Yale researchers report on study of the efficacy of the Alcat Test for patients with irritable bowel syndrome and the pathomechanisms of food sensitivity

 

Good evening and welcome everyone to this evening’s webinar entitled:  Yale researchers report on the study of the efficacy of the Alcat Test for patients with IBS (irritable bowel syndrome).

 

We appreciate that everyone is taking their time away from their busy schedules to join us this evening. Our speaker today is Dr. Wajahat Mehal.

 

Dr. Mehal is a gastroenterologist and immunologist. He received his medical degree and his D.Phil in immunology from Oxford University in England. Dr. Mehal did his fellowship and residency at Yale School of Medicine, where he is a full professor and very involved in several of Yale’s programs, such as the Digestive Disease program, Hepatology, Metabolic Health, and Weight Loss.

 

Dr. Mehal became interested in studying the Alcat Test through his colleague, Dr. Ather Ali, after Dr. Ali conducted an NIH funded study that surveyed health care practitioners about their use of unconventional medical tests and found that many practitioners reported positive outcomes using the Alcat Test.

 

This presentation about the study was recorded earlier this week at Dr.Mehal’s lab, and Dr. Mehal is with us this evening live for questions and answers that will immediately follow. Any questions that we don’t get to answer tonight can be addressed later on. Thank you.

 

 

Presentation

 

Dr. Mehal

Good afternoon everybody.

I’d like to take you through some of the background and the findings of the study. This presentation is in memory of Dr. Ather Ali, who was the key driver for this project.

 

So as an introduction, irritable bowel syndrome or IBS, this is a functional disorder of the gastrointestinal tract and is characterized by chronic abdominal pain and altered bowel habits.

In the United States, perhaps between 8-20% of the population meets the diagnostic criteria, and IBS accounts for 25-50% of all referrals to gastroenterologists. So, it’s a very common condition. Because it’s common, IBS is associated with an estimated $1,5 billion annual costs- direct and indirect. And the majority of patients, interestingly, report food related symptoms, which was one reason why we obviously wanted to study the effects of food and diet on IBS.

 

Slide 2

The epidemiology is, as I’ve said before, the North America approximately 10-15%. There is a female predominance, and the prevalence of IBS was 25% lower in those aged over 50, as compared with those below 50.

 

Slide 3

So, the diagnosis of IBS is based or suspected in patients with chronic abdominal pain and altered bowel habits (constipation and/or diarrhea).

The most widely used diagnostic criteria is the Rome IV criteria.

 

Slide 4

By this set of criteria, IBS is defined as recurrent abdominal pain on average occurring at least one day per week in the last three months, and is associated with two or more of the following criteria:

• The pain is related to defecation
• Is associated with a change in stool frequency
• And is associated with a change of stool form or appearance.

 

Slide 5

There is a sort of a qualitative scale that is used with sort of type 1 being a very hard stool, and at the other end of the spectrum type 7 basically being watery stool. And that way patients can gauge and quantify whether they’ve had a change in their stool characteristics.

 

Slide 6

IBS also has within itself certain subtypes. Some patients have IBS predominantly with constipation, and these patients report abnormal bowel movements and usually constipation type 1 and type 2 types of stool. Other patients have IBS predominantly with diarrhea, and some patients have a very mixed form of this disease. And a few patients don’t easily fall into any of these categories.

 

Slide 7

So, current therapies for IBS are very broad. They fall into dietary modifications, physical activity, and then adjunctive pharmacological therapies.

 

Slide 8

The dietary modifications are mostly trying to identify which food items are exacerbating the disease. So, typically there is an attempt to exclude gas-producing foods. These are things like beans, and onions, and celery, and also to try and exclude or minimize alcohol and caffeine. Avoid lactose-containing foods, and then try and eat foods with low fermentable capacities. So oligo, di-, mono- saccharides and polyols – so the FODMAP diet, avoid gluten in IBS-D or diarrheal type disease, and then the flip side would be to use fiber in patients with IBS of a more constipating characteristic.

 

Slide 9

Within this world of trying multiple different things, some of which is actually quite empiric and definitely hit-and-miss, there are some commercial food intolerance tests. And these tests will try and individually identify which food items will improve patients’ symptoms. The data on these is that, up til now, they’ve been predominantly anecdotal and unvalidated.

