Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT03868267 |
| Other study ID # |
5278 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
May 9, 2019 |
| Est. completion date |
December 1, 2023 |
Study information
| Verified date |
February 2021 |
| Source |
Hamilton Health Sciences Corporation |
| Contact |
Paul Moayyedi, MD,PhD,FRCP |
| Phone |
+1-905-521-2100 |
| Email |
moayyep[@]mcmaster.ca |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational [Patient Registry]
|
Clinical Trial Summary
The purpose of this prospective cohort study is to compare upper GI symptoms and endoscopy
findings in Canada with Japan and Iran, and correlate this with the upper GI microbiome. The
investigators plan to recruit 500 new patients referred for upper GI endoscopy in Canada
(McMaster University) and 500 in Japan (Tohoku University Hospital) and 500 from Iran (Tehran
University of Medical Sciences). Written consent will be obtained from all participants.
Patients will complete three symptom questionnaires and a demographic one before endoscopy.
Then saliva collection device will be applied for collecting saliva and microbiota from the
oral cavity. Esophagogastroduodenoscopy (EGD) will be performed thereafter and brushing of
the esophagus, stomach, and the duodenum will be done using a sterile sheathed brush (one for
each site) to sample collect gut microbiota and gastric biopsies will be done for assessing
H.pylori status.
In addition, a group of these patients will undergo measurement of nitrate reductase activity
(NRA) in their oral cavity. This will be done on twenty erosive gastro-esophageal reflux
disease (GERD) patients, twenty non-erosive GERD patients, and twenty patients without any
endoscopic or clinical GERD. This latter part of the study will be done at the Canadian and
Iranian sites only. Bacterial community profiling of the 16S rRNA gene will be carried out
using paired end reads of the V3 region. Triplicate amplifications will be pooled for 150 or
250 nt paired-end Illumina sequencing in the McMaster Genome Center. For specific substudies
analysis of the mycome will also be carried out.
Description:
Objective The purpose of the trial is to compare upper GI symptoms and endoscopy findings in
Canada with Japan and Iran, and correlate this with the upper GI microbiome.
The primary outcomes is the bacterial microbiome in patients with FD compared to control
patients without upper GI symptoms and a normal endoscopy. The investigators will do a
planned subgroup analysis according to country of recruitment as well as sex and age based
subgroups (50 years)
Secondary outcomes are
1. The microbiome in esophagitis patients compared to patients without esophagitis. To
assess the oral nitrate reducing bacteria on the dorsum of the tongue of erosive GERD
patients, patients with non-erosive GERD and comparing them with those of the normal
controls enrolled in the JUICE study.
2. The microbiome in H. pylori negative peptic ulcer disease (PUD) patients compared to H.
pylori positive PUD patients and asymptomatic normal endoscopy controls.
3. The microbiome in those with anxiety patients versus controls without anxiety in the FD
group.
4. The microbiome in those with depression versus controls without depression in the FD
group.
5. The microbiome in those with anxiety patients versus controls without anxiety in the
patients without upper GI symptoms and a normal endoscopy.
6. The microbiome in those with depression versus controls without depression in the
patients without upper GI symptoms and a normal endoscopy
7. Comparison of upper GI pathology (inflammation, dysplasia, malignancy) and the
microbiome in the Canadian versus the Japanese populations.
Methodology Study population, inclusion & exclusion criteria This is a prospective
international cohort study in Canada, Japan, and Iran. The investigators will recruit adult
patients (≥18 years of age) who are undergoing esophago-gastro duodenoscopy (EGD) for any
reason and are consenting to be enrolled in the study during the study period. The control
group will be patients with no or minimal upper GI symptoms who are referred for EGD.
Exclusion criteria will be the patients who cannot speak English in Canada, who cannot speak
Japanese in Japan, and cannot speak Persian in Iran.
Methods Written consent will be obtained from all participants. The consent by clinician for
medical reasons includes standard endoscopy and gastric biopsies for assessing H.pylori
status. The research consent contains saliva collection with specific device called "Oracol
plus®" and tissue brushing at the oesophagus, stomach (body and antrum) and distal duodenum.
