Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: Study protocol.

BACKGROUND: The performance of the microbiota is observed in several digestive tract diseases. Therefore, reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, tests and therapies in hepatobiliopancreatic diseases.
METHODS: Bile samples will be collected in endoscopic retrograde cholangiopancreatography patients (case group) and living liver transplantation donors (control group). We will characterize the microbiome based on two types of sequence data: the V3/V4 regions of the 16S ribosomal RNA (rRNA) gene and total shotgun DNA. For 16S sequencing data a standard 16S processing pipeline based on the Amplicon Sequence Variant concept and the qiime2 software package will be employed; for shotgun data, for each sample we will assemble the reads and obtain and analyze metagenome-assembled genomes.
RESULTS: The primary expected results of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. In addition, it seeks to demonstrate the existence of changes in the case of illness and also possible disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases. TRIAL REGISTRATION: NCT04391426. Registered 18 May 2020, https://clinicaltrials.gov/ct2/show/NCT04391426.

Introduction

Microbioma is the set of microorganisms that occurs naturally in a particular site, such as the human gastrointestinal tract. Typically, it has trillions of microbes, including fungi, viruses and bacteria [1], which coexist with human cells.

Normally, the microbiome bacteria interact with the epithelial barrier, with immune cells modulating their response, in addition to influencing local metabolism through their own metabolites. This maintains homeostasis [2]. Thus, an imbalance of the microbiota, such as the use of antibiotics or due to bacterial translocation, can lead to the development of diseases. It does this through the proliferation of pathogenic bacteria, for example, which can greatly affect the host and have potential pathological implications [2-4].

Studies have shown a close relation between dysbiosis and the outbreak of infections or chronic diseases. In 2019, Saus et al. [5] gathered data about the relation between the intestinal microbiota and the development of colorectal carcinoma (CRC), the most studied since the 1990s. Currently, it is known that in patients with this neoplasm there is a co-abundance of pro-inflammatory factors, opportunistic pathogens and other microbes. This is associated with metabolic dysfunction and the depletion of butyrate-producing bacteria, an important factor in intestinal homeostasis. With these studies, interest was raised in investigating other sites, such as the biliopancreatic tract (BPT). In 2015, Mitsuhashi et al. showed the association of the oral microbiota with the pancreatic carcinogenesis process. In periodontal diseases, Fusobacterium can be translocated via lympho-hematogenous pathways, leading to pancreatic dysbiosis. This would be associated with malignancy in the progression of pancreatic adenocarcinoma and worse prognosis [4].

Thus, research was conducted in rats, finding an association between the components of the tumour microbiota and the speed of progression of biliopancreatic disease [6]. In pancreases of rats and humans with pancreatic duct adenocarcinoma (PDAC), a greater abundance of Malassezia spp. was found compared to bowel or pancreas controls without the disease. Due to the presence of the fungus, there is greater activation of mannose-binding lectin (MBL) and, consequently, the complement cascade is activated, leading to greater inflammation in the pancreas, which accelerates tumour progression [6, 7].

Traditionally, BPT neoplasia are diagnosed at an advanced stage, despite the improvement in the quality of diagnostic imaging. For early diagnosis, Mendez et al. conducted an experiment with PDAC-mutated mice before they developed the disease. By DNA sequencing of the fecal microbiota bacteria, it was found that, with the progression of pancreatic carcinogenesis, there was a change in the bacterial composition. The metabolites of these bacteria associated with the tumour promote greater production of polyamines, which increases as the neoplasia develops. Then, the dosage of polyamines could be used as a biomarker to track the progression of adenocarcinoma [8]. The analysis of the fecal microbiota, therefore, would be a possibility for early diagnosis of PDAC, prompting research in human patients at high risk for carcinogenesis.

