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

BACKGROUND: Severe acute respiratory syndrome caused by the novel coronavirus (SARS-CoV-2) is frequently associated with gastrointestinal manifestations. Herein we evaluated the interest in measuring the intestinal fatty acid-binding protein (I-FABP), a biomarker of intestinal injury, in COVID-19 patients.
METHODS: Serum I-FABP was analyzed in 28 consecutive patients hospitalized for a PCR-confirmed COVID-19, in 24 hospitalized patients with non-COVID-19 pulmonary diseases, and 79 patients admitted to the emergency room for abdominal pain.
RESULTS: I-FABP serum concentrations were significantly lower in patients with COVID-19, as compared to patients with non-COVID-19 pulmonary diseases [70.3 pg/mL (47-167.9) vs. 161.1 pg/mL (88.98-305.2), respectively, p = 0.008]. I-FABP concentrations in these two populations were significantly lower than in patients with abdominal pain without COVID-19 [344.8 pg/mL (268.9-579.6)]. I-FABP was neither associated with severity nor the duration of symptoms. I-FABP was correlated with polymorphonuclear cell counts.
CONCLUSIONS: In this pilot study, we observed a low I-FABP concentration in COVID-19 patients either with or without gastrointestinal symptoms, of which the pathophysiological mechanisms and clinical impact remain to be established. Further explorations on a larger cohort of patients will be needed to unravel the molecular mechanism of such observation, including the effects of malabsorption and/or abnormal lipid metabolism.

Introduction

The SARS-CoV-2 infection is associated with an extensive range of symptoms, including first-line respiratory manifestations and gastrointestinal manifestations [1]. Indeed, in a study including 164 COVID-19 patients of Chinese descent, Zhang et al. evidenced gastrointestinal signs in one-third of the patients [1].

Furthermore, the SARS-CoV-2 receptor is the Angiotensin-Converting Enzyme 2 (ACE2), which is mainly expressed by enterocytes brush border of the intestinal epithelium, despite its strong expression by lung epithelial cells [2]. Therefore, identifying intestinal-derived molecules released upon SARS-CoV-2-induced damages could serve as biomarkers to monitor the pathology, particularly in long-term and/or severe patients.

We recently found that in patients with COVID-19, plasma citrulline concentration inversely correlates with systemic inflammation. Patients that presented low plasma citrulline concentrations also showed higher systemic inflammation. Moreover, low citrulline and gastrointestinal symptoms were associated with more severe diseases [3].

Indeed COVID-19 is associated with a wide range of intestinal pathologies and alterations in gut microbiota [4]. Citrulline, a non-proteinogenic amino acid, is a marker of short bowel enterocyte mass and function. This marker has also been shown to be a marker of acute mesenteric ischemia [5]. Therefore, the decrease of citrulline concentration in COVID-19 patients suggests that the infection might alter the enterocyte function, leading to a loss of function.

Thus, we are convinced that identifying circulating biomarkers of digestive complications is mandatory to improve patients’ management and provide insight into the infection’s pathophysiological mechanisms.

Intestinal Fatty Acid-Binging Protein (I-FABP) is a 15 kDa cytosolic protein known to be involved in the uptake and trafficking of fatty acids in the small and large intestine. The protein is mainly expressed by enterocytes located at the tips of the intestinal villi. This cytoplasmic protein is released by mature enterocytes as soon as the cell membrane integrity is compromised. I-FABP is thus the reflection of the extent of gut damage, and it is therefore used as a biomarker of mucosal injury and other diseases affecting the intestine [6].

Indeed, since enterocytes are the first affected in ischemic intestinal injuries, I-FABP has been proposed as a diagnostic marker of acute intestinal ischemia [7]. Moreover, its circulating concentration has been evidenced to present a prognostic value in patients admitted in intensive care units (ICU) in the context of several pathological conditions, including septic shock and postoperative cardiac surgery intensive care [8, 9]. To expand the possible readouts of enterocyte function and viability, we aimed at analyzing serum I-FABP in patients with COVID-19.

