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Multi-Donor Fecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure

Future Pharmacol. 2022, 2(1), 55-63; https://doi.org/10.3390/futurepharmacol2010005

by Veronika Řehořová^1,2,†, Ivana Cibulková^2,3,†, Hana Soukupová^2,4 and František Duška^1,2,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Future Pharmacol. 2022, 2(1), 55-63; https://doi.org/10.3390/futurepharmacol2010005

Submission received: 22 December 2021 / Revised: 20 February 2022 / Accepted: 28 February 2022 / Published: 4 March 2022

Round 1

Reviewer 1 Report

Manuscript ID futurepharmacol-1545099

Title: Multi-donor Fecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure

Journal: Future Pharmacology

Authors: Řehořová et al

The manuscript describes a standardized operating procedure of fecal microbial transplantation for critically ill patients prepared by a multidisciplinary team of intensivists, gastroenterologists, and microbiologists, and based on based on feedback from State Institute for Drug Control in the Czech Republic. The above manuscript is very interesting. It should be noted, however, that the manuscript presents some errors and shortcomings. Deficiencies and errors presented below should be corrected before the manuscript could be accepted for publication.

List of deficiencies and errors:

The authors should use the same type of English spelling throughout the manuscript, with the American spelling preferred. In the title, the authors used form “fecal”, in the remaining parts of the manuscript the form “faecal” was used. This should be unified. There are also some minor typing errors, for example: Institue” in line 24.
Line 24. The authors used a form “State Institue of Drug Control”; whereas in line 118 they used a form “the State Institute for Drug Control”. Both forms should be the same.
Section “Dysbiosis and Diarrhoea in Intensive Care Patients”, lines 62-63. The authors stated that ‘The main risk factors of diarrhea in ICU patients are antibiotics therapy, enteral nutrition[23]”. The authors should write some write a few sentences about the relationship between enteral feeding and the possibility of developing diarrhea and complication in critically ill patients. In theory, enteral nutrition should be of benefit. Under normal condition, food introduced into the digestive system provides nutrients to the body and stimulates the renewal of the mucosa in the digestive tract by acting on it directly, as well as by releasing gastrointestinal hormones. Hormones such as gastrin (PMID: 7589156), cholecystokinin (PMID: 11787760) and ghrelin (PMID:19439811; PMID: 24622834; PMID: 26769837) protect the mucosa against damage in the gastrointestinal tract and accelerate its healing., which improves the mucosal barrier and prevents uncontrolled penetration of factors from the lumen of gastrointestinal tract into the body. On the other hand, the lack of enteral feeding or administration of antagonists for gastrin of cholecystokinin lead to the atrophy of the mucosa and organs of gastrointestinal system (PMID: 1665365; PMID: 1714408). However, in critically ill patients, especially those with injures of central nervous system, the protective mechanisms of enteral feeding are disrupted. In critically ill patients, circulation is centralized. To ensure sufficient blood flow to vital organs (the heart and brain), in other organs, including the gut, peripheral vascular resistance increases and blood flow in these organs decreases. The digestive system is very sensitive to reduction in organ blood flow and hypoxia. Damage of the mucosa in the stomach and other parts of gastrointestinal tract is associated with a decrease in mucosal blood flow whereas the protection and healing are associated with an increase in mucosal blood flow (PMID: 15680274; PMID: 22534700; PMID: 26769837). In addition, small bowel hypoxia leads to gut barrier failure associated with bacterial translocation, systemic inflammation and the development of multiple organ dysfunction syndrome (PMID: 26808138). These mechanisms explain why, in the study quoted by the authors (23), enteral feeding worsened the condition of critically ill patients with traumatic brain injury. In addition, it indicates that the rectal route of fecal microbial transplantation is probably associated with a lower risk of adverse events.
Line 107 The abbreviation “MDRO”. All abbreviations should be presented in their full name in the place where they appear for the first time. In addition, the link to reference 43 is not active.
Lines 118-121. The authors stated that “For example, the State Institute for Drug Control (SUKL) of the Czech Republic decided in 2019 to consider FMT transplant a drug, and decided to apply the same principles of regulation and oversight as is in place for drug trials”. This statement does not explain where the acronym SUKL came from. The authors should consider replacing this statement with the following sentence: “For example, the State Institute for Drug Control (Statni ustav pro kontrolu leciv - SUKL) of the Czech Republic decided in 2019 to consider FMT transplant a drug, and decided to apply the same principles of regulation and oversight as is in place for drug trials”.
Line 24-25. The authors should replace “authority of one European Union country (SUKL)” with authority of the Czech Republic, SUKL”.
Line 127. Correct “clin ical”.
Line 135. Correct 50 mlL.
Line 135. The authors have written about seven 50 mL aliquots from seven donors. What was the original stool mass in each 50 mL aliquot? This information should be provided in the manuscript.
Line 136-138. The authors stated that “In addition to standard guidelines-driven precautions[49] in donors to prevent transmissible infection, the deep-frozen transplant mixed with glycerol is quarantined for at least 2 months before use.” First, authors should use the current guidelines (PMID: 31563878; PMID:33151137). Secondly, they should state in the manuscript, in accordance with cited guidelines, what criteria must be applied for the qualification of stool donors, as well as for serological and stool tests.

