Epidemiological and laboratory profile of patients confirmed with Covid-19 and admitted to a reference hospital

The new coronavirus, SARS-CoV-2, which causes COVID-19 is easily transmitted from person to person. About 15% develop severe pneumonia and of these, 6% progress to severe acute respiratory syndrome (SARS) and multiple organ failure. Identify biomarkers that can predict which individuals are likely to be affected by severe disease and are at risk of death is crucial. However, the pathophysiology of the disease is not completely elucidated and the characterization of the clinical profile of people infected with SARS-CoV-2 remains unclear. Therefore, the objective of this study was to evaluate the epidemiological and laboratory profile of patients confirmed with COVID-19 and admitted to the University Hospital Lauro Wanderley, based on the correlation of laboratory tests to the evolution of the disease, determination of the most influential variables in the worsening of the disease. Data consisted of complete blood count and serum biochemical analyzes. Overall, 74 patients met all criteria of this study and were included, 40 from the Intensive Care Unit (ICU), and 34 from the Infectious Diseases Infirmary (IDP), both exclusively for patients with COVID-19. Based on these findings, patients with increased WBC count, ALT, AST, and LDH should be closely monitored as these factors predict ICU admission and mortality. Among other laboratory parameters, patients admitted to the ICU have higher levels of D-dimer compared to IDI patients. The results suggests that higher levels of D-dimer on admission is related to a worse prognosis of the disease.


Introduction
Coronaviruses are enveloped RNA viruses; these viruses belong to the Coronaviridae family. Among the seven species that cause respiratory infections in humans, two are responsible for causing severe acute respiratory syndrome with high mortality rates, namely SARS-CoV and MERS-CoV (Rojas et al., 2020).
The new coronavirus, SARS-CoV-2, which causes COVID-19 (Coronavirus disease) was detected on December 31, 2019 in Wuhan, China. The virus is easily transmitted from person to person, mainly through respiratory secretions, saliva droplets or fomites. With a high rate of transmissibility, WHO confirmed, by June 27, 2021, 181 million cases and 3.93 million deaths worldwide (WHO), 2021). In Brazil, in the same period, 18.4 million of cases and 514,000 deaths were reported.
Most people infected with the virus are asymptomatic or have mild to moderate respiratory disease. However, about 15% develop severe pneumonia and of these, 6% progress to severe acute respiratory syndrome (SARS) and multiple organ failure . It is believed that advanced age, the presence of comorbidities, such as cardiovascular diseases, diabetes, chronic respiratory diseases and cancer, besides being male are the main risk factors for the development of severe forms of the disease and the need for intensive therapy support (WHO), 2021).
Therefore, identifying biomarkers that can predict which individuals are likely to be affected by severe disease and are at risk of death is crucial (Keski, 2021). This approach can favor early diagnosis, as well as optimize the use of limited resources in the appropriate treatment and recovery of these patients .
However, the pathophysiology of the disease is not completely elucidated and the characterization of the clinical profile of people infected with SARS-CoV-2 remains unclear. Recent studies suggest that these patients have hematological and biochemical changes (Ferrari et al., 2020;Lippi & Plebani, 2020). Changes in the blood count have been described in the literature (Pan et al., 2020), such as a reduction in the erythrocyte count and hemoglobin levels; in the leukogram (Ai et al., 2020), as an increase in the number of leukocytes, including neutrophilia and monocytosis and a lymphocytic reduction; and on the platelet count , such as thrombocytopenia and elevation of D-dimer, considered one of the main markers of mortality.
In addition, as it is considered a systemic infection, there is evidence that Covid-19 can cause liver and kidney damage. In the liver, an increase in aspartate aminotransferase (AST / TGO) enzymes was observed, alanine aminotransferase (ALT/TGP) e gamma-glutamyl transferase (GGT), and total bilirubin levels (Y. Zhao et al., 2020). Authors also report elevated lactate dehydrogenase (LDH) (Miao et al., 2020), decreased levels of albumin, and elevated levels of inflammatory markers such as C-reactive protein (CRP) (Hsih et al., 2020;Mardani et al., 2020;Zhu et al., 2020), IL-6, and procalcitonin (Ai et al., 2020). On the other hand, in the kidneys, some markers also appear to vary in their levels. Among them, we can highlight an increase in creatinine and serum urea (X.  and a reduction in the glomerular filtration rate .
Thus, the deepening of clinical differences, based on laboratory parameters, is necessary for a better understanding of COVID-19, which, consequently, will assist in patient management decisions. Therefore, the objective of this study was to evaluate the epidemiological and laboratory profile of patients confirmed by Covid-19 and admitted to the University Hospital Lauro Wanderley, based on the correlation of laboratory tests to the evolution of the disease, determination of the most influential variables in the worsening of the disease.

