The role of IL-6, ferritin, and coagulopathy in Covid-19 clinical progression

Background In COVID-19, the release of pro-inflammatory mediators in the cytokine storm, primarily interleukin-6 (IL-6), has been hypothesized to induce pulmonary intravascular coagulation. However, the relationship between IL-6 and coagulopathy remains unclear in COVID-19 progression. We aimed to investigate the correlation of IL-6 with D-dimer, fibrinogen, prothrombin time (PT), and ferritin. Furthermore, we also analyzed the effect of those parameters on the worsening of COVID-19 patients. Methods A prospective cohort study was conducted in moderate and severe COVID-19 patients from June 2020 to January 2021. A serial evaluation of IL-6, D-dimer, fibrinogen, ferritin, and PT was performed and correlated with the patient's condition at admission and on the 14th day. The outcomes (improvement, worsening, or discharged patients) were recorded during the study. Results Of 374 patients, 73 study subjects (61 severe and 12 moderate COVID-19) were included in this study. A total of 35 out of 61 severe and one out of 12 moderate illness subjects had experienced worsening. Spearman-rank correlation of IL-6 with with ferritin, D-dimer, fibrinogen, and PT was 0.08 ( p=0.5), −0.13 ( p=0.27), 0.01 ( p=0.91), and 0.03 ( p=0.77), respectively. In ROC analysis, D-dimer (74,77%) and IL-6 (71,32%) were the highest among other variables (>60%). Conclusions In COVID-19 patients, there was a correlation between elevated IL-6 and D-dimer levels with disease deterioration. There was no correlation between elevated IL-6 levels with ferritin, D-dimer, fibrinogen, and PT levels. Therefore, changes in IL-6 and D-dimer can predict worsening in moderate and severe COVID-19 patients.


Introduction
2][3] One known pathological inflammatory response triggered by Covid-19 infection is cytokine storm. 2,3Cytokine storm is a fatal immune response characterized by unregulated activation of immune cells and massive generation of cytokines and chemical mediators. 2,3Covid-19 infection can induce massive inflammatory response by rapidly activating pathogenic Th1 cells which produce proinflammatory cytokines, one of which is Interleukin-6 (IL-6). 2,3][10] IL-6 also play a role in amplifying the coagulation system by activating the epithelial cells, monocytes, and neutrophils, 11 which are supposed to inhibit anticoagulant protein S and antithrombin and enhance the activity of clotting factor VII, von Willebrand, and fibrinogen.A study by Gomez-Mesa, et al. stated that about 20%-50% of hospitalized patients with COVID-19 have elevated D-dimer, prolonged PT, thrombocytopenia, and/or low fibrinogen levels. 1 Therefore, in this study, we aim to seek the association between coagulopathy, which is measured by D-dimer, fibrinogen and PT level, along the Covid-19 progression.In this study, we also seek the IL-6 correlation with coagulation in Covid-19 to seek if IL-6 could have predictive value of Covid-19 associated Coagulopathy (CAC). 12rritin level is reported elevated in recent studies and is associated with hemophagocytic lymphohistiocytosis/ macrophage activation syndrome in Covid-19. 5,13However, the absence of five out of eight characteristics in the previous Covid-19 study explains the differences between the disease. 146][17][18][19] In this prospective cohort study, we aimed to investigate the role of these parameters in Covid-19 disease progression.

s the differe
ces between the disease. 146][17][18][19] In this prospective cohort study, we aimed to investigate the role of these parameters in Covid-19 disease progression.

According to studies of natural course of Covid-19, disease progression typically occurs within 7-14 days after the onset of symptoms. 20Therefore, we use the cut-off within 14 days after symptoms to evaluate disease progression in this study and stop the observation afterward to make the sample homogenous.Spo 2 /FiO 2 is one component of the Covid-19 severity index and is simple, non-invasive, and readily available tool in most clinical settings in the world. 21Spo 2 /FiO 2 reflects directly the respiratory function.Spo 2 /FiO 2 also reported by many studies to have predictive value of invasive mechanical ventilation 22 and mortality in Covid-19. 21Thus, we also try to seek the correlation between IL-6, PT, fibrinogen, and ferritin with Spo 2 /FiO 2 variables.


Methods


Study design

This was a prospective cohort study conducted from July 2020 to January 2021 at the Pertamina Central Hospital Modular Extension Simprug (Simprug Modular Extension Hospital, SMEH, Jakarta, Indonesia).Inclusion criteria were patients with Covid-19 confirmed by positive SARS-CoV-2 polymerase chain reaction (PCR) testing who were admitted from July 2020 to January 2021, aged 18 years or older; classified clinically moderate or severe Covid-19 (according to WHO interim guidance) 23 ; and willingness to provide blood sample.Exclusion criteria were history of chronic bleeding; prior anticoagulant therapy, undergoing hemodialysis; undergoing plasma convalescent clinical trial, or taking immunomodulation therapy (particularly IL-6 and intravenous immunoglobulin therapy) before or throughout the observation period.


Sample collection and storage

First blood sample were drawn from patients who fulfilled the criteria, on the first day upon admission.The second blood sample were collected at the end of observation period which is on 14 th day upon admission.The collected blood samples were stored in the fridge at -20°Celsius at Clinical Pathology laboratory of Pertamina Central Hospital.


REVISED Amendments from Version 1

Major grammar revision was done and restructuring sentences to fix inconsistencies and contradicting statements.


Any further responses from the reviewers can be found at the end of the article

The blood samples were analyzed for IL-6, D-dimer, ferritin, fibrinogen, CRP, and prothrombin time.IL-6 was analyzed in the Integrated Laboratory of the According to studies of natural course of Covid-19, disease progression typically occurs within 7-14 days after the onset of symptoms. 20Therefore, we use the cut-off within 14 days after symptoms to evaluate disease progression in this study and stop the observation afterward to make the sample homogenous.Spo 2 /FiO 2 is one component of the Covid-19 severity index and is simple, non-invasive, and readily available tool in most clinical settings in the world. 21Spo 2 /FiO 2 reflects directly the respiratory function.Spo 2 /FiO 2 also reported by many studies to have predictive value of invasive mechanical ventilation 22 and mortality in Covid-19. 21Thus, we also try to seek the correlation between IL-6, PT, fibrinogen, and ferritin with Spo 2 /FiO 2 variables.

Study design
This was a prospective cohort study conducted from July 2020 to January 2021 at the Pertamina Central Hospital Modular Extension Simprug (Simprug Modular Extension Hospital, SMEH, Jakarta, Indonesia).Inclusion criteria were patients with Covid-19 confirmed by positive SARS-CoV-2 polymerase chain reaction (PCR) testing who were admitted from July 2020 to January 2021, aged 18 years or older; classified clinically moderate or severe Covid-19 (according to WHO interim guidance) 23 ; and willingness to provide blood sample.Exclusion criteria were history of chronic bleeding; prior anticoagulant therapy, undergoing hemodialysis; undergoing plasma convalescent clinical trial, or taking immunomodulation therapy (particularly IL-6 and intravenous immunoglobulin therapy) before or throughout the observation period.

Sample collection and storage
First blood sample were drawn from patients who fulfilled the criteria, on the first day upon admission.The second blood sample were collected at the end of observation period which is on 14 th day upon admission.The collected blood samples were stored in the fridge at -20°Celsius at Clinical Pathology laboratory of Pertamina Central Hospital.

REVISED Amendments from Version 1
Major grammar revision was done and restructuring sentences to fix inconsistencies and contradicting statements.

