New study raises questions about use of the test to determine diet adherence in celiac disease
By Amy Ratner, director of scientific affairs
The reliability of urine tests to determine how much gluten might be getting into the gluten-free diet is questioned in a new study.
The tests, which measure gluten immunogenic proteins (GIP) excreted in urine, are frequently positive in people who are following the gluten-free diet and negative in people who are consuming a significant amount of gluten, according to a study by researchers from Italy and the Mucosal Immunology and Biology Research Center at Massachusetts General Hospital in Boston.
But other scientists defend the accuracy of the tests based on the results of previous studies. Additionally, they note that the tests are useful as an objective measure of how much and how frequently those on the gluten-free diet are exposed to gluten, often despite their best efforts.
Meanwhile, authors of the study published recently in the journal Clinical and Translational Gastroenterology concluded that minute traces of gluten in the diet can result in a positive test and that false negatives are common.
Thirty four percent of study participants had positive urine test results while following a gluten-free diet, according to the study. Twenty five percent of those given large doses of gluten in a challenge had negative urine test results.
The performance of the test in monitoring the gluten-free diet was “poor,” the study authors wrote.
“Due to the impossibility of standardizing the test in normal conditions” it seems unlikely that determination of GIP in urine is a reliable tool for assessing compliance with the gluten-free diet, the study says.
In addition to being used in research, urine tests are available to physicians and for at-home use by consumers to determine how much gluten above a certain level might be getting into an ostensibly gluten-free diet, most often due to cross-contact.
The study set out to determine how well the urine test measures GIP and how the amount of gluten consumed is related to the quantity of GIP recovered. The tests have been seen as a promising tool to measure compliance with the gluten-free diet, the study says. A small fraction of gluten peptides is absorbed and excreted in urine or excreted in stool, the authors wrote.
Twenty-five pediatric residents at the Polytechnic University of Marche volunteered for the study. They did not have celiac disease but followed the gluten-free diet for three days each before consuming varying amounts of a gluten and for 24 hours after. Urine samples were collected before each of six gluten challenges in amounts ranging from zero to 1,000 milligrams (mg). All urine excreted in the 24 hours afterward was tested for GIP.
The gluten-free diet followed by study participants included foods labeled and certified gluten-free, which contained less than 20 parts per million of gluten, the standard for gluten-free foods in the United States and Europe. A dietitian knowledgeable about celiac disease oversaw the diet and study participants kept a food diary. The likelihood of cross-contact from restaurant meals was reduced because of COVID-19 restrictions in place in 2020 when the study was done, the authors note. he diet was followed for three days before the gluten challenges, the study says, because data has shown that urinary GIP is undetectable from 16 to 34 hours from the complete removal of gluten from the diet.
In urine samples taken after the three days of the gluten-free diet but before any gluten challenge, 51 of 150 were positive for GIP.
When the challenge contained zero gluten, seven of 17 samples were positive. However, in the challenges of 10 to 1,000 mg, only 55 of 81 samples were positive and there was no significant change in the 24-hour GPI when the amount of gluten increased from 10 to 1,000 mg, the study says.
In a second part of the study, 12 participants went on to follow the stricter gluten-contamination-elimination diet, which consists of only whole, fresh, unprocessed gluten-free food. All urine samples taken were negative for GIP.
Study participants were then given gluten challenges of 5 or 10 mg. These tiny amounts of gluten are tolerable and may be found in a standard gluten-free diet, the study says. Eight of 24 samples were positive for GIP.
All samples taken after the gluten elimination diet but before the gluten challenge were negative for GIP, and 8 of 24 were positive after challenges of 5 or 10 mg.
“A true zero-gluten diet was constantly associated with a negative urinary GIP test, whereas traces of gluten that may be found in commercially available gluten-free food yielded a positive result in a significant portion of cases,” the authors wrote. “The previously reported high rate of positive GIP tests in patients with celiac disease on a gluten-free diet should not be interpreted as evidence of poor compliance with the gluten-free diet.”
They note that this has “raised many concerns in the real life of celiac disease patients.” The authors add that the use of the urine test might be useful in monitoring hypersensitive celiac disease patients on the elimination diet.
A 2020 study that examined the gluten content of the gluten-free diet being followed by those with celiac disease in a real-world setting found that two thirds were consuming some level of gluten. The results were based on urine and stool tests and questionnaires filled out by study participants.
