Hydrogen and Methane Breath Test: The Asian Neurogastroenterology and Motility Association Monograph

Abstract

Despite of the widespread use of hydrogen and methane breath test, the variability in testing protocols, gas measurement techniques, and interpretation criteria continues to challenge the reproducibility and comparability across centers, especially in the Asia-Pacific region. The Asian Neurogastroenterology and Motility Association hence presents the first Asian monograph guiding application and interpretation of breath test. The monograph was formulated according to the framework of indications, preparatory process, performance, and interpretation of results, as well as future direction for research.

Background

Breath test has emerged as a non-invasive diagnostic for many gastrointestinal disorders since the early 1970s.¹ Hydrogen and methane breath test quantifies gases produced in the intestines, which then are absorbed into the bloodstream and exhaled via the lungs.

Intestinal gas volume averages 100-115 mL in healthy adults and primarily comprises hydrogen (H₂), methane (CH₄), carbon dioxide (CO₂), oxygen (O₂), and nitrogen (N₂); with trace (< 1%) amounts of odoriferous compounds including sulfur-containing gases, volatile amines, and short-chain fatty acids.² Intestinal CO₂ is mainly derived from acid-bicarbonate neutralization, O₂ comes from swallowed atmospheric air, and N₂ from outward diffusion of dissolved intravascular gas.³

Microbiota fermentation of substrates such as glucose, is the only source of intestinal H₂ and CH₄.^4-7 Excessive microbial activity in small intestinal bacterial overgrowth (SIBO), leads to increased fermentation and causes elevated breath hydrogen or methane levels.^8,9 Similarly, in carbohydrate malabsorption, incomplete digestion or absorption provides increased substrates for fermentation.^10,11 Resultant breath signatures allow non-invasive diagnosis of these conditions which are not only prevalent,¹⁰ but often exhibiting symptoms such as bloating, abdominal discomfort, diarrhoea, and flatulence which overlap with disorders of gut-brain interaction (DGBI).

Despite their widespread use, breath test remains the subject of ongoing debate concerning standardisation, diagnostic accuracy, and clinical utility. Variability in testing protocols, gas measurement techniques, and interpretation criteria continues to challenge their reproducibility and comparability across centers. To address the need for a standardized Asian perspective, and to introduce Asia-specific recommendations reflecting local population characteristics, the Asian Neurogastroenterology and Motility Association (ANMA) in collaboration with the Gastrointestinal Motility Group of the Chinese Society of Gastroenterology, Chinese Medical Association, presents the first Asian monograph guiding usage and interpretation of breath test. In this ANMA monograph, a key question of broad clinical relevance is explored: when to think about hydrogen and methane breath test. The monograph also provides a horizontal comparison of breath test protocols for SIBO and carbohydrate malabsorption in relation to earlier consensus guidelines. Additionally, special focus has also been placed on the feasibility of implementing breath test within the Asian region.

Methodology

Twelve members from the Asia-Pacific region formed a task force well-equipped with comprehensive knowledge and clinical expertise. This diverse representation underscores the commitment to ensuring culturally relevant and regionally applicable recommendations. The monograph was developed using a modified consensus development framework whereby a collaborative discussion approach based on a pre-drafted framework of core recommendations was employed. The following steps summarize the process:

• (1)Pre-draft preparation: systematic literature review, consolidation of best practices, and stakeholder surveys were undertaken to establish the evidentiary foundation and identify key thematic domains.
• (2)Monograph formulation: a comprehensive draft was produced through critical synthesis of the collated data, serving as the framework for subsequent expert deliberation.
• (3)Expert review and iterative discussion: iterative refinement was conducted through a combination of virtual meetings and electronic correspondence by the task force.
• (4)Consensus finalization: the final content was ratified during an in-person consensus conference, applying a structured consensus development methodology.

The task force performed a systematic literature review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement.¹¹ Relevant studies were identified through searches in MEDLINE, Embase, Scopus, PsycINFO, CINAHL, and the Cochrane Library using search terms ([Hydrogen breath test] OR [methane breath test] OR [Hydrogen sulphide breath test]). Additional grey literature searches were conducted in Google and Baidu. References of all included studies were also screened to identify additional articles not retrieved during the initial database search. The systematic literature search identified 3393 citations, after excluding duplicates and irrelevant articles, a total of 601 articles were reviewed. The methodological quality of the identified studies was assessed according to the method of the Grades of Recommendation, Assessment, Development, and Evaluation Working Group, where applicable (Table 1).¹²

Table 1.

Grades of Evidence

Category	Description
Grade of evidence
High	Further research is unlikely to change our confidence in the estimate of effect.
Moderate	Further research is likely to have an important impact on our confidence in the estimate of effect and might change the estimate.
Low	Further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate.
Very low	Any estimate of effect is very uncertain.

Pre-meeting surveys were completed by task force members to address knowledge deficits and consolidate best practices. Costs and logistical considerations specific to various healthcare systems were also collated. Survey results were summarized and circulated before discussion.

The monograph was formulated according to the framework of indications, preparatory process, performance and interpretation of results as well as future direction for research. Preliminary discussion was conducted virtually on 20 January 2025. Based on the discussion, a series of consensus positions were drafted which formed the core recommendations of the monograph. Final consensus was reached following an in-person meeting on 21 June 2025 at Zhengzhou, Henan Province, China.

Indications for Breath Test

Hydrogen and Methane Breath Test Is Recommended for Patients With a Strong Clinical Suspicion of Small Intestinal Bacterial Overgrowth, Particularly Those With Established Risk Factors

SIBO manifests with a wide spectrum of presentations ranging from symptoms such as abdominal discomfort, bloating, flatulence, and diarrhea, to severe malabsorption syndrome including nutritional deficiencies, steatorrhea and weight loss.¹³ Many gastrointestinal and systemic conditions have been linked to SIBO, as well as risk factors including chronic proton pump inhibitor use and history of abdominal surgery (Table 2).^14-33

Table 2.

