diagnostic accuracy of a breath test for pancreatic exocrine insufficiency..., Dominguez-Munoz [/bib_ref] To answer this question, subjects are allowed to continue with their usual enzyme replacement therapy. In contrast to these "static" liver function tests, "dynamic" quantitative tests measure the elimination of a substance, which is cleared and/or metabolized almost exclusively by the liver via specific metabolic pathways in subcellular compartments, for example, by cytochromes for microsomal liver function or cytosolic or mitochondrial enzymes. 151 Accordingly, these tests constitute a more accurate measure of the specific aspects of liver function.
Established dynamic quantitative liver function tests are the indocyanine green clearance test and the galactose elimination capacity test. C-LFBT also represent dynamic tests with oral consumption or intravenous application of the marker substance and measurement of the end product of hepatic metabolism, that is exhaled 13 CO 2 . Some modifications have been shown to detect early changes in liver metabolic capacity in patients, prior to the presence of structural damage to the liver (i.e., inflammation, fibrosis), 152 though with limited sensitivities and specificities. [bib_ref] The utility of the 13 C-galactose breath test as a measure of..., Saadeh [/bib_ref] [bib_ref] 13 C-galactose breath test and 13 C-aminopyrine breath test for the study..., Giannini [/bib_ref] [bib_ref] 13 C-methacetin breath test as liver function test in patients with chronic..., Braden [/bib_ref] [bib_ref] The non-invasive (13)C-methionine breath test detects hepatic mitochondrial dysfunction as a marker..., Banasch [/bib_ref] [bib_ref] Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis, Portincasa [/bib_ref] [bib_ref] 13 C-aminopyrine breath test accurately predicts long-term outcome of chronic hepatitis C, Rocco [/bib_ref] [bib_ref] Liver function breath tests for differentiation of steatohepatitis from simple fatty liver..., Tribonias [/bib_ref] [bib_ref] Noninvasive estimation of disease activity and liver fibrosis in nonalcoholic fatty liver..., Korkmaz [/bib_ref] Still, the dynamic nature of 13 C-LFBT, their possible versatility in terms of assessing a range of different liver functions, and the ease with which they can be repeated to follow relative changes in liver function with time, generally imply a marked potential for clinical application. [bib_ref] Clinical utility of 13 C-liver-function breath tests for assessment of hepatic function, Afolabi [/bib_ref] When should 13 C-LFBT be utilized? C-aminopyrine, yl- C]-caffeine) and cytosolic ( 13 C-phenylalanine) function. [bib_ref] 13 C-aminopyrine demethylation is decreased in cirrhotic patients with normal biochemical markers, Afolabi [/bib_ref] [bib_ref] Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis, Portincasa [/bib_ref] [bib_ref] Liver function breath tests for differentiation of steatohepatitis from simple fatty liver..., Tribonias [/bib_ref] [bib_ref] Noninvasive estimation of disease activity and liver fibrosis in nonalcoholic fatty liver..., Korkmaz [/bib_ref] [bib_ref] The characterisation of hepatic mitochondrial function in patients with non-alcoholic fatty liver..., Afolabi [/bib_ref] [bib_ref] Longitudinal effects of hepatitis C virus treatment on hepatic mitochondrial dysfunction assessed..., Banasch [/bib_ref] [bib_ref] The role of 1 3C-methacetin breath test for the noninvasive evaluation of..., Fierbinteanu-Braticevici [/bib_ref] [bib_ref] 13 C-Methacetin breath testing in patients with nonalcoholic fatty liver disease, Kempinski [/bib_ref] [bib_ref] 13 C-methacetin-breath test compared to also noninvasive biochemical blood tests in predicting..., Dinesen [/bib_ref] [bib_ref] Relationship between 13 C-aminopyrine breath test and the MELD score and its..., Giannini [/bib_ref] [bib_ref] Validity of the 13 C-caffeine breath test as a noninvasive, quantitative test..., Park [/bib_ref] [bib_ref] Evaluation of 13 C-phenylalanine and 13 C-tyrosine breath tests for the measurement..., Ishii [/bib_ref] These have been mainly used in patients with liver fibrosis and cirrhosis due to nonalcoholic fatty liver disease, [bib_ref] 13 C-aminopyrine demethylation is decreased in cirrhotic patients with normal biochemical markers, Afolabi [/bib_ref] [bib_ref] Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis, Portincasa [/bib_ref] [bib_ref] Liver function breath tests for differentiation of steatohepatitis from simple fatty liver..., Tribonias [/bib_ref] [bib_ref] Noninvasive estimation of disease activity and liver fibrosis in nonalcoholic fatty liver..., Korkmaz [/bib_ref] [bib_ref] The characterisation of hepatic mitochondrial function in patients with non-alcoholic fatty liver..., Afolabi [/bib_ref] [bib_ref] The role of 1 3C-methacetin breath test for the noninvasive evaluation of..., Fierbinteanu-Braticevici [/bib_ref] [bib_ref] 13 C-Methacetin breath testing in patients with nonalcoholic fatty liver disease, Kempinski [/bib_ref] [bib_ref] Non-alcoholic fatty liver disease causes dissociated changes in metabolic liver functions, Lykke Eriksen [/bib_ref] [bib_ref] 13 C-methionine breath test detects distinct hepatic mitochondrial dysfunction in HIV-infected patients..., Banasch [/bib_ref] chronic hepatitis C, [bib_ref] Longitudinal effects of hepatitis C virus treatment on hepatic mitochondrial dysfunction assessed..., Banasch [/bib_ref] [bib_ref] 13 C-methacetin-breath test compared to also noninvasive biochemical blood tests in predicting..., Dinesen [/bib_ref] cirrhosis, [bib_ref] Relationship between 13 C-aminopyrine breath test and the MELD score and its..., Giannini [/bib_ref] [bib_ref] Noninvasive diagnosis and prognosis of liver cirrhosis: a comparison of biological scores,..., Forestier [/bib_ref] and hepatocellular carcinoma. [bib_ref] Liver function as assessed by breath tests in patients with hepatocellular carcinoma, Palmieri [/bib_ref] Additional potential fields of application are in steatohepatitis, [bib_ref] The non-invasive (13)C-methionine breath test detects hepatic mitochondrial dysfunction as a marker..., Banasch [/bib_ref] [bib_ref] Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis, Portincasa [/bib_ref] [bib_ref] Liver function breath tests for differentiation of steatohepatitis from simple fatty liver..., Tribonias [/bib_ref] fatty liver, [bib_ref] Noninvasive estimation of disease activity and liver fibrosis in nonalcoholic fatty liver..., Korkmaz [/bib_ref] [bib_ref] The role of 1 3C-methacetin breath test for the noninvasive evaluation of..., Fierbinteanu-Braticevici [/bib_ref] [bib_ref] Non-alcoholic fatty liver disease causes dissociated changes in metabolic liver functions, Lykke Eriksen [/bib_ref] the effect of transarterial chemoembolization on liver function has been monitored by C-methacetin test, [bib_ref] Monitoring liver function of patients undergoing transarterial chemoembolization (TACE) by a 13..., Barzakova [/bib_ref] and several studies suggest that this test could also be used for planning of hepatic resections. [bib_ref] First intraoperative measurement of liver functional capacity during liver surgery using the..., Makridis [/bib_ref] [bib_ref] Reductions in post-hepatectomy liver failure and related mortality after implementation of the..., Jara [/bib_ref] [bib_ref] The LiMAx test: a new liver function test for predicting postoperative outcome..., Stockmann [/bib_ref] Given that, as yet, few studies have been performed involving small groups of children with rare diseases, 178-181 the use of [Corrections added on June 28, 2021 after first online publication: In [fig_ref] TABLE 9: Examples of validated test protocols for 13 C-liver function breath tests in... [/fig_ref] , typos have been corrected. In the 3rd column, 2nd row, "postprandially" has been deleted from "At baseline and at 10 min [fig_ref] TABLE 1: Descriptors of grading 1 [/fig_ref] and demonstrate the very broad potential applicability of C-breath test technology.
[formula] intervals [/formula]