 

Slide 10

The adjunctive pharmacological therapy is that treatment should be based on the predominant symptom type- and this is really a symptom relief approach – and make incremental changes in therapy at 2 to 4 week intervals.

 

Slide 11

So, for IBS-C (constipation) not surprisingly osmotic laxatives, Polyethylene glycol, has been suggested and is actually recommended at some level by the AGA, or the American Gastroenterological Association, but keeping in mind that the evidence for this is low quality evidence in terms of the clinical trial quality of data.

And then a number of other medications, such as long acting chloride channel activators- again moderate to low quality evidence; and then guanylate cyclase agonists to stimulate intestinal fluid secretion and serotonin receptor agonists.

 

Slide 12

For IBS-D, antidiarrheal agents are often the first line therapy, not surprisingly, so loperamide. Again, AGA recommended, but very low quality evidence. Bile acid sequestrants, cholestyramine for example, and then serotonin antagonists.

 

Slide 13

Other symptomatic therapies… symptomatic therapies that can be used are antispasmodic agents, antidepressants, and antibiotics, all with either low- or moderate-quality evidence, as well as probiotics.

 

Slide 14

So, going into the actual study, the title of the study is “Efficacy of individualized diets in patients with irritable bowel syndrome: a randomized controlled trial”.

 

And the trial design was very rigorous. So this was a parallel group, double-blind, randomized (1:1) controlled trial. The inclusion criteria were adults between the ages of 18 and 75 years meeting Rome III criteria and also concurrent IBS medication, as long as the dosing was stable for 30 days prior to enrollment.

 

The exclusion criteria were a history of other organic intestinal disease, particularly inflammatory bowel disease, significant abdominal surgeries, radiation proctitis, recent antibiotic use, and significant dietary changes in the last few weeks.

 

The trial design prior to initiation, the primary outcome was identified as a difference between groups in the Global Improvement Scale (GIS), which is a scale used to quantify IBS symptoms. Secondary items were three additional scales: one was a Symptom Severity Scale (SSS), one was Adequate Relief Scale (AR), and the third was Quality of Life (QOL) scores.

The sample size of 46 participants was calculated with 80% power to detect group differences of 1.1 in the GIS scale.

 

Slide 16

The test that was used to personalize the diets was a leucocyte activation test. For this, peripheral venous blood was collected in sodium citrated tubes, and was sent to Cell Science Systems in Florida for testing.

There, the leucocytes were separated from whole blood using a density gradient. Neutral buffer and autologous plasma were added back into the cells, and 200 separate individual food samples or extracts were individually tested with peripheral blood leukocytes. And then the cell count read outs were performed using the electric sensing zone method. The results from this test for each of the 200 foods were reported either as positive, meaning that a severe or moderate reaction was identified in the test, or negative meaning that a mild or no reaction was identified.

 

Slide 18

An additional analysis was performed in a sub group. This analysis was an Aptamer based proteomic analysis from the plasma. And aptamers are a synthesized and constructed sequence of nucleic acids, which are designed to bind to specific biomarkers and allow detection of biomarkers in a biological samples. So, it’s essentially it’s a powerful discovery tool and the power lies in the fact that in the small volumes, such as 65 microliters, between 1,300 and 5,000 individual proteins can be identified and quantified.

 

From this patient population, 6 individuals who were strong responders were identified and plasma prior to the removal of a positive food item and after the removal of a positive food item was then subsequently analyzed by this Aptamer based test and this was done by sending the samples to SomaLogic.

 

Slide 19

So, the diet assignments were as follows. There were two groups. The experimental group, or the intervention group, were given individualized diets consistent with the leukocyte test and the patients were instructed to avoid positive foods for 4 weeks. They were allowed to consume mild foods every fourth day and negative foods as they wished. And again keeping… just to make the point, that the patients didn’t know whether the foods they were asked to avoid were positive or negative, and the staff member instructing them also didn’t know– so this was entirely double-blinded.

 

The control of the comparison group were also instructed to avoid foods for 4 weeks. But these were foods that had been identified as negative, based on the leukocyte test. And they were allowed to consume mild foods as well as positive foods as they wished.

 

So, going into this, the null hypotheses was, the assumption was that there would be no differences between the two groups. Which is what one would expect if the leukocyte test was not providing any specific information.

