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Corresponding author and Reprint Requests: Rafael Simone Saia, Department of Physiology, Ribeirão Preto Medical School/USP. Avenida dos Bandeirantes, 3900. 14049-900 - Ribeirão Preto, SP, Brazil. Fax: +55 16 3315-0221 Phone: +55 16 3315-3199.
Laboratory of Intestinal Pathophysiology, Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
Laboratory of Intestinal Pathophysiology, Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
Division of Intensive Care Medicine, Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, BrazilIntensive Care Unit, Fundação Hospital Santa Lydia, Ribeirão Preto, São Paulo, Brazil
Division of Intensive Care Medicine, Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
Division of Intensive Care Medicine, Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
SARS-CoV-2 exhibits tropism for the gastrointestinal tract; however, lesions in enterocytes and their correlation with disease severity and patient prognosis are still unknown.
Methods
SARS-CoV-2 patients were enrolled in 5 medical centres in São Paulo, Brazil and their clinical characteristics and laboratory findings recorded. At admission, day 7 and day 14 of hospitalisation, plasma and urine samples were collected, and cytokine levels and intestinal fatty acid-binding protein (I-FABP) concentrations measured.
Results
COVID-19 patients displayed ≈48-, 74- and 125-fold increased urinary I-FABP levels at admission (n=283; P<0.001), day 7 (n=142; P<0.01) and day 14 (n=75; P<0.01) of hospitalisation. Critically ill patients and nonsurvivors showed higher I-FABP concentrations compared with patients with less severe illness. At admission, infected patients demonstrated enhanced production of plasma interferon (IFN)-γ and interleukin (IL)-6. The receiver operating characteristic curve suggested I-FABP as a biomarker for COVID-19 disease severity at admission (P<0.0001; Youden index=6.89; area under the curve=0.699). Patients with I-FABP ≥6.89 showed higher IL-6 and C-reactive protein levels (P<0.001) at admission and had a prolonged length of hospital stay.
Conclusions
Our findings revealed damage to enterocytes in SARS-CoV-2 infection, which is associated with illness severity, poor prognosis and exacerbated inflammatory response.
Gastrointestinal (GI) symptoms, including vomiting, abdominal discomfort and diarrhoea, have been reported in COVID-19 infections at the onset of hospitalisation, affecting 10%–30% of patients (
). COVID-19 patients with ongoing diarrhoea have displayed enhanced concentration of faecal calprotectin (a marker of gut inflammation), as well as systemic interleukin (IL)-6 production, regardless of viral load (
). A basal level of urinary I-FABP represents the physiological turnover rate of the epithelium, whilst its increment may indicate enterocytes cell damage. In sepsis and inflammatory bowel disease (IBD), the increased urinary concentration of I-FABP has been confirmed as a reliable biomarker and predictor of disease reactivation, worse prognosis and clinical complications (
). Therefore, our study assessed the levels of I-FABP in hospitalised patients with SARS-CoV-2 infection and investigated its role as a predictor of disease severity and poor prognosis.
This prospective cohort study enrolled 283 inpatients from 5 medical centres (Hospital das Clínicas, Hospital Santa Lydia, Hospital Unimed and Santa Casa de Misericórdia in Ribeirão Preto city and also Hospital e Maternidade Santa Isabel in Jaboticabal city), São Paulo State, Brazil (Supplementary methods). SARS-CoV-2 infection was confirmed through viral RNA detection in nasopharyngeal swabs by real-time reverse transcriptase-polymerase chain reaction, and positive cases were recruited from April 23rd to September 25th 2020. Comorbidities such as GI disorders (IBD, celiac disease and cancer), HIV, and alcohol and drugs addictions were exclusion criteria. At admission, day 7 and day 14 of hospitalisation (in infirmary or intensive care unit), venous blood and urine samples were collected in the morning (06:00–08:00), chilled and transferred to the research laboratory. Total blood and urine were centrifuged (1200 g, 15 min, 4 °C), the supernatants aliquoted and kept at -70 °C for further analysis. Healthy volunteers were seronegative for the presence of anti-SARS-CoV-2 immunoglobulin (Ig)M and IgG antibodies (Supplementary methods).
I-FABP and cytokines [tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-6] were quantified by enzyme-linked immunosorbent assay, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Urinary data were normalised to creatinine concentrations (LabTest, Lagoa Santa, MG, Brazil). Continuous variables were expressed as median (interquartile range [IQR]) or 10–90 percentile range and then compared using Mann-Whitney U-test or Kruskal-Wallis test (Supplementary results). Categorical variables were compared by Fisher's exact test or Pearson χ2 test. Statistical significance was considered different when P<0.05.