## Conclusions and future perspectives
This consensus-based clinical practice guideline aims to assist physicians and provide them with the information required to perform high quality 13 C-breath tests for patients with various GI symptoms and diseases. In a consensus process, in which representatives from all regions of Europe and specialists representing European scientific societies participated, the available evidence was evaluated, taking account of local facilities, diverse clinical practice, and health care environments. Patient involvement was not covered in our guideline because of its focus on diagnostic recommendations. However, after publication of the guideline, a qualitative and quantitative assessment of guideline adoption into clinical practice is planned, including patient and public involvement.
The guideline gives an overview over general methodology of C-breath testing and provides recommendations for the use of -617 parameters, concomitant diseases, and/or medication affecting these processes may confound breath test results. In fact, studies have shown that the influence of concomitant diseases on many 13 Cbreath tests is small or can be prevented by adaptation of the breath test protocol. Information on general 13 C-breath test methodology as detailed in the guideline will help clinicians to optimize patient selection and preparation and to better understand and interpret breath test results for individual patients.
As depicted in , 13 C-UBT has excellent sensitivity and specificity for diagnosis of H. pylori infection, superior to most other tests, it is highly standardized and well established in the clinic. C-GEBT and 13 C-PFBT are validated and accepted tests for diagnosis of gastric emptying disturbances and PEI, respectively. So far, their wide spread use has been hampered by the lack of standardization among centers, which we aim to improve by this guideline. Moreover, the tests are currently not commercially available in Europe, so that test meals need to be prepared on site. C-LFBT are currently used by highly specialized centers, only. They have the potential to measure various aspects of liver function, including assessment of prognosis in chronic liver disease. However, their clinical role, in general, and applicability in pediatric patients, in particular, still need to be established.
Apart from identification of this and other areas of future research, the guideline should improve pan-European harmonization of diagnostic approaches to symptoms and disorders, which are very common in specialist and primary care gastroenterology practice, both in adult and pediatric patients.