Julio C. Bai, MD, professor emeritus at the Research Institutes Universidad del Salvador, who was not involved with new study, said previous research has led to questions about the sensitivity of the urine test. Sensitivity measures how often a test correctly gives a positive result when, in this case, someone has consumed above a set limit of gluten.
But he noted that the new study also questions the test’s specificity. Specificity measures how often a test gives a correct negative result, when in this case, someone has not consumed above a set limit of gluten.
“This, in my opinion, remains debatable, as it is extremely difficult to achieve a [zero milligram] gluten intake. Complete elimination of gluten from the diet has been shown to be almost impossible to achieve in celiac patients, and it is likely to be even more difficult in healthy subjects, as those enrolled in this study,” he said.
Bai has done research using tests for GIP in urine and stool to determine real world exposure to gluten in those with celiac disease on a gluten-free diet.
He noted that more than 60 studies using the tests have been published to date, revealing that involuntary and voluntary gluten exposure is very common in those with celiac disease on a gluten-free diet. “Some of these studies indicated 40 percent of treated celiac disease patients are exposed to more than 50 mg of gluten per day, and 24 percent are exposed to more than 100 mg,” Bai said.
Questions remain about how to best use the test, which is the first generation of its kind, in clinical practice, according to Bai, “because there is no precedent for GIP detection kits, there is no available ‘gold standard’ to compare against.” Also, the detection and quantification of GIP in urine is complicated by the dynamics of absorption, with testing for GIP in urine done with the understanding that its presence only represents gluten exposure of no more than the previous 24 hours.
Still, Bai said the research community welcomes key studies like the newly published study, which “uncover controversies which should stimulate more research to determine the best timing and conditions for the routine use of these innovative GIP tests in clinical practice.”
Ángel Cebolla, PhD, chief executive officer and chief scientific officer at Biomedal, the biotechnology company that makes the urine test and a stool test for GIP, said the new study has “too many deficiencies” compared to previous research. Most concerning is the lack of controls regarding how well study participants followed the gluten-free diet, he said.
For example, previous studies using the urine tests have also analyzed food to confirm it was gluten free. Stool tests, which are more sensitive than urine tests, were used to verify that a strict gluten free diet was being followed.
“We believe that those high rate of positives [among study participants following a typical gluten-free diet] could be due to low adherence to the diet of the volunteers or technical mistakes in the use of the tests,” he said. “Objective controls to confirm or discard any feasible explanation are missing.”
When the gluten elimination diet was used, the 34 percent of results identified in the first part study as false positive disappeared, he said. These two results seem to contradict each other, Cebolla noted.
A 2015 study done by Cebolla and colleagues concluded that GIP is detected in urine after gluten consumption, enabling a new and non-invasive method to monitor gluten-free diet compliance and transgressions. The method was sensitive, specific and simple enough to be convenient for clinical monitoring of patients with celiac disease as well as for basic and clinical research applications including drug development, the study says.
A false negative is feasible, but a false positive has never been reported, he said. Cebolla was an author of a 2021 study of the range of time and the amount of GIP excreted in urine after specific gluten ingestions. That study concluded an increase in the frequency of urine tests may be a suitable approach to avoid false negative results.
The ability to capture a biomarker of gluten consumption in a sample of urine is a convenient, noninvasive procedure, Cebolla’s study says. “Therefore, urinary GIP detection provides a supplemental tool to evaluate gluten exposure in individuals following a [gluten-free diet}, the authors wrote, noting that the results provide additional knowledge about gluten metabolism and GIP excretion, which could be useful to fine-tune the application of GIP determination in the follow-up of patients with celiac disease and gluten-related disorders
As it has become clearer that more repeat testing gives a more accurate picture of gluten consumed, Biomedal has launched a kit with five tests for at-home use, reducing the price per test, according to Cebolla. This also helps with detection of a low amount of gluten accidentally ingested when dining out, he said.
When used in a doctor’s office, multi-testing of three urine samples from different days would give a clearer picture of intestinal damage or healing, Cebolla said. Three negative urine tests from three different days would mean a 97 percent possibility of no villus atrophy, according to one study. Research has found the concentration of GIP and repeated positive urine test results are significantly related to damage to the finger-like absorbing surface of the small intestine, called the villi, seen in celiac disease.
You can read more about the new study here.
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