Hyperferritinemia as a factor associated with poor prognosis in COVID-19 patients

Summary

Worldwide, hundreds of millions of people have been infected with COVID-19 since December 2019; however, about 20% or less developed severe symptoms. The main aim of the current study was to  assess  the  relationship  between  the  severity of Covid-19 and different clinical and laboratory parameters. A total number of 466 Arabs have willingly joined this prospective cohort. Out of the total number, 297 subjects (63.7%) had negative COVID-19 tests, and thus, they were recruited as controls, while 169 subjects (36.3%) who tested positive for COVID-19 were enrolled as cases. Out of the total number of COVID-19 patients, 127 (75.15%) presented with mild symptoms, and 42 (24.85%) had severe symptoms. The age range for the participants was 20 to 82 years. Compared with controls, the severity of the disease was associated with significantly high ferritin levels (P < 0.001). The severity of the disease was also associated with a significant increase in C-reactive protein (P < 0.001), D-dimer (P < 0.001), white blood cell count (WBC) (P < 0.01), IgM (P < 0.001), and Granulocytes (P < 0.01). In addition, severe COVID-19 symptoms in the current study were associated with a significant decrease in lymphocytes (P < 0.01). There was a four-fold increase in serum ferritin levels in COVID-19 patients presented with severe symptoms upon admission. The former was associated with significantly high levels of CRP and D-dimer. Thus, hyperferritinemia, together with high CRP and D-dimer concentrations, may serve as reliable predictors for disease severity and poor prognosis in Arabs with COVID-19.

Keywords:

Ferritin – COVID-19 – mild symptoms – severe symptoms – poor prognosis

Introduction

The sudden outbreak of the Coronavirus in 2019 (COVID-19) has caused an unimaginable shockwave acrosstheworld. Ithasspreadrapidlyfrom Chinatoevery corner of the globe, resulting in a disastrous increase in the number of infections and deaths1). Complications associated with severe symptoms involve severe respiratory syndrome, assisted mechanical ventilation, and increasing death incidence rate1, 2). Clinical features of patients with severe COVID-19 symptoms include a significant decrease in lymphocytes3–5) which is usually associated with a significant increase of C-reactive protein, D-dimer, IgM6), white blood cell count (WBC), granulocytes7, 8), and ferritin levels1, 2). Nevertheless, the prognostic role of these biomarkers in COVID-19 patients is ambiguous. Studies regarding the importance of D-dimer as a potential biomarker in patients with COVID-19 are conflicting as some reported significant correlation with poor outcomes1, 2, 9), while others showed such a relationship is limited only to venous thromboembolism events10). Although, these results have limited D-dimer advantages as reliable biomarker in different COVID-19 scenarios, however it continued to be used to screen for patients with venous thromboembolism and identifies those at risk10). Ferritin was another biomarker that has been targeted during COVID-19 in order to evaluate the fluctuating levels of this biomarker during different disease stages and severity1, 2).

Clinically, high levels of ferritin or hyperferritinemia has been used as biomarker of uncontrolled inflammation11);  thereby  serum  ferritin  level  was  suggested as biomarker to examine the effectiveness of treatment and to monitor patients’ response11–14). In addition, high ferritin level is considered as an immune-suppressant and hyperferritinemia plays a major role in the immune system’s dysregulation, directly as an immune suppressor and indirectly as a vital pro-inflammatory contributor towards cytokine storm15–17). In laboratory medicine, ferritin is considered to be one of the most requested tests to evaluate body iron status and the most frequently requested test in laboratory medicine in primary care and referral settings12, 18). Ferritin levels are not always seen within the normal range, but low ferritin level is a reliable marker for iron deficiency13, 18) and mild elevation is commonly reported in clinical practice13, 14). It is estimated that around 10% of individuals with hyperferritinemia are due to iron overload, while the rest is due to specific conditions mostly related to inflammation13). Previous1, 2) and most recent studies15) have observed high ferritin levels in COVID-19 patients presented with severe symptoms. The search for molecular markers for COVID-19 comes in great demand to decrease disease side effects and increase survival rate. It would be essential to elucidate the causes of severe COVID-19 and to identify reliable markers associated with disease severity. Therefore, the current study was designed to evaluate biochemistry alterations in COVID-19-infected people and to examine the possible correlation between clinical features and candidate biochemistry biomarkers.