Symptoms and Conditions Associated With Small Intestinal Bacterial Overgrowth

Symptoms possibly associated with SIBO	Prevalence of SIBO in clinical conditions
	GI conditions	Prevalence	Non-GI conditions	Prevalence
Diarrhea (including steatorrhea)	Irritable bowel syndrome	31.0-36.7%14,15	Systemic sclerosis	15-38%26
Bloating	Functional dyspepsia	17.2-53.4%16	Diabetes mellitus	24-35%27
Flatulence	Chronic constipationa	73%17	Hypothyroidism	54%28
Abdominal pain	Inflammatory bowel disease	22.3%18	Obesity	37.6 %29
Constipationa	Atrophic Gastritis	57.5%19	Alzheimer’s disease	49%30
-Nutritional deficiencies	Celiac disease	8.7-23.3%20	Parkinson’s disease	33-52%31
-Weight loss	Pancreatitis	38.6%21	Chronic proton pump inhibitor use	65.7- 88.6%32,33
	Non-alcoholic fatty liver disease	35.0%22
	Liver cirrhosis	40.8%23
	Post-abdominal surgery (including cholecystectomy)	41.1-46.8%24,25

^aAssociated with intestinal methanogen overgrowth.

SIBO, small intestinal bacterial overgrowth; GI, gastrointestinal.

Small bowel aspirate and culture is the most direct method for diagnosing SIBO but is invasive and resource-intensive. Furthermore, distal small bowel access is often challenging, which limits sampling to the proximal small bowel and risk false negative results.⁹ Conversely, false positive cultures may occur due to contamination from oral flora.³⁴ Breath test is a low-risk, low-cost indirect test that assesses intestinal bacterial fermentation after administration of a substrate by measuring hydrogen and methane in expired alveolar air.^10,35,36

Patients with DGBI are more likely to have a positive test for SIBO as compared with healthy controls. Patients with diarrhea-predominant IBS more often have SIBO when compared with other subtypes.¹⁵ The coexistence of SIBO had a significant negative impact on IBS patients in terms of symptom severity and health-related quality of life.³⁷ A previous study suggested that positive baseline hydrogen breath test result predicted a better treatment response to rifaximin in diarrhea-predominant IBS.³⁸ However, another study did not show the role between hydrogen breath test and efficacy of rifaximin treatment.³⁹ Patients with functional dyspepsia have higher prevalence of SIBO than controls as demonstrated in a systematic review and meta-analysis.¹⁶ A randomized clinical trial has found that treatment with 2 weeks of rifaximin could lead to adequate relief of global dyspeptic symptoms.⁴⁰ On the other hand, intestinal methanogen overgrowth (IMO) is associated with a higher rate and severity of constipation.⁴¹ Studies have shown improvement in constipation following treatment of IMO with rifaximin or neomycin.⁴² Finally, breath test-guided therapy may be more cost-effective than empiric rifaximin use in IBS.⁴³

Breath Test May Be Used for Diagnosing Lactose, Fructose, and Xylitol Malabsorption.

In lactose malabsorption, breath test is a proven non-invasive, investigation with good sensitivity (mean value of 77.5%) and excellent specificity (mean value of 97.6%).^44-47 The role of breath test for fructose, sorbitol, and xylitol intolerance is emerging, though further research is required.^48,49 False negative results in carbohydrate breath test may occur in subjects with low hydrogen excretion or prolonged GI transit while false positives may occur due to SIBO or rapid GI transit.^50-53

Asians have a high prevalence of adult-onset lactose malabsorption, with most populations averaging at least 60-80%. Exceptions are the Bedouins in Jordan and Saudi Arabia (Table 3).^54-83 Provocation of symptoms following carbohydrate ingestion is separately termed as intolerance and may occur in the absence of malabsorption. The majority of intestinal symptoms occurs in the first 4 hours after carbohydrate load.⁵⁷ Very early onset of development < 10 minutes should prompt consideration of other pathologies including functional dyspepsia and SIBO. Occurrence of typical symptoms 30-90 minutes after ingestion may be sufficient to establish diagnosis of malabsorption in populations with elevated pre-test probability and a positive and long-lasting response to dietary intervention may confirm the diagnosis of carbohydrate intolerance without need for further investigation. In this context, symptom assessment and dietary intervention may be considered before lactose breath test in the Asian population due to an elevated baseline prevalence of primary lactase deficiency. Clinicians should be mindful of the dose-dependent relationship between lactose intake and symptom development as well as response to milk elimination.⁸⁴

Table 3.

Prevalence of Lactose Malabsorption in Asia-Oceania

Location	Prevalence
Afghanistan	83.0%54
Australia	84.0%,58 10.0-42.0%59
Bangladesh	58.2%,60 85.5%,61 82.5%62
Bhutan	No reliable published data
Brunei	No reliable published data
Cambodia	0.0% lactase persistence63 No reliable published data
China	Han: 86.0-92.0%, Mongols 88.0%, Kazakhs 76.0%, Uyghurs 33.0-43.0%54 76.6% (CI, 75.0-78.2%)55,57,64-66
India	Lactose malabsorption in 78.9% of southern Indians and in 57.1% of northern Indians Lactose intolerance in 88.2% of southern Indians and in 66.2% of northern Indians67
Indonesia	91.0%,54 69.0-81.2%68 In an Indonesian study, the prevalence of symptomatic hypolactasia at 3 years of age was 9.1%. The prevalence rose to 28.6% at 5 years, and 73.0% at 1214 years of age68
Iraq	86.0%54 < 25.0% in Bedouins54
Israel	81.0%54 < 25.0% in Bedouins54
Japan	73.0-87.0%,54 89.0%69
Jordan	75.0-78.0%,54 24% in Jordan Bedouins
Lebanon	75.0-78.0%54
Malaysia	87.1%70 88% Malay/91% Chinese/83% Indian71 85.7%72
Mongolia	87.9%73
New Zealand	9.0-64.0%74
Pakistan	45.3%75 60.0%76
Papua New Guinea	77.0%,77 98.0%78
Russia	41.0-94.0% with wide variance based on region54
Saudi Arabia	< 25.0% in Bedouins54
Singapore	81.0%,79 95.0%,56 100.0%80
South Korea	0.0% lactase persistence63
Sri Lanka	73.0%54
Taiwan, China	70.0%81
Thailand	97.0-100.0%,54 77.0%,82 51.0%83
Turkey	71.0%54
Vietnam	100.0%,54 0.0% lactase persistence63

To note: countries or regions without reliable published data were omitted.