Material and methods

In a single academic center (Beaujon Hospital, APHP, France), from April 14 to April 24, 2020, we prospectively enrolled twenty-eight consecutive patients hospitalized for a PCR-confirmed COVID-19 on nasopharyngeal swabs associated with pneumonia. The study was approved by the Ethics Committee of Paris-Nord Val de Seine University Hospitals, and patients gave their written informed consent.

All COVID-19 patients exhibited pneumonia. Following the ethical committee’s standard (IRB 00,006,477), we collected clinical and biological data. Routine baseline clinical characteristics and biological data were collected upon admission for all patients. Patients were included either from the medical ward or directly from the Intensive Care Unit (ICU). Patients from ICU (n = 6) were either immediately included or hospitalized in ICU within 48h after their admission at the hospital.

Blood samples were drawn at the time of COVID-19 diagnosis, collected in appropriate tubes before immediate centrifugation at 3,000 rpm for 15 min at room temperature, and subsequent storage at -80°C until further analysis. Biological current assays were performed at the time of inclusion on blood collected on heparin (C-Reactive Protein, I-FABP, Citrulline) or EDTA (circulating polymorphonuclear cells, lymphocytes).

As control populations, we first analyzed 24 currently hospitalized patients with non-COVID-19 pulmonary diseases, including infectious disease (4%), neoplastic (8%), inflammatory or degenerative diseases (88%).

We then studied 79 patients admitted to the emergency room for abdominal pain, initially suspected of acute mesenteric ischemia, but finally discarded after CT Scan angiography. Those patients instead presented infectious (23%), inflammatory (19%), mechanical (31%), or extra-intestinal causes (27%) of abdominal pain.

I-FABP concentrations were measured in serum using the ELISA kit from Hycult Bioteck (HK40602, Uden, The Netherlands, linearity range: 47–3,000 pg/mL) following the manufacturer’s instructions.

Briefly, samples and standards were incubated in 96-well microtiter plates coated with antibodies recognizing human I-FABP. The standard curve included six points ranging from 47 pg/mL to 3000 pg/mL (S1 Fig).

The biotinylated secondary antibody was then added to the wells. After washes, the streptavidin-peroxidase conjugate was added and then reacted with tetramethylbenzidine (TMB) substrate. The absorbance was then measured with a spectrophotometer at 450 nm (Infinite® 200 PEO, TECAN). I-FABP was assayed by batch at the end of the inclusion period. We used two quality controls (sample pool)–at the beginning and the end of the plates.

Citrulline plasma concentrations were assayed using an ultra-performance liquid chromatography-mass spectrometer (UPLC-MS; Xevo TQS, Waters®).

The retention time was 1.43 min on the Xevo TQS, Waters®.

The results are presented as an average of two measurements (duplicate). If a coefficient of variation higher than 10% was observed between two measures, the sample was reanalyzed.

CRP and other common biological markers were routinely performed in the central laboratory onto Architect C8000 (Abbott) or XN-3000 (Sysmex).

For each of the continuous variables, we report the mean and standard deviation, or median and Interquartile range when indicated. Categorical variables are expressed as the number of observations and percentages. Quantitative data were analyzed with the Mann-Whitney U test. Subgroup analyses were performed with the use of the Kruskal-Wallis tests for skewed distributions. All graphs and statistical analysis were performed in Graphpad Prism 9.

Results

Among our COVID-19 population mean age was 62 years (standard deviation -SD-: 13.94). Twenty-five percents were females. Thirteen patients presented elevated blood pressure, 14 diabetes, eight dyslipidemia. Only two exhibited abdominal pain, nausea, vomiting, 15 a recent loss of appetite, whereas 11 complained of diarrhea. As a sum, 14 out of the 28 patients exhibited almost one digestive sign. The average body mass index (BMI) was 28 (standard deviation: 3.9). The main features are summarized in Table 1. We separated patients as severe or non-severe on two criteria: hospitalization in ICU/ and or death. Five patients deceased during the hospitalization. Among patients with pulmonary diseases, the mean age was also 62 years (SD: 12.36), with 45.8% of females, whereas in patients with abdominal pain, the mean age was 51 years years (SD:19.9), with 39% of females.