Author Response

Editor-in-Chief

Future Pharmacology

Re: Revision of paper Rehorova et al. (futurepharmacol-1545099R1)

Dear Editor,

We thank both reviewers for their insightful comments to paper Rehorova et al. Multi-donor Fecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure.

In the letter below, we explain step-by-step the changes we made to the manuscript. We sincerely hope the revised form will now be suitable for publication in Future Pharmacology.

All the best

Frantisek Duska – on behalf of the authors

Detailed response to reviewers’ comments

Reviewer #1

List of deficiencies and errors:

The authors should use the same type of English spelling throughout the manuscript, with the American spelling preferred. In the title, the authors used form “fecal”, in the remaining parts of the manuscript the form “faecal” was used. This should be unified. There are also some minor typing errors, for example: Institue” in line 24.

Answer/Changes made to the manuscript: This has ´been corrected and American spelling used throughout.

Line 24. The authors used a form “State Institue of Drug Control”; whereas in line 118 they used a form “the State Institute for Drug Control”. Both forms should be the same.

Answer/Changes made to the manuscript: Thanks, we now consistently use the term, which is at institutions website (https://www.sukl.eu/index.php?lang=2)

Section “Dysbiosis and Diarrhoea in Intensive Care Patients”, lines 62-63. The authors stated that ‘The main risk factors of diarrhea in ICU patients are antibiotics therapy, enteral nutrition[23]”. The authors should write some write a few sentences about the relationship between enteral feeding and the possibility of developing diarrhea and complication in critically ill patients. In theory, enteral nutrition should be of benefit. Under normal condition, food introduced into the digestive system provides nutrients to the body and stimulates the renewal of the mucosa in the digestive tract by acting on it directly, as well as by releasing gastrointestinal hormones. Hormones such as gastrin (PMID: 7589156), cholecystokinin (PMID: 11787760) and ghrelin (PMID:19439811; PMID: 24622834; PMID: 26769837) protect the mucosa against damage in the gastrointestinal tract and accelerate its healing., which improves the mucosal barrier and prevents uncontrolled penetration of factors from the lumen of gastrointestinal tract into the body. On the other hand, the lack of enteral feeding or administration of antagonists for gastrin of cholecystokinin lead to the atrophy of the mucosa and organs of gastrointestinal system (PMID: 1665365; PMID: 1714408). However, in critically ill patients, especially those with injures of central nervous system, the protective mechanisms of enteral feeding are disrupted. In critically ill patients, circulation is centralized. To ensure sufficient blood flow to vital organs (the heart and brain), in other organs, including the gut, peripheral vascular resistance increases and blood flow in these organs decreases. The digestive system is very sensitive to reduction in organ blood flow and hypoxia. Damage of the mucosa in the stomach and other parts of gastrointestinal tract is associated with a decrease in mucosal blood flow whereas the protection and healing are associated with an increase in mucosal blood flow (PMID: 15680274; PMID: 22534700; PMID: 26769837). In addition, small bowel hypoxia leads to gut barrier failure associated with bacterial translocation, systemic inflammation and the development of multiple organ dysfunction syndrome (PMID: 26808138). These mechanisms explain why, in the study quoted by the authors (23), enteral feeding worsened the condition of critically ill patients with traumatic brain injury. In addition, it indicates that the rectal route of fecal microbial transplantation is probably associated with a lower risk of adverse events.

Answer/Changes made to the manuscript: We have added a new paragraph which also contains seven suggested new references.

Line 107 The abbreviation “MDRO”. All abbreviations should be presented in their full name in the place where they appear for the first time. In addition, the link to reference 43 is not active.

Answer/Changes made to the manuscript: Thank you for spotting this. We have defined MDRO abbreviation at its first occurrence. We also double-checked and updated reference 43, now 50, and the web link is working well.

Lines 118-121. The authors stated that “For example, the State Institute for Drug Control (SUKL) of the Czech Republic decided in 2019 to consider FMT transplant a drug, and decided to apply the same principles of regulation and oversight as is in place for drug trials”. This statement does not explain where the acronym SUKL came from. The authors should consider replacing this statement with the following sentence: “For example, the State Institute for Drug Control (Statni ustav pro kontrolu leciv - SUKL) of the Czech Republic decided in 2019 to consider FMT transplant a drug, and decided to apply the same principles of regulation and oversight as is in place for drug trials”.

Answer/Changes made to the manuscript: Thank you, we have adopted this suggestion.

Line 24-25. The authors should replace “authority of one European Union country (SUKL)” with authority of the Czech Republic, SUKL”.

Answer/Changes made to the manuscript: Thank you, we have adopted this suggestion.

Line 127. Correct “clin ical”.

Answer/Changes made to the manuscript: Done.

Line 135. Correct 50 mlL.

Answer/Changes made to the manuscript: Done.

Line 135. The authors have written about seven 50 mL aliquots from seven donors. What was the original stool mass in each 50 mL aliquot? This information should be provided in the manuscript.

Answer/Changes made to the manuscript: We use 1:3 weight/weight dilution. Tis has now been added to main manuscript body.

