Viral load and gastrointestinal inflammation in COVID-19 patients

Aditya Riadi Syafei; Titong Sugihartono; Iswan Abbas Nusi; Poernomo Boedi Setiawan; Herry Purbayu; Ummi Maimunah; Ulfa Kholili; Budi Widodo; Husin Thamrin; Amie Vidyani; Hasan Maulahela; Yoshio Yamaoka; Muhammad Miftahussurur

doi:10.12688/f1000research.120611.1

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Research Article

Viral load and gastrointestinal inflammation in COVID-19 patients

[version 1; peer review: 2 not approved]

Aditya Riadi Syafei¹, Titong Sugihartono², Iswan Abbas Nusi², [...] Poernomo Boedi Setiawan², Herry Purbayu², Ummi Maimunah², Ulfa Kholili², Budi Widodo², Husin Thamrin², Amie Vidyani², Hasan Maulahela³, Yoshio Yamaoka⁴, Muhammad Miftahussurur^2,5

Aditya Riadi Syafei¹, Titong Sugihartono², [...] Iswan Abbas Nusi², Poernomo Boedi Setiawan², Herry Purbayu², Ummi Maimunah², Ulfa Kholili², Budi Widodo², Husin Thamrin², Amie Vidyani², Hasan Maulahela³, Yoshio Yamaoka⁴, Muhammad Miftahussurur^2,5

PUBLISHED 12 Jul 2022

Author details Author details

¹ Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga, Surabaya, Jawa Timur, 60286, Indonesia
² Gastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga,, Surabaya, Jawa Timur, 60286, Indonesia
³ Division of Gastroenterology, Department of Internal Medicine, Faculty of Medicine-Cipto Mangunkusumo Teaching Hospital, University of Indonesia, Jakarta, 10430, Indonesia
⁴ Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, Japan
⁵ Helicobacter pylori and Microbiota Study Group, stitute of Tropical Disease, Universitas Airlangga, Surabaya, Jawa Timur, 60115, Indonesia

Aditya Riadi Syafei
Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Titong Sugihartono
Roles: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing

Iswan Abbas Nusi
Roles: Methodology, Resources

Poernomo Boedi Setiawan
Roles: Data Curation, Validation

Herry Purbayu
Roles: Methodology, Writing – Review & Editing

Ummi Maimunah
Roles: Methodology, Writing – Original Draft Preparation

Ulfa Kholili
Roles: Validation, Writing – Review & Editing

Budi Widodo
Roles: Conceptualization, Writing – Review & Editing

Husin Thamrin
Roles: Methodology, Resources

Amie Vidyani
Roles: Data Curation, Validation

Hasan Maulahela
Roles: Data Curation, Investigation

Yoshio Yamaoka
Roles: Data Curation, Supervision

Muhammad Miftahussurur
Roles: Conceptualization, Supervision, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Pathogens gateway.

This article is included in the Emerging Diseases and Outbreaks gateway.

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Abstract

Background: ACE-2 receptors are well-known as binding receptors to spike protein of SARS-CoV-2 highly expressed in the gastrointestinal system. The Role of SARS-CoV-2 viral load and its effect on gut inflammation in COVID-19 patients are still not well-understood. This study aims to determine the impact of SARS-CoV-2 viral load on gastrointestinal inflammation.
Methods: A total of 44 inpatient subjects who fulfilled eligibility criteria were examined for cycle threshold values from nasopharyngeal swab samples collected from several nucleic levels based on fluorescence signal and calprotectin levels of stool samples using Calprotectin enzyme-linked immunosorbent assay (ELISA) kit.
Results: Of 44 subjects, 52.3% were male, with a median age of 52.5 years. Hypertension or diabetes was found in 26 patients. The median cycle threshold value was 31.3 with a value range of 10.9-40.0, median cycle threshold was significantly lower in subjects with comorbidity with P = 0.01. The median fecal calprotectin level was 42 μg/g with a value range of 5.1-1,393.7 μg/g, with median fecal calprotectin significantly higher in subjects with gastrointestinal symptoms with P = 0.008 with a relative risk (RR) of 5.5. There was a significant correlation between cycle threshold and fecal calprotectin in subjects with comorbidity with P <0.05, a coefficient contingency of 0.414.
Conclusion: Subjects with comorbidity are prone to have higher viral loads paralleled with gastrointestinal inflammation. Subjects with overt gastrointestinal manifestations had a five-fold higher degree of gut inflammation.