Among the therapies, the most commonly used treatments for neoplasia are chemotherapy and radiotherapy. In BPT carcinomas, however, these methods have low sensitivity [9], which can be attributed, according to studies, to tumor dysbiosis. As previously mentioned, unchanged symbiotic microbiota mediates the immune response. Thus its imbalance decreases the expression of genes related to inflammation, phagocytosis, antigen presentation and adaptive immune response. On the other hand, genes related to tissue development, cancer and metabolism are stimulated [10]. Thus, the use of chemotherapeutic drugs loses effectiveness due to this negative regulation of the immune system's anti-tumor capacity. An example of this is oxaliplatin, whose effect is to stimulate the production of reactive oxygen species to promote DNA damage and tumor cell apoptosis. In mice injected with colon carcinoma cells, their cytotoxic effect decreased after being treated with antibiotics. [9, 10].

There have been several advances in the introduction of new chemical compounds that interfere in specific signalling pathways of carcinogenesis, also affected by the microbiome. Because of this association, microbial agents and their metabolites are being tested to develop treatments that can reduce the tumor and are potentially preventive [11]. This was observed in studies with species of Lactobacillus, which modulates the expression of some enzymes such as beta-glucuronidase. The action of the enzyme is reduced by bacilli. This acts in the disjunction of carcinogenic agents, converting pro-carcinogens into their active form [12]. Lenoir et al. also demonstrated that L.casei has anti-tumor properties by decreasing the T-reg response and increasing Th17, promoting a decrease in CRC in rats. Thus, the microbe proved to be a protector and a new therapeutic alternative to carcinoma [11, 13].

Even with current treatments, mortality in some groups of BPT malignancies remains high, with low survival rates. However, in recent studies in patients with pancreatic duct adenocarcinoma, it was found that the greater variety of the tumor microbiota and the predominance of specific bacterial genera are related to a longer survival time when treated surgically [6]. Considering this recent progress, improvement is expected for the coming decades.

Thus, the performance of the microbiota is observed in all clinical and pathological stages of carcinogenesis, from its development, diagnosis and treatment, including prognosis and survival. However, there is a lack of studies on biliary microbiota and its relation with hepatobiliopancreatic diseases. Therefore, further investigation is necessary, since researching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, interventions and therapies in hepatobiliopancreatic diseases.

Aim

In this study, our aim will be to characterize the specific composition of the biliary microbiota in patients with hepatobiliopancreatic diseases compared to healthy controls, using 16S ribosomal RNA (rRNA) pyrosequencing methods.

Materials and methods

Medical costs

Medical costs or other cash payments to donors and families will no be offered and participation will be voluntary. All procedures will be performed in the public health system, at Hospital das Clínicas University of Sao Paulo School of Medicine (HCFMUSP), entirely free of charge.

Study design and patients

This is a case-control study that will be carried out at the Department of Gastroenterology of Clinicas Hospital in the Faculty of Medicine of the University of São Paulo.

Patients who will undergo endoscopic retrograde cholangiopancreatography (ERCP) and donors previously selected to interventional liver transplantation will be recruited for the collection of bile, configuring case and control groups, respectively. The project outline is illustrated in Fig 1.

Fig 1

Study scheme.

External validation

All patients who will not be included, with a condition for which a procedure is planned, such as ERCP or hepatectomy for liver transplantation, will have their records noted and stored, including information on date, sex, age, Body Mass Index (BMI) and the reason for non-inclusion or exclusion.

Patients over 18 years old

Patients previously scheduled for ERCP

Cannulation of the bile duct, via the transpapillary route, with the aid of a papillotome with an end kept sterile until contact with the papilla

Use of antibiotics during ERCP or in the last 2 months prior to the procedure

Emergency ERCP

Pregnancy

Uncorrected coagulopathy

Patients who will undergo ERCP (case group)

Exclusion criteria

History of previous ERCP will not be considered an exclusion criteria.

ERCP technical description

The procedure will be performed under conscious sedation or general anesthesia, at the discretion of the medical team responsible for carrying out the procedure.

Before the beginning of ERCP, the end of the papillotome will be covered with sterile surgical plastic (plastic cover for videolaparoscopy), in order to avoid its contamination during passage through the working channel of the duodenoscope and contact with the digestive tract, up to the greater duodenal papilla.

The duodenoscope will be introduced according to the usual technique and positioned in front of the greater duodenal papilla. The papillotome with a sterile end will then be passed through the working channel of the duodenoscope. Before attempting cannulation of the papilla, air will be injected through the papillotome injection channel to remove the sterile plastic from its end.

