Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

Laura A Bolte; Marjolein A Y Klaassen; Valerie Collij; Arnau Vich Vila; Jingyuan Fu; Taco A van der Meulen; Jacco J de Haan; Gerbrig J Versteegen; Aafje Dotinga; Alexandra Zhernakova; Cisca Wijmenga; Rinse K Weersma; Floris Imhann

doi:10.1371/journal.pone.0249405

. 2021 Apr 8;16(4):e0249405. doi: 10.1371/journal.pone.0249405

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

Laura A Bolte ^1,^2,^‡, Marjolein A Y Klaassen ^1,^2,^‡, Valerie Collij ^1,², Arnau Vich Vila ^1,², Jingyuan Fu ^1,³, Taco A van der Meulen ⁴, Jacco J de Haan ⁵, Gerbrig J Versteegen ⁶, Aafje Dotinga ⁷, Alexandra Zhernakova ², Cisca Wijmenga ², Rinse K Weersma ¹, Floris Imhann ^1,^2,^*

Editor: Nikhil Pai⁸

¹Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

²Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

³Department of Pediatrics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

⁴Department of Oral and Maxillofacial Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

⁵Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

⁶Department of Medical Psychology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

⁷Lifelines Cohort Study, Groningen, The Netherlands

⁸McMaster University, CANADA

Competing Interests: Floris Imhann received a speaker fee from Abbvie. Rinse Weersma received speaker fees from Abbvie, MSD, and Boston Scientific, a consulting fee from Takeda Pharmaceuticals and unrestricted research grants from Pfizer, Takeda, Ferring and Tramedico. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

‡ These authors share first authorship on this work.

^✉

* E-mail: f.imhann@rug.nl

Roles

Laura A Bolte: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft

Marjolein A Y Klaassen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

Valerie Collij: Data curation, Project administration, Resources, Software, Writing – original draft

Arnau Vich Vila: Data curation, Supervision, Writing – review & editing

Jingyuan Fu: Data curation, Funding acquisition, Writing – review & editing

Taco A van der Meulen: Data curation, Resources, Writing – review & editing

Jacco J de Haan: Data curation, Resources, Writing – review & editing

Gerbrig J Versteegen: Conceptualization, Investigation, Methodology, Writing – review & editing

Aafje Dotinga: Data curation, Investigation, Writing – review & editing

Alexandra Zhernakova: Data curation, Funding acquisition, Resources, Writing – review & editing

Cisca Wijmenga: Data curation, Funding acquisition, Resources, Writing – review & editing

Rinse K Weersma: Conceptualization, Data curation, Funding acquisition, Methodology, Resources, Supervision, Writing – review & editing

Floris Imhann: Conceptualization, Data curation, Investigation, Methodology, Supervision, Visualization, Writing – original draft

Nikhil Pai: Editor

PMCID: PMC8031379 PMID: 33831035

Abstract

Faecal sample collection is crucial for gut microbiome research and its clinical applications. However, while patients and healthy volunteers are routinely asked to provide stool samples, their attitudes towards sampling remain largely unknown. Here, we investigate the attitudes of 780 Dutch patients, including participants in a large Inflammatory Bowel Disease (IBD) gut microbiome cohort and population controls, in order to identify barriers to sample collection and provide recommendations for gut microbiome researchers and clinicians. We sent questionnaires to 660 IBD patients and 112 patients with other disorders who had previously been approached to participate in gut microbiome studies. We also conducted 478 brief interviews with participants in our general population cohort who had collected stool samples. Statistical analysis of the data was performed using R. 97.4% of respondents reported that they had willingly participated in stool sample collection for gut microbiome research, and most respondents (82.9%) and interviewees (95.6%) indicated willingness to participate again, with their motivations for participating being mainly altruistic (57.0%). Responses indicated that storing stool samples in the home freezer for a prolonged time was the main barrier to participation (52.6%), but clear explanations of the sampling procedures and their purpose increased participant willingness to collect and freeze samples (P = 0.046, P = 0.003). To account for participant concerns, gut microbiome researchers establishing cohorts and clinicians trying new faecal tests should provide clear instructions, explain the rationale behind their protocol, consider providing a small freezer and inform patients about study outcomes. By assessing the attitudes, motives and barriers surrounding participation in faecal sample collection, we provide important information that will contribute to the success of gut microbiome research and its near-future clinical applications.

Introduction

Gut microbiome research is being conducted using ever greater sample sizes to elucidate the role of gut microbiota in the pathogenesis of Inflammatory Bowel Diseases (IBD) and other immune-mediated inflammatory diseases [1–4]. The results of these studies hold great promise for clinical applications that use microbiome features as diagnostic biomarkers [5], determinants of disease activity [3] and predictors of individual drug response [6, 7]. The microbiome itself may also be a treatment target for prebiotic, probiotic, antibiotic and dietary interventions [2, 8, 9]. Moreover, as clinical interest grows in the use of faecal microbiota transplantation (FMT) to treat dysbiosis-related disorders such as recurrent Clostridium difficile‒associated diarrhoea and IBD, so will the need for voluntary stool donors [10–12].

As a consequence, there is a growing demand for stool samples collected by both patients and healthy volunteers. However, little is known about participant perspectives on collecting faecal samples for microbiome research and future care. Several studies have examined participant experiences with the faecal occult blood test (FOBT) used in colorectal cancer screening, the results of which mainly capture experiences coloured by the fear of having cancer [13, 14]. Other studies report barriers to faecal sample collection in general practice, including difficulty with the process, embarrassment and concerns around hygiene [14, 15]. Despite these barriers, most patients in the clinical setting do provide faecal samples because they are unwell and it has been recommended that they do so [15]. While personal benefit has been identified as the main motive for collecting and returning a stool sample in clinical care [14], there is no direct personal benefit for voluntary stool donors for FMT or microbiome research, who may face similar barriers.

In contrast to FMT or clinical tests such as the FOBT, at-home collection of faecal samples for microbiome research requires participants to follow sampling protocols and to store the sample in their home freezer in order to avoid post-collection bias in microbial composition, and this storage aspect may present an additional hurdle for volunteers. The accepted best-practices for microbiome studies involve freezing the sample to -80°C within 15 minutes of production and storage in a domestic frost-free freezer for fewer than 3 days. Samples taken for metabolomics studies, in particular, require that stool be frozen without preservatives and the freezing of live bacteria in glycerol preservative for culturing [16–18]. Since the stool samples used for research are collected by IBD patients at home, researchers need these patients to fully understand how to collect the sample. However, patient willingness to provide a faecal sample or to store it in the home freezer for research, their motives for and experiences with participation in microbiome research and the potential barriers they encounter, or how these barriers can be overcome, have thus far not been described.

Here, we explore the motives for and barriers to faecal sample collection given by 780 patients and healthy volunteers, including participants of one of the largest IBD gut microbiome cohorts to date. Our findings allow us to make recommendations for researchers and clinicians that will allow them to better account for participant attitudes when designing gut microbiome studies for research and clinical applications.