Keywords

COVID-19, cycle threshold, fecal calprotectin, gastrointestinal symptoms, infections

Corresponding authors: Titong Sugihartono, Yoshio Yamaoka

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2022 Syafei AR et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Syafei AR, Sugihartono T, Nusi IA et al. Viral load and gastrointestinal inflammation in COVID-19 patients [version 1; peer review: 2 not approved]. F1000Research 2022, 11:784 (https://doi.org/10.12688/f1000research.120611.1) First published: 12 Jul 2022, 11:784 (https://doi.org/10.12688/f1000research.120611.1) Latest published: 12 Jul 2022, 11:784 (https://doi.org/10.12688/f1000research.120611.1)

Introduction

Coronavirus Disease 2019 (COVID-19) has been declared a global pandemic by World Health Organization (WHO) since March 2020.¹ COVID-19 is caused by infection of Coronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the genus of Betacoronavirus and family of Coronaviridae, similar to SARS-CoV and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) that cause respiratory problems. The virus infects host cells that express Angiotensin-Converting Enzyme 2 (ACE-2) receptor, it is found higher in the lungs and gastrointestinal system. Infection of SARS-CoV-2 activates the innate immune system and then releases cytokines, chemokines, and pro-inflammatory mediators.²

The current diagnostic test suggested by WHO is based on Nucleic Acid Amplification Test (NAAT), such as Reverse-Transcription Polymerase Chain Reaction (RT-PCR). The cycle threshold is the number of replication cycles required by the fluorescence signal to pass the threshold. Cycle threshold inversely correlated to quantitative viral loads detected in the sample, although PCR testing does not distinguish live viruses from nonviable viruses.³^,⁴ Several studies showed the clinical utility of cycle threshold value and high viral load correlated with disease severity, but other studies show no correlation in different samples settings.³^,⁵^,⁶ Clinical symptoms of COVID-19 are fever, cough, respiratory symptoms, and currently, extra-pulmonary clinical manifestations, including gastrointestinal, have been reported to vary in more than 20% of hospitalized patients. One study in 2020 showed the presence of the SARS-CoV-2 genome in stool samples.⁶^,⁷ This proves that the SARS-CoV-2 infection in the gastrointestinal tract is thought to be caused by the expression of ACE-2 in the digestive tract. Transmembrane Serine Protease 2 (TMPRSS2) is expressed including in gastrointestinal epithelium, that facilitates cell entry by priming of the spike protein.⁸

Several studies found virus RNA in stool specimens and anal swabs, however, it remains unclear when the virus starts shedding in the gastrointestinal tract and takes a longer duration of viral shedding than in the respiratory tract. Gastrointestinal manifestations in SARS-CoV-2 infected patients provide clues whether inflammation occurs in gastrointestinal tissue. Fecal calprotectin, a protein released by neutrophils and used as a direct marker of mucosal inflammation of any etiology, sensitive and non-invasive biomarker in mucosal inflammation.⁹^,¹⁰ Diarrhea in cases of viral infection can naturally be inflammatory or non-inflammatory, based on the research data, it includes the results of non-inflammatory mechanisms. Another study in 2021 showed an increase in fecal calprotectin levels in 66.7% of COVID-19 patients in asymptomatic gastrointestinal tract patients.¹¹^,¹²

The gastrointestinal system in SARS-CoV-2 infected patients occur gut microbiome alteration characterized by a decrease in diversity with shifting toward pathogenic bacteria and away from beneficial symbionts. The virus enters cells by recognizing receptor ACE-2 to invade the host cell via TMPRSS2 and initiate an immune response that results in increased gut permeability and dysbiosis.¹² Gut dysbiosis and ACE-2 impairment lead to a leaky gut. In a viral infection, high levels of circulating pro-inflammatory cytokines can alter the gut microbiome and disturb intestinal integrity. Increased inflammation in the intestine leads to a leaky gut allowing bacterial antigens and toxins to translocate to the systemic circulation, leading to worsening clinical outcomes in COVID-19 patients.¹²^,¹³

Studies analyzing fecal calprotectin levels in SARS-CoV-2 infection are relatively still limited.¹¹ Some studies mention the association between cycle threshold values and disease severity, while other studies show significant fecal calprotectin increase results in COVID-19 patients, but studies on the association between the two have yet to be understood.⁵^,⁶^,¹¹ We determined a correlation between the cycle threshold value of nasopharyngeal swab and fecal calprotectin in COVID-19 patients.

Methods

Study design and participants

This research was an observational analytic study with a cross-sectional design. We conducted consecutive sampling at Dr. Soetomo Teaching Hospital Surabaya from 1 September to 30 November 2020. Since there was no prior study of these specific subjects we decided to count the minimum sample size using infinite population formula. We analyzed 44 samples from non-ICU that meet this research eligibility criterion. Inclusion criteria were age 18 or above, a confirmed case of COVID-19, and signed participants’ agreement of this study. Exclusion criteria were the patient's refusal to participate in this study, gastrointestinal malignancies, liver cirrhosis, End-Stage Renal Disease, and Inflammatory Bowel Disease. Written informed consent was obtained from all patients and the research protocol approved by Faculty of Medicine, Universitas Airlangga-Dr. Soetomo Teaching Hospital Surabaya, Indonesia health research ethics committee with certificate number 0065/KEPK/IX/2020. Patients confirmed COVID-19 based on PCR results from nasopharyngeal swab specimens. The RT-PCR cycle threshold value was measured from an analytical phase of a nasopharyngeal swab specimen using a nucleocapsid (N) gene target with a negative value >40. Analytical phase was conducted in bio-safety level 2 facility in close system with automated process, using rt-PCR kit Biocov, Acon and light cycler Roche or Gentier (China). Cycle threshold value data were collected from clinical microbiologists at The Department of Clinical Microbiology Faculty of Medicine Universitas Airlangga, Surabaya. Fecal calprotectin level is calprotectin measured in the patient's feces sample. Stool preparations are taken by the nurse or by the patient. Stool with a minimum of five grams was analyzed using the PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim). Calprotectin remained stable in feces for six days with a normal reference value of <50 μg/g. We took 44 samples that were eligible for this study and analyzed all samples as subjects fulfilled all research variables.

