Characterization of Resistance Against Live-attenuated Diarrhoeagenic E. Coli

Study Overview
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Brief Summary
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Although the existing diarrhoeagenic Escherichia coli (E. coli) challenge model is already suitable for dietary interventions in its current form, further characterization of the working-mechanism of the attenuated strain and further optimization of the study design will enable the investigators to better select those ingredients that affect the key pathophysiological processes. The aim of the CORAL study is to further characterize and increase the discriminative power of the diarrhoeagenic E. coli challenge model.

Detailed Description
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Primary Objective: In the CORAL study the investigators want to determine whether increasing the inoculation dose of diarrhoeagenic Escherichia coli (E. coli) to 5E10 Colony Forming Units (CFU) (at day 14) and addition of a second challenge 1E10 CFU (at day 35) will result in an increased effect-size and duration of measurable outcomes and in an expansion of the relevant clinical and biomarker readouts of the challenge model. Secondary Objective: By extensive biomarker and transcriptome analysis of blood and fecal samples, the investigators aim to explore the working mechanism of the non-toxin producing diarrhoeagenic E. coli strain and the kinetics of the host response to this infection. In addition, the investigators want to determine whether adding extended fasting and addition of a standardized evening meal, prior to the inoculation day, will result in a decreased between-subject variation. Study design: The CORAL study is a parallel 7-weeks intervention study. Subjects will be randomly assigned to one of two inoculation dosages of a live attenuated diarrhoeagenic E. coli (n=20 per group). Subjects will be instructed to maintain their usual pattern of physical activity and their habitual food intake, but to standardize their dietary calcium intake. After a standardized evening meal and an overnight fast, subjects will be orally infected with a live, but attenuated, diarrhoeagenic E. coli (strain E1392-75-2A; collection NIZO food research; dose will be either 1E10 CFU (n=22) or 5E10 CFU (n=22) at study day 14). At study day 35, after a standardized evening meal and an overnight fast, all subjects will receive a second inoculation of 1E10 CFU of the ETEC vaccine (n=44). At various time points before and after both diarrhoeagenic E. coli challenges an online diary will be kept to record all food and drinks consumption to assess dietary macronutrient intake. Moreover, subjects will report information on stool consistency, frequency and severity of symptoms. At various time points before and after both diarrhoeagenic E. coli challenges venous blood and (complete) stool samples will be collected. Blood and fecal samples are collected to quantify several infection- and immune system markers. Study population: Healthy male subjects, 18-55 years of age who fulfil all of the inclusion criteria and none of the exclusion criteria will participate in the CORAL study. Intervention: At study day 14 and 35, after a standardized evening meal and an overnight fast, all subjects will receive an inoculation of the diarrhoeagenic E. coli (1E10 CFU (n=22) or 5E10 CFU (n=22) at study day 14; 1E10 CFU (n=40) at study day 35) Subjects will be instructed to maintain their habitual diet, except for their dairy intake. Dairy has a high calcium content and contributes significantly to total daily calcium intake. These dietary guidelines will limit calcium intake on average to 500 mg/day. From our previous studies, we know that calcium can significantly reduce the gastro-intestinal symptoms induced by the E. coli strain. Main study parameter: 1. Percentage of faecal dry weight (% determined by freeze-drying) Secondary study parameters: Total faecal wet weight (faecal weight in g/day) Time to first diarrhoeal stool (reported by the subjects in the online diary) Stool consistency (Bristol Stool Scale reported by the subjects in the online diary Number of stools with Bristol Stool Scale >4 (Bristol Stool Scale reported by the subjects in the online diary) Stool frequency (Stools per day reported by the subjects in the online diary) Incidence and duration of WHO-defined diarrhoea (Calculated from the Bristol Stool Scale and the Stool frequency reported by the subjects in the online diary) The incidence, duration and severity of Gastro-intestinal symptoms (Gastro-intestinal Symptom Rating Scale reported by the subjects in the online diary). In addition, the study contains an explorative phase consisting of biomarker and transcriptome analysis, in order to further explore and identify the mechanism and kinetics of the host response to the infection.

Official Title
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Characterization Of Resistance Against Live-attenuated Diarrhoeagenic E. Coli

Conditions
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Diarrhea, Gastroenteritis, Bacterial Infections, Escherichia Coli Infections

Intervention / Treatment
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* Biological: E. coli strain E1392-75-2A