Methods

Cohorts and participants

In total, we contacted 1250 individuals, including IBD patients, patients with other disorders and healthy volunteers. A questionnaire (S1 Table) was sent in January 2017 to 772 patients who had previously been recruited at the University Medical Center Groningen in the Netherlands for gut microbiome studies for which they needed to provide a faecal sample. These patients had been included in four disease-specific cohorts for IBD (n = 660), melanoma (n = 9), Sjögren’s syndrome (n = 55) and systemic lupus erythematosus (SLE) (n = 48) (Fig 1). The latter three cohorts only comprised the participants who joined the gut microbiome studies. The questionnaire was aimed at obtaining patient experiences and identifying barriers encountered during the collection process. With the IBD cohort, we were also able to send out questionnaires to patients who previously refused to participate in gut microbiome research. The questionnaire recipients in the IBD cohort therefore comprised both patients who were previously willing to collect a stool sample for research (n = 577, IBD-Willing) and those who were not willing to do so (n = 83, IBD-Unwilling), indicating a willingness rate of 87.4% of the IBD microbiome study prior to this survey.

Fig 1 — Chart depicts the cohorts, diseases, departments and respondents in this study. IBD inflammatory bowel disease; SLE systemic lupus erythematosus; n number. From top to bottom: Source, Department, Cohort, Sub-cohort, Assessment Method, Responses by Cohort, Responses to Questionnaire, Total Responses. ^a IBD-Willing: patients who previously indicated their willingness to collect faecal samples for research. ^b IBD-Unwilling: patients who previously indicated that they were not willing to collect faecal samples for research. ^c Total responses include 5 individuals who did not fill in their participation number and could not be assigned to a cohort.

In addition, we interviewed a random selection of participants (n = 478) from the Lifelines general population cohort, of whom 9,547 individuals participated in the faecal sample collection project DAG3, using a brief questionnaire to analyse their opinions in the faecal sampling collection process (S2 Table) [19].

Ethics approval and consent to participate

The collection of faecal samples was previously approved by the Institutional Review Board (IRB) of the University Medical Center Groningen (IRB number 2008.338). All participants who participated in the faecal sample collection studies provided a signed informed consent form. For a single questionnaire study, no additional IRB approval was required according to Dutch medical research law. The questionnaire and the interview to assess the attitudes towards faecal sampling of patients and the general population, respectively, have been designed specifically for this study and are not published elsewhere. Consent to participate was integrated in the questionnaire.

Questionnaire design and processing

The questionnaire was designed in collaboration with a psychologist from the IBD Centre in Groningen. It covered eight distinct areas: (A) general information, including living situation, (B) prior experiences with faecal sample collection, (C) information about the type of toilet and freezer at home, (D) perceptions of the collection process, (E) perceptions of storing faecal samples in their freezer, (F) experience with the pick-up of the faecal samples from the participant’s home by hospital employees, (G) satisfaction with the information provided by our university medical centre and (H) future willingness to collect faecal samples for clinical care purposes. An English translation of the Dutch questions and the answers to the questionnaire and the interview can be found in the S1 and S2 Tables, respectively.

In our questionnaire, we addressed both patients previously willing to participate in faecal sample collection for microbiome research (IBD-Willing, melanoma, SLE and Sjögren’s Syndrome) and patients not willing to participate (IBD-Unwilling). The IBD-Unwilling cohort was asked to answer questions about their reasons for not participating despite their willingness to participate in research in general. Patients who had participated in faecal sample collection for research were asked about their experiences. Of the 347 respondents to our questionnaire, 45 gave inconsistent answers to questions, indicating they had not correctly understood the instructions. We chose to exclude these 45 questionnaires, resulting in a final sample of 302 respondents (39.1% response rate). To ensure that exclusion of these 45 questionnaires did not introduce bias, we performed our analyses on both the full set (347) and the final set (302) for comparison purposes and found similar results.

Statistical analyses

Descriptive statistics were determined for each question using the statistical software package R [20] (S1 Table). Chi-Square tests and Fisher’s exact tests were performed to determine statistically significant differences between counts (Table 1).

Table 1. Patient willingness to collect and freeze faecal samples and associated factors.

		n (%)
Motivation to participate in faecal sample collection for microbiome research		All patients
	Benefit for other patients	170 (57.0%)
	Both, benefit for self and others	48 (16.1%)
	Benefit for self	38 (12.8%)
	Other options/combinations	27 (9.1%)
	Did not fill in	15 (5.0%)
	Total	298 (100%)
Willing to collect faecal samples for future healthcare	Willing to collect	IBD cohort	Non-IBD cohort
Split by IBD / No IBD	Yes	224 (89.6%)	43 (89.6%)	P = 0.673
	No	15 (6.0%)	4 (8.3%)
	Did not fill in	11 (4.4%)	1 (2.1%)
	Total	250 (100%)	48 (100%)
Willing to collect faecal samples for future healthcare	Willing to collect	GI-disorder	No GI-disorder
Split by GI-disorder / No GI-disorder	Willing to collect	GI-disorder	No GI-disorder
	Yes	205 (81.0%)	38 (90.5%)
	No	34 (13.4%)	4 (9.5%)	P = 0.564
	Did not fill in	14 (5.5%)	0 (0.0%)
	Total	253 (100%)	42 (100%)
Willing to collect for future healthcare	Willing to collect	Living alone	Living together
Split by living alone/living together	Yes	49 (16.4%)	213 (71.5%)	P = 0.543
	No	2 (0.7%)	16 (5.4%)
	Did not fill in	18 (6.0%)
	Total	298 (100%)
Was the collection process easy?		All patients
	Yes	253 (84.9%)
	No	35 (11.7%)
	Did not fill in	10 (3.4%)
	Total	298 (100%)
Time between sample collection and storage in the freezer		All patients
	1–5 minutes	186 (62.4%)
	5–10 minutes	74 (24.8%)
	10–15 minutes	20 (6.7%)
	>15 minutes	4 (1.3%)
	Did not fill in	14 (4.7%)
	Total	298 (100%)
Unpleasant to store faecal samples in home freezer?		All patients
	Yes	73 (24.5%)
	No	215 (72.1%)
	No answer	10 (3.4%)
	Total	298 (100%)
Maximum time patients want to store faecal samples in their freezer		All patients
	I do not want that	2 (0.9%)
	1 to 3 days	33 (14.6%)
	1 week	57 (25.3%)
	2 to 4 weeks	25 (11.1%)
	>1 month	6 (2.7%)
	I do not mind	92 (40.9%)
	No answer	10 (4.4%)
	Total	225 (100%)
Was it clear why faecal samples need to be frozen?		All patients
	Yes	224 (75.2%)
	No	57 (19.1%)
	Did not fill in	17 (5.7%)
	Total	298 (100%)
Clarity of instruction manual vs. Willing to collect faecal samples	Clarity of instruction	Willing to collect	Not willing to collect
	Yes, very clear	95 (31.9%)	5 (1.7%)
	Yes, clear	157 (52.7%)	11 (3.7%)
	Neither clear nor unclear	8 (2.7%)	1 (0.3%)
	No, unclear	4 (1.3%)	1 (0.3%)	P = 0.046
	No, very unclear	0 (0.0%)	1 (0.3%)
	Did not fill in	15 (5.0%)
	Total	298 (100%)
Knowing the purpose of freezing vs. Willing to freeze	Willing to freeze	Knowing the purpose of freezing	Not knowing the purpose of freezing
	Willing to freeze	200 (67.1%)	42 (14.1%)	P = 0.003
	Not willing to freeze	23 (7.7%)	15 (5.0%)	P = 0.003
	Did not fill in	18 (6.0%)
	Total	298 (100%)

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IBD inflammatory bowel disease; GI gastrointestinal; SLE systemic lupus erythematosus; n number; % percentage of total

The following five associations were calculated:

Willingness to collect faecal samples for future screening and care vs. Gastrointestinal disease (Fisher’s exact test) to test if disease presence (Gastrointestinal disease or No gastrointestinal disease) is associated with willingness;
Willingness to collect faecal samples for future screening and care vs. Home situation (Fisher’s exact test) to test if having co-habitants is associated with willingness;
Willingness to collect faecal samples for future screening and care vs. Clarity of the instruction manual (Fisher’s exact test) to test if understanding the protocol properly is associated with willingness;
Willingness to collect faecal samples for future screening and care vs. Clarity of oral instruction (Fisher’s exact test) to test if understanding the protocol properly is associated with willingness;
Willingness to store faecal samples in the home freezer for future screening and care vs. Knowing the purpose of freezing the samples (Chi-Square test of independence with Yate’s continuity correction) to test if understanding the reason for freezing is associated to increased willingness of storing the samples in the home freezer.