Statistical analysis

Data were analyzed using the IBM SPSS Statistics version 25 (IBM Corp., USA). Demographic data and clinical characteristics are presented descriptively, frequency and percent for categorical data types (nominal and ordinal), while mean, standard deviation, median, and minimum-maximum for continuous data (interval and ratio). The independent variable is the cycle threshold value as numerical data (ratio and interval). The dependent variable is fecal calprotectin level as numerical data (ratio and nominal). Correlational numerical analytic tests carried out the association between variables in this study. The correlation test was performed using Pearson. The P-value that is considered statistically significant was <0.05 and the confidence interval (CI) was 95%.

Results

Characteristics of subjects

Subjects based on gender were male with 52.3% and female was 47.7%. The median age of subjects was 52.5 years with the youngest age of 18 years and the oldest of 65 years. The highest number of subjects was based on a range of age 51-60 years at 29.54%. Eighteen subjects were without comorbidity (40.9%) and the highest comorbidity in subjects was diabetes mellitus and hypertension in 14 patients (31.8%), followed by diabetes mellitus 18.2% and hypertension 9.1%. The characteristics of subjects based on clinical manifestations were cough 75%, fever 70.5%, shortness of breath 68.2%, and anosmia 9.1%. The characteristics of subjects based on gastrointestinal manifestations were anorexia 63.6%, nausea 38.6%, diarrhea 31.8%, vomiting 29.5%, and abdominal pain 11.4%. Mean symptom onset was 4.02 ± 2.64 days with a median of three days. The shortest onset was one day and the longest onset was 14 days.

Table 1 shows median systolic and diastolic blood pressures of 130 and 80 mmHg, the median pulse of 88 times per minute, median respiratory rate of 21.5 times per minute, median axillary temperature of 36.7°C, and peripheral oxygen saturation of 97%. Laboratory characteristics of subjects showed a median lymphocyte value of 1,295/mm³ slightly below the normal range of 1,300-4,000/mm³, the median neutrophil-lymphocyte ratio was 4.9348, median ferritin was 588.4 μg/L, and median D-dimer was 1,150 μg/L.

Table 1. Clinical and laboratory characteristics.

Characteristics	Total (n = 44)
Characteristics	Median	Min	Max
Systolic blood pressure (mmHg)	130	90	170
Diastolic blood pressure (mmHg)	80	58	100
Pulse (times per minute)	88	61	110
Respiratory rate (times per minute)	21.5	18	26
Temperature (^oC)	36.7	36	38
Peripheral oxygen saturation (%)	97	72	100
Hemoglobin (g/dl)	12.4	7.5	14.9
Leukocytes (/mm³)	9,280	4,090	25,380
Absolute neutrophils (/mm³)	6,930	2,670	20,510
Absolute lymphocyte (/mm³)	1,295	250	2,690
Neutrophil-lymphocyte ratio	4.9348	1.98	44.59
Platelets (/mm³)	330,000	107,000	620,000
CRP (mg/dl)	0.8	0.1	7.8
Ferritin (μg/L)	588,400	24.6	3,000
D-dimer (ng/ml)	1,150	190	35,200
Procalcitonin (μg/L)	0.09	0.01	5.37

Nasopharyngeal swab RT-PCR cycle threshold value in subjects

The measurement of cycle threshold value was obtained based on fluorescence with RT-PCR technique used to detect SARS-CoV-2 genes in upper respiratory tract specimens of subjects. This value is obtained based on a real-time amplification curve based on signal changes and then determines the quantitative detection of SARS-CoV-2 at the nucleic acid level. The mean cycle threshold value for all subjects was 30.33 ± 6.07 with a median of 31.31, a minimum value of 10.09, and a maximum of 40.00. The median cycle threshold value in subjects with comorbidity was 30.4 significantly lower (P = 0.01). Comorbidity consists of diabetes mellitus, hypertension, or a combination of these. Distribution of subjects with gastrointestinal symptoms and subjects with diarrhea symptoms did not significantly differ in cycle threshold value (Mann-Whitney test, P = 0.921, and P = 0.521, respectively). Distribution of cycle threshold value in subjects group based on WHO severity classification also did not show a significant difference between mild, moderate, severe, or critical (the Kruskal-Wallis test, P = 0.436).