Any further responses from the reviewers can be found at the end of the article
The blood samples were analyzed for IL-6, D-dimer, ferritin, fibrinogen, CRP, and prothrombin time.IL-6 was analyzed in the Integrated Laboratory of the Faculty of Medicine, Universitas Indonesia.During transferring IL-6 samples were maintained at constant temperature -20°Celsius according to Guidance on regulations for the Transport of Infectious Substances Guidance on regulations for the Transport of Infectious Substances 2007.IL-6 was analyzed using an ELISA IL6 Vmax microplate reader.Complete blood count, CRP, D-dimer, fibrinogen, and prothrombin time (PT) were analyzed using Sysmex CS2100i.Ferritin was analyzed using Cobas 601 Roche in the Clinical Pathology laboratory of Pertamina Central Hospital.
aculty of Medicine, Universitas Indonesia.During transferring IL-6 samples were maintained at constant temperature -20°Celsius according to Guidance on regulations for the Transport of Infectious Substances Guidance on regulations for the Transport of Infectious Substances 2007.IL-6 was analyzed using an ELISA IL6 Vmax microplate reader.Complete blood count, CRP, D-dimer, fibrinogen, and prothrombin time (PT) were analyzed using Sysmex CS2100i.Ferritin was analyzed using Cobas 601 Roche in the Clinical Pathology laboratory of Pertamina Central Hospital.

Other variables recorded in this study are non-invasive SpO 2 and FiO 2 , which ratio is one parameter of Covid-19 disease severity index. 21The data was recorded from a noninvasive pulse oximeter monitor.These varia Other variables recorded in this study are non-invasive SpO 2 and FiO 2 , which ratio is one parameter of Covid-19 disease severity index. 21The data was recorded from a noninvasive pulse oximeter monitor.These variables are then correlated with the IL-6, ferritin, D-dimer, and PT level to seek the association.CRP level also recorded in this study and also analyzed to IL-6, D-dimer, ferritin, coagulopathy marker and SpO 2 /FiO 2 ratio but not the main focus of the study.

n correlated
ith the IL-6, ferritin, D-dimer, and PT level to seek the association.CRP level also recorded in this study and also analyzed to IL-6, D-dimer, ferritin, coagulopathy marker and SpO 2 /FiO 2 ratio but not the main focus of the study.


Assessment patients' severity

The severity of the illness was classified according to the WHO interim guidance (published 13 th March 2020) 23 as mild, moderate, severe, or critical disease.Moderate case was defined as patient with clinical signs and symptoms of pneumonia but SpO 2 levels is > 93% with room air.Severe cases was defined as patient with clinical signs and symptoms of pneumonia, and one of the following: 1) respiratory rate > 30 times per minute, 2) signs of respiratory distress, or SpO 2 < 93% with room air. 23,24tients included in this study were patients who i

Assessment patients' severity
The severity of the illness was classified according to the WHO interim guidance (published 13 th March 2020) 23 as mild, moderate, severe, or critical disease.Moderate case was defined as patient with clinical signs and symptoms of pneumonia but SpO 2 levels is > 93% with room air.Severe cases was defined as patient with clinical signs and symptoms of pneumonia, and one of the following: 1) respiratory rate > 30 times per minute, 2) signs of respiratory distress, or SpO 2 < 93% with room air. 23,24tients included in this study were patients who initially classified as moderate or severe disease.Patients were then observed up to day 14th or concluded earlier if the patients improved and discharged earlier, or worsened and passed away before day 14th.Worsening was defined as clinical disease severity progression from moderate to severe, or to critical disease or death.All patients received antibiotics, antiviral, corticosteroids, and anticoagulants (Fondaparinux, LMNH, or UFH) according to the standard hospital therapy based on WHO Covid-19 clinical guideline. 23Detailed information regarding covid-19 therapy given is described in result section.

tially classified as moderate
r severe disease.Patients were then observed up to day 14th or concluded earlier if the patients improved and discharged earlier, or worsened and passed away before day 14th.Worsening was defined as clinical disease severity progression from moderate to severe, or to critical disease or death.All patients received antibiotics, antiviral, corticosteroids, and anticoagulants (Fondaparinux, LMNH, or UFH) according to the standard hospital therapy based on WHO Covid-19 clinical guideline. 23Detailed information regarding covid-19 therapy given is described in result section.


Statistical analysis

All collected data were analyzed using Anaconda package program 25 and reported in the text, table, or figures.All p-values < 0.05 were considered statistically significant.Correlation test was used to analyze correlation between IL-6 with ferritin and coagulopathy variables (PT, D-dimer, and Fibrinogen).Method of cutoff points obtained using area under curve method.Cutoff points by receiver operator characteristic obtained by calculating the optimum sensitivity a

Statistical analysis
All collected data were analyzed using Anaconda package program 25 and reported in the text, table, or figures.All p-values < 0.05 were considered statistically significant.Correlation test was used to analyze correlation between IL-6 with ferritin and coagulopathy variables (PT, D-dimer, and Fibrinogen).Method of cutoff points obtained using area under curve method.Cutoff points by receiver operator characteristic obtained by calculating the optimum sensitivity and specificity.

Results
During the study period from July 2020 to January 2021, there were 374 moderate to severe Covid-19 patients treated at the SMEH.Of those, 117 patients were randomly selected, 42 were excluded due to incomplete data, one received convalescent plasma therapy, and 1 had lysis of blood samples.The remaining 73 patients were further analyzed.

e received convalescent plasma
therapy, and 1 had lysis of blood samples.The remaining 73 patients were further analyzed.

The characteristics of the study subjects are summarized in Table 1.Of the 73 Covid-19 patients, 61 (84%) were classified as having severe Covid-19 and 12 (16%) were classified as having moderate Covid-19.The majority of the patients were male and the mean age of patients was 61 years (SDAE12.74).Most of the patients were overweight, hospitalized at day 6 th of the illness with duration of 7 days.As the final observation outcomes, most of the subjects with moderate disease experienced improvement, and subjects with severe disease experienced worsening within 14 days of the illness.All subjects were given antiviral therapy either remdesivir, favipiravir, or oseltamivir based on clinical judgement and according to Indonesia National Guideline of Covid-19. 24One patient was given oseltamivir because remdesivir or favipiravir was contraindicated (GFR <30 ml/min/1,73 m 2 ), and one patient was not in either antiviral because both remdesivir and favipiravir were contraindicated and oseltamivir was not available.

Patients suspected with secondary infection or sepsis were The characteristics of the study subjects are summarized in Table 1.Of the 73 Covid-19 patients, 61 (84%) were classified as having severe Covid-19 and 12 (16%) were classified as having moderate Covid-19.The majority of the patients were male and the mean age of patients was 61 years (SDAE12.74).Most of the patients were overweight, hospitalized at day 6 th of the illness with duration of 7 days.As the final observation outcomes, most of the subjects with moderate disease experienced improvement, and subjects with severe disease experienced worsening within 14 days of the illness.All subjects were given antiviral therapy either remdesivir, favipiravir, or oseltamivir based on clinical judgement and according to Indonesia National Guideline of Covid-19. 24One patient was given oseltamivir because remdesivir or favipiravir was contraindicated (GFR <30 ml/min/1,73 m 2 ), and one patient was not in either antiviral because both remdesivir and favipiravir were contraindicated and oseltamivir was not available.
Patients suspected with secondary infection or sepsis were started empiric antibiotic(s) (either meropenem, levofloxacin, azithromycin, or combinations) and were changed later according to bacterial culture.Corticosteroid (dexamethasone 6 mg/day or methylprednisolone 32 mg/day for 10 days) was given to all severe cases, and selected moderate cases based on clinical judgement.Anticoagulants (unfractionated heparin, enoxaparin, or fondaparinux) were also given according to according to Indonesia National Guideline of Covid-19. 24All patients not proven to have VTE, received thromboprophylaxis dose of anticoagulant, either UFH 2Â5000 units subcutaneously, enoxaparin 1Â4000 unit of anti-Xa (40 mg), or fondaparinux 1Â2.5 mg.The dose of anticoagulant was adjusted based on clinical judgement and the increase of D-dimer level.

tarted empiric antibi
tic(s) (either meropenem, levofloxacin, azithromycin, or combinations) and were changed later according to bacterial culture.Corticosteroid (dexamethasone 6 mg/day or methylprednisolone 32 mg/day for 10 days) was given to all severe cases, and selected moderate cases based on clinical judgement.Anticoagulants (unfractionated heparin, enoxaparin, or fondaparinux) were also given according to according to Indonesia National Guideline of Covid-19. 24All patients not proven to have VTE, eceived thromboprophylaxis dose of anticoagulant, either UFH 2Â5000 units subcutaneously, enoxaparin 1Â4000 unit of anti-Xa (40 mg), or fondaparinux 1Â2.5 mg.The dose of anticoagulant was adjusted based on clinical judgement and the inc

ase of D
dimer level.

