Association between D-dimer levels and post-acute sequelae of SARS-CoV-2 in patients from a tertiary care center

SARS-CoV-2 belongs to the Coronaviridae family is known to cause the respiratory disease, COVID-19. It is the third largest RNA virus in the family. This disease-causing virus was first observed in Wuhan, China, in 2019 and rapidly spread around the globe [1]. The first wave in India started in March 2020, peaked in September 2020 and reduced in February 2021. In that first wave, India did not record a high mortality rate as in other countries [2]. The second wave in India began in March 2021 and peaked in April 2021, with several concerns such as fewer hospital beds, an inability to provide treatment guidelines, a demand in essential requirements for treatment and mortality in young populations. The reason for increased mortality was mainly double and triple mutation in the virus strain, which become more pathogenic and resistant to the vaccines [3]. Unlike in the first wave, in the second wave the symptoms were varied and inflammatory markers such as C-reactive protein (CRP), D-dimer, lactate dehydrogenease and creatinine phosphokinase were significantly higher.

D-dimer is one of the important biomarkers to determine the severity of the patient admitted due to COVID-19. Normally, D-dimer levels in the blood will be <0.5 μg/ml and the value varies due to aging and pregnancy [4]. During infection, the coagulation pathway will be activated due to the immune response and the activation of thrombin also activates other pathways leading to the imbalance of procoagulant–anticoagulant, which results in various complications with increased levels of D-dimer [5]. In COVID-19 infection, the level of D-dimer increases rapidly and results in venous thromboembolism (VTE); in later stages, it leads to thromboinflammation. Thus, the D-dimer value is one of the important markers when adjusting the therapeutic dose of anticoagulant to reduce patient mortality [6]. Multiple randomized controlled trials were carried out to optimize the anticoagulant dose to lessen the complications of COVID-19, including the multiplatform adaptive-design trial (MPT), the INSPIRATION trial and the HEP-COVID trial. These three trials were performed to study the efficacy of prophylactic and intermediate doses of heparin in patients affected with varying degrees of COVID-19. The trials suggested use of a standard prophylactