Inflammatory laboratory findings associated with severe illness among hospitalized individuals with COVID-19 in Medan, Indonesia: a cross-sectional study

Background: Coronavirus disease (COVID-19) remains a global health problem. COVID-19 patients with severe pneumonia have a higher risk for critical illness, mostly complicated by acute respiratory distress syndrome. The inflammatory response is critical, and the cytokine storm increases the severity of COVID-19. Many factors could be associated with a cytokine storm but they are incompletely understood. This study presents characteristics of COVID-19 patients and explore the clinical and inflammatory parameters of severe and critically ill COVID-19 patients in the intensive care unit (ICU). Method: This cross-sectional study was conducted in all severe COVID-19 patients admitted to the ICU. Peripheral blood was taken for laboratory examination within 24 hours of admission. Haematologic parameters, serum electrolyte, renal function, liver function, pancreas enzyme, D-dimer, inflammatory cytokines interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), and C-reactive protein (CRP) were assessed in this study. Comparative analyses were done between sex, existing comorbidities, body mass index (BMI), and COVID-19 vaccination status. Results: A total of 80 subjects were included in the study. The most frequent comorbidities found among the subjects were obesity (36.35%) and diabetes (22.5%). Only 13.75% of subjects were vaccinated. Laboratory results indicated leucocytosis and neutrophilia, with a neutrophil-lymphocyte-ratio (NLR) of 7. The mean inflammatory findings (IL-6, IL-10, TNF-alpha, IFN-gamma, MCP-1), D-dimer, CRP, and lipase increased. Lipase levels were higher in men (p = 0.003) and in comorbidity groups. No significant differences were found among different BMI groups. Lipase, IL-6, and MCP-1 levels were significantly higher (p=0.019, <0.0001, and 0.03, respectively) in the non-vaccinated group. Conclusions: Most patients with severe COVID-19 have comorbidities and increased inflammatory markers.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the cause of the catastrophic coronavirus disease (COVID-19) pandemic that began in January 2020. 1,2It has claimed 4.4 million human lives as of August 22, 2021. 3By August 2021, 4,043,736 cases were reported in Indonesia with 130,182 deaths. 4COVID-19 has a fatality rate of 2.3%, less than the outbreaks of severe acute respiratory syndrome coronavirus outbreak (SARS-CoV) (9.5%) in 2003 and the Middle East respiratory syndrome coronavirus outbreak (MERS-CoV) (34.4%) in 2012. 5The SARS-CoV-2 outbreak initially was linked to the Hua Nan seafood and wet animal market in Wuhan. 6e SARS-CoV-2 infection varies from asymptomatic, mild upper respiratory tract illness, to severe pneumonia with respiratory failure and death. 7Patients with severe COVID-19 usually present with respiratory rates greater than 30 breaths/minutes, oxygen saturation (SpO 2 ) less than 93%, and greater than 50% lung infiltrates, and are at higher risk for clinical deterioration and critical illness. 8Acute respiratory distress syndrome (ARDS) was the most common complication occurring in 60% to 70% of patients admitted to the intensive care unit (ICU). 9ARDS occurs most often in the setting of pneumonia, sepsis, aspiration of gastric contents or severe trauma and is present in ~10% of all patients in ICU worldwide. 10This wide range of differences is presumably due to the atypical disease process in ARDS, suggesting the non-effectivity of mechanical ventilation in reducing lung injury. 11[14][15] The inflammatory response plays a critical role in COVID-19.The inflammatory cytokine storm increases the severity of COVID-19. 16,172][23] The cytokine storm is crucial to the progression of COVID-19 and might lead to ARDS and death. 24Patients who survive from cytokine storms tend to suffer long-term lung damage and fibrosis, causing impairment in pulmonary function and lower quality of life. 25 Indonesia, resources for the management of COVID-19, particularly laboratory parameters, remains constrained.This issue heightened the need for simple approaches to detect cytokine storms in patients with COVID-19, which could help stratify the risk of morbidity and mortality in COVID-19 patients at the time of hospitalisation.This study presents details of patients with COVID-19 hospitalised in the ICU of Mitra Medica General Hospital in Medan, North Sumatera, Indonesia.We aim to explore the clinical and inflammatory parameters of severe and critically ill COVID-19 patients in the ICU.