The variable distributions were analyzed according to disease severity and the worsening or improvement of patients' condition, as shown in Table 2.

Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels (Table 3).

Figure 1 and Table 4 displayed The variable distributions were analyzed according to disease severity and the worsening or improvement of patients' condition, as shown in Table 2.
Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels (Table 3).
Figure 1 and Table 4 displayed the analysis of the receiver operating characteristic (ROC) curve (AUC) for correlation between elevated IL-6, ferritin, fibrinogen, D-dimer, and PT levels with Covid-19 patients' deterioration.We found no correlation between IL-6 and other variables.Thus, as one of the disease severity index components, we aimed to investigate the correlation between the oxygen saturation (SpO 2 )/fraction of inspired oxygen (FiO 2 ) ratio and IL-6, ferritin, fibrinogen, D-dimer, and PT (Figures 2 and 3).As a result, our study demonstrated the correlation between IL-6, ferritin, fibrinogen, D-dimer, and PT and SpO 2 /FiO 2 ratio as the severity determinants.The clinical course of the Covid-19 disease have been reported comprehensively, with the median O-A interval was 7 days, and illness onset to dyspnea interval was 8 days. 26Subjects in this study had earlier median O-A interval which was 6 days.Peng et al. reported that the O-A interval less than 7 days predict the likelihood of disease progression in moderate-severe disease Covid 19. 27But limitations were stated in aforementioned study that the health-seeking behavior of the patient and the domestic health system might influence the admission interval, aside from selection bias and confounding factors.
he analysis of the receiver operating characteristic (ROC) curve (AUC) for correlation between elevated IL-6, ferritin, fibrinogen, D-dimer, and PT levels with Covid-19 patients' deterioration.We found no correlation between IL-6 and other variables.Thus, as one of the disease severity index components, we aimed to investigate the correlation between the oxygen saturation (SpO 2 )/fraction of inspired oxygen (FiO 2 ) ratio and IL-6, ferritin, fibrinogen, D-dimer, and PT (Figures 2 and 3).As a result, our study demonstrated the correlation between IL-6, ferritin, fibrinogen, D-dimer, and PT and SpO 2 /FiO 2 ratio as the severity determinants.The clinical course of the Covid-19 disease have been reported comprehensively, with the median O-A interval was 7 days, and illness onset to dyspnea interval was 8 days. 26Subjects in this study had earlier median O-A interval which was 6 days.Peng et al. reported that the O-A interval less than 7 days predict the likelihood of disease progression in moderate-severe disease C vid 19. 27But limitations were stated in aforementioned study that the health-seeking behavior of the patient and the domestic health system might influence the admission interval, aside from selection bias and confounding factors.

In this study, most subjects with moderate disease experienced improvement (11 subjects out of 12 [91%]) and most subjects with severe disease experienced worsening (35 subjects out of 61 [59%]).Almost half of severe group has no comorbidities, thus three fourth of the severe group had at least one comorbidity.While in moderate group, about 80% of subjects had at least one comorbidity.All of subjects in moderate group were survived, while in severe group, only 55% subjects survived despite therapies given.This study found that the comorbidity was not associated with the patient's deterioration.

Co In this study, most subjects with moderate disease experienced improvement (11 subjects out of 12 [91%]) and most subjects with severe disease experienced worsening (35 subjects out of 61 [59%]).Almost half of severe group has no comorbidities, thus three fourth of the severe group had at least one comorbidity.While in moderate group, about 80% of subjects had at least one comorbidity.All of subjects in moderate group were survived, while in severe group, only 55% subjects survived despite therapies given.This study found that the comorbidity was not associated with the patient's deterioration.
Comparison of initial level of IL-6, ferritin, and coagulopathy marker with the level of the end of observation Overall subjects came with increased initial D-dimer, fibrinogen, and ferritin levels.Those markers also higher in severe group compared to moderate group.9][30][31][32][33] Furthermore, they showed increased IL-6, ferritin, and fibrinogen reflecting the acute phase response (APR) of the disease.Subjects who visited the emergency department for more than 6 days (mean 7.5 days) had almost three times greater risk of worsening than those who came for less than 6 days (mean 4 days).
parison of initial level of IL-6, ferritin, and coagulopathy marker with the level of the end of observation Overall subjects came with increased ini ial D-dimer, fibrinogen, and ferritin levels.Those markers also higher in severe group compared to moderate group.9][30][31][32][33] Furthermore, they showed increased IL-6, ferritin, and fibrinogen reflecting the acute phase response (APR) of the disease.Subjects who visited the emergency department for more than 6 days (mean 7.5 days) had almost three times greater risk of worsening than those who came for less than 6 days (mean 4 days).

Levels of IL-6 at the first sample collection (Table 1) were similar to the previous study. 30In contrast to the studies by Jin Zhang et al. 34 and Awasthi et al., 35 in this study, subjects did not show a significant difference in IL-6 levels at the first measurement between moderate to severe illness.However, IL-6 level in worsening group had rising trend, while in contrast, IL-6 in improving group had down trend.This demonstrates the changes in IL-6 level might held more predictive value in disease progression than the initial value per se.


Correlation between increased levels of IL-6 and ferritin

In acute phare response (APR), IL-6 is reported to be a pro-inflammatory cytokine that increase and determine infectionassociated ferritin levels. 32,36However, we did not found the aforementioned correlation between ferritin levels and IL-6 in the first Levels of IL-6 at the first sample collection (Table 1) were similar to the previous study. 30In contrast to the studies by Jin Zhang et al. 34 and Awasthi et al., 35 in this study, subjects did not show a significant difference in IL-6 levels at the first measurement between moderate to severe illness.However, IL-6 level in worsening group had rising trend, while in contrast, IL-6 in improving group had down trend.This demonstrates the changes in IL-6 level might held more predictive value in disease progression than the initial value per se.

Correlation between increased levels of IL-6 and ferritin
In acute phare response (APR), IL-6 is reported to be a pro-inflammatory cytokine that increase and determine infectionassociated ferritin levels. 32,36However, we did not found the aforementioned correlation between ferritin levels and IL-6 in the first and second sample collection (Figure 3).

d second sa
ple collection (Figure 3).