Results

Of the 772 patients who received the questionnaire, 302 patients responded (39.1%). When combined with the 478 Lifelines interviewees, we had information from 780 individuals in total (Fig 1).

Of the patients who responded to the written questionnaire, 97.4% had collected a faecal sample for prior gut microbiome research projects. Unfortunately, response from the IBD patients who did not want to participate in gut microbiome research was very low: only three of the 83 IBD-Unwilling patients responded to the questionnaire, making it hard to draw broad conclusions from their answers. Nevertheless, extensive and valuable information could be obtained from the participants who did respond (Table 1, S1 Table).

Respondent motivations for participating in research projects were mainly altruistic, as future benefits for other patients (57.0%) was mentioned much more often than future benefits for themselves (12.8%) or future benefits for both themselves and others (16.1%). Most of the patients who responded (82.9%) and the population controls who were interviewed (95.6%) indicated that they were willing to collect faecal samples for future screening or research. We had anticipated that respondents with gastrointestinal disorders, who are more accustomed to handling stool, would be more willing to collect a stool sample. However, we found that having a gastrointestinal disorder was not related to the willingness to do so, with all groups showing similarly high levels of willingness to participate in future collections (IBD-cohort, willing: 224 of 250 (89.6%) vs. Non-IBD-cohort, willing: 43 of 48 (89.6%), P = 0.673, Fisher’s exact test). Only 26.2% of the patients who responded felt the collection of faecal samples was dirty, and most of the population controls interviewed perceived faecal sample collection as ‘not inconvenient at all’ (49.8%) or ‘not inconvenient’ (28.7%).

Most patients thought the collection process was easy (84.9%), immediately succeeded in collecting the sample (89.0%) and were able to store their faecal sample in the freezer within 15 minutes (93.9%) as required, with 62.4% of these respondents reporting only needing 5 minutes to do so. Most respondents (72.1%) did not mind storing the stool samples in their home freezer. However, while most patients were willing to store a stool sample in their freezer, many were only willing to do so for a brief period of time: maximum 1 to 3 days (14.6%), 1 week (25.3%), or 2 to 4 weeks (11.1%). 40.9% said that they did not mind storing faecal samples for a longer time. Some patients even reported clearing the entire freezer before the stool sample collection and keeping it empty until the sample was picked up on dry ice by our collection team.

Household composition did not influence willingness to collect and store stool samples in a home freezer, as we saw no difference in attitude between participants living alone versus those living with a partner, children, parents or roommates (P = 0.543, Fisher’s exact test). A minority of respondents (19.1%) did not understand why the faecal sample needed to be frozen. This is an important observation because the clarity of the written instructions was associated with future willingness to collect stool samples (P = 0.046, Fisher’s test) and knowing the purpose of freezing stool (stopping bacterial growth) was associated with future willingness to freeze the stool samples (P = 0.003, Chi-square test).

More than half of the patients (58.3%) did not know how the stool samples would be processed and investigated, even though most patients (80.2%) indicated that they would like to learn more about the results of the gut microbiome research they were participating in, and some felt very disappointed about not being briefed afterwards.

Discussion

In this study, we investigated the attitudes towards faecal sampling of participants in one of the largest IBD gut microbiome cohorts and compared them to those of other patient cohorts and healthy volunteers [21]. By assessing the attitudes, motives and barriers surrounding participation in faecal sample collection, we are able to provide important information that will contribute to the success of gut microbiome research and its near-future clinical applications.

Gut microbiome researchers setting up new cohorts or clinicians trying new faecal tests should not shy away from doing so and should focus on providing adequate subject information

Our study demonstrates that stool sample collection for gut microbiome studies and future clinical applications is acceptable to the majority of IBD patients and even to population controls. Most IBD patients (87.4%) were willing to participate in our previous stool sample collection (IBD-Willing, n = 577), and most respondents (82.9%) and interviewees (95.6%) indicated that they were willing to participate again.

Other studies have assessed patient willingness to receive or donate stool samples for FMT, the transfer of faecal material containing microbiota from a healthy donor into a diseased patient. One study found that 77% of patients visiting the gastroenterologist would undergo FMT if medically indicated [22], whereas only 36.9% of IBD patients were willing to undergo FMT in a report by Zeitz et al. [23]. Familiarity with the gut microbiome might contribute to the higher willingness to participate in gut microbiome research in our study compared to FMT. Previous studies had found that only 46.5% of IBD patients [23] and 12% of patients visiting a gastroenterologist [22] knew about FMT. Interestingly, the willingness rate of the IBD patients to undergo FMT almost doubled after an information leaflet was provided [23]. Recognition of FMT in postgraduate medical students has been shown to be similarly low [24]. While nearly half of these students had not heard about FMT, the majority recognised that disrupting and restoring the gut microbiota played an important role in the pathogenesis and prevention of diseases. In the same study, willingness to undergo FMT or donate samples was significantly higher among those who were familiar with FMT [24].

Researchers and clinicians should inform participating patients and healthy volunteers about the outcome of the research

In our study, patients were very interested in the outcome of the study they contributed to and were disappointed when they were not informed about the results. Most of our participants also indicated a desire to know more about the study and its outcome. This is in line with a previous report of the attitudes of 400 patients towards participation in clinical trials conducted at an internal medicine ward [25]. Positive feedback on how FMT can help patients has also been shown to be a motivator for donating faecal samples for FMT [10]. Based on the responses to our questionnaire, our team of microbiome researchers wrote a newsletter for participants about our scientific findings and publications. We recommend future researchers and clinicians provide similar feedback when possible.

An emphasis on the public benefit of the research could help with establishing large cohorts for microbiome research

The main driver for participation in gut microbiome research reported by our respondents was the possibility that the research could benefit others with disease (57%). The motivation to contribute to research for the next generation of patients affected by the disease has also been reported to rank highly in other studies of research participation [26, 27]. McSweeney et al. also identified altruism as the main motive to donate faecal samples for FMT, and many patients who were willing to donate faecal samples said they did so to help those who were ill and to contribute to progress in scientific research [10]. As expected, this differs from the motives of patients who collect or receive faecal samples as part of their clinical care, where personal benefit is the main incentive [14]. Despite concerns around hygiene, logistics and privacy, most patients return their faecal sample to their doctor because it was recommended that they do so for their own health [15]. Similarly, other studies have shown that the majority of patients would undergo FMT if it was medically indicated and recommended by the doctor [22] and that willingness to undergo FMT was positively associated with disease severity and previous TNF-treatment in IBD patients [23, 28]. These factors are not relevant when recruiting volunteers for microbiome research or healthy donors for FMT, making it even more crucial to inform potential volunteers about the process and to remove any barriers in order to obtain sufficient sample sizes or guarantee cost-effectiveness. Our study suggests that an emphasis on the public benefit of the research could help with establishing large cohorts for microbiome research [27].