Data source
A cross-sectional observational study was developed with data collected from patients confirmed with  admitted to the University Hospital Lauro Wanderley (HULW), located in João Pessoa, Paraíba, Brazil, covering the period from March 20, 2020, to January 31 of 2021. All data, including age, gender, length of stay and laboratory tests were extracted from the electronic system of the Clinical Analysis Laboratory Unit (ULAC). Laboratory data consisted of complete blood count (total white blood cell, lymphocyte, and platelet count) and serum biochemical analyzes (C-reactive protein, lactate dehydrogenase, d-dimer, aspartate aminotransferase and alanine aminotransferase. Finally, 74 patients met all criteria of this study and were included, 40 from the Intensive Care Unit exclusively for Covid-19, and 34 patients from the Infectious Diseases Infirmary (IDP), exclusively for patients with Covid-19. This study was previously approved by the Committee of

Participants
For the formation of the groups, the following eligibility criteria were used: patients with a positive result in the RT-PCR test and admitted to hospital admission in the service sector exclusively for patients with COVID-19.
The flowchart, with the total number of patients considered, until reaching the 74 patients selected for the study is shown in Figure 1. During the period from March 20, 2020, to January 31, 2021, 2221 samples were collected from nasopharyngeal/oropharyngeal swab for analysis by the RT-PCR methodology. Of these, 1575 had a "Not detectable" result, and were excluded from the survey. Inclusion and exclusion criteria were then applied to the remaining 646 patients. 572 were excluded for not being suitable for this research, as they were patients admitted to other clinics, employees, caregivers, and duplicate results. At the end, the N of 74 patients admitted to clinics dedicated to the management of Covid-19 was obtained, including COVID-ICU-Covid (n=40) and COVID-IDI (n=34).

Laboratory tests
Patients admitted to inpatient clinics for COVID-19, according to the medical protocol followed, generally underwent exams every 2 days or at every change in their health conditions.

Statistical analysis
Continuous variables were expressed as median (IQR) and compared with the t-test or Mann-Whitney test; categorical variables were expressed as number (%) and compared by the χ² test or Fisher's exact test among patients who received care in the ICU and IDI (infectious diseases infirmary). Results were considered significant when the p value was less than or equal to 0.05. Statistical analyzes were performed using GraphPad Prism Software, version 6.01.

Results
The study sample consisted of 74 patients with COVID-19: 40 hospitalized in the ICU (intensive care unit) and 34 in the IDI (infectious diseases infirmary). The characteristics of the study participants are shown in Table 1. Results expressed as median ± standard deviation and 95% confidence interval (95% CI). bResults expressed as median and interquartile interval (P25% -P75%). All variables were categorized, and the results are expressed in n (%) or n / N (%), where N is the total number of patients with available data. p1 (p value referring to the independent t test or Mann-Whitney test), p2 (p value referring to the Chi-square test or Fisher's exact test), ICU (intensive care unit) and IDI (infectious diseases infirmary). *p≤0.05. Source: prepared by the author.
The results of this study demonstrate that there is no significant difference between ICU patients and IDI patients in relation to age, gender and length of hospital stay, not even when the variables were categorized. However, the majority of patients were men, aged 40-60 years (Table 1).
Regarding to laboratory parameters ( patients. 74 patients were tested for C-reactive protein and 48 were tested for D-dimer, 64 (86.5%) and 17 (35.4%) of whom, respectively, had levels above the normal range. Thus, it was observed that the levels of CRP and ALT were similar in both groups. However, D-dimer levels were significantly higher in patients in the ICU in comparison to the IDI group (p1 = 0.05).