Table 1 lists the demographic, clinical characteristics, and laboratory findings of SARS-CoV-2 patients (survivors and nonsurvivors) and healthy volunteers. Nonsurvivors showed higher number of underlying comorbidities (cardiovascular and pulmonary diseases, diabetes, degenerative and malignancies), as well as deviations of the laboratory parameters (anaemia, leucocytosis, lymphocytopenia, D-dimers, C-reactive protein (CRP), lactate dehydrogenase, serum creatinine and urea), requirement of mechanic ventilation and intensive care at admission. At day 7 and day 14 of hospitalisation, aging, pre-existing diseases, leucocytosis, and the need for mechanical ventilation were relevant factors in nonsurvivor patients.
Table 1Demographic, clinical characteristics and laboratory parameters of healthy volunteers and SARS-CoV-2 infected patients (Survivors and Nonsurvivors)
Noteworthy, COVID-19 patients displayed ≈48-, 74- and 125-fold increased urinary I-FABP levels at admission, day 7 and day 14 of hospitalisation, respectively (Figure 1A; P<0.001). Critically ill patients showed higher I-FABP concentrations compared with those in the infirmary within the same hospital stay periods (Figure 1B). Likewise, nonsurvivor patients were distinguished by increased I-FABP levels in contrast to survivors (Figure 1C). At admission, the inflammatory response was characterised by augmented plasma IFN-γ and IL-6 levels in SARS-CoV-2 patients (Figure 1D). Interestingly, urinary IL-6 concentrations were elevated in infected patients and nonsurvivors (Figure 1E). The receiver operating characteristic (ROC) curve analysis indicated I-FABP as a good biomarker for disease severity and predictor for poor prognosis of COVID-19 at admission (Figure 1F; Youden index=6.89; p<0.0001) and prolonged hospital stay (Figure 1G). Patients with I-FABP ≥6.89 ng/mg showed higher plasma and urine IL-6 and serum CRP concentrations, suggesting an interaction between inflammation and damage to enterocytes (Figure 1G).
Figure 1Urinary intestinal fatty acid-binding protein (I-FABP) as a prognostic biomarker of SARS-CoV-2 disease severity. (A) Urinary I-FABP concentration determined by enzyme-linked immunosorbent assay (ELISA) in healthy volunteers and SARS-CoV-2 infected patients at admission, day 7 and day 14 of hospitalisation (Kruskal-Wallis test and Dunn's multiple comparison post-test). (B) Urinary I-FABP concentration in SARS-CoV-2 patients in non-critical and critical clinical conditions at admission, day 7 and day 14 of hospitalisation (Mann-Whitney test). (C) Urinary I-FABP concentration in SARS-CoV-2 survivor and nonsurvivor patients at admission, day 7 and day 14 of hospitalisation (Mann-Whitney test). (D) Plasma cytokines levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6 determined by ELISA at admission (Mann-Whitney test). (E) Urinary IL-6 concentration at admission in healthy volunteers, SARS-CoV-2 infected patients, survivors and nonsurvivors (Mann-Whitney test). (F) Comparison of receiver operating characteristics (ROC) curve analysis of urinary I-FABP (area under the curve (AUC)=0.699, 95% CI=0.636–0.762, P<0.0001), urinary IL-6 (AUC=0.573, 95% CI=0.476–0.671, P=0.14), plasma IL-6 (AUC=0.509, 95% CI=0.436–0.582, p=0.82), plasma IFN-γ (AUC=0.523, 95% CI=0.445–0.602, P=0.54) and serum CRP (AUC=0.591, 95% CI=0.519–0.662, P=0.01) for SARS-CoV-2 survivor and non-survivor patients. Cr: creatinine. (G) Plasma and urinary IL-6 concentrations, serum C-reactive protein levels at admission and length of hospital stay in SARS-CoV-2 infected patients divided according to the Youden index=6.89 ng/mg creatinine (low I-FABP <6.89 and high I-FABP ≥6.89 ng/mg) obtained from the ROC curve in (F) (Mann-Whitney test). Results shown as bars and violin plots are expressed as median and 10–90 interquartile range. n represents the number of patients per group. P values indicate the statistical significance between groups.
