Decreased Copper/Selenium Ratio Among Non-Responder Healthcare Workers to SARS-CoV-2: An Evidence of High Copper/Selenium Ratio Effects on the Immune Response to COVID-19 and Symptoms

Background: The relationship between strong immune response to infections and trace elements such as selenium (Se) and copper (Cu) is well documented. Furthermore, Se and Cu behave as negative and positive acute phase reactants under infectious conditions, respectively. Since SARS-CoV-2 causes systemic inammation, this study was conducted to evaluate the association of Se and Cu serum levels with symptoms and immune response to SARS-CoV-2, and then assess the Cu/Se ratio in this matter. Methods: Blood samples and nasopharyngeal swabs were obtained from 126 SARS-CoV-2 participants with mild and severe clinical symptoms. The SARS-CoV-2 infection and immune response to the virus were conrmed by RT-PCR and anti-SARS-CoV-2 IgG, respectively. The measurement of Se and Cu serum levels were analyzed by atomic absorption spectrophotometry and colorimetric assay, respectively. Finally, data were analyzed and a P-value < 0.05 was considered statistically signicant. Results: The mean Se levels were higher in patients with mild symptoms (108.73 ± 5.38 μg/L, P-value = 0.0012) and IgG non-responders (110.33 ± 3.38 μg/L, P-value < 0.001), whereas, the mean Cu was higher in participants with severe symptoms (111.055 ± 11.98 μg/dL, P-value = 0.045) and IgG responders (112 ± 9.98 μg/dL, P-value = 0.0058). The Cu/Se ratio was lower (ratio < 1) in participants with no immune responses to infection and mild symptoms versus immune responder patients with severe symptoms (P-value < 0.001). Conclusion: Our results suggest that Cu/Se ratio may be considered as a nutritional biomarker of severity and immune response in SARS-CoV-2-infected patients.


Background
Coronavirus disease 2019 (COVID-19) is a condition caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was rst identi ed in Wuhan, China, in December 2019. The COVID-19 has spread rapidly all over the world and consequently COVID-19 pandemic has seriously affected human health and social life, and the World Health Organization (WHO) declared a global pandemic for COVID-19 on March 11, 2020 [1]. The SARS-CoV-2 can cause a spectrum of clinical features from mild signs and symptoms to acute respiratory distress syndrome, severe pneumonia, and death [2]. There is growing evidence that points to the relationship between in ammation and immune response to the SARS-CoV-2 virus [3].
Su cient amounts of micronutrients are required for immune system development, maintenance, functions, surveillance and nally defense against infections such as SARS-CoV-2. It is therefore believed that trace elements and vitamins including selenium (Se), copper (Cu), zinc (Zn), vitamin C, and vitamin D play an important role in an effective immune response to SARS-CoV-2 infection [4][5][6]. These micronutrients have an impact especially on cell-and humoral-mediated immune response against infections [7]. De ciencies of trace elements cause susceptibility to infections [8], and also infections such as SARS-CoV-2 or systemic in ammation status such as COVID-19 could induce a reduction of Se serum level concentration during the acute phase response [9][10][11]. This event may be due to the redistribution of the serum Se into the damaged tissues [12]. Besides, acute infections or in ammation status lead to an increase of Cu serum level concentration [13]. Interaction of this process results in an increased serum Cu/Se ratio.
The importance of trace element balance such as Cu/Zn ratio in autoimmune diseases [14], in ammation [15], infections such as SARS-CoV-2 [16], and cancers [17,18] is well documented. In this regard, the high Cu/Se ratio has also been reported in recurrent aphthous stomatitis, as an in ammatory condition [19].
To the best of our knowledge, no studies were reported the possible relation between Cu and Se balance (Cu/Se ratio) in SARS-CoV-2-infected patients with immune response. Therefore, we aimed to evaluate the relationship between the levels of Cu, Se, and Cu/Se ratio and immune response to the SARS-CoV-2 infection in healthcare workers (HCWs) at Rafsanjan University of Medical Sciences (RUMS), Rafsanjan, Iran in the third quarter of 2021.