Discussion
The results of the present study demonstrated a greater number of men than women in the 74 cases of COVID-19 infection. MERS-CoV and SARS-CoV have also been found to infect more this gender (Badawi & Ryoo, 2016). According to Jaillon, Berthenet and Garlanda (2019) (Jaillon et al., 2019) the protection of the X chromosome and sex hormones may be responsible for the reduced susceptibility of women to viral infections, as they play an important role in innate and adaptive immunity.
Furthermore, similar counts of leukocyte, lymphocyte and platelet and significantly elevated D-dimer, AST and LDH levels were found in ICU patients when compared with IDI patients. In this sense, the results suggest the presence of biochemical alterations, but not hematological in ICU patients, considering all these variables as predictors of prognosis in COVID-19.
Previous studies have shown that patients with COVID-19 have leukocyte levels in the blood. It is described that, in the severe forms compared to the mild or asymptomatic forms of the disease, the patients present leukocytosis, however a reduction in the number of circulating lymphocytes percentage (Tan et al., 2020; . Our findings, although there were no differences between groups, corroborate this evidence. Most patients, both in the ICU and IDI, have a leukocyte elevation rate and a lymphocytic reduction. It is hypothesized that these changes in the white blood series may happen due to the fact that (1) the SARS-CoV-2 virus can directly infect lymphocytes, since they express the ACE2 receptor, resulting in lymphocyte death; (2) the virus can destroy directly lymphatic organs; or (3) inflammatory cytokines, as tumor necrosis factor (TNF) and interleukin (IL)-6, remained disordered, inducing apoptosis and lymphocyte deficiency (Tan et al., 2020;Liao et al., 2002;Xu et al., 2020).
Therefore, the platelet elevation present in some ICU and / or IDI patients may be related to the sustained inflammatory response, which leads to the activation of coagulation (D. Wang et al., 2020). All of these pathological mechanisms may be associated with a worse prognosis and /or death in patients with COVID-19.
In a meta-analysis, Zhang et al., 2020 identified five significant markers when it comes to the admission of patients with COVID-19 to the ICU whose result is high: leukocyte count, ALT, AST, LDH and procalcitonin.
Elevated LDH levels are predictive of the development of an acute respiratory syndrome and elevated WBC count associated with elevated LDH count has been found to predict mortality . Among the biochemical markers, LDH was the only one that significantly predicted the development of an acute respiratory syndrome, admission to the ICU, and mortality. LDH is released from cells after damage to the cytoplasmic membrane and functions as a prognostic biomarker of immune surveillance Kuang et al., 2020). LDH promotes the production of lactate, which leads to an increase in immunosuppressive cells and inhibition of cytolytic cells (Ding et al., 2017). These changes can weaken the immune response mounted against infection caused by SARS-CoV-2, increasing the severity of the disease in patients with elevated LDH levels.
The clinical association of higher levels of LDH with severe cases of COVID-19 indicates that an excessive inflammation is related to an unfavorable clinical outcome. Inflammasomes often participate in the induction of inflammatory processes in the host cell . The NLRP3 inflammasome promotes inflammation through the cleavage and activation of inflammatory molecules including active caspase-1 (Casp1p20), IL-1β and IL-18. The presence of cell death and inflammasome-derived products, such as IL-1β, IL-18 and LDH in sera from patients with COVID-19, reveals the involvement of the inflammasome in the pathogenesis of the disease .
The increased inflammatory characteristics of COVID-19 and the correlation of disease severity with LDH, a typical marker of pyroptosis, suggest that SARS-CoV-2 triggers inflammasome activation. Rodrigues et al. (2020), found higher levels of Casp1p20 and IL-18 in the serum of COVID-19 patients, indicating active inflammasomes. In addition, SARS-CoV-2 triggered the release of LDH in monocytes in a process that is independent of priming and requires viable SARS-CoV-2.
MCC950, a potent and selective inhibitor of NLRP3, did not affect LDH release, suggesting that NLRP3-independent responses operate to induce a lytic form of virus-induced cell death. Finally, positive associations of Casp1p20 and/or IL-18 levels with inflammatory markers, C-reactive protein, LDH and ferritin were detected30. Therefore, elevated LDH levels can be a valuable marker of SARS-CoV-2 infection (Rodrigues et al., 2021).
Regarding the liver biomarkers, although not significant between groups, our data show that C-reactive protein and ALT levels were increased, unlike AST, in both ICU and IDI patients. The increase in ALT and AST in severe COVID-19 may be a result of liver damage caused by direct binding of SARS-CoV-2 to ACE2 positive cholangiocytes (Chai et al., 2020).
In another meta-analysis, Tian et al. (2020) found that C-reactive protein (+66.3 µg/mL) were higher in non-survivors, and regarding liver function tests, ALT (+5.7 U / L) and AST (+15.2 U / L) also were found at higher levels in this group. These results indicate impaired liver function at admission in non-survivors compared to survivors. Mortality has also been associated with lower platelet counts and elevated levels of D-dimer, suggesting a possible coagulopathy in these patients .
On the other hand, in severe cases of COVID-19 it has been identified that patients have infection-induced coagulopathy and secondary hyperfibrinolysis (Ji et al., 2020). Our findings reaffirm these observations and showed that patients admitted to the ICU have higher levels of D-dimer compared to IDI patients. This is corroborated by another study that demonstrates that the higher level of D-dimer on admission is related to a worse prognosis of the disease .
Therefore, studies indicate that pulmonary thrombosis and deep vein thrombosis in cardiovascular diseases may be responsible for the elevation of D-dimer levels (Giannitsis et al., 2017;Z. F. Wang et al., 2011). Thus, having observed that non-surviving patients have higher levels of D-dimer compared to survivors, treatment with anticoagulants may be feasible in severe cases of COVID-19.

Conclusion
Based on these findings, patients with increased WBC count, ALT, AST, and LDH should be closely monitored as these factors predict ICU admission and mortality. A major factor in the risk stratification of patients with COVID-19 is the initial assessment of their biochemical and hematological parameters, since these markers demonstrate organ dysfunction, inflammation or coagulopathy and brings a higher risk of an unfavorable outcome. In general, variations in the level of Ddimer are correlated with the prognosis of the disease and the anticoagulant treatment may benefit severely ill patients with COVID-19. Further studies are needed to better understand whether abnormal baseline levels predispose an individual to a higher risk of mortality or whether virus alters marker levels directly.