If the patient is eligible for this study, the investigators will record demographics (age,
sex, education, race, past medical history, medication taking - especially PPIs and
antibiotics), and the main reason for endoscopy. Patients will complete questionnaires; upper
GI symptoms measured by Short Form Leeds Dyspepsia Questionnaire (SFLDQ), quality of life
measured by EQ-5D, and anxiety and depression measured by The Hospital Anxiety and Depression
Scale (HADS). Then saliva collection device; Oracol plus® (Malvern Medical Developments,
Worchester, UK) will be applied to collect saliva. The device is designed to be used in a
similar way to a toothbrush. Saliva is collected by rubbing the sponge swab firmly along the
gum (at the base of the teeth if present) as well as the dorsum of the tongue until the
sponge is wet, this takes about 1 minute. Once the saliva has been taken it is secure within
the container, a microtube is incorporated within the device so that the saliva is
centrifuged directly into the micro tube provided. This reduces the risk of aerosol
contamination. This process is aimed to document the microbiomes in oral cavity in order to
confirm the resident microbiomes in upper GI tract.
The oral nitrate reductase activity (NRA) measurement
Considering that erosive GERD is rather infrequent in Japan, this part of the study will be
performed in Hamilton and Tehran as follows:
Twenty erosive GERD patients, twenty non-erosive GERD patients enrolled in JUICE from Canada
and twenty controls with no upper GI symptoms are enrolled after informed consent is
obtained. In addition to the procedures in described in JUICE, the following is done for
these groups:
1. An Oracol plus® swab is brushed over the gums and the dorsum of the tongue for 60
seconds as described in the main protocol. It is processed in the same way and the
microbiome study is done in the same way described for the JUICE protocol proper.
2. NRA is measured with consecutive oral cavity samplings at one minute intervals for three
minutes as follows: cases and controls are asked to fast overnight and avoid
high-nitrate foods (including lettuce, cabbage, celery, turnip, beetroot, radish, and
rhubarb) for at least 8 h. Mouth NRA assay is done between 7:30 and 10:00 am. On the
morning of the test, the individual is initially asked to rinse his/her mouth with 22 ml
of deionized, sterile water. Then he/she is asked to hold 22 ml of a 10 mg nitrate-N/L
solution in the mouth for 3 min. The subject is instructed to mix the solution in the
mouth at the beginning and every 1 min over the 3 min, and 1.5 ml of the solution
incubated in the mouth is sampled with a sterile syringe after each mixing (i.e., at the
beginning and at 1-min intervals). The rest of the solution is discarded. Each sample is
immediately transferred into an Eppendorf tube containing 56.25 microliter 0.8 M NaOH
(to destroy vitamin C and top further nitrite production). Then 18.0 microliter 1.4 M
ZnSO4 is added to each tube, shaken for 2 s and left at room temperature for 30 min. The
samples are then centrifuged at 13,000g at 25℃ for 7 min. This treatment removes the
proteins which could possibly interfere with the Griess reaction used for nitrite
measurement. Nitrite is measured by adding 250 microliter of Griess reagent A [0.2%
(w/v) sulfanilamide in 5% (v/v) concentrated H3PO4] to 500 microliter of the supernatant
of the centrifuged sample in a 1,000-microliter cuvette and reading absorbance at 540 nm
using a spectrophotometer (WPA BiowaveII, UK). This reading is named ''A1.'' Then, 250
microliter of Griess reagent B [0.02% (w/v)
N-(1-naphtyl)-ethylenediamine-dihydrochloride solution] is added to the cuvette. The
cuvette is then kept in the dark at room temperature for 10 min and the absorbance was
again read at 540 nm and the reading named ''A2.'' The value (A2-A1) is read against a
standard curve developed by testing known concentrations of NaNO2 and the final nitrite
concentration determined. A 500-microliter sample of the de-ionized sterile water is
treated exactly like the saliva sample in each instance and serves as blank for quality
control. Nitrite values at 0, 1, 2, and 3 min are plotted against time and the slope of
the trend line calculated. This slope represents the nitrite formed per person per
minute. The slope is multiplied by 22 (the volume of nitrate solution) to calculate the
''nitrate reductase activity'' and reported as microgram nitrite-N formed per person per
minute.