In studies where a time-series of many stool samples needs to be collected, researchers should consider providing participants with a small freezer

Only a minority of our participants (26.2%) felt the collection of faecal samples was dirty or inconvenient. However, the need to store samples in a participant’s home freezer can be a barrier to participation in faecal sample collection, especially when participants have to store samples for a prolonged period. While most patients were willing to store a stool sample in their freezer (72.1%), many were only willing to do so for a brief period of time, from a few days to a maximum of 1 month. In another study in which patients were interviewed about providing faecal samples to their general practitioner, a much larger proportion of patients mentioned embarrassment and concerns about hygiene and contamination, discretion and privacy [14]. Fear of infectious diseases and disgust about the procedure were also identified as the most common concerns of patients about undergoing FMT [22, 23]. This is underlined by the finding that IBD patients would choose a colonoscopy as the preferred route of FMT rather than an enema or nasogastric tube [22, 23]. Even post-graduate medical students considered donating faeces troublesome because it hampered their privacy, and they also expressed concerns about the acceptability among patients [24]. Privacy was not a big concern in our study. While screening for FMT requires the donor to provide a lot of private information, as not all stool samples are suitable, participants in gut microbiome research might feel more anonymous. We also hypothesized that IBD patients are more accustomed to handling faecal samples, resulting in fewer perceived barriers. However, we could not find significant differences in the willingness to collect and store faecal samples between our IBD cohort and the other cohorts.

Collection process perceived as easy

Most patients thought the collection process was easy (84.9%) and reported that they immediately succeeded in collecting the sample (89.0%) and in storing it in the freezer within 15 minutes according to the collection protocol (93.9%), which indicates that faecal sampling does not present a significant logistical challenge for individuals. Other studies have identified barriers towards faecal sampling in clinical care, including difficulty with the collection process, lack of information given by doctors and inability to return the sample to the institution [14, 15]. The difficulty of collecting the faecal sample was one of the major factors impacting FOBT response in a South African study [29]. Higher donation frequency, the logistics of collection or transport of faecal samples, the screening process, lack of public awareness and negative social perception have also been identified as deterrents to donating stool for FMT [10]. One reason why most of our participants perceived the collection system as easy could be that we provided an instruction sheet with the collection kit.

Gut microbiome researchers and clinicians should explain why their collection protocol was designed in a specific way

We show that understanding the purpose of our procedures is associated with increased willingness to collect and freeze stool samples. Explaining the procedures and the reasons why they need to be carried out in a specific way increases participant willingness to collect and freeze a faecal sample. This may be particularly important in populations with lower health literacy, with one study showing that higher education levels are strong predictors of FMT acceptance in patients [22]. Similar to our findings, other studies have indicated that patients collecting faecal samples for clinical care value an information leaflet provided with the stool collection kit [14] and that screening compliance for FOBT is significantly improved when patients obtain this information [30].

Household composition did not affect willingness to collect and store a faecal sample

We saw no significant difference in attitude between participants living alone versus those living with partners, children, parents or roommates. Of those willing to collect faecal samples for research, 16.4% lived alone and 71.5% lived with others. Another study even showed that having children and being married were strong predictors of FMT acceptance in patients [22]. It is possible that patients with children are more likely to embrace FMT, even though it may be unappealing in nature, because of their responsibility towards their family [27].

Strengths and limitations

Our questionnaire study was limited by knowing only the answers of the respondents. IBD patients who previously declined to participate in our gut microbiome studies (IBD-Unwilling, n = 83) were also less likely to respond to the questionnaire (n = 3), making it difficult to assess their reasons for refusing participation. Overall, the 39.1% response rate to our questionnaire is in line with the recognised 40% average response rate for postal surveys [31]. Another survey of IBD patients investigating their perspectives on FMT obtained a similar response rate of 31.4% [23]. The positive attitudes towards faecal sample collection in our study may not always be representative of other patients, and attitudes may differ depending on the reason for stool sample collection, e.g. samples collected for research vs. those collected for diagnosis of a potential disease (a process that may be accompanied by fear), or the health care setting, e.g. secondary vs. routine primary care. The strength of our study is that we were able to obtain information on the attitudes, motives and barriers surrounding participation in gut microbiome research for 780 patients with different disorders and for healthy volunteers, a group who have not been assessed to date. We obtained enough information to formulate the following conclusions and recommendations for both gut microbiome researchers and clinicians.

Conclusions

Targeting the gut microbiome will soon be part of the diagnostic process and treatment of IBD and other diseases associated with microbial dysbiosis [5, 6, 32, 33], requiring repeated sampling from patients [34]. Here, we assessed the perspectives of patients and healthy volunteers on faecal sampling for gut microbiome research.

We derive the following recommendations for gut microbiome researchers and clinicians:

Gut microbiome researchers setting up new cohorts and clinicians trying new faecal tests should not shy away from doing so.
Gut microbiome researchers and clinicians should explain to participants why their collection protocol was designed in a specific way.
In studies where a time-series of many stool samples needs to be collected, researchers should consider providing participants with a small freezer.
Researchers and clinicians should inform participating patients and healthy volunteers about the outcome of the research.

Supporting information

S1 Table. Patient questionnaire outcome.

Translation of the questionnaire and descriptive statistics for each question. IBD inflammatory bowel disease; SLE systemic lupus erythematosus; n number; % percentage of total. a. IBD-Willing: IBD patients who previously indicated willingness to collect faecal samples for research. b. IBD-Unwilling: IBD patients who previously indicated not being willing to collect faecal samples for research. c. No ID-number: patients who did not fill in their participation number and could not be assigned to a cohort. d. All-Willing: all patients who previously indicated willingness to collect faecal samples for research, i.e. all except for the IBD-Unwilling cohort. e. All-Unwilling: all patients who previously indicated not being willing to collect faecal samples for research.

(XLSX)

Click here for additional data file.^{(32.2KB, xlsx)}

S2 Table. Interview outcome.

Translation of the interview and descriptive statistics for each question.

(XLSX)

Click here for additional data file.^{(14.1KB, xlsx)}

S1 File. Supplementary methods.

Additional information on statistical analyses and results of the interview conducted in the general population cohort.

(DOCX)

Click here for additional data file.^{(20KB, docx)}

Acknowledgments

We would like to thank the participants of this study for filling in the questionnaire and participating in the interviews, Lifelines for providing data on the population control participants, Wilma Westerhuis for collection logistics, Esther Bos and Jurya Glansbeek for help sending the questionnaires and Kate McIntyre for language editing.

Data Availability

The summary statistics for each question in the questionnaire are available in Supplementary Tables S1 and S2. Using these statistics, all tests in this study can be replicated. Our cohorts comprise specific groups of patients from a specific geographical area and the content of the questionnaire comprises household composition, education level and home details about the toilet and the freezer. As a consequence, individual level data could lead to identification of patients. Therefore, European privacy law prohibits us from making individual level data publicly available. Data requests should be sent to the Research Office Lifelines (research@lifelines.nl) and the Medical Ethics Review Board of the UMCG (metc@umcg.nl).