 

Slide 20

The results are as follows…

 

Slide 21

So, in this flow diagram you will see that 126 individuals were assessed for eligibility. 68 were excluded because they didn’t meet inclusion criteria (n=46). 13 declined to participate (n=13), 9 for some other reasons (n=9).

 

A total of 58 were randomized, 29 into each arm. And then the rest of it you can see there that 29 were randomized at 4 weeks.

On the allocation side 4 had discontinued, on the comparison side none had discontinued. Finally for analysis we had 26 on the intervention side and 29 on the comparison side.

 

Slide 22

The demographics of the two populations were as follows: they were essentially identical age, a female predominance was identical, 97% and 86% were Caucasian . And, as we go to the rest of the data set they very well matched.

Looking at the foods that were most frequently restricted we can see that, and I’ll just run through the names on the left side, so strawberry, cinnamon, almonds, apple, onions, pear, buckwheat, chickpea were all restricted at approximately at 19-26%. And a number of them, particularly almonds, apples, onions, and pears are part of the high FODMAP, diet but the vast majority are not part of the high FODMAP diet. And patients, if they had been following the high FODMAP diet, would have continued to consume these.

 

Slide 23

So, as I mentioned earlier the primary outcomes were to examine the IBS Global Improvement Scale (GIS) before restricting foods and after restricting foods.

And the secondary outcomes were three additional scales. One was the Symptom Severity Scale (SSS), one was the Adequate Relief (AR) scale, and one was the Quality of Life (QOF).

 

Slide 24

So, in the treatment outcomes, these are sort of numerical data, just as a reference, but in then in the next slide I’ll show you what was actually found.

 

Slide 25

So, the Global Improvement Scale (GIS), which was a primary outcome, showed that the individuals in whom there was an intervention, meaning they had been asked to not eat food items which were given a positive reading, actually had a much better Global Improvement Scale as compared to the comparators – the comparators are in red. So, this was significant at 4 weeks and it was even more significant in 8 weeks at 0.02 p value (less than 2% possibility of the results occurring by chance).

 

The symptoms of severity scale also showed significance at 4 weeks and at 8 weeks at a value of 0.03. And Adequate Relief and the Quality of Life, both of which was secondary outcome, did not show a significant difference between the 2 populations.

 

Slide 26

The Aptamer data is as follows: So, there was paired analysis of 12 samples. So, these were from 6 individuals and 6 samples were taken pre food restrictions and 6 post food restriction. All of these 6 individuals were in the intervention group. So, from the plasma of these individuals at pre and post times, 1128 proteins were analyzed. 87 of them were significantly different at a p value of less than 0.05 between 4 weeks and the baseline samples.

However, since so many proteins were assayed to correct for multiple analysis these data were analyzed using an appropriate correction. And following the appropriate correction a single pre-post difference remains significant: and this was in neutrophil elastase, which was reduced in individuals who had a strong response.

 

Slide 27

So, the strength of the study by design- it was adequately powered, it was entirely randomized with a matched comparison diet, it was rigorously blinded. The subjects as well as the staff were blinded as to whether they were guiding on removing a positive food item or negative food item. The outcomes measures are validated by the studies and there was a high level of dietary adherence and minimal dropout. And also, the analysis was performed in an intention to treat manner.

 

The limitations are that ethnically the populations was very homogenous and almost entirely Caucasian. The intervention was relatively short at 4 weeks. However, since the findings are positive this is somewhat less of a limitation. And other factors that can affect IBS were not accounted for. And obviously there are many as such factors such as changes in the gut microbiota, psychological stress, or travel. However, although they were not accounted for, they were controlled for, because there was a controlled group in the study.

 

So, the overall conclusions from these data are that the leukocyte activation test can be used to develop an individualized diet that can alleviate symptom burden in IBS. These dietary changes may be less restrictive than a low-FODMAP diet. And hence, may result in better long-term adherence.

And importantly, keep in mind, that as with all complex clinical studies, future studies, ideally multisite with larger sample size to confirm these results, and in particular to confirm them with other dietary interventions, would be the ideal way to proceed from now.

 

Slide 28

That’s my last summary slide, and again I would like to finish with just reminding everybody that this presentation is in memory of Dr. Ather Ali, who was the prime driver for this.