Table 1

Main COVID19-patients characteristics.

		N = 28
Age (mean +/- SD)		61.2 +/- 13.8
Sex (female (n, %))		7 (25%)
Time from symptoms onset (days)		14 +/- 9.88
Cardiovascular risk factors	Elevated blood pressure	13 (46.4%)
	Diabetes	14 (50.0%)
	Dyslipidemia	8 (28.5%)
	Cardiovascular history	2 (7.1%)
Pulmonary diseases	COPD	2 (7.1%)
	Asthma	2 (7.1%)
History of IBD		0 (0%)
Smoking		1 (3.5%)
Medications	ARA2	4 (14.2%)
	Steroids	0 (0%)
	NSAIDS	0 (0%)
Clinical features	Fever	18 (64.4%)
	Caughing	22 (78.6%)
	Shortness of breath	13 (46.4%)
	Anosmia	8 (28.6%)
	Agueusia	9 (40.8%)
	Arthromyalgia	9 (32.1%)
	Recent loss of appetite	14 (53.6%)
	Nausea/vomiting	3 (10.7%)
	Abdominal pain	2 (7.1%)
	Diarrhea	11 (39.3%)
	Digestive symptoms	14 (50.0%)
Physical characteristics	Baseline body mass index	28 +/- 3.9
Outcome	ICU	6 (19.2%)
	Orotracheal intubation	3 (10.7%)
	Death	4 (3.9%)

IBD: inflammatory bowel disease. ICU: Intensive Care Unit. ARA2: angiotensin 2 antireceptor. COPD: chronic obstructive pulmonary disease

As shown in Fig 1, I-FABP serum concentrations were significantly decreased in patients with COVID-19, as compared to control patients with non-COVID-19 pulmonary diseases [Med: 70.3 pg/mL (IQ: 47–167.9) vs. 161.1 pg/mL (IQ: 88.98–305.2), respectively, p = 0.008].

Fig 1

Serum I-FABP concentration is decreased in COVID-19 patients.

I-FABP concentrations were measured in COVID-19 patients (n = 28) and in patients with abdominal pain or pulmonary disease (n = 79 and 24, respectively). ANOVA and Mann-Whitney tests were used for statistical comparison. CRTL: control.

I-FABP concentrations in these two populations were significantly lower than in patients with abdominal pain without COVID-19 [Med: 344.8 pg/mL (IQ: 268.9–579.6)].

We next assessed whether the severity of the disease was associated with serum I-FABP concentration. We found no statistically significant difference in I-FABP concentrations between patients from the ICU and those from the medical ward or between patients who died from the disease compared with patients who survived (Fig 2). No correlation was found between I-FABP concentrations and the duration of symptoms (S2 Fig).

Fig 2

Serum I-FABP concentration is not associated with severe forms of COVID-19.

I-FABP concentrations were measured in COVID-19 patients from medical ward (n = 22) and ICU (n = 6) (left panel) and patients that live (n = 24) or deceased (n = 4; right pannel). Mann-Whitney test was used for statistical comparison.

We then analyzed the correlation of several biological markers, including biomarkers of systemic inflammation (CRP), current biological markers (circulating polymorphonuclear cells (PMN), Lymphocytes), and citrulline with I-FABP. The correlation table is presented in Fig 3. We found a strong, statistically significant correlation of I-FABP concentrations with the number of PMN. There was no correlation between I-FABP and citrulline or between I-FABP and systemic inflammatory biomarker (CRP) or lymphocytes.

Fig 3

Serum I-FABP correlated with the number of polymorphonuclear cells in COVID-19 patients.