Funding Statement

LAB and RKW are supported by a research grant from the Seerave Foundation. RKW is supported by the collaborative TIMID project (LSHM18057-SGF) financed by the PPP allowance made available by Top Sector Life Sciences & Health to Samenwerkende Gezondheidsfondsen (SGF) to stimulate public-private partnerships and co-financing by health foundations that are part of the SGF. JF and AZ are supported by the Gravitation grant ExposomeNL from the from the Dutch Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek, NWO) (024.004.017). AZ is further supported by a European Research Council (ERC) Starting Grant (715772) and a NWO-VIDI grant (016.178.056). JF is supported by the NWO Gravitation Netherlands Organ-on-Chip Initiative (024.003.001) and the ERC Consolidator grant (101001678). JF and AZ are further supported by a grant from the Dutch Heart Foundation (CardioVasculair Onderzoek Nederland, CVON) (2018-27). CW is supported by the NWO Gravitation grant (024.003.001), a Spinoza award (NWO SPI 92-266) and a grant from the Netherlands’ Top Institute Food and Nutrition (GH001).

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PLoS One. doi: 10.1371/journal.pone.0249405.r001

Decision Letter 0

Nikhil Pai

4 Jan 2021

PONE-D-20-24145

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

PLOS ONE

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Thank you for your clear, and practical manuscript assessing the attitudes of patients towards faecal sampling for gut microbiome studies. The results of this paper are derived from an impressive survey sample, and the practical guidance offered in the conclusion of this piece are valuable. Please attend to the minor revisions suggested by the Reviewer. I also want to personally thank you for your patience. It has been challenging to find reviewers for certain publications, and this is not reflective of the quality of the work that you have submitted. Kindly attend to these minor revisions and I look forward to seeing a revised submission for assessment. Again, our thanks for your patience with this process.

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Reviewer #1: Dear authors

Thank you for writing such a clear and interesting article on the patient perspective towards stool collection. I have a few minor comments

1. introduction

Paragraph 1: Authors state ‘little is known about participant perspectives on collecting

faecal samples for microbiome research and future care, with available literature

currently limited to several studies examining participant experiences with the faecal

occult blood test (FOBT) used in colorectal cancer screening, the results of which mainly

capture experiences coloured by the fear of having cancer’ and reference 2 other studies.

I agree that literature on this topic is limited however it is not only limited to FOBT studies. The Lecky et al study for example examined patients both with and without previous experience of providing a stool sample and does not state that recruited participants had collected samples for FOBT.

Other work in this arena, not just linked to FOBT, also include

McNulty, C.A., Lasseter, G., Newby, K. et al. Stool submission by general practitioners in SW England - when, why and how? A qualitative study. BMC Fam Pract 13, 77 (2012). https://doi.org/10.1186/1471-2296-13-77

Breanna McSweeney, Jessica R. Allegretti, Monika Fischer, Huiping Xu, Karen J. Goodman, Tanya Monaghan, Carmen McLeod, Benjamin H. Mullish, Elaine O. Petrof, Emmalee L. Phelps, Roxana Chis, Abby Edmison, Angela Juby, Ralph Ennis-Davis, Brandi Roach, Karen Wong & Dina Kao (2020) In search of stool donors: a multicenter study of prior knowledge, perceptions, motivators, and deterrents among potential donors for fecal microbiota transplantation, Gut Microbes, 11:1, 51-62, DOI: 10.1080/19490976.2019.1611153

Please make this more clear in your introduction.

2. Discussion paragraphs 2 and 3

These paragraphs are more suited to introducing the topic and the need for stool collection rather than discussing the findings of this study and its relevance and/or implications

3. As previously mentioned, other research has been carried out in this areas therefore the discussion would benefit from more comparison to this literature highlighting similarities and differences and potential reasons for this.

Reviewer #2: The topic of the manuscript is interesting and important to tell researchers who are going to collect stool samples. This study highlighted the difficulty for achieving stool sample for microbial study. The “dirty” is one of the important factors affecting the attitudes to response the survey and collect stool sample. The lower response rate for this survey is the main limitation of this study, which might be the bias of the survey. However, authors did not clearly state this difficulty in INTRODUCTION or discuss this pint in DISCUSSION. As I know, the similar survey on patients’ or physicians’ attitude to process fecal transplant have the similar difficulty in practice. These should be discussed for supporting authors’ findings. The following reports should be included for discussion:

Patients' views on fecal microbiota transplantation: an acceptable therapeutic option in inflammatory bowel disease? Eur J Gastroenterol Hepatol 2017.

Perceptions of fecal microbiota transplantation for Clostridium difficile infection: factors that predict acceptance. Ann Gastroenterol. 2017.

The recognition and attitudes of postgraduate medical students toward fecal microbiota transplantation: a questionnaire study. Therap Adv Gastroenterol. 2019

Minor comments as below:

1. In introduction, microbiota is target for fecal microbiota transplantation too. This is very important. Please add it in the sentence.

2. Methods - There were nearly 40 questions in your questionnaire. Why do you only calculate the five questions which you mentioned?

3. In table 1, the number of patients in “Maximum time patients want to store fecal samples in their freezer” should subtract 73 as shown in “Unpleasant to store fecal samples in home freezer?”. Seventy-three patients are unpleasant to store fecal samples in home freezer. So they are not necessary to consider the time to store fecal sample.

4. Results - 97.4% had collected a faecal sample for prior gut microbiome research projects. Is this data for question eight in the Supplementary Table 1? An error?

5. Results - According to your definition in the Supplementary Method, the patients with gastrointestinal disease includes “IBD willing + IBD unwilling + no identification number” participants. You mentioned in the results that 250 patients had GI-disorder. This may be a statistical error. In additon, this is a little different from the data in the questionnaire. Because you asked participants in question eight if they had been diagnosed with intestinal disease, and only 42 patients answered no.

6. Limitation should better not be included in conclusion. Please put it to the part of discussion.

7. Conclusions include too much content. Please try to use summative words in this part and put the current content to suitable part.

8. The structure of the manuscript is unconventional. Please refer the regular style of the journal.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Apr 8;16(4):e0249405. doi: 10.1371/journal.pone.0249405.r002

Author response to Decision Letter 0

19 Feb 2021

Additional journal requirements

1) PLOS ONE's style requirements: The journal’s requirements were adopted.

2) Questionnaire validation: The questionnaire was specifically designed for this study together with the Department of Health Psychology of the University Medical Center Groningen. The questionnaire has not been validated in a separate study.

3) Data availability and reproducibility: The summary statistics for each question in the questionnaire are available in Supplementary Tables S1 and S2. Using these statistics, all tests in this study can be replicated. Our cohorts comprise specific groups of patients from a specific geographical area and the content of the questionnaire comprises household composition, education level and home details about the toilet and the freezer. As a consequence, individual level data could lead to identification of patients. Therefore, European privacy law prohibits us from making individual level data publicly available. For collaborations please contact the Research Office Lifelines (research@lifelines.nl) and the Medical Ethics Review Board of the UMCG (metc@umcg.nl).

4) ORCID iD of corresponding author: In the editorial manager we were only able to add the ORCID iD of the submitting author (Laura Bolte). The ORCID iD of the corresponding author (Floris Imhann) is: 0000-0001-5278-903X.

5) Supporting file captions and citations: Supporting file captions and file names have been adapted according to the journals’ requirements and matched with in-text citations.