Line 136-138. The authors stated that “In addition to standard guidelines-driven precautions[49] in donors to prevent transmissible infection, the deep-frozen transplant mixed with glycerol is quarantined for at least 2 months before use.” First, authors should use the current guidelines (PMID: 31563878; PMID:33151137). Secondly, they should state in the manuscript, in accordance with cited guidelines, what criteria must be applied for the qualification of stool donors, as well as for serological and stool tests.

Answer/Changes made to the manuscript: Thanks for this: we have replaced ref. 49 (now 56) with the newest 2021 consensus report from a multidisciplinary UEG working group. This has not been published when we first drafted the manuscript. We have also added a reference to table in Supplementary appendix, when all criteria and tests are listed in full.

Reviewer 2

The article “Multi-donor Fecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure” by Veronika Řehořová , Ivana Cibulková , Hana Soukupova , František Duška describes the rationale for this SOP and the process of its development in detail and releaser the full proposed SOP is the form of an online appendix which developed for experimental use in critically ill patients by a multidisciplinary team of intensivists, gastroenterologist and microbiologist based on feedback from regulatory authority (State Institue of Drug Control of the Czech Republic). I suggest need to minor revise and provide more information:

The introduction suggests adding more references to describe gut microbiota associate with metabolism or health disease, such as gut-liver axis, brain-gut axis

Answer/Changes made to the manuscript: We have added suggested text incl. 2 references to gut-liver axis and brain-gut axis

“We recommend the use of normal saline as opposed to sterile water as it provides a better outcome”, some research reports suggest using buffer, can we use the buffer in this system?

Answer/Changes made to the manuscript: We appreciate this interesting suggestion which makes sense from physiological standpoint as hyperchloridemia induced in the transplantate induced by using a solution containing 152 mEq/L of chloride can -in theory – affect the viability of microbial species in the specimen. Yet, we recommend the use of normale saline as this is, to our knowledge, the only solvent that has so far been experimentally tested in this context.

“We have chosen 350 mL volume as a compromise between 500 mL that demonstrated a 97% success rate, and 200 mL successful in 80% of cases”, please provide more information to explain the volume design

Answer/Changes made to the manuscript: Explanation added into the manuscript text.

“We have chosen administration by an enema, despite its lower efficiency, compared to colonoscopy. With appropriate patient positioning, we found graft distribution throughout the colon acceptable, reaching the ascendent colon (Figure 1).” Is there more evidence that such an approach is sufficient to apply?

Answer/Changes made to the manuscript: Unfortunately not at present. Important objective in the upcoming trials will be not only to provide more data on the distribution of the graft in the recipient’s colon, but also the efficacy of donor microbiome engraftment. We have added a comment in the revised paper admitting this important limitation.

The supplement step 3 “The labeled samples are stored at -80 °C for 3 to 12 months”, how to check stored at -80°C can keep to 12 months, please explain.

Answer/Changes made to the manuscript: We agree with the reviewer that experimental data supporting 12 months storage are lacking and there is only 1 Chinese study on clinical outcomes of FMT (PMID: 32594727). We recognise this weakness, SUKL has requested an ex vivo viability study which we are now conducting. This information has been added to the revised manuscript.

We thank all the reviewers for their detailed insightful reviews, which we hope have helped to improve the manuscript.

Reviewer 2 Report

The introduction suggests adding more references to describe gut microbiota associate with metabolism or health disease, such as gut-liver axis, brain-gut axis
“We recommend the use of normal saline as opposed to sterile water as it provides a better outcome”, some research reports suggest using buffer, can we use the buffer in this system?
“We have chosen 350 mL volume as a compromise between 500 mL that demonstrated a 97% success rate, and 200 mL successful in 80% of cases”, please provide more information to explain the volume design
“We have chosen administration by an enema, despite its lower efficiency, compared to colonoscopy. With appropriate patient positioning, we found graft distribution throughout the colon acceptable, reaching the ascendent colon (Figure 1).” Is there more evidence that such an approach is sufficient to apply?
The supplement step 3 “The labeled samples are stored at -80 °C for 3 to 12 months”, how to check stored at -80°C can keep to 12 months, please explain.

Author Response

Please see the reply letter.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Manuscript ID futurepharmacol-1545099 the review 2

Title: Multi-donor Fecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure

Journal: Future Pharmacology

Authors: Řehořová et al

The new version of the manuscript is almost ready for publication. However, there are a few errors that should be corrected. The main problem is the reference list. The authors should check point by point the references comparing them with data shown in PubMed:

Some examples of errors in References:

Reference 1. Due to a large number of the authors, the names of authors are replaced with “Human Microbiome project Consortium”.
Reference 2. Names of authors should be written using capital letters. No page numbers of the article.
Reference 3. No name of the journal, no number of volume, wrong page number.
Reference 4. No name of the journal.

These problems are observed almost in all references. Moreover, there are some problems with references related to in the case of sentences presented in lines 75-82. References 35 should be the article shown in lines 352-353 in the second part of references 34. And the numbering of next references should be shifted by 1.

Line 79. According to American English spelling “ischaemia” should be written as “ischemia”.

