ASSOCIATIONS BETWEEN SERUM LEVELS OF C3, C4, AND TOTAL CLASSICAL COMPLEMENT ACTIVITY IN COVID-19 PATIENTS AT THE TIME OF ADMISSION AND CLINICAL OUTCOME


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Abstract

In the present study, we investigated the association between complement system status at the time of admission and clinical outcomes in COVID-19 patients. This single-center study was carried out with sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of Medical Sciences (Bojnurd, Iran) with less than three days passage since onset of COVID-19 symptoms. Twenty-three healthy volunteers with demographic features similar to the patient group (matched by age and gender) were included in the study as a control group. Patient information including demographic information, demographic data, clinical characteristics, and clinical outcomes were obtained from electronic medical records. Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female, and the average age was 48.7 ± 8.8 years. The healthy control group included 23 cases (11 (47.8%) female, 12 (52.1%) males, mean age 46.4 ± 4.4 years). Twenty-one of the 61 patients (34.4%) were admitted to the ICU, and sixteen of them (26.2%) died. Thirty-three (54.10%) patients with COVID-19 were hospitalized for less than 7 days, and 28 (45.90%) of them were hospitalized for ≥7 days. Our results show that length of hospital stay in the no-ICU group was significantly lower than the ICU admission or death groups (6.49 ± 0.24 vs. 8.85 ± 1.59 and 10.53 ± 1.80, p =0.0002). The levels of C3, C4, and CH50 were determined through the immunoturbidimetric method and single-radial-haemolysis plates, respectively, on serum samples obtained from patients at the time of admission or those in the control group. Our results indicate that C3, C4 and CH50 levels were markedly lower in COVID-19 patients than in the control group. We also found that complement parameter levels in COVID-19 patients who died or were admitted to ICU were significantly lower than in non-ICU COVID-19 patients.  In general, it seems that serum level of C3, C4, and CH50 at admission may predict disease progression or adverse clinical outcome in COVID-19 patients.

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Associations between serum levels of C3, C4, and total classical complement activity in COVID-19 patients at the time of admission and the clinical outcomes

Abstract

In present study we investigated the association between complement system status at the time of admission and clinical outcomes in COVID-19 patients. This single-center study was carried out on sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of medical sciences (Bojnourd, Iran) and less than three days passed since onset of symptoms of COVID-19. Twenty-three healthy volunteers, demographic findings were similar to the patient group (matched on age and gender), were included in the study as a control group. Patients' information including demographic information, demographic data, clinical characteristics, and clinical outcomes obtained from electronic medical records. Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female and the average age was 48.7 ± 8.8 years. The healthy control group included 23 cases (11 (47.8%) female and 12 (52.1%) males, mean age 46.4 ± 4.4 years). Twenty-one of the 61 patients (34.4%) were admitted to the ICU and sixteen of them (26.2%) died.33 (54.10%) patients with COVID-19 were hospitalized for less than 7 days and 28 (45.90%) of them were hospitalized for more than ≥7 days. Our results showed that length of hospital stay in no-ICU group significantly lower than ICU admission and death groups (6.49 ± 0.24 Vs. 8.85 ± 1.59 and 10.53 ± 1.80, P =0.0002). The levels of C3, C4, and CH50 were determined through immunoturbidimetric method and single-radial-haemolysis plates, respectively, on serum samples obtained from patients at the time of admission and control group. Our results indicate that levels of C3, C4 and CH50 markedly lower in COVID-19 patients than control group. We also found that levels of these values of complement parameters in COVID-19 patients who died or admitted to ICU significantly lower than COVID-19 patients who did not admit to ICU.  In general, it seems that serum level of C3, C4, and CH50 at admission may predict disease progression and adverse clinical outcomes in COVID-19 patients

Keywords: COVID-19; Complement System; Clinical outcome; Intensive Care Units; Discharge; Mortality;

 

 

 

 

 

 

Introduction

More than a year after its emergence, COVID-19, the disease caused by SARS-CoV-2, continues to plague the world and dominate our daily lives [1]. Patients with COVID-19 may develop symptoms such as fever, dry cough, pharyngeal pain, tiredness, abdominal pain, diarrhea, conjunctivitis, muscle fatigue, and pneumonia and may be left with serious side effects or even die [2]. Previous studies reported that 14.1–33.0% of COVID-19 infected patients are prone to develop into severe cases, and the mortality rate of critical cases is 61.5%, increasing sharply with age and underlying comorbidities [3, 4]. In more severe cases of COVID-19, patients can develop acute respiratory distress syndrome (ARDS), leading to a worse prognosis [5].The deregulated activation of multiple adaptive and innate immune pathways, including the T cell activation, cytokine expression profile from macrophages and neutrophils, the complement system, and several procoagulant and thrombogenic pathways, is believed to fuel a hyperinflammatory state that drives ARDS and may lead to multiple organ injury and finally death in COVID-19 [6, 7].