When performing cannulation of the bile duct, with the aid of endoscopic and radioscopic vision, the route of the papillotome injection until bile return will be aspirated with a sterile 5 ml syringe, to confirm the correct positioning of the instruments in the bile duct. Then, 1 ml of bile will be aspirated, which will be sent for analysis of the microbiota.

If there is contamination of the papillotome prior to contact with the papilla, the patient will be excluded from the protocol.

Living liver transplantation donors (control group)

The correct selection of donors for living donor liver transplantation is essential not only to decrease the risk of complications for donors, but also to increase graft and recipient survival.

First, there must be ABO blood type compatibility. Then, the compatibility between the weight and height of the patient and the donor is analysed. Subsequently, the size of the liver to be donated is evaluated, and it is necessary to calculate the relation between the weight of the liver (donor) with the recipient. Finally, the entire anatomy of the donor and recipient is evaluated, such as veins, arteries and bile ducts.

Patients over 18 years old up to 55 years old

Previously selected patients with scheduled surgery

BMI: 18 kg/m² to 28 kg/m²

Blood typing identical to the recipient

Absence of significant medical, psychiatric problems or previous abdominal surgery

Normal laboratory tests: liver function tests, blood count, coagulogram, pregnancy test and serology for hepatitis B, C and HIV

Normal imaging exams: CT of the abdomen and pelvis with liver volume (remaining volume—30–40% of the total liver volume), MRI with cholangioresonance

Use of antibiotics in the last 2 months prior to the procedure

Pregnancy

Uncorrected coagulopathy

è Description of the interventional liver transplantation technique

The hepatectomy procedure for related living donor liver transplantation, left lobe or right lobe will be performed under general anesthesia.

Started by a Makuuchi incision (“J” incision), followed by positioning the retractor and cavity inventory, and dissecting the cystic artery and cystic duct.

The cystic duct will be opened and its catheterization and aspiration of 1 ml of bile will be carried out, which will be sent for analysis of the microbiota.

Subsequently, intraoperative cholangiography will be performed to study the intra and extrahepatic biliary anatomy, followed by anterograde cystic-funicular cholecystectomy and left or right hepatectomy as clinical indication.

Ethics approval and consent to participate

The study protocol was approved by the Hospital Ethics Committee (Faculty of Medicine, University of São Paulo—FMUSP—CAAE: 29547920.9.0000.0068) and informed consent will be obtained from all individual participants included in the study. The study was registered at ClincalTrials.gov (Identifier: NCT04391426).

None of the transplant donors will be from a vulnerable population and all donors or next of kin will have provided written informed consent that was freely given.

The participant will discuss with Dr. Alberto Meyer or a member of his team about the decision to participate in this study. In this conversation, the purposes of the study will be explained as well as the procedures to be performed, their discomforts and risks, the guarantees of confidentiality and permanent clarifications. It will also be clarified that the participation is free of charge, nor will there be any payments, and that the participant will have guaranteed access to hospital treatment when necessary. The participants will voluntarily agree to participate in this study and will be able to withdraw consent at any time, before or during it, without penalty or loss of any benefit that he may have acquired, or in his service at HCFMUSP. They will sign a consent form and receive a copy initiated by the researcher.

Processing of samples

Sample collections will be sent to the Molecular Biology Sector of the Central Laboratory Division (LIM-03), which will be responsible for DNA extraction, construction of 16S libraries and sequencing of microbiomes.

DNA extraction

DNA extraction will be performed from 1 ml of the content of the e-swab, using the DNA Kit Zymobiomics MiniPrep, according to the manufacturer's recommendations. The DNA will be stored immediately at -20°C until use.

Sequencing of microbiomes

The microbiomes will be studied based on two types of sequencing data. The first is the V3/V4 region of the 16S unit of the ribosomal gene; the other is total (shotgun) DNA. In the case of 16S, we will use the 16S Metagenomic Sequencing Library protocol (Illumina, San Diego, USA). In the case of total DNA, Genomic DNA libraries will be built using Nextera® XT DNA Sample Preparation Kit (Illumina) from an input of 1 μg of DNA, according to the manufacturer's recommendations. The 16S and genomic DNA libraries will be sequenced in the MiSeq sequencer (Illumina, San Diego, USA) (or some more modern Illumina sequencer at the time samples become available), using the MiSeq® Reagent Kit v3 (600 cycles; Illumina, San Diego, USA).