 

Materials and methods

In the current study, we have performed a hospital perspective cohort study including 466 participants (169 COVID-19 cases and 297 controls) who willingly joined the study when they were admitted to the Ramadi General Hospital, the province of Al-Anbar, Iraq, between April and December 2020.

Ethical approval

The study protocol was approved ethically by the Scientific Research Ethics Committee at the University of Anbar (No: 123SC-2020), and every participant signed a written consent form after listening to a brief presentation about the project. The study was performed in conformity with the World Medical Association Declaration of Helsinki.

Definition of cases and controls

In the COVID-19 group, only those confirmed by fast test and real-time polymerase chain reaction or quantitative polymerase chain reaction (PCR) as positive cases were enrolled in the current study as cases. Furthermore, all patients were examined using lung ultrasound and chest CT scan. Controls were approached the hospital complaining of symptoms similar to that of COVID-19; however, their tests were negative, as confirmed through clinical examination by specialists and PCR testing. COVID-19 patients (n = 169) were then divided into two groups based on the disease severity as follows: the first group included 127 COVID-19 patients with mild symptoms, while the critically ill or patients with severe symptoms group included only 42 COVID-19 subjects.

Collecting of the cases

All cases of the current study were collected from Ramadi General Hospital, Ramadi City, Iraq. The severity of the COVID-19 patients was classified into mild and severe based on symptoms upon admission and the need for assisted ventilation, hospitalization, and intubation. While both groups shared symptoms like fever, cough, tiredness, loss of smell and/or taste, headache, bone aches, runny nose, loss of appetite, and some cases reported diarrhea and red, irritated eyes or achy eyes. Besides these symptoms, severe cases were suffering from shortness of breath, chest pain, loss of speech, loss of mobility, and difficulty breathing. All suspected and mild cases were sent home after giving their details, including a full description of their conditions and full contact details to get feedback within 24 hours to confirm their health status after getting the PCR results. These individuals were asked to isolate themselves for the next 24 hours until getting feedback regarding the test’s result. All mild cases were given remedies as prescribed in COVID-19 treatments, protocol with complete instruction to manage their symptoms at home. Severe cases were admitted to the hospital instantly, and cases with difficulty breathing were supplied with oxygen. Survived cases stayed from 6 hours to 7 days in the hospital before they were discharged. Some very severe cases were requested to stay home, while clinicians made the urgent visit with an unbalance and brought these patients to hospitals when needed.

Collecting the controls

All controls in this study approached the same hospital where cases were recruited as they were suspected to have COVID-19 and had symptoms suspected to be COVID-19. However, their test results were negative. These results were confirmed after a quick check-up by clinicians at the hospital before going through a PCR test to confirm that they were COVID-19 negative. Following PCR, all controls had to wait 24 hours after performing before officially being enrolled as controls.

Inclusion criteria

Participant living in the province of Al-Anbar, mostly from Ramdi city and outskirt, who had a positive PCR result confirming the infection with COVID-19, were Arab and 20 years or older at the time of the commencement of the study was included as cases in the currents study. Those who had negative PCR results have been included as controls. The age range for cases and controls was 20 to 82 years.

Exclusion criteria

We excluded all individuals with chronic obstructive pulmonary disease and those with a history of liver damage such as cirrhosis, liver failure, hepatitis B virus (HBV), and hepatitis C virus (HCV). Because the study was designed to target Arabs and in order to avoid any interruption with genetic predisposition, we have excluded all other ethnic groups. Finally, individuals above or below the selected age range of the current study were also excluded.

Samples collection and related measurements Samples from COVID-19 patients were collected at a separate section out of the hospital. Patients then were directed to the next door room which was prepared for blood withdrawal with the help of another nurse. White blood count (WBC), serum values of C-reactive protein (CRP), COVID-IgM, lymphocytes, D-dimer, Gran and ferritin were quantified in all patients and controls enrolled in the study. Serum concentrations of CRP was evaluated using a specific automated protein analyzer (PA120) provided by (Shenzhen Genius Electronics Co., Ltd. China 2019). Measuring IgM was performed using Vidas technique (ELISA) which is designed to measure the titer of IgM. Other biochemistry biomarkers included: complete blood count (CBC), measured using the Medonic technique; CRP measured using Biolis (Biochemistry); ferritin and D-dimer measured using the Vidas technique.

