This is a single centre, cross-sectional study conducted at Government Tirunelveli Medical College, a Tertiary Care Centre in Southern India from March to July 2020. All those who had a contact history with COVID positive cases or clinical signs or symptoms were traced by the Public Health Care Workers and nasopharyngeal swab test was done in the ICMR approved Microbiology Laboratory of Tirunelveli Medical College. Those who tested positive for Real Time Polymerase Chain Reaction (RT-PCR) were admitted in the hospital and included in this study irrespective of their age. All those who were negative for RT-PCR were excluded. Thus, we enrolled 217 patients (N). This study was approved by the Ethics Committee of the Institute (REF NO: 1792/PATH/2020) and data was collected retrospectively. Based on the clinical data, they were classified. According to the “Clinical Management Protocol: COVID 19” by the Ministry of Health and Family Welfare and Director General of Health Services [2]. The clinical classification was as follows: 1) Asymptomatic: Contacts or patients with travel history tested swab positive by PCR, without any clinical signs like fever, sore throat, dry cough, dyspnoea; 2) Mild: Patients with uncomplicated upper respiratory tract infection may have mild symptoms such as fever, cough, sore throat, nasal congestion, malaise, headache without evidence of breathlessness or hypoxia (normal saturation); 3) Moderate: Pneumonia with no signs of severe disease. For adolescents or adults with presence of clinical features of dyspnoea and or hypoxia, fever, cough, including SpO2 <94% (range 90-94%) on room air, respiratory rate is more or equal to 24 breaths per minute. For children with presence of clinical features of dyspnoea and or hypoxia, fever, cough, including SpO2 <94% (range 90-94%) on room air, respiratory rate is more or equal to 40 breaths per minute. Fast breathing is defined as (in breaths/min): <2 months: ≥60; 2-11 months: ≥50; 1-5 years: ≥40; 4) Severe: Severe Pneumonia with clinical signs of pneumonia plus one of the following; respiratory rate >30 breaths/min, severe respiratory distress, SpO2 <90% on room air for adolescent or adult and child with cough or difficulty in breathing, plus at least one of the following: central cyanosis or SpO2 <90%; severe respiratory distress (e.g., grunting, chest indrawing); signs of pneumonia with any of the following danger signs: inability to breastfeed or drink, lethargy or unconsciousness, or convulsions; with any other following signs of pneumonia: chest indrawing, fast breathing (in breaths/min): <2 months ≥60; 2-11 months ≥50; 1-5 years ≥40. acute respiratory distress syndrome, sepsis, septic shock and death [2].

The patients travel history, exposure history, contact history, demographic data, signs and symptoms, laboratory and radiological data were collected. Peripheral venous blood samples were collected as per standard protocol in sodium citrate containers. D-dimer was estimated in the separated plasma, using latex based assay using semi-automated coagulation analyser (ECL-50, ERBA). Based on the Guan W et al., study (February 2020), the cut-off value for D-dimer level was fixed as 500 ng/mL and the study population are divided into two groups to compare their clinical severity [24]. The throat samples were tested for SARS COV2, with Labgun RT-PCR kit (Labgenomics, Republic of Korea) in the hospital laboratory. All collected samples were handled following strict biosafety measures and biomedical waste management guidelines.

Statistical Package for the Social Sciences (SPSS) version 16.0 (SPSS Inc. Chicago, IL) was used. Percentages were used for categorical variables and Median±IQR for continuous variables. Chi-square test was used to compare the D-dimer values between symptomatic and asymptomatic groups. A value of p<0.05 was considered statistically significant.

A total of 217 COVID-19 cases were included in the study population. A 87.5% (n=190) of the study population were adults and among them 52.9% (115) were male and 34.5% (75) were female [Table/Fig-1]. About 27 were children and in 31-40 and 41-50 age group there were 44 patients [Table/Fig-2]. Among the 217 cases 88.9% were asymptomatic cases, 8.8% were presented with mild clinical severity and 2.3% were with moderate clinical presentation [Table/Fig-3]. There was a statistically significant difference in age, Neutrophil count (%), RBC count and Neutrophil Lymphocyte (NL) Ratio between the symptomatic and asymptomatic cases [Table/Fig-4].

