High-Resolution Measurements of Diet-Microbiome Interactions

Irritable Bowel Syndrome (IBS) affects an estimated 25-45 million people in the United States, accounts for roughly 20-40% of gastroenterology visits, and imposes a major societal burden, with annual U.S. costs estimated at approximately $1.7-$10 billion in direct medical spending and approximately $20 billion in indirect costs from lost productivity (Hulisz 2004; Leong et al. 2003; Canavan et al. 2014). Despite this burden, IBS remains poorly understood, and no validated biochemical diagnostic or curative therapy currently exists (Lacy et al. 2021; Kim et al. 2017).

In most individuals, a high-fiber diet supports a healthy microbiome and improves health outcomes without adverse symptoms (Fu et al. 2022). In patients with IBS, however, fermentable fiber is known to worsen symptoms such as bloating, flatulence, and abdominal pain; patients are often advised to restrict fermentable fiber intake (e.g., a low-FODMAP diet), and antibiotics are sometimes used therapeutically to suppress fermentation (Halmos et al. 2014; Lacy et al. 2021). These clinical observations motivate the mechanistic hypothesis underlying this protocol: that IBS symptoms may arise, at least in part, from disordered microbial fermentation and impaired intestinal gas handling.

Progress toward this goal has been limited by the absence of direct clinical tools capable of quantifying fermentation physiology in vivo and by intestinal region. Current clinical testing (e.g., hydrogen/methane breath testing) provides only an indirect, systemic proxy for fermentation and remains variably standardized (Rezaie et al. 2017; Pitcher et al. 2022).

The human intestine is more than 30 feet in length and is home to regionally distinct microbial communities that closely track local gut physiology (Martinez-Guryn et al. 2019). Recently developed non-invasive capsule-based microbiome sampling has shown that the small intestine and proximal colon harbor communities that are distinct from stool and disproportionately altered in IBS (Shalon et al. 2023; Leite et al. 2020; Chung et al. 2016).

This protocol leverages two complementary ingestible capsule technologies - a real-time gas-sensing capsule (Atmo) and a regional fluid/microbiome-sampling capsule (CapScan) - together with stool, urine, breath, and blood-based measurements, to establish regional gas production and consumption profiles in healthy individuals and patients with IBS.

The Healthy and IBS cohorts are defined by baseline clinical status and are not randomized. Within each cohort, participants undergo a randomized two-period crossover of meal order. Each participant receives both the low-fiber control meal and the 15 g inulin meal, with the order assigned at random.

• Inulin (Dietary supplement): Participants consume a standardized breakfast shake containing 15 g of inulin at one of the two study visits, according to the randomized meal sequence. At the other visit, they consume the same base shake without added inulin as the low-fiber control. Visits occur approximately one week apart.
• Low-fiber control breakfast shake (Other): Participants consume the standardized low-fiber base breakfast shake without added inulin at one of the two study visits, according to the randomized meal sequence. This condition serves as the within-participant control. Visits occur approximately one week apart.
• Atmo Gas-Sensing Capsule (Device), also known as Atmo Gas Capsule System: At each of the two study visits, participants will swallow one Atmo gas-sensing capsule with the assigned standardized breakfast shake at t=0. As it transits the gastrointestinal tract, the wireless capsule records regional luminal hydrogen (H2), carbon dioxide (CO2), oxygen-related signals, temperature, and transit-related data. Data are transmitted by radiofrequency to a wearable receiver for analysis. Capsule passage will be monitored after each visit.
• CapScan Regional Fluid-Sampling Capsule (Device), also known as CapScan, Envivo Bio Collection Device: At each of the two study visits, participants will swallow one CapScan capsule at t=4 hours. CapScan is a single-use, passive ingestible device that collects regional gastrointestinal luminal fluid as it transits the intestine. The collected samples will be used for ex vivo microbiome and multiomic analyses. The device passes in the stool and is retrieved by the participant using the provided retrieval materials for subsequent processing.

Primary outcomes

• Successful Acquisition of a Complete Regional Multiomic Fermentation-Physiology Dataset [up to 12 months]: To successfully obtain a complete regional multiomic fermentation-physiology dataset - defined as paired Atmo gas capsule data (H2, CO2, O2), CapScan regional microbiome/fluid sampling, stool microbiome sequencing, and breath testing (H2, CH4, H2S) with serum SCFA measurements - from at least 30 of the 40 enrolled participants (15 healthy, 15 IBS), across both study visits. Primary outcome measure: the number and proportion of enrolled participants from whom a complete multiomic dataset is successfully obtained across both study visits.

