ABSTRACT
Irritable bowel syndrome (IBS) is one of the most common conditions in gastroenterology. The lack of characteristic endoscopic findings and specific organic biomarkers relegated IBS to a “functional condition” (rather than an organic disease) diagnosed based solely on the assessment of symptoms in an approach that requires the initial exclusion of other organic conditions. Over the last few decades multiple hypotheses have been proposed for the pathophysiology of IBS. In the last 15 years a prevailing hypothesis has emerged suggesting that IBS is due, at least in part, to changes in the gut microbiome. These changes can be detected in stool but data consistently demonstrate that there are changes in small bowel flora. This has been validated by findings from breath testing, small bowel culture, and deep sequencing of small bowel aspirates. These changes in intestinal microbial colonization in IBS form the basis for the recent FDA approval of rifaximin for this condition. However, the reason for these microbial changes remained unknown. Many prospective studies have shown that acute gastroenteritis is a precipitating cause of IBS. Studies using an animal model have since shown that infection with Campylobacter jejuni can result in phenotypes similar to those seen in humans with IBS, and have been used to study the development and diagnosis of IBS. In this article, we examine the development of the first serum biomarker and underlying pathophysiological mechanisms in IBS. This new serologic marker suggests that the majority of subjects with IBS have an organic basis for their disease.
Irritable bowel syndrome (IBS) is one of the most common conditions in gastroenterology 1,2, and has also been one of the most challenging for clinicians. This is in part due to the lack of specific biological markers for the condition, as well as the lack of hallmark findings on endoscopy. In attempting to elucidate the pathophysiology of IBS, experts have investigated a number of avenues including the role of stress 3,4, alterations in the brain-gut axis 5-8, changes in bile acid metabolism 9,10, and altered intestinal motor function 11,12, amongst others. However, two concepts have proven to have great significance in IBS. These include the findings of altered small bowel microbiota in IBS 13,14, and that acute gastroenteritis can precipitate the development of IBS 15-17.
Burden of IBS
Irritable bowel syndrome represents a significant health burden. In the US, data suggest that this condition affects over 40 million individuals 2,18. In clinical practice, up to 50% of visits to the gastroenterologist may be due to IBS 19,20 and, based on the AGA burden of illness assessment from over a decade ago, this condition represents a tremendous economic burden to the healthcare system and society 21. This was due in part to the lack of good therapies, and more importantly, the lack of specific diagnostic tools. Tools to definitively diagnose and treat IBS would be of tremendous benefit to patients, and would also substantially reduce the global health care costs associated with IBS.
Diagnosing IBS
For more than 3 decades, the diagnosis of IBS has been based on a set of symptom criteria. These included the Kruis 22, Manning 23 and Rome criteria 22-28. Although the Manning criteria were the most validated set of criteria, the majority of studies have relied on the expert consensus-based criteria that have been developed by the Rome committee. The most recent iteration of these are the Rome IV criteria 28.
There are number of significant problems with the symptom criteria-based approach to IBS. First, the Rome criteria are vague and non-specific. This leads to a great number of false positives in gastrointestinal disease conditions. Although the criteria have merit when comparing to healthy controls, healthy subjects are generally not seeking care from a gastroenterologist. In studying patients with inflammatory bowel disease, more than 70% of these non-IBS subjects would meet the symptom criteria for IBS using the Rome criteria 29,30. As a result, IBS has become a “diagnosis of exclusion”. This framework essentially means that the Rome criteria have become the most expensive test for IBS, since it is imperative to first rule out other conditions first. Thus, at the discretion of the clinician, subjects may undergo endoscopy, celiac testing, blood panels, stool testing, radiologic studies and other invasive, time-consuming and expensive testing prior to being diagnosed with IBS 31,32.
Compounding this direct expense, the Rome criteria do not provide the patient with confidence in a diagnosis. To tell a patient they have IBS based on criteria may lower confidence and a patient may be motivated to seek further opinions. This pattern of testing and doctor shopping substantiates data that it can take up to 6 years to secure a diagnosis of IBS 33.
Microbes and IBS
In the last 20 years, there has been a growing understanding that microbes play a role in IBS. It has been demonstrated that subjects with IBS have small intestinal bacterial overgrowth (SIBO). This has now been confirmed by breath testing 13,14,34-36, meta-analyses 37, small bowel culture 38,39, qPCR 40 and deep sequencing of the duodenal flora 41. This is true even after controlling for use of proton pump inhibitors 42. The concept that the pathophysiology of IBS has a microbial basis was further supported by the recent approval of rifaximin for diarrhea-predominant IBS (D-IBS) by the FDA 43. Although the concept of SIBO is well established in a subset of IBS, the root cause of these changes remained uncertain.
