According to the 2020 WHO guidelines, in areas where the COVID-19 virus is widely spread, RT-PCR screening is considered sufficient11. However, one or more negative results do not exclude the possibility of infection by the virus. Several factors can lead to a negative result in an infected individual, including poor sample quality, insufficient collected content, collection performed late or early in the course of the infection, or the sample not being handled and shipped adequately. Due to possible failures in the performance of the RT-PCR test, the National Health Commission of China recommended computed tomography (CT) as the main type of diagnostic screening, emphasizing that radiological examinations would be important for the early detection of the disease12,13. The criteria used in the selection of patients assessed in this study, related to computed tomography, were based on the standards duly recognized in the literature as characteristics of viral infection for COVID-198,14.
As far as gender is concerned, the reported cases of COVID-19 vary in different countries. In Brazil, in relation to cases of Severe Acute Respiratory Syndrome (SARS) by COVID-19, 55% of those affected are male. In this study, there was a predominance of 65.6% of males. In absolute numbers, China had more cases reported in men, while in South Korea there is a higher frequency in women. In Spain, the frequency of cases is similar for both genders. Initially, it was more frequent in men, but later, the magnitude of the numbers ranged from equal to increasing in women15. Based on published studies, the mean age of patients was 56 years, ranging from 55–65 years, with men being the most often affected. This fact would probably be related to the high levels of Angiotensin-Converting Enzyme II (ACE2)16. Data from the Ministry of Health of Brazil, in 2021, reported that the most affected age group was 60 to 69 years old, with 22.2% of cases. In this study, the mean age of the assessed patients was 65 years17.
The pandemic situation has boosted science to a better understanding of the mechanisms of SARS-CoV-2 virulence factors, aiming to identify them and develop barriers to their transmission and development in the human body, as well as to know their pathogenicity and clinical evolution of the patients in the different phases of this disease, through the employed tests, with the objective of providing safer and more successful therapeutic approaches in controlling the virus spread.
The knowledge about the course of the disease is very important when correlating the obtained results and the hospitalization period of the assessed patients. According to the literature, initially, SARS-CoV-2 binds to the host’s ACE2 receptor with the help of transmembrane serine protease-2 (TRMPSS2), a co-receptor, cleaving the viral protein. In the asymptomatic phase, infection of the host cell, viral diffusion in the human body and the production of virions predominate. The innate immunity, mediated by natural killer cells, neutrophils and monocytes/macrophages, reacts to viral replication, causing cytopathic effects with the release of pro-inflammatory mediators, triggering the onset of signs and symptoms. Cell immunity, mediated by B cells, CD4 and CD8 T cells, develops, increasing the intensity of these symptoms. There is an imbalance between effective and hyperactivated immune responses that can result in a cytokine storm, increasing lung injury, leading to respiratory failure. In this phase, protective neutralizing antibodies can enhance the activation of the antibody-dependent response and activate the complement system classical pathway, with an increase in viral replication and release of pro-inflammatory cytokines. The imbalance between inflammation and coagulopathy, as well as the SARS-CoV-2 infection of blood vessel walls cells determine thrombotic events causing organ damage. These uncontrolled processes trigger a strong pathological cycle, which eventually lead to systemic and organic cell dysfunction7. The patients in the present study were assessed from the onset of the first symptoms, during which time they were admitted for hospitalization.
In general, in the initial days after SARS-CoV-2 infection, clinical or laboratory markers do not demonstrate a severity factor. Approximately 5 days after the development of viral pneumonia, the cytokine storm can occur, which represents the period of greatest inflammatory stress and severity in the patient’s clinical status. Therefore, the need for serial measurements of inflammatory and thrombotic markers when monitoring the disease evolution would be justified18.
COVID-19 has shown to be an infectious and inflammatory disease that mainly affects the lungs, but which also involves damage to multiple organs with different injury pathways. Hemoglobinopathy, hypoxia and iron overload play an additional role in these injuries. Hemoglobin dysfunction can occur due to hemoglobin denaturation and deregulation of iron metabolism19. The observed alterations regarding the RDW (red cell distribution width) indicate the variability in the sizes of the erythrocytes of the assessed patients, which may provide complementary information for the etiological diagnosis of anemia, even before iron deficiency is identified20. In this study, a significant reduction in hemoglobin levels was observed, as well as in hematocrit, in addition to an increase in RDW throughout 90 days of hospitalization.
A meta-analysis showed that increased neutrophil and decreased lymphocyte levels are persistent findings in patients hospitalized with COVID-198, similar to the data found in this study with 90 days of hospitalization. Another study showed a significant relationship between the increase in total leukocytes, about 1.5-fold greater, higher neutrophil levels, with an approximately 1.7-fold greater count, and reduction in the number of lymphocytes, 0.9-fold lower in patients who showed disease worsening, requiring intensive care21. The abovementioned meta-analysis showed as predictive values of worsening for patient admission at the ICU an increase in leukocytes of about 2-fold, an increase in neutrophils of up to 4.4-fold, and the reduction of lymphocytes, of 0.4-fold8. In this study, although in the beginning of the second half of the hospitalization period the total leukocyte levels were high, the analysis of the 90 days of hospitalization showed no significant change in these levels. Eosinophils are circulating leukocytes found in tissues that have potent pro-inflammatory effects on several diseases. The patients assessed here had increased levels of eosinophils during the second half of the hospitalization period (from the 43rd to the 53rd day), for about 10 days. It was demonstrated that these cells have several other functions, including immunoregulation and antiviral activity. Their granules contain cytotoxic proteins, eosinophil peroxidase and two RNAses (the eosinophil cationic protein and the eosinophil neurotoxin). There is little indication that eosinophils have a protective role during SARS-CoV-2 infection; however, eosinopenia seems to be a prognostic indicator for more severe COVID22.