Secondary outcomes

• Change in Regional Intestinal Gas Concentrations Following Inulin Challenge [Through completion of both study visits, 2 week]: Regional hydrogen (H2), carbon dioxide (CO2), and oxygen (O2) concentration-time profiles will be measured using the Atmo gas-sensing capsule. Changes in gas concentrations between the low-fiber control visit and the 15 g inulin visit will be compared within participants and between healthy and IBS groups, including regional differences between the small intestine and colon.
• Regional Intestinal Microbiome Composition Following Dietary Fiber Challenge [Through completion of sample collection, up to 72 hours after each study visit]: Microbial composition will be characterized from regional intestinal fluid samples collected using the CapScan capsule and from stool samples. Microbiome profiles will be evaluated across intestinal regions and compared between the low-fiber and inulin visits and between healthy and IBS participants.
• Change in Breath H2, CH4, and H2S Concentrations Following Inulin Challenge [8 hours during each study visit]: Breath hydrogen (H2), methane (CH4), and hydrogen sulfide (H2S) concentrations will be measured every 30 minutes during each 8-hour study visit. Concentration-time profiles will be compared between the low-fiber and inulin visits and between healthy and IBS participants.
• Change in Serum Short-Chain Fatty Acid Concentrations Following Inulin Challenge [8 hours during each study visit]: Serum short-chain fatty acid (SCFA) concentrations will be measured from serial blood samples collected every 30 minutes during each 8-hour study visit. SCFA concentration-time profiles will be compared between the low-fiber and inulin visits and between healthy and IBS participants.
• Change in Gastrointestinal Symptom Severity Following Inulin Challenge [8 hours during study visit]: Participant-reported gastrointestinal symptoms, including bloating/distention, abdominal pain/cramping, flatulence, nausea, and urgency, will be assessed throughout each study visit. Symptom trajectories will be compared between the low-fiber and inulin visits and between healthy and IBS participants.
• Glucose Response Following Dietary Fiber Challenge [Through CGM monitoring throughout 2 weeks including both study visits.]: Continuous glucose monitor (CGM)-derived glucose profiles will be evaluated following the low-fiber and inulin meal challenges. Glucose responses will be compared within participants across study visits and between healthy and IBS groups.
• Atmo Capsule Gastrointestinal Transit Time [From capsule ingestion through passage, confirmation up to 72 hours after clinic visit.]: Quantify the gastrointestinal transit time of the Atmo capsule after participant ingestion.

Sex: All, ages: 18 Years to 75 Years, accepts healthy volunteers

Inclusion Criteria:

1. Age 18-75 years.
2. Able to provide written informed consent.
3. Able and willing to swallow capsules and complete all study procedures.
4. For the Healthy cohort: no history of a diagnosed chronic GI disorder and no ongoing GI symptoms warranting clinical evaluation.
5. For the IBS cohort: meets Rome IV diagnostic criteria for IBS, with a prior clinical hydrogen/methane breath test on record sufficient to assign a breath-test subtype.
6. Willing and able to complete a virtual (Zoom) screening visit and two in-clinic study visits approximately one week apart.

Exclusion Criteria:

1. Known or suspected gastrointestinal stricture or physiological/mechanical gastrointestinal obstruction.
2. Gastrointestinal fistula.
3. Dysphagia or other swallowing disorder affecting the ability to swallow food, pills, or capsules.
4. Gastrointestinal surgery within the past 3 months.
5. Prior gastrointestinal surgery that alters luminal anatomy (e.g., bowel resection or bariatric surgery).
6. Diverticulitis.
7. Gastric bezoar.
8. Radiation enteritis.
9. Pacemaker or other implanted or portable electromechanical medical device.
10. Pregnancy or breastfeeding.
11. BMI ≥40 kg/m².
12. Use of antibiotics, probiotics, or prebiotics within 4 weeks prior to enrollment.
13. Use of proton-pump inhibitors (e.g., omeprazole, lansoprazole, esomeprazole \[Nexium®\]) within 7 days prior to the study procedure.
14. Use of histamine blockers, motility-altering medications, narcotic analgesics, or anticholinergic medications within 3 days prior to the study procedure.
15. Use of antiemetics/5-HT3 antagonists, macrolide antibiotics, or 5-HT4-modulating medications within 2 days prior to the study procedure.
16. Use of antacids (e.g., Gaviscon®, Mylanta®, TUMS) within 1 day prior to the study procedure.
17. Diabetes or use of medications affecting glucose handling.
18. Known allergy or intolerance to inulin or other study meal components.
19. Current participation in another interventional study.

• Sean P Spencer, MD PhD, Principal investigator, Stanford University
• Michael Fischbach, PhD, Study director, Stanford University
• Benjamin Good, PhD, Study director, Stanford University
• Jonas Cremer, PhD, Study director, Stanford University