Irritable bowel syndrome with diarrhea: is colonoscopy mandatory? Opportunistic use of colon mucosal organ culture to diagnose celiac disease
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Irritable bowel syndrome with diarrhea: is colonoscopy mandatory? Opportunistic use of colon mucosal organ culture to diagnose celiac disease
CC BY 4.0
Introduction
According to the American Society of Gastrointestinal Endoscopy (ASGE) and the European Panel on the Appropriateness of Gastrointestinal Endoscopy guidelines, most colonoscopies are performed to screen for colon cancer, fecal occult blood, hematochezia, unexplained iron deficiency anemia, chronic diarrhea, follow‑up ulcerative colitis, and unexplained weight loss. Nevertheless, several studies published in the United States, Europe, and the Middle East indicate that 20%–50% of colonoscopies are performed due to inappropriate reasons. Interestingly, the number of colonoscopies carried out has also increased for purely defensive reasons. Consequently, most of the tests give negative results due to their inappropriateness, thus constituting a waste of resources.1-5
A recent analysis based on a national database showed that about a quarter of all colonoscopies performed in the United States are conducted due to the presence of symptoms generally related to irritable bowel syndrome (IBS).1-3
One of the main clinical concerns in IBS patients is microscopic colitis (MC), which usually affects approximately 1% of these individuals.3 Importantly, the presence of MC is usually underestimated, and many such patients are instead diagnosed with IBS. Furthermore, in recent years, several studies have reported an association between celiac disease (CeD) and MC. The concomitance of CeD and MC should always be taken into consideration when gastrointestinal symptoms persist despite following a gluten‑free diet in patients with CeD. Likewise, the association should also be considered when treatment with budesonide does not appear to be successful in patients with MC.
CeD is a chronic autoimmune gastrointestinal inflammatory disease triggered by ingestion of gluten. It only affects genetically predisposed individuals of any age and sex. Over the last 30 years, estimates point to an increasing prevalence of CeD from 1% to over 1.5%.8,9
The CeD diagnosis is based on the presence of specific autoantibodies (antitransglutaminase antibodies [anti‑tTG] and antiendomysial antibodies [EMAs]) on a gluten‑containing diet, and atrophy of the intestinal mucosa.5,6
Although the diagnostic process is clearly defined, delayed CeD diagnoses are still common. One of our previous studies has quantified how many diagnostic tests are performed unnecessarily before reaching the correct diagnosis. We demonstrated that the duodenal mucosa produces specific antibodies, as shown by their detection in the culture supernatants of duodenal biopsies.7These works have shown that oral mucosa and colon mucosa can also be used to diagnose CeD as sources of material for the organ culture system.5
The aim of this study was to use the organ culture of colon biopsies to raise a diagnostic suspicion of CeD in the context of IBS with diarrhea (IBS‑D).
Patients and methods
Over the past 3 years, we studied 78 patients who underwent rectosigmoidocoloscopy (RSCS) for different reasons, including blood in stool, rectorrhagia, follow‑up of IBS, family history of colorectal cancer, anemia and weight loss, pain / abdominal swelling, and diarrhea. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Council of the Department of Translational and Precision Medicine, Sapienza University of Rome (8.7.0 10/12/2020). Informed consent was obtained from all patients involved in the study.
Rectosigmoidocoloscopy / organ culture
According to the ASGE guidelines, biopsies of the colonic mucosa were performed for diagnostic purposes for each of the 78 patients included in the study. One colon biopsy was used for organ culture. Samples testing positive for anti‑tTG were confirmed by EMA positive results. In the patients showing anti‑tTG/EMA positive results in the culture supernatants we also tested serum samples for the same antibodies. All these patients were advised to perform esophagogastroduodenoscopy (EGDS) to obtain a biopsy of the duodenal mucosa for histologic examination (diagnostic gold standard).1,5 Anti‑tTG immunoglobulin A (IgA) antibodies were measured in the culture supernatants (diluted 1:5) with enzyme‑linked immunosorbent assay (ELISA) in microtiter‑plate wells coated with recombinant human tTG (Eurospital, Trieste, Italy). Absorbance was measured in an ELISA plate reader at 450 nm. As previously reported, 0.3 U/ml was used as a cutoff to identify anti‑tTG positive results.8,9
Organ culture system
The culture medium, prepared with 17 ml of the Roswell Park Memorial Institute 1640 medium, 3 ml of fetal bovine serum, 0.2 ml of L‑glutamine (200 mM), 2 ml of penicillin (10 000 IU/ml)-streptomycin (10 000 μg/ml), and 0.04 ml of gentamicin (10 mg/ml) (Gibco / Invitrogen, Carlsbad, California, United States), was preventatively stabilized at pH 7.4 and sterilized by filtration with a 0.22 μm pore size filter (Sigma Chemical, St. Louis, Missouri, United States). Thereafter, the culture supernatants were collected and stored at −20 °C until use. All steps were performed in sterile environment.
Antiendomysium antibody detection
IgA EMAs were sought in undiluted culture supernatants by indirect immunofluorescence analysis on cryostat sections of monkey esophagus (Antiendomysium Biopsy, Eurospital). Positive EMA results were identified by a typical honeycomb‑like staining pattern along muscularis mucosae (endomysium), which marks the collagenous matrix of type 3 connective tissue surrounding the smooth muscle fibers of the primate esophagus.
