Irritable bowel syndrome (IBS)—a condition characterized by altered bowel habits (either predominately constipation, predominately diarrhea, or both) and chronic abdominal pain—can be quite debilitating. Estimates vary widely from country to country, but the prevalence of IBS in industrialized countries is approximately 11%, with women affected more than men at a ratio of 1.67 (1).
However, perhaps because of the touchy subject of discussing their own bowel habits, only about 15% of individuals with IBS seek medical help (2). Clinicians sometimes refer to IBS as a functional disorder given that anatomical alterations (e.g. inflammation or villous atrophy) or dysfunction (e.g. enzyme deficiency) are typically not observed in patients’ gastrointestinal (GI) systems. This makes IBS a diagnosis of exclusion since the syndrome shares clinical symptoms with other organic diseases including malignancy, infection, and autoimmune bowel diseases.
Given the lack of obvious etiology, treatments for IBS often focus on symptom relief: laxatives for constipation, anti-diarrheal agents for diarrhea, or smooth muscle relaxants for pain. Unfortunately, only treating the symptoms neither diagnoses the problem nor resolves the underlying dysfunction.
No guideline-endorsed biochemical or imaging tests to diagnose IBS exist, so diagnosis relies on clinical symptoms. Rome diagnostic criteria require recurrent weekly abdominal pain observed over the last 3 months and at least two of the following: 1) pain during defecation, 2) change in stool frequency, or 3) change in stool form or appearance (3). For physicians to use the Rome criteria, they first must exclude other organic GI disorders, the symptoms of which would easily fulfill the Rome criteria (4). Given the wide range of differential diagnoses for all type of IBS, this article focuses on laboratory testing only for patients presenting with chronic diarrhea and no occult blood.
Laboratory evaluation of a patient with chronic diarrhea
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Infections: Acute diarrhea from bacterial, viral, or parasitic infections is often self-limiting within 1–3 weeks, but chronic diarrhea of >4 weeks may warrant laboratory evaluation. Excluding infection requires bacterial stool culture testing (Salmonella, Shigella, and Campylobacter), parasitic protozoa and worm testing, various antigen detection methods (Cryptosporidium, Giardia, and rotavirus), and polymerase chain reaction (PCR)-based methods (norovirus and adenovirus 40/41). New multiplex PCR assays provide a rapid and cost-effective option in place of multiple individual microbiology tests (5).
Disorders of intestinal inflammation: Organic inflammatory disorders of the GI tract can cause chronic diarrhea. One test to consider for assessing intestinal inflammation is serum c-reactive protein (CRP); however, CRP expression is nonspecific and variable, and the clinical sensitivity of elevated CRP for inflammation-associated diarrhea is limited (49%) (6). This makes biomarkers released from the inflamed intestinal mucosa into the stool more useful.
Fecal calprotectin helps distinguish IBS from irritable bowel disease (IBD), specifically ulcerative colitis and Crohn’s disease, and other chronic inflammatory conditions of the digestive tract. Calprotectin is the most abundant cytosolic protein in neutrophils, and when neutrophils are activated, it accumulates in feces. With high sensitivity but low specificity, a fecal calprotectin concentration <50 μg per gram of stool helps rule out inflammatory causes, while a high result is consistent with GI inflammation (7). Similarly, elevated lactoferrin in the stool indicates fecal leukocytes and intestinal inflammation with sensitivity and specificity of 82% and 79%, respectively (6).
Diet-induced diarrhea: Some food-induced diarrhea can be identified without laboratory testing by simply avoiding the suspected food sources (e.g. artificial sweeteners or lactose) and monitoring for symptom relief. However, other dietary triggers of diarrhea may signify a more serious underlying condition in which laboratory testing can be helpful. Patients may experience diarrhea when they eat fatty foods if they have disorders involving either inadequate pancreatic enzyme production required for fat digestion or the inability to absorb dietary fats. Pancreatic-damaging diseases, such as chronic pancreatitis or cystic fibrosis, can cause exocrine pancreatic insufficiency (EPI), while damage to the intestinal absorptive function may result from infections, celiac disease, or small intestinal Crohn’s disease.
