Abstract

IBD, a disease that is difficult to detect in its early stages and more difficult to treat the later it is detected, has become a problem mainly in developed countries. There are two main reasons for the delay in detection: first, the high hurdle of having to make the decision to go to a doctor for a consultation, and second, the difficulty in distinguishing IBD from IBS, a less serious disease with symptoms similar to those of IBD.

Therefore, we have developed a test that lowers the hurdle to testing and at the same time makes it possible to distinguish IBD from IBS. By marketing it as a readily available over-the-counter drug, we will shorten the time required for a definitive diagnosis and reduce the various burdens involved in the treatment of IBD.

What is IBD

Inflammatory bowel diseases (IBD) are chronic relapsing immune-mediated disorders of the intestine. IBD is classified into two major subtypes: ulcerative colitis (UC) and Crohn's disease (CD), which can cause serious complications.

IBD has become a global disease with rising incidence across every continent. The prevalence of IBD is expected to grow exponentially in the next decade because of increasing incidence of young-onset IBD combined with the expansion of the aging IBD population. To prepare for the growing IBD disease burden, we must develop an efficient method to promptly diagnose. However, the diagnosis of IBD can be challenging because this disease may be mistaken for the more benign irritable bowel syndrome (IBS) [1].

Median time to diagnosis from first seeking medical care for symptoms was 1 year for both CD and UC, and assessed by date of diagnosis was similar over the past 24 years. More than half of the patients sought emergency care prior to diagnosis. Approximately one-third of patients had undergone gastrointestinal surgery. Approximately 40% required a stoma ,A new excretory outlet for stool and urine created in the abdomen as a result of surgery [2] ,following surgery.

Only about 25% of patients have remission of the disease in a short period of time such as three months, and many relapse or are hospitalized. Many people require long-term treatment with steroids and antidepressants, and once the disease has progressed, long-term treatment is necessary.

IBD causes problems with schoolwork and work, and some people even lose their jobs. Despite increased awareness of IBD allied to a rising disease burden, there appeared to be no improvement in the time to diagnosis over the past 25 years. Many patients had to seek emergency care prior to diagnosis. In one study, 45% of respondents received their final diagnosis after more than a year, 67% attended an emergency department prior to diagnosis, and 7% more than 10 times.

Problems with delayed detection of IBD[A]

A significant delay between the time from initial primary care provider visit to final IBD diagnosis. Although both patients with CD and UC presented to their primary care provider within a month of symptom onset, it took 6 additional months for patients with CD—as opposed to 2 additional months for patients with UC—to be evaluated by a specialist. Overall, the median time to diagnosis for CD was approximately 10 months compared with 3 months in UC. A quarter of our patients with CD were not diagnosed until after 26 months of symptom onset.

Diagnostic delay may negatively impact disease outcomes. CD progresses over time from an inflammatory disease to a stricturing and/or penetrating disease. Patients with CD with longer time to diagnosis had increased odds of developing complications and intestinal strictures at the time of diagnosis in an incremental fashion. The correlation between longer time to diagnosis and increased rate of strictures and surgery persisted after diagnosis indicating that early diagnosis might impact on disease progression and clinical outcomes—likely because therapy is initiated at an earlier time.

It is shown that the probability of remaining stricture and surgery free in patients with CD depending on time to diagnosis in Figure 1[3].

Odds of developing any complication at the time of diagnosis in patients increase with longer time to diagnosis[4]. 63% of patients with CD diagnosed after 26 months of symptom onset had at least one complication at the time of diagnosis compared with 25% of patients diagnosed within 4 months of symptom onset.

The number of predicted complications at diagnosis as a function of diagnostic time followed a positive rising trajectory as illustrated in Figure 2[5].

Why is early detection of IBD so difficult?

IBD is difficult to detect early because its symptoms are very similar to those of IBS, which has a global incidence of 11.2%[1]. IBD patients with IBS-like symptoms are often misdiagnosed as having IBS during medical interviews, resulting in delayed detection[6]. The diagnosis of IBD is suspected based on characteristic findings from the medical interview and physical examination, and the diagnosis is confirmed by endoscopy which is a hurdle for patients to overcome in order to distinguish between IBD and IBS. This may be one of the reasons why it has taken so long from symptom awareness and visit to the doctor to confirm the diagnosis.

Conventional Approach

Currently, a tool to investigate the early detection of IBD is the “fecal calprotectin test” (henceforth FCT). Calprotectin is a protein found in neutrophils and monocytes, a type of white blood cell. It is mainly associated with inflammatory reactions in the intestine. By measuring the calprotectin concentration in feces, the presence and degree of intestinal inflammation can be evaluated.

Fecal Calprotectin Test (FCT): Merits and Demerits

Merits

Non-invasive Test: As a stool test, it is less burdensome for patients.
Inflammation Activity Assessment: It reflects IBD activity through quantitative testing, helping in disease management and monitoring treatment effectiveness.
Differentiation between IBD and IBS: Since IBS does not involve inflammation, calprotectin levels are usually low, making it effective in distinguishing from IBD.

