D-dimer level and inflammatory markers in COVID-19 patients with and without diabetes mellitus - A comparative study during the first wave of the pandemic

Introduction and Aim: High prevalence of diabetes mellitus makes it crucial to understand the distinctive inflammatory features of COVID-19 infection. Hyperglycemia and diabetes mellitus are independent prognosticators for mortality and morbidity in SARS-CoV-2 infection. This study compares D-dimer and inflammatory markers in COVID-19 patients with and without T2DM during the first wave of the pandemic. Materials and Methods: A retrospective study was conducted at a tertiary care hospital. D-dimer, IL-6, Ferritin, CRP and LDH levels in COVID-19 patients with and without T2DM were compared. D-dimer values were correlated with the IL-6, Ferritin, CRP, LDH and checked for statistical evidence. HbA1c levels were assessed for association of the severity of COVID-19 infection. Results: Serum levels of inflammatory biomarkers of 205 COVID-19 patients were compared in which 106 had T2DM and 99 were non-diabetic. Ferritin and LDH showed statistically significant elevation in diabetic COVID-19 patients. D-dimer showed positive correlation with all the inflammatory markers both in diabetic and non-diabetic COVID-19 patients. HbA1c showed statistically significant positive correlation only with Ferritin in COVID-19 patients with T2DM. Conclusion: Ferritin was significantly associated with severity of diabetes as indicated by correlation to HbA1c. D-dimer, Ferritin, IL-6 and LDH levels were significant in COVID 19 patients having diabetes and those without.


INTRODUCTION
he COVID-19 (coronavirus disease 2019) initially began in China and the infection has affected millions of people globally, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a formerly unidentified beta coronavirus (1)(2)(3)(4)(5).The categories of presentations of this disease based on the severity of respiratory distress and associated leukocytosis and increased inflammatory markers was described in a comparison report of patients having SARS-CoV-2 infection (6).Uncontrolled inflammation responses, releases tissue injury enzymes and hypercoagulable state is related to poor regulation of glucose metabolism.Serum level of inflammatory markers such as IL-6, coagulation index, D-dimer, C-reactive protein, and serum ferritin, were considerably increased (p < .01) in patients having diabetes compared to those without diabetes mellitus, which suggested, patients having diabetes are further responsive to an inflammation which can progressively worsen COVID-19 infection (7).
Diabetes and related complications increase the chances of morbidity and mortality because of repressed humoral and innate immune functions.The levels HbA1c > 9% linked to 60% greater chances of hospital stay and severity of developing pneumonia during bacterial infection.High prevalence of diabetes mellitus makes it more crucial to study the distinctive aspects of COVID-19 infection in such patients (8).
Hyperglycemic state results in vascular abnormalities leading to rise in inflammatory responses and oxidative stress (9).A reviewing study involving 41 COVID-19 patients, has shown that 32% were with inherent diseases, amid which 20% had diabetes showing that diabetic patients are potentially at greater risk of COVID-19 with poor prognosis (10).Some patients having COVID-19 pneumonia also developed acute respiratory distress syndrome (ARDS), and some of them died due to multiple organ failure (9).It has been found that there was a significant increase in D-dimer level in patients presented with diabetes, indicating the susceptibility to a higher coagulable state than patients without diabetes, is an important factor for bad prognosis of COVID-19 infection (10).Increase in Ddimer concentration of more than 1.0 μg/ml helps in predicting the possibility of thromboembolism and frequent occurrence of thromboembolic events.There was decreased mortality in COVID-19 patients seen with the use of heparin, signifying that treatment of thrombo embolism is captious in the management of COVID-19 (11).Several studies have demonstrated that there is a greater predisposition to few other infectious diseases in diabetes because of a dysregulated immune system.Diabetes and increased blood glucose levels are independent prognosticators for mortality and morbidity in patients having SARS infection (12).There is activation of inflammatory cascade ensuing sensing virus' RNA and its structural proteins by the inflammatory sensors.Patients with T COVID-19 develop pathological and clinical manifestations due to inflammatory cytokine storms caused by NOD-like receptor protein 3 (NLRP3) and interleukin-6 (IL-6) (13).In reaction to inflammation, C-reactive protein is produced in the liver which is an acute phase reactant.Through the initial phase of COVID-19, plasma C-reactive protein levels can show the extent of lung lesions and severity of the disease and also helps in assessing clinical evaluation index.Early detection of pneumonia can be made by estimating C-reactive protein and high CRP levels are seen in patients with severe pneumonia (14).In patients with acute or chronic inflammation, malignant tumors and infections, elevated serum ferritin levels are seen, as ferritin is an acute phase reactant.IL-6 and serum ferritin are found to be significantly increased in COVID-19 (15).A meta-analysis showed that increased serum ferritin CRP, D-dimer levels were related with increased poor outcomes like ARDS, mortality, severe COVID-19 and the need for ICU care in such patients (14) Recent data in patients with COVID-19 has suggested substantial difference in LDH levels with varied severity of the disease (15).LDH enzyme found intracellularly in almost all the tissues.Conventionally, when there is cardiac damage, LDH has been used.However, abnormal increase in LDH level can result from tissue damage, due to multiple organ injury and consequent anaerobic glycolysis (16).In addition, research has shown that COVID-19 infected patients have low haemoglobin levels and elevated levels of ferritin (17).Increased ferritin levels may be prognostic of an impending inflammatory reaction in COVID-19 affecting iron metabolism.Iron is an essential micronutrient for both human cells and pathogens and an immune response triggered to control its usage by the pathogens may restrict iron utilization during infections and leading to anemia, which in turn, decreases uptake of oxygen to tissues, ensuing in multi-organ failure (18)(19)(20).Hence the present study is to compare the inflammatory markers -CRP, ferritin, IL-6, LDH with D-dimer in COVID-19 patients having Type 2 diabetes mellitus (T2DM) to those without and to understand their role in the severity and prognosis of the disease.