Data analysis

The data analyses will be performed at the Bioinformatics Laboratory (Setulab), located in the Biochemistry Department, at the Chemistry Institute of the University of São Paulo.

16S data. Sequencing the 16S amplicons (bacteria) from the different samples will result in sets of reads. The data will be sent to Setulab servers, where the analyses will be carried out, which will mainly follow the steps and programs available in the QIIME 2 package [14]. Briefly, the steps include quality control, to remove short reads or reads with poor average quality; determination of amplicon sequence variants (ASVs) using the deblur [15] and / or DADA2 [16] programs; taxonomic classification of ASVs; alpha and beta diversity analyses; rarefaction analysis; and associated statistical analyses, seeking to show differences in the microbial composition between control and case samples. We will also determine microbial taxa associated with the main differences using ANCOM [17].

Statistical tests. Wilcoxon tests will be used to compare mean differences between case and control samples for phylum, genus and ASV log abundances. Kruskal-Wallis tests will be performed to compare differences in the means between both groups for alpha diversity. We will use PERMANOVA and ANOSIM [18] to compare beta-diversity differences between groups using three distance metrics: weighted UniFrac, unweighted UniFrac and Bray-Curtis.

Total DNA data. these analyses will be carried out according to the following steps:

Quality control for the removal of short reads or average quality below a threshold.

Quality control to separate DNA from the microbiota from any human DNA that may have been sequenced.

Classification of reads using kraken2 [19].

Use of the metaWrap pipeline [20] to recover genomes (generation of Metagenome-Assembled Genomes, or MAGs). The application will be separated by sample, and the results will be compared later.

Classification of MAGs using the GTDB-tk program [21].

Annotation of MAGs by the NCBI PGAP pipeline [22].

Evaluation of the representativeness of MAGs in the samples by comparison of taxonomic classification of MAGs with those of reads (step 3).

The results obtained will also be compared with results from the literature, in particular through the eHOMD website [23].

Data management

Once this study is completed, the biological material will receive a code and will be filed in the biorepository anonymously, and may be used for other academic studies, without commercial purpose, as long as approved by the Research Ethics Committee, in accordance with the guidelines of the national body that coordinates the principles of research in our country, the National Commission for Ethics in Research.

Safety and risks regarding adverse events

Bile collection, both in the case group and in the control group, carries minimal risk and discomfort. However, serious adverse events related to examination (ERCP) and / or hepatectomy surgery will be documented on a form throughout the course of the study and will be reported to the principal investigator within 24 hours of observation. If the event is considered to be related to the collection of bile by the principal investigator, he will send a report to the local ethics committee within 3 days.

Timetable

The research is estimated to last twenty-four months, according to the following schedule in Table 1.

Table 1

Research schedule.

Month	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24
Bibliographic Survey	X	X	X	X	X	X
Sample collection				X	X	X	X	X	X	X	X	X	X	X	X	X	X
Sample processing								X	X	X	X	X	X	X	X	X	X
Data tabulation															X	X	X	X
Results analysis																		X	X	X	X	X
Completion of work																							X	X

Statistical analysis

All data will be presented as average and standard deviation. Student’s t-test will be performed with SPSS version 20 for Windows and the Mann-Whitney test will be performed using R software and Python scripts. These tests will be used for comparison, as appropriate. A PCA signal will be developed as discriminative analysis.

The sample size calculation, according to the population under investigation, was estimated at 40, with 20 from each group, based on the exposure ratio between cases and controls. In the ratio of 1: 1, with an effect size of 0.98, type I error of 5% and power of 80%. The calculation was based on the distribution of relative abundance between cases and controls, based on the non-parametric Mann-Whitney two-tailed test [24, 25].

For the final calculation, confounding variables (gender, age and body mass index (BMI)) will be considered, as these can influence the variation of the microbiome and, consequently, in its analysis [26].

