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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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 [2]. Patients with COVID-19 may develop symptoms such as fever, dry cough, pharyngeal pain, tiredness, abdominal pain, diarrhea, conjunctivitis, muscle fatigue, or pneumonia; some are left with serious side effects or even die [14]. Previous studies have reported that 14.1–33.0% of COVID-19 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 [22, 23]. In more severe cases of COVID-19, patients can develop acute respiratory distress syndrome (ARDS), leading to a worse prognosis [9]. Deregulated activation of multiple adaptive and innate immune pathways (including T cell activation, cytokine expression 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 [13, 19].

The complement system is a key part of the innate immune system which plays an important role in defense against foreign pathogens such as viruses but, in addition to being an important part of the immune defense system, it plays a critical role in promoting the inflammatory process that leads to organ dysfunction [21]. Although several studies have been carried out on the complement system in COVID-19 and its relationship with clinical outcomes [5, 6, 8, 12, 25], 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 [5]. They suggested that measurement of C3 and C4 levels cannot be used to show severity disease [5].

Controversially, Ghazavi et al. found that the levels of C3 and C4 in non-severe COVID-19 patients were significantly higher than in severe-COVID-19 patients [8]. A previous study by Fang et al. also indicated decreased complement C3 levels are associated with poor prognosis in COVID-19 patients [6]. Java and colleagues claimed that the role of the complement system in COVID-19 patients is time dependent, wherein complement activation in the first week of infection can serve as a “friend”, and its activation in the second or third weeks of infection can be a “foe” [12]. Zinellu et al., in a systematic review, concluded that lower concentrations of C3 and C4 are significantly associated with higher COVID-19 severity and mortality [25]. 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 [25].

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 the role of the complement system for prediction of the risk of developing critical COVID-19.

Materials and methods

Study design and participants. 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 (Bojnourd, Iran) from April 20 to August 5, 2021 with less than three days passage since the onset of COVID-19 symptoms. All patients with a positive RT-PCR test for SARS-CoV-2 and common CT imaging findings associated with COVID-19 were included in the study. Patients also had no history of SARS-CoV-2 infection or 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 indicates that age and comorbidities could possibly confound the association between complement system status and clinical events [16]. Therefore, we adjusted for sex, age and comorbidities. 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.

Data collection and assessment of serum complement levels and 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 for 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 a negative PCR-test for COVID-19. Blood samples were centrifuged immediately, and serum was obtained and frozen at –70°C until use.

Serum C3 and C4 concentrations were determined using kits from Roche Diagnostics (Indianapolis, IN, USA) according to the immunoturbidimetric method [18]. The CH50 test was evaluated using single-radial-haemolysis plates (Biogen, Iran) as described elsewhere [18]. All values were compared to the normal ranges which were reported as: 89–187 mg/dL for C3; 10–40 mg/dL for C4; and 70–130 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 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%]), or 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, 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 non-ICU group was significantly lower than in the ICU admission or 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 the healthy control group. As shown in Table, serum C3 and C4 concentrations and CH50 assay were markedly lower in COVID-19 patients than in the healthy control group. Further statistical analysis showed that serum levels of C3, C4, and CH50 in the non-ICU admission group were statistically higher than in the death and ICU admission groups. We did not observe a significant difference in C3, C4, and CH50 levels between ICU admission and death groups of COVID-19 patients (p > 0.05).

 

Table. Comparison of serum C3, C4 and CH50 levels at the time of admission between COVID-19 patients with different clinical outcomes and the 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

 

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

 

Figure. Comparison of hospital stay length in COVID-19 patients with serum levels of C3, C4 and CH50 at the time of admission

Note. Data are mean±SD. *p < 0.05 was considered statistically significant.

 

Discussion

The complement system efficiently recognizes and eliminates viral pathogens via several mechanisms including: opsonization of viruses; lysing of virus-infected cells; induction of an antiviral immunoinflammatory state; boosting of virus-specific immune responses; and directly neutralizing cell-free viruses [1]. On the other hand, complement activation in viral infections may play a critical role in pathogenesis, clinical manifestation, and disease severity [3, 10].