MATERIALS AND METHODS
This study was approved by the Institutional Ethics Committee with protocol no.IECKMCMLR-10/2020/302.This study was conducted at the Central Laboratory of a tertiary care hospital.Male and female subjects above 18 years, with or without diabetes mellitus were included in the study.Pregnant women and subjects with malignancies and other neoplastic conditions were excluded from the study.205 subjects diagnosed with COVID-19 by RT PCR and XPERT SARS CoV-2 were involved in the study, of which 106 were known cases of T2DM and 99 were non-diabetic.Details of blood investigations and history and duration of diabetes were taken from the medical records of the subjects.D-dimer was estimated by particle-enhanced immunoturbidimetric assay.Ferritin and IL-6 were analyzed by Sandwich principle.CRP was estimated by slide agglutination.HbA1c was estimated by HPLC method by using D10 hemoglobin variant instrument and UV assay was used for the estimation of LDH.D-dimer values were correlated with the results of inflammatory markers of COVID -19 patients with and without T2DM.HbA1c levels were scrutinized with the risk of severity of COVID-19 infection.

Statistical analysis
Data analysis was done using SPSS software version 20.Data with skewed distribution presented as Median (Interquartile range) and comparison among groups was done with Mann-Whitney U nonparametric test.Data with normal distribution displayed as Mean ± Standard deviation.Box-plot diagram was used to show the comparison of inflammatory markers between non-diabetic and diabetic patients (Fig. 1).Spearman's correlation was applied for both D-dimer and HbA1c respectively, with the inflammatory biomarkers Ferritin, IL-6, CRP and LDH in COVID-19 patients with and without T2DM.Correlation of HbA1C with inflammatory markers was shown by Scatter-plot diagram (Fig 2).
p value <0.05 was considered statistically significant.