Measuring fecal fat is the first-line test to detect impaired fat absorption and steatorrhea. The test requires challenging patients with a high fat diet (approximately 100 grams per day) and measuring fat in their stool collected over 48–72 hours. The traditional method for assessing fecal fat involves labor-intensive lipid extraction and gravimetric or titrimetric measurement techniques. More recently, direct nuclear magnetic resonance detection of fecal fats or near-infrared reflectance spectroscopy have shown equivalent results to traditional gravimetric methods (8). Healthy individuals excrete less than 6 grams of fat per day, but those with chronic diarrhea may excrete up to 14 grams per day even without a malabsorption pathology. Clinicians usually consider steatorrhea when fecal fat excretion exceeds 20 grams per day. Concomitant pancreatic enzyme testing aids in confirming EPI.
Celiac disease/gluten sensitivity: Patients with other chronic conditions such as celiac disease (CD) may have IBS-like symptoms including abdominal pain, bloating, and chronic diarrhea without overt malabsorption and weight loss. Laboratory-based identification of CD is important in the decision to implement a gluten-free diet. CD testing begins with serologic measurement of patient autoantibodies by enzyme-linked immunosorbent assay (ELISA) to identify candidates for confirmatory intestinal biopsy (9). Testing usually begins with serum tissue transglutaminase (TTG) antibodies, most commonly the IgA subtype. While anti-TTG-IgA is >90% sensitive for CD, this biomarker is not useful in patients with selective IgA deficiency (i.e. patients with extremely low or nonexistent IgA concentrations via immunonephelometry). In this subset of patients TTG IgG antibody testing is more appropriate. In patients already consuming a gluten-free diet, the positivity rate on serologic tests will be diminished. Here, initial testing for expression of DQ2 or DQ8, HLA markers that confer genetic susceptibility for CD, is an effective way to rule out CD if the immunogenotype is not identified.
Non-celiac gluten sensitivity (NCGS), formerly called gluten intolerance, also causes symptoms suggestive of IBS with diarrhea (IBS-D) (10). Similar to those with CD, NCGS patients sometimes experience increased stool frequency after they ingest gluten. However, they do not develop CD’s characteristic serum antibodies or duodenal mucosa lesions. Importantly, there are no NCGS-specific laboratory tests, and NCGS also does not have the same dependence on genetic susceptibility as CD, as only 50% of NCGS patients express HLA DQ2 or HLA DQ8. While this genetic prevalence is higher than that observed in the general population (~30%) this NCGS genetic connection pales in comparison with that of patients with CD (>97%). Therefore, clinicians identify NCGS through negative serologic CD tests and by patients’ symptoms lessening after they implement gluten-free diets.
Scenarios in which all the above considerations are excluded
After a physician excludes other causes of chronic diarrhea he or she is left with the diagnosis of IBS-D. The pathophysiology of IBS-D is not well understood, but the symptoms traditionally have been assumed to be psychogenic along with some abnormalities of the gut smooth muscle, visceral hypersensitivity, and hypervigilance of the central nervous system—all of which are difficult to treat directly. Many IBS-D treatment options take a “try it and see” approach to symptom reduction (e.g. probiotics, opioid receptor antagonists, tri-cyclic antidepressants, antispasmodic drugs, etc.) without any patient preselection. However, recent efforts to subcategorize IBS-D patients into treatable populations have shown some success (12,13).
Bile acid diarrhea: Approximately 25% of patients with IBS-D have excess bile acids in their stool, termed bile acid diarrhea (BAD) (14). The liver synthesizes and releases bile acids into the stomach where they solubilize dietary fats and facilitate lipid absorption in the small intestine. The primary bile acids—chenodeoxycholic acid and cholic acid—are reabsorbed by active transport in the terminal ileum: Microbiota deconjugate and dehydroxylate the remainder in the colon to form secondary bile acids, mainly lithocholic acid and deoxycholic acid (DCA), which are excreted in the stool (See figure: Life Cycle of Bile Acids).
The small intestine and colon reabsorb approximately 95% of bile acids, signaling the liver to downregulate bile acid synthesis via negative feedback inhibition. Some bile acid species induce fluid secretion and increased mucosal permeability and cause chronic diarrhea. Therefore, BAD is found in several disorders that impede the body’s ability to efficiently produce, reabsorb, and recycle bile acids (e.g. diseases of ileal dysfunction or ileal resection, post-cholecystectomy, small intestine infections, celiac disease, or chronic pancreatitis).
