Evaluation of serum complement C4 among COVID-19 patients in Khartoum state: a case control-based study

Background: The complement system is made up of an abundance of unique plasma proteins that play an important role in innate immunity and inflammation, aiding in the fight against pathogenic microbes and viral diseases. The purpose of this study was to evaluate the serum complement C4 concentration in COVID-19 patients in Khartoum and compare them to healthy controls. Methods: A total of 100 samples were collected, 50 samples from COVID-19 patients who presented as cases and 50 samples from people who were evidently healthy. Overall, 33 (66%) the patient populations in the case group were not in the hospital’s intensive care unit (ICU), compared to 17 (34%) who were. The concentrations of C4 in each serum sample were calculated in milligrams per deciliter. SPSS version (20) was used to analyze the data. Results: The means level of complement C4 (mg/dL) were 37.44±18.618, 23.90±10.229 in the case group and in the control group, respectively. There was a statistically significant difference in complement C4 level between case and control (p-values ≤0.01). In addition, the mean complement C4 level in the ICU and non-ICU case groups was 25.00±17.85 and 43.85±15.712 mg/dL, respectively. There was a statistically significant variance in complement C4 level between ICU and non-ICU (p-values ≤0.01). Furthermore, the cases were divided into four age groups: 20-40, 40-60, 60-80, and over 80 years old. The one-way ANOVA test showed no statistically significant differences between age categories in complement C4 level (P = 0.735) Conclusions: The case group had a higher mean level of complement C4 than the control group, which could be understood by the stimulation of the complement cascade during the COVID-19 illness. Furthermore, the complement C4 level in severe COVID-19 patients was lower than in non-severe COVID-19.


Introduction
In the last twenty years, endemic cases of the Middle East respiratory syndrome (MERS)-CoV in 2012 and severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 both occurred, with case mortalities ranging from 14-15% to 35%. 1 At the end of December 2019, a new coronavirus was determined to be the root of a cluster of pneumonia cases with an unknown cause in Wuhan, Huanan Fresh fish Supplier Market, the city in China's Hubei Province that served as the initial site where cases of coronavirus disease 2019 (COVID-19) were identified. 2 The novel coronavirus spread rapidly, causing an epidemic in China, and a pandemic with rising incidence in many other nations all over the globe. 2 Symptomless infection, mild upper airway disease, severe and highly contagious pneumonia, respiratory distress, and sometimes even fatality are all on the clinical phenotype of SARS-CoV-2 infection. 3itially, a link to a seafood restaurant in Wuhan that sold live animals and was frequented or worked at by the majority of the previous pneumonia patient populations was noticed.But as the pandemic disease spread, person-to-person transmission took over as the main way it was spread.Large droplets released during sneezing and coughing by symptomatic patients help spread the COVID-19 infectious disease, but transmission can also occur from people who are symptomless before developing symptoms. 4The COVID-19 virus can cause clinical and pathological lung lesions that resemble those seen in other types of acute respiratory distress syndrome (ARDS) because it enters lung tissue after sticking to viral Spike proteins (S) and ACE2 receptors. 5 order to eliminate invasive pathogens, complement pathways, a crucial part of the innate immune system, are activated by coronaviruses. 6The main complement molecules C3 and C4 are cleaved by the proteolytic process when the complement system is activated, resulting in the cleavage products C3a, C3b, C4a, and C4b, which can cause the recruitment of inflammatory cells and the activation of neutrophils. 7Patients with mild COVID-19 have shown higher levels of C3 and C4 complement, whereas those with severe COVID-19 had lower levels, which might be clarified by higher consumption from the formation of immune complexes. 8 light of this, the goal of this study was to compare the serum complement C4 levels of COVID-19 patients in Khartoum to those of healthy controls.Additionally, we compared the COVID-19 patients' levels of C4 based on the severity of their illnesses.

Ethics approval and consent to participate
The Ethics Committee of Sudan University of science and technology approved this research (approval number: 112BNC/12/2021; approval date: 01/12/2021).In addition, written approval was obtained from the Jabra hospital of Emergency and Trauma manager (approval number: JET 36 in December 2021).Respondents provided written informed consent after being guaranteed that only investigators would have access to the data and that it would only be used for investigation.Respondents could fill the questionnaire privately.All procedures in this study were carried out in conformity with the rules and regulations specified in the Helsinki Declaration that were applicable.

Study setting
During the period from January to April 2022 a total of 100 subjects were involved in this study, of which 50 were cases confirmed to have COVID-19 infection and another 50 apparently healthy control were the control group.Cases were matched in sociodemographic characters.Cases and control were randomly selected from patients attending Jabra hospital of Emergency and Trauma.Gender was informed based on the self-administered questionnaire filling.

Inclusion and exclusion criteria
The study included patients diagnosed with COVID-19 and apparently healthy controls.Patients who had received any care before being admitted other than antipyretics, had any recorded coinfections, or had immune dysfunction or malignant neoplasms were excluded.