Some authors discuss the association between critically-ill patients with COVID-19 and their specific coagulation profile, particularly D-dimer elevation, prolonged prothrombin time, and low platelet counts. According to the authors, these changes reflect the hypercoagulable state observed in critically-ill patients, which can promote microthrombi in the lungs or other organs23. More studies are necessary to improve the understanding between the hematological function of platelets and Covid-19, and different stages of the disease, gender, age, and preexisting comorbidities should be considered.
The increase in the activated partial thromboplastin time of the patients analyzed in this study is mostly due to the heparin anticoagulant therapy (low molecular weight) required by patients with COVID-19. The thrombotic complications of these patients seem to resemble systemic coagulopathies in severe infections, such as sepsis-induced coagulopathy or disseminated intravascular coagulation. Data in the literature show that patients with COVID-19 have elevated D-dimers and fibrinogen levels, similar to what was found in this study. There were no significant changes in prothrombin activity time in the patients studied herein. Patients with disseminated intravascular coagulation usually have prolonged prothrombin time and thrombocythemia24.
Many patients with COVID-19 have characteristic coagulation alterations, such as high fibrinogen levels, as well as increased D-dimer levels, which can help in the screening for the identification of hypercoagulable patients. Increased D-dimer occurs in parallel with fibrinogenemia in many patients25, as shown in this study. There are reports of an increase in these parameters in non-severe patients26, whereas other authors have reported worsening with increased values of D-Dimer, of around 2.5-fold; similarly, other authors have shown significantly higher values of D-Dimer (2-fold increase) in more severe cases, when compared to milder cases21,27.
In this study, the values of serum urea levels showed a slight increase, without statistical significance; however, serum creatinine levels, especially after the 50th day of hospitalization, showed a significant increase. There are reports of altered creatinine values, of around 1.1-fold lower21. Reports point to a direct relationship between high creatinine levels and other markers of renal function with severity and mortality in patients with COVID-1928.
There is a study that shows an increase in both transaminases, aspartate aminotransferase (AST), with an increase of about 1.8-fold, and ALT, with an increase of about 1.5-fold21. In another study, an increase of 1.8-fold in the ALT levels was observed in the patients8. This study observed that between approximately 25 and 30 days of hospitalization, there was an approximately 2-fold increase in the ALT levels, and an increase of approximately 4-fold between days 45 and 50 of hospitalization.
The presence of elevated troponin in hospitalized patients was associated with higher mortality in patients with COVID-1929,30. Authors showed a 2.2-fold increase in the troponin levels of the assessed patients21. In this study, there was no significant change in troponin levels during the 90 days of hospitalization.
Similarly to the high levels of lactate dehydrogenase (LDH) observed in the patients in this study during their 90 days of hospitalization, other authors also found high levels of LDH, around 2.1-fold higher21,26. Elevated LDH values are associated with worsening of the disease, need for hospitalization and mortality in patients31.
Studies have shown a significant reduction in serum levels of sodium, potassium and calcium in patients with COVID-19, although chlorine levels did not show any significant changes32. In the patients analyzed in this study, although the serum levels of sodium and calcium showed a certain decrease in their values, it was not significant. Moreover, serum chloride and potassium levels did not show any significant changes. The patients showed a reduction in serum magnesium levels as of the 22nd day of hospital admission, but without significance, differently from what was reported by other authors33. It is important to evaluate the patients’ electrolyte levels so that corrective therapeutic actions can be carried out quickly and effectively.
This study showed a reduction in the patients’ albumin levels analyzed throughout the 90 days of hospitalization. Authors have shown a reduction of approximately 0.8-fold in the albumin values of their assessed patients8.
The C-reactive protein levels of the patients assessed in this study showed an elevation of approximately 10-fold or higher in their serum levels. The data obtained in the present study are in agreement with other studies; however, some of them evaluated patients with non-severe COVID 1926,27.
When directly destroyed by the virus, the hemoglobin releases iron into the circulation, which can lead to a loss of the hemoglobin’s capacity to bind to oxygen, causing tissue hypoxia and organ failure. Additionally, the increased free iron can cause oxidative damage to the lungs and other organs, leading to inflammation and immune dysfunction. Excess iron causes blood hyperviscosity, leading to thrombotic phenomena and a state of circulatory hypercoagulation. On the other hand, in a compensatory way, there is an increase in ferritin, a protein that stores iron. This is a predictor of COVID-19 severity, as it contributes to immune dysfunction, inflammation and hypercoagulation34. In this study, an increase in serum ferritin levels was observed in the assessed patients.