Result parameters

The primary purpose of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. For this, the microbiota of patients with hepatobiliopancreatic diseases will be compared with that of healthy controls. In addition, it seeks to demonstrate the existence of changes in the case of illness.

Further investigation includes disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases.

Dissemination policy

MICROBILIO results should be presented at international medical conferences on corresponding areas of interest, for example, gastroenterology. Written publications will be submitted to surgical or endoscopic scientific journals. The authorship of written publications must be confirmed unequivocally by all leading researchers.

Protocol version

This manuscript refers to the first version of the complete study protocol, made on June 4th, 2020. Modifications to the protocol will be reported to all investigators, the local Research Ethics Committee, all participants and the journal.

Discussion

The composition and role of the microbiota of the gastrointestinal tract is an increasing focus of study. There is even evidence that these microbes are related to cancer and other chronic diseases [5]. Biliary dysbiosis, for example, has been shown to be a protagonist in the pathogenesis of several diseases in this tract, as has been recently described. However, given the proximity and connection between the pancreatic, hepatic and biliary tracts, it may be that the influence of this microbiota is more extensive, reaching the entire hepatobiliopancreatic tract [27].

On the other hand, the bile microbiota is still little studied, being sequenced in healthy individuals only recently [24]. Little is known about its composition in malignant diseases [28], with biliary lithiasis being the most studied and best characterized disease [24, 29].

This study aims to consolidate existing knowledge and deepen it with additional information. Thus, we seek to broaden the panorama about the different compositions of the biliary microbiota and, possibly, to shed light on a new form of study for hepatobiliopancreatic diseases, as well as strategies on how to deal with such conditions. In the future, more longitudinal studies will be necessary, since those may reveal time-varying compositional changes in the microbiome, both in hepatobiliopancreatic diseases and healthy people.

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6 Oct 2020

PONE-D-20-22918

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: study protocol

PLOS ONE

Dear Dr. Alberto Meyer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.  The Reviewers found merit in your study and we encourage resubmisison.

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When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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2. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

3.We note that your study will involve tissue/organ transplantation. Please provide the following information regarding tissue/organ donors for transplantation cases analyzed in your study. Even if your study will be retrospective or focused on transplant recipient outcomes, the requested details about the donors are needed to clarify whether the procedures will meet international standards for tissue/organ transplantation.

1. Please state in your response letter and ethics statement whether the transplant cases for this study will involve any vulnerable populations; for example, tissue/organs from prisoners, subjects with reduced mental capacity due to illness or age, or minors.

- If a vulnerable population will be used, please describe the population, justify the decision to use tissue/organ donations from this group, and clearly describe what measures were taken in the informed consent procedure to assure protection of the vulnerable group and avoid coercion.

- If a vulnerable population will not used, please state in your ethics statement, “None of the transplant donors will be from a vulnerable population and all donors or next of kin will have provided written informed consent that was freely given.”

2. In the Methods, please provide detailed information about the procedure by which informed consent will be obtained from organ/tissue donors or their next of kin. In addition, please provide a blank example of the form used to obtain consent from donors, and an English translation if the original is in a different language.

3. Please discuss whether medical costs will be covered or other cash payments will be provided to the family of the donor. If so, please specify the value of this support (in local currency and equivalent to U.S. dollars).

Thank you for your attention to these requests.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

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Reviewer #1: Partly

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This is a "Registered Report Protocol", where the investigative team plans to conduct an observational, case-control study to understand the composition of the biliary microbiota in patients with hepatobiliopancreatic diseases, compared to health controls. The study design is appropriate, given that well-designed observational studies are often found to be effective, compared to RCTs (randomized controlled trials).

I do have some additional comments that may require attention:

1. Sample size/Power: The sample size/power statement should explicitly mention the "name" of the statistical test used, and the "response" that was used to calculate this. The authors' mentioned effect sizes, but never mentioned the name of the test, whether it was one-tailed, or two-tailed, etc. a two-tailed test. Better to be specific (what test?).

2. Writing style: Even before presenting the sample size/power, I found lot of words and sentences used in describing 16S rRNA method, but I never found "what is the response" they will be using? Microbiome data is usually compositional in nature; see article below:

https://www.frontiersin.org/articles/10.3389/fmicb.2017.02224/full

The writeup needs to clearly describe the type of responses to be used, specifically, are they using the compositional data, or some "summary" of the compositional outcomes to conduct the comparison between disease and controls.