Sample collection and analysis using DIRUI CS-T180
A volume of 3 mL of blood was collected in plain containers from both patients and controls at Jabra Hospital of Emergency and Trauma.Then the samples were centrifuged, serum separated and stored at -80°C until analysis.C4 ELISA kit was obtained from Abcam (United Kingdom).All the protocols were done according to the manufacturer instruction.After that all reagents were brought to 37°C for warming, then 800 μL from R1 (Diluent) were added to 20 μL from the sample or calibrator and 200 μL from R2 (Antibody); finally, the absorbance of calibrator and sample had been read.The concentration of C4 was obtained by calculating the differences between calibrator and sample reading.

Data analysis
The Statistical Package for Social Science (SPSS) Version 20 was used for data analysis.The data was presented as mean and standard deviation, frequency; Pearson's correlation, Ttest results, and one way Anova have been used as specific tests with a significant threshold value of 0.05 (P-value≤0.05).
Males and females in the case group had average complement C4 levels of 40.15AE20.095and 32.18AE14.466respectively.There was no discernible gender difference in the complement C4 level among the cases group (P-value=0.153,Table 4).Based on their age, the cases were divided into four groups: the first group was made up of patients that were between the ages of 20 and 40; the second group was made up of patients between the ages of 40 and 60; the third group was made up of cases between the ages of 60 and 80; and the fourth group (80-100 years).The level of complement C4 did not significantly differ between age groups, according to a one-way ANOVA test (P-value=0.735,Table 5).The case group's average complement C4 levels were 25.00AE17.85mg/dL and 43.85AE15.712mg/dL respectively.There was a statistically significant difference in the complement C4 level between ICU and non-ICU P-value was 0.00 (Table 6).

Discussion
Immunoassays are frequently used in clinical practice to measure complement C4 and determine and track complement activation.A decrease in serum C4 levels brought on by increased immune system C4 consumption.Complement C4 testing in COVID-19 patients may be able to shed light on how clinical risk is balanced between normal and abnormal complement activation. 9In the current study, in serum samples, complement C4 levels were assessed in 50 COVID-19 patients and 50 healthy individuals.The findings showed that there was a significant difference between the case group and control in terms of the mean complement C4 level (P-value=0.00).Zinellu and Mangoni (2021), showed there was a noticeably higher level of complement C4 in the case than in the control, which supports this finding (P-value less than 0.05).In the current study, patients with severe COVID-19 disease had lower complement C4 levels than patients with less severe COVID-19 disease, and this difference was significant (P-value=0.00).This finding concurs with that of Ghazavi and colleagues in Iran, who discovered that complement C4 levels were lower in COVID-19 patients with severe disease (P=0.014) and higher in those without severe disease (P-value 0.012). 8Al-Hakeim and his colleagues also discovered in Iraq that patients who spent longer than two weeks in the hospital had lower complement C4 levels than those who were discharged earlier (P-value less than 0.05). 10This result supported the findings of Al-Hakeim and his colleagues in Iraq, who found no correlation between the mean complement C4 levels across age groups and sex (P=0.681). 10Males and females in the case group had average complement C4 levels of 40.15AE20.095and 32.18AE14.466respectively.There was no discernible gender difference in the complement C4 level among the cases group.Prior clinical evidence has revealed that, while males and females share a comparable illness rate, males are more likely to be hospitalized, experience a more severe illness progression, and die at a greater rate than females. 11study with a large number of patients in the asymptomatic and ICU groups using COVID-19 is recommended to generate clearer and more significant results.Due to funding limitations, this research was limited to 50 COVID-19 patients.As a result, it is recommended that similar studies be conducted on a greater number of COVID-19 cases.Also, research on the other complement components would be valuable in order to acquire a better understanding of the complements pathway's involvement.There is considerable debate over how soon complement overuse must be monitored and which components of complement consuming must be evaluated.

Conclusions
Comparing the case group to the control, there was a higher mean level of complement C4, which can be attributed to the activation of the complement system during COVID-19 infection.Furthermore, the complement C4 level was lower in patients with severe COVID-19 particularly in comparison to those with non-severe COVID-19, which could be because the immune system of patients with severe infection consumes more complement C4.
Authors' contributions YA performed main experiments, YA, NM, MY, BG collected' samples and information.MY, designed the experiments and wrote the manuscript.All authors read and approved the final manuscript.The project contains the following underlying data: -Mohammed Yahya data.xlsx(13.57kB).
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Andang Miatmoko
Universitas Airlangga, Surabaya, East Java, Indonesia This manuscript provides supporting data regarding the profiles of complement C in healthy subjects and COVID-19 patients with different severity of illnesses.Some information needs to be clarified or confirmed to improve the quality of the manuscript.Please give more details about the roles of complement C in the progression of infectious diseases so the manuscript's main idea can be clearly defined.In addition, the authors have cited research that shows the profile of complement c in COVID-19 (Ref.8).So, what is this study's new information or added value?Please define it clearly. 1.
In this study, the authors did not provide details of medication taken or consumed by the subjects.Please add the details since using different classes of antipyretics may affect the complement c levels.