RESULTS
The clinical laboratory data of patients diagnosed with COVID-19, with and without T2DM are given in Table 1.A total of 205 COVID-19 positive patients involved in the current study, of which 106 were known cases of T2DM and 99 were non-diabetic patients.Serum levels of inflammatory biomarkers IL-6, D-dimer, CRP, LDH, ferritin in COVID-19 patients having T2DM and those without T2DM were measured and associated.Fig. 1 shows the median values of all inflammatory markers and was raised in COVID-19 patients with T2DM compared to non-diabetic patients.However, among them elevations of only ferritin (p=0.012) and LDH (p=0.002) were statistically significant (Table 1).

DISCUSSION
Poor prognosis of coronavirus infections has been associated with diabetes.A retrospective study proposed that hyperglycemia and pre-existing diabetes were independent predictors for death and morbidity in SARS patients (21).Pro-inflammatory states existing in diabetes could bring out the cytokine storm, and subsequently, multi-organ failure and ARDS in patients having COVID-19 (22).However, there is an increased rate of infection in diabetes, and several studies have shown incidence of diabetes comparable to that in patients with COVID-19 without diabetes.Although several inflammatory markers have been recognized, and the detection of hypercoagulability made accessible, the comparison of them in COVID-19 patients having diabetes and those without may support the management process.The present study compared the inflammatory markers like CRP, ferritin, LDH, IL6 with D-dimer in COVID-19 patients having diabetes mellitus and those who do not, to understand their role in the severity and prognosis.
Varikasuvu et al., indicated that elevated levels of hypercoagulability and inflammatory markers were seen in COVID-19 patients having diabetes when compared to COVID-19 patients not having diabetes in the meta-analysis.Several studies suggest that activation T-cell differentiation and immune system imbalance is seen in diabetes (23).In the present study, D-dimer showed statistical significance and positive correlation with ferritin, CRP, IL-6 and LDH in all the COVID-19 patients.However, HbA1C showed statistically significant positive correlation with ferritin only.This highlighted that the hypercoagulability markers may significantly increase in COVID-19 patients having diabetes mellitus and those who do not.Identification of an ideal inflammatory marker is important in regulation of COVID-19 patients having diabetes.
A retrospective study by Tamaryn et al., showed cardiovascular risk factors such as hypertension and chronic kidney disease higher in COVID-19 patients with T2DM compared to those without diabetes (29).
In our study, the inflammatory role of LDH and ferritin revealed a significant finding in all COVID 19 patients.However, ferritin was the main inflammatory marker associated with T2DM in COVID-19 patients.The IL -6 levels showed a lesser significance of the ongoing process, with a probable role of other comorbidities along with T2DM.The role of good glycemic control is a key factor associated with the progression of COVID-19.

CONCLUSION
The clinical scenario of COVID-19 disease is progressively clear from significant studies.Many studies have shown that diabetes as a distinguishing comorbidity related with more severity and manifestation of acute respiratory distress syndrome and increased mortality.Hence, along with the current guidelines and recommendations for COVID-19, assessment of the role of inflammatory markers is important.Ferritin was significantly associated with severity of diabetes as indicated by correlation to HbA1c in COVID-19 infection.D-dimer showed positive correlation with ferritin, CRP and LDH in all COVID 19 positive patients.

Table 1 :
Descriptive statistics of inflammatory markers and HbA1c values from the laboratory investigations of COVID -19 patients with and without T2DM

Table 3 :
Correlation of HbA1c with D-dimer and other inflammatory markers in COVID-19 positive T2DM patients DOI: https://doi.org/10.51248/.v43i6.3665Biomedicine-Vol.43 No. 6: 2023 specific inflammatory marker study on LDH by Shi et al., gives a clue on the significance of its increase as the COVID-19 disease progresses.The study showed a considerable difference of substantial risk of disease progression in patients with high values of LDH, to normal range of LDH during admission.This multicenter nested case-control study also noted that the increase in age and increased LDH level were