Figure 1Urinary intestinal fatty acid-binding protein (I-FABP) as a prognostic biomarker of SARS-CoV-2 disease severity. (A) Urinary I-FABP concentration determined by enzyme-linked immunosorbent assay (ELISA) in healthy volunteers and SARS-CoV-2 infected patients at admission, day 7 and day 14 of hospitalisation (Kruskal-Wallis test and Dunn's multiple comparison post-test). (B) Urinary I-FABP concentration in SARS-CoV-2 patients in non-critical and critical clinical conditions at admission, day 7 and day 14 of hospitalisation (Mann-Whitney test). (C) Urinary I-FABP concentration in SARS-CoV-2 survivor and nonsurvivor patients at admission, day 7 and day 14 of hospitalisation (Mann-Whitney test). (D) Plasma cytokines levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6 determined by ELISA at admission (Mann-Whitney test). (E) Urinary IL-6 concentration at admission in healthy volunteers, SARS-CoV-2 infected patients, survivors and nonsurvivors (Mann-Whitney test). (F) Comparison of receiver operating characteristics (ROC) curve analysis of urinary I-FABP (area under the curve (AUC)=0.699, 95% CI=0.636–0.762, P<0.0001), urinary IL-6 (AUC=0.573, 95% CI=0.476–0.671, P=0.14), plasma IL-6 (AUC=0.509, 95% CI=0.436–0.582, p=0.82), plasma IFN-γ (AUC=0.523, 95% CI=0.445–0.602, P=0.54) and serum CRP (AUC=0.591, 95% CI=0.519–0.662, P=0.01) for SARS-CoV-2 survivor and non-survivor patients. Cr: creatinine. (G) Plasma and urinary IL-6 concentrations, serum C-reactive protein levels at admission and length of hospital stay in SARS-CoV-2 infected patients divided according to the Youden index=6.89 ng/mg creatinine (low I-FABP <6.89 and high I-FABP ≥6.89 ng/mg) obtained from the ROC curve in (F) (Mann-Whitney test). Results shown as bars and violin plots are expressed as median and 10–90 interquartile range. n represents the number of patients per group. P values indicate the statistical significance between groups.
Epithelial cells from ileum and colon express angiotensin-converting enzyme-2, the host cell entry receptor of coronaviruses, invading them for replication (
). Soluble protein mediators, such as IFN-γ and IL-6, are key drivers of inflammation-associated enterocyte damage and responsible for increased gut mucosal permeability (
). Indeed, our findings confirmed the detrimental contribution of SARS-CoV-2 to the loss of enterocyte membrane integrity, which is correlated with increased I-FABP levels (up to day 14 after hospitalisation) and patient clinical condition. Moreover, patients with I-FABP ≥6.89 ng/mg also showed exacerbated IL-6 production, a predictive cytokine of disease progression and acute respiratory distress syndrome complication (
). Our results revealed that increased I-FABP levels at admission may predict poor prognosis and SARS-CoV-2 illness severity, such as need for intensive care, mechanic ventilation and prolonged hospital stay.
This study was supported by The Brazilian National Council for Scientific and Technological Development (CNPq, Grant #401337/2020-0), Ministry of Health and Ministry of Science, Technology and Innovation; The São Paulo Research Foundation (FAPESP, Grant #2020/10097-5); Foundation for Support to Education, Research and Assistance (FAEPA, Grant #416/2020 and #827/2021). The manuscript content is solely the responsibility of the authors and does not necessarily represent the official view of the Research Foundations.
Ethics approval
The study protocol was approved by the Research Ethics Committee of Clinics Hospital of Ribeirão Preto Medical School, University of São Paulo (protocol CAAE 30794820.7.0000.5440) and all patients or family members gave their informed consent to participate in this research.
Patient and public involvement
Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Acknowledgments
We are grateful to the excellent support of the medical teams (doctors, nurses, physiotherapists and psychologists) in the hospitals involved in this study: 1) Santa Lydia Hospital: Dr. Marcelo César Carboneri, Dr. Walther de O. Campos Filho, Bárbara R. Bauduíno Brandão, Eliane Bernardes de Queiróz, Luís F. de Melo Bougleux, Paula C. Trugillo Silva Neves, Renata C. Braulio da Silva, Silmara Miamoto, Sílvio Tolomeotti; 2) Unimed Ribeirão Preto Hospital: Dr. Gustavo Ribeiro de Oliveira, Dr. Plínio José E. de Camargo, Adriana de Fátima S. dos Santos, Bruna Maritan da Costa, Giovana A. Faggion Nomellini, Larissa Mil-Homens Albergaria; 3) Santa Casa de Misericórdia de Ribeirão Preto: Dr. Luiz C. Fontes Mega, Mariana C. Corrêa Marcolla, Rafael Cruz da Silva; and 4) Hospital e Maternidade Santa Isabel: Dr. Pedro Miguel Verardino, Edson Alves Julião, Nayara A. Poltronieri Correa.
Contributors
Study concept and design: RSS, HG, FLS, ABF. Involved in patient care: FLS, MAM, KMLM, AMD, MRC. Patient recruitment: RSS, HG, FLS, KJBP, MAM, AMD, MRC. Laboratory Analyses: RSS, HG. Data collection: RSS, HG, FLS, KJBP. Statistical analysis: RSS, ABF. Drafting the manuscript: RSS. All the authors edited the manuscript and have approved the final version.
Preprint Server
The present results have not been published as a preprint version.
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