There are patients with persistent symptoms despite reported dietary intervention. Carbohydrate breath test may hence be considered as establishing a proven link between carbohydrate challenge and malabsorption may spur tighter compliance and symptom improvement. Breath testing may also benefit patients who face nutritional and/or quality of life concerns due to carbohydrate restriction, by ensuring carbohydrate restriction is only applied to those with confirmed malabsorption. Furthermore, there is benefit from ruling out malabsorption during the diagnostic workup of a persistently symptomatic patient by shunting focus towards other differentials. Finally, it has been shown that in symptomatic patients with IBS, gas production by carbohydrate fermentation does increase symptoms due to visceral hypersensitivity, even though volume of gas produced is the same as a normal person.⁸⁵ Hence, there is role to test for carbohydrate malabsorption in the setting of persistent symptoms, because it may suggest benefit from low-fermentable oligo-, di-, mono-saccharides, and polyol (FODMAP) diet in improving IBS symptoms.

Predicting Response to Dietary Intervention

Diets high in FODMAP can trigger IBS symptoms by increasing intestinal gas production and transit time, leading to abnormal contractions and hypersensitivity.⁸⁶ Low FODMAP diet is an effective treatment for many IBS. However, it may result in significant dietary restrictions and up to 50% of patients do not respond to a low FODMAP diet,⁸⁷ the prediction of low FODMAP responsive patients is essential to treatment success as an accurate prediction bolsters physician confidence in recommending the diet to appropriate patients and improves adherence.

Breath test following a meal challenge may help predict response to low-FODMAP dietary intervention. A previous study has shown that patients with positive lactose or fructose breath test had greater symptom relief on a low-FODMAP diet, indicating breath test may guide dietary intervention.⁸⁸ A recent retrospective study including 62 IBS patients showed that higher lactulose-nutrient challenge meal-induced hydrogen in the breath was associated with greater low-FODMAP diet treatment response.⁸⁹ Thus, a hydrogen breath test following a high-FODMAP meal challenge, so-called FODMAP meal challenge test (FMCT), has been developed to assess its predictive capacity for response to low-FODMAP diet. Ghoshal et al⁹⁰ enrolled 40 Rome III IBS patients and 20 asymptomatic volunteers, who underwent an 8-hour hydrogen breath test following consumption of isocaloric (450 kcal) test meals: a low-FODMAP formulation (rice, brinjal, corn, and banana) and a high-FODMAP composition (wheat, kidney bean, pulse, and cardoon). They found that IBS patients and controls produced more breath hydrogen after high- (greater in IBS) than low-FODMAP meal. The FMCT demonstrated clinically actionable predictive performance, achieving 78.6% sensitivity, 66.6% specificity, and 75.6% overall diagnostic accuracy in identifying responders to low-FODMAP dietary intervention, thereby establishing its utility in personalized IBS nutritional management protocols.⁹⁰ A Spot Breath Test following a structural individual low FODMAP dietary advice protocol has also been developed and it was found that a hydrogen level of more than 8 parts per million (ppm) and methane of more than 2.25 ppm could predict the response to low FODMAP dietary advice in funtional gastrointestinal disorder patients with bloating symptoms with high sensitivity and specificity.^91,92

While this concept is promising, further studies are needed for validation and quantification of a formal dietary challenge protocol and the composition of a low FODMAP test meal. Clinicians are recommended to reference the above cited studies for sample protocols in the interim.

Oro-cecal Transit Time

Oro-cecal transit time (OCTT) is the time between the ingestion of an unabsorbable fermentable carbohydrate and the start of a quantifiable rise of H₂ concentration in end-expiratory breath, which represents the sum of esophageal, gastric, and small bowel transit. Multiple factors including the test meal as well as the type and dose of the test substrate, may affect the reproducibility of breath test for OCTT.^93-96 Currently, breath test for assessment of OCTT has no definite indications in clinical practice.

Clinical Approach: Role of Breath Test

Breath test has a role in the evaluation of persistent symptoms of diarrhea, abdominal discomfort, flatulence, bloating, and nausea. Breath test should be part of a secondary assessment after significant conditions such as malignancy and inflammatory bowel disease have been excluded. Whether breath test is performed before other tests, for example celiac screening or SeHCAT scintigraphy, needs to be contextualized to each patient and health system resource availability.

The diagnostic algorithm in Figure, contextualized for Asian practice, directs breath test towards 3 key pathways based on regional prevalence and evidence. The SIBO pathway targets patients with established risk factors, supported by high SIBO prevalence in IBS and functional dyspepsia cohorts.¹⁵ The carbohydrate malabsorption pathway is particularly relevant given Asia's high rates of primary lactase deficiency (Table 3), aiding confirmation in symptomatic patients despite initial dietary measures.⁸⁴ Finally, predicting DGBI dietary response is supported by regional studies where FODMAP meal challenge tests identified likely responders,^90,92 aligning with the need for cost-effective nutritional management amid diverse Asian diets.

Figure 1.

When to think about breath test. SIBO, small intestinal bacterial overgrowth.