Several reports have shown that the 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 or serum complement activity can be useful to predict adverse clinical outcomes in COVID-19 patients. Therefore, this study was designed to assess the relationship between serum levels of C3, C4, and total classical complement activity in COVID-19 patients at the time of admission and clinical outcomes. The results of this study indicate that serum levels of C3 and C4 and CH50 assay were markedly lower in COVID-19 patients than in the 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 were ICU admitted were significantly lower than in COVID-19 patients who were not ICU admitted.

Conversely, Keshavarz and colleagues showed that serum levels of the C3 and C4 factors have no significant change between patients and healthy individuals [15]. In another study, Zhang et al. showed that complement C3 cannot predict disease progression [24]. 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 [5]. Henry et al. have also shown that complement hyperactivation failed to predict progression to severe COVID-19 [11]. In addition, 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 outcomes [11]. These inconsistencies may be due to differences in study design, limited sample size, characteristics of COVID-19 patients, time between the onset of symptoms and obtaining the blood sample, or confounders.

However, this study supports the findings from previous observations [6, 8, 15]. Fang et al. in a retrospective cohort study investigated C3 levels in COVID-19 patients [6]. They showed that C3 levels in the non-survival group were significantly lower than in the survival group; they concluded that decreased complement C3 levels are associated with poor prognosis in COVID-19 patients [6]. The finding also match earlier findings by Ghazavi et al., which showed that C3 and C4 levels were markedly decreased in severe COVID-19 patients in comparison with non-severe COVID-19 patients [8]. 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 [15].

The decreased levels of C3 and C4 in COVID-19 patients (in comparison to the healthy group), and significant differences between the non-ICU admission group and the 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. These may trigger severe inflammatory responses in numerous organs [4]. In support of this possibility, Brandon and colleagues have demonstrated that C3a and the C3a/C3 ratio are significantly elevated in severe COVID-19 patients presenting to the emergency department compared to mild or moderate severity COVID-19 patients [11]. On the other hand, Fletcher-Sandersjöö et al. suggested that overactivation of the complement cascade in COVID-19 patients is associated with activation of coagulation systems and consequent severe complications [7]. It is possible, therefore, that overproduction of C3a and C4a following complement hyperactivation is associated with adverse outcomes in patients with SARS-CoV-2 infection through activation of the coagulation cascade and thrombus formation.

Taken together, these results suggest that the levels of complement C3, C4, and CH50 at admission may predict disease progression and adverse clinical outcome 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 [17, 20]. Thus, we suggest that future studies include: investigation of alteration of C3 and C4 at different stages of the disease; determination of the relationship between serum levels of C3 and C4 and inflammatory biomarkers; and design of clinical trials with complement inhibitors, such as a C3 inhibitor.

Acknowledgments

This work was supported by the North Khorasan University of Medical Sciences, Bojnurd, Iran. The authors thank the members of the Department of Pathobiology and Medical Laboratory Sciences, North Khorasan University of Medical Sciences, Bojnurd, Iran for their technical assistance. We would also like to thank the clinical staff of Imam Hassan Hospital for assisting us in this research project.

Ethics approval

The study was performed based on an informed consent and was approved by the Ethics Committee of North Khorasan University of Medical Sciences, Bojnourd, Iran (IR.NKUMS.REC.1399.021).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflict of interest.

About the authors

A. Razi

North Khorasan University of Medical Sciences

Email: raknv@mail.ru

PhD, MD (Medicine), Assistant Professor

Иран, Bojnurd

A. Azimian

North Khorasan University of Medical Sciences

Email: raknv@mail.ru

PhD, MD (Medicine), Associate Professor, Faculty Member, Department of Pathobiology and Laboratory Sciences

Bojnurd

R. Arezumand

North Khorasan University of Medical Sciences

Email: raknv@mail.ru

PhD, MD (Medicine), Assistant Professor, Faculty Member, Department of Pathobiology and Laboratory Sciences

Иран, Bojnurd

A. Solati

North Khorasan University of Medical Sciences

Email: raknv@mail.ru

PhD, MD (Philosophy), Associate Professor, Faculty Member, Department of English Language

Иран, Bojnurd

H. N. Ahmadabad

North Khorasan University of Medical Sciences

Author for correspondence.
Email: namdar360@gmail.com

PhD, MD (Medicine), Associate Professor, Faculty Member, Department of Pathobiology and Laboratory Sciences

Иран, Bojnurd

References

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