Statistical analysis

Collected data were analyzed using analysis of variance (ANOVA) to assess any differences between cases (COVID-19 groups) and controls. Data are expressed as mean ± standard deviation (SD) or n (%). As illustrated in Figures 1, 2, 3, 4, 5, and 6, a comparison between COVID-19 patients and their controls has been performed after dividing the COVID-19 group into two sub-groups (Mild and Severe) based on the severity of the symptoms at time upon admission. Thus, we have divided the cases into two groups: patients who developed mild symptoms (75.15%) and were sent back home and patients with severe symptoms (24. 75%) who had been hospitalized and their health status over the period of hospitalization either improved or worsened. To examine the effect of age progression on disease severity, we have divided COVID-19 cases into three age groups (age ranges; 20 to 39, 40–59, and ≥ 60 years old) as described in Table 1. For continuous variables, differences between two groups or more were assessed by t-test or Mann–Whitney test and ANOVA or Kruskal–Wallis test with the correspondent post-tests, according to the variable distribution. To compare differences between proportions of categorical variables, we used a two-sided chi-square test. The lowest level of statistical significance was set as long as the P value was less than 0.05.

 

 

Table 1. COVID-19 cases of the current study after been divided by age ranges and disease severity

Age range	No. of cases	Mild symptoms	Severe symptoms
20–39	78	69	9
40–59	55	44	11
≥ 60	36	14	22
Total	169	127	42

 

Results

Regrouping COVID-19 patients according to their ages showed a critical increase in the number of severe cases with age (Table 1). Thus, the increasing age of the patients is associated with a sharp increase in the  probabilities to have poorer outcomes. Compared with controls, the current study has shown a more than fourfold increase in serum ferritin levels in patients with severe COVID-19 (P < 0.001). Significant differences were also observed between COVID-19 patients presented with mild symptoms compared with control (P < 0.01).

The significant increase in serum ferritin level in patients with severe COVID-19 was correlated with a significant increase in CRP level (P < 0.001), as shown in Figure 1. Expectedly, there was a significant increase (P < 0.01) in WBC in patients with severe COVID-19 compared with the control group, and this was in parallel with the significant increase in serum ferritin level (Fig. 2). Although WBC has increased in patients presented with mild symptoms, this increase was not significant and was within the normal range (Fig. 2). Compared with their counterparts in the control group, as shown in Figure 3, severe COVID-19 patients presented with high serum ferritin levels have witnessed an increase in D-dimer. However, that increase in D-dimer was highly significant in patients who presented with severe symptoms (P < 0.001). In accordance with all infectious diseases, there was a significant increase in IgM in patients with severe COVID-19 compared with controls (Fig. 4). These differences were observed in both groups, mild (P < 0.01) and severe symptoms group (P < 0.001). A significant increase in granulocytes’ concentrations was observed in patients presented with severe COVID-19 symptoms compared with controls (P < 0.01) as shown in Figure 5. Compared with controls, the significant increase in granulocytes in COVID patients presented with mild symptoms was also detected at lower level of significance (P < 0.05).

The significant increase of serum ferritin level in COVID-19 patients presented with severe symptoms was associated with a significant decrease in lymphocytes in comparison with the control group (P < 0.01). Although, patients with mild symptoms have witnessed a decrease in lymphocytes compared with their counterpart controls, however these differences were not significant (Fig. 6).