Antitissue transglutaminase antibody detection
IgA anti‑tTG antibodies were measured in the culture supernatants diluted 1:5, using ELISA on microtiter‑plate wells coated with recombinant human tTG (INOVA Diagnostics, San Diego, California, United States; distributed by Instrumentation Laboratory, Milan, Italy). Absorbance was read at 450 nm and, as previously reported,10 the 0.3 U/ml antibody level was used as a cutoff to identify anti‑tTG positive results in duodenal cultures. The mean serum antibody level of EMA and anti‑tTG negative patients + 2 SD (0.2) was used as a cutoff to identify anti‑tTG positive results in sigmoid material cultures. Reproducibility of the ELISA was calculated by intra- and interassay coefficient of variation (CV). For the intra‑assay variation, 10 reference samples were tested 4 times in the same run; CV range was 0.8%–3.1%. For the interassay variation, the same 10 reference samples were tested 1 time in 3 different runs; CV range was 1.9%–8.2%.
Results
As many as 11 out of 78 patients (8.6%) undergoing RSCS tested positive for anti‑tTG IgA in the culture supernatants of colon biopsies. Positive results for anti‑tTG IgA ranged from 0.3 U/ml to 0.8 U/ml. They were confirmed by EMA detection.
Of those 11 patients, only 8 agreed to blood sampling for anti‑tTG/EMA detection, and they all tested positive. Then, the 8 patients underwent EGDS to perform duodenal biopsy with organ culture, which showed duodenal mucosal atrophy and EMA positivity in culture fluids.
Discussion
CeD represents the main cause of malabsorption, which often causes growth delay in children, and several systemic symptoms in adults. CeD, whose prevalence in the general population ranges from 1% to 2%, is often associated with the presence of other autoimmune diseases. Many studies have already shown that a late diagnosis of CeD can negatively affect well‑being and increase the risk of developing gastrointestinal lymphoma.11,12 The importance of an early diagnosis is therefore crucial. Additionally, a failure to diagnose CeD results in high costs for the patient and the health care provider.
Previous works have already demonstrated that EMA and anti‑tTG antibodies are detectable in culture supernatants of the sigmoid mucosa as well as the duodenal and oral mucosa.8,12,13
Although the small intestine mucosa is the primary target organ of CeD, other parts of the gastrointestinal tract may also be sensitive to gluten. Immunologic changes have, in fact, been observed in the oral and rectal mucosa of celiac patients after local administration of gliadin.4
Several studies have already demonstrated that the organ culture of the intestinal mucosa of celiac patients could also be a useful diagnostic tool.10
According to current literature, CeD could also be considered a subset of IBS due to its heterogeneous clinical presentation.14 Several epidemiologic studies have in fact shown that patients with typical IBS‑D symptoms frequently, and often inappropriately, undergo colonoscopy.1,15-20
In our study, the sigmoid organ culture additionally allowed us to detect CeD in patients undergoing colonoscopy, as they suffered from IBS‑D.
In conclusion, we managed to make an opportunistic diagnosis of CeD in 11 of the 78 patients (8.6%) included in our study. These patients tested positively for EMA‑IgA and anti‑tTG IgA in organ cultures of sigmoid mucosal biopsies, with values ranging from 0.3 U/ml to 0.8 U/ml.
Of the 11 patients who tested positive for anti‑tTG IgA in colon biopsy culture supernatants, only 8 agreed to be tested for specific serum antibodies, and all of them had positive results. They all underwent EGDS for duodenal biopsy, which showed atrophy of the duodenal mucosa, and tested positive for EMA in the organ cultures.
Although further studies are needed to confirm the results presented here, in all cases of IBS‑D, especially if accompanied by diarrhea and anemia, where diagnostic procedures involve colonoscopy, the use of organ culture could become an unexpectedly effective diagnostic tool.
Although organ culture is usually considered only a confirmatory test, its more routine use could reduce the inaccuracies related to the routine diagnostic process. The use of appropriate specific commercial diagnostic kits already available (Antiendomysium Biopsy) could also simplify this opportunistic diagnostic process.
In all cases of IBS‑D, the use of intestinal mucosal organ culture could, therefore, immediately suggest a diagnosis of CeD and avoid further diagnostic tests that are often time‑consuming and expensive.
Finally, this work could improve the quality of life of patients with IBS‑D and offer them more viable therapeutic options.
- Chey WD, Nojkov B, Rubenstein JH, et al. The yield of colonoscopy in patients with non‑constipated irritable bowel syndrome: results from a prospective, controlled US trial. Am J Gastroenterol. 2010; 105: 859‑865. | Crossref
- Vulpoi RA, Luca M, Ciobanu A, et al. The potential use of artificial intelligence in irritable bowel syndrome management. Diagnostics. 2023; 13: 3336. | Crossref
- Lieberman DA, Holub J, Eisen G, et al. Utilization of colonoscopy in the United States: results from a national consortium. Gastrointest Endosc. 2005; 62: 875‑883. | Crossref
- Bergman D, Khalili H, Lebwohl B, et al. Celiac disease and risk of microscopic colitis: a nationwide population‐based matched cohort study. United European Gastroenterol J. 2023; 11: 189‑201. | Crossref
- Singh AD, Ellias S, Singh P, et al. The prevalence of the celiac disease in patients with dyspepsia: a systematic review and meta‑analysis. Dig Dis Sci. 2022; 67: 3067‑3079. | Crossref
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