In the context of management, patients with persistent symptoms despite attempted carbohydrate elimination diet, or with concerns for dietary intervention may benefit from breath testing. Breath test may also be used to predict response to dietary modification for patients with DGBI.^89,92,97

Protocol for Breath Test

Preparation Measures of Breath Test

Various factors such as medication use, smoking, physical exercise, and diet have shown to impact breath gas composition. Previous studies have required avoiding these factors in order to minimize the influence on breath test. However, the prescribed avoidance durations vary considerably across different research protocols (Supplementary Table1)^39,98-125 and consensus guidelines (Supplementary Table 2).^126-128 The ANMA monograph provides updated recommendations on the preparation measures for breath test (Table 4).

Table 4.

Preparation Measures Prior to Breath Testing

Factors	Preparation measures prior to breath test
Antibiotics	Avoid for 4 weeks
Colonic cleansing (for colonoscopy/bowel surgery)	Avoid for 4 weeks
Laxative agents	Avoid for 1 week
Probioticsa	Avoid for 1 week (ideally 4 weeks)
Motility agents	Avoid for 72 hours
PPIsa	No requirement for PPIs to be routinely withheld prior to testing.
Restricted diet	Poorly absorbable carbohydrates and dietary fibers should be avoided for 24 hours prior to breath test.
Fasting	Avoid food intake, other than sips of water, for 8 hours
Smoking	Avoid for 2 hours prior to and during breath test
Alcohol	Avoid for 24 hours
Physical activity	Avoid for 2 hours prior to and during the breath test.
Oral cavity cleansing	Brush teeth on morning before going for breath test Oral cavity cleansing with chlorhexidine/antiseptic mouth gargle just before breath test. Dental kit should be brought for breath test in the event repeat oral cleansing is required.

^aUse of these agents during stipulated washout period should be reflected in test report.

Antibiotics

The influence of antibiotic treatment on gut microflora and the resultant change in hydrogen and methane levels has been shown by previous studies.^42,129-133 Duration of this effect is less well defined (Supplementary Table 1). Studies suggest that microbiota restoration largely occurs 4 weeks after antibiotics.^134,135 Furthermore, most publications and prior guidelines recommend avoidance for 4 weeks prior to breath test (Supplementary Table 2). Hence, in the current ANMA monograph, we recommend antibiotic therapy to be avoided for 4 weeks prior to the breath test (Table 4).

Colonic cleansing

Thorough cleansing of the gut before colonoscopy or surgery modifies breath hydrogen excretion and increases risk of false negative breath test.¹³³ We recommend that colonic cleansing (for colonoscopy or bowel surgery) should be avoided for 4 weeks prior to the breath test (Table 4).

Laxatives

Laxatives agents interfere with the stability of colonic flora.¹³³ Required duration for stopping laxative agents before the performance of breath test in published studies varies widely from 1 day to 4 weeks (Supplementary Table 1). As a 4-week gap may not be practical in patients with chronic constipation, we recommend that laxatives be avoided for 1 week prior to the breath test (Table 4).

Probiotics

Probiotics alter gut microbiota and motility,^136-138 however, formulations are heterogeneous, and the effect cannot be generalized under a single umbrella. Recommendations vary widely from 3 days to 4 weeks in different studies, with the majority falling between 1-2 weeks (Supplementary Table 1). We recommend that probiotics be stopped for 4 weeks prior to breath test, however, given that probiotics relieve symptoms in many patients who may not tolerate prolonged cessation, the duration of avoidance may hence be reduced to 1 week. Duration of cessation should be recorded in the test report.

Motility agents

Motility agents affect gut motility and may alter microbial flora, both of which can affect breath test results.¹³⁹ Motility agents were historically withheld for 4 weeks but newer studies have reduced the duration of cessation. Based on the pharmacokinetic half-life of motility agents, a discontinuation period of 72 hours is recommended prior to the breath test in the current ANMA monograph (Table 4).

Proton pump inhibitors

Although PPIs have been shown to affect the intestinal bacteria by some studies,^140,141 there is a lack of conclusive data on stopping or continuing PPIs prior to breath test. Subjects on regular PPI with a clinical suspicion of PPI-related SIBO should not stop PPI prior to testing. Those who are not on regular PPI are not recommended to start PPI prior to breath testing. We recommend to record the use of PPI in the test report (Table 4). Further studies are required to quantify effect of potassium competitive acid blockers on breath test.

Diet

A high fasting level of breath H₂ might impair the detection of a rise in H₂ concentration by fermentation of the test carbohydrate, and therefore should be minimized. High fasting levels may be the result of a high fiber meal on the previous evening.¹⁴² A low fiber diet or a diet containing FODMAP decreases breath H₂ excretion.⁸⁸ There’s no clear data about how long of restricted diet as well as fasting period before the breath test should be. The precise duration requires to be determined in future studies. The majority of existing studies implement dietary restrictions of up to 24 hours preceding breath test (Supplementary Table 1). Prior consensuses have consistently recommended restricted diet 24 hours prior to analysis. Notably, a standardized pre-test diet (eg, white rice with steamed chicken breast or plant-based alternatives) rather than solely prohibiting specific foods might enhance result reproducibility. Nevertheless, the practical implementation of such standardized dietary protocols may present logistical challenges in real-world clinical settings. Hence, at the least, this ANMA monograph propose avoiding diet containing poorly absorbable fermentable carbohydrates and dietary fibers for 24 hours before the test. Besides, an overnight fasting period of 8-12 hours is also recommended to keep a low fasting level of breath hydrogen.^126-128 We recommend a minimum 8-hour fasting period before the test (Table 4).

Smoking

Previous data has demonstrated the effect of smoking on the breath hydrogen results.^143,144 Smoking also increases gastric transit time.¹⁴⁵ No clear data has investigated the precise time-course. The duration required for avoiding smoking before the test varies from half an hour to 24 hours in different studies, mostly 2 hours (Supplementary Table 1). The North American consensus recommends avoiding smoking on the day of testing.¹²⁷ With the consideration of how long a smoker might tolerate refraining from smoking, the United European Gastroenterology (UEG) consensus recommends avoiding smoking for 2 hours from a practical standpoint.¹²⁸ We recommend avoiding smoking for 2 hours prior to and during the breath test (Table 4).