Discussion

The current study has shown that ferritin level is significantly higher in COVID-19 patients than in controls. In addition, when taking into consideration subdividing COVID-19 patients according to the severity of the disease, these differences become even higher when comparing ferritin levels in patients with severe COVID-19 and controls (P < 0.001). Hyperferritinemia is frequently observed in COVID patients with severe symptoms, and significant differences was detected between these patients and COVID patients presented with mild symptoms (P < 0.01). These results suggest crucial roles of ferritin in the pathogenesis of COVID-19. In addition, increasing the incidence of hyperferritinemia among patients with severe symptoms may indicate a direct relationship between ferritin and disease severity. Nevertheless, when dividing COVID-19 cases by age range (Table 1), we have observed that more than three quarters of the severe cases were 40 years and older, while more than half of the severe cases (52%) were 60 years and older. Furthermore, the number of severe cases in older age groups was more than double compared with similar cases in younger age groups, despite the fact that older age groups have less than half of the total number of the cases that the younger ages had. The significant increase in COVID-19 severity with age progression is in line with previously

 

published data19–21). Older age is associated with immune dysregulation due to physiological changes that affect the immune system19, 21, 22). A dysregulated immune status occurs when the immune system cannot control or restrain an immune response21, 22). This causes a greater susceptibility to infectious disease19–22). Compared with younger individuals, previous studies have shown that older COVID-19 patients are in urgent need for hospitalization, admission to the intensive care unit (ICU) and/or mechanical ventilation19–21). This group also had the highest death rate due to COVID-19 compared with younger age groups19–21, 23). Such deterioration in the immune system contributes crucially not only to autoimmune diseases but also to cardiovascular, metabolic and neurodegenerative diseases of aging19–23). The human immune system undergoes crucial aging-related changes and loses the ability to protect against infectious and noninfectious diseases including cardiometabolic diseases and different types of cancer. Consequently, older age has been considered as a risk factor for many diseases including cardio-metabolic diseases24, 25) and different types of cancers26–29). While the COVID-19 pandemic was an indirect risk factor for weight gain and obesity in children30) and adults31) due to physical inactivity, sedentary lifestyle, and poor eating patterns, on the other hand, obesity and other cardiometabolic diseases are both associated with increased susceptibility to COVID-19 and worst outcomes32). This could be due to the proinflammatory state of cardiometabolic disease and obesity32).

To our knowledge, this is the first study examining ferritin levels in Arabs with COVID-19 and the relationship between older age and disease severity. In agreement with these findings, previous and most recent studies have shown that COVID-19 is associated with significantly high serum ferritin level upon hospital admission1, 2). Furthermore, among those who presented with severe symptoms, serum ferritin levels continue elevating throughout their hospital stay and only exceeded the upper level of detection after 16 days of hospitalization2).

In another word, ferritin levels continue escalating following the detection of COVID-19 and the highest level of ferritin was observed in those with longer periods in hospital. Most importantly, the highest level of ferritin was associated even with further deterioration in health status of patients diagnosed with severe COVID-19 and was closely related to high death rates2). Thus, increases in serum ferritin upon admission of patients with COVID-19 is an excellent predictor of poor prognosis. Unfortunately, in the current study, we did not perform any follow-up to evaluate escalating levels of ferritin as we have relied on one point time test which was upon patients’ admission to the hospital. Increasing ferritin levels in patients with COVID-19 is of a high importance and may have a great prognostic value since it is associated with poor prognosis. The higher ferritin level in COVID-19 patients was associated with significant increase in CRP (P < 0.001) and D-dimer (P < 0.001). Among patients with severe symptoms, there was more than six fold increase in CRP levels compared with controls. Similarly, increased ferritin level was associated with almost fourfold increase in D-dimer concentrations. These results in agreement with previous published data showed that increased CRP and D-dimer upon admission in COVID-19 individuals is associated with the need to intensive care support and were strongly related to assisted ventilation1, 2, 6, 9, 33). A previous meta-analysis and meta-regression study examined the relationship between poorer outcomes and D-dimer values has reported high sensitivity but relatively low specificity in detecting COVID-19-related venous thromboembolism events10). Furthermore, the relationship between COVID-19 severity, death rate and D-dimer value was found to be restrained10). This is probably realistic since the reliance on more biomarkers gives a clearer image than the reliance on a single test in regards to disease prognosis. On the other hand, our results suggest that plasma ferritin together with CRP and D-dimer tests can be considered as first line tests when diagnosing and evaluating patients with COVID-19 upon admission.

In routine clinical practice, individuals with hyperferritinemia usually had multiple conditions, and the diagnosis of hyperferritinemia most often occurred incidentally as a result from laboratory screening or follow-up test17). Serologically, the significant increase in ferritin level or hyperferritinemia is difficult to interpret without the existence of observable clinical milieu. This is because hyperferritinemia is associated with a wide etiological spectrum and common concomitant existence of numerous etiologies ranged from malignancy to arthritis34). A previous study had reported that hyperferritinemia is a consequence of non-human immunodeficiency virus infection17), while others found that malignancy is the most frequent cause of hyperferritinemia34). A part from its known role as an iron storage protein, ferritin can be undertaking other functions, since it might be actively secreted at the site of infection. Previous studies have considered ferritin as a signaling molecule and as an active contributor to the cytokine storm through it is proinflammatory effect and is consider as a key mediator of immune dysregulation directly through its suppressive effect especially under extreme hyperferritinemia16, 18).

Hyperferritinemia in the current study was associated with a significant increase in WBC and IgM in COVID-19 patients presented with severe symptoms. Although, WBC has increased in COVID patients with mild symptoms, however this increase was insignificant and stayed within the normal range. Previous research showed that autoantibodies are directed to WBC and such antibodies may deplete T cells and B cells which are important to fight the virus8). In another word, significant decrease in leukocytes causes a state of immunodeficiency, weakening the body and doubling the risk associated with COVID-19. There was a significant increase in granulocytes in the current study and these results are in line with previously published data7). A previous study suggested that granulocytes can be used to identify COVID-19 severity7). Nevertheless, data regarding the roles of granulocytes in the pathogenesis of COVID-19 are scarce.

Hyperferritinemia in COVID-19 patients of the current study was associated with a significant reduction in lymphocytes, and our results are in agreement with previously published studies. Decreased lymphocytes or lymphopenia have been considered as one of the major serological characteristics of severe COVID-19 patients3–5). Clinically, lymphocyte’s test is usually performed during routine medical encounters. Accumulative evidence showed that age plays an important role in the development lymphopenia. Thus, compared with children, lymphopenia is more common in elderly35). In line with these observations, recent studies reported a high incidence rate of lymphopenia in older COVID-19 patients compared with children36). A significant reduction in lymphocytes is known to play a crucial role in the pathogenesis of acute myocardial infarction and congestive heart failure37, 38). Thus, it is not surprising that lymphopenia in patients with severe COVID-19 is associated with decreased survival rate.

Finally, this study has several drawbacks, which include the reliance on one time point when collecting blood samples from patients with COVID-19. As a result, we could not have a proper image on the highest ferritin level in patients with severe symptoms in relation to infection progression nor we were able to detect the gradual decrease in ferritin levels and other studied biomarkers in relation to the treatment response and viral clearance. Second, we relied on relatively small sample size, which makes it not feasible to generalize our conclusion. Due to cultural and religious reasons, our previous studies39, 40) have faced enormous challenges to collect the needed number of females, in contrast females have willingly joined the COVID-19 project and this can be considered among the strengths of the current study.

Conclusion

Hyperferritinemia is associated with poor prognosis in Arabs diagnosed with severe COVID-19. Severe symptoms of patients presented with COVID-19 included: difficulties in breathing, assisted ventilation, long stay in hospital, ICU admission, long COVID and decreased survival rate. In addition, the high ferritin level was found to be associated with hyper reaction of the immune system, resulting in a significant increase in serum CRP, WBC, IgM, granulocytes and D-dimer. In contrast, hyperferritinemia was associated with a significant reduction of lymphocytes. The results of the current study suggest that ferritin together with CRP and D-dimer concentrations can be considered as markers upon admission to predict disease severity and poor prognosis in Arabs diagnosed with COVID-19. These findings may limit the number of biochemistry tests for individuals diagnosed with COVID-19, as they may provide a better image for patients who are in need for hospitalization and ICU admission. However, a larger cohort is needed in order to generalize this conclusion.

Acknowledgments

We would like to thank the clinicians, nurses and laboratory technologists in the Ramadi General Hospital for their assistance in this study. We also thank the Faculty of Science, University Of Anbar, Iraq, for technical support. Finally, we would like to express our gratitude to all the participants of this study.

Conflict of interest: none.

 

Faisal Al-ani • Osamah Al-ani (*)
Faculty of Medicine, Odessa National Medical University
Odessa 65000, Ukraine
e-mail: oalani@europe.com