Fecal calprotectin levels in subjects

Based on the kit used in the laboratory, fecal calprotectin cut-off value <50 μg/g was considered negative. In this study, 26 subjects (59.1%) with fecal calprotectin levels <50 μg/g, subjects with fecal calprotectin levels ≥50 μg/g were 18 patients (40.9%). The mean fecal calprotectin level in all subjects is 124.51 ± 240.54 μg/g with a median of 42 μg/g and a minimum value of 5.1 μg/g and a maximum of 1,393.7 μg/g. Table 2 showed the results of the Mann-Whitney test, statistically significant differences in fecal calprotectin increase in the group of subjects with gastrointestinal symptoms (61.70 vs 21.55 μg/g, P = 0.008, respectively). This study also found that fecal calprotectin correlates with hemoglobin levels with P < 0.05 and a coefficient correlation of -.307. The median value of fecal calprotectin levels was found higher in groups of subjects with symptoms of diarrhea than in subjects without symptoms of diarrhea (143.45 vs 24.4 μg/g, P < 0.0001, respectively). Subjects with symptoms of diarrhea tend to have fecal calprotectin levels ≥50 μg/g with a relative risk (RR) of 5.571 (95% CI; IQR 2.47-12.56) with P < 0.0001 in the Chi-Square test. This study also found that fecal calprotectin correlates with D-dimer in subjects with symptoms of diarrhea with P = 0.024 and a coefficient correlation of.598 (normal reference of D-dimer <500 ng/ml).

Table 2. Fecal calprotectin levels in subjects with gastrointestinal symptoms.

Subjects (n = 44)	Fecal calprotectin (μg/g)
Subjects (n = 44)	Mean ± SD	Median	Min	Max	P*
With gastrointestinal symptoms	170.65 ± 280.64	61.70	5.1	1,393.7	0.008
Without gastrointestinal symptoms	25.64 ± 15.92	21.55	5.1	50.2	0.008
With diarrhea	298.0 ± 365.47	143.45	47.2	1,393.7	<0.0001
Without diarrhea	43.55 ± 67.88	24.4	5.1	326.5	<0.0001

* Mann-Whitney test; SD: Standard Deviation.

Cycle threshold and fecal calprotectin

The non-parametric Spearman correlation test showed no significant correlation between the cycle threshold value and fecal calprotectin levels (P = 0.613; r = 0.078) in this study. Spearman's non-parametric test showed no statistically significant relationship between the cycle threshold value and fecal calprotectin levels in subjects with gastrointestinal symptoms and diarrhea symptoms (P = 0.353 and P = 0.817, respectively). However, in subjects with comorbidity, based on Fisher’s exact test there was a sufficient correlation between cycle threshold value with fecal calprotectin cut-off of ≥50 μg/g with P < 0.05, contingency coefficient of 0.414.

Discussion

This study found that viral load correlates with gastrointestinal inflammation in the population with comorbidity. This study also showed patients with comorbidity have higher viral loads than subjects without comorbidity. These findings suggest that comorbidity becomes an independent risk factor for gastrointestinal inflammation and an infection risk factor.¹⁴ Studies showed that people with comorbidity are at higher risk of infection and have more severe disease manifestations. Several studies suggested that patients with comorbidity had compromised immune systems to infection.¹⁵ This study found that subjects with comorbidity had a lower cycle threshold than subjects without comorbidity suggesting that patients with comorbidity are prone to have more viral load and inadequate immune response to infection. A study in 2020 found that the risk of infection in a population with comorbidities such as diabetes mellitus or hypertension is associated with the up-regulation of ACE-2. Another study also showed a risk of mortality relatively higher in populations with comorbidity.¹⁵^,¹⁶

Widely known SARS-CoV-2 is attached to ACE-2 receptors in the respiratory or gastrointestinal system. Up-regulation of ACE-2 receptors in comorbidity population, attached with the virus with inappropriate immune response causing ineffective defense mechanism against the virus and replicating at higher levels.¹⁵^,¹⁶ A study in 2020 stated that patients with comorbidity had a decrease in gut microbiome diversity. The gut microbiome had several benefits as normal flora in gastrointestinal systems, such as a defense mechanism against pathologic entities. Several pathomechanisms disrupt gastrointestinal mucosal homeostasis.¹⁷ This study found in subjects with diarrhea symptoms, fecal calprotectin correlates with D-dimer. COVID-19 that occurs hypercoagulable state, caused by endothelial dysfunction that causes fibrin to degrade and release D-dimer protein component in the infected patient was found significantly increased. This pro-thrombotic state could induce tissue ischemia, including intestinal mucosa layer, activation of a local gut immune response, and induce calprotectin release.¹⁸^,¹⁹ This study also found that fecal calprotectin negatively correlates with hemoglobin. This founding shed a new perspective on the role of thrombosis and the consequences of intestinal damage. Gastrointestinal inflammation correlates with more severe anemia due to colitis or micro gastrointestinal bleeding need further study to investigate. Hyper-inflammatory state in COVID-19 not only causes alteration in gastrointestinal homeostasis led to inflammation but also alters the metabolism of iron by producing hepcidin, in response to IL-6 activity, that causes reduced transferrin and inhibits iron absorption in the intestinal ultimately causing iron deficiency. The limited understanding of gastrointestinal inflammation and its effect on systemic iron metabolism needs further investigation.¹⁸^–²¹

This study also found fecal calprotectin potential as a non-invasive biomarker to determine disruption of gastrointestinal mucosal homeostasis, whether due to direct viral infection on gut epithelial cells or secondary to systemic inflammation response. This study also found that gut inflammation happened more severely in subjects with an overt gastrointestinal manifestation such as abdominal pain, diarrhea, nausea, vomiting, and anorexia. One study suggests viral infect enteroids, disrupt intestinal permeability resulting in enterocyte dysfunction.²² Studies in 2020 also support these findings of significantly increasing fecal calprotectin in patients who suffered diarrhea.²³ One study suggests that symptoms may be direct mucosal pathology of viral infection or secondary to virus-induced inflammation, in turn, due to the entry of inflammatory cells into intestinal mucosa, including neutrophils and lymphocytes, and thus disruption of gut microbiota. This also found higher viral loads in stool specimens in subjects with diarrhea symptoms.²⁴ One study in 2021 found specific Immunoglobulin A (IgA) of Receptor Binding Domain (RBD) specific to SARS-CoV-2 in stool, this suggesting that immune response to inflammation in a gut system can be primary or secondary from a systemic response.²⁵

Several studies found that viral clearance in the gut system takes longer than in the respiratory system. Virus clearance in the gut system varies up to several days to weeks after seroconversion from upper respiratory samples.²⁶ A study conducted in 2020 stated that patients with gastrointestinal symptoms have more extended duration virus clearance and are more likely to have positive stool tests. This suggests the potential risk of fecal-oral transmission and raises the question of whether this prolonged delayed clearance in the gut system causes persistent gastrointestinal inflammation or becomes one of the long COVID-19 manifestations that needs further investigation.²⁷

There were several limitations of this study. The monocentric study with a small sample of this study might not be enough to represent the general population. Therefore, further investigation with more samples collected might be needed. In addition, this study did not consider the time of sample collecting and did not analyze with cycle threshold from anal swabs or stool specimens.

Conclusion

There were significantly higher viral loads in subjects with comorbidity in this study. Subjects with overt gastrointestinal manifestations had a five-fold higher degree of gut inflammation. Viral loads and gut inflammation were correlated in subjects with comorbidity.

Data availability

Figshare. Raw Data of Viral Load and Gastrointestinal Inflammation Research in COVID-19 Patients.xlsx. DOI: https://doi.org/10.6084/m9.figshare.19769218.v2.²⁸

This project contains the following underlying data:

- The raw material data contain much information about general biodata patients, the observational result of vital signs, head/neck, thoracal, abdominal, and extremity.
- In addition, the data include Peripheral Complete Blood Count, Inflammation markers, CT value to see the viral load, and the fecal calprotectin

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Author contribution

Syafei, AR: Conceptualization, Methodology, Writing—original draft preparation; Writing—review and editing; Sugihartono, T: Conceptualization, Writing—original draft preparation, Writing—review and editing; Nusi, IA: Methodology, Resource; Setiawan, PB: Data accuration, Validation; Purbayu, H: Methodology, Writing—review and editing; Maimunah, U: Methodology, Writing—original draft preparation; Kholili, U: Writing—review and editing, Validation; Widodo, B: Conceptualization, Writing—review and editing; Thamrin, H: Methodology, Resource; Vidyani, A: Data curration, Validation; Maulahela, H: Data accuration, Investigation; Yamaoka, Y: Data Accuration, Supervision; Miftahussurur, M: Conceptualization, Supervision, Writing—original draft preparation.

Acknowledgments

The authors would like to thank to Faculty of Medicine Universitas Airlangga, Surabaya, East Java, Indonesia.

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Version 1

VERSION 1 PUBLISHED 12 Jul 2022

Author details Author details

Aditya Riadi Syafei
Roles: Conceptualization, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Titong Sugihartono
Roles: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing

Iswan Abbas Nusi
Roles: Methodology, Resources

Poernomo Boedi Setiawan
Roles: Data Curation, Validation

Herry Purbayu
Roles: Methodology, Writing – Review & Editing

Ummi Maimunah
Roles: Methodology, Writing – Original Draft Preparation

Ulfa Kholili
Roles: Validation, Writing – Review & Editing

Budi Widodo
Roles: Conceptualization, Writing – Review & Editing

Husin Thamrin
Roles: Methodology, Resources

Amie Vidyani
Roles: Data Curation, Validation

Hasan Maulahela
Roles: Data Curation, Investigation

Yoshio Yamaoka
Roles: Data Curation, Supervision

Muhammad Miftahussurur
Roles: Conceptualization, Supervision, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

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The author(s) declared that no grants were involved in supporting this work.