Correlation table between serum concentration of I-FABP, citrulline, C reactive protein (CRP), and the number of leukocytes. Pearson correlation test was used. The correlation index is indicated. A negative sign indicates a negative correlation.

Discussion

In the present study, we observed that I-FABP concentrations were significantly decreased in patients with COVID-19, compared to patients with non-COVID-19 pulmonary diseases and in patients with abdominal pain without COVID-19. I-FABP is a biomarker of enterocyte injury. To date, although we did not reevaluate them in the present study, values in healthy controls for I-FABP with ELISA Hycult were previously evaluated less than 90 pg/mL, thus slightly higher than what we observed for COVID-19 patients. This result was unexpected. We also evaluated these results taking into account the potential impact of pulmonary diseases and of marked abdominal pain.

I-FABP is known to participate in fatty acid metabolism. Notably, it has been suggested that I-FABP could be involved in dietary lipid sensing and signaling, as indicated by results obtained in -IFAPB -/- mice [10]. It has been shown that lipid metabolism is modified in COVID-19 patients. Hypolipidemia is associated with the severity of COVID-19, with LDL cholesterol and Total cholesterol concentrations lower in all COVID-19 patients and HDL cholesterol [11, 12], and hypertriglyceridemia in severe patients [13]. This could partially explain our observation due to an active mechanism rather than a free passive diffusion of the marker.

On the other hand, malabsorption is a common feature in COVID-19 patients with digestive symptoms. A decrease in I-FABP may be due to fat malabsorption through enterocytes, as evidenced in coeliac disease [14]. However, possibly due to the population’s small size, we did not evidence any difference in I-FABP concentrations between patients with or without gastrointestinal signs (data not shown).

Patients with systemic inflammatory response syndrome (SIRS) have increased intestinal permeability. Thus, severe COVID-19 patients are at risk of developing increased intestinal permeability with prognosis worsening. However, we did not evidence a marked difference related to the prognosis in the present study.

I-FABP has also been studied in other infective conditions, such as Clostridioides difficile infection [15]. Severe sepsis in ICU patients has been shown to be associated with an increase in I-FABP concentrations [16]. In both observations, I-FABP was increased in patients. But no study, to the best of our knowledge, evaluated I-FABP in acute viral infections to date.

Intriguingly, one of the four patients who died presented a much higher concentration of I-FABP compared to other COVID-19 patients. This patient also presented numerous other comorbidities, such as diabetes, hypertension and a recent loss of appetite. This observation might suggest that the increase of I-FABP in some COVID-19 patients might depend on the combination of pathological conditions that are in fine associated with marked alterations of the intestinal barrier, or perhaps related to bacterial diseases rather than viral diseases.

I-FABP does not correlate with the citrulline pattern we previously observed in COVID-19 patients. This observation may have several explanations, including differential evolution and half-life for both markers, a difference between the reduction of enterocyte mass and function in COVID-19 patients and the alteration of the intestinal mucosa. I-FABP is also not correlated with CRP, which reflects the inflammation climate. The correlation we observed between PMN and I-FABP is puzzling. This positive correlation is highly significant (S1 Table). Our initial hypothesis was that I-FABP might be associated with intestinal damage in severe COVID-19 patients. The role of PMN in COVID-19 pathogenesis is still unclear. Indeed, an expanded myeloid compartment and some subtypes of PMN have been associated with the prognosis in some studies [17].

This study presents some limitations.

The number of patients we included is small, especially for the group presenting severe symptoms. I-FABP concentrations should be analyzed in larger COVID-19 patients’ populations with and without gastrointestinal symptoms. More extensive studies should be undertaken to determine whether I-FABP could be used either as a prognosis factor in COVID-19 or peculiar phenotypes. Notably, it has been shown that acute mesenteric ischemia is a complication in severe forms of COVID-19 requiring hospitalization in the ICU [18]. Whether I-FABP should be used in this setting remains to be elucidated.