2.
Regarding mild and severe cases, the authors defined them based on ICU and non-ICU cases.However, ICU patients often have comorbidities, causing poor prognosis of the disease.This study focuses on complement C and the severity level of COVID-19, so it needs specific data regarding the severity of COVID-19, for example, Ct values or any other COVID-19 markers.

3.
Please describe when the blood sampling was conducted in the method section.In addition, the incubation period of infection may affect the levels of complement c, and it can be analyzed from the day patients suffered from the symptoms and the day they were admitted to the hospital.Please add the data about this.Or, could the author provide the references that the incubation period does not affect the levels of complement c.

4.
Since the profiles of serum complement levels between control and case, and mild case and severe case had different results, which is affected by the immune response, please add the data regarding blood neutrophils so the analysis can be carried out well.

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?Yes

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.
Reviewer Expertise: Pharmaceutical Sciences I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
This manuscript includes a case-control study that attempted to correlate complement C4 levels with COVID-19 infection pathogenesis and outcome.The complement C4 level in the case group was lower than in the controls.The manuscript material could be viewed as additional support for the previous research highlighting the role of complement in the pathophysiology of COVID-19.
Despite the fact that the manuscript is nicely written, and adds to the current literature, I have some comments and questions that should be addressed by the author: The introduction is excessively brief and lacks critical information about the correlation between complement and COVID-19 pathogenesis, with only one paragraph detailing this crucial part.As a result, authors should cite some recent papers to validate their findings.

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Section of the methodology: 2. The optimal Cases to Control ratio should be 1:4, but the authors used 1:1 why?
3. Why were patients with immunological dysfunction or malignant neoplasm excluded from the study?Inclusion and exclusion criteria section?It should be clearer or deleted.
Results: This section is well-written, and the statistical analysis and interpretation are precise.

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In the discussion part, ''Prior clinical research has demonstrated that, whereas males and females share a comparable sickness rate, males are more likely to be hospitalized, experience a more severe illness progression, and die at a higher rate than females'' -is this comment a criticism or an interpretation?
○ Conclusions: Well written and accurately reflects the author's findings.

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? Yes
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.

Reviewer Expertise: Microbiology
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Thanks you for your time to review our manuscript In the introduction part 1. Regarding the introduction part we will make the required modifications which will appear in the second version of the manuscript.
In the methodology part 2. Regarding the sample size I would like to inform you that we used a total coverage sampling technique therefore we involved all COVID-19 patients in specific time manner and we use 1:1 case to ratio because this is the least ratio that can be applied for sampling the case control study population and it's acceptable in many previous investigations.
3. patients with immune dysfunction or malignant neoplasm were excluded be uses such disorders will affect the immune Status of cases and controls and consequently will affect on the complement level including C4 and according this will lead to bias in the results therefore we excluded them from the cases and controls.

in the discussion part
Prior clinical research has demonstrated that, whereas males and females share a comparable sickness rate, males are more likely to be hospitalised, experience a more severe illness progression, and die at a higher rate than females.This section is interpretative part.

Adnan Mezher Maysam
College of Pharmacy, Tikrit University, Tikrit, Saladin Governorate, Iraq I think this article is an important addition to the literature, because the complement is a vital component of innate immunity where it forms the first line of defense against potentially harmful microbes, but its role in COVID-19 is still not clear.In this article, the results showed that case group had a higher level of complement C4 than the control group, which means that the complement plays an important role in the severity of disease, and the C4 complement should be considered as a diagnostic marker.
I hope the authors intend to evaluate level of anti-pro inflammatory cytokine, such as IL-37 and IL-38, soon in severe and non-severe COVID-19 cases.
I have carefully read this manuscript and below are my comments: Are the authors discussing the role of complement in pathogenesis of COVID-19 patients or in immune system?Clear this point.

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Indicate the aim of this paper more clearly and the conclusion also needs improvement.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

○ 1 .
How and at what power was the sample size estimated?
Interests: Authars have no any competing interests Reviewer Report 04 July 2023 https://doi.org/10.5256/f1000research.145606.r182340© 2023 Mezher Maysam A. 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.

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Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Clinical immunology, COVID-19

Table 4 .
Comparison of complement C4 level among cases group based on their sex.

Table 3 .
Comparison of complement C4 level between case and control.

Table 2 .
Sex frequencies in ICU and non-ICU in case group.

Table 1 .
Mean AE SD of age and gender distribution among the study groups.

Table 6 .
Comparison of complement C4 level among cases group according to the severity of disease.

Table 5 .
Comparison of complement C4 level among cases age groups.