The complement system is a key part of the innate immune system that plays an important role in defense against foreign pathogens such as viruses but, in addition to being an important part of the immuno-defense system, it plays a critical role in promoting the inflammatory process that leads to organ dysfunction [8]. Although several studies have been carried out on complement system in COVID-19 and its relationship with clinical outcomes [9-13], they did not indicate a clear protective or adverse effect of this system. Dheiret al. in a retrospective study showed there is no significant difference in terms of C3 and C4 levels in both ICU and non-ICU COVID-19 patients [9]. They suggested that measurement of C3 and C4 levels cannot be used to show severity disease [9].Controversially, Ghazavi and co-works found that the levels of C3 and C4 in non-severe-COVID-19 patients significantly higher than severe-COVID-19 patients [10]. Previous study by Fang et al. also indicated decreased complement C3 levels are associated with poor prognosis in patients with COVID-19 [11]. Java and collagenous claimed that role of the complement system in COVID-19 patients is time dependent, when complement activation in the first week of COVID-19 infection can sever as a “friend” and its activation in the second or third weeks of infection can be a “foe” [12]. Zinelluet al. in a system review concluded that lower concentrations of C3 and C4 are significantly associated with higher COVID-19 severity and mortality [13]. They suggested that additional studies are required to determine whether measurement of complement components can be useful to predict adverse clinical consequences in COVID-19 patients [13]. Therefore we conducted a study to assess the association between complement system status at the time of admission and clinical outcomes (e.g., length of stay, ICU admission, discharge, mortality) in COVID-19 patients. Understanding this association will help us elucidate role of complement system for the prediction of the risk of developing critical COVID-19.

 

Materials and methods

Study design and participants

This single-center study was carried out on sixty-one adult patients with COVID-19 who were hospitalized at Imam Hassan Hospital of North Khorasan University of medical sciences (Bojnourd, Iran) from April 20 to August 5, 2021 and less than three days passed since onset of symptoms of COVID-19. All the patients who had positive RT-PCR test for SARS-CoV-2 and common CT imaging findings associated with COVID-19 included in the study.  Patients also had no history of SARS-CoV-2 infection and COVID-19 vaccination. Available data suggest that patients with mild-to-moderate COVID-19 remain infectious no longer than three days after symptom onset. Recent evidence indicated that age and comorbidities could possibly confound the association between complement system status and clinical events [14], therefore we adjusted for the sex, age and comorbidities.

Twenty-three healthy volunteers, demographic findings were similar to the patient group (matched on age and gender), were included in the study as a control group.

Data collection and assessment of serum levels C3, C4 and serum complement activity

We obtained demographic data, exposure history, chronic medical histories, clinical symptoms or signs, clinical outcomes, and hospitalization duration from electronic medical records. We also collected serum samples from PCR-confirmed COVID-19 patient samples sent to Imam Hasan hospital laboratory on the first day of admission to analysis of serum levels of C3, C4, and total classical complement activity (CH50 assay) present in the serum. We also collected Blood samples from the healthy controls with negative PCR-testing for COVID-19. Then blood samples centrifuged immediately; serum was obtained and frozen at -70oc until used.

Serum C3 and C4 concentrations were determined using kits from Roche Diagnostics (Indianapolis, IN, USA) according to the immunoturbidimetric method [15]. The CH50 test was evaluated using the single-radial-haemolysis plates (Biogen, Iran) as described elsewhere [15].  All values were compared to the normal ranges which were reported as 89-187 mg/dL for C3, 10-40 mg/dL for C4, and70-130 mm for CH50.

Statistical analysis

All statistical analyses were carried out using GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA). Data distribution was analyzed by a Kolmogorov-Smirnov test. According to the results of the normality test, a one-way ANOVA followed by Dunn’s or Tukey’s post-hoc test or a non-parametric Kruskal-Wallis test were used for statistical comparisons. Analyses were adjusted for the age, sex and comorbidities. Data were expressed as mean ± standard deviation (SD). Values of p < 0.05 (*) were considered significant.

