2.4.1 Stream 1 primary care.

Primary Care sites across Norfolk will act as Patient Identification Centres (PIC) sites to identify suitable participants by searching their electronic patient record against the study inclusion and exclusion criteria. The PIC site will send an introductory pack to the potential participant, who will respond to the letter or contact the research team by phone /email if they are interested in participating in the study.

2.4.2 Stream 2 BCSP.

In England, all patients aged 60 to 74 registered with a GP are automatically sent a faecal immunochemical test (FIT) kit every 2 years to detect faecal occult blood which could indicate abnormalities in the bowel which may have the potential to develop into cancer. Patients with a positive FIT result are referred for further investigation by colonoscopy. Patients invited for a colonoscopy are contacted by mail in advance of their appointment scheduled as part of their routine care. In addition to this mail out, the BCSP will send these patients within Norfolk an introductory pack for the MOTION study. If the patient is interested in participating in the study, they are asked to respond to the letter or contact the research team by phone /email. Stream 2 will potentially identify participants for cohorts 1, 2 & 3.

2.4.3 Stream 3 NSFT.

The Norfolk and Suffolk NHS Foundation Trust (NSFT) Memory Assessment Services receive 200 GP referrals each week in Norwich, South Norwich, North Norwich/Norfolk, Great Yarmouth and Lowestoft. When referred patients are seen for a memory assessment by the Community Teams for each area, they are asked to sign a Consent to Research form which allows the NSFT research team to approach them for potential research studies. The NSFT research team will approach those patients with a diagnosis of MCI that have completed this form and will provide the patient with an introductory pack to the study. Patients who are interested in participating in the study will be asked to respond to the letter or contact the research team by phone /email. Stream 3 will mainly identify participants suitable for cohort 3.

2.4.4 Additional recruitment methods.

Participants may also be identified by expressing an interest on the Quadram Institute’s website using the online form provided for the study. Additionally, any individuals who have signed up to the Quadram Institute Bioscience Participant Database who meet the eligibility criteria, will be sent the study information pack and asked to respond to the letter, or contact the research team by phone/email if they are interested in participating in the study.

2.6.2 Blood samples.

Blood samples will be collected at each study visit appointment at QI-CRF.

At baseline and study visits 2,4,6 and 8, two 10mL blood samples will be taken in EDTA blood collection tubes and two 10mL blood samples will be taken in Serum Separation Tubes. Samples will be processed and stored in QIB laboratories. Analyses will include, for example, assays to characterise immunoglobulins and antibodies; metabolites; inflammatory mediators (e.g. cytokines); and the presence of microbes and microbial products using established protocols within QIB.

At study visits 1, 3, 5, and 7, 15mL of blood will be used to determine total red cell counts, haemoglobin concentration and for assessment/quantitation of key biochemical markers of organ function and health (e.g. bone, liver, heart, kidney and thyroid). All of these have been identified as significant covariates for microbiome composition [18].

2.6.3 Physical measurement collection.

Height, body composition measurements, blood pressure and hand grip strength measurements will be taken by a trained member of staff at the QICRF at study visits 2, 4, 6 & 8. Data will be recorded on the study case report form.

2.6.4 Health questionnaire.

At the baseline appointment, participants will be asked to complete a health questionnaire which asks questions related to demographics, lifestyle, behaviour and hygiene. This questionnaire has been used in previous longitudinal studies of the human gut microbiota and identifies key environmental factors that are known to impact on the structure and function of gut microbes [19].

At subsequent appointments, participants will be asked to complete a shorter version of this questionnaire to collect information about changes in health status, medication use and any other changes in the routine of the participants at the time of producing follow up samples.

2.6.5 Cognitive tests and associated questionnaires.

The cognitive tests and associated questionnaires used in the MOTION study are:

• Mini-Addenbrooke’s Cognitive Examination (Mini-ACE) [14]- a short standard cognitive screening test well validated and used in dementia screening.
• Cognitive Change Index (CCI) [15]—a short validated self-completed measure of the participant’s own perception of their cognitive decline.
• Patient Health Questionnaire (PHQ9) [20]—brief self-report instrument for screening the severity of depression symptoms in the participant.
• Supermarket Test [21]—a computer- and tablet-based assessment of spatial orientation using an ecological supermarket environment.
• Sea Hero Quest [22]—novel computer, tablet, or virtual reality measure of spatial navigation within the context of a video game.
• Cambridge Behavioural Inventory Revised (CBI-R) [23]—a self-rated measure of changes in the participant’s behaviour. These domains include memory and orientation, everyday skills, self-care, abnormal behaviour, mood, beliefs, eating habits, sleep, stereotypic and motor behaviours and motivation and frequency of these behaviours.
• Body Perceptions Questionnaire (BPQ) [24]—a self-report questionnaire assessing the participant’s degree of interception ability.
• Rey Complex Figure Test (RCFT) [25]—a short measure of visual memory and visuospatial constructional ability which includes a 3-minute delayed recall.
• Trail Making Test (TMT) [26]—a short test of processing speed, attention and set-switching.

Participants will be asked to complete these at their Baseline visit and study visits 1, 3, 5, 7.

The mini-ACE will give a general overview of cognitive intactness in participants via an objective test. This is complemented by the CCI which allows participants themselves to report whether they have any concerns in regard to their cognition.

In addition, more specialised cognitive tests, targeted towards the first changes commonly seen in dementia, will be employed to measure specific cognitive domains. For spatial navigation, which is currently emerging as one of the earliest cognitive sign of dementia disease pathological processes, we will use the Supermarket and Sea Hero Quest tests. The RCFT allows a stricter assessment of episodic memory in participants, which is currently still seen as the gold standard of dementia cognitive changes. Finally, the TMT allows to detect more slowing down of response and problems switching tasks, which is commonly found in more vascular brain changes and vascular dementia.

These tests will be complemented by 3 additional questionnaires, PHQ-9 allows establishment of depressive symptoms which can impact on cognitive performance, CBI-R allows establishment of any behavioural/neuropsychiatric symptoms which can impact on cognition; finally, BPQ is a questionnaire which taps into introception, i.e. how well people are aware of their bodily functions. The BPQ will be therefore important to how people are aware of their gut and has been suggested to be a cognitive surrogate marker of gut-brain interaction levels.

2.6.6 Retinal morphology OCT/OCT(A) scans.

There is a growing literature on the use of Optical Coherence Tomography (OCT) scanning to measure both the Retinal Nerve Fibre Layer (RNFL) and the Inner Ganglion Cell Layer (iGCL) of the retina and correlating these measurements with the risk of dementia [13, 27]. Embryologically the eye is an out pouching from the brain and contains neural tissue. It is possible that if the brain is undergoing a degenerative atrophy as seen in dementia then there might also be measurable changes in the retina, particularly the retinal nerve fibres of the ganglion cells. This might mean that OCT of the RNFL or iGCL becomes a useful non-invasive way of detecting those at higher risk of dementia, or quantitatively measuring the response to novel treatment strategies.

The OCT scan will be performed following the Baseline Visit, around Study Visit 4 and prior to Study Visit 8 in all participants.

We will use the newer technique of Optical Coherence Tomography Angiography (OCT A) at the Baseline Visit & Visit 8 in subgroup 1 (the same 90 participants– 30 from each cohort who undergo a colonoscopy) which, in addition to the OCT scan, measures blood flow in the retina, again in a non-invasive manner. We wish to get OCT (A) scans on the participants in whom we have the most information about the gut microbiome and this will be those in Subgroup 1.

There is minimal data on correlating OCT (A) findings with dementia, or indeed how to quantify and analyse the large amount of data that OCT (A) scans generate [28]. However, the MOTION study is unique in collecting OCT(A) data in ageing participants with and without MCI which will be examined by an Ophthalmologist who is blinded to participants cognitive and clinical data.to see if there is any change in the vascular pattern in participants with MCI (cohort 3), looking at both the retinal and choroidal blood supply, measuring when possible the vascular density and looking for other changes such as enlargement of the foveal avascular zone which might reflect progressive microvascular changes.

2.6.6.1 Outcome measures. Outcomes will be extracted from OCT(A) data by an Ophthalmologist who is blinded to participants cognitive and clinical data. Peripapillary RNFL in superior, inferior, temporal and nasal quadrants and mean quadrants will be measured. Macular thickness will be recorded form the OCT and inner ganglion cell layer thickness will be recorded by outer, inner and foveal segments.