Although subjects did not show raised IL-6 level at admission, we found an increase in ferritin and fibrinogen levels, indicating that inflammation was great in initial state.Furthermore, other factors contributing to increased ferritin levels, such as epithelial damage, were also considered.Since a d Although subjects did not show raised IL-6 level at admission, we found an increase in ferritin and fibrinogen levels, indicating that inflammation was great in initial state.Furthermore, other factors contributing to increased ferritin levels, such as epithelial damage, were also considered.Since a decrease in the SpO 2 /FiO 2 ratio was due to an acute respiratory distress syndrome (ARDS) manifestation, 37,38 the correlation between SpO 2 /FiO 2 ratio with ferritin and fibrinogen was investigated to determine whether the epithelial damage affected IL-6 and other inflammatory variables.Our result showed a statistically significant correlation between SpO 2 /FiO 2 ratio with ferritin and fibrinogen (Figure 2).This correlation showed that inflammation affected the SpO 2 /FiO 2 ratio from the onset of the illness.However, since the increase of IL-6 was not correlated with SpO 2 /FiO 2 ratio, we assumed that other factors contributing to ferritin release in this study were greater than induced by IL-6. 19,39According to a study by Zhi et al., several possibilities lead to the increase of ferritin: 1) pro-inflammatory cytokines stimuli (i.e., IL-1β, tumour necrosis factor α (TNF-α), and IL-6) caused an inflammatory reaction that damaged the cells, 2) intracellular ferritin leakage due to cell damage and inflammation, and 3) the ferritin leakage from the injured cells further triggers the damage of other cells via Fenton and Haber-Weiss's reaction. 13rrelation between levels of IL-6 with D-dimer, fibrinogen, and PT Inflammation will trigger the coagulation system characterized by changes in the value of D-dimer, fibrinogen, and PT at the time of viral entry. 40Thus, we investigated the correlation between levels of IL-6 and markers of coagulopathy and inflammation.
crease in the SpO 2 /FiO 2 ratio was due to an acute respiratory distress syndrome (ARDS) manifestation, 37,38 the correlation between SpO 2 /FiO 2 ratio with ferritin and fibrinogen was investigated to determine whether the epithelial damage affected IL-6 and other inflammatory variables.Our result showed a statistically significant correlation between SpO 2 /FiO 2 ratio with ferritin and fibrinogen (Figure 2).This correlation showed that inflammation affected the SpO 2 /FiO 2 ratio from the onset of the illness.However, since the increase of IL-6 was not cor elated with SpO 2 /FiO 2 ratio, we assumed that other factors contributing to ferritin release in this study were greater than induced by IL-6. 19,39According to a study by Zhi et al., several possibilities lead to the increase of ferritin: 1) pro-inflammator cytokines stimuli (i.e., IL-1β, tumour necrosis factor α (TNF-α), and IL-6) caused an inflammatory reaction that damaged the cells, 2) intracellular ferritin leakage due to cell damage and inflammation, and 3) the ferritin leakage from the injured cells further triggers the damage of other cells via Fenton and Haber-Weiss's reaction. 13rrelation between levels of IL-6 with D-dimer, fibrinogen, and PT Inflammation will trigger the coagulation system characterized by changes in the value of D-dimer, fibrinogen, and PT at the time of viral entry. 40Thus, we investigated the correlation between levels f IL-6 and markers of coagulopathy and inflammation.

Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded that coagulopathy in Covid-19 patients could occur disregard to IL-6 and ferritin levels.Such explanations for this finding are: 1) ARDS pathophysiology in the infected subjects with moderate and severe illness is related to a massive loss of the angiotensin-converting enzyme (ACE)-2 enzyme, causing damage to the alveolar epithelium and vascular endothelium, 41 2) compartmentalization of the inflammatory cascade, as described by Chow and Tisoncik et al., 42,43 and 3) an acute phase response (APR) increase may be due to ot Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded that coagulopathy in Covid-19 patients could occur disregard to IL-6 and ferritin levels.Such explanations for this finding are: 1) ARDS pathophysiology in the infected subjects with moderate and severe illness is related to a massive loss of the angiotensin-converting enzyme (ACE)-2 enzyme, causing damage to the alveolar epithelium and vascular endothelium, 41 2) compartmentalization of the inflammatory cascade, as described by Chow and Tisoncik et al., 42,43 and 3) an acute phase response (APR) increase may be due to other pro-inflammatory cytokines, such as TNF-α or IL-1, 31,39,40,44,45 and the aforementioned vicious cycle of cellular destruction.
er pro-inflammatory cytokines, such as TNF-α or IL-1, 31,39,40,44,45 and the aforementioned vicious cycle of cellular destruction.

An analysis was carried out on the second blood collection after patients had a worsening during treatment to determine whether the incidence of coagulopathy correlates with IL-6 levels, in which the effect of IL-6 and cytokine storm was the utmost.The data from the second blood collection showed a significant correlation between the SpO 2 /FiO 2 ratio and the D-dimer, ferritin, PT, and IL-6 (Figure 3).Furthermore, th An analysis was carried out on the second blood collection after patients had a worsening during treatment to determine whether the incidence of coagulopathy correlates with IL-6 levels, in which the effect of IL-6 and cytokine storm was the utmost.The data from the second blood collection showed a significant correlation between the SpO 2 /FiO 2 ratio and the D-dimer, ferritin, PT, and IL-6 (Figure 3).Furthermore, the D-dimer, fibrinogen, and PT were correlated with ferritin, while there was no correlation with IL-6.Our result demonstrated that inflammation correlates with coagulopathy, but conceivably not via a direct IL-6 pathway.To explain this finding, according to a study described by Sinha et al. that the interaction of mediators and pathways involved is not always constantly linear or uniform. 46Worsening subjects showed significant changes in D-dimer, fibrinogen, and PT.Since there was no correlation between IL-6, ferritin and PT, fibrinogen, and D-Dimer, we determined the changes in the variables' mean values between the first and second blood collections.Furthermore, Wilcoxon's non-parametric test was performed since the data was not normally distributed (Figure 4).

D-dimer, fibrinogen, and PT were correlated with ferritin,
while there was no correlation with IL-6.Our result demonstrated that inflammation correlates with coagulopathy, but conceivably not via a direct IL-6 pathway.To explain this finding, according to a study described by Sinha et al. that the interaction of mediators and pathways involved is not alw ys constantly linear or uniform. 46Worsening subjects showed significant changes in D-dimer, fibrinogen, and PT.Since there was no correlation between IL-6, ferritin and PT, fibrinogen, and D-Dimer, we determined the changes in the variables' mean values between the first and second blood collections.Furthermore, Wilcoxon's non-parametric test was performed since the data was not normally distributed (Figure 4).

In worsening subjects, the second IL-6 blood collection had a higher value than the first one, albeit not statistically significant.Possible reasons include: 1) cytokines other than IL-6 affect the increase in D-dimer, fibrinogen, and ferritin levels, or 2) external factors, such as administration of anti-inflammatory corticosteroids and heparin, also have antiinflammatory effects, 47 resulting in an insignificant increase of IL-6 and ferritin.During the study period, corticosteroids and heparin were administrated following the standard therapy for moderate and severe Covid-19 patients, or 3) large amounts of soluble IL-6 receptors production binds free IL-6 to reduce the concentration, as described by Garbers et al., 48 or 4) the presence of other cytokines (IL-6 cytokine family), including IL-11, 49 that also has a pro-inflammatory effect. 50e disease severity was determined using WHO criteria for oxygen saturation, respiratory rate, and radiological findings.Most subjects were admitted to the emergency department with severe illness (83%), with a median SpO 2 / FiO 2 ratio was 97.In moderate illness, there were no differences in IL-6 at the baseline.

Our study found a negative correlation between D-dimer, fibrinogen, and PT levels with the SpO 2 /FiO 2 ratio, reflecting the disease severity.This finding demonstrated that coagulopathy had a role in the patien In worsening subjects, the second IL-6 blood collection had a higher value than the first one, albeit not statistically significant.Possible reasons include: 1) cytokines other than IL-6 affect the increase in D-dimer, fibrinogen, and ferritin levels, or 2) external factors, such as administration of anti-inflammatory corticosteroids and heparin, also have antiinflammatory effects, 47 resulting in an insignificant increase of IL-6 and ferritin.During the study period, corticosteroids and heparin were administrated following the standard therapy for moderate and severe Covid-19 patients, or 3) large amounts of soluble IL-6 receptors production binds free IL-6 to reduce the concentration, as described by Garbers et al., 48 or 4) the presence of other cytokines (IL-6 cytokine family), including IL-11, 49 that also has a pro-inflammatory effect. 50e disease severity was determined using WHO criteria for oxygen saturation, respiratory rate, and radiological findings.Most subjects were admitted to the emergency department with severe illness (83%), with a median SpO 2 / FiO 2 ratio was 97.In moderate illness, there were no differences in IL-6 at the baseline.
Our study found a negative correlation between D-dimer, fibrinogen, and PT levels with the SpO 2 /FiO 2 ratio, reflecting the disease severity.This finding demonstrated that coagulopathy had a role in the patient's deterioration even though SpO 2 /FiO 2 ratio showed no correlation with IL-6.The worsening lung function was due to pre-existing inflammation before the increase in IL-6 may explain the correlation with the coagulation system.
's deterioration even though SpO 2 /FiO 2 ratio showed no correlation with IL-6.The worsening lung function was due to pre-existing inflammation before the increase in IL-6 may explain the correlation with the coagulation system.