# Acknowledgments
This guideline was developed with the support of an UEG Activity Grant. [bib_ref] Correlation of a [ 13 C]glucose breath test with surrogate markers of..., Banerjee [/bib_ref] [bib_ref] Non-invasive 13 C-glucose breath test using residual gas analyzermass spectrometry: a novel..., Ghosh [/bib_ref] [bib_ref] 13 C]glucose breath testing provides a noninvasive measure of insulin resistance: calibration..., Hussain [/bib_ref] [bib_ref] A novel, noninvasive 13 C-glucose breath test to estimate insulin resistance in..., Jetha [/bib_ref] [bib_ref] Investigation of metabolism of exogenous glucose at the early stage and onset..., Kawagoe [/bib_ref] [bib_ref] Comparison of the [ 13 C] glucose breath test to the hyperinsulinemic-euglycemic..., Lewanczuk [/bib_ref] [bib_ref] The 13 C-glucose breath test for insulin resistance assessment in adolescents: comparison..., Maldonado-Hernandez [/bib_ref] [bib_ref] Assessment of insulin resistance by a 13 C glucose breath test: a..., Mizrahi [/bib_ref] [bib_ref] The 13 C-glucose breath test is a valid non-invasive screening tool to..., Salas-Fernandez [/bib_ref] [bib_ref] )C-glucose breath tests: a non-invasive method for detecting early clinical manifestations of..., Takemoto [/bib_ref] Insulin resistance, metabolic syndrome 13 C-glucose + unmarked glucose (e.g., 75 g) dissolved in water, outcome parameters:

## Conflict of interest
Cumulative 13 CO 2 -exhalation, maximal DOB-value, C-exhalation at specific time point C-exhalation is significantly decreased in patients with impaired insulin resistance/ metabolic syndrome, site of labeled carbon atom in glucose molecule is relevant for metabolic results
Breath test described as valid surrogate index of clamp-derived measures of insulin resistance, with good accuracy and precision, but controversial findings regarding applicability as screening tool (insufficient accuracy) in minority of studies Non-invasive tool for early diagnosis and follow up of patients in high-risk groups [bib_ref] Use of the lactose-[ 13 C]ureide breath test for diagnosis of small..., Berthold [/bib_ref] [bib_ref] The use of 13 C-labelled glycosyl ureides for evaluation of orocaecal transit..., Wutzke [/bib_ref] [bib_ref] Gastrointestinal handling of glycosyl [ 13 C]ureides, Morrison [/bib_ref] [bib_ref] Lactose-[ 13 C]ureide breath test: a new, noninvasive technique to determine orocecal..., Van Den Driessche [/bib_ref] SIBO, OCTT Lactose-[ 13 C]ureide, moderate sensitivity (66.7%), high specificity (100%) for SIBO in comparison with culture from jejunal aspirates, glucose-[ 13 C]ureide could be used alternatively Bacterial metabolism of marker is prerequisite for 13 CO 2 -exhalation, increase caused by SIBO or arrival of the marker substance in the colon Predosing with unlabelled ureide is frequently part of the protocol, induces enzyme induction, increases rate of 13 CO 2 -recovery but complicates procedure [bib_ref] 13 Cstarch breath test--comparative clinical evaluation of an indirect pancreatic function test, Loser [/bib_ref] [bib_ref] Feasibility of a breath test with a substrate of natural 13 C-abundance..., Jonderko [/bib_ref] [bib_ref] 13 C-Labeled-Starch breath test in congenital sucrase-isomaltase deficiency, Robayo-Torres [/bib_ref] [bib_ref] 13 C-breath tests for sucrose digestion in congenital sucrase isomaltase-deficient and sacrosidasesupplemented..., Robayo-Torres [/bib_ref] PEI (amylase deficiency)

## Congenital sucrase-isomaltase deficiency
Maize starch is naturally enriched with 13 C, 50-100 g used as pancreatic function test, but limited sensitivity (up to 77%) and specificity (up to 74%) in comparison with "gold standard" (secretin cerulein test) CO 2 release following labelled starch or sucrose is decreased in patients with sucraseisomaltase deficiency [bib_ref] 13 C-erythromycin breath test as a noninvasive measure of CYP3A activity in..., De Wildt [/bib_ref] [bib_ref] 13 C]pantoprazole breath test as a predictor of the anti-platelet function of..., Furuta [/bib_ref] [bib_ref] C]-pantoprazole breath test to predict CYP2C19 phenotype and efficacy of a proton..., Furuta [/bib_ref] [bib_ref] The effect of proton pump inhibitors on the CYP2C19 enzyme activity evaluated..., Modak [/bib_ref] [bib_ref] Evaluation of a [ 13 C]-dextromethorphan breath test to assess CYP2D6 phenotype, Leeder [/bib_ref] [bib_ref] The use of the 13 C-dextromethorphan breath test for phenotyping CYP2D6 in..., Opdam [/bib_ref] [bib_ref] Evaluation of CYP2D6 enzyme activity using a 13 Cdextromethorphan breath test in..., Safgren [/bib_ref] [bib_ref] Rapid identification of dihydropyrimidine dehydrogenase deficiency by using a novel 2-13 C-uracil..., Mattison [/bib_ref] [bib_ref] The uracil breath test in the assessment of dihydropyrimidine dehydrogenase activity: pharmacokinetic..., Mattison [/bib_ref] Activity of drug metabolizing enzymes 13 C-erythromycin applied iv (or orally)  hepatic (and intestinal) cytochrome P450 3A4
and 3A5 (CYP3A4/5) activity in vivo, pilot studies 13 C-pantoprazole  CYP2C19 activity, can predict the anti-platelet efficacy of clopidogrel (which is metabolized by CYP2C19 to its active metabolite) and high metabolization rate of PPI C-dextromethorphan  CYP2D6 activity (e.g., relevant for activation of the prodrug tamoxifen), C]uracil  dihydropyrimidine dehydrogenase (DPD) deficiency to predict 5-fluorouracil dose-related toxicity Abbreviations: DOB,  over baseline; OCTT, oro-cecal transit time; PEI, pancreatic exocrine insufficiency; PPI, proton pump inhibitor; SIBO, small intestinal bacterial overgrowth.