6) Competing Interests: Floris Imhann received a speaker fee from Abbvie. Rinse Weersma received speaker fees from Abbvie, MSD, and Boston Scientific, a consulting fee from Takeda Pharmaceuticals and unrestricted research grants from Pfizer, Takeda, Ferring and Tramedico. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Reviewer 1

Q.1.1: Thank you for writing such a clear and interesting article on the patient perspective towards stool collection. I have a few minor comments.

Introduction, Paragraph 1: Authors state ‘little is known about participant perspectives on collecting faecal samples for microbiome research and future care, with available literature currently limited to several studies examining participant experiences with the faecal occult blood test (FOBT) used in colorectal cancer screening, the results of which mainly capture experiences coloured by the fear of having cancer’ and reference 2 other studies. I agree that literature on this topic is limited however it is not only limited to FOBT studies. The Lecky et al study for example examined patients both with and without previous experience of providing a stool sample and does not state that recruited participants had collected samples for FOBT. Other work in this arena, not just linked to FOBT, also include:

McNulty, C.A., Lasseter, G., Newby, K. et al. Stool submission by general practitioners in SW England - when, why and how? A qualitative study. BMC Fam Pract 2012, 13:77.

McSweeney B, Allegretti JR, Fischer, M et al. In search of stool donors: a multicenter study of prior knowledge, perceptions, motivators, and deterrents among potential donors for fecal microbiota transplantation, Gut Microbes 2020, 11:1, 51-62.

Please make this more clear in your introduction.

R.1.1: We thank the reviewer for the positive feedback and suggestions to improve our manuscript. We agree that our findings connect well with other disciplines such as the diagnosis of intestinal infectious diseases as part of clinical routine, or the search for stool donors for fecal microbiota transplantation. We now refer to these articles in the introduction.

Page 3, lines 9-12:

“Moreover, as the clinical interest grows in the use of fecal microbiota transplantation (FMT) for dysbiosis-related disorders such as recurrent Clostridium difficile associated diarrhea and IBD, so will the need for voluntary stool donors.[10–12]”

[10] McSweeney B, Allegretti JR, Fischer M, Xu H, Goodman KJ, Monaghan T, et al. In search of

stool donors: a multicenter study of prior knowledge, perceptions, motivators, and deterrents

among potential donors for fecal microbiota transplantation. Gut Microbes 2019; 11: 51-62.

[11] Moayyedi P, Surette MG, Kim PT, Libertucci J, Wolfe M, Onischi C, et al. Fecal Microbiota

Transplantation Induces Remission in Patients With Active Ulcerative Colitis in a Randomized

Controlled Trial. Gastroenterology 2015; 149: 102-109.e6.

[12] Vrieze A, Nood EV, Holleman F, Salojärvi J, Kootte RS, Bartelsman JFWM, et al. Transfer of

Intestinal Microbiota From Lean Donors Increases Insulin Sensitivity in Individuals With

Metabolic Syndrome. Gastroenterology 2012; 143: 913-916.e7.

Page 3, lines 18-24:

“Other studies report patient barriers to fecal sample collection in general practice, including difficulty with the process, embarrassment and concerns around hygiene. [14, 15] Despite these barriers, most patients do provide fecal samples in the clinical setting, because they are unwell and have been recommended to do so. [15] While personal gain has been identified as the main motive for collecting and returning a stool sample in clinical care,[14] there is no direct personal benefit for voluntary stool donors for FMT or microbiome research, who may face similar barriers.”

[14] Lecky DM, Hawking MK, McNulty CA. Patients’ perspectives on providing a stool sample to

their GP: a qualitative study. Br J Gen Pract 2014; 64: e684–e693.

[15] McNulty CA, Lasseter G, Newby K, Joshi P, Yoxall H, Kumaran K, et al. Stool submission by

general practitioners in SW England - when, why and how? A qualitative study. BMC Family

Practice 2012; 13: 77.

Q.1.2: Discussion paragraphs 2 and 3: These paragraphs are more suited to introducing the topic and the need for stool collection rather than discussing the findings of this study and its relevance and/or implications.

R.1.2: We agree with the reviewer that the need for stool collection is more suitable to introduce the topic and have moved these paragraphs to the introduction.

Page 3, lines 5-12:

“The results of these studies hold great promise for clinical applications that include the use of microbiome features as diagnostic biomarkers,[5] determinants of disease activity,[3] and predictors of individual drug response.[6,7] The microbiome itself may also be a treatment target for prebiotic, probiotic, antibiotic and dietary interventions. [2,8,9] Moreover, as clinical interest grows in the use of fecal microbiota transplantation (FMT) for dysbiosis-related disorders such as recurrent Clostridium difficile infection and IBD, so will the need for voluntary donors.[10–12]”

Q.1.3: As previously mentioned, other research has been carried out in these areas therefore the discussion would benefit from more comparison to this literature highlighting similarities and differences and potential reasons for this.

R.1.3: We agree with the reviewer and added a comparison of the identified attitudes towards fecal sampling for gut microbiome research in our study with previous reports on the perspectives on collecting or donating fecal samples for clinical care or FMT.

Page 12, lines 16-19:

“This is in line with a previous report of the attitudes of 400 patients towards participation in clinical trials conducted at an internal medicine ward.[25] Positive feedback on how FMT can help patients were also motivators to donate fecal samples for FMT.[10]”

Page 13, line 7-13:

“McSweeney et al. also identified altruism as the main motive to donate fecal samples for FMT and many patients who were willing to donate fecal samples said to do so to help those who were ill and contribute to progress in scientific research. [10] As expected, this differs from the motives of patients who collect or receive fecal samples as part of their clinical care, where personal gain is the main incentive. [14] Despite concerns around hygiene, logistics and privacy, most patients return their faecal sample to their doctor because they were recommended to do so for their own health.[15]”

Page 15, line 5-12:

“Other studies identified barriers towards fecal sampling in clinical care, including difficulty with the collection process, lack of information given by doctors and inability to return the sample to the institution. [14, 15] The difficulty of collecting the fecal sample was one of the major factors impacting FOBT response in a South African study. [29] Higher donation frequency, logistics of collection or transport of fecal samples, screening process, lack of public awareness and negative social perception were identified as deterrents to donate stool for FMT. [10]”

Page 15, line 20 – Page 16, line 2:

“Similar to our findings, studies indicate that patients collecting fecal samples for clinical care value an information leaflet provided with the stool collection kit [14] and that screening compliance for the FOBT is significantly improved when patients obtain this information. [30]”

[10] McSweeney B, Allegretti JR, Fischer M, Xu H, Goodman KJ, Monaghan T, et al. In search of

stool donors: a multicenter study of prior knowledge, perceptions, motivators, and deterrents

among potential donors for fecal microbiota transplantation. Gut Microbes 2019; 11:51-62.

[14] Lecky DM, Hawking MK, McNulty CA. Patients’ perspectives on providing a stool sample to

their GP: a qualitative study. Br J Gen Pract 2014; 64: e684–e693.

[15] McNulty CA, Lasseter G, Newby K, Joshi P, Yoxall H, Kumaran K, et al. Stool submission by

general practitioners in SW England - when, why and how? A qualitative study. BMC Family

Practice 2012; 13: 77.

[29] Price CL, Szczepura AK, Gumber AK, Patnik J. Comparison of breast and bowel cancer

screening uptake patterns in a common cohort of South Asian women in England. BMC Health

Services Research 2010; 10: 103.

