Clinical Trial Details
— Status: Not yet recruiting
Administrative data
| NCT number |
NCT06423508 |
| Other study ID # |
EK_01149_3/05/2024 |
| Secondary ID |
|
| Status |
Not yet recruiting |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
June 3, 2024 |
| Est. completion date |
September 30, 2025 |
Study information
| Verified date |
May 2024 |
| Source |
University of Vienna |
| Contact |
David Gomez Varela PhD. Senior Scientist, PhD |
| Phone |
1427755361 |
| Email |
david.gomez.varela[@]univie.ac.at |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
The study aims to assess the effectiveness of various commercial and non-hazardous buffers
for the storage of human gut fecal samples over time. This evaluation will be conducted by
comparing the performance of these buffers against directly frozen samples using
metaproteomic analysis. The study is motivated by the need for standardized protocols for
sample preservation in metaproteomic research, particularly focusing on protein preservation
in fecal samples. By investigating proteomic, taxonomic, and functional identifications, the
research seeks to provide insights into the reliability of these buffers as storage
solutions. Additionally, the study plans to explore inter- and intra-individual variabilities
at the proteome level by periodically collecting fecal samples from volunteers, complementing
existing knowledge in metaproteomics. Overall, the study addresses a critical gap in the
field and has the potential to enhance reproducibility and comparability across metaproteomic
studies
Description:
A. Introduction:
Metaproteomics studies the entire protein complement of a microbial community and offers
insight into intricate ecosystems like the gut microbiome. Metaproteomics allows researchers
to delve deep into gut microbiomes, providing insights into these microbial inhabitants,
abundance and functional activities. This knowledge has been instrumental in elucidating the
mechanisms underlying various diseases, such as inflammatory bowel disease, and obesity.
Researchers can develop targeted strategies for personalized medicine and precision
healthcare based on the underlying biology by identifying key protein signatures.
In gut microbiome studies, standardized sample collection and preservation are particularly
crucial to preserve the integrity of the microbial community and its proteome. Indeed, to
obtain reliable results it must be considered the susceptibility to environmental influences
like temperature and oxygen amount. For gut microbiome metaproteomic studies, non-invasive or
minimally invasive methods are preferred to minimize disruption, or contamination, and
preserve the native microbial composition and protein profiles. Also, the sample handling and
storage strategies affect the degradation of proteins and the microbial population stability.
For this reason, immediate freezing, or preservation at low temperatures (e.g., -80°C) is
often recommended to maintain sample integrity until analysis. However, using a cold chain
for storage and transportation is not compatible with an on-site sampling strategy that is
necessary for large-scale and delocalized studies. Moreover, it is less ideal, more
expensive, and logistically tedious than potential room temperature options.
Furthermore, present knowledge of the fecal sample collection/preservation methods is based
on preserving nucleic acids or metabolites during the transportation to the laboratory for
analysis. However, protein preservation requires different strategies and studies are scarce
and rely on adopting the devices or reagents used in the case of nucleic acids or metabolites
analysis. Thus, tailored, and standardized protocols together with metadata collection are
essential for ensuring reproducibility and comparability across metaproteomic studies.
The present study aims to evaluate the fitness of several commercial and non-hazardous
buffers for the storage of human gut fecal samples over time. Fecal samples will be stored in
five different buffers and will be benchmarked with directly frozen samples. Using
metaproteomics the investigators will compare the proteomic, taxonomic, and functional
identifications to understand the potential of these buffers as a reliable storage solution
for human feces. This research is unique and to the best of our knowledge, no similar study
has addressed this demanding question.
Preliminary evidence in humans has shown inter- and intra-individual variability of the
proteomes of the host and microbiomes. In particular, the intra-individual taxonomic and
functional variation in the gut microbiome seems to be lower than inter-individual variation
over time. However, metatranscriptomics profiles exhibited comparable variability within and
between subjects. Therefore, using metaproteomics, investigators aim to assess the inter- and
intra-individual variabilities at the proteome level by studying fecal samples periodically
collected from participants.
B. The academic and social relevance:
The first part of the study aims to define the most effective storage buffer for preserving
protein integrity during sample transportation from the site of collection to the site of
processing - a key factor in the future of metaproteomics applied to health and disease.
Despite its importance, a similar evaluation has not been performed for proteomic
applications. This knowledge will be critical for academic purposes to be used for
large-scale human fecal proteomic studies. For people to have their gut health examined
through metaproteomics and unlock the potential of microbial communities-based personalized
medicine and therapeutic interventions there will be a requirement for consistent sample
collection and this study will provide it.
The second part of the study is designed to understand the variability that can be observed
in the gut microbiome composition and functions from the proteomics perspective. The gut
microbiome is dynamic and is known to be altered by multiple factors such as diet, age,
genetics, environment, etc. These variations can be broadly classified into inter- and
intra-individual. Inter-individual variability refers to the differences observed between
individuals within a population or group, and they can encompass a wide range of
characteristics, including genetic makeup, physiological parameters, lifestyle factors, and
environmental exposures. Understanding inter-individual variability from people of the same
"community" is essential in fields such as personalized medicine, where tailored
interventions account for individual differences to optimize treatment outcomes.