Alcohol

Since alcohol has been shown to affect the GI transit time,^95,146 some studies have required avoiding alcohol 24 hours before the breath test (Supplementary Table 1). However, no recommendation has been made concerning avoiding alcohol before breath test in prior consensus. We recommend avoiding alcohol for 24 hours prior to the breath test (Table 4).

Physical activity

Hyperventilation reduces the breath H₂ concentrations,¹⁴⁷ thus exercise can affect the breath hydrogen and methane levels.^148-150 Previous consensus have recommended limiting physical activity before and during the test.^126-128 We recommend avoiding physical activity for 2 hours prior to and during the breath test (Table 4).

Oral cavity cleansing

Hydrogen may also be produced by the oropharyngeal bacteria. The early peak of breath hydrogen concentration may be the consequence of the interference of oral bacteria, which can be prevented by mouth-washing with a chlorhexidine solution.^151,152 Oral cavity cleansing with an antiseptic solution (eg, chlorhexidine) is recommended before the breath test in the UEG consensus.¹²⁸ In the ANMA monograph, we recommend oral cavity cleansing with chlorhexidine/antiseptic mouth gargle before the breath test. Participants are advised to perform thorough dental hygiene, including morning tooth brushing prior to breath test. To optimize test validity, participants are required to bring their oral care kits and implement contingency protocols whereby subjects conduct supervised oral cleansing and undergo repeat testing when baseline hydrogen concentration is high (Table 4).

In order to standardize the preparation measures and ensure the consistency among institutes, it is recommended to use a standardized patient education manual in various languages (Supplementary Figure). This manual can provide patients with clear and unified guidance, thus improving the quality and efficiency of breath test.

Quality Control

Elevated baseline hydrogen and methane ≥ 20 ppm should prompt repeat of breath test after oral cleansing with chlorhexidine gargle; if elevated baseline levels persist, the test should be repeated with full adherence to preparatory measures.

High basal hydrogen concentrations can occur as a result of oral fermentation and/or improper preparation; consequently, this may increase false negative results. Prior guidelines recommend not to continue the test if baseline levels exceed cutoffs ranging from 20 ppm for H₂.^{127, 128} Breath test should be repeated after oral cleansing with chlorhexidine gargle¹⁵³; if baseline levels are still elevated, the test should be rescheduled with full adherence to preparatory measures.¹⁵⁴ If baseline levels are still elevated despite complete adherence to preparatory measures, an alternative means of testing should be sought.

Standard reporting of results should include any deviations which occurred during preparation and/or performance of the breath test.

It may not be feasible to ensure complete adherence to all recommended preparation and/or performance measures during conduct of the breath test (Table 4). All deviations should be clearly recorded in the report, to facilitate contextualization of the results during interpretation. Examples include if pharmacotherapy agents were not stopped for the recommended duration, or if the test had to be repeated due to a high baseline.

Performance of Breath Test

Small intestinal bacterial overgrowth

A. Substrate and dose

The most frequently used substrates in breath test for SIBO diagnosis are glucose and lactulose. Glucose is a monosaccharide which is completely absorbed in the small bowel, whereas lactulose is a non-digestible disaccharide that reaches the colon.¹⁵⁵ A recent study found that the jejunal culture was positive in 14/40 (35.0%) subjects, the lactulose breath test (LBT) was positive in 18/40 (45.0%) subjects, and the glucose breath test (GBT) was positive in 12/40 (30.0%). The GBT showed good agreement (κ = 0.659) and LBT showed poor agreement (κ = 0.588).³⁶ Meta-analysis further correlates that GBT has better accuracy than LBT for diagnosis of SIBO (GBT pooled sensitivity 54%, interquartile range [IQR], 48-61% and specificity 83%; IQR, 79-87%; compared to LBT sensitivity 42%, IQR 32-53% and specificity 7%, 62-78%).¹⁵⁶ Glucose is hence the better substrate and recommended over lactulose as the sole substrate for SIBO breath test. A 75 g glucose dose is recommended and is harmonized with the dose used for diabetic oral glucose tolerance test. It was found that 75 g glucose dose produces more positive SIBO results than 50 g glucose.⁹⁹ Ghoshal et al¹⁵⁷ performed paired glucose hydrogen breath test among 40 patients with DGBI on 2 separate days using 50 g and 75 g glucose, and found no difference between 50 g and 75 g of glucose, indicating that use of 75 g glucose did not increase the detection rate of SIBO compared to 50 g dose. Lactulose may accelerate the small bowel transit, which increases the risk of false positive results.¹⁵⁸ Thus, lactulose, at a dose of 10 g, may be considered only if glucose is not available or contraindicated––for instance in patients with uncontrolled hyperglycaemia or rare cases of glucose-carbohydrate malabsorption.

B. Test duration

A test duration of 120 minutes, with a sampling interval of 15-minutes, is recommended in the investigation of small intestinal bacterial overgrowth by glucose H₂ breath test (Table 5).

Table 5.

Protocols of Breath Test for Small Intestinal Bacterial Overgrowth Diagnosis in Prior Consensuses and Asian Neurogastroenterology and Motility Association Monograph

Test protocol	Rome Consensus	North American Consensus	UEG Consensus	ANMA Monograph
Substrates	Lactulose 10 g and glucose 50 g/250 mL GBT is recommended.	Lactulose 10 g/ glucose 75 g + 1 cup water	Lactulose 10-20 g and glucose 50 g/250 mL	glucose 75 g
Test duration	120 min	120 min	120 min	120 min
Sampling intervals	15 min	Not mentioned	15 min	15 min

UEG, United European Gastroenterology; ANMA, Asian Neurogastroenterology and Motility Association; GBT, glucose breath test.