 

Results

Demographic data and clinical characteristics

Of 61 hospitalized patients with COVID-19, 28 (47.54%) were female and the average age was 48.7 ± 8.8 years. The most commonly self-reported symptoms at onset of illness were fever (n = 41 [67.2%]), cough (n = 39[63.9%]), fatigue or myalgia (n = 19 [31.4%]), diarrhea (n = 9 [14.7%]), and headache (n = 6[9.8%]). Twenty-six (42.6%) patients had comorbidities, including cardiovascular disease (n = 10 [38.4%]), diabetes (n = 6 [23.0%]), hypertension (n = 7 [26.9%]), chronic kidney disease (n = 3 [11.5%]), and endocrine system diseases (n = 2 [7.6%]). The healthy control group included 23 cases (11 (47.8%) female and 12 (52.1%) males, mean age 46.4 ± 4.4 years).

Twenty-one of the 61 patients (34.4%) were admitted to the ICU and sixteen of them (26.2%) died.33 (54.10%) patients with COVID-19 were hospitalized for less than 7 days and 28 (45.90%) of them were hospitalized for more than ≥7 days. Our results showed that length of hospital stay in no-ICU group significantly lower than ICU admission and death groups (6.49 ± 0.24 Vs. 8.85 ± 1.59 and 10.53 ± 1.80, P =0.0002).

 

 C3, C4, and CH50 levels in COVID-19 patients and healthy control group

As shown in Table 1, serum C3 and C4 concentrations and CH50 assay were markedly lower in COVID-19 patients than healthy control group. Further statistical analysis showed that serum levels of C3, C4, and CH50 in the no-ICU admission group were statistically higher than the death and ICU admission groups. We did not observe significant difference in C3, C4, and CH50 levels between ICU admission and death groups in COVID-19 patients (P˃0.05). 

We also analyzed relationship between C3, C4, and CH50 levels in COVID-19 patients and hospital length of stay. As shown in Fig. 1, serum levels of C3, C4, and CH50 at the time of admission in COVID-19 patients who were hospitalized more than or equal to 7 days were statistically lower than COVID-19 patients who were hospitalized lower than 7 days (P< 0.05).

 

Discussion

The complement system efficiently recognizes and eliminates viral pathogens via several mechanisms include: opsonizing viruses, lysing virus-infected cells, inducing an antiviral immunoinflammatory state, boosting virus-specific immune responses, and directly neutralizing cell-free viruses [16]. On the other hand, complement activation in viral infections may play a critical role in pathogenesis, clinical manifestation, and disease severity [17, 18].

Several reports have shown that concentration of complement components and serum complement activity change in the COVID-19 patients. However, they did not definitively determine whether measurement of complement components and serum complement activity can be useful to predict adverse clinical outcomes in the COVID-19 patients. So this study was designed to the relationship between serum levels of C3, C4, and total classical complement activity in COVID-19 patients at the time of admission and the clinical outcomes. The results of this study indicate that serum levels of C3 and C4 and CH50 assay markedly lower in COVID-19 patients than healthy control group. The most interesting finding was that serum levels of C3, C4, and total classical complement activity in COVID-19 patients who died or admitted to ICU significantly lower than COVID-19 patients who did not admit to ICU.

Conversely, Keshavarz and co-workers showed that serum levels of C3 and C4 factors have no significant change between patients and healthy individuals [19]. In another study, Zhang et al. showed that complement C3 cannot predict disease progression  [20]. Our findings are also contrary to that of Dheirs et al. who found there was no significant difference in terms of C3 and C4 levels in both ICU and non-ICU COVID-19 patients [9]. Henry and co-workers have also shown that complement hyperactivation failed to predict progression to severe COVID-19 [21]. Additionally, They also showed that there are no significant differences in total classical complement activity (or CH50 level) at the time of admission between COVID-19 patients with different clinical outcome [21]. These inconsistencies may be due to differences in study design, limited sample size, characteristics of COVID-19 patients, and time between the onset of symptoms and obtaining the blood sample, or confounders.