## -

## United european gastroenterology journal

# Data availability statement
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

## Orcid

## Jutta keller
https://orcid.org/0000-0002-5884-1115
Heinz F. Hammer https://orcid.org/0000-0001-8683-4951
Mark R. Fox https://orcid.org/0000-0003-4394-5584

## Figure 2
General principle, performance and clinical role of 13 C-breath tests used in gastroenterology. C-urea used for detection of H. pylori ( 13 C-UBT, marked in green) is metabolized by bacterial urease to produce 13 CO 2, which is absorbed, transported to the lung (broken green arrow) and exhaled. For 13 C-gastric emptying breath tests ( 13 C-GEBT, marked in red), C-pancreatic function breath test ( 13 C-PFBT, marked in brown), and 13 C-liver function breath tests ( 13 C-LFBT, marked in blue), orally applied substrates or their metabolites are absorbed in the small intestine. Subsequently, they are transported to the liver where they undergo further metabolization with production of 13 CO 2 , which is transported to the lung and exhaled [Corrections added on June 28, 2021 after first online publication: (image and caption) has been revised.]
[fig] Recommendation 4. 6: Drugs with potential influence on gastrointestinal transit and/or lipid digestion and absorption should be avoided before the test. The duration of withdrawal depends on the halflife of the drug, 48-72 h are usually sufficient (91%, 3%; Q:C S:B). [/fig]
[fig] 13: C-LIVER FUNCTION BREATH TESTSEstablished parameters for the assessment of liver function under routine clinical conditions are measurement of bilirubin, albumin, liver enzymes and parameters of coagulation factor synthesis in serum or plasma, respectively. Clinical prognostic grading systems (e.g., Child-Pugh score, Model for End-stage Liver Disease score) combine several of these biochemical parameters including clinical symptoms of advanced liver cirrhosis.150 [/fig]
[table] TABLE 1: Descriptors of grading 1 [/table]
[table] TABLE 2: Six-point Likert-scale 1 [/table]
[table] TABLE 3: Indications for H. pylori testing in adults and children4,5 antibody-based ELISA is used.4 Some serology tests have high sensitivity and specificity, but these tests may perform differently in different geographic locations according to the antigenic composition of the circulating strains. Thus, the Maastricht V/Florence Consensus recommends that only locally validated tests should be used.4 [/table]
[table] Table 4: As discussed above, alterations of marker substance, dose or other components of the test meal/solution can markedly influence test results. Hence, it is of pivotal importance that tests are validated in representative patient populations and that clinicians adhere to strictly standardized study protocols. Since the original description of the 13 C-UBT by Graham et al., 57 several changes of the test protocol have been proposed affecting the dose of 13 Curea, type of test meal/solution, time of breath collection, cut-off values, and measuring device. 58 Logan et al. first introduced a modified version with only one breath sample to be analyzed, however this was pooled from several samples collected over 30 min at 5 min intervals. 59 Currently, most studies report DOB values of a single sample collected 30 min after application of 13 Curea with a threshold of >4 for diagnosis of H. pylori infection. At minority of studies used sampling periods of 10 or 20 min and thresholds between DOB>3 and DOB>6. 50 13 C-urea was usually applied with a citric acid solution; however, orange juice 54or semiliquid meals have also been used.55 Examples for wellestablished test protocols are given inTable 4. [/table]
[table] C -: LFBT for clinical purposes cannot be recommended in the pediatric population, so far. should 13 C-LFBT be performed?Recommendation 5.3 13 C-liver function breath tests performed for clinical purposes have to adhere strictly to standardized and adequately validated study protocols. This refers to preparation of the patient, test meal/solution, test performance and evaluation of test results (for examples compareTable 9) (100%, 0%; Q:C S:A). [/table]
[table] TABLE 9: Examples of validated test protocols for 13 C-liver function breath tests in adults Abbreviations: HC, healthy controls; NAFLD, non-alcoholic Fatty Liver disease; NASH, nonalcoholic steatohepatitis; NPV, negative predictive value; PPV, positive predictive value; pp, postprandial; SENS, sensitivity; SPEC, specificity. [/table]