Concurrent to the evolving SIBO hypothesis in IBS, data emerged suggesting that acute gastroenteritis was a definitive precipitant of IBS. In fact, based on multiple large-scale prospective and longitudinal studies, gastroenteritis is the only proven root cause of IBS 44-48. Some suggest that a source event of gastroenteritis can only be identified in a small subset of the total population of IBS. However, recall bias and lead time bias may impair a full understanding of the prevalence of IBS that derives from a post-infectious event. A recent study used a mathematical model to calculate the number of IBS patients in the US that could result from acute gastroenteritis, using data from the CDC and the breadth of published work at the time on rates of IBS from gastroenteritis and remission rates 37. Based on the study assumptions, a steady state would be reached at 15 years with a point prevalence rate for IBS of 9.1% of the US population derived solely from acute gastroenteritis. Although this study is a hypothetical analysis based on a series of assumptions, it raises the possibility that the majority of IBS results from acute gastroenteritis.
To better understand the pathophysiology of IBS on the basis of two distinct microbial concepts (SIBO and post-infectious IBS), animal models were needed. Existing animal models were based on various pathogens such as the parasite Trichinella spiralis 49,50 . An important challenge with these models was that pathogens such as Trichinella spiralis are not common in humans. One of the first descriptions of post-infectious IBS in humans was based on infection with Campylobacter jejuni in Europe. Since then, most outbreaks of post-infectious IBS result from infections with C. jejuni 51,52, pathogenic Escherichia coli 53, Salmonella 54 and Shigella 55, which are among the most common causes of gastroenteritis. Besides symptom development, the only marker for post-infectious IBS in humans is an elevation in rectal intra-epithelial lymphocytes 51.
To more accurately represent post-infectious IBS as seen in humans, a new animal model was developed based on infection with C. jejuni 81-176 56. In the first validation of this model, rats were initially infected with C. jejuni, were allowed to recover from the acute illness and then assessed 90 days after C. jejuni clearance from the stool. In this post-infectious period, rats were found to have developed altered stool form, increased rectal lymphocytes and SIBO when compared to controls, all of which are seen in human subjects with post-infectious IBS 56. These findings, for the first time, unified the concepts of SIBO and the role of bacterial pathogens in post-infectious IBS.
Autoimmunity and IBS
This validated animal model has allowed the discovery of pathways linking gastroenteritis to the development of IBS. The four major bacterial pathogens that cause gastroenteritis all produce cytolethal distending toxin (Cdt). Cdt is a heterotrimeric complex of three subunits, CdtA, CdtB, and CdtC 57-59. Of these, CdtB is the active toxin subunit and CdtA and CdtC are proposed to collaborate to facilitate the binding and entry of CdtB into mammalian cells. While the role of CdtB is not fully understood, it has been implicated in causing G-2 phase arrest in vitro 58. In a follow-up study, rats infected with C. jejuni were compared to rats infected with a mutant C. jejuni strain with an insertional deletion of CdtB 60. Rats infected with the mutant C. jejuni lacking CdtB exhibited a near-normal post-infectious phenotype 60, suggesting that CdtB plays a key role in the pathophysiology of post-infectious IBS.
Building on this work, immunohistochemical analyses of sections from the gastrointestinal tracts of these rats were performed. These studies revealed a striking reduction in the numbers of the interstitial cells of Cajal (ICCs) in rats which had been infected with C. jejuni, particularly the deep muscular plexus 61. This was significant as ICCs, which are also reduced in human IBS subjects, have been described as intestinal pacemakers, and are required for normal intestinal motility, including phase III of interdigestive motor activity 62. Consistent with the fact that an absence or reduced frequency of these contractions is known to induce SIBO 11,62, the reduction in ICCs correlated with the development of SIBO in these rats 61. Further immunohistochemical analyses revealed that antibodies to CdtB were binding to elements of the neuromuscular apparatus of the gut, including ICCs and myenteric ganglia 63. This pattern of staining was also seen in small intestinal sections from rats that were never exposed to C. jejuni, and from human subjects who did not have IBS 63, suggesting that antibodies to the CdtB toxin were binding to a neuronal protein in the host gut, likely as a result of molecular mimicry.
Through a series of studies in this animal model and in human tissue sections, it became clear that the endogenous protein with which host antibodies to CdtB were cross-reacting was the cytoskeletal protein vinculin 63. Vinculin is a cytoplasmic actin-binding protein and an important component of focal adherens junctions (FAJ) and tight junctions 64-67, with roles in cell adhesion 68, neuronal cell motility and contractility 69, as well as structural integrity 70 and epithelial barrier formation 71. Vinculin knockout mouse models demonstrate failure of neuronal migration 72. Vinculin consists of a globular head domain that binds to talin and α-actinin, and a tail region that binds to F-actin, paxillin, and lipids; in its inactive form, the head and tail regions of vinculin bind together 73,74. Interestingly, molecular mimicry of the vinculin-talin interaction is the mechanism by which both the IpA toxin of Shigella 75 and the Rickettsia surface cell antigen 4 76 achieve cell entry, activating vinculin and interacting with the actin cytoskeleton. Significantly, we found that C. jejuni-infected rats exhibited reduced vinculin expression in the small intestinal wall, and that levels of antibodies to CdtB correlated both with the degree of loss of vinculin, and with the development of SIBO in these animals 63.