For Ishihara test score, choroidal thickness will be recorded from the OCT. OCT (A) data will be analysed for area of Foveal Avascular Zone (FAZ).

Data reported from the right eye unless the OCT grader reports segmentation failure. In that case, data from the other eye will be used if no segmentation failure is recorded for that eye.

Participants with ophthalmic conditions that would interfere with the OCT measurements will be excluded from analysis. This would include diabetic retinopathy, retinal vein occlusion, uveitis and glaucoma.

2.6.7 MRI scans.

Participants undergoing the MRI scans will attend an MRI scan within 6 weeks of their baseline visit, and again within 6 weeks of the final study visit (study visit 8).

The 60-minute scan will capture both structural and functional, 3D T1-weighted, sequences.

2.6.8. Colon biopsies.

Microbe populations that are specifically and intimately associated with the lining of the gut wall are ideally situated to influence the host and intestinal cells, but they can be underrepresented or absent in stool samples. For this reason, colon tissue samples are important to: 1) fully identify the microbes that are in most intimate contact with the host 2) identify and analyse how the cells within the tissue respond and react to these microbes and their products.

For colonoscopy biopsies, 6–8 pinch biopsies each measuring 3mm³ will be taken from the large bowel during the BCSP colonoscopy. These biopsies are taken to examine the microbiome that is adhering directly to the mucosal lining of the colon.

Fresh tissue will be immediately used for organ or cell culture systems, or for cell isolation and analysis. Other immediate means of processing tissue include those for (immuno) histology; isolating cells (e.g. immune and epithelial cells) for detailed characterisation (e.g. repertoire and subset identification and functional properties); proteins isolation/quantification (e.g. cytokines and other inflammatory or immunoregulatory proteins); and/or nucleic acids (DNA/RNA) extraction and microbial/host cell gene expression analysis.

For long term storage, biopsy samples will be preserved by, for example, snap freezing in liquid nitrogen (particularly useful for preserving RNA) and/or immersion in formalin (ideal for preserving tissue histology).

2.6.9 Electronic frailty index data.

The electronic frailty index (eFI) helps identify and predict adverse outcomes for older patients in Primary Care. The eFI is made up of 36 deficits comprising around 2,000 Read codes and is a ‘cumulative deficit’ model, which measures frailty on the basis of the accumulation of a range of deficits, which can be clinical signs (e.g. tremor), symptoms (e.g. vision problems), diseases, disabilities and abnormal test values [29]. Many of these deficits are associated with age-associated changes in gut microbe populations. Data will be requested from the participant’s GP retrospectively at the end of the study. Data will be collected for a single timepoint at the end of the study.

2.8.1 Interim analysis/ monitoring.

Interim reports will focus on monitoring the progress of the study against the main objectives to inform any necessary changes in protocol. These will include, as relevant at each stage of the study:

• Monitoring the rate of eligible participants from each stream,
• Participation rate of GPs
• Participation rate among different patient groups
• Rate of accruals from each stream
• Rate of accruals into each cohort
• Proportion consenting to each element of the study
• Demographic and clinical characteristics of the cohorts
• The completeness and quality of data and samples being received
• Patient’s adherence to the sample collection protocols
• Response rate to sub-studies
• Attrition rate across different patient groups in each wave
• Rate of adverse events

2.8.2 Cross-sectional analyses (following completion of baseline assessments).

Following the completion of baseline data collection, we will have data on the composition of the gut microbiome and associated clinical, demographic and lifestyle data on all participants. This can be used to address the following questions:

1. What is the association between age and the composition of the gut microbiome, and what is the variation in gut microbiome composition and function among middle aged and older people in Norfolk?
2. How do differences in lifestyle between age-groups account for these differences?
3. Is the microbiome associated with cognitive function and associated symptoms, physical function or other markers of successful ageing?
4. Can age or lifestyle differences account for any associations identified in 3 above, or
5. Does the gut microbiome mediate the effects of age and lifestyle on health?

2.8.3 Longitudinal analyses (after successive waves and following completion of all data collection).

Detailed analyses plans will be prepared following data collection but ahead of any analysis after each visit, building on the cross-sectional findings, but in brief planned analyses will address the following questions:

1. 6. What is the intra-individual variation and trajectory of different components of the gut microbiome with age?
2. 7. How does the baseline composition of the gut microbiome predict future changes in clinical outcomes? E.g., among people with mild cognitive impairment, can analysis of the gut microbiome be used to predict future cognitive change.
3. 8. How do changes in the gut microbiome correlate with changes in clinical outcomes?