[30] O’Carroll RE, Steele RJ, Libby G, Brownlee L, Chambers JA. Anticipated regret to increase

uptake of colorectal cancer screening in Scotland (ARTICS): study protocol for a randomised

controlled trial. BMC Public Health 2013; 13: 849.

Reviewer 2

Q.2.1: The topic of the manuscript is interesting and important to tell researchers who are going to collect stool samples. This study highlighted the difficulty for achieving stool sample for microbial study. The “dirty” is one of the important factors affecting the attitudes to response the survey and collect stool sample. The lower response rate for this survey is the main limitation of this study, which might be the bias of the survey. However, authors did not clearly state this difficulty in INTRODUCTION or discuss this point in DISCUSSION. As I know, the similar survey on patients’ or physicians’ attitude to process fecal transplant have the similar difficulty in practice. These should be discussed for supporting authors’ findings. The following reports should be included for discussion:

Patients' views on fecal microbiota transplantation: an acceptable therapeutic option in inflammatory bowel disease? Eur J Gastroenterol Hepatol 2017.

Perceptions of fecal microbiota transplantation for Clostridium difficile infection: factors that predict acceptance. Ann Gastroenterol. 2017.

The recognition and attitudes of postgraduate medical students toward fecal microbiota transplantation: a questionnaire study. Therap Adv Gastroenterol. 2019

R.2.1: We thank the reviewer for the thorough inspection of our manuscript. We agree that the non-response bias should be stated more clearly as a limitation in the discussion section. Moreover, we agree that the above-mentioned literature should be described and compared to our results more elaborately in the discussion.

Therefore, we have largely rewritten our discussion including the literature and limitations mentioned by the reviewer.

Page 11, line 15 – Page 12, line 11:

“Other studies assessed patient willingness to receive or donate stool samples for FMT, the transfer of fecal material containing microbiota from a healthy donor into a diseases patient. One study found that 77% of patients visiting the gastroenterologist would undergo FMT if medically indicated. [22] Conversely, only 36.9% of IBD patients were willing to undergo FMT in a report by Zeitz et al. [23] Familiarity with the gut microbiome might contribute to a higher willingness to participate in gut microbiome research in our study compared to FMT. Only 46.5% of IBD patients [23] and 12% of patients visiting a gastroenterologist [22] knew about FMT. Interestingly, the willingness rate of IBD patients to undergo FMT almost doubled after an information leaflet was provided. [23] Recognition of FMT in postgraduate medical students was equally low. [24] While nearly half of the students had not heard about FMT, the majority recognized that disrupting and restoring gut microbiota played an important role in the pathogenesis and prevention of diseases. In the same study, willingness to undergo FMT or donate samples was significantly higher among those who were familiar with FMT. [24]”

[22] Park et al. Perceptions of fecal microbiota transplantation for Clostridium difficile infection: factors that predict acceptance. Ann Gastroenterol 2017; 30: 83–88.

[23] Zeitz et al. Patients’ views on fecal microbiota transplantation: an acceptable therapeutic option in inflammatory bowel disease? Eur J Gastroenterol Hepatol 2017; 29: 322–330.

[24] Wu et al. The recognition and attitudes of postgraduate medical students toward fecal

microbiota transplantation: a questionnaire study. Therap Adv Gastroenterol 2019; 12:

1756284819869144.

Page 13, lines 14-20:

“Similarly, studies showed that the majority of patients would undergo FMT if it was medically indicated and recommended by their doctor [22] and willingness to undergo FMT was positively associated with disease severity and previous TNF-treatment in IBD patients. [23, 28] Naturally, recommendation by your own doctor is not part of when recruiting volunteers for microbiome research or healthy donors for FMT, making it even more crucial to inform volunteers about the process and remove any barriers in order to obtain sufficient sample sizes or to guarantee cost-effectiveness.”

[28] Kahn et al. Fecal bacteriotherapy for ulcerative colitis: patients are ready, are we? Inflamm Bowel Dis 2012; 18: 676–684.

Page 14, lines 11-19:

“Fear of infectious diseases and disgust about the procedure were also identified as the most common concerns of patients to undergo FMT. [22, 23] This is underlined by the finding that IBD patients would choose a colonoscopy as the preferred route of FMT rather than an enema or nasogastric tube. [22, 23] Even post-graduate medical students considered donating feces troublesome because it hampered their privacy and also showed concerns about the acceptability among patients. [24] Privacy was not a big concern in our study. While screening for FMT requires the donor to provide a lot of private information as not all stool samples are suitable, participants in gut microbiome research might feel more anonymous.”

Page 15, lines 20-22:

“This may be particularly important in populations with a lower health illiteracy, with a study showing that higher education levels are strong predictors of FMT acceptance in patients. [22]”

Page 16, lines 7-11:

“Another study even showed that having children and being married were strong predictors of FMT acceptance in patients. [22] It is possible that patients with children are more likely to embrace FMT, even though it may be unappealing in nature, because of their responsibility towards their family. [27]”

[27] Prainsack and Buyx. A solidarity-based approach to the governance of research

biobanks. Medical Law Review 2013; 21: 71–91.

Page 16, line 12 – Page 17, line 5:

“Strengths and limitations

Our questionnaire study was limited by knowing only the answers of the respondents. IBD patients who previously disagreed to participate in our gut microbiome studies (IBD-Unwilling, n=83) were also less likely to respond to the questionnaire (n=3), making it difficult to assess their reasons to refuse participation. Overall, the 39.1% response rate to our questionnaire is in line with the recognised average response rate for postal surveys (40%). [31] Another survey of IBD patients to investigate their perspectives on FMT, obtained a response rate of 31.4%. [23] The positive attitudes towards fecal sample collection in our study may not always be representative of other patients, and attitudes may differ depending on the reason for stool sample collection, e.g. samples collected for research vs. those collected for diagnosis of a potential disease (a process that may be accompanied by fear), or the health care setting, e.g. secondary vs. routine primary care. The strength of our study is that we were able to obtain information on the attitudes to, motives for and barriers to participation in gut microbiome research of 780 patients with different disorders and healthy volunteers, which has not been assessed to date. We obtained enough information to formulate the following conclusions and recommendations for both gut microbiome researchers and clinicians.”

Q.2.2: Minor comments as below: In introduction, microbiota is a target for fecal microbiota transplantation too. This is very important. Please add it in the sentence.

R.2.2: We agree with the reviewer. The other reviewer has also addressed this issue (Q.1.1). We have now added the prospect of fecal transplantation in the introduction.

Page 3, lines 9-12:

“Moreover, as clinical interest grows in the use of fecal microbiota transplantation (FMT) for dysbiosis-related disorders such as recurrent Clostridium difficile infection (CDI) and IBD, so will the need for voluntary donors.”

Q.2.3: Methods - There were nearly 40 questions in your questionnaire. Why do you only calculate the five questions which you mentioned?

R.2.3: For all factors assessed by the questionnaire (38 questions), we calculated the average (SD) and counts (%), respectively (S1 Table). This way, an overview of the participants’ answers on all the assessed factors was displayed, including views on the participants’ home situation, whether they felt free to refuse participation, their intrinsic motivation to collect feces, and logistic aspects such as type of toilet, freezer and time they had to wait until the sample was picked up.

After presenting these descriptive statistics of the participants’ perspectives on the fecal sample collection, we wanted to answer a limited number of specific questions that we think would help microbiome researchers conduct better studies, rather than perform statistical analyses comparing every question to every factor in order to find any statistically significant differences.