Population, setting and procedures
This study was approved by the ethics committee of RUMS, Rafsanjan, Iran (Ethical code: IR.RUMS.REC.1400.024). Also, this trial procedure was conducted under the ethical standards of the Iranian Ministry of Health and Medical Education on human experimentation and in concordance with the Helsinki Declaration. 126 HCWs were participated in this study. All participants completed a questionnaire containing sociodemographic data (i.e., age, sex, height, weight), clinical information such as exposure to con rmed COVID-19 cases, professional information (i.e., shift, hospital department, and occupation), and self-report of COVID-19 related symptoms (i.e., fever, dry cough, fatigue and weakness, sore throat, the impaired sense of smell and taste) after obtaining written informed consent. Five milliliters (ml) vein blood samples and nasopharyngeal swabs were collected from participants (HCWs in medical centers and HCWs in administrative departments) with clinical symptoms, under aseptic and standard conditions. The exclusion criteria were: taking any form of dietary trace elements or multivitamins/supplements, pregnancy, history of cancer, autoimmune disease and taking immunosuppressive medication. All collected bloods were immediately transferred to the laboratory for the next steps. To select participants, we predicted exposure to SARS-CoV-2 according to clinical symptoms. For this aim, based on participants' self-report of clinical symptoms, two clinical criteria (major and minor) were considered [20], and then the participants were divided into two subgroups, high and medium to low possibility to COVID-19, according to clinical criteria (Table 1).

Measurement of Se and Cu serum levels
After centrifugation of the bloods for 5 min at 5000 rpm, serum was obtained and stored at -20 ºC until Se and Cu assessment. The Se and Cu serum levels were measured by atomic absorption spectrophotometry and colorimetric assay, respectively. According to the instruction manual of the kit, the reference range for serum Se was 46-143 µg/L and for Cu was 60-153 µg/dL. The remaining serum was used for IgG antibody measurement.

Assessment of immune response to the SARS-CoV-2 infection
About one month after clinical symptoms and con rmed infection, the immune response to SARS-CoV-2 infection was evaluated by measuring serum anti-SARS-CoV-2 IgG antibody. For this purpose, serum IgG antibody to SARSCoV2 was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Pishtazteb Co., Tehran, Iran) according to the manufacturer's instructions. The cutoff index 0.9, 1.1 and 0.9-1.1 was considered as negative, positive and borderline result, respectively. The ELISA diagnostic kit sensitivity and speci city for IgG was 94.1% and 98.3%, respectively.

Data Analysis
Data analysis was performed using the IBM SPSS 22 statistical software. Data was reported as mean ± standard deviation (SD). The difference in serum Se and Cu levels between subgroups was analyzed using independent sample t-tests. Pearson correlation test was used to analyze the mutual relationships between elements. A P-value < 0.05 was considered statistically signi cant.

Demographic data
The ow diagram of study selection and outcomes are shown in Figure 1. 126 HCWs in medical centers and administrative departments with a mean range of age 37.7±5.0 (76 female (60%) and 50 male (40%)) were enrolled in this study. There were statistical age difference (P-value = 0.026) and no statistical gender difference (P-value = 0.42) among participants. The results of the COVID-19 prediction based on clinical symptoms according to sex, age, and other demographic data are shown in Table 2. 96 of the participants (76%) were HCWs in medical centers, and the others (24%) worked in the administrative departments. The HCWs who worked in medical centers had more contact with COVID-19 patients (P-value < 0.5), which results in a high possibility of COVID-19.   Figure 1 and Table 2). The outcome results of the molecular and serological tests were statistically different between the two subgroups (P-value < 0.5) ( Table 2).