Intra-individual variability, on the other hand, refers to variations observed within the
same individual over time or under different conditions and can arise from biological
fluctuations, such as circadian rhythms, hormonal fluctuations, or physiological responses to
stimuli. Recognizing and characterizing intra-individual variability is crucial for
accurately interpreting research findings and assessing the reliability of biological
measurements, particularly in longitudinal studies or clinical monitoring scenarios. This
study aims to understand these variabilities through the proteomics data collected from
participants' fecal samples and define the variables detected in the gut microbiome.
C. Objectives:
1. To identify the effect of five different buffers used for the storage and transportation
of collected human fecal material, by metaproteomic profiles.
2. To determine the variability in microbiota and host proteomes at the inter- and
intra-individual levels.
D. Study Design:
For the first phase, the investigators will deploy the following protocol for on-site fecal
sampling and transport preservation: The participants (recruited among the members of our
laboratory/Division) will be provided with 18 tubes (numbered 1-18; 15 ml conical centrifuge
tubes; Ref# 339650, Thermo Scientific and 18 easy-to-use commercial swabs (C1052-50, Zymo
Research) or spatulas (DNAGenotek). The 18 tubes correspond to three storage time points of 6
different experimental conditions: an empty tube (without preservation liquid) as a control
sample and 5 different non-hazardous fecal preservation liquids: (a) In-house buffer (called
LB) with SDS and Urea, commercially obtained (b) Zymo DNA/RNA Shield (R1100, Zymo Research),
(c) Copan Amies buffer (480CE, Copan Italia SpA), (d) OMNIgene•GUT (OM200, DNAGenotek), (e)
OMNImet•GUT (ME200, DNAGenotek). Briefly, LB, Zymo Shield, and OM200 are known to contain
detergents that help to preserve the biomolecules, Copan Amies buffer is a nutrient-free
media known to keep the cells viable and shown to have applications in culturomics, and ME200
is solvent based useful in metabolomic applications. In addition, the participants will
receive a commercial stool catcher that is displayed over the toilet for facilitating fecal
sampling (Servoprax Stuhlfaenger, Reference number H7 61000-50 from www.medundorg.de) and a
little box with ice packs in which the three Control samples will be stored and transported
to the lab. These ice-stored Control samples are key to evaluating the performance of the
different storage buffers. The participants will bring the samples to our laboratory in the
following 24 hours. Upon reception in the lab, the Control samples will be stored at -80°C
until further biochemical processing using established protocols. The rest of the samples
will be stored at +20°C inside an orbital shaker incubator for a total of 2, 5, and 10 days
(simulating different storage/transport times, thereof). At the end of these time points, the
samples will be stored at -80°C until further biochemical processing using established
protocols. The metaproteomic profiles (indicating the taxonomical and functional changes in
the gut microbiome and the host physiology; as in the previous experiment from these samples
will inform us about the best preservation liquid (defined as the one where the microbiome
composition and function are more like the Control sample). This experiment is necessary for
initiating Phase II of the study, and it is novel in the field as no guidelines are
established for best in-house fecal sampling practices in metaproteomics. Six healthy
participants (among the members of the Systems Biology of Pain laboratory, Division of
Pharmacology & Toxicology, Department of Pharmaceutical Sciences) will be recruited. This
is a randomized study with no participant groups. Samples will be collected once and
distributed to 18 tubes representing different conditions. A sample size of 6 subjects will
be enough to find the best preservation solution and the sampling will be performed one time
under normal bowel movement by the participants. In addition, the participants will fill out
a questionnaire (attached) that includes information like age, sex, Body Mass Index, the
gastrointestinal symptoms rating scale (GSRS), administration of antibiotics in the last 30
days before sample collection, the Bristol stool form scale (BSFS), and the approximate time
needed for sample collection in the 18 tubes (to account for potential effects due to
oxidation processes).
For the second phase, the participants will be expected to collect fecal samples for 4 weeks
during their regular defecation (ten different times maximum). They will be provided with an
adequate number of 15ml conical centrifuge tubes (339650, Thermo Scientific) containing the
best-performing preservation buffer (as defined in the first phase of the study, see above),
and a commercial collection device with a stool catcher. Upon reception in the lab, the
samples will be stored at -80°C until further biochemical processing using established
protocols. In addition, the participants will fill out a questionnaire (see above).
Participants will take part voluntarily in the experiment after receiving an explanation of
all the risks and benefits of participating in the present study, and after signing the
written informed consent form according to the Declaration of Helsinki (see the consent form,
attached). The participants will be instructed and provided with necessary sampling devices
to collect stool samples (see above) and submit the samples to the Department of Pharmacology
and Toxicology at the earliest opportunity. Participants will be expected to complete
self-administered questionnaires that include information like the one described above. For
the second phase, 46 healthy participants (among the members of our laboratory/Division) will
be recruited from both sexes (23 males and females) considering a possible dropout rate of
20%. This is a randomized study with no participant groups. The sample size was calculated
based on the threshold of Eggerthella estimates with time. The statistics procedure used is F
tests - two-way ANOVA with repeated measures with the following values Cohens'd = 0.75, 1-β=
0.80, and α= 0.05. A total of 400 - 460 samples are expected to be collected.