Additionally, COVID-19 patients frequently present diarrhea, which may affect the circulated concentration of I-FABP due to increased passage. I-FABP should also be measured in stool samples, although there is no current assay appropriate for such measurement. Finally, correlations between I-FABP with other biomarkers of COVID-19 prognosis should be informative, for instance, with Growth differentiation factor 15 (GDF-15) is associated with severe COVID-19, hypoxemia, and SarsCov2 viremia [19]. Whereas intestinal mucosal injury has been evidenced in COVID-19 [20], our first study failed to evidence I-FABP as a prognosis marker or a marker associated with gastrointestinal signs in COVID-19. However, our results are in line with those obtained by Hoel et al. [21], which showed no elevation of I-FABP in COVID-19 patients with cardiac involvement. Further investigations will be needed to decipher such observation’s molecular mechanism, in line with our previous data on citrulline.

Standard curve of ELISA assay.

Hycult ELISA assay was used as recommended by the manufacturer.

(TIF)

Click here for additional data file.

Linear regression between I-FABP concentration and symptom duration.

No significant association was seen (R2 = 0.06327).

(TIF)

Click here for additional data file.

(XLSX)

Click here for additional data file.

P-value of the correlation between I-FABP and current biological values.

(DOCX)

Click here for additional data file.

2 Feb 2021

PONE-D-20-39264

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

PLOS ONE

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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

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Mar 2021

Dear Editor,

On behalf of all the co-authors, I would like to thank you and the reviewers for the valuable comments and the significant heed in improving our manuscript.

You will find detailed answers to all of the comments, including the comments made by the editor and those made by the reviewers. Modifications suggested by the editor and/or reviewers appear in blue in the manuscript. We hope you will find the manuscript is now suitable for publication.

No ethical or legal restrictions on the diffusion of data after anonymization. We provide the corresponding files.

Answer to editor comments

" “Comité d’Evaluation de l’Ethique des projets de Recherche Biomédicale (CEERB) Paris Nord” (Institutional Review Board -IRB 00006477- of HUPNVS, Paris 7 University, AP-HP".

Please amend your current ethics statement to confirm that your named institutional review board or ethics committee specifically approved this study.

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We have now introduced this statement in the text. The project was approved by the CEERB.

The study was approved by the Ethics Committee of Paris-Nord Val de Seine University Hospitals.

3.Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

All patients gave their informed consent for participating in the study. All patients get a preliminary written information on the protocol. These points are now precsised in the text.

4.We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

We have identified no ethical or legal restrictions on the diffusion of data, after anonymization. However, IFABP values in controls with abdominal pain are currently submitted as controls in another study, and thus, we provided only some minimal data.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please

see http://journals.plos.org/plosone/s/data-availability#loc-recommended- repositories.

We have now added these data. However, those data are presented at this time in french...Please precise if an english version is awaited.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Answer to reviewers

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.

We have added a more detailed description of the material and methods

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?

We aimed to study I-FABP and designed the study to perform so. In comparison, COVID19 patients were extensively explored. We previously published data on citrulline in a previous manuscript, and

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.

We have now added the curve in supplementary data, and completed the text.

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

We have added some missing information concerning statistical analysis.

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.

We have now presented the results separately from the discussion and developed this latter one.

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/

We have now included these two points in the discussion section.

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/). We have now included this evidence in the discussion section.

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.

We have now changed the discussion.

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.

We have included a section on limitations and gave some perspectives.

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 January 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.

We explored whether the Lymphocytes ratio on PMN ratio was correlated with I-Fabp or different within the different populations. It was not significant, and we did not include it in the final version. We have added the GDF15 perspective in the perspective section.

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

There is no information about statistical analysis.

It has been now added in the method section.

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.

These perspectives have been included at the end of the discussion.

The discussion is too short.

We have further discussed the results, including the hypothesis for our observation.

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 rewrite the sentence in order to better explain the behaviour of this marker in the mentioned conditions.