[Table/Fig-1]:

Distribution of study population.

In our study population, the Mean±SD and Median±IQR of D-dimer values (in ng/mL) were 223.4±230.6 and 157.0±187.7, respectively. The mean D-dimer value was 212.7 ng/mL in asymptomatic cases which increases to 243.7 ng/mL in mild cases and 525.8 ng/mL in patients with moderate clinical presentation. The mean D-dimer value was found to increase as the category of our study group ascended from asymptomatic patients to mild, moderate clinical cases [Table/Fig-5]. Around 91.1% of the cases who had normal D-dimer values (<500 ng/mL) were asymptomatic cases [Table/Fig-6]. The Chi-square test showed that it was statistically significant and the odds of patients with high level of D-dimer were supposed to be clinically symptomatic cases which were 5.5 times more than that of the odds of patients with normal levels of D-dimer [Table/Fig-7].

A recent article by Iba T et al., presents a better overview about the possible pathogenesis. Persistent inflammation results in the formation of Interleukin-1β and Interleukin-6 which are known to cause thrombocytosis and hyperfibrinogenemia. In the early stage of disease, inflammation and coagulation are limited to the lungs. But as disease progresses, these features become systemic and later on present as Disseminated Intravascular Coagulation (DIC). Alveolar macrophages release urokinase type Plasminogen Activator (u-PA) which promotes local fibrinolysis and D-dimer elevation. Moreover, direct infection of endothelium by the SARS-CoV2 virus via its receptor, Angiotensin-Converting Enzyme 2 (ACE-2), results in a surge in plasminogen activator release. As disease severity progresses, increased fibrinogen levels and activated platelets aggravates procoagulant state. Pulmonary microthrombi formation is promoted by Plasminogen Activator Inhibitor 1 (PAI-1) which leads to suppression of fibrinolysis. In normal vascular endothelium ACE-2 mediates anticoagulant activities. But once SARS-CoV2 virus binds to it, ACE-2 causes cell damage followed by increased expression of tissue factor and downregulation of the protein C system. Ultimately coagulation and thrombotic events may occur even in the absence of secondary complications like tissue hypoxia and superadded infections [25].

Studies conducted in Netherland and France show increased incidence of thrombotic complications in patients with severe disease [26,27]. A retrospective study on 183 patients by Tang N et al., established that 71.4% of non-survivors and around 0.6% of the survivors showed features of overt DIC. The median time from admission to development of DIC was four days [7]. Autopsy findings published by Wichmann D et al., brings to notice a high incidence of thromboembolism in COVID-19 patients and the need to consider pulmonary embolism in cases of haemodynamic instability [28]. A state of dynamic hypercoagulation as evidenced by the presence of microthrombi in various organs accompanied by reduced platelet levels is seen in COVID patients, especially those with comorbidities. Plasmin mediated hyperactive fibrinolysis may also lead to haemorrhage and marked elevation of circulating fibrin degradation products in such patients [29].

