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Stool tests reveal it’s common for unintended gluten to get into a strict gluten-free diet

June 30, 2023

Study explores how to interpret test results in celiac disease management and clinical trials

By Amy Ratner, director of scientific affairs

Even celiac disease patients who were following a gluten-free diet and felt well were still unintentionally consuming some amount of gluten that was detected in stool tests, according to preliminary results of a study recently presented as a poster at Digestive Disease Week (DDW).

Australian researchers had set out to determine how tests for gluten in stool can best be used as an objective tool to monitor the gluten-free diet both by physicians treating patients and in clinical trials. They were interested in determining the best time to test for gluten and the lowest amount of gluten that could reliably be detected in a stool test.

Their study was designed to evaluate tests for gluten in stool in a real world scenario that mimicked “accidental, low-dose gluten exposure.” It also aimed to compare stool testing to traditional ways of evaluating the adequacy of the gluten-free diet, such as blood tests for celiac disease antibodies. Fifty two study participants who had been on the gluten-free diet for at least one year were recruited for the study.

The study called for participants to eat a cookie containing one of several amounts of gluten, followed by measurement of gluten immunogenic peptides (GIP) found in all stool samples collected for one week.

But even before study participants were given the cookies, preliminary tests found that 87 percent had one or more stools samples containing gluten, which researchers called “high background gluten excretion.” This was despite the fact that preliminary blood tests for gluten antibodies, excellent gluten-free diet adherence test scores, and daily symptom monitoring indicated that gluten was not getting into the patients’ diets.  

Those findings prompted researchers from the Walter and Eliza Hall Institute and colleagues to follow-up their original study with a second investigation into background gluten exposure with 12 of the celiac disease patients from the first study.

Study one

For the first study, researchers included 29 participants who had no detectable gluten in stool samples taken immediately before the gluten challenge was initiated.

Participants were given 50, 250, 500 or 1000 milligrams (mg) of gluten or a placebo in a cookie. They then collected samples of all stool for one week.  Celiac disease blood tests were done at the beginning and end of the study and symptoms were monitored daily, Additionally, study participants completed a food diary and a behavior questionnaire that asked about lifestyle, knowledge of the gluten-free diet and diet practices.

GIP was detected in stool samples of all participants except those who got the placebo.

The amount of gluten in the cookie did not correlate with the amount of gluten in the stool, but higher amounts were detectable for a longer time. For example, gluten was detectable for up to six days following a 1000 mg challenge, but only for two days with a 50 mg challenge. Additionally, acute gastrointestinal symptoms increased significantly after the 1000 mg dose but did not occur consistently with less gluten.

Study results lead researchers to conclude that the optimal testing window after a suspected gluten exposure is one to two days.

Study two

In the follow-up study, researchers set out to determine the sources of gluten in the regular gluten-free diet of 12 original study participants and to test whether they were consistently being exposed to gluten over time. Researchers selected participants to cover a range of high to low gluten detection in the earlier, pre-study stool tests.

Study participants followed their regular gluten-free diet and collected stool samples three times a week for two weeks.  Test results showed that GIP detections “remained remarkably consistent” for each participant over time from the first study to the second.

If a person had gluten in all or most samples during the initial monitoring, they had the same levels in the follow up study one to two years later, according to the authors. Similarly, if someone was negative in the first study, they likely remained negative.

“This indicates gluten exposures are more than random occurrences and may reflect long-term lifestyle and dietary behaviors,” the authors note.

In addition to stool tests, study participants again reported on their diet and filled out the lifestyle questionnaire. The questionnaire revealed a trend in which people who rarely dined out or only dined out in gluten-free accredited restaurants tended to have lower levels of GIP in stool tests, the study says. However, matched food diaries reviewed by a dietitian “did not reveal any obvious sources of gluten exposure in participants with high GIP levels,” the authors wrote.

While dining practices are an important consideration, more data is needed to understand sources of unintended gluten exposure, the study concludes. The researchers are currently doing studies examining GIP detection in stool and gluten in food samples, particularly as these relate to celiac disease patients who have inexplicably high levels of gluten in stool samples despite seemingly being seemingly excellent in follow the gluten-free diet.

 A previous study in the United States and Canada compared gluten from samples of food to gluten in stools. When food tested positive for gluten peptides, it was associated with a positive stool test in 83 percent of cases.

Overall conclusions

Results of the two studies suggest that gluten excretion and therefore gluten consumption is common in people with celiac disease who are seemingly healthy. Ninety four percent of study participants self-reported high adherence to the gluten-free diet and all had negative blood tests for celiac disease antibodies.

If used to monitor a celiac disease patient’s gluten-free diet, more than one stool test would be needed, the study concludes.

However, the high sensitivity of tests for GIP in stool means some positive test results might not be significant in managing treatment for those with celiac disease. More study into the relationship between GIP in stool and intestinal damage is required to appropriately interpret a positive GIP result, the study says.

“A negative GIP result may be more informative than a positive result,” researchers concluded, noting that a negative result could be a way to rule out gluten exposure in celiac disease patients who continue to have symptoms on the gluten-free diet.

Additionally, since positive GIP might not be an indication of clinically significant gluten exposure, how to use stool tests in clinical trials has yet to be determined, according to the study. A positive GIP result alone might not be adequate reason to exclude a person from participating in a clinical trial, the authors note.

DDW is the largest international gathering of physicians, researchers and academics in the fields of gastroenterology, hepatology, endoscopy and gastrointestinal surgery. Studies presented at DDW as posters are often preliminary and give an early look at investigations that are likely to include more details as they progress toward publication in a peer reviewed journal. Posters selected to be presented at DDW go through a review process.


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