Thank you so much.

 

 

Q&A session

 

I am Amy Pieczarka, Director of Nutrition Services for PreviMedica and I am happy to be here with you this evening. We did get a lot of questions and that’s very exciting because that shows a lot of interest.

 

First question for you Dr. Mehal:

With regard to the neutrophil elastase, could you go a little bit more into that and explain what that is for the people who are on the webinar this evening and not familiar with that, and what the significance of the drop in the neutrophil elastase was among the strong responders and did that surprise you?

 

Answer Dr. Mehal:

Sure, I’ll be happy to. So neutrophils are an innate immune cell, which becomes activated by a number of stimuli. And when they are activated they release a number of enzymes, of which elastase is one of them, but they release many, many different enzymes. These enzymes function to trap bacteria, kill bacteria, but they can also cause damage to tissues. So, like a lot of the sort of attack molecules, they are not entirely specific for bacteria. They can cause self-damage. So, the fact that neutrophil elastase was reduced would be certainly consistent with less tissue damage after removal of positive food items, which are obviously something that we would all like to see. And in some studies which have actually biopsied intestinal tissue in patients with IBS they have found increased numbers of neutrophils. Neutrophils, again consistent with the fact that in IBS, there is a genuine inflammation occurring.

 

So, the tissue biopsies studies show increased neutrophils in the intestines. And these tests showed increased neutrophil elastase in the peripheral blood. So, admittedly, different studies done in different times and different patients, but one way to put them together would be that by removing the positive food items that there is less intestinal neutrophil infiltration, activation, and release of neutrophil elastase. But that’s putting together data from 2 to 3 different studies, and that’s a possibility.

 

20:09

Amy:

Thank you! Do your finding have implications for other inflammatory disorders of the gut such as inflammatory bowel disease (IBD), or celiac disease, or even eosinophilic esophagitis?

 

Dr. Mehal:

So, possibly. The etiology of irritable bowel disease is not known. One possibility is that there is increased permeability in the gut. So, if we imagine that for some unknown reason… there is increased permeability, but if we say that we don’t currently understand what it’s due to, but the increased permeability will resolve in potentially greater amounts of food antigens getting inside the gut epithelium and then activating immune cells, which then can cause inflammation and cause tissue damage. And the reason that I mentioned increased permeability is that that’s a common feature of a number of conditions, including inflammatory bowel disease, and celiac disease, and possibly even eosinophilic esophagitis. So, all of these conditions could have increased permeability as a common feature, resulting in increased amounts of food getting into the body in a non-processed way and causing immune cell activation.

 

21:06

Amy:

1. In your presentation you mentioned that the Alcat Test measures peripheral leukocytes. Could you explain a bit more about the Alcat Test, actually how it works and how it might be different from antibody testing?

 

Dr. Mehal:

Sure. So, the Alcat Test clearly is a cell based test. So, in fact, once the blood sample is obtained, the sample..the cells are washed such that one is left with the cells. And, in fact, the immoglobulins are in fact removed from the assay system. So that part is clear. This has nothing to do with measuring antibodies or an antibody-mediated test. It’s an immune cell based test. Now, when immune cells undergo activation to a variety of mechanisms obviously many, many things change in them. And one of things that changes is their size. They become larger. So they become roughly 1.5-1.7 times larger than their resting state. And that affects a number of things including the conductance through the cell.

And so, the Alcat test is measuring a combination of changes in immune cells related to change in size and conductance with activation.

 

1. Deutsch:

If I may I just want to clarify… one point there… when the Alcat Test is performed the autologous plasma is still present in the sample. So, whether an antibody is involved in the process or not, it would still result as an end point in changes in cell size, number, morphology, activation, or non-activation.

 

Amy:

Dr. Mehal, were there other biomarkers or pathways that you saw to be associated with the positive reaction to the foods or multiple foods?

 

Dr. Mehal:

Right, so the proteomic aspect was a discovery approach, which means that we in a very unbiased way actually assayed 1,128 proteins using an Aptamased proteomic assay. This was done externally from our lab by SomaLogic company. And we actually found a total of 87 separate proteins that read significance at a p-value at 0.05. However of course, when one does multiple tests purely statistically one could find- or purely by chance – one could find statistical associations, so a number of statistical corrections have to be made, which after they were made we were left with neutrophil elastase. However, we have to keep in mind that the sample size was extremely small. There were essentially 6 individuals with 6 pre and 6 post samples. And it will be very interesting to see if these preliminary results, with 87 kits, if any of them once a sample size is a more reasonable sample, could be confirmed.