There is a pretty robust literature on conducing microbiome data analysis using compositional data, using standard freeware (like R); the authors are suggested to consider that, versus doing some rudimentary/half-hearted analysis. I strongly suggest addition of a qualified statistician/bioinformatician within the research team to produce nice data analysis results, and not some point and click.

3. Abstract/Statistical Analysis Methods: That brings me to the abstract & statistical analysis proposed. I see names of tests, like Student's t, and Man-Whitney tests. Again, it reads weird, when no responses are mentioned corresponding to the proposed tests, i.e., on which quantities those tests will be used.

4. Longitudinal Design: From the current writeup, study doesn't look longitudinal (correct me if I am wrong!). Plans to consider a longitudinal study maybe crucial (can be part of the Discussion/Conclusion Section), given that those may reveal time-varying compositional changes between the two groups.

Reviewer #2: In the current manuscript, Nascimento et al. reported a registered study protocol aiming at evaluating the difference of microbiota in bile samples between healthy people and those with hepatobiliopancreatic diseases. The authors well described the study procedure in human samples and the processing of DNA preparation for sequencing. However, the authors did not well justify several factors including aging and gender which significantly contribute to the difference of microbiota profiles in their statistical analysis. Based on previous studies (Gut microbiota and aging, Science 04 Dec 2015:Vol. 350, Issue 6265, pp. 1214-1215; Intestinal Microbiota Is Influenced by Gender and Body Mass Index, PLoS One. 2016 May 26;11(5):e0154090.doi: 10.1371/journal.pone.0154090. eCollection 2016.), the proposed statistical analysis which did not consider age, body mass index, and gender as variance factors may not obtain significant outcomes.

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29 Oct 2020

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

A: Dear reviewers, the necessary style changes were made, according to the requirements of the journal, following the models presented.

2. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

A: Dear reviewers, the ethics statement was previously on a separate topic, but it has been modified so that it appears in the Methods section, as requested.

3.We note that your study will involve tissue/organ transplantation. Please provide the following information regarding tissue/organ donors for transplantation cases analyzed in your study. Even if your study will be retrospective or focused on transplant recipient outcomes, the requested details about the donors are needed to clarify whether the procedures will meet international standards for tissue/organ transplantation.

1. Please state in your response letter and ethics statement whether the transplant cases for this study will involve any vulnerable populations; for example, tissue/organs from prisoners, subjects with reduced mental capacity due to illness or age, or minors.

- If a vulnerable population will be used, please describe the population, justify the decision to use tissue/organ donations from this group, and clearly describe what measures were taken in the informed consent procedure to assure protection of the vulnerable group and avoid coercion.

- If a vulnerable population will not be used, please state in your ethics statement, “None of the transplant donors will be from a vulnerable population and all donors or next of kin will have provided written informed consent that was freely given.”

A: Dear reviewers, none of the transplant donors will be from a vulnerable population and all donors or next of kin will have provided written informed consent that was freely given, being duly established in the Ethics statement, as requested.

2. In the ethods, please provide detailed information about the procedure by which informed consent will be obtained from organ/tissue donors or their next of kin. In addition, please provide a blank example of the form used to obtain consent from donors, and an English translation if the original is in a different language.

A: Dear reviewers, in Methods, under the topic Ethics approval and consent to participate, it was informed how consent will be obtained from the organ donors. In this submission, the model of the informed consent form was also attached in its original language (Portuguese) and its English translation in Supporting Infomation.

3. Please discuss whether medical costs will be covered or other cash payments will be provided to the family of the donor. If so, please specify the value of this support (in local currency and equivalent to U.S. dollars).

A: As for costs and payments to donors and families, no payment will be offered and participation will be voluntary. In addition, there will be no medical costs, as all procedures will be performed in the public health system, in the teaching hospital Hospital das Clínicas of the Faculty of Medicine of the University of São Paulo (HCFMUSP), entirely free of charge.