Author Response

See enclosed

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In their manuscript, Rehorova et al. described the background regarding the use of fecal microbiota transfer (FMT) in critically ill patients. Moreover, the establishment of standard operational procedures (SOP) within this context is outlined and discussed.

In addition to several points with regards to content, context and style (see below), the overall message of the manuscript is at least partly limited in scope as it reflects a Czech Republic-centered view of the investigated topic. With regards to legislative but also regulative issues, the outlined SOP is not approximately capable to stand respective regulations in other European countries (e.g. Germany). Therefore, the benefit for readers outside of the Czech Republic has to be disputable.

Finally, I do not think this manuscript is capable of being published in its current form.

To improve, please see my specific comments below:

Major points:

English grammar and style: the manuscript has to be revised by an English native speaking person due to relevant concerns and a lot of typos have to be corrected/changed throughout the manuscript
line 46 ff: the authors pointed out a variety of disease entities that have been associated with microbiome alterations => since this listing is far from complete: is there any specific reason behind the entities named? If not, could the authors state these associations more in general? Moreover, the listing needs to be re-organized and grammatically adapted
line 53ff: this conclusion is over-simplified, incomplete and finally wrong (by only focussing on SCFA!) => please adapt
overall, references used seem out-dated => the authors should add more current literature
the relevance of bacteriophages, the virome and mycobiome should be named and discussed in the context of FMT (=> probably relevant effect size for FMT success)
the specific risk of transmitting multi-resistant organisms/bacteria should be named and discussed, separately (=> see also respective FDA-safety warnings: https://www.fda.gov/safety/medical-product-safety-information/fecal-microbiota-transplantation-safety-alert-risk-serious-adverse-events-likely-due-transmission)
line 90: although FMT as procedure in the treatment regimen of rCDI is now widely recommend by multiple society guidelines, the statement that “... the method gradually became the golden standard for treatment of recurrent and refractory Clostridium difficile infection (CDI)” should be attenuated since there is still a lack of long term data regarding overall efficacy but especially potential side effects!
line 93 ff: this listing is also very inconclusive (see above)
line 104/105 => a reference is mandatory!
line 111 ff: besides the legislative status in the Czech Republic, prospective use of FMT is still very limited (=> study could only be performed under good clinical practice, GCP conditions leading to immens regulatory restrictions and costs of such studies). This topic has to be discussed more in detail and especially within a broader context (out of the Czech perspective)
Figure 1: is the admixture of contrast solution part of the SOP => how influences by the contrast solution to the FMT graft have been ruled out and based on which data this has been verified?
line 180 f: effect sizes regarding the FMT application by enema vs. via colonoscopy should be discussed differentially as the latter way of application seems to hold the highest effect size
line 182: the normal small intestine IS NOT STERILE!

Minor points:

line 37: the plural of microbiome (“microbioma”) is uncommon => adapt
line 44: the same is true for “pathobiom”, that should be pathobiomE? If the authors want to use this term then a specific reference should be given for it

Author Response

To:

Editor-in-chief

Biomolecules

Re: Response to reviewers with step-by-step description of changes made to the manuscript Rehorova et al.: Multi-donor Faecal Microbial Transplantation for Critically Ill Patients: Rationale and Standard Operating Procedure

Dear Editor,

Dear Reviewers,

Thank you for insightful review of our above-named manuscript. In this letter, we describe the changes we have made to our paper and explain them. The revised manuscript in “track changes” format is attached as well as the final revised version.

REVIEWER #1

R1Q1: English grammar and style: the manuscript has to be revised by an English native speaking person due to relevant concerns and a lot of typos have to be corrected/changed throughout the manuscript

Answer: The text has been extensively revised as suggested. All corrections can be found in the track-changes version of the manuscript

R1Q2: line 46 ff: the authors pointed out a variety of disease entities that have been associated with microbiome alterations => since this listing is far from complete: is there any specific reason behind the entities named? If not, could the authors state these associations more in general? Moreover, the listing needs to be re-organized and grammatically adapted

Answer: There was a missing bracket in the original sentence. We added more general statement and only list those diseases as example.

R1Q3: line 53ff: this conclusion is over-simplified, incomplete, and finally wrong (by only focussing on SCFA!) => please adapt

Answer: We changed this part of article as suggested.

R1Q4. Overall, references used seem out-dated => the authors should add more current literature

we use more current literature

Answer: We respectfully disagree. Most references are <5 years old and by going step-by-step through older ones, it is difficult to update those without omitting classical landmark paper or using secondary citations (e.g. recent review paper instead of the original paper).

R1Q5: The relevance of bacteriophages, the virome and mycobiome should be named and discussed in the context of FMT (=> probably relevant effect size for FMT success)

Answer: We agree and thank for this comment. We have added a section on FMT efficacy to the text, listing all parts of the microbiome. We added this at later section (line 84).

R1Q6: The specific risk of transmitting multi-resistant organisms/bacteria should be named and discussed, separately (=> see also respective FDA-safety warnings: https://www.fda.gov/safety/medical-product-safety-information/fecal-microbiota-transplantation-safety-alert-risk-serious-adverse-events-likely-due-transmission)

Answer: This specific risk is now mentioned in the revised text incl. the reference to FDA warning.