Carbohydrate malabsorption

A. Substrate and dose

Lactose. Cow’s milk contains approximately 5 g of lactose per 100 mL, amounting to 25 g in 500 mL, which is equivalent to roughly 2 servings. Higher doses such as 40-50 g significantly exceeds typical dietary intake. Clinically significant malabsorption is better diagnosed using a lesser, physiological dose. A study showed that positive breath test using 25-g dose better predicted symptom resolution than by 50-g and 12-g of lactose.⁸⁴ A dose of 25-g of lactose is hence the recommended dose.

Fructose. Tests on healthy subjects show most have the capacity to absorb up to 25 g of fructose, with 25 g fructose the recommended test dose.¹⁵⁹

Xylitol/D-xylose. A dosage of 25 g has been commonly used for D-xylose/Xylitol breath test in previous studies.^160-163

B. Test duration and sampling interval

Significant variation exists in the literature in terms of the required length of study for assessment of carbohydrate malabsorption, ranging from 2 hours to 5 hours.¹⁶⁴ The raison d'être for breath test in carbohydrate malabsorption is fermentation of unabsorbed substrate by colonic flora, it is hence crucial to provide sufficient duration for colonic transit. Rao et al¹⁵⁹ reported that the average time for reaching peak gas concentration was 77 minutes (range = 30-180 minutes) for an abnormal fructose breath test, thus suggesting that 180 minutes is sufficient to detect colonic fermentation.

The majority of studies use a sampling interval of 15-60-minutes. A short 15-minute sampling interval provides greater resolution but doubles the logistic requirement compared to 30 minutes.

Balancing practicality with test accuracy, the recommendation is hence for a test duration of at least 180 minutes with a 30-minute sampling interval.

C. Symptom evaluation

Symptom provocation by carbohydrate challenge forms an integral part of breath test and underpins dietary intervention. All symptoms should be recorded together with a time stamp and quantification of severity. Symptom manifestations may differ across clinical populations and hence the use of existing questionnaires, such as the functional gastrointestinal-checklist¹⁶⁵ which requires individualized adaptation to specific healthcare settings. Majority of intestinal symptoms occur in the first 4 hours after a carbohydrate load,⁵⁷ hence documentation should be extended up to 3-5 hours after substrate challenge (Table 6).

Table 6.

Protocols of Breath Test for Carbohydrate Malabsorption Diagnosis in Prior Consensuses and Asian Neurogastroenterology and Motility Association Monograph

Test Protocol	Rome Consensus	North American Consensus	UEG Consensus	ANMA Monograph
Substrates	Lactose: 1 g/kg up to max 25 g in 10% water solution	Lactose: 25 g + 1 cup water Fructose: 25 g + 1 cup water	Lactose: 25 g in watery solution Fructose: 20-25 g in watery solution	Lactose: 25 g Fructose: 25 g Xylitol: 25 g
Test duration	4 hr	Over 3 hr	3-5 hr, or shorter if positive diagnosis for malabsorption and intolerance is confirmed.	Over 3 hr
Sampling intervals	30 min	Not mentioned	15-60 min	30 min
Symptom evaluation	During the test and 8 hr after	Not mentioned	Longer than 3-5 hr	The use of a standardized questionnaire or the systematic documentation of a predefined symptom cluster is recommended

UEG, United European Gastroenterology; ANMA, Asian Neurogastroenterology and Motility Association.

Interpretation of Breath Test

Small Intestinal Bacterial Overgrowth

Rise of ≥ 12 ppm from baseline in hydrogen is considered a positive test suggesting the presence of Small Intestinal Bacterial Overgrowth.

Differences in recommended thresholds for SIBO breath test exist across current guidelines. A 12 ppm cutoff has been adopted by 2022 Asian-Pacific consensus on SIBO¹⁶⁶ as well as 2009 Rome Consensus,¹²⁶ whereas ASENEM-SEPD 2023,¹⁶⁷ AGA 2020,¹⁶⁸ and North American 2017¹²⁷ consensus adopt the ≥ 20 ppm threshold.

Meta-analysis synthesized results of 14 studies benchmarking breath test against jejunal aspirate culture, to determine the optimal cutoff value.¹⁵⁶ Results showed that a threshold value lower than 20 ppm provides better accuracy compared to the ≥ 20 ppm proposed by the North American and European consensus. GBT cut off ≥ 20 ppm provided a pooled sensitivity of 47.3% and specificity of 80.9%, positive likelihood ratio 1.95, negative likelihood ratio 0.66, diagnostic odds ratio 3.35, area under the curve (AUC) 0.7, whereas a cutoff < 20 ppm showed improved pooled sensitivity 61.7% and specificity 86.0%, positive likelihood ratio 3.2, negative likelihood ratio 0.54, diagnostic odds ratio 8.11, AUC 0.79. Given most trials with cutoff < 20 ppm, particularly those from Asia, used the threshold of 12 ppm, the cutoff of 12 ppm hence was selected for this monograph.

Conceptually, glucose undergoes complete fermentation in the small intestine, where the lower microbial load compared to the colon produces less hydrogen, hence supporting a lower diagnostic threshold for hydrogen detection. Furthermore, given the role of breath test as a noninvasive first-line SIBO diagnostic supports the requirement for a more sensitive threshold is preferable (Supplementary Table 3).^8,35,169-180

Of note, though a positive GBT is confirmatory of the presence of SIBO, a negative test may not mean the absence of it. Persistent positive GBT results, particularly if unresponsive to therapy, may suggest rare causes such as isomaltase deficiency,¹⁸¹ warranting further evaluation with genetic testing and use of alternative diagnostics, such as small bowel aspirate and culture for SIBO.

Early peak criterion may result in false positive from rapid cecal transit and should not be relied on for diagnosis of small intestinal bacterial overgrowth, particularly with lactulose hydrogen breath test. Accuracy of breath test may be improved with concomitant assessment of orocecal transit.

False positive diagnosis of SIBO may occur with rapid transit, for instance in patients with short transit from past gastrointestinal tract surgery.¹⁸² Rapid small bowel transit is common in IBS and increased with non-absorbable lactulose test substrate compared to glucose. Early-peak criterion whereby peak occurring within 90 minutes of lactulose ingestion hence should not be accepted unless there is concomitant independent measure assessment of orocecal transit. Combination of breath testing with an independent measurement of orocecal transit time, for example scintigraphy, can increase the accuracy of breath test,^52,158 if this is available.