Ethical approval
This study was approved by the ethics committee of Universitas Sumatera Utara (Ethical clearance number 453/KEP/ USU/2020).The ethics committee is in charge of the North Sumatera province including this study location.Informed consent was obtained before data collection.In this study, written informed consent was obtained from each patient's proxy if the patient was unconscious.Otherwise, written informed consent was obtained from the corresponding patient.

Study and patients
This cross-sectional study was conducted in all COVID-19 cases (confirmed by the RT-PCR test) admitted to the ICU of Mitra Medica General Hospital Medan, Indonesia, between May and June 2021.Inclusion criteria were all subjects classified as severe COVID-19 according to the World Health Organisation guidelines. 26The diagnosis of severe COVID-19 was made if subjects met one or more of the following criteria: dyspnoea, respiratory rate of 30/min, SpO 2 of 93%, PaO 2 /FiO 2 ratio less than 300 mm Hg, greater than 50% lung infiltrate on CT scan within 24 to 48 hours, and those with respiratory failure, septic shock, and/or multiple organ dysfunction. 26

Data collection
Demographic data, clinical history, and vaccination status of patients were collected from their medical records, including COVID-19 vaccination status.BMI data were calculated from the patient's weight and height.A total of 10 mL peripheral

REVISED Amendments from Version 1
We have fixed grammatical errors and added statistical test results in supplementary data.

Statistical analysis
Statistical analysis was done using GraphPad Prism version 8.0.A normality test with the Kolmogorov-Smirnov test was conducted to determine the distribution normality of the data.Parametric data were presented in as means AE standard deviations, while non-parametric data were presented as medians and interquartile ranges.Data were compared between genders, subjects with comorbidities and without comorbidities, BMI, and vaccination status.Patients' BMIs were classified as underweight, normal weight, overweight, and obese based on BMI criteria for Asia. 27The differences between the two groups were tested with the independent t-test and the Mann-Whitney test.The t-test was utilised for parametric data and the Mann-Whitney test for non-parametric data.Meanwhile, differences between more than two groups were done with the one-way ANOVA test for parametric data and otherwise with the Kruskal Wallis test.Statistical analysis was performed within 95% confidence intervals.Significance was established based on p-values of <0.05.

Patient demographics and clinical features
A total of 80 subjects were included in the study.The demographic data are presented in Table 1.The mean ages of all the subjects was 59 years old, and most were male.The most frequent comorbidity found among the subjects was obesity (36.35%), followed by diabetes (22.5%).Only 11 subjects (13.75%) were vaccinated in this study.

Laboratory findings
Table 2 presents the laboratory results from this study.The leucocyte and neutrophil percentages increased in the subjects.The neutrophil to lymphocyte ratio (NLR) was 7. The inflammatory findings were increased in severe COVID-19 patients compared with normal value in the study.D-dimer as a coagulopathy parameter increased above the normal range in this study.The subjects also had increased CRP, ALT levels, AST levels, and lipase.Other parameters including serum electrolyte levels and renal function, were within normal reference values.Results of comparison analysis between males and females are shown in Figure 1.Lipase levels were higher in men (129.5 (AE52.32),p = 0.003).Analyses of BMI groups are presented in Table 3.There are no significant differences found between different BMI groups.As for the analysis regarding non-comorbid and comorbid groups, lipase levels were higher in groups with comorbidity compared with those without comorbidity (shown in Figure 2).Between the vaccinated and non-vaccinated groups, results indicated a significantly higher level of lipase, IL-6, and MCP-1 (p-values = 0.019, <0.0001, and 0.03, respectively) in the non-vaccinated group (Figure 3).