Article Versions (1)

version 1

Published: 12 Jul 2022, 11:784

https://doi.org/10.12688/f1000research.120611.1

© 2022 Syafei AR et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Syafei AR, Sugihartono T, Nusi IA et al. Viral load and gastrointestinal inflammation in COVID-19 patients [version 1; peer review: 2 not approved] F1000Research 2022, 11:784 (https://doi.org/10.12688/f1000research.120611.1)

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Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 1

VERSION 1

PUBLISHED 12 Jul 2022

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Reviewer Report 04 Apr 2023

Ener Cagri Dinleyici, Department of Pediatrics, Faculty of Medicine, Eskisehir Osmangazi University, Eskişehir, Turkey

Not Approved

https://doi.org/10.5256/f1000research.132472.r165778

This is a prospective study which aims to evaluate gastrointestinal involvement in patients with COVID 19, during the first phase of the pandemic in 2020. Although the idea was original at the time of the study, it has been more than 3 years since the pandemic and a vast knowledge of SARS-CoV-2 has emerged. The sample size is too small to make some conclusions, in addition to this, patient population is so heterogenous to evaluate GI inflammation. The authors make some conclusions about the GI invıolvement and presence of co-morbidities, but the sample size is not enough to make this conclusion. Fecal calprotectin is a marker for GI inflammation however not specific for one disease and might be increased in different conditions including FGID.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Microbiota, Intestinal microbiota composition

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 09 Aug 2022

Yen-Po Wang, Institute of Brain Science, Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan

Not Approved

https://doi.org/10.5256/f1000research.132472.r145103

This is a prospective study evaluating GI symptoms, diarrhea, and laboratory values in patients with COVID-19 infection. The idea was sound but the statistical analysis wasn't complete and therefore some issues need further clarification.

Many diseases

Many diseases or conditions may be related with fecal calprotectin. The underlying disease of patients should be added in the demogrpahic data. How many patients had functional GI disease prior to COVID-19 infection? How many patients received abdominal surgery prior to COVID-19 infection?
In table 1, ferritin level should be further checked. Neurophil-lymphocyte ratio digit number should be identical. Standard deviation may be added on the table.
It seemed that many patietns had increasd fecal calprotectin in this study. How many patients had fecal calprotectin level >250 ug/g? What were their findings in colonsocopy? Were any of them found to have IBD?
In patients with diarrhea, did they receive stool culture exam? Are there any patients found with enteric infection, or even Clostridium difficile symptoms?
What did 'gastrointestinal symptom' mean and include in this study?
In patients with comorbidity, fecal calprotectin was related with cycle threshold value. What is the difinition of comorbidity? How many patients had comorbidity? Did it mean GI comorbiidity or also other comorbidity? The authors stated that comorbitidy is an independent risk factor for GI inflammation, however, there is no multivariate analysis or regression analysis in evluating this issue in this study.
When did the patients receive stool tests? What is the durgation between NP swab and stool tests?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: functional GI disease, IBD, GERD, endoscopy, small bowel endoscopy, EUS

CITE

Report a concern

Author Response 02 Sep 2022

Muhammad Miftahussurur, Gastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga,, Surabaya, 60286, Indonesia

02 Sep 2022

Author Response
Response to Reviewer 1:
1. Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria
... Continue reading
Response to Reviewer 1:

Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria that we believed relate to fecal calprotectin level as we mentioned the exclusion criteria in Methods section Study design and participants page 10, we excluded patients with history of gastrointestinal malignancies and Inflammatory Bowel Disease. History of abdominal surgery we did not include in the criteria, therefore we will add it in the study limitations.

Thank you for your suggestion. We will resolve the mis-type in ferritin and also add the mean and standard deviation as per the reviewer's suggestion.

Thank you for your question. In this study we were using laboratory tool cut-off point from PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim) with value of <50 μg/g considered negative. Regarding samples with fecal calprotectin level >250 μg/g, there were six patients but due to limited resources and safety concerns, patients have not undergone colonoscopy, as we will add in the limitations.

Thank you for your question. The total number of subjects involved in our study is 44 patients, none of them received stool culture examination nor stool examination. We will disclose this as limitations.

Thank you for your question. We defined gastrointestinal symptoms as nausea, vomitting, anorexia, diarrhea and abdominal pain. We will add this definition in the Characteristic subjects.

Thank you for your question and comment. In this study we grouped comorbidity as diabetes mellitus, hypertension, or both. In this study we found 26 patients with comorbidity. After we did regression we found no significant correlation between comorbidity and gut inflammation, therefore we will rephrase the statement ”comorbidity becomes an independent risk factor for gastrointestinal inflammation” to “comorbidity becomes a risk factor for an infection”.