R1Q7. Line 90: although FMT as procedure in the treatment regimen of rCDI is now widely recommend by multiple society guidelines, the statement that “... the method gradually became the golden standard for treatment of recurrent and refractory Clostridium difficile infection (CDI)” should be attenuated since there is still a lack of long term data regarding overall efficacy but especially potential side effects!

Answer: Agreed. We downtoned the statement from “golden standard” to “well established treatment”.

R1Q8. Line 93 ff: this listing is also very inconclusive (see above) change the text Answer: line 104/105 => a reference is mandatory!

Answer: We added a reference.

R1Q9. Line 111 ff: besides the legislative status in the Czech Republic, prospective use of FMT is still very limited (=> study could only be performed under good clinical practice, GCP conditions leading to immens regulatory restrictions and costs of such studies). This topic has to be discussed more in detail and especially within a broader context (out of the Czech perspective)

Answer: We agree with that and changed this paragraph from very Czech Republic-centered to more general. We added: In collaboration with regulatory authority of one Eeuropean Union country (SUKL) we have developed such an SOPs, which we describe and explain below. Although directly applicable in only one EU country, we believe that it can be useful in its current form or after minor modification in other countries and help to standardize FMT procedure in the trials in the critically ill patients.

R1Q10: Figure 1: is the admixture of contrast solution part of the SOP => how influences by the contrast solution to the FMT graft have been ruled out and based on which data this has been verified?

Answer: Thanks for this point. We recognise that we only have anecdotal data and no robust evidence. We have changed Figure 1 saying: Note: Addition of contrast agent is normally not a part of FMT and it should be noticed that there are no robust data on how FMT spreads after rectal administration. We have also down toned the respective part of the methodology (line 189) saying that we still cannot be sure about the spread after administration via rectal tube.

R1Q11: Line 180 f: effect sizes regarding the FMT application by enema vs. via colonoscopy should be discussed differentially as the latter way of application seems to hold the highest effect size.

Answer: We have revised the text and mention lower effect size.

R1Q12 Line 182: the normal small intestine IS NOT STERILE!

Answer: Words “normally sterile” were deleted from this sentence.

R1Q13: Line 37: the plural of microbiome (“microbioma”) is uncommon => adapt

Answer: We have changed this.

R1Q14 Line 44: the same is true for “pathobiom”, that should be pathobiomE? If the authors want to use this term then a specific reference should be given for it

Answer: The word is not in use in the revised text, only in Ref 31 in the form of “Pathobiome”.

REVIEWER #2

This paper try to describe a standardized operating procedure of multi-donor faecal microbial transplantation in ICU patients with critical dysbiosis. FMT indeed attracted much attention recent years on accout for its therapeutical effect in dysbiosis.

R2Q1: However, the application of FMT in ICU patients needs to be treated with great caution. Since the safety of the FMT procedure is still controversial. In 2020, a case of drug-resistant bacteremia after FMT was reported. The immunity of ICU patients are usually low which may can not endure the FMT operation. There is no evidence that the SOP procedure introduced in this paper is safe for use.

Answer: We think this comment is important. Although we have never stated in the manuscript that the FMT is safe, publishing a “cook book” for it might give exactly that impression. Consequently, we added a sentence: This SOPs aim to make FMT in critically ill patients as safe as possible, but it should be stressed that good quality data on FMT safety in critically ill patients are still not available.

R2Q2: The faecal sample from the donor will be frozen before use in this paper, however, most of the FMT cases reported prefer fresh samples. After frozen, bothe benefical and harmful bacteria in the sample may inactivated.

Answer: We agree although the viability of most microbial species is reduced by freeying-storage-thawing cycle, the FMT still remains highly efficacious. Most trials report a reduction of achieving clinical success of FMT to around 70%. We have chosen frozen samples due to

Safety (Sample can be quarantined until the donor is re-tested for transmittable diseases)
Logistics (Frozen samples are readily available in ICU whenever a critically ill patients needs them)
Standardisation (Use of multi-donor FMT increases the homogeneity of investigational products.

In acute care, searching for and testing the donor is so time consuming, that before the fresh FMT could be delivered it would have been already too late for the patient. Although in theory fresh samples could (and probably will) be more efficient, we see little point to test an intervention, which is not feasible in real life ICU.

R2Q3: There are many spell mistakes in this paper. For example: in line 82, line 95, line 143…

Answer: See also R1Q1. We have extensively revised the text to correct typos and spelling errors.

Q2Q4: In line 90 and 94, the bacteria name of the Clostridium difficile should be italic. And the name of the genus Clostridium has been updated.

Answer: We have changed to italic. Also in lines 154 and 227.

Answer:

REVIEWER #3

R3Q1: Řehořová, Cibulková and colleagues present a standard operating procedure for fecal microbiota transplantation (FMT) in critically ill patients. While this is an accepted modality with several controlled trials showing promising results in the treatment of Clostridioides difficile infections (CDI), there have been important safety sentinel events that the authors do not adequately cite, including deaths directly related to FMT (e.g. PMID 31665575). In describing the SOP to focus on several key features, there are several aspects of the SOP that require additional justification or clarity

Answer: Thank you. This is extremely important point – the two deaths resulting from transmition of MDR Enterobacteriaceae from the donor were now added into the revised manuscript, together with reference to FDA warning against this risk (see also answer to R1Q6).