 

Amy:

Ok. Does you data suggest that an individualized approach can be combined with a more generalized approach such as one within a low FODMAP plan?

 

Dr. Mehal:

So, the data clearly supports that removal of positive food items results in symptomatic improvement. So, that part is clear. The study wasn’t designed to combine removal of positive food items with an additional dietary manipulation. It would be reasonable to conclude that it would be helpful because, you know, if the two separate things are helpful and one combines them, as a clinician, it would be reasonable to assume that there would be increasingly beneficial in combination. However, the study design doesn’t actually allow us to directly address that, but in general seeing these patients I would expect it would be helpful.

 

Amy:

Ok. Do you think that following a personalized eating pattern like the one that was formulated from the Alcat results would possibly change the microbiota?

 

Dr. Mehal:

So, the microbiota is sensitive to all sorts of environmental changes– and certainly dietary changes, it’s extremely sensitive. So, I think the simple answer is “yes”, I would expect to see a change in the microbiota. The question in my mind would be that any changes that are seen, are they greater than would be expected by randomly changing food intake for a particular item?

Right, so are there specific changes, you know, presumably or most interestingly in the beneficial direction by removing a pos Alcat – a positively identified food item – that are different from removing either a negative or some random food item. So, I think there would be definitely changes but the real answer of what that means would probably be more complex than other changes that could be. How do the changes related to removing a food item compare with removing a random item or removing a negative item.

 

Amy:

Do you think that irritable bowel syndrome has links to other disorders? So meaning, could it possibly signal other inflammatory disease processes that are occurring that really have not gotten to the point where it’s diagnosable?

 

Dr. Mehal:

Sure, so this area is under active investigation. So there are no absolute conclusive associations. But, you know, people have studied the large databases – patients with irritable bowel, compared them with a control group in the same database without irritable bowel, and then examined to see if the group with irritable bowel has additional disease associations. And a number of studies have found increased disease associations, including particularly celiac disease, inflammatory bowel disease, and colon cancer in those statistical sort of associations. So, there is certainly data out there suggesting that patients with IBS have increased prevalence of other organic GI diseases. The data isn’t at the level where these associations have been consistently found in every study to the point of being widely acceptable, but there are certainly very strong hints there.

 

Amy:

1. Do you have further interest in studying other aspects of the Alcat Test?

 

Dr. Mehal:

So I think so. I think, you know, these results are so encouraging … and again you have to recall that “we” went into this, by “we”  I have to say that the other senior author, Dr. Ather Ali, who was really somebody who initiated this and was the clinician predominately who ran this and I was providing laboratory support. So, we must remember Dr. Ather’s contribution to all this. But you know, Dr. Ali and I really went into this with what would be called a null-hypothesis. We didn’t have any strong presumption or bias which way the results would go. And, frankly, the expectation of the null-hypothesis approach is that one would not see any difference in the two groups- the group that had a positive food removal and the groups that had, you know, the negative food removal.

So, we were very pleasantly surprised to find the results that are actually clearly showed that in this study, which was very carefully randomized, blinded, double-blinded, that removal of positive Alcat Test foods resulted in significant improvement in IBS.

 

So, the reason I’m going into that is that having walked into this in an entirely neutral way,  now we are in a totally different place, because we have positive data so that’s clearly very stimulating and raises lots of questions about other disease states that are food related where the Alcat Test might be helpful.

 

One obvious thing… I run a weight loss program. So one obvious question would be whether removal of positive Alcat Test foods can improve inflammation in people with obesity because it’s really the inflammation that does damage in individuals who are obese… damage to the liver, damage to other organs.  We know how difficult it is to lose weight and it’s not clear that the weight loss is the crucial feature that needs to be improved, it’s really removal of the inflammation. So, I think it would be fascinating to test different people with a BMI of greater than 30, if removal of positive food items can result in reduced inflammation – irrespective of whether they do or don’t lose weight.

