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

Abstract Introduction 13C‐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 13C‐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 13C‐breath testing and provides recommendations for the use of 13C‐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 13C‐breath testing are summarized briefly. The recommendations specifically detail when and how individual 13C‐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 13C‐breath tests.


INTRODUCTION
Breath tests are valuable, noninvasive diagnostic tests that are widely applied for the assessment of gastroenterological symptoms and diseases. 13 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. 1 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 13 C-breath test group.
The following key questions were addressed in the guideline: 1. What is the role of 13 Table 1. 1 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 Table 2 1 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, 1 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). 1 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, 3 stable isotope 13 C-breath tests have gained considerable importance and  have been recommended for distinct diagnostic purposes by national and international guidelines and expert consensus papers. [4][5][6][7] This is due to the fact that they are not only reliable, but also noninvasive, relatively simple and safe diagnostic tools. 13 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 13 C was achieved over prolonged periods of time without negative effects in adult animals and without signs of teratogenicity or embryotoxicity. [8][9][10] Thus, the small requirements of 13 C as a tracer in most clinical studies, particularly relative to its naturally high abundance, precludes any discernible risk of toxicity. Accordingly, 13 13 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. 29 All of these functions as well as the process under investigation can be influenced by demographic and physiological parameters, concomitant diseases and drug intake. [30][31][32][33][34][35][36][37][38][39][40] 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 Figure 1 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 13 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. 41,42 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. 35 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. 43 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. 36 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 [44][45][46] and the 13 C-OABT correlated well with scintigraphy in patients who were critically ill. 47 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.

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. Table 3

summarizes important indications for
H. pylori testing in adults and children recommended by current European guidelines. 4,5 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. 50  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. 18,60 Accordingly, it appears to be prudent to perform the 13 C-UBT in fasting conditions. 60 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. 61 Therefore, a fasting period of 4 h is suggested, including in young children.

Recommendation 2.7
Prior to the 13   patients. 6,66 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. 6 Specific indications for gastric emptying testing in adults as suggested by international guidelines and expert consensus papers are given in Table 5. 6,65,71 For pediatric patients, there are no generally accepted guideline recommendations on gastric emptying testing. The recommendations given in Table 5 are 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?  Table 6).
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. 78,88,89 The lack of standardization is of concern because it makes    Scintigraphic equivalent values or breath test data corrected for scintigraphy according to Ghoos et al. 1993. 77 [Corrections added on June 28, 2021 after first online publication: Typos have been corrected in Table 6.] KELLER ET AL. -609 patients. 90 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. 13 C-acetate has been used as a marker for acaloric and caloric liquids such as formula diets. 81,87,[91][92][93] In young children or children dependent on gastrostomy feeding, the test meal often consists of milk or milk-based formulas. 94 Apple juice has also been used. 32 Results of the test closely correlate with those of scintigraphy in adults and children. 81  The Wagner-Nelson method has been suggested for analysis of 13 C-GEBT by Sanaka et al. 106 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. 106 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. 43 Indications for pancreatic function testing in adults and children as recommended by international guidelines are given in Table 7. 7,[121][122][123][124] When should 13 C-PFBT be utilized?  128 Currently, the original 127 or modified versions 11,12,14,133 of the 13  with specificity ranging between 80% and 90% in adults. 121,137 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). 12 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. 7,11,132,133,138 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. 139 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. 137 Direct comparisons between 13 C-MTGBT and fecal elastase favor the 13 C-MTGBT for diagnosis of steatorrhea, 140 and generally in patients with chronic pancreatitis after pancreatic resections. 141 In infants with cystic fibrosis, sensitivity of the 13 C-MTGBT for diagnosis of steatorrhea was high, but specificity was low. 142    Compared to 13 C-GEBT, 13  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. 148 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 Table 8].

-
UNITED EUROPEAN GASTROENTEROLOGY JOURNAL PERT. 11,132 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. 150 13 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. [153][154][155][156][157][158][159][160] 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. 40 When should 13 C-LFBT be utilized?  13 C-aminopyrine, [3-meth-yl- 13 C]-caffeine) and cytosolic ( 13 C-phenylalanine) function. 152,157,[159][160][161][162][163][164][165][166][167][168] These have been mainly used in patients with liver fibrosis and cirrhosis due to nonalcoholic fatty liver disease, 152,157,[159][160][161]163,164,169,170 chronic hepatitis C, 162,165 cirrhosis, 166,171 and hepatocellular carcinoma. 172 Additional potential fields of application are in steatohepatitis, 156,157,159 fatty liver, 160,163,169  the effect of transarterial chemoembolization on liver function has been monitored by 13 C-methacetin test, 174 and several studies suggest that this test could also be used for planning of hepatic resections. [175][176][177] Given that, as yet, few studies have been performed involving small groups of children with rare diseases, 178-181 the use of 13   [Corrections added on June 28, 2021 after first online publication: In Table 9, typos have been corrected. In the 3rd column, 2nd row, "postprandially" has been deleted from "At baseline and at 10 min  Table 10 and demonstrate the very broad potential applicability of 13 C-breath test technology.

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 13 C-breath testing and provides recommendations for the use of 13  -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 Figure 2, 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. 13 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. 13 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.

CONFLICT OF INTEREST
Cumulative 13 CO 2 -exhalation, maximal DOB-value, 13 C-exhalation at specific time point 13 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 (195)(196)(197)(198) 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 (136,(199)(200)(201) PEI (amylase deficiency)

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.

FIGURE 2
General principle, performance and clinical role of 13 C-breath tests used in gastroenterology. 13 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), 13 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: Figure 3 (image and caption) has been revised.]