R3Q2: What are the baseline inclusion/exclusion criteria for the modality in FMT recipients? Are all ICU patients to be considered as potential candidates? I would have ethical concerns about including the following sorts of patients:

Neutropenic patients
Recent GI surgery
Known perforated viscus / abdominal free air
Patients unable to give informed consent

Answer: We agree and we have added points 1-3 among specific contraindications to line 229. With regards inability of informed consent, this would excluded almost all critically ill patients as most of them are receiving sedation to facilitate endotracheal intubation or have other conditions impairing decision making capacity. Therefore, most trials in ICU patients are rely on a surrogate decision maker, such as the next of kin. Consequently, we do not consider the lack of capacity to give consent a contraindication, rather, this aspect should be reviewed with the respective research ethics board for each trial using FMT in ICU patients.

R3Q3: In addition to the above, the authors need to comment explicitly on the principal limitation of FMT in the ICU setting: engraftment. How is an FMT procedure expected to succeed / engraft in the setting of a patient population where wide-spectrum antibiotic administration is the rule, not the exception? What is the external validity of FMT in a population if antibiotic administration becomes an exclusion criterion? If antibiotics become an exclusion criterion, for how long after discontinuation will FMT be excluded given evidence that some antibiotics remain in the gut for days (i.e. neomycin, enteral vancomycin)?

Answer: Again, extremely relevant point and it is our fault that we have not made it clearer in the text that we advise to only use FMT at least 48 hours after stopping antibiotics. Although some antibiotics are detectable as long as 6 days after last administration (https://www.tandfonline.com/doi/pdf/10.3109/08910609209141594), we are more pragmatic using the fact, that the profound diarrhoea (the main indication for FMT) is likely to increase the clearance of antibiotics from the gut. It is then the concentration in the blood, which influences the concentration of antibiotics in intestinal lumen. Most common antibiotics used in intensive care, such as broad-spectrum beta-lactams, aminoglycosides, or iv. glycopeptides have relatively short plasma half lives and therefore 48 hours is well above 5 biological half-lifes and it is also consistent with the practice in non-critically ill patients (https://www.hopkinsmedicine.org/gastroenterology_hepatology/clinical_services/advanced_endoscopy/fecal_transplantation.html).

In the revised manuscript, we have added a paragraph discussing these important aspects.

R3Q4. Why is a semi-rigid rectal irrigation tube necessary, and why do the authors feel that extensive patient positioning is truly necessary to "[distribute] the transplantate well throughout the length of the colon"? A quick review of successful enema administration of FMT for CDI does not show that this was done in several RCTs, only a simple retention enema. What will the approach be in a patient who cannot participate in these positioning manuevers in the context of critical illness?

Answer: Retention enema is a technique dependent on voluntary controls of sphincters, and this is not the case for most critically ill patients, who are under influence of sedatives. Consequently, we used

semi-rigid rectal irrigation tube + positioning with inflated balloon of faecal collection system as less invasive alternative to colonoscopy. Of note, patients’ positioning does not require active participation by patients themselves and is safe to perform if the patient is haemodynamically stable, which is a prerequisite (See paragraph on Safety)

R3Q5: The ID screening outlined in the supplementary procedure is not adequate, particularly given the plan to pool feces from 7 donors in each aliquot. In particular:

HIV screening as outlined does not address the window period problem unless the labs as outlined are collected at baseline and again at the end of the 3 month quarantine period. This is not specified.
Window period infections are also not addressed for the hepatitis virus screening, which does not include NAAT testing or a plan to repeat at any interval.

Answer: The original document was designed to comply with the recommended local requirements for stool donors (https://www.infekce.cz/DPFMT18.htm), which combine serology testing with questionnaires to detect risk behaviour. Nonetheless, but we agree with the Reviewer #3, that rules should be even stricter for multi-donor FMTs. Consequently, we revise the SOP to mandate full serology testing every 2 months during the period of donation. This also better reflect the established practice of “daily donations” of limited group of long term donors.

R3Q6: No donor deferral criteria are provided, nor is the questionnaire given to the donor for risks of infectious diseases. Would the following donors be deferred:

History of travel to India 6 months ago?
History of travel to India 18 months ago but for a total stay duration of 4 months?
A donor with >1 sexual partner in the last 12 months?
Donor with history of IBS?
Patients with an extensive family history of GI malignancies, peptic ulcer disease, or autoimmune disease?

Answer: We now attach full donor refusal criteria in the Supplementary data file, that are compliant with most of the above.

R3Q7: Are donors compensated? If so, what measures are in place to prevent conflicts of interest in assessing the donor deferral criteria?

Answer: Yes, the donors have the inconvenience and lost time compensated a small amount (equivalent EUR 40) for every donation, whilst no compensation is offered for blood test etc. This makes the process still an act of altruism rather than way of earning money for living. The donor cannot work in healthcare or share a household with anyone who does. The donor also have to adhere to some dietary restrictions (see also R3Q18).