The gold standard test for BAD is the 23-seleno 25-homotaurocholic acid retention (75SeHCAT) test, which measures the rate of bile acid turnover after ingestion of a gamma emitter selenium conjugated synthetic bile acid. The retention of 75SeHCAT, as measured by whole body gamma counting at day 7 compared with day zero following oral administration, identifies individuals with low retention (i.e. decreased bile acid reabsorption). This test is available in many countries worldwide with the notable exception of the United States. However, laboratory tests that measure total fecal bile acids or the percent of primary fecal bile acids help identify patients with IBS-D, many of whom have BAD (15). Compared with the stool of healthy individuals, patients with BAD have increased total bile acids and a disproportionately high ratio of primary to secondary bile acids. Moreover, studies have found that treating BAD patients with bile acid sequestrants improves stool form, symptom severity scores, and stool frequency (16,17).
Laboratories can use enzymatic assays to measure total fecal bile acids—but these methods do not measure individual bile acid species (18). Chromatographic assays offer more specific and sensitive results for total and individual bile acids(12). These methods measure the most prominent bile acid species found in stool and report a total bile acid concentration, including the relative proportions of primary to secondary bile acids.
Fecal bile acid testing and fecal fat testing have the same patient preparation and sample collection requirements: a high fat diet and a timed stool sample. This means that one stool sample will suffice for both tests. However, for patients who bristle at the idea of collecting, storing, and shipping their own stool back to the laboratory, an alternative test for BAD may be more acceptable. Specifically, one of the metabolic intermediates in the synthesis of bile acids from cholesterol, 7-alpha-hydroxy-4-cholesten-3-one (7aC4), is elevated in the serum of patients with BAD owing to decreased reabsorption of colonic bile acids and subsequent lack of negative feedback regulation of bile acid synthesis in the liver. Laboratories accurately measure 7aC4 concentrations in serum using mass spectrometry techniques; however, diurnal variation may affect results, so measurements should be performed on a morning fasting sample. Serum 7aC4 test results are proportional to both 75SeHCAT test results and fecal bile acid test results, making it an attractive screening test for BAD without the need for a multi-day stool collection (20).
Serotonergic antagonists: Serotonin is a monoamine neurotransmitter derived from tryptophan that modulates a wide range of neuropsychological processes. However, most of it resides in enterochromaffin cells of GI tract mucosa where it regulates bowel motility and secretion via a complicated mechanism of interaction with multiple cell surface receptor subtypes, some of which have opposing effects. That said, increased circulating serotonin (or 5-hydroxytryptamine [5-HT]) presumably released from enteroendocrine cells, has been observed in IBS-D patients compared with lower concentrations in IBS-C (IBS with constipation) patients (21,22). Subsequently, selective serotonergic antagonists, specifically 5-HT3 receptor antagonists, benefit some patients with IBS-D. Whether 5-HT concentrations can be used to select candidates for treatement has yet to be shown.
What’s on the horizon?
While the diagnostic work-up we describe here is what most gastroenterologists currently use, is it the best we can do? A new “IBSchek” test claims in the literature to identify IBS-D in the general population of patients with chronic diarrhea. This ELISA system measures two newly identified markers found to be elevated in animal models of IBS-D, namely anti-cytolethal distending toxin B (anti-CdtB) and anti-vinculin (23). In gastroenteritis, bacterial pathogens such as Campylobacter jejuni excrete the toxin CdtB. Subsequent to the host defense, anti-CdtB antibodies can be found in the gut where they interact with the actin filament binding protein vinculin, likely via molecular mimicry. Therefore, the test identifies both anti-CdtB and anti-vinculin antibodies in circulation and may be useful in excluding conditions with overlapping symptoms such as IBD and CD. Anti-CdtB was shown to be 91.6% specific for IBS-D versus IBD, while anti-vinculin was 83.8% specific for the same discrimination. However, both tests displayed less ability to distinguish IBS-D from CD—a more frequent differentiation required in clinical practice. Whether this test is specific enough to meet the clinical needs for GI specialists remains to be determined.
Conclusion
Diarrhea-predominant IBS is not a straightforward diagnosis, so physicians consider a multitude of diarrhea-causing etiologies during clinical evaluations (24). Some laboratory testing aids in this process, however most tests in the laboratory armamentarium identify “not-IBS-D” conditions.
References
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Leslie J. Donato PhD, DABCC, is co-director of the cardiovascular laboratory medicine, co-director of the hospital clinical laboratory and point-of-care testing at Mayo Clinic in Rochester, Minnesota. +Email: [email protected]