However, this study supports the findings from the evidence from previous observations [11, 10, 19].Fang et al. in a retrospective cohort study investigated C3 levels in COVID-19 patients [11]. They showed that C3 levels in non-survival group was significantly lower than survival group and concluded that decreased complement C3 levels are associated with poor prognosis in patients with COVID-19 [11]. The finding also accords with earlier findings by Ghazavi et al., which showed that C3 and C4 levels markedly decrease in severe COVID-19 patients in comparison to non-severe-COVID-19 patients [10].  In agreement with our data, Keshavarz et al. indicated that mean CH50 activity level in COVID-19 patients is significantly reduced compared to healthy individuals [19].

The decreased levels of C3 and C4 in COVID-19 patients in comparison to healthy group and significant differences between no-ICU admission group and death and ICU admission groups may be explained by the fact that hyperactivation of the complement system leads to proteolytic cleavage of the key complement molecules C3and C4 leading to cleavage products including C3a, C3b, C4a, and C4b, which may trigger severe inflammatory responses in numerous organs [22]. In support of this possibility, Brandon and collagenous have demonstrated that C3a and C3a/C3 ratio are significantly elevated in sever COVID-19 patients presenting to the emergency department in comparison to mild or moderate-COVID-19 patients [21]. On the other hand, Fletcher-Sandersjöö and colleagues suggested that overactivation of the complement cascade in COVID-19 patients is associated with the activation coagulation systems and consequent severe complications [23]. It is possible, therefore, that overproduction of C3a and C4a following the complement hyperactivation is associated with adverse outcomes in patients with SARS-CoV-2 infection through activation of coagulation cascade and thrombus formation.

Taken together, these results suggest that at level of complement C3, C4, and CH50 at admission may predict disease progression and adverse clinical outcomes in patients with SARS-CoV-2 infection. These findings may be somewhat limited by several confounders, such as different viral strains, sociodemographic factors, and various types of drug therapy during hospitalization [24, 25].  Thus, we suggest that future studies include investigation of alteration of C3 and C4 at different stages of the disease, determination of relationship between serum levels of C3 and C4 and inflammatory biomarkers, and design of clinical trials with complement inhibitors, such as C3 inhibitor.

 

 

Table 1. Comparison of serum levels of C3, C4 and CH50 at the time of admission between COVID-19 patients with different clinical outcome and healthy control group.

Lab Variable

Healthy Control

No-ICU admission

ICU admission

Death

P value

C3, mg/dl

137.1 ± 25.82

98.76 ± 19.09

72.62 ± 13.92

71.63 ± 14.28

˂0.0001

C4, mg/dl

28.50 ± 6.25

24.46 ± 5.87

17.71 ± 6.96

18.79 ± 5.44

˂0.0001

CH50, mm

94.43 ± 13.66

91.58 ± 17.89

77.24 ± 12.66

78.50 ± 7.64

0.007

 

 

 

Figure. 1. Comparison of length of hospital stay in COVID-19 patients with serum levels of C3, C4 and CH50 at the time of admission. Data are the mean ± SD. *P < 0.05 was considered statistically significant.

 

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About the authors

Abdollah Razi

North Khorasan University of Medical Sciences, Bojnurd Iran.

Email: abdolahrazi@gmail.com

Assistant Professor, MD, Faculty member, Department of Urology, Imam Ali Hospital

Iran, Islamic Republic of

Amir Azimian

North Khorasan University of Medical Sciences, Bojnurd, Iran.

Email: amir_azimian2003@yahoo.com

PhD, Associate Professor, Faculty member, Department of Pathobiology and Laboratory Sciences, School of Medicine

Roghaye Arezumand

North Khorasan University of Medical Sciences, Bojnurd, Iran.

Email: r.arezumand@kums.ac.ir

Assistant Professor, PhD, Faculty member, Department of Pathobiology and Laboratory Sciences, School of Medicine

Iran, Islamic Republic of

Akbar Solati

North Khorasan University of Medical Sciences, Bojnurd, Iran.

Email: great1351sowlati@gmail.com

Assistant Professor, PhD, Faculty member, Department of English Language, School of Medicine

Hasan Namdar Ahmadabad

North Khorasan University of Medical Sciences, Bojnurd, Iran.

Author for correspondence.
Email: namdar360@gmail.com

PhD, Associate Professor, Faculty member, Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran

Iran, Islamic Republic of

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Copyright (c) Razi A., Azimian A., Arezumand R., Ghasemzadeh-Moghaddam H., Solati A., Namdar Ahmadabad H.

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