Leisman 45 and Sinha 46 proposed that the pathophysiology of moderate and severe Covid-19 ARDS differs from typical ARDS, where the inflammatory system activation marked by increased IL-6 occurred only in a few Covid-19 ARDS patients.Our study showed that only 15.1% of subjects had IL-6 elevation at the initial examination, with an increase of 10-times the lower standard limit.Furthermore, if Covid-19 ARDS is considered a hyperinflammatory type, the IL-6 will be much lower than the value found in studies of the hyperinflammatory typ Leisman 45 and Sinha 46 proposed that the pathophysiology of moderate and severe Covid-19 ARDS differs from typical ARDS, where the inflammatory system activation marked by increased IL-6 occurred only in a few Covid-19 ARDS patients.Our study showed that only 15.1% of subjects had IL-6 elevation at the initial examination, with an increase of 10-times the lower standard limit.Furthermore, if Covid-19 ARDS is considered a hyperinflammatory type, the IL-6 will be much lower than the value found in studies of the hyperinflammatory type that showed an increase of more than 100-times the lower standard limit.
that showed an increase of more than 100-times the lower standard limit.

The second blood collection sample showed that IL-6, ferritin, D-dimer, and PT had a statistically significant negative correlation with the SpO 2 /FiO 2 ratio, suggesting that deteriorating lung function is also correlated with inflammation and coagulopathy.However, we found no statistically significant correlation between IL-6 with D-dimer, fibrinogen, PT, and ferritin.As mentioned previously, this phenomenon may be caused by administering anti-inflammatory drugs that can reduce APR levels.

Fibrinogen is a soluble glycoprotein synthesized by the liver, and the activation can produce insoluble fibrin in the plasma.This process occurs via intrinsic and extrinsic pathways, and the activity is assessed by measuring PT.

Fibrinogen level is elevated in inflammatory conditions. 39In patients who experienced worsening, we found that the fibrinogen levels at admission were higher than normal, with no changes in PT values.Thachil argued that the initial increase of fibrinogen would regulate inflammation.However, when the D-dimer levels continue to elevate with decreased fibrinogen levels, the protective role of fibrino The second blood collection sample showed that IL-6, ferritin, D-dimer, and PT had a statistically significant negative correlation with the SpO 2 /FiO 2 ratio, suggesting that deteriorating lung function is also correlated with inflammation and coagulopathy.However, we found no statistically significant correlation between IL-6 with D-dimer, fibrinogen, PT, and ferritin.As mentioned previously, this phenomenon may be caused by administering anti-inflammatory drugs that can reduce APR levels.
Fibrinogen is a soluble glycoprotein synthesized by the liver, and the activation can produce insoluble fibrin in the plasma.This process occurs via intrinsic and extrinsic pathways, and the activity is assessed by measuring PT.
Fibrinogen level is elevated in inflammatory conditions. 39In patients who experienced worsening, we found that the fibrinogen levels at admission were higher than normal, with no changes in PT values.Thachil argued that the initial increase of fibrinogen would regulate inflammation.However, when the D-dimer levels continue to elevate with decreased fibrinogen levels, the protective role of fibrinogen ceases, and thrombus formation begins. 51In our finding, the initial fibrinogen levels in moderate and severe Covid-19 were increased but decreased at the second blood collection sample analysis, which showed a tendency towards normal compared to the worsening group.This result demonstrated that hypercoagulable conditions might have a role in the pathology of the worsening group, a condition that differs from the hypothesis as reported by Thachil. 51[54][55][56] The correlation between ferritin levels with D-dimer, fibrinogen, and PT As part of APRs, ferritin was negatively correlated with the SpO 2 /FiO 2 ratio (Figure 2).We assumed that the inflammation process had occurred prior to the patients' hospitalization, which explains the presence of a correlation between ferritin and the disease severity (moderate and severe).In the early phase, ferritin levels did not correlate with D-dimer, PT, and fibrinogen levels, which unlikely indicate that inflammation triggers coagulopathy.
en ceases, and thrombus formation begins. 51In our finding, the initial fibrinogen levels in moderate and severe Covid-19 were increased but decreased at the second blood collection sample analysis, which showed a tendency towards normal compared to the worsening group.This result demonstrated that hypercoagulable conditions might have a role in the pathology of the worsening group, a condition that differs from the hypothesis as reported by Thachil. 51[54][55][56] The correlation between ferritin levels with D-dimer, fibrinogen, and PT As part of APRs, ferritin was negatively correlated with the SpO 2 /FiO 2 ratio (Figure 2).We assumed that the inflammation process had occurred prior to the patients' hospitalization, which explains the presence of a correlation between ferritin and the disease severity (moderate and severe).In the early phase, fe ritin levels did not correlate with D-dimer, PT, and fibrinogen levels, which unlikely indicate that inflammation triggers coagulopathy.

On the initial observation, there was an elevation in ferritin levels in the subjects with moderate and severe illness (Table 1).In the second blood collection sample, ferritin level On the initial observation, there was an elevation in ferritin levels in the subjects with moderate and severe illness (Table 1).In the second blood collection sample, ferritin levels increased in the worsening but decreased in the improved group.Finally, we found the most significant changes in the improved group of moderate illness subjects (Table 2), as also reported by Zhi et al. 13 To explain this finding, we hypothesized that 1) pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 can increase ferritin synthesis, 2) damage to the cellular level due to inflammation will release intracellular ferritin, 31 and 3) in acidosis, increased production of reactive oxygen species induce the release of iron from the ferritin.In addition, Fenton and Haber-Weiss reaction increases the concentration of hydroxyl radicals and eventually damages the cells.Overall, this process will initiate a vicious cycle of cell damage by increasing ferritin concentration.
increased in the worsening but decreased in the improved group.Finally, we found the most significant changes in the improved group of moderate illness subjects (Table 2), as also reported by Zhi et al. 13 To explain this finding, we hypothesized that 1) pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 can increase ferritin synthesis, 2) damage to the cellular level due to inflammation will release intracellular ferritin, 31 and 3 in acidosis, increased production of reactive oxygen species induce the release of iron from the ferritin.In addition, Fenton and Haber-Weiss reaction increases the concentration of hydroxyl radicals and eventually damages the cells.Overall, this process will initiate a vicious cycle of cell damage by increasing ferritin concentration.

Rosario, 57 explained that ferritin has immunosuppressant and pro-inflammatory effects.The immunosuppressant properties were found in the ferritin H fraction, suppressing B lymphocyte antibodies' production.Additionally, ferritin also reduces granulocytes' phagocytosis, regu Rosario, 57 explained that ferritin has immunosuppressant and pro-inflammatory effects.The immunosuppressant properties were found in the ferritin H fraction, suppressing B lymphocyte antibodies' production.Additionally, ferritin also reduces granulocytes' phagocytosis, regulates the production of granulocytes-monocytes, and induces the production of IL-10 in lymphocytes.Moreover, it also inhibits the function of CXCR4 (chemokine receptor 4), an activator of the mitogen-activated protein kinase system that plays a role in the proliferation, differentiation, and migration of the cells.
ates the production of granulocytes-monocytes, and induces the production of IL-10 in lymphocytes.Moreover, it also inhibits the function of CXCR4 (chemokine receptor 4), an activator of the mitogen-activated protein kinase system that plays a role in the proliferation, differentiation, and migration of the cells.

The role of ferritin in the inflammatory process has been described in hepatic stellate cells.For example, increased nuclear factor κB can promote the expression of inflammatory media The role of ferritin in the inflammatory process has been described in hepatic stellate cells.For example, increased nuclear factor κB can promote the expression of inflammatory mediators, including IL-1β, and induce the production of nitric oxide synthase.This immunomodulatory process belongs to ferritin H and L properties.However, the role of ferritin as an inducer of inflammation or anti-inflammatory in Covid-19 infection has not been proven since the elevated ferritin type is still unknown. 31,32,57e correlation between ferritin with fibrinogen, D-dimer, and PT in Covid-19 patients is recognized as a damageassociated molecular pattern that can modulate IL-6 concentrations. 31ere was no correlation between decreased SpO 2 /FiO 2 ratio and IL-6 in the first blood collection sample, but SpO 2 /FiO 2 ratio was correlated with acute-phase protein ferritin and fibrinogen.Albeit D-dimer, fibrinogen, and PT were correlated with the SpO 2 /FiO 2 ratio, we found no correlation between ferritin and those.Based on this finding, we demonstrated that the increase in APR does not correlate with the incidence of coagulopathy in Covid-19 since the condition has different mechanisms, including epithelial damage due to the entry of the SARS-CoV-2 virus and the increased production of NETs by neutrophils, as previously described.
ors, including IL-1β, and induce the production of nitric oxide synthase.This immunomodulatory process belongs to ferritin H and L properties.However, the role of ferritin as an inducer of inflammation or anti-inflammatory in Covi -19 infection has not been proven since the elevated ferritin type is still unknown. 31,32,57e correlation between ferritin with fibrinogen, D-dimer, and PT in Covid-19 patients is recognized as a damageassociated molecular pattern that can modulate IL-6 concentrations. 31ere was no correlation between decreased SpO 2 /FiO 2 ratio and IL-6 in the first blood collection sample, but SpO 2 /FiO 2 ratio was correlated with acute-phase protein ferritin and fibrinogen.Albeit D-dimer, fibrinogen, and PT were correlated with the SpO 2 /FiO 2 ratio, we found no correlation between ferritin and those.Based on this finding, we demonstrated that the increase in APR does not correlate with the incidence of coagulopathy in Covid-19 since the condition has different mechanisms, including epithelial damage due to the entry of the SARS-CoV-2 virus and the increased production of NETs by neutrophils, as previously described.

Alteration of IL-6, ferritin, fibrinogen, PT, and prediction of Covid-19 severity Theoretically, inflammatory (IL-6 and fe Alteration of IL-6, ferritin, fibrinogen, PT, and prediction of Covid-19 severity Theoretically, inflammatory (IL-6 and ferritin) and coagulopathy (D-dimer, fibrinogen, and PT) markers are correlated with patients' deterioration. 4,5,13,58Interleukin-6 regulates the inflammatory process, and the elevation is associated with the worsening of Covid-19 patients. 6,16In accordance with recent studies, this study also proved that increased levels of IL-6 can predict the deterioration of subjects.
ritin) and coagulopathy (D-dimer, fibrinogen, and PT) markers are correlated with patients' deterioration. 4,5,13,58Interleukin-6 regulates the inflammatory process, and the elevation is associated with the worsening of Covid-19 patients. 6,16In accordance with recent studies, this study also proved that increased levels of IL-6 can predict the deterioration of subjects.

Inflammation due to Covid-19 can activate epithelium, endothelium, macrophages, and neutrophils.Massive recruitment of neutrophils to the lungs and releas Inflammation due to Covid-19 can activate epithelium, endothelium, macrophages, and neutrophils.Massive recruitment of neutrophils to the lungs and release of NETs regulates coagulation and IL-6 activation. 54,59We found that alteration in D-dimer levels can predict the patients' deterioration.Therefore, the availability of D-dimer in hospitals can justify its function as a routine examination.
of NETs regulates coagulation and IL-6 activation. 54,59We found that alteration in D-dimer levels can predict the patients' deterioration.Therefore, the availability of D-dimer in hospitals can justify its function as a routine examination.

Although the alteration of ferritin and fibrinogen cannot predict the patient's deterioration, these APR markers can be elevated in inflammatory conditions. 32,39Thus, administration of corticosteroid therapy or anti-inflammatory agents, such as heparin, can reduce the inflammation process, 47,60 as marked in our study by the unaltered ferritin and decreased fibrinogen levels (Table 2).

Prothrombin time is the period required for plasma to clot after adding tissue factor.The reaction velocity is the result of coagulation factors activation that consist of coagulation factors XII to X. Furthermore, Yu Zhang et al. found that PT had a sensitivity of 83.54% and specificity of 65.22% for sepsis patients at the cut-off 20.
Although the alteration of ferritin and fibrinogen cannot predict the patient's deterioration, these APR markers can be elevated in inflammatory conditions. 32,39Thus, administration of corticosteroid therapy or anti-inflammatory agents, such as heparin, can reduce the inflammation process, 47,60 as marked in our study by the unaltered ferritin and decreased fibrinogen levels (Table 2).
Prothrombin time is the period required for plasma to clot after adding tissue factor.The reaction velocity is the result of coagulation factors activation that consist of coagulation factors XII to X. Furthermore, Yu Zhang et al. found that PT had a sensitivity of 83.54% and specificity of 65.22% for sepsis patients at the cut-off 20. 61Our study showed a 75% quartile of 12 in severe Covid-19 subjects, describing that levels of PT were insensitive to measure patient's deterioration.
61Our study showed a 75% quartile of 12 in severe Covid-19 subjects, describing that levels of PT were insensitive to measure patient's deterioration.

A similar finding was also reported in a retrospective study by Long et al., 58 where the initial measurement of PT was not correlated with the disease severity.This phenomenon may demonstrate the effect of hypercoagulation as the dominant pathophysiology in the early phase of the disease, in contrast to DIC in sepsis. 62,63r study limitation is that the IL-6's diurnal variation cannot be eliminated since the use of a consecutive sampling A similar finding was also reported in a retrospective study by Long et al., 58 where the initial measurement of PT was not correlated with the disease severity.This phenomenon may demonstrate the effect of hypercoagulation as the dominant pathophysiology in the early phase of the disease, in contrast to DIC in sepsis. 62,63r study limitation is that the IL-6's diurnal variation cannot be eliminated since the use of a consecutive sampling method and the disease phase factor.
ethod and the disease phase factor.


Conclusion

In moderate and severe Covid-19 patients, there was a correlation between elevated IL-6 and D-dimer levels with disease deterioration.Groups with worsening outcome had higher presenting IL-6, Ferritin, D-dimer, and fibrinogen levels.IL-6 and D-dimer can be independent predictor for poor prognosis in Covid-19 disease progression, depicted by the upward


Introduction:

The introduction is very brief.Basic concepts regarding pathophysiological aspects of inflammation in COVID-19 may be described here.The role of parameters included in metadata and their behaviour in COVID-19 may be elucidated.


1.

The treatment protocol pertaining to cor

Conclusion
In moderate and severe Covid-19 patients, there was a correlation between elevated IL-6 and D-dimer levels with disease deterioration.Groups with worsening outcome had higher presenting IL-6, Ferritin, D-dimer, and fibrinogen levels.IL-6 and D-dimer can be independent predictor for poor prognosis in Covid-19 disease progression, depicted by the upward

Introduction:
The introduction is very brief.Basic concepts regarding pathophysiological aspects of inflammation in COVID-19 may be described here.The role of parameters included in metadata and their behaviour in COVID-19 may be elucidated.

1.
The treatment protocol pertaining to corticosteroids, low molecular weight heparin or UFH, and antiviral medications e.g.remdesivir etc. should be mentioned.And the modifications of these drugs according to the severity of disease.
icosteroids, low molecular weight heparin or UFH, and antiviral medications e.g.remdesivir etc. should be mentioned.And the modifications of these drugs according to the severity of disease.