 

30:10

1. Deutsch:

Speaking of inflammation Dr. Mehal… we know that you authored of a very interesting paper in Scientific American a couple of years ago entitled  “Cells on Fire”. And it was a very, very clear explanation about inflammatory processes that occur in cells and clearly elucidated the steps of the inflammasome. And I am wondering if you see, or at this moment can speculate, on any connections between the gross cellular changes that are observed through this rapid clinical scan called the Alcat Test and other inflammatory pathways that you are familiar withthrough your work.

 

31:00

Dr. Mehal:

So, certainly… I think, you know the similarities with the inflammasome would be that the Alcat, most of the cells of the Alcat Test would be innate immune cells. Those are the cells in which inflammasome activation is predominantly active. So, there is a general overlap in terms of we’re speaking about likely the same cell population. And then also, many non-specific stimuli can activate the inflammasome and, you know, food antigens may be a form of non-specific stimulus as well.

 

So, I think there are a number of overlaps. It could actually be interestingly tested. What I mean by that is that it would be quite possible to use, to perform the Alcat Test as you normally do, and then also perform an identical test, but in the presence of an inflammasome inhibitor. There are specific chemicals that inhibit the inflammasome and if you were to find that by including an inflammasome inhibitor, something like caspase 1 inhibitor for example, that the reactivity of the Alcat test was reduced, then that would be direct evidence that the Alcat test and the inflammasome activation pathway was in fact one and the same.

 

1. Deutsch:

And would you say that the same holds true with other inflammatory pathways aside from the inflammasome activation itself.

 

Dr. Mehal:

Potentially, but it would have to be tested. There are number of inflammatory pathways; the various kinases that are required for inflammation. So one could certainly design up an experimental plan where individual, each of the major inflammatory pathways is inhibited individually, to try and get to the bottom of which one might be most relevant for the reactivity of the Alcat Test that you’re seeing.

 

Amy:

Well, we did get quite a few other questions. A lot of them had to do with individual situations and symptoms that they are experiencing, and their loved ones and so forth and specific questions about the Alcat Test and degrees of reactivity and so forth. And we want to assure everyone that we will definitely address them all. But we think it’s best to address them individually.

We did get a lot of questions regarding celiac disease. So, I am wondering if maybe this one question that actually just came in..

Do you have a sense of about what percentage of patients with long-term well-controlled celiac disease still actually present with IBS-D?

 

Dr. Mehal:

I would have to look that up. I think, you know, both celiac and IBS have obviously predominantly GI symptoms. And, in general, once celiac disease is diagnosed, symptoms tend to be attributed to the celiac disease because there are strong diagnostic tests for celiac disease. Whereas IBS tends to be much more of a functional diagnosis. So, I think it’s difficult… it’s just a sort of the bias that people have, is that if you have a strong diagnostic test and you can sort of get a handle on that disease, that tends to be the predominant disease that one deals with.

So, I don’t have the data to give you a concrete answer to that.

 

34:20

Amy:

1. Alright.

 

1. Deutsch:

Is it reasonable to think that an early step in the disease process that leads to celiac in patients that are susceptible would be activation of innate immune cellular responses?

 

Dr. Mehal:

Potentially. So, celiac disease, you know, clearly is a very specific immune reaction against protein components in wheat and such like. So, that certainly exists. It does cause epithelial damage, particularly in the small bowel. However, following that, there could be additional levels of injury. For example, by following an increase of gut permeability there may be additional entry of food antigens completely separate from gliadin, which are causing additional levels of symptomatic worsening.

 

So, you know, when the system is damaged in one way it can certainly expose it to additional types of damage in multiple other ways. So, celiac disease is a well-defined immunological disease entity but there may be role for additional levels of food intolerances on top of the well-known celiac disease food intolerance.

 

35:42

1. Deutsch:

Thank you

 

 

Amy:

Thank you very much!

 

 

1. Deutsch:

So again, I think there are a number of questions that we didn’t get answered today, and some of those are personal, and those people would be able to get access to our dietitians to address those questions.

And certainly, Dr. Mehal, if we get more questions for you we’ll pass them on if you have time.

 

Dr. Mehal

Yeah sure, you can pass them on by email. Thank you so much.

 

All:

Thank you. Thank you very much.

 

Dr. Mehal:

Have a good evening.

 

All:

Thank you, you as well, thank you.