Demerits

Limited Specificity: Calprotectin responds to general inflammation, meaning levels can rise due to non-intestinal inflammatory diseases such as infections or cancer, making it not specific to IBD.
Inability to Identify Specific Inflammation Sites: While elevated calprotectin levels indicate overall intestinal inflammation, they do not pinpoint specific sites or the extent of inflammation.
False Positives and Negatives: Some patients might have inflammation without elevated calprotectin levels (false negatives) or elevated levels due to non-IBD causes (false positives).
Cost and Accessibility: In some regions or facilities, the cost of calprotectin testing is high and not readily available.
Difficulty in Stool Collection: Patients might be reluctant to collect stool samples and the quality or timing of the sample can affect results.

Our Approach

The period between recognizing symptoms and actually visiting a doctor contributes significantly to diagnostic delays. Even after seeing a doctor, stool tests and endoscopic examinations are necessary to confirm a diagnosis. These tests are time-consuming and present a high barrier to patients. To address this, we are developing an over-the-counter IBD test kit that allows for easy testing through oral administration, thereby lowering the barriers to initial consultation and testing and promoting early detection.

Additionally, we have focused on the distinction between IBD, an inflammatory disease, and IBS, a non-inflammatory condition. Our goal is to aid in differentiating between IBD and IBS by orally administering E. coli that can detect nitric oxide (NO), a compound produced in large quantities during inflammation.Although this test does not have the sensitivity and specificity for a definitive diagnosis, it serves as a good catalyst for reducing the time it takes to visit a doctor and undergo tests.

By enabling earlier treatment, the difficulty of treatment and the risk of complications can be reduced, saving time and money.

Comparison of Gassle and Fecal Calprotectin Test

Common Merits

Both are non-invasive tests resulting in less burden on patients.
Both can detect inflammation, helping to differentiate between IBD and IBS.

Advantages of Gassle

Ease of Use: FCT requires stool collection with sample quality and timing potentially affecting results. Gassle, on the other hand, does not require this, making it more convenient.
Cost: FCT is quite expensive, costing around 139 CAD [7]in Canada where the IBD prevalence is high. Gassle aims to be an oral test, potentially offering a lower cost.
Accessibility: FCT's market share is skewed with 42% in North America, 26% in Europe, and 19% in Asia-Pacific[8]. In contrast, Gassle, as an oral test, can be distributed to underdeveloped regions lacking sufficient facilities, promoting early detection of IBD in more countries and regions.
Test Duration: FCT takes about two weeks for professionals to collect and analyze data[9]. Gassle, as an oral test, reaches the colon in about 8 hours and shows results within 2 to 3 days at the latest.
Specificity: FCT detects inflammation in non-colonic regions as well, whereas Gassle is specific to the colon.

Challenges of Gassle

Pre-test Restrictions: There are restrictions on taking non-steroidal anti-inflammatory drugs before the test.
Inability to Identify Specific Inflammation Sites: It cannot specify the sites or extent of inflammation within the colon.
Low Sensitivity: There is a possibility of false positives and negatives.
Inability to Perform Quantitative Assessment: The test relies on odor indicators, making quantitative assessment of inflammation activity impossible, thus not useful for disease management or monitoring treatment effectiveness.

Based on the above

the purpose of Gassle is not to make a definitive diagnosis but to encourage patients to visit a hospital, consult a doctor, and undergo a colonoscopy. Therefore, as long as the possibility of false negatives is minimized, it meets its goal as an easy-to-use oral test.

HSV-TK/GCV System

Since genetically modified E. coli is introduced into the body, it must be prevented from passing the modified E. coli through the sewage system to the natural world through excretion or other means. We also need to control the production of propionic acid in the colon. We propose a control system using Thymidine Kinase (HSV-TK) and Ganciclovir (GCV) as a solution to these problems.

HSV-TK is an enzyme expressed from a gene derived from Herpes simplex virus 1 and selectively eliminates TK+ cells because it converts ganciclovir to a toxic substance [10][11]. Because of this property, HSV-TK is also called a suicide gene.

Because the HSV-TK, GCV system is used for apoptosis of human cancer cells, few experiments have been conducted with E. coli, which do not need apoptosis. Therefore, there is no information to indicate whether this system also works properly within E. coli, so we need to find out for ourselves. we will do by introducing HSV-TK into E. coli and see if it works in the presence of Ganciclovir.

Another problem is that E. coli can be flushed out by defecation before the GCV can control the number of E. coli. Those taken into the body by oral administration are excreted in the stool within 12-72 hours [12]. By then, the recombinant E. coli must be wiped out. As a solution to this, we use mathematical modelling to predict the optimal GCV dosing time by predicting the rate at which E. coli flow through the body, the amount of propionic acid produced and the number of E. coli in the GCV dose.

Hardware

For our purposes, E. coli Gassle must be delivered to the large intestine alive. However, if Gassle is simply swallowed, it will be killed by stomach acid. Therefore, we create "Hardware" to deliver live E. coli Gassle to the large intestine.

Our capsule consists of a layer that protects the capsule from stomach acid, a layer that specifically disintegrates in the colon, and a culture layer and nutrition to transport Gassle at maximum density.

Once the capsule disintegrates in the colon, a sufficient number of E. coli are released, allowing for rapid testing. Learn more... Hardware