Thank you for your question. The patients received stool test within 24 hours after patients had confirmed positive results from nasopharyngeal swab.
Response to Reviewer 1:

Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria that we believed relate to fecal calprotectin level as we mentioned the exclusion criteria in Methods section Study design and participants page 10, we excluded patients with history of gastrointestinal malignancies and Inflammatory Bowel Disease. History of abdominal surgery we did not include in the criteria, therefore we will add it in the study limitations.

Thank you for your suggestion. We will resolve the mis-type in ferritin and also add the mean and standard deviation as per the reviewer's suggestion.

Thank you for your question. In this study we were using laboratory tool cut-off point from PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim) with value of <50 μg/g considered negative. Regarding samples with fecal calprotectin level >250 μg/g, there were six patients but due to limited resources and safety concerns, patients have not undergone colonoscopy, as we will add in the limitations.

Thank you for your question. The total number of subjects involved in our study is 44 patients, none of them received stool culture examination nor stool examination. We will disclose this as limitations.

Thank you for your question. We defined gastrointestinal symptoms as nausea, vomitting, anorexia, diarrhea and abdominal pain. We will add this definition in the Characteristic subjects.

Thank you for your question and comment. In this study we grouped comorbidity as diabetes mellitus, hypertension, or both. In this study we found 26 patients with comorbidity. After we did regression we found no significant correlation between comorbidity and gut inflammation, therefore we will rephrase the statement ”comorbidity becomes an independent risk factor for gastrointestinal inflammation” to “comorbidity becomes a risk factor for an infection”.

Thank you for your question. The patients received stool test within 24 hours after patients had confirmed positive results from nasopharyngeal swab.
Competing Interests: We have no conflicts of interest to disclose. Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 02 Sep 2022

Muhammad Miftahussurur, Gastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga,, Surabaya, 60286, Indonesia

02 Sep 2022

Author Response
Response to Reviewer 1:
1. Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria
... Continue reading
Response to Reviewer 1:

Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria that we believed relate to fecal calprotectin level as we mentioned the exclusion criteria in Methods section Study design and participants page 10, we excluded patients with history of gastrointestinal malignancies and Inflammatory Bowel Disease. History of abdominal surgery we did not include in the criteria, therefore we will add it in the study limitations.

Thank you for your suggestion. We will resolve the mis-type in ferritin and also add the mean and standard deviation as per the reviewer's suggestion.

Thank you for your question. In this study we were using laboratory tool cut-off point from PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim) with value of <50 μg/g considered negative. Regarding samples with fecal calprotectin level >250 μg/g, there were six patients but due to limited resources and safety concerns, patients have not undergone colonoscopy, as we will add in the limitations.

Thank you for your question. The total number of subjects involved in our study is 44 patients, none of them received stool culture examination nor stool examination. We will disclose this as limitations.

Thank you for your question. We defined gastrointestinal symptoms as nausea, vomitting, anorexia, diarrhea and abdominal pain. We will add this definition in the Characteristic subjects.

Thank you for your question and comment. In this study we grouped comorbidity as diabetes mellitus, hypertension, or both. In this study we found 26 patients with comorbidity. After we did regression we found no significant correlation between comorbidity and gut inflammation, therefore we will rephrase the statement ”comorbidity becomes an independent risk factor for gastrointestinal inflammation” to “comorbidity becomes a risk factor for an infection”.

Thank you for your question. The patients received stool test within 24 hours after patients had confirmed positive results from nasopharyngeal swab.
Response to Reviewer 1:

Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria that we believed relate to fecal calprotectin level as we mentioned the exclusion criteria in Methods section Study design and participants page 10, we excluded patients with history of gastrointestinal malignancies and Inflammatory Bowel Disease. History of abdominal surgery we did not include in the criteria, therefore we will add it in the study limitations.

Thank you for your suggestion. We will resolve the mis-type in ferritin and also add the mean and standard deviation as per the reviewer's suggestion.

Thank you for your question. In this study we were using laboratory tool cut-off point from PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim) with value of <50 μg/g considered negative. Regarding samples with fecal calprotectin level >250 μg/g, there were six patients but due to limited resources and safety concerns, patients have not undergone colonoscopy, as we will add in the limitations.

Thank you for your question. The total number of subjects involved in our study is 44 patients, none of them received stool culture examination nor stool examination. We will disclose this as limitations.

Thank you for your question. We defined gastrointestinal symptoms as nausea, vomitting, anorexia, diarrhea and abdominal pain. We will add this definition in the Characteristic subjects.

Thank you for your question and comment. In this study we grouped comorbidity as diabetes mellitus, hypertension, or both. In this study we found 26 patients with comorbidity. After we did regression we found no significant correlation between comorbidity and gut inflammation, therefore we will rephrase the statement ”comorbidity becomes an independent risk factor for gastrointestinal inflammation” to “comorbidity becomes a risk factor for an infection”.