R3Q8: Insufficient detail is provided about "laboratory exclusion of the presence of ABX-resistant microorganisms." The specific methodology and organisms screened for need to be specified, but at a minimum I would suggest vancomycin-resistant Enterococcus, extended spectrum beta lactamase Enterobacterales, carbapenem-resistant Enterobacterales, and methicillin-resistant Staphylococcus aureus. The authors should also strongly consider screening/excluding healthy donors colonized with Clostridioides difficile OR specifically specify why they feel including these donors in the pool is expected to be low-risk. Donors also need to be screened for norovirus.

Answer: We thank the reviewer for this comment. In the revised supplementary materials, we provide more details of donor screening, which is fully compliant with recent Czech, European, and British guidelines. In addition to that, we included test for Enteropathogenic E.coli (EPEC,EAEC,ETEC,EIEC),Vibrio cholerae, MDRO (VRE, ESBL,CRE), PCR detection of CMV, Rotavirus and Norovirus, and standard microscopic parasitological exam (to detect Giardinia lamblia, Cryptosporidium parvum, Isospora, Microsporidia, Entamoeba histolytica). Further, we included virological tests via. Faecal occult blood test will be also performed. A nasopharyngeal swab – PCR will be performed to rule out COVID-19 infection

R3Q9: The authors, as most in the FMT field, are exclusively focused on bloodborne pathogen and classical enteric infection risk, but I would encourage mention of a plan to screen for the following infections which are routinely shed in stool, saliva, urine, and tears. This can be done either with direct screening of feces (i.e. CMV and HSV1, HSV-2) or screening of the donor (i.e. anal Pap smears for HPV or rectal swabs for HSV) or serological screening of the donor. Donor serological screening to assess risks for donor-derived infection, particularly in immunosuppressed patients, could also be outlined:

Helicobacter pylori
Herpes simplex virus 1 and 2
Cytomegalovirus (CMV)
Human papillomavirus (HPV)
JC virus

Answer: From above listed pathogens, we have added CMV tests. The donor screening is performed in agreement with Czech, European and British guidelines published up to date. See citations at R3Q13.

R3Q10: An informed consent framework and draft informed consent document should be provided. How many of the above considerations are communicated in writing to prospective patients?

Answer: The informed consent for the transplant acceptor contains potential FMT risks, including infection transmission, changes in the metabolic properties of the guts, caused by the mikroflóra composition changes are also described (weight changes, insulin resistance changes, mood changes etc.). Other potential risks, described in the consent are bowel perforation or bleeding, connected to the tube insertion.

Answer: We provide an example of informed consent and its English translation as “Supporting material to review process”. Relevant risks – as listed above – are indeed listed.

R3Q11: In the SOP for "preparation of the transplant," it is not acceptable to call this an SOP and make a statement like "no strict specifications on the mixing process are given." It is also not acceptable to use a kitchen hand-held blender. How could this be used and not result in cross-contamination between samples? How would the blender be disinfected or, preferably, sterilized between samples? Overall, I am not satisfied that the methodology outlined here is safe, as there is no detail about good manufacturing process details given.

Answer: Thanks for this relevant comment. We originally used hand-held blender treated with hot water, detergent and afterwards 2% chlorhexidine wipes. Yet, we agree with the reviewer that for formal SOPS a more robust technique is required to standardise the blending and – more importantly – sterilisation to prevent cross-cointamination. In the revised SOPs, we changed the recommended blender to https://www.grainger.com/product/WARING-COMMERCIAL-Lab-Blender-45H280) and recommend mixing time of 2 mis and full sterilization of stainless steel removable jar part.

R3Q12: The "final preparation of multi-donor transplant" section also sounds unsafe, as there is additional opportunity to contaminate the final product in a 37C water bath. Overall, the authors have not given sufficient detail to describe measures that will be used to prevent contamination of the FMT product during preparation.

Answer: We agree that graft thawing and mixing process was not described in sufficient details. In the revised SOPs we included more details, incl. the bath sanitation procedure.

R3Q13: Other donor selection considerations: What is the justification of the exclusion of donors >60yo?

Answer: This age threshold has been recommended by European consensus conference on FMT (See Cammarota, G. et al. European consensus conference on faecal microbiota transplantation in clinical practice. Gut 66, 569–580, 2017)., as well as by the British joint guidelines (Mullish, B. H. et al. The use of faecal microbiota transplant as treatment for recurrent or refractory Clostridium difficile infection and other potential indications: Joint British Society of Gastroenterology and Healthcare Infection Society guidelines. Gut 67, 1920–1941, 2018) and it based on the fact that microbiome diversity declines with aging, whilst microbiome-associated co-morbidities increase ( Mangiola, F., Nicoletti, A., Gasbarrini, A. & Ponziani, F. R. Gut microbiota and aging. Eur. Rev. Med. Pharmacol. Sci. 22, 7404–7413, 2018). Therefore, we also decided to limit donor age to <60 years.

R3Q14. Are investigators planning to include/exclude donors based on body mass index?

Answer: Yes. Body weight and height measurement is a part of the donor physical examination. Only persons with BMI between 20 and 30 will be accepted as donors.

R3Q15: Is diet recorded for donors, and what are the safety provisions for avoiding use of FMT from donors consuming certain donors in recipients with life-threatening food allergies?