Discussion
More men were included in our study group with severe COVID-19, suggesting that they suffer from the severe form of COVID-19 compared with women.Also, recent studies suggested that men also tend to present more severe forms of the disease and have a higher mortality rate. 28The number of men who died is 2.4 times that of women.While men and women had the same susceptibility, men were more prone to dying. 28,29Potential risk factors have been suggested, including different behaviours between genders, genetic and hormonal factors, and the influence of sex genetics in viral pathogenesis. 29Risky behaviours such as smoking and alcohol consumption have been reported in more men than women. 30These behaviours increase risks for hypertension, cardiovascular disease, and chronic pulmonary disease, which could exacerbate the severity and susceptibility to COVID-19.The mechanism of SARS-CoV-2 infection is regulated by the expression of ACE-2 and TMPRSS2 genes.These factors are often associated with sex.For instance, the ACE-2 gene is found on the X-chromosome.Inactivation of this gene has been associated with the incidence of All data presented as median (interquartile range).
COVID-19 in males and females. 31The gene that transcribes TMPRSS2 is influenced by androgens, and the presence of androgens promotes the expression of TMPRSS2.TMPRSS2 acts as a co-receptor for SARS-CoV-2 cell invasion, and its high expression will lead to increased susceptibility for COVID-19. 32,33e mean age of all the subjects in our study was 59 years old, with the youngest subject over 50 years old.Older people were more sensitive to SARS-CoV-2 infection and had a higher positive rate than younger individuals. 34Also, older subjects were associated with increased mortality from COVID-19 due to poorer lung function and the likelihood of having comorbidities when compared with younger patients. 34,35Ageing is associated with an increase in proinflammatory cytokine levels (CRP, D-dimer, procalcitonin, and IL-10). 36Older patients typically experience a decline in physiological immune function and immunosuppression, thus making it difficult for them to control pro-inflammatory responses. 35esity is one comorbidity related to severe COVID-19. 37Subjects with a BMI of less than 18.5 kg/m 2 and greater than 25 kg/m 2 have a higher risk for a fatal illness. 38In this study, a total of 36.35% were obese and 6.25% were underweight based on Asian BMI criteria.Obesity is one of the risk factors for cardiometabolic disease and is reported to cause immune system dysregulation.Obese patients have the highest risk for longer hospitalisation and death due to COVID-19. 39Adipocytes could increase the inflammatory response by stimulating macrophages to produce interleukins (IL-1, IL-6, IL-8, IL-10) and TNF-alpha.Meanwhile, underweight COVID-19 patients are at risk of developing acute kidney injury, worsening the patient's condition. 40In this study, laboratory parameters were not significantly different within various BMI groups.This might also be influenced by other patient condition.
Diabetes mellitus is also widely associated with the incidence of COVID-19. 41Increased glucose metabolism in patients with diabetes could directly enhance the replication of SARS-CoV-2.Increased glucose escalates the production of mitochondrial reactive oxygen species and activates hypoxia-inducible factor 1α. 42 Insulin resistance itself is associated with an impaired response to IFN type 1, thus generating a high viral load and inhibiting the body's inflammatory response. 43ccination is part of the prevention program against SARS-CoV-2.The vaccine promotes antibody production to prevent COVID-19. 44Also, vaccine administration effectively reduces disease severity. 23In this study, 86.25% of patients were reported as non-vaccinated.Several parameters were significantly different between the vaccinated and non-vaccinated groups.Lipase, IL-6, and MCP-1 were higher in the non-vaccinated group.Increased lipase and IL-6 indicate an inflammatory response and a more severe disease. 7On the other hand, MCP-1 is suspected of inhibiting of .Laboratory parameters between genders.Lipase levels were significantly higher in men (129.5 (AE52.32),p = 0.003).C-reactive protein, aspartate aminotransferase levels, and interferon-gamma were also higher in men.Women, though not significant, had higher levels of D-dimer, alanine Aminotransferase levels, amylase, tumour necrosis factor-alpha, interleukin-6, interleukin-10, and monocyte chemoattractant protein-1.Laboratory parameters between non-comorbid and comorbid groups.Lipase levels were significantly higher in patients with comorbidities.Most laboratory parameters were higher in patients with comorbidities, except for aspartate aminotransferase levels and interferon-gamma.