Definitions:
For practical purposes, GERD is defined clinically according to the Montreal summit as
troublesome regurgitation or heartburn occurring at least once a week. This definition will
be used to enroll the non-erosive patients as well as excluding GERD in controls. It will
also be used to assess the erosive patients clinically. The controls need not only to be
asymptomatic (according to the above-mentioned definition), but also to have no gross
endoscopic sign of reflux.
Esophagogastroduodenoscopy and sampling for the microbiome EGD will be performed and a
sterile sheathed brush will be used to collect gut microbiota. For each patient, tissue
brushing will be performed first from the the middle and lower esophagus with one brush, then
the stomach (greater and lesser curvature of body and antrum) with a second brush, the
duodenal cap and distual duodenum with a third brush. This will be conducted as the endoscope
is introduced and in each case the collection will be obtained by gently brushing the
appropriate area. The material collected on the brush surface will be re-suspended into RNA
later in a sterile tube. Samples were allowed to incubate at room temperature for up to 2
hours, then frozen and stored at -80 °C. Japanese frozen microbiome samples will ship or
bring to McMaster Genome Centre by one of investigators. The Iranian samples will be
processed in Tehran and the extracted DNA samples will be transferred to McMaster for
sequencing. The last part i.e. the analysis of the sequenced DNA samples can either be done
in Tehran or at McMaster. Two biopsies each from antrum and body of stomach will be obtained
to assess H. pylori status as a standard of care of both cohorts.
Microbiome analysis DNA extraction and molecular profiling will be carried out using standard
protocols. Bacterial community profiling of the 16S rRNA gene will be carried out using
paired end reads of the V3 region. Triplicate amplifications will be pooled for 150 or 250 nt
paired-end Illumina sequencing in the McMaster Genome Center. This approach provides
overlapping sequence reads of the V3 region, which can be used for correcting poor quality
base calls and increasing sequencing accuracy. Multiplexing the run with barcoded primers
will provide approximately 25,000 sequence reads per sample. The sequence data will be
processed by an in-house bioinformatics pipeline that incorporates quality filtering with
cutadap PandaSeq, AbundantOTU+, mothur, and QIIME. Output will include clustered sequences in
operational taxonomic units (OTUs) using AbundantOTU+ and taxonomic assignments using the RDP
classifier classifier using the Greengenes training set. Microbiome analysis will include
α-diversity metrics for each sample and β-diversity measures (weighted and unweighted
unifrac, Bray-Curtis, nonmetric multidimensional scaling) and other statistical analysis
using QIIME25 and PhyloSeq. Functional properties of the microbiota will be inferred using
Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt)
and further compared using Statistical
Sample size The investigators plan to recruit 500 new patients referred for upper GI
endoscopy in Canada (McMaster University) and 500 in Japan (Division of Gastroenterology,
Tohoku University Hospital) and 500 from Iran (Tehran University). This is based on
preliminary data from 20 patients that have had assessment of the microbiome in the
esophagus, stomach and duodenum and assumes that the odds ratio for the operational taxonomic
unit will be 2 fold between organic disease and healthy control (EGD with no GI symptoms e.g.
for evaluation of anemia where no abnormality is found) and assumes that there will be at
least 50 healthy controls and 50 patients with a given disease. 500 patients will be needed
as the investigators will be evaluating gastric ulcer, FD, esophagitis separately and will
need to adjust for multiple testing.
For NRA measurement, the investigators need 18 patients among GERD, NERD and control arm.
This assumes a Z-alpha of 1.96 (two-sided), 1-beta=0.8416, Sigma of 0.99, and mean difference
of 0.9310.
Statistical analysis Baseline descriptive data will be analyzed and reported as means and
standard deviations for continuous variables, and percentage and frequency for categorical
variables. Dichotomous variables will be analyzed suing Chi squared (x2) test and continuous
using Student's t test (for two groups) and ANOVA (for more than two groups) unless numbers
are < 50 in a group when the investigators will use Mann Whitney U test for two groups and
Kruskal-Wallis for more than two groups. SPSS version 21.0 (SPSS (Japan) Co., Ltd., Tokyo,
Japan) for Windows systems will be used with differences considered significant at the <0.05
level for the primary outcomes and <0.01 for the secondary to adjust for multiple testing.