Level of ≥ 10 ppm after substrate challenge is considered positive for intestinal methanogen overgrowth.

Excess methane production in the human gut is predominantly contributed by Methanobrevibacter.¹⁸³ However, there are patients with high basal levels CH₄ due to reasons other than intestinal microbial fermentation. Hence, while some guidelines have recommended spot CH₄, evidence is still nascent and IMO should be diagnosed by a rise of ≥ 10 ppm.

IMO has distinct phenotypical characteristics: Experimental and clinical studies indicate that methane inhibits GI motility and hence its concentration may inversely correlate with stool form and frequency.¹⁸⁴ Patients with CH₄-predominant methanogen overgrowth may have increased prevalence of abdominal bloating and abdominal distension with constipation; while IBS patients with constipation have increased prevalence of methane-positive breath test.²⁰ Reduction of methane production in these patients with rifaximin improves constipation.¹⁸⁵

Carbohydrate Malabsorption

Rise of ≥ 20 ppm from baseline in hydrogen during the test should be considered positive for carbohydrate malabsorption for lactose, fructose, and xylitol.

Cutoff value of ≥ 20 ppm rise in hydrogen within 3 hours as threshold for positive diagnosis of carbohydrate malabsorption has consistently been applied for prior consensus.^126-128,186 False negative results may occur due to inability of gut flora to produce H₂ or after recent antibiotic sterilization. False positive results may occur due to SIBO.

Lactose. The 20 ppm cutoff provides a sensitivity of 60% with specificity close to 100%.^47,187 Reducing the cutoff to 10 ppm increases sensitivity at the expense of specificity and hence is not recommended.^45,47

Fructose. Use of the 20 ppm cutoff has been shown to correlate with symptoms of intolerance.^49,188,189 However, repeatability of breath test to diagnose malabsorption has been called into question.⁴⁸ Hydrogen breath test has also not been shown to be predictive of response to fructose-free diet.^49,189 Overall, breath testing for fructose intolerance may provide answers to previously unexplained symptoms but further research is required.

Sorbitol/xylitol. Tests on 25 healthy individuals showed that malabsorption was 84.0% for sorbitol and 12.0% for xylitol, 57.0% of sorbitol and 100.0% of xylitol exhibited symptoms.¹⁶³ A larger study of 36 patients with functional abdominal bloating found that 50.0% of those tested had fructose plus sorbitol malabsorption using cutoff of 20 ppm; Symptoms of sugar intolerance were observed in 17 out of 26 (65.0%) patients with malabsorption.¹⁸⁹ There has been correlation of sorbitol with gastrointestinal symptoms in other studies as well.^190-192 Impact of dietary restriction of sorbitol/fructose on symptoms has not been proven.¹⁸⁹ Hence, while testing may provide answers to previously unexplained symptoms, further research on clinical implications is required.

There is insufficient evidence to recommend routine concomitant measurement of orocecal transit to refine accuracy of breath test for carbohydrate malabsorption.

Rapid and/or prolonged orocecal transit may result in false positive/negative results respectively.⁵² Concomitant measurement with scintigraphy or other means (eg, motility capsules) in theory may refine performance of breath test and has been suggested in other guidelines¹²⁸ as an adjunct to increase test sensitivity through lowering of cutoff value to 10 ppm. However, these test increases costs and logistical concerns. Furthermore, there have been no studies directly assessing the impact on carbohydrate malabsorption diagnostic thresholds and outcomes. Hence, a firm recommendation cannot be provided at this point.

Increase in methane ≥ 10 ppm above baseline may indicate carbohydrate malabsorption.

Breath methane excretion provides an important alternative target for intestinal gas breath excretion measurement in the subgroup of hydrogen nonproducers, thus improving the test accuracy.¹⁹³ Addition of methane measurement during breath test may increase test performance. A rise of ≥ 10 ppm was considered positive for excessive methane production and has been recommended by the North American¹²⁷ and European¹²⁸ Consensus.

Inclusion of methane improves the precision of semiquantitative measurements of carbohydrate malabsorption using AUC calculation; cutoff of rise of 10 ppm was recommended.¹⁹⁴ A further 16.0% of patients with normal lactose digestion on H₂ breath test were identified on a study benchmarked to ¹³C/H₂ breath test¹⁹⁵ using cutoff of 5 ppm. Further studies corroborate that including methane assessment improves accuracy of carbohydrate malabsorption tests. Agreement between breath test and genetics is modest,¹⁹⁶ though of note this study used a cutoff of 20 ppm for CH₄.

CH₄ level at baseline, forming the basis of spot methane as substitute for complete breath test. Cutoff values however have not been conclusive determined and range from 3 ppm to ≥ 10 ppm.^197-200 In this situation, further studies both for spot CH₄ and response of CH₄ to carbohydrate challenge, especially in the Asian population are required. Until further evidence is available, spot CH₄ is not suggested as a diagnostic criterion for carbohydrate malabsorption.

Overall, core recommendations of the indications and interpretation of breath test are summarized below (Table 7).

Table 7.