2.8.4 Numbers of participants.

The study will recruit 360 participants. Over four years we anticipate that roughly 20% of participants will die or drop out of the study, reflecting rates seen in national cohort studies in similar populations over similar periods of time, hence 300 participants would be available for analysis at the end of the study.

A primary aim of this study is to estimate the link between microbiome composition and longitudinal change in clinical outcomes. A simulation study was conducted to estimate power for this objective, using as inputs the dynamics of cognitive change estimated from The Irish Longitudinal Study on Ageing [30]. There are many unknowns with respect to the prevalence of the microbiome features that might be associated with cognitive decline, the size of their effects, and variation in outcome measures. There is no prior data on our primary cognitive outcome measure (Mini- ACE), and so a generic measure of cognitive function (Mini-Mental State Examination, MMSE) was used, which is likely to have similar measurement properties. The standard deviation in MMSE change over four years was 2.2 points in a dataset with similar characteristics to the planned MOTION study. The smallest clinically meaningful difference in MMSE score has been reported as 1.4 [31], although much smaller effects are likely to be of scientific or public health importance. The power to detect effects equivalent to 1.5, 1.0, 0.8 and 0.5 MMSE points (corresponding to standardised effect sizes of 0.68 standard deviations to 0.22 standard deviations) over 4 years were estimated by simulation (at p<0.05), assuming prevalence of the specific microbial feature causing the effect is between 5% and 50%. A mixed effects model was used, with five evenly spaced time points, fixed effects of baseline microbiome and its interaction with time, and random intercept and slope accounting for between participant differences in cognitive change. The power quoted was the power to detect the interaction between the baseline microbiome and time on cognitive change, assuming this effect was constant over the study period. These are shown in Table 2, showing that under reasonable assumptions the MOTION study will have enough power to detect moderate effects of relatively prevalent features, and large effects of rare features.

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Table 2. Power of the MOTION study to detect difference in cognitive function between groups defined by features of the microbiome.

https://doi.org/10.1371/journal.pone.0276118.t002

The simulation was conducted using the simr package in R statistical software [32, 33].

2.9.1 Trial management group.

The Trial Management Group including the Chief Investigator (Prof Simon Carding), the study research team, NHS Principal Investigators and Clinical Research Network’s (CRN) Research Nurses will be responsible for the day-to-day management of the trial. They will monitor all aspects of the conduct and progress of the trial, ensure that the protocol is adhered to and take appropriate action to safeguard participants and the quality of the trial itself.

2.9.2 Trial management oversight group.

Management of the study will be overseen by a Trial Management Oversight Group whose membership is made up of representatives from;

• Health, Safety, Environment and Quality Assurance.
• QIB Sponsor Representative.
• Gut Microbes and Health QIB Programme Manager.
• QIB Statistician.
• Information Technology Security Specialist.
• NRP Biorepository.
• Patient and Public Involvement.
• QIB Bioinformatician.

The Trial Management Oversight Group will be responsible for overseeing the running of the study. They will ensure the monitoring and facilitating the progress of the study, and ensure the study is delivered within the projected timelines. Recruitment targets, success of data collection, and any specific issues arising will be addressed.

2.10.1 Declaration of helsinki and relevant regulations.

The Investigator will ensure that this study is conducted in accordance with the principles of the Declaration of Helsinki. The proposed research will be conducted in accordance with the conditions and principles of the International Conference on Harmonisation Good Clinical Practice, and in compliance with national law. The research will meet the requirements of the new EU General Data Protection Regulation (GDPR), UK Data Protection Act 2018 and relevant sponsor’s policies.

2.10.2 Expenses and benefits.

Reasonable travel expenses for all study-related visits will be paid.

As a thank you to participants, each participant will receive a shopping voucher worth £10 at Study Visit 4 and a further voucher to the value £15 upon study completion at Study Visit 8.

It is not anticipated that any post-trial care will be required. If any participant is harmed whilst taking part in this clinical research study as a result of negligence on the part of a member of the study team, QIB holds liability insurance for such circumstances.