2.


Methods

The exclusion criteria should explain whether patients on oral anticoagulants, liver dysfunction were included or not.As a large size of study population had diabetes and hypertension, underlying coronary artery disease is likely to be present.Such population may have been on oral anticoagulant therapy prior to COVID-19.

1.

C-reactive protein have been also studied and detailed in result section.It is a new parameter which is described by

Methods
The exclusion criteria should explain whether patients on oral anticoagulants, liver dysfunction were included or not.As a large size of study population had diabetes and hypertension, underlying coronary artery disease is likely to be present.Such population may have been on oral anticoagulant therapy prior to COVID-19. 1.
C-reactive protein have been also studied and detailed in result section.It is a new parameter which is described by authors in results bearing no mention in methods.
uthors in results bearing no mention in methods.


2.

The criteria of worsening of patients need to be described well according to citation used or modification, if any.


3.


Results:

"The patients were randomized" is not correct as selection of patients and their 1.

classification was the decision of authors.

The part of discussion, " Fig 4.Variable alterations between the first and second sample collection in the worsening group" should be explained in the result section.


2.

With usi

2.
The criteria of worsening of patients need to be described well according to citation used or modification, if any.

Results:
"The patients were randomized" is not correct as selection of patients and their 1.
classification was the decision of authors.
The part of discussion, " Fig 4.Variable alterations between the first and second sample collection in the worsening group" should be explained in the result section.

2.
With using terms like "most" and "majority", the percentages must be mentioned next to them.
g terms like "most" and "majority", the percentages must be mentioned next to them.


3.

Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.


4.

Attempt may be made to include data on patients who worsened.5.

The normal reference laboratory values should be given for all parameters used in the study.


6.


Discussion:

T

3.
Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.

4.
Attempt may be made to include data on patients who worsened.5.
The normal reference laboratory values should be given for all parameters used in the study.

Discussion:
This section needs to be rewritten as there are many grammatical errors.The authors must seek professional help.
is section needs to be rewritten as there are many grammatical errors.The authors must seek professional help.

1.

Many lines and paragraphs are not correct.e.g., "This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination.Additionally, uncorrelated IL-6 with coagulopathy markers, perhaps due to t 1.
Many lines and paragraphs are not correct.e.g., "This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination.Additionally, uncorrelated IL-6 with coagulopathy markers, perhaps due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems,32 inducing amplification of the cascade".
e ability of IL-6 to activate the inflammatory cascade in multiple body systems,32 inducing amplification of the cascade".


2.

The part, "Although subjects did not experience an inflammatory process at admission" is not acceptable.Subjects do not experience inflammatory process they suffer with symptoms.


3.

In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopa

2.
The part, "Although subjects did not experience an inflammatory process at admission" is not acceptable.Subjects do not experience inflammatory process they suffer with symptoms.

3.
In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded.The word "concluded" may be changed to "assumed".Authors should mention e.g., "in the first sample analysis..." and then start elaboration.The entire part does not fit well with subsequent portions of discussion.Authors are advised to write in flow oriented manner.
hy, we concluded.The word "concluded" may be changed to "assumed".Authors should mention e.g., "in the first sample analysis..." and then start elaboration.The entire part does not fit well with subsequent portions of discussion.Authors are advised to write in flow oriented manner.


4.

The authors are also advised to discuss

4.
The authors are also advised to discuss the results and its implications with a statistician.5.
the results and its implications with a statistician.5.


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


Is

e study des
gn 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?not the main focus of this study thus we did not describe CRP comprehensively in the method section.3. "The patients were randomized" is not correct as selection of patients and their classification was the decision of authors.

we agreed and have revised the grammar ○ 4. The pa

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?not the main focus of this study thus we did not describe CRP comprehensively in the method section.3. "The patients were randomized" is not correct as selection of patients and their classification was the decision of authors.
we agreed and have revised the grammar ○ 4. The part of discussion, " Fig 4.Variable alterations between the first and second sample collection in the worsening group" should be explained in the result section.We added more explanations about fig 4

of discussion, " Fig 4.Var
able alterations between the first and second sample collection in the worsening group" should be explained in the result section.We added more explanations about fig 4

○ 5.With using terms like "most" and "majority", the percentages must be mentioned next to them.
○ 5.With using terms like "most" and "majority", the percentages must be mentioned next to them.
We agreed and added percentages next to them ○ 6. Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.we reconstructed the statements and reduced repetitions ○ 7. Attempt may be made to include data on patients who worsened.we added some narratives about worsened patients in discussion ○ 8.The normal reference laboratory values should be given for all parameters used in the study.

e agreed and added percentag
s next to them ○ 6. Repetition of many result parameters in discussion e.g., "males were most commonly affected in this study" is not appropriate.we reconstructed the statements and reduced r

etitions
○ 7. Attempt may be made to include data on patients who worsened.we added some narratives about worsened patients in discussion ○ 8.The normal reference laboratory values should be given for all parameters used in the study.

We added normal reference laboratory values as footnotes below the table ○ Discussion:

1.This section needs to be rewritten as there are many grammatical errors.The authors must seek professional help.We already consulted with professional translator and we made corrections in discussion.We hope the corrected version will convey the study result more clearly ○ 2. Many lines and paragraphs are not correct.e.g., "This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination.Additionally, uncorrelated IL-6 with coagulopathy markerss due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems,32 inducing amplification of the cascade"., we reconstr We added normal reference laboratory values as footnotes below the table ○ Discussion: 1.This section needs to be rewritten as there are many grammatical errors.The authors must seek professional help.We already consulted with professional translator and we made corrections in discussion.We hope the corrected version will convey the study result more clearly ○ 2. Many lines and paragraphs are not correct.e.g., "This study found that increased levels of IL-6 can predict the deterioration of subjects, but it is insufficient to justify it as a routine examination.Additionally, uncorrelated IL-6 with coagulopathy markerss due to the ability of IL-6 to activate the inflammatory cascade in multiple body systems,32 inducing amplification of the cascade"., we reconstructed the statements ○ 3. The part, "Although subjects did not experience an inflammatory process at admission" is not acceptable.Subjects do not experience inflammatory process they suffer with symptoms.we reconstructed the statement ○ 4. In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we concluded.The word "concluded" may be changed to "assumed".Authors should mention e.g., "in the first sample analysis..." and then start elaboration.The entire part does not fit well with subsequent portions of discussion.Authors are advised to write in flow oriented manner.we tried to reconstruct the structure and add more conjunctive narrations.
cted the statements ○ 3. The part, "Although subjects did not experience an inflammatory process at admission" is not acceptable.Subjects do not experience inflammatory process they suffer with symptoms.we reconstructed the statement ○ 4. In subheading "Correlation between levels of IL-6 with D-dimer, fibrinogen, and PT", "Since there was no correlation found between IL-6, ferritin, and the incidence of coagulopathy, we conclude .The word "concluded" may be changed to "assumed".Authors should mention e.g., "in the first sample analysis..." and then start elaboration.The entire part does not fit well with subsequent portions of discussion.Authors are advised to write in flo oriented manner.we tried to reconstruct the structure and add more conjunctive narrations.

○ 5.The authors are also advised to discuss the results and its implications with a statistician.


Introduction

The introduction needs to be revised to better contextualize on the work performed.Provide rationale of evaluating only prothrombin time and D-dimer instead of adding partial thromboplastin time too whereas it was mentioned in introduction that von Willebrand activity also enhanced.


1.

The term "effectiveness of parameters" in Introduction, para 2 is inappropriate.2.

Provide rationale of evaluating SpO2/FiO2 ratio as the chosen severity index to compared with selected inflammatory markers.


3.


Methods


This section requires substantial revision and improvement

Define the term "BALF" in Study design.