Participation Criteria
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Eligibility Criteria
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Inclusion criteria: Ability to follow verbal and written instructions; Age between 18 and 55 years; Availability of internet connection; BMI ≥20 and ≤27 kg/m2; Healthy as assessed by the NIZO food research medical questionnaire; Male subjects; Signed informed consent; Voluntary participation; Willing to accept disclosure of the financial benefit of participation in the study to the authorities concerned; Willing to accept use of all encoded data, including publication, and the confidential use and storage of all data for at least 15 years; Willing to comply with study procedures; Willingness to abstain from high calcium containing products. Willingness to abstain from medications that contain acetaminophen, aspirin, ibuprofen, and other nonsteroidal anti-inflammatory drugs, (OTC) antacids and antimotility agents (eg, loperamide) on the three days before, during and 3 days after diarrhoeagenic E. coli challenge. Willingness to abstain from alcoholic beverages three days before, during and three days after diarrhoeagenic E. coli challenge. Willingness to give up blood donation starting 1 month prior to study start and during the entire study; Exclusion criteria: Disease of the GI tract, liver, bile bladder, kidney, thyroid gland (self-reported); Diarrhoeagenic E.coli strain (as used in the study) detected in fecal sample at screening; Evidence of current excessive alcohol consumption or non-therapeutic drug (ab)use); Evidence of IgA deficiency (serum IgA < 7 mg/dL or below the limit of detection of assay). High titer serum antibodies against CFA-II diarrhoeagenic E.coli strain (as used in the study) at screening; History of microbiologically confirmed ETEC or cholera infection in last 3 years. Known allergy to the following antibiotics: ciprofloxacin, trimethoprim-sulfamethoxazole, and penicillins. Mental status that is incompatible with the proper conduct of the study; Not having a general practitioner, not allowing disclosure of participation to the general practitioner or not allow to inform the general practitioner about abnormal results. Occupation involving handling of ETEC or Vibrio cholerae currently, or in the past 3 years. Participation in any clinical trial including blood sampling and/or administration of substances starting 1 month prior to study start and during the entire study; Personnel of NIZO food research, their partner and their first and second degree relatives; Reported average stool frequency of <1 or >3 per day; Symptoms consistent with Travelers' Diarrhoea concurrent with travel to countries where ETEC infection is endemic (most of the developing world) within 3 years prior to dosing, OR planned travel to endemic countries during the length of the study. Use of antibiotics, norit, laxatives (up till 6 months prior to inclusion), cholestyramine, antacids H2 receptor antagonists or proton pump inhibitors or immune suppressive agents (up till 3 months prior to inclusion); Vaccination for or ingestion of ETEC, cholera, or E coli heat labile toxin within 3 years prior to inclusion; Vegetarians and vegans

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 55 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: 1E10 CFU Escherichia coli (E. coli)<br>1E10 Colony Forming Units (CFU) Diarrhoeagenic E. coli (strain E1392-75-2A; collection NIZO food research). Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonization Factor Antigen II (CFA/II) and provides 75% protection against challenge with an LT, ST, CFA/II strain. At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 1E10 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A<br>* At study day 14, after a standardized evening meal and an overnight fast, subjects will receive a single oral dose of the attenuated diarrhoeagenic E. coli strain E1392-75-2A (dose will be either 1E10 CFU (n=22) or 5E10 CFU (n=22)). Oral challenge will occur at 10.00 AM. Under supervision of the project team, subjects will get a NaHCO3 solution (100 ml 2% NaHCO3) to neutralize the gastric acid. After 5 minutes, they get a fruit juice (100 ml) containing the attenuated diarrhoeagenic E. coli strain at the above-mentioned dose. Subjects go home, but are not allowed to drink or eat for 1 hour. At study day 35, after a standardized evening meal and an overnight fast, all subjects will receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli.<br>* Other names: E1392-75-2A;|
| Experimental: 5E10 CFU Escherichia coli (E. coli)<br>5E10 Colony Forming Units (CFU) Diarrhoeagenic E. coli (strain E1392-75-2A; collection NIZO food research). Diarrhoeagenic E. coli strain E1392/75-2A serotype O6:H16 belongs to Pathogen class 2 . The strain has a deletion of genes encoding the heat-labile (LT) and heat-stable (ST) toxins and can not produce any toxins. However, it continues to express Colonization Factor Antigen (CFA/II) and provides 75% protection against challenge with an LT, ST, CFA/II strain. At day 14, after a standardized evening meal and overnight fast, subjects receive a single oral dose of 5E10 CFU of the attenuated diarrhoeagenic E. coli strain E1392-75-2A. At day 35, after a standardized evening meal and overnight fast, all subjects receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli. | Biological: E. coli strain E1392-75-2A<br>* At study day 14, after a standardized evening meal and an overnight fast, subjects will receive a single oral dose of the attenuated diarrhoeagenic E. coli strain E1392-75-2A (dose will be either 1E10 CFU (n=22) or 5E10 CFU (n=22)). Oral challenge will occur at 10.00 AM. Under supervision of the project team, subjects will get a NaHCO3 solution (100 ml 2% NaHCO3) to neutralize the gastric acid. After 5 minutes, they get a fruit juice (100 ml) containing the attenuated diarrhoeagenic E. coli strain at the above-mentioned dose. Subjects go home, but are not allowed to drink or eat for 1 hour. At study day 35, after a standardized evening meal and an overnight fast, all subjects will receive a second inoculation 1E10 CFU of the diarrhoeagenic E. coli.<br>* Other names: E1392-75-2A;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in percentage of faecal dry weight from baseline | % of faecal dry weight determined by freeze-drying | Day 14-17 and Day 35-38 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in total faecal wet weight from baseline | Total faecal weight in g/day | Day 14-17 and Day 35-38 | 
| Time to first diarrhoeal stool | Time to first diarrhoeal stool reported by subjects | Day 14-15 and Day 35-36 | 
| Change in Stool frequency from baseline | Stools per day reported the subjects | Day 14-17 and Day 35-38 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Experimental challenge trial