We have rewritten this sentence as follow :

We recently found that in patients with COVID-19, plasma citrulline concentration inversely correlates with systemic inflammation: Patients that presented low plasma

citrulline concentrations also showed higher systemic inflammation. Moreover, low citrulline and gastrointestinal symptoms were associated with more severe diseases.

- I-FABP has also been studied in other infective conditions, such as Clostridioides difficile (see Oliva et al, Open Forum Infect Dis. 2019 December 3;7(1):ofz507) We have introduced this idea and this reference in the discussion

Methods

- "Time of study inclusion" means time of COVID-19 diagnosis?

Yes, and blood samples were drawn at the time of COVID-19 diagnosis. We have now precise this point in the discussion.

- How was diagnosis of COVID-19 made? PCR-based methods on which sample? We prospectively enrolled twenty-eight consecutive patients hospitalized for a PCR- confirmed COVID-19 on nasopharyngeal swabs.

We have now precise this point.

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

All patients exhibited pneumonia. This notion has been introduced in the text.

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

We separate patients as severe or non-severe on two criteria: hospitalization in an ICU/ and or death. This classification is now introduced in the text.

- "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.

We first compared our results with patients with abdominal pain of various origins. As COVID19 patients' results were lower, we wanted to know whether the pulmonary disease could be associated with a significant decrease in I-FABP. Still, we did not found this data in the literature. We have now precise the type of pulmonary disease.

- 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 to evaluate whether COVID-19 patients have values similar to those observed in healthy controls.

We have no healthy subject population. We did not get any authorization to include healthy subjects in the present study. According to the literature, normal values for IFABP are less than 90 pg/mL using the Hycult ELISA (Guzel et al, Surg Today 2014). Thus these data are in the range of those observed in COVID-19 patients.

- 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.

We initially did not match controls and patients according to age and sex. However, the mean age and the sex ratio are now precised in both populations.If necessary we can now perform the matching according to the age and sex.

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

Blood samples were drawn at the time of COVID-19 diagnosis, collected in appropriate tubes before immediate centrifugation at 3,000 rpm for 15 min at room temperature for sera, and subsequent storage at -80°C until further analysis.

- 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.

We have now precise the selection. ICU-group were patients. Regarding the ICU patients, there were both

- Statistical analyses description are absent in the method.

We have added this information.

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.

Among the whole COVID19 population, fourteen patients exhibited gastrointestinal symptoms. We have now precise this point in the text.

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

We have now included a table to present variables on patients.

- How many patients had COVID-19 associated pneumonia?

All patients exhibited pneumonia. This point has been added to the text.

- 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?

We found no correlation between I-FABP concentration and duration of symptoms. We have now added these data as supplemental Figure 2.

- 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?

We have corrected in the text: it was, in fact, a recent loss of appetite and not anorexia. So we have modified the text, since I is not a pathological preexisting condition.

- "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.

These has been added in the method section.

- 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 correlation is positive, and we have included the notion in the text.

- 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.

We have rewritten the discussion. Our hypothesis is that an increase of I-FABP is associated with some peculiar phenotypes of COVID-19, possibly those associated with bacterial infections.

- "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 rewritten The conclusions are not supported by the results of the study.

Authors should rewrite the last sentence of the study.

We have completely change the discussion and conclusion. We really think these modifications improve the lwhole manuscript.

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

We have added this information both in table 1 and figure legends.

- A table with study population characteristics would be appropriate

We have included the table with patients characteristic.

Figures

- Figure 1. The number of patients with abdominal pain is 80; the text is 79. Please correct.

We have corrected this mistake, 79 patients were included.

- Figure 3. It seems that only the coefficient is presented. Authors should also include the p-value.

We have added the value in supplemental table 1.

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.

25 Mar 2021

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

PONE-D-20-39264R1

Dear Dr. Guedj,

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6 Apr 2021

PONE-D-20-39264R1

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

Dear Dr. Guedj:

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