1.

Clarify what happen if enrolled patients received IL-6 inhibitor or Ig later part of hospital stay?Example before day 14.Were they withdrawn or included in the analysis?2.

CRP was not mentioned in the sample collection and tables but is showed in the graphs.


3.

Provide rationale and appropriate reference of using cut off point 14 days for assessment in the Assessment of patient's severity.What if patient stay longer than that and changed in clinical status?


4.

Revise the term "or considered complete" used in Assessment of patient's severity.Perhaps use 'censored'?


5.

Provide reference and appropriate citations used for definition of COVID-19 disease severity progression or worsening.


6.

Provide more information on the standardized approach of administering antibiotics, antivirals, corticosteroids and anticoagulant in the study center.This is to understand the different types of these agents used and possible interaction with the variables assessed.


7.

The term "coagulopathy" used in Statistical analysis, para 1 should be defined earlier.


8.

The term "randomized" in Results, para 1 is incorrect because the study population was not randomized.Perhaps 'randomly selected' is more suitable?9.


Results

For Table 1; should add normal reference range as foot note.1.

Statement "subjects with severe disease experienced worsening on day 14..." should be "subjects with severe disease experienced worsening within 14 days of..."


2.

Percentage of hypertension should be reflected in the table 1; can be as footnote.


3.

The statement "All subjects were given the combination of remdesivir, favipiravir, and oseltamivir" is not consistent with the actual number of patients received it.


4.

Author should describe the approach or basis of how different types of antibiotics given to these patients.This important to understand fa ○ 5.The authors are also advised to discuss the results and its implications with a statistician.

Introduction
The introduction needs to be revised to better contextualize on the work performed.Provide rationale of evaluating only prothrombin time and D-dimer instead of adding partial thromboplastin time too whereas it was mentioned in introduction that von Willebrand activity also enhanced.

1.
The term "effectiveness of parameters" in Introduction, para 2 is inappropriate.2.
Provide rationale of evaluating SpO2/FiO2 ratio as the chosen severity index to compared with selected inflammatory markers.

This section requires substantial revision and improvement
Define the term "BALF" in Study design.

1.
Clarify what happen if enrolled patients received IL-6 inhibitor or Ig later part of hospital stay?Example before day 14.Were they withdrawn or included in the analysis?2.
CRP was not mentioned in the sample collection and tables but is showed in the graphs.

3.
Provide rationale and appropriate reference of using cut off point 14 days for assessment in the Assessment of patient's severity.What if patient stay longer than that and changed in clinical status?

4.
Revise the term "or considered complete" used in Assessment of patient's severity.Perhaps use 'censored'?

5.
Provide reference and appropriate citations used for definition of COVID-19 disease severity progression or worsening.

6.
Provide more information on the standardized approach of administering antibiotics, antivirals, corticosteroids and anticoagulant in the study center.This is to understand the different types of these agents used and possible interaction with the variables assessed.

7.
The term "coagulopathy" used in Statistical analysis, para 1 should be defined earlier.

8.
The term "randomized" in Results, para 1 is incorrect because the study population was not randomized.Perhaps 'randomly selected' is more suitable?9.

Results
For Table 1; should add normal reference range as foot note.1.
Statement "subjects with severe disease experienced worsening on day 14..." should be "subjects with severe disease experienced worsening within 14 days of..."

2.
Percentage of hypertension should be reflected in the table 1; can be as footnote.

3.
The statement "All subjects were given the combination of remdesivir, favipiravir, and oseltamivir" is not consistent with the actual number of patients received it.

4.
Author should describe the approach or basis of how different types of antibiotics given to these patients.This important to understand factors may influence the inflammatory markers evaluated in different category of patients.

ors may inf
uence the inflammatory markers evaluated in different category of patients.


5.

Statement "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT" should be, "Correlation coefficient

5.
Statement "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT" should be, "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels".

6.
Disease severity index was not mentioned in introduction and methodology.7.
Discussion; Characteristics of the study subjects Para 3, line 2: statement "In this phase, most patients begin to feel shortness of breath accompanied by hypoxia" is this refers to all mild cases?If so, then it is incorrect statement.

inflammator
The argument using Zhi et al., findings was rather contradicting to author's finding of no correlation with IL-6.

Conclusion
Perhaps should include differences in inflammatory and hemostatic response between initial presentation and as patient became worse.
these patients.This important to understand factors may influence the inflammatory markers evaluated in different category of patients.We added explanations in results section regarding the selection of type of antibiotics given to the patients ○ Statement "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT" should be, "Correlation coefficient analysis using the Spearman method showed p≥0.05 between IL-6 and ferritin, fibrinogen, D-dimer, and PT levels".

itin,
We revised the sentence like you suggested ○ Disease severity index was not mentioned in introduction and methodology.We added explanations regarding disease severity index in introduction ○ Discussion; Characteristics of the study subjects Para 3, line 2: statement "In this phase, most patients begin to feel shortness of breath accompanied by hypoxia" is this refers to all mild cases?If so, then it is incorrect statement.We revised the statement.What we meant was most patients begin to feel chest discomfort and in moderate or severe cases, begin to feel shortness of breath.

Conclusion
Perhaps should include differences in inflammatory and hemostatic response between initial presentation and as patient became worse We added more explanations regarding the difference in inflammatory and coagulation marker between initial and later sample.

○
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Figure 2 .
Figure 2. Correlation between IL-6, ferritin, D-dimer, and fibrinogen variables with SpO 2 /FiO 2 ratio at the initial observation.Spearman correlation test.CRP: C-reactive protein.Notes: A-D: correlation between SpO2 /FiO 2 ratio and inflammatory markers CRP, IL-6, ferritin, and fibrinogen with a statistical significance was found between SpO 2 /FiO 2 ratio and inflammatory markers CRP, ferritin, and fibrinogen, but not with IL-6.FigureE and F: correlation between SpO 2 /FiO 2 ratio and coagulation markers D-dimer and prothrombin Time with a statistical significance was found between SpO 2 /FiO 2 ratio and D-dimer, but not with prothrombin time.

Figure 3 .
Figure 3. Correlation between IL-6, ferritin, D-dimer, and fibrinogen variables with SpO 2 /FiO 2 ratio at the end of observation.Spearman correlation test.CRP: C-reactive protein.Notes: A-D: correlation between SpO 2 /FiO 2 ratio and inflammatory markers CRP, IL-6, ferritin, and fibrinogen with a statistical significance was found between SpO 2 / FiO 2 ratio and inflammatory markers CRP and ferritin, but not with IL-6 and fibrinogen.Figure E and F: correlation between SpO 2 /FiO 2 ratio and coagulation markers with a statistical significance was found between SpO 2 /FiO 2 ratio and D-dimer and prothrombin time.

2 .
Discussion: Correlation between levels of IL-6 with D-dimer, fibrinogen, and PTIt is difficult to analyze and to explain what really happened among worsening patients as no clear definition of this event and what type of treatment they received?1.

○Discussion:
Correlation between increased levels of IL-6 and ferritin Para 2, line 1: statement should be "...patient did not show raised inflammatory markers...".The argument using Zhi et al., findings was rather contradicting to author's finding of no correlation with IL-6.We reconstructed the statements.

○Discussion:
Correlation between levels of IL-6 with D-dimer, fibrinogen, and PTIt is difficult to analyze and to explain what really happened among worsening patients as no clear definition of this event and what type of treatment they received?We tried to explain the management protocol used in the result section and explain what happened in worsening group based on the markers analysed ○

Table 1 .
Characteristics of the study subjects.

Table 2 .
Variables' distribution according to disease severity and outcomes.

Table 3 .
Correlation between IL-6 and other variables.

Table 4 .
Area under ROC curve for the variable differences and Covid-19 patients' deterioration.
Notes: AUC: Area under the ROC curve, ROC: Receiver operator characteristic, CI: Confidence interval.