Q.2.4: In table 1, the number of patients in “Maximum time patients want to store fecal samples in their freezer” should subtract 73 as shown in “Unpleasant to store fecal samples in home freezer?”. Seventy-three patients are unpleasant to store fecal samples in home freezer. So they are not necessary to consider the time to store fecal sample.

R.2.4: We understand the remark of the reviewer and have subtracted these 73 participants from this equation in the results, Table 1 (page 10) and Supplementary Table 1.

Page 8, line 9 – 11:

“However, while most patients were willing to store a stool sample in their freezer, many were only willing to do so for a brief period of time: maximum 1 to 3 days (14.6%), 1 week (25.3%), or 2 to 4 weeks (11.1%). 40.9% said that they did not mind storing faecal samples for a longer time.”

Q.2.5: Results - 97.4% had collected a faecal sample for prior gut microbiome research projects. Is this data for question eight in the Supplementary Table 1? An error?

R.2.5: Indeed, 97.4% had collected a faecal sample for gut microbiome research before (summing up answers A to D in question 8 “Did you ever participate in research for which you had to collect and deep-freeze a small stool sample?”: A) yes, 1 time + B) yes, 2-5 times + C) yes, 6-10 times + D) yes, >10 times). Only in the IBD cohort we were also able to send questionnaires to patients who previously refused to participate in gut microbiome studies, of which 3 patients responded (IBD-Unwilling, n=3). The other cohorts (melanoma, Sjögren’s syndrome, SLE) were comprised of gut microbiome research participants. We agree that this needs a better explanation in the methods section.

Page 4, lines 17-30:

A questionnaire (S1 Table) was sent in January 2017 to 772 patients who had previously been recruited at the University Medical Center Groningen in the Netherland for gut microbiome studies for which they needed to provide a faecal sample. These patients had been included in four disease-specific cohorts: IBD (n=660), melanoma (n=9), Sjögren’s syndrome (n=55) and systemic lupus erythematosus (SLE) (n=48) (Fig 1). The latter three cohorts only comprised participants who joined the gut microbiome studies. The questionnaire was aimed at obtaining their experiences and identifying barriers encountered during the collection process. With the IBD cohort, we were also able to send out questionnaires to patients who previously refused to participate in gut microbiome research. Questionnaire recipients in the IBD cohort therefore comprised both patients previously willing to collect a stool sample for research (n=577, IBD-Willing) and patients previously not willing to do so (n=83, IBD-Unwilling), indicating a willingness rate of 87.4% of the IBD microbiome study prior to this survey.”

We also asked if patients were willing to participate again after their experiences.

Page 11, lines 10-14:

“Our study has demonstrated that stool sample collection for gut microbiome studies and future clinical applications is acceptable to the majority of IBD patients and even to population controls. Most IBD patients (87.4%) were willing to participate in our previous stool sample collection (IBD-Willing, n = 577) and most respondents (82.9%) and interviewees (95.6%) indicated that they were willing to participate again.”

Q.2.6: Results - According to your definition in the Supplementary Method, the patients with gastrointestinal disease includes “IBD willing + IBD unwilling + no identification number” participants. You mentioned in the results that 250 patients had GI-disorder. This may be a statistical error. In addition, this is a little different from the data in the questionnaire. Because you asked participants in question eight if they had been diagnosed with intestinal disease, and only 42 patients answered no.

R.2.6: We thank the reviewer for his remark. This calculation was indeed performed based on the participants belonging to the IBD cohort. The reviewer is referring to the answers to question 6 in the questionnaire (S1 Table), where we asked the participants specifically about the presence of any intestinal diseases. Indeed, this data is different from the definition by cohort (IBD cohort vs. No IBD cohort). We have now also performed the analysis using question 6 to define presence or absence of intestinal disease: There was no correlation between GI-disease (Q6) and willing to participate in future faecal sample collection (p = 0.564, Table 1, page 9).

Q2.7: Limitation should better not be included in conclusion. Please put it to the discussion.

R2.7: The limitations now appear at the end of the discussion on page 16, lines 1-13.

Q2.8: Conclusions include too much content. Please try to use summative words in this part and put the current content to suitable part.

R.2.8: The conclusion has been adapted. Page 25, lines 7-21:

“Targeting the gut microbiome will soon be part of the diagnostic process and treatment of IBD and other diseases that are associated with microbial dysbiosis [5,6,31,32] requiring repeated sampling from patients.[33] Here, we assessed the perspectives of patients and healthy volunteers on faecal sampling for gut microbiome research. We derive the following recommendations for gut microbiome researchers and clinicians:

(1) Gut microbiome researchers setting up new cohorts or clinicians trying new faecal tests should not shy away from doing so.

(2) Gut microbiome researchers and clinicians should explain why their collection protocol was designed in a specific way.

(3) In studies where a time-series of many stool samples needs to be collected, researchers should consider providing participants with a small freezer.

(4) Researchers and clinicians should inform participating patients and healthy volunteers about the outcome of the research.”

Q.2.9: The structure of the manuscript is unconventional. Please refer the regular journal style.

R.2.8: We have changed the structure of our manuscript accordingly.

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PLoS One. doi: 10.1371/journal.pone.0249405.r003

Decision Letter 1

Nikhil Pai

18 Mar 2021

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

PONE-D-20-24145R1

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This study highlighted the difficulty for achieving stool sample for microbial study. The “dirty” is one of the important factors affecting the attitudes to response the survey and collect stool sample. This kind of study focus on stool should be put on the better position for more attention.

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PLoS One. doi: 10.1371/journal.pone.0249405.r004

Acceptance letter

Nikhil Pai

31 Mar 2021

PONE-D-20-24145R1

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

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Supplementary Materials

S1 Table. Patient questionnaire outcome.

(XLSX)

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S2 Table. Interview outcome.

Translation of the interview and descriptive statistics for each question.

(XLSX)

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S1 File. Supplementary methods.

Additional information on statistical analyses and results of the interview conducted in the general population cohort.

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Data Availability Statement

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PERMALINK

Patient attitudes towards faecal sampling for gut microbiome studies and clinical care reveal positive engagement and room for improvement

Laura A Bolte

Marjolein A Y Klaassen

Valerie Collij

Arnau Vich Vila

Jingyuan Fu

Taco A van der Meulen

Jacco J de Haan

Gerbrig J Versteegen

Aafje Dotinga

Alexandra Zhernakova

Cisca Wijmenga

Rinse K Weersma

Floris Imhann

Roles

Abstract

Introduction

Methods

Cohorts and participants

Fig 1. Cohort selection and responses.

Ethics approval and consent to participate

Questionnaire design and processing

Statistical analyses

Table 1. Patient willingness to collect and freeze faecal samples and associated factors.

Results

Discussion

Gut microbiome researchers setting up new cohorts or clinicians trying new faecal tests should not shy away from doing so and should focus on providing adequate subject information

Researchers and clinicians should inform participating patients and healthy volunteers about the outcome of the research

An emphasis on the public benefit of the research could help with establishing large cohorts for microbiome research

In studies where a time-series of many stool samples needs to be collected, researchers should consider providing participants with a small freezer

Collection process perceived as easy

Gut microbiome researchers and clinicians should explain why their collection protocol was designed in a specific way

Household composition did not affect willingness to collect and store a faecal sample

Strengths and limitations

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nikhil Pai

Roles

Author response to Decision Letter 0

Decision Letter 1

Nikhil Pai

Roles

Acceptance letter

Nikhil Pai

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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