In our study, we noted that the mean D-dimer value increases as the category of our study group moves up from asymptomatic patients to mild, moderate and severe clinical cases. The mean D-dimer value was 212.7 ng/mL in asymptomatic cases which increases to 243.7 ng/mL in mild cases and 525.8 ng/mL in patients with moderate clinical presentation. These findings are in concordance with a multicentre retrospective study conducted in China by Guan W et al., [24]. According to it, 260 of the 560 enrolled patients amounting to 46.4% had elevated D-dimer levels (0.5 mg/L) and this rise in value was more pronounced among severe patients when compared to non-severe ones. They defined the degree of severity of COVID-19 as severe cases if patients admitted with any one of the following major criteria: septic shock with need for vasopressors; respiratory failure requiring mechanical ventilation or any three or more of the following minor criteria: respiratory rate >30 breaths/min; PaO2/FIO2 ratio <250; multilobar infiltrates; confusion/disorientation; uraemia (blood urea nitrogen level >20 mg/dL); leukopenia (white blood cell count, 4,000 cells/mL); thrombocytopenia (platelet count, 100,000/mL); hypothermia (core temperature, 36°C); Hypotension requiring aggressive fluid resuscitation [24]. Study by Tang N et al., in march 2020, showed that patients with severe disease had a 3.5 fold increase in D-dimer levels when compared to those without it along with higher mortality rates [7]. Similar findings were also reported by Yao Y et al., who observed a high level of serum D-dimer level in non-survivors when compared to the survivors [30].

A recent guidance report by International Society of Thrombosis and Haemostasis (ISTH) for recognition and management of coagulopathy in COVID-19 states markedly raised D-dimers levels, that is, three-four folds increase as a criteria for admission even though patient has no other severity symptoms [31]. We further established two groups in our study, one whose D-dimer value was above 500 ng/mL and another with values below 500 ng/mL. In our study, population, it was noted that 91.1% of the cases who had normal D-dimer values were asymptomatic. The chi-square test showed that it was statistically significant and the odds of a patient with high D-dimer levels turning out to be clinically symptomatic were 5.5 times more than that of the odds of a patient with normal D-dimer levels.

High D-dimer levels have been reported as one of the risk factors to predict occurrence of acute respiratory distress syndrome and also its progression to death in COVID-19 patients [32]. Wang D et al., noted that patients in need of admission to intensive care units had significantly higher D-dimer levels than those not in need of it [33]. Also, D-dimer levels show a sequential rise with time in non-survivors as against values in those who survived [6,33]. Zhang L et al., established a cut-off value of 2.0 μg/mL as an independent predictor for in-hospital death in patients [5].

Initiation of DIC in patients with sepsis is well-documented [34]. The pathophysiology behind this is considered to be endothelial dysfunction along with altered immune regulation [35]. Giannis et al., attributed this to endothelial dysfunction characterised by high levels of von Willebrand (vW) factor, a procoagulatory state due to activation of tissue factor pathway and toll-like receptor activation causing systemic inflammation in cases of coronavirus infections [36]. Increase in D-dimer levels in COVID-19 may be explained by excess production of thrombin and early shutdown of fibrinolytic pathway secondary to any infectious stimuli as noted in past studies [37]. Another possible explanation for this is hypoxia in severe cases of COVID-19 leading to thrombus formation via increased blood viscosity and hypoxia inducible transcription factor dependent signalling pathway [38]. Some suggest that high levels of circulating proinflammatory cytokines implicated in the cytokine storm noticed in severe cases of COVID-19 might lead to direct activation of coagulation cascade [29].

COVID-19 is a pandemic disease for which prevalence rate is not known. Due to the emerging need for the new classifying factors and new prognostic factors, data over the period of five months have been documented in this study. The current study was conducted at a single centre with limited sample size. Hence, further study may be needed to extrapolate the findings to wider patient population.

Elevated D-dimer levels associated with the severity of clinical course of patients infected with SARS CoV-2 when compared to patients with mild or asymptomatic clinical presentations. Hence, this reinforces the fact that D-dimer levels at the time of diagnosis helps to predict the clinical course of the disease and is a definite guide for early management of complications. The prevalence of COVID-19 is not clearly documented and our present knowledge confines to only patients with moderate and severe clinical presentation who reported to hospital for treatment and diagnosed as COVID positive. This enhanced level of D-dimer is a definite indicator of the pathogenetic mechanisms underlying severe clinical presentations and a subject matter of deeper analysis. The role of vW Factor in the increase of D-dimer levels in severely affected COVID-19 patients needs further study.