Table 3. Inflammation laboratory findings of hospitalised patients infected with SARS-Cov
IFN-signalling. 45IFNα and IFN-β have antiviral activity; thus non-vaccinated individuals tend to have poorer immune response due to low antiviral activity. 46bjects in this study had an increased leukocyte count and lymphopenia.This finding was also observed in the metaanalysis by Huang et al., which reported that patients with severe COVID-19 tended to have higher leukocyte counts and lower lymphocyte counts compared to non-severe illness. 47Leucocytosis may be present due to co-infection with bacterial pneumonia.Steroid medication given to those with severe illness induces leucocytosis or variability in the immune response. 48Lymphopenia might be directly induced by lymphoid tissue destruction, inflammatory cytokines or a metabolic disorder that caused by COVID-19 infection.TNF-alpha, IL-6, and other inflammatory cytokines could induce a lymphocyte deficiency. 49utrophilia was also reported in this study, with an NLR of 7. In patients with COVID-19, NLR may reflect the severity of inflammation.Neutrophil percentages have been mainly within the normal range in non-severe cases but were increased in the severe form of illness. 48Older and critical patients tend to present with neutrophilia, suggesting that this condition related to the cytokine storm. 50,51A predictive risk model by Liu et al. suggested that the incidence of severe disease was 50% in patients with aged 50 years or older and NLR of greater than or equal to 3.13 compared with 9.1% in patients with an age 50 years or older and NLR of less than 3.13. 52In our study, subjects were >50 years with an NLR greater than 3.13, thus correlating with the risk of severe disease.
Inflammatory parameters (IL-6, IL-10, TNF-alpha, IFN-gamma, MCP-1) were increased in COVID-19 patients with severe illness.These findings were also found in recent studies. 16,20,24,53,54Patients with COVID-19 had high amounts of pro-inflammatory cytokines (IFN-gamma, TNF-alpha, IP-10, IL-1B, MCP-1).Patients requiring ICU admission had higher cytokines, suggesting that a cytokine storm was associated with ARDS progression and severe illness.However, COVID-19 patients also present with increased anti-inflammatory cytokines (IL-4, IL-10), that differ from those in SARS-CoV infection. 24,53The Univariate Cox Analysis by Yang et al. indicated that circulating IL-6 significantly predicted the progression of COVID-19 infection.Serum IL-6 was higher in COVID-19 patients with pneumonia than those without pneumonia.Increased IL-6 might induce tissue-damaging-inflammation and cause alveolar cell injury. 20atients with IL-6 levels greater than 32.1 pg/mL were more likely to have severe complications. 16IL-6 trans-signalling could enhance the production of IL-8, MCP-1, and IL-10. 54dimer increased in our subjects, with a mean of 800 ng/mL.D-dimer is a fibrin degradation product widely used as a biomarker for thrombotic disorders.D-dimer value of less than 500 ng/mL is usually considered normal.D-dimer can predict severe and fatal cases of COVID-19 with moderate accuracy (sensitivity 77%, specificity 71%). 55In a multicentre meta-analysis by Paliogiannis et al., D-dimer concentrations in patients with severe COVID-19 were significantly higher than those with non-severe forms. 56In the analysis by Ozen et al., threshold D-dimer value of 370 ng/ml was calculated to have 74% specificity and 77% sensitivity for predicting lung involvement in COVID-19 patients. 57A cut-off of 1500 ng/mL is the optimal value of admission D-dimer for predicting mortality in COVID-19 patients. 58P levels increased almost eightfold above reference values in this study.CRP is an active regulator of host innate immunity and induces the classical complement pathway.Therefore, it can mediate inflammation. 59A significant increase in CRP was found in COVID-19 patients, with average levels from 20 to 50 mg/L. 60CRP usually is not elevated in viral infections, but the macrophage activation syndrome may explain the high serum CRP and poorer disease progression.Elevated CRP may also indicate co-infections of bacterial aetiology. 61Up to 86% of patients with a severe COVID-19 had increased CRP in higher concentration than mild or non-severe patients. 62The risk of developing severe events is increased by 5% for every one-unit increase in CRP levels in COVID-19 patients. 63e mean level of lipase was significantly higher in men in our study.This finding was similar to the study by Barlass et al., who showed that increased lipase indicated possible pancreatitis and was connected with a poor prognosis. 64Although there was higher lipase activity in the male animal model, there was no definite explanation for higher lipase activity in men. 65Lipase levels were also significantly different in the groups of patients with and without comorbidities.After adjusting the analysis with groups of comorbidities, there was no significant difference.Perhaps comorbidities could interfere with the body's physiological processes and induce stress in various organs, including the pancreas.
This study has limitations because we did not compare the laboratory profiles between severe groups and mild/moderate groups.However, for COVID-19 patients, the presence of comorbidities and elevated inflammatory markers should raise healthcare providers' awareness for the risk of severe disease course.There are also interesting results that could be important for future treatment.Lipase, IL-6, and MCP-1 results were found significantly different between the vaccine and non-vaccine groups.Elevated lipase may indicate possible pancreatic involvement that may be a consideration for managing COVID-19.In the method section, the authors mentioned that among data collected was "vaccination status".Please elaborate whether this refers to COVID-19 vaccination (or others).
In the results, clinical and laboratory parameters were compared to normal reference values.Do these values represent the population where the study was performed (i.e.Indonesian?)In this case, ideally, samples measured from healthy controls and/or patients with milder disease should be included in the study to provide a better comparison.