Answer: The description of the dietary limitations is a part of the donor informed consent and adherence to dietary restrictions is regularly checked. Because this is reliant on self-reported data, recipients with severe food allergies will be excluded (See exclusion criterion: „History of severe anaphylactic food allergy”).

Dear editor,

We again want to thank all reviewers for extremely helpful and insightful notes and sincerely hope that them the revised manuscript is now acceptable for publication in Biomolecules.

Best regards

František Duška – on behalf of authors

Reviewer 2 Report

However, the application of FMT in ICU patients needs to be treated with great caution. Since the safety of the FMT procedure is still controversial. In 2020, a case of drug-resistant bacteremia after FMT was reported. The immunity of ICU patients are usually low which may can not endure the FMT operation. There is no evidence that the SOP procedure introduced in this paper is safe for use.
The faecal sample from the donor will be frozen before use in this paper, however, most of the FMT cases reported prefer fresh samples. After frozen, bothe benefical and harmful bacteria in the sample may inactivated.
There are many spell mistakes in this paper. For example: in line 82, line 95, line 143…
In line 90 and 94, the bacteria name of the Clostridium difficile should be italic. And the name of the genus Clostridium has been updated.

Author Response

To:

Editor-in-chief

Biomolecules

Dear Editor,

Dear Reviewers,

REVIEWER #1

Answer: The text has been extensively revised as suggested. All corrections can be found in the track-changes version of the manuscript

Answer: There was a missing bracket in the original sentence. We added more general statement and only list those diseases as example.

R1Q3: line 53ff: this conclusion is over-simplified, incomplete, and finally wrong (by only focussing on SCFA!) => please adapt

Answer: We changed this part of article as suggested.

R1Q4. Overall, references used seem out-dated => the authors should add more current literature

we use more current literature

R1Q5: The relevance of bacteriophages, the virome and mycobiome should be named and discussed in the context of FMT (=> probably relevant effect size for FMT success)

Answer: We agree and thank for this comment. We have added a section on FMT efficacy to the text, listing all parts of the microbiome. We added this at later section (line 84).

Answer: This specific risk is now mentioned in the revised text incl. the reference to FDA warning.

Answer: Agreed. We downtoned the statement from “golden standard” to “well established treatment”.

R1Q8. Line 93 ff: this listing is also very inconclusive (see above) change the text Answer: line 104/105 => a reference is mandatory!

Answer: We added a reference.

R1Q11: Line 180 f: effect sizes regarding the FMT application by enema vs. via colonoscopy should be discussed differentially as the latter way of application seems to hold the highest effect size.

Answer: We have revised the text and mention lower effect size.

R1Q12 Line 182: the normal small intestine IS NOT STERILE!

Answer: Words “normally sterile” were deleted from this sentence.

R1Q13: Line 37: the plural of microbiome (“microbioma”) is uncommon => adapt

Answer: We have changed this.

R1Q14 Line 44: the same is true for “pathobiom”, that should be pathobiomE? If the authors want to use this term then a specific reference should be given for it

Answer: The word is not in use in the revised text, only in Ref 31 in the form of “Pathobiome”.

REVIEWER #2

Safety (Sample can be quarantined until the donor is re-tested for transmittable diseases)
Logistics (Frozen samples are readily available in ICU whenever a critically ill patients needs them)
Standardisation (Use of multi-donor FMT increases the homogeneity of investigational products.

R2Q3: There are many spell mistakes in this paper. For example: in line 82, line 95, line 143…

Answer: See also R1Q1. We have extensively revised the text to correct typos and spelling errors.

Q2Q4: In line 90 and 94, the bacteria name of the Clostridium difficile should be italic. And the name of the genus Clostridium has been updated.

Answer: We have changed to italic. Also in lines 154 and 227.

Answer:

REVIEWER #3

Neutropenic patients
Recent GI surgery
Known perforated viscus / abdominal free air
Patients unable to give informed consent

In the revised manuscript, we have added a paragraph discussing these important aspects.

R3Q5: The ID screening outlined in the supplementary procedure is not adequate, particularly given the plan to pool feces from 7 donors in each aliquot. In particular:

HIV screening as outlined does not address the window period problem unless the labs as outlined are collected at baseline and again at the end of the 3 month quarantine period. This is not specified.
Window period infections are also not addressed for the hepatitis virus screening, which does not include NAAT testing or a plan to repeat at any interval.

R3Q6: No donor deferral criteria are provided, nor is the questionnaire given to the donor for risks of infectious diseases. Would the following donors be deferred:

History of travel to India 6 months ago?
History of travel to India 18 months ago but for a total stay duration of 4 months?
A donor with >1 sexual partner in the last 12 months?
Donor with history of IBS?
Patients with an extensive family history of GI malignancies, peptic ulcer disease, or autoimmune disease?

Answer: We now attach full donor refusal criteria in the Supplementary data file, that are compliant with most of the above.

R3Q7: Are donors compensated? If so, what measures are in place to prevent conflicts of interest in assessing the donor deferral criteria?

Helicobacter pylori
Herpes simplex virus 1 and 2
Cytomegalovirus (CMV)
Human papillomavirus (HPV)
JC virus