PONE-D-20-39264

I-FABP is decreased in COVID-19 patients, independently of the prognosis

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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5. Review Comments to the Author

Reviewer #1: Authors evaluated serum levels of the intestinal fatty acid-binding protein (I-FABP), a biomarker of intestinal injury, in COVID-19 patients. It’s an important study considering the digestive system involvement in COVID-19, however, it presents a negative result against the authors’ assumed hypothesis. Authors showed (I-FABP) is significantly decreased in COVID-19 patients for which they are not able to explain the patho-physiological mechanisms and the clinical significance.

Major concerns:

1. Materials and method section is very short, more elaboration is necessary.

2. Materials and method: Authors write they correlated several biological markers, including biomarkers of systemic inflammation, current biochemical markers, and citrulline with I-FABP in COVID-19 patients. The descriptions of these markers are missing in the paper. Please provide complete list, describe the methods used in their estimation, and detailed results of the study for these markers. Why these markers were not studied for the control groups?

3. Materials and method: Include standard curve for the ELISA estimation. Also include details for how many times ELISA was performed and how many multiples were used for each sample.

4. Materials and method: Details for statistical analysis is completely missing, please include necessary details.

5. Results: As it’s not a brief communication, present results separately from the discussion. All patients with COVID-19 don’t have digestive symptoms, analyzing the results separately for those with digestive symptoms may be more informative.

6. Discussion: Even if authors are not able to explain pathophysiological basis of decreased expression of I-FABP, they can present some assumptions based on the literature evidence, that will add clinical significance to this study. Malabsorption is a common feature in COVID-19 in COVID-19 patients with digestive symptoms. It’s possible that a decrease in I-FABP may be due to fat malabsorption through enterocytes. Ref: Decreased expression of Intestinal I- and L-FABP levels in rare human genetic lipid malabsorption syndromes. https://link.springer.com/article/10.1007/s00418-007-0302-x

Hypolipidemia is associated with the severity

of COVID-19: https://pubmed.ncbi.nlm.nih.gov/32430154/

7. Discussion: Authors write that “I-FABP does not follow the citrulline pattern in COVID-19, which suggests that the reduction of enterocyte mass and function in COVID-19 patients is not associated with an alteration of the intestinal mucosa.” This is a far reaching interpretation of their results. In contrast of their claim, current evidence suggests clear cut intestinal mucosal injury in COVID-19 (Ref: COVID-19 and the Digestive System:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273952/).

8. Discussion: Authors should avoid reaching this conclusion with their limited investigation: “we can conclude that the interest of I-FABP to highlight the risk of intestinal complications in these patients is however unlikely, according to our results.” Instead they should conclude with what were the main outcomes of this study.

9. Discussion: Although, finding of this study is important, this reviewer believes that a single ELISA based assay is not sufficient to validate results of this study. Also sample size of the study is small, especially for the test group presenting patients with severe symptoms. Please include a section of limitations and provide future directions how the results can be further validated.

Reviewer #2: Plasma level of I- FABP is used as a validated prognostic marker of gut damage for IBDs, acute ischemia and HIV. (Ref Isnard S. Plasma Levels of C-Type Lectin REG3α and Gut Damage in People With Human Immunodeficiency Virus. J Infect Dis. 2020 Jan 1;221(1):110-121). As ACE is strongly expressed in gut investigators assessed serum I-FABP in small groups of patients with COVID-19.

Contra intuitively COVID patients had only half mean value when compared to controls. They did not observed correlation between I-FABP and citrulline or CRP). IFABP is known to participate in fatty acid metabolism, and GDF15 is known to decreased HDL and LDL cholesterol and hypertriglyceridemia in severe COVID patients.

Myhre PL. Growth Differentiation Factor 15 Provides Prognostic Information Superior to Established Cardiovascular and Inflammatory Biomarkers in Unselected Patients Hospitalized With COVID-19. Circulation. 2020 Dec;142(22):2128-2137.

Lymphocytes ratio on PMN or neutrophil should also be presented as one of easy-to-get prognosis for COVID for I FABP, CRP and citrulline correlation.

Assessment of GDF15 will be of interest.

Interesting hypothesis generating observation.

Reviewer #3: The idea is quite interesting, however there are some important methodological issues.

There is no information about statistical analysis.

I-FABP concentration should be analyzed in COVID-19 patients with and without gastrointestinal symptoms.

Additionally, majority of COVID-19 patients present diarrhea, which may affect concentration of I-FABP due to increased passage. Then I-FABP should be also measured in stool samples.

The discussion is too short.

Reviewer #4: The authors presented a potentially interesting study with the aim of evaluating the value of circulating I-FABP in patients with COVID-19, as expression of intestinal damage. Although interesting, since a not negligible rate of patients presents with intestinal symptoms and eventually might have intestinal damage even in the absence of intestinal symptoms, the manuscript lacks of fundamental aspects that compromise its entire value.

