PONE-D-23-26927Enteroaggregative Escherichia coli in persons with diarrhoea and healthy controls in mid-Norway: A prospective, case control studyPLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

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5. Review Comments to the Author

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Reviewer #1: Review PONE-D-23-26927

TITLE: Enteroaggregative Escherichia coli in persons with diarrhoea and healthy controls in mid-Norway: A prospective, case control study

The study describes the EAEC findings from patients at a university hospital in Mid-Norway with comparisons to healthy subjects.

The findings in the control group are, as the authors themselves comment, less frequently described in a group with no immediate intention to travel; in traveller studies the pre-travel samples have mostly been collected at travel clinics, and those with an intention to travel are also likely to have travelled in the past. However, Bruijnesteijn van Coppenraet et al. have a rather similar study setting in the Netherlands in their 2015 article.

The main problem with the study lies in the control group setting. Nontravellers should not be used as controls: Among asymptomatic travellers, EAEC is found in 16-28% of stool samples (Paschke et al. 2011, Lääveri et al. 2016, Lertsethtakarn-Ketwalha et al. 2018). By contrast, as the authors observe, EAEC is a rare finding in pre-travel stool samples in many traveller studies.

The authors state that nontravellers were defined as not having travel history documented. This does not automatically equal to having no travel history since unless this was actively documented as “no travel history” instead of information on travel history missing i.e. not documented at all.

To my knowledge, the interplay between the various gastrointestinal pathogens has been poorly described in clinical settings particularly among travellers. Furthermore, the association of EAEC with age is not independent from history of travel to low- and middle-income countries; EAEC can be found in stools for several weeks after travel. Therefore, in addition to using nontravellers as controls, I don’t agree with the use of a multivariable regression model used for the identification of the role of EAEC. A more practical approach would have been “only EAEC” vs. “no EAEC” vs “EAEC together with other pathogens. In addition, asymptomatic colonization with Salmonella and even ETEC is relatively common among travellers; this has been described in many studies with control group design (e.g. Paschke et al.2011, Lääveri et al.2016, Lertsethtakarn-Ketwalha et al. 2018). Moreover, the aetiology of diarrhoea among nontravellers is generally considered to be of viral origin although norovirus findings among travellers are more frequent in more recent traveller studies than e.g. 30 years ago.

Unfortunately, due to the issues mentioned above, I cannot recommend accepting this article. However, I encourage the authors to change this into a more descriptive study instead of trying to assess the role of EAEC in diarrhoea; for that purpose they would need to analyze travellers and nontravellers separately and the proportion of travellers in the control group appears too small for statistical analyses.

Reviewer #2: 1. The title needs to be modified, as the case control study itself reflects the inclusion of healthy as well as infected individuals, the usage twice may be avoided.

2. The geo co-ordinates of the study setting may be given for appropriate location.

3. Cite suitable references for methodology. For instance, the sample processing, isolation of EAEC and molecular detection. Alike, the gene sequence and PCR conditions.

4. ‘maximum number of pathogens detected in a single sample was five (n=16)’: interesting to find it in text.

5. Representation of the data year wise or season wise would also fetch more interesting findings.

6. Minor typos and grammatical errors need to be addressed.

Reviewer #3: The study was well designed, and the manuscript is generally well-written and I nearly found no concern. At the discretion of the editor, authors should clarify on the following;

- There is no justification / rationale for conducting the study in mid-Norway, in Sør-Trøndelag County

- You did one extraction for the assays targeting both nucleic acids? Yet, some viruses targeted e.g. Astrovirus, Sapovirus etc are RNA so clarify on whether the kit achieves extraction of RNA and DNA simultaneously.

Reviewer #4: Please note : There is no novelty in the study. The fact of EAEC associated with travellers diarrhoea is known . The study undertaken on huge sample size highlights the prevalence in that region. What is the additional information gained .The clinical presenation and duration of illness is not mentioned. The treatment and follow up of the patients is not looked into.There are kits for multiplex PCR on the stool to pick up the pathogens . There is no new finding other than the prevalence of EAEC in that region.

Reviewer #5: Well done on the well written piece of work. Just one or two comments

1) On Lines 267 – 270 unnecessary speculation. If it was not done why mention it?

2) Figure 3 - what is the unit on the Y-axis? What exactly is this Figure showing? And can the results be explained for the 60 -69 & 70 - 79 age groups.

3) All Figures seem to be missing legends.

Reviewer #6: I have reviewed the manuscript and found several grammar mistakes that should be corrected. Below are some specific points that require attention

Methods: Study populations

1. The age group of patients and healthy individuals included in the study should be described.

2. Line 105-106: “multiple samples from the same person as duplicates” The authors need to justify why they excluded repeat samples from the same patient, given that gastro pathogens are known to shed intermittently in the stool. A repeat sample is generally recommended in diarrheal cases to increase the chances of detecting a pathogen. Also, what is the rationale for selecting a 3-month window period?

3. Line 107: The meaning of "did not reveal a previously undiagnosed pathogen" is not very clear.

4. Line 117: exclusion criteria: Did the authors consider antibiotic consumption history in the control group before sample collection (as it can take up to four weeks for the normal flora to establish after a 7 days antibiotic course)? if so what period before sample collection was considered?

5. Did the authors follow up with the control group participants especially those who tested positive for a pathogen for the development of any diarrheal symptoms after the sample collection? Some of the control group participants may have been in the infection development state at the time of sample collection.

6. The authors need to briefly describe the real-time PCR parameters used, such as whether it was SYBR green-based/Taqman probe-based, and the machine used.

7. Did the authors use any positive controls or perform the assay in duplicates/triplicates to confirm the reproducibility of results?

Travel destinations:

8. The authors should specify the duration of travel that was considered a travel case. Also, was travel to a foreign country the only consideration, or was travel within the country also considered?

Sample collection:

9. Did the authors differentiate hospital-acquired diarrhoea from community-acquired diarrhoea? The authors should explain what the hospitalized patients represent.

10. The lysis buffer composition or brand name of the reagents used in the study where available should be provided.

Results:

11. Line 215: The authors should explain how they decided on the CT value cutoffs. Did the Multiplex PCR assay provide them?

12. The values described in Line 222-224 should be either ascending or descending.

13. The authors have not described whether any seasonal patterns were observed in the study but have included this as a strength of the study.

14. It would be interesting to know the authors' thoughts on the discovery of mosaicism of virulence factors in diarrheagenic E. coli pathotypes based on genomic studies.

Discussion:

15. The authors should consider reducing the number of discussion points.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: David Patrick Kateete

Reviewer #4: No

Reviewer #5: No

Reviewer #6: No

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Enteroaggregative Escherichia coli in mid-Norway: A prospective, case control study

PONE-D-23-26927R1

Dear Dr. Haugan,

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.

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Kind regards,

Furqan Kabir

Academic Editor

PLOS ONE

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