Core Recommendations of Breath Test in Asian Neurogastroenterology and Motility Association Monograph

Statement	GRADE rating	Explanation
Hydrogen and methane breath test is recommended for patients with a strong clinical suspicion of SIBO, particularly those with established risk factors.	High	Extensive studies show relationship between risk factors and SIBO. Breath testing is a low cost, non-invasive test for diagnosis of SIBO
Breath test may be used for diagnosing lactose malabsorption.	High	Extensive studies utilizing a wide range of comparators support the accuracy of breath testing for diagnosis of lactose malabsorption
Breath test may be used for diagnosing fructose and xylitol malabsorption.	Low	Limited studies show a promising role of breath testing for malabsorption in these substrates
Breath test may be considered to guide response to dietary intervention for irritable bowel syndrome.	Low	Two cohort studies and one well-designed controlled trial support the novel implementation of breath testing to guide dietary intervention in IBS. However, more studies are required.
Elevated baseline hydrogen and methane ≥ 20 ppm should prompt repeat of breath test after oral cleansing with chlorhexidine gargle; if elevated baseline levels persist, the test should be repeated with full adherence to preparatory measures.	Moderate	Supported by in vivo studies showing hydrogen production by oropharyngeal bacteria. More studies are required to determine the magnitude of impact, reproducible cutoffs and correction factors.
Rise of ≥ 12 ppm from baseline in hydrogen is considered a positive test during suggesting the presence of SIBO.	High	Meta-analysis of multiple trials supports a threshold value below 20 ppm with 12 ppm cutoff based on 3 Asia-pacific studies.
Early peak criterion may result in false positive from rapid cecal transit and should not be relied on for diagnosis of SIBO.	Low	Evidence derived from in vivo studies comparing breath testing to gastrointestinal motility studies
Accuracy of breath test for SIBO may be improved with concomitant assessment of orocecal transit.	Very low	Benefit has been suggested by a few recent studies, with an intuitive physiological backing. However, more research is required to determine clinical impact.
Level of ≥ 10 ppm after substrate challenge is considered positive for intestinal methanogen overgrowth	Low	More research is required provide an objective cutoff and reproducible definition criteria.
Rise of ≥ 20 ppm from baseline in hydrogen during the test should be considered positive for carbohydrate malabsorption for lactose	High	Supported by multiple high-quality studies. Threshold has been consistently adopted by major societal guidelines.
Rise of ≥ 20 ppm from baseline in hydrogen during the test should be considered positive for carbohydrate malabsorption for fructose and xylitol	Low	Threshold utilized by existing studies. More research is required to determine ideal cutoff.
Increase in methane ≥ 10 ppm above baseline may indicate carbohydrate malabsorption	Low	More research is required provide an objective cutoff and reproducible definition criteria.
There is insufficient evidence to recommend routine concomitant measurement of orocecal transit to refine accuracy of breath test for carbohydrate malabsorption	Very low	Benefit has been suggested by a few recent studies, with an intuitive physiological backing. However, more research is required to determine clinical impact.

SIBO, small intestinal bacterial overgrowth; IBS, irritable bowel syndrome; ppm, parts per million.

To note: preparatory measures and protocols should adhere to standards set in Tables 4-6.

Unmet Needs

By providing standardized recommendations on preparatory measures, test protocols, and interpretation criteria, this monograph constitutes a critical first step toward improving inter-center reproducibility and comparability of breath test results across Asia, and the Working Group has identified the following unmet needs regarding the breath test as the key areas for future multicenter research to validate the proposed standards.

Standardization and Methodological Refinements

Lack of universal protocols

There exists significant variations in methodological aspects, for instance in pre-test preparations, substrate dosing protocols, and sampling intervals. These inconsistencies hinder reliable cross-study comparisons and limit the broader applicability of findings. Additional questions to consider include whether prolonging breath test beyond 120 minutes to 180 minutes for SIBO, or if shortening the test interval for carbohydrate malabsorption, significantly improves test accuracy and cost effectiveness.

Improved detection technologies

Current breath test devices lack the sensitivity to detect low levels of hydrogen and methane. Implementing advanced detection technologies such as gas chromatography offers the potential for improved sensitivity and more precise quantification, thereby enhancing the reliability of test results. Finally, hydrogen sulfide is an emerging field for further research.

Confounding factors

The influence of confounding factors such as oral microbiome activity, smoking, physical exercise, diet, and medication use on breath gas levels remains inadequately quantified especially in the Asian setting. Standardized correction protocols with oxygen and carbon dioxide measurements during breath test should also be developed to optimize reproducibility.

Clinical Relevance

The clinical relevance of the breath test remains a subject of ongoing debate and investigation. Studies have yielded conflicting results regarding the correlation between gas profiles; for instance, methane-dominant compared to hydrogen-dominant phenotypes and specific gastrointestinal symptoms such as constipation or diarrhea. Furthermore, while elevated methane levels are often linked to slower intestinal transit, their direct causative role in clinical outcomes remains unclear. Additionally, more evidence is required to support the use of breath test as a reliable measure of gastrointestinal transit time.

Research Gaps

Current studies have focused on diagnostic applications, beyond which, scientific gaps persist especially in gut microbiomics and metabolomics. Precise pathophysiological mechanisms linking breath gas profiles to specific gut microbiome compositions such as methanogens and sulfate-reducing bacteria remain poorly understood. Oral flora composition and compensation for the resultant gas production is another area of study. Finally, the modulation of gas production over time by different diets, specifically low-FODMAP and high-fiber regimens, requires investigation.

Expanding Clinical Applications

Therapeutic guidance represents the next frontier for breath testing, as highlighted by emerging evidence and which is newly endorsed by this monograph. Recent studies have found the potential in predicting response to antibiotics and dietary treatment. In particular, the low FODMAP meal challenge is a novel application with promising results; however, there lacks an objective standard to quantify the composition of a low FODMAP test diet. Furthermore, multicenter international studies are recommended to ascertain reproducibility. Breath test to predict response to carbohydrate dietary intervention or even to personalize intake thresholds is an additional area for study.

Beyond the gut, there are potential associations between breath gases and metabolic diseases such as obesity and diabetes, neurological conditions such as Parkinson disease and dementia, as well as systemic dysfunction including chronic liver and kidney disease. Elucidating these links may offer expanding value to uncover novel diagnostic or pathophysiological insights.

Ethics Statements

Patient consent and ethics board approval are not applicable to this publication.

Supplementary Materials

Note: To access the supplementary tables and figure mentioned in this article, visit the online version of Journal of Neurogastroenterology and Motility at http://www.jnmjournal.org/, and at https://doi.org/10.5056/jnm25185.