This series of studies now support a potential pathophysiologic pathway in the development of post-infectious IBS (Figure 1). Acute gastroenteritis with resulting systemic exposure to CdtB leads to positive seroconversion. Subsequently, homology between CdtB and vinculin leads to the generation of autoantibodies to vinculin in a subset of subjects and ongoing reductions in ICC numbers amongst other effects. These in turn lead to changes in neuromuscular function, ultimately resulting in SIBO and the development of IBS. This forms the basis for IBS-D being an antibiotic-responsive disease.
Development of IBS Biomarker
The results from the above-described studies suggested that serum biomarkers for IBS based on previous gastroenteritis as a causative agent could be feasible. To examine this, an enzyme-linked immunosorbent assay (ELISA) was developed to accurately measure the levels of anti-CdtB and anti-vinculin in humans 29. These biomarkers were tested in a large-scale (180 center) study of IBS-D, comparing to groups of subjects with celiac disease, Crohn’s disease, ulcerative colitis and healthy controls 29. In this study, these two markers were found to be highly useful in identifying IBS-D compared to other causes of diarrhea. Based on a pre-test probability of IBS in a gastroenterologist practice 77, the test characteristics produce a post-test probability of IBS of nearly 95% 29.
Although these biomarkers were highly specific for IBS, they had some limitations. Despite the greater than 90% specificity, the sensitivity was below 50% at the selected cutoff levels. These cutoffs were selected for optimization of the likelihood ratio and to improve test accuracy. In other words, a positive test meant there was a high likelihood that the patient had a definitive diagnosis. This cutoff selection was designed reduce the need for additional testing.
The second important issue was whether the test would only identify subjects with IBS-D, or would also identify subjects with IBS-M and IBS-C. In a second study, anti-CdtB and anti-vinculin titers were assessed in these three subject groups. The results of this analysis demonstrated that anti-CdtB and anti-vinculin titers had a statistically significant negative gradient from IBS-D to IBS-M, then to IBS-C and healthy individuals (p<0.001) 78. There was no statistical difference between antibody levels in subjects with IBS-C as compared to healthy individuals. Based on the definition of a positive test, the test can be used to identify subjects with IBS-D and IBS-M, but not IBS-C 78.
Implications of a Plasma Biomarker for IBS
The measurement of anti-CdtB and anti-vinculin antibodies as a means of diagnosing IBS represents an important breakthrough for a number of reasons (Table 1). This represents the first test that makes IBS a “diagnosis of inclusion”. A positive test is seen in 56% of subjects with IBS-D, giving these patients a positive diagnosis. These patients would no longer require the additional testing to rule out other diseases required by the Rome criteria. Furthermore, they could proceed to receiving appropriate therapy for their IBS more rapidly. Another aspect is that these biomarkers represent part of a pathophysiologic mechanism in IBS. Previous biomarker tests were based on computer algorithms primarily involving blood markers that would rule in Crohn’s or ulcerative colitis 79. This new test validates IBS as a disease (and not simply a condition) which anchors patients in their diagnosis.
One final important aspect of these biomarkers for diagnosing IBS-D (and recently IBS-M) is that a positive diagnosis using these tests could lead to a significant reduction in health care costs. In a recent study, based on the test characteristics, it was determined that more routine use of these markers could save more than $500 per patient in health care costs. Some patients have described up to $20,000 out-of-pocket after insurance costs for lists of normal tests such as colonoscopy 80.
In summary, the newly discovered plasma biomarkers anti-CdtB and anti-vinculin are diagnostic for IBS-D and now IBS-M (but not IBS-C). The use of this test has led to IBS becoming a diagnosis of inclusion based on a pathophysiologic mechanism that includes post-infectious IBS and SIBO. While future studies may use this test to predict therapy, these have yet to be conducted. In the meantime, these biomarkers provide patients with validation of their disease and appear to demonstrate significant cost benefits.
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Table 1: Merits of anti-CdtB and anti-vinculin testing in the diagnosis of IBS-D
| Merits of Testing |
| Testing is highly specific for IBS-D |
| Positive testing assures patient of their diagnosis |
| Positive tests speaks to the pathogenesis of their IBS (post-infectious) |
| Leads to early IBS therapy due to early diagnosis |
| Reduces unnecessary test burden in patients |
| Results in a decrease in health care costs |
Figure Legends
Figure 1: Potential pathophysiologic pathway in the development of post-infectious IBS.
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