2.
In table 3, the authors compared inflammatory marker levels in patients with different BMI categories and showed no significant differences between the groups.Did the author also look at the correlation between the BMI values and levels of these markers to further justify this finding? 3.
Figure 2 shows the comparison of laboratory parameters between the non-comorbid and comorbid groups.However, in this study, the assessed comorbidities have completely different mechanisms, and hence, characteristics (for example, those with pulmonary diseases such as COPD or tuberculosis, those with CKD, and those with metabolic or cardiovascular diseases would have different immune background).Can the authors justify why in figure 2 the comorbidities were categorized into one entity and not analyzed as separate? 4.
In figure 3, is the duration post-vaccination (I would assume COVID-19 vaccination?) and numbers of vaccines known?Please elaborate in the methods section.

5.
In the methods section, the authors only mentioned that samples were collected after 24 hours of ICU admission.Is the duration of illness known?The authors should add this in the methods section, or elaborate in the discussion. 6.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment.A qualified statistician is required.

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

Kurnia Fitri Jamil
Department of Orthopaedics and Traumatology, Universiti Kebangsaan Malaysia, Kuala Lumpur, Indonesia The suggestions that we can give to the manuscript are: The grammatical errors in the text were quite large in number.Authors need to send the manuscript for proofreading and correction. 1.
As highlighted by the authors, the time of study was short.2.
It is better if the authors can provide more updates or additional facts that can be obtained from this study compared to what has already been published in the literature.

3.
I suggest that the inflammation laboratory findings, especially C-reactive protein, are compared when the patient is admitted to the hospital and at the end of the treatment, and differentiated by each stage of COVID-19 disease.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
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-2 stratified by body mass index.data presented as median (interquartile range).

Figure 2 .
Figure2.Laboratory parameters between non-comorbid and comorbid groups.Lipase levels were significantly higher in patients with comorbidities.Most laboratory parameters were higher in patients with comorbidities, except for aspartate aminotransferase levels and interferon-gamma.
Underlying data Figshare: COVID Master Data ICU.xlsx, https://doi.org/10.6084/m9.figshare.18027170. 66Intan Mauli Warma Dewi Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands With interest, I read the study entitled "Inflammatory laboratory findings associated with severe illness among hospitalized individuals with COVID-19 in Medan, Indonesia: a cross-sectional study" by Darmadi et.al.In this study, the authors present the clinical and inflammatory characteristics of critically ill COVID-19 patients in the ICU.The results highlight the increased inflammatory profiles in severe COVID-19 patients and in those with comorbidities.Please find some minor comments below:

Reviewer Report 21
December 2021 https://doi.org/10.5256/f1000research.78544.r102157© 2021 Jamil K.This is an open access peer review report 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.

Table 2 .
Clinical and laboratory findings of severe SARS-CoV-2.