Cu/Se serum levels and COVID-19 symptoms
The associations between Cu, Se and Cu/Se ratio serum levels with COVID-19 symptoms were estimated. The Se serum levels were in the normal range among all participants (Up to 124 µg/L), while, some individuals had high Cu serum levels (up to 169.1 µg/dL). The mean Se serum level was signi cantly higher in HCWs with mild symptoms (108.73 ± 5.38 µg/L, P-value = 0.0012), whereas, the mean Cu serum level was statistically higher in individuals with severe symptoms (111.055 ± 11.98 µg/dL, P-value = 0.045) ( Figure 2, Table 2). In terms of the Cu/Se ratio, a higher statistical difference in the ratio was found in participants with severe symptoms (P-value < 0.001) (Figure 2).

Cu/Se serum levels and response to SARS-CoV-2
The associations between Cu, Se and Cu/Se ratio serum levels and immune response to SARS-CoV-2 were also evaluated. For this aim, the individuals with a cut-off index of anti-SARS-CoV-2 IgG ≥ 1.1 were considered as a COVID-19 immune responder, while, the participants with a cut-off index of anti-SARS-CoV-2 IgG ≤ 0.9 were considered as non-responders to the COVID-19. The results indicated that the mean Se serum level was statistically higher in COVID-19 immune non-responders (110.33 ± 3.38 µg/L, P-value < 0.001), whereas in responders participants, the Cu serum level was statistically higher (112 ± 9.98 µg/dL, P-value = 0.0058) (Figure 3, Table 3). The Cu/Se ratio was higher in HCWs with IgG response to the infection with statistically different (P-value < 0.001) (Figure 3, Table 3). For the determination of correlation between Se and Cu with response/non-response to SARS-CoV-2 infection, the Pearson correlation analysis was performed. The results showed that a negative signi cant correlation of Se with non-responders (r = -0.774, P-value = 0.014), whereas, the correlation of Cu was positive with COVID-19 immune responders (r = 0.601) but not statistically difference (P-value = 0.05) ( Table 4).