Thank you for your question. The patients received stool test within 24 hours after patients had confirmed positive results from nasopharyngeal swab.
Competing Interests: We have no conflicts of interest to disclose. Close
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Version 1

VERSION 1 PUBLISHED 12 Jul 2022

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	1	2
Version 1 12 Jul 22	read	read

Yen-Po Wang, National Yang-Ming University School of Medicine, Taipei, Taiwan
Ener Cagri Dinleyici, Eskisehir Osmangazi University, Eskişehir, Turkey

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Reviewer Report

2 Views

04 Apr 2023 | for Version 1

Ener Cagri Dinleyici, Department of Pediatrics, Faculty of Medicine, Eskisehir Osmangazi University, Eskişehir, Turkey

2 Views Cite this report Responses(0)

Not Approved

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiota, Intestinal microbiota composition

Respond to this report

Responses (0)

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Reviewer Report

23 Views

09 Aug 2022 | for Version 1

Yen-Po Wang, Institute of Brain Science, Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan

23 Views Cite this report Responses(1)

Not Approved

Many diseases or conditions may be related with fecal calprotectin. The underlying disease of patients should be added in the demogrpahic data. How many patients had functional GI disease prior to COVID-19 infection? How many patients received abdominal surgery prior to COVID-19 infection?
In table 1, ferritin level should be further checked. Neurophil-lymphocyte ratio digit number should be identical. Standard deviation may be added on the table.
It seemed that many patietns had increasd fecal calprotectin in this study. How many patients had fecal calprotectin level >250 ug/g? What were their findings in colonsocopy? Were any of them found to have IBD?
In patients with diarrhea, did they receive stool culture exam? Are there any patients found with enteric infection, or even Clostridium difficile symptoms?
What did 'gastrointestinal symptom' mean and include in this study?
In patients with comorbidity, fecal calprotectin was related with cycle threshold value. What is the difinition of comorbidity? How many patients had comorbidity? Did it mean GI comorbiidity or also other comorbidity? The authors stated that comorbitidy is an independent risk factor for GI inflammation, however, there is no multivariate analysis or regression analysis in evluating this issue in this study.
When did the patients receive stool tests? What is the durgation between NP swab and stool tests?

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

functional GI disease, IBD, GERD, endoscopy, small bowel endoscopy, EUS

Respond to this report

Responses (1)

Author Response

02 Sep 2022

Muhammad Miftahussurur, Gastroentero-Hepatology Division, Department of Internal Medicine, Faculty of Medicine-Dr. Soetomo Teaching Hospital, Universitas Airlangga,, Surabaya, 60286, Indonesia

Response to Reviewer 1:

Thank you for your suggestion and questions. We did not put underlying disease into account, furthermore we excluded patients from samples based on criteria that we believed relate to fecal calprotectin level as we mentioned the exclusion criteria in Methods section Study design and participants page 10, we excluded patients with history of gastrointestinal malignancies and Inflammatory Bowel Disease. History of abdominal surgery we did not include in the criteria, therefore we will add it in the study limitations.
Thank you for your suggestion. We will resolve the mis-type in ferritin and also add the mean and standard deviation as per the reviewer's suggestion.
Thank you for your question. In this study we were using laboratory tool cut-off point from PhiCal© Calprotectin enzyme-linked immunosorbent assay (ELISA) kit (Immundiagnostik AG, Stubenwald-Allee 8a, D-64625 Bensheim) with value of <50 μg/g considered negative. Regarding samples with fecal calprotectin level >250 μg/g, there were six patients but due to limited resources and safety concerns, patients have not undergone colonoscopy, as we will add in the limitations.
Thank you for your question. The total number of subjects involved in our study is 44 patients, none of them received stool culture examination nor stool examination. We will disclose this as limitations.
Thank you for your question. We defined gastrointestinal symptoms as nausea, vomitting, anorexia, diarrhea and abdominal pain. We will add this definition in the Characteristic subjects.
Thank you for your question and comment. In this study we grouped comorbidity as diabetes mellitus, hypertension, or both. In this study we found 26 patients with comorbidity. After we did regression we found no significant correlation between comorbidity and gut inflammation, therefore we will rephrase the statement ”comorbidity becomes an independent risk factor for gastrointestinal inflammation” to “comorbidity becomes a risk factor for an infection”.
Thank you for your question. The patients received stool test within 24 hours after patients had confirmed positive results from nasopharyngeal swab.

View more View less

Competing Interests

We have no conflicts of interest to disclose.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Viral load and gastrointestinal inflammation in COVID-19 patients

Abstract

Keywords

Introduction

Methods

Study design and participants

Statistical analysis

Results

Characteristics of subjects

Table 1. Clinical and laboratory characteristics.

Nasopharyngeal swab RT-PCR cycle threshold value in subjects

Fecal calprotectin levels in subjects

Table 2. Fecal calprotectin levels in subjects with gastrointestinal symptoms.

Cycle threshold and fecal calprotectin

Discussion

Conclusion

Data availability

Author contribution

Acknowledgments

References

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