Overall, the manuscript is poor and sometimes lacks of clarity.

The methods are poorly represented. The control groups lack crucial information.

The authors considered their study as a research article. However, it is more appropriate to consider the paper as a research note or a short communication. In fact, the paper is very short, only 8 references are presented and overall the manuscript is quite poor.

In detail:

Introduction

- In the introduction, authors presented some results on plasma levels of citrulline in COVID-19 and acute intestinal ischemia. However, the sentence is not very clear. Therefore, I would suggest authors to re-write the sentence in order to better explain the behaviour of this marker in the mentioned conditions.

- I-FABP has been also studied in other infective conditions, such as Clostridioides difficile (see Oliva et al, Open Forum Infect Dis. 2019 Dec 3;7(1):ofz507)

Methods

- “Time of study inclusion” means time of COVID-19 diagnosis?

- How was diagnosis of COVID-19 made? PCR-based methods on which sample?

- Were patients with COVID-19 with or without pneumonia? Please include in the method.

- How authors assessed COVID-19 disease severity? It must be stated in the method section along with the appropriate reference.

- “As control populations, we analyzed 24 currently hospitalized patients with non-COVID-19 pulmonary diseases.”: Why did the author chose non-COVID pulmonary diseases as control population? Was the decision based on the possibility of the gut-lung axis? Were these pulmonary diseases infective conditions (such as pneumonia) or non infective? There is no mention in the method section. Results might differ according to these conditions.

- How the authors choose the controls? How were the levels of I-FABP in healthy controls? Healthy subjects might be included as a control group in order to evaluate whether COVID-19 patients have values similar to those observed in healthy controls.

- I believe that controls group should have been matched with study population: for instance, age and sex might influence the value of I-FABP. Values might be very different if a patient with COVID-19 is a young female and control patient with pulmonary condition is a 90-year old man.

- How were the sample stored in the period comprised between sample collection and analysis? Please explain.

- I suppose that patients were divided into ICU and non-ICU and died/survived groups, respectively. However, no mention in the method section is present. Would the author consider ICU-group as patients admitted to ICU during hospitalization or directly from the Emergency Room Department? Please specify.

- Statistical analyses description are absent in the method.

Results and discussion

- Overall, how many patients presented with gastrointestinal disease? Authors described single symptoms, however, it could be interesting to know the total number of patients with gastrointestinal involvement.

- Overall, results (i.e. comorbidities) might be better presented and described.

- How many patients had COVID-19 associated pneumonia?

- It could be interesting to know the duration of symptoms before I-FABP collection. Is there a correlation between I-FABP levels and duration of symptoms?

- Authors should better describe the patient who died with high levels of I-FAB, preferably in the discussion. What does it mean anorexia? How diagnosis of anorexia was made?

- “This observation might suggest that the increase of I-FABP in COVID-19 patients might depend on the combination of pathological conditions that are in fine associated with increased alterations of the intestinal barrier”: this could be partly true. However, the authors should better explain and argue this sentence. Accordingly, authors should insert appropriate references.

- “We then analyzed the correlation of several biological markers, including biomarkers of systemic inflammation, current biochemical markers, and citrulline with I-FABP”. This part belongs to the methods section. Which biological markers were analyzed? Please specify. How was analyzed citrulline? Please specify in the method.

- Is the correlation between I-FABP and PMN positive or negative? This is not clear from the text; therefore, reading the discussion regarding this finding is quite difficult.

- The authors hypothesis was that in COVID-19 patients an intestinal damage is present, leading to the expectation of high I-FAB levels. However, they found lower level of I-FABP. How did the authors motivate and explain their findings? With this regard, the discussion is very poor and does not support the study results.

- “While the link of I-FABP levels and lipid metabolism should be further investigated in COVID-19 patients, we can conclude that the interest of I-FABP to highlight the risk of intestinal complications in these patients is however unlikely, according to our results.” This sentence is not clear. Was the aim of the authors to evaluate the possible intestinal damage in COVID-19 patients or the evaluation of intestinal complication in COVID-19 patients? If the aim, as I understood, is to evaluate the possible intestinal damage in COVID-19 patients, this sentence should be re-written The conclusions are not supported by the results of the study. Authors should re-write the last sentence of the study.

- No mention on the number of death or ICU admission is present in the result section.

- A table with study population characteristics would be appropriate

Figures

- Figure1. The number of patients with abdominal pain is 80; in the text is 79. Please correct.

- Figure3. It seems that only the coefficient is presented. Authors should also include the p value.

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Reviewer #1: Yes: Ashutosh Kumar

Reviewer #2: Yes: Jean-Pierre Routy

Reviewer #3: No

Reviewer #4: No

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I-FABP is decreased in COVID-19 patients, independently of the prognosis

PONE-D-20-39264R1

Dear Dr. Guedj,

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