Discussion
Our results indicate that HCWs with severe symptoms may have shown a better immune response to SARS-CoV-2 infection. In ammation caused by severe symptoms such as fever, chills, dry cough or shortness of breath, fatigue, and weakness causes a decrease and increase of Se and Cu, respectively, resulting in an imbalance in serum Cu/Se levels. Interestingly, decreased Cu/Se ratios in non-responders to SARS-CoV-2 infection indicate mild in ammation and weak immune responses. Therefore, Cu/Se ratio could represent an in ammatory and immune status in SARS-CoV-2-infected HCWs.
Despite the normal levels of Se in all HCWs, the Se serum levels in patients with severe symptoms were found to be lower than in patients with mild symptoms. In complication status such as COVID-19 disease, the severe symptoms indicate in ammation and involvement of the immune system in the body.
In ammation, pro-in ammatory cytokines, hypoxia, and many stress signals can affect the Se serum level, which led to aggravating and accelerating Se decline [21,22]. The COVID-19 infection is characterized by systemic in ammation especially in the lung, which resulting hypoxia [1]. In a recent study by Sun et al., reduced Se status was reported in patients with systemic sclerosis, as an autoimmune disease with systemic in ammation, vasculopathy, brosis, and hypoxia [23]. Also, in another study by Younesian et al., for assessment of Se serum levels in COVID-19 patients with severe and mild symptoms, it was shown that patients with severe conditions had lower Se serum levels compared with patients with mild symptoms [10]. It seems that a decline in Se serum level is occurred in an in ammatory condition and considered as a negative phase reactant in these situations [24,25].
In the current study, increased Cu serum level was also shown in HCWs with severe symptoms, such that some individuals had higher Cu serum levels versus normal range (up to 169.1 µg/dL). Unlike selenium, studies were shows that Cu is increased in infections, such that progressed rising in Cu serum level is the hallmark of infections [26,27]. In another hand, the in ammatory conditions signi cantly affect Cu serum level, such that increased Cu is known as a positive phase reactant [28,29]. Since the pathophysiology of SARS-CoV-2 is characterized by systemic in ammation, so the elevated Cu serum level in patients with severe symptoms is reasonable. In two studies by Bagher Pour et al., and Skalny et al., for estimate association of trace elements with severity and clinical outcomes of COVID-19 patients, it was shown that high Cu serum level is associated with severe symptoms, hospitalization, and mortality [16,30].
Our results also indicated that HCWs with severe symptoms had a high Cu/Se ratio compared with mild symptoms subjects. Normal levels of trace elements cause maintain general health, but the imbalance between them could have serious consequences. Some trace elements are antagonists, which means they may compete against each other. In this regard, the association of Cu and Zn balance with in ammation is well known. Several studies have shown a correlation of high Cu/Zn ratio with an in ammatory condition such as oxidative stress [14], autoimmune diseases [31], infections such as SARS-CoV-2 [16], and cancers [17,18]. In another hand, a low Cu/Zn ratio is associated with an increased risk of incident infections [32]. In terms of Cu and Se balance, Cu has also been shown to be a Se antagonist [33]. Thus, elevated Cu levels in in ammatory conditions can affect Se and lead to an imbalance in the Cu/Se ratio. The high Cu/Se ratio has also been reported in in ammatory conditions such as recurrent aphthous stomatitis [19], pulmonary arterial hypertension in systemic sclerosis patients [23], acute ischemic stroke [34]. So, it seems that a high Cu/Se ratio may be considered as an in ammatory situation such as SARS-CoV-2 infection.
Our results also demonstrate that HCWs with an IgG response to SARS-CoV-2 belonged to the group with severe symptoms. Positive and negative correlations were found between Cu and IgG responders, and Se and IgG non-responders. Also, the Cu/Se ratio was higher in IgG responders compared with IgG nonresponder subjects. The in ammation during infections such as SARS-CoV-2 is caused by the strong activation of the innate immune system. SARS-CoV-2 activates innate immune cells, which led to the production and release of pro-in ammatory and in ammatory cytokines named cytokine storm. Although cytokine storm leads to severe symptoms, hospitalization, and mortality in patients with COVID-19, it can also activate the e cient adaptive immune responses [35,36]. The link between innate and adaptive immunity is well known, and numerous studies have indicated strong an innate response lead to the full activation of adaptive immunity [37]. In a study by Hackler et al., it was reported that surviving COVID-19 patients showed higher Cu and Se serum levels in comparison to COVID-19-dead patients, and Cu and Se serum levels are contributed to a good prediction of survival [38]. It seems that the Cu/Se ratio could be considered as the status of immune response activation and survival in patients with COVID-19 disease.
The limitation of the current study was the small population, and authors suggest this nding estimate in a large population with severe symptoms and strong immune response against SARS-CoV-2 infection. As well as, we measured only anti-SARS-CoV-2 IgG, so other antibodies, such as long-term IgM and IgA, may be involved in the long-term immune response to COVID-19 and protect HCWs against re-infection. In another hand, we did not estimate cellular immunity in this matter, and individuals with severe symptoms, and without humoral response, may have e cient cellular immunity against infection, and further research is needed.

Conclusion
Our results suggest that the normality of Se and Cu serum levels are important in in ammatory conditions such as response to SARS-CoV-2 infection. Besides, the balance between them, in the in ammatory condition such as COVID-19, is also more important. Hence, the Cu/Se ratio may be considered as a nutritional biomarker for in ammation and immune response in SARS-CoV-2-infected patients. Flow diagram of study selection and outcomes. The levels of Se and Cu were in the normal range among participants with mild symptoms, while, HCWs with severe symptoms had normal and high serum levels of Se and Cu, respectively (Pos: Positive, Neg: Negative).

Figure 2
The results of Se, Cu, and Se/Cu ratio in HCWs with severe and mild symptoms. Se and Cu serum levels were statistically difference between two group with P-value = 0.0012 (A) and P-value = 0.045 (B), respectively. The Se/Cu ratio was also statistically different between the two groups with severe and mild symptoms with P-value < 0.001 (C).