6. Review Comments to the Author

1. Sample size/Power: The sample size/power statement should explicitly mention the "name" of the statistical test used, and the "response" that was used to calculate this. The authors' mentioned effect sizes, but never mentioned the name of the test, whether it was one-tailed, or two-tailed, etc. a two-tailed test. Better to be specific (what test?).

A: Thank you, dear reviewers, for your comment, highlighting this mistake. It has already been corrected and added in Methods, in the sub-item Statistical Analysis, the name of the test, and its due characteristics, as requested, better specifying the details, as the reviewer can check.

2. Writing style: Even before presenting the sample size/power, I found lot of words and sentences used in describing 16S rRNA method, but I never found "what is the response" they will be using? Microbiome data is usually compositional in nature; see article below:

https://www.frontiersin.org/articles/10.3389/fmicb.2017.02224/full

The writeup needs to clearly describe the type of responses to be used, specifically, are they using the compositional data, or some "summary" of the compositional outcomes to conduct the comparison between disease and controls.

There is a pretty robust literature on conducting microbiome data analysis using compositional data, using standard freeware (like R); the authors are suggested to consider that, versus doing some rudimentary/half-hearted analysis. I strongly suggest addition of a qualified statistician/bioinformatician within the research team to produce nice data analysis results, and not some point and click.

A: Thank you for your detailed comments and for providing the reference. As explained in the Methods, our raw data will be sequencing reads of two kinds: rRNA 16S and total (shotgun) DNA. As such, as the reference cited says, these data are compositional. All of the methods and statistical tests that we plan to use take this into account. One of the authors of the present study is Dr. J.C. Setubal, who is the team’s bioinformatician.

3. Abstract/Statistical Analysis Methods: That brings me to the abstract & statistical analysis proposed. I see names of tests, like Student's t, and Man-Whitney tests. Again, it reads weird, when no responses are mentioned corresponding to the proposed tests, i.e., on which quantities those tests will be used.

A: We thank the reviewer for pointing this out. Due to an oversight on our part the Methods section of the abstract was garbled. This has been corrected. For the statistical tests that we plan to use, please refer to the Methods section.

4. Longitudinal Design: From the current writeup, study doesn't look longitudinal (correct me if I am wrong!). Plans to consider a longitudinal study maybe crucial (can be part of the Discussion/Conclusion Section), given that those may reveal time-varying compositional changes between the two groups.

Reviewer #2: In the current manuscript, Nascimento et al. reported a registered study protocol aiming at evaluating the difference of microbiota in bile samples between healthy people and those with hepatobiliopancreatic diseases. The authors well described the study procedure in human samples and the processing of DNA preparation for sequencing. However, the authors did not well justify several factors including aging and gender which significantly contribute to the difference of microbiota profiles in their statistical analysis. Based on previous studies (Gut microbiota and aging, Science 04 Dec 2015:Vol. 350, Issue 6265, pp. 1214-1215; Intestinal Microbiota Is Influenced by Gender and Body Mass Index, PLoS One. 2016 May 26;11(5):e0154090.doi: 10.1371/journal.pone.0154090. eCollection 2016.), the proposed statistical analysis which did not consider age, body mass index, and gender as variance factors may not obtain significant outcomes.

A: We appreciate your suggestion. Longitudinal studies would provide significant information regarding the variation of the microbiome composition of both groups over time, therefore, it is crucial to highlight the need to carry out this kind of study to better understand the microbiota of a site still so little known. This recommendation is best described in the Discussion.

Reviewer # 2's suggestion was also of great value to us to improve and enrich this study protocol. In Methods, in the field of statistical analysis, it is described that these possible confounding variables will be considered, which may, in fact, influence the composition of microbiomes, both in cases and controls.

Submitted filename: Response to the Reviewers.docx

Click here for additional data file.

5 Nov 2020

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: study protocol

PONE-D-20-22918R1

Dear Dr. Meyer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors addressed my previous comments to a greater degree of satisfaction. I have no further comments.

Reviewer #2: Thanks for your responses. All my concerns have been well addressed. I look forward to the outcomes of your study.

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Reviewer #1: No

Reviewer #2: No

10 Nov 2020

PONE-D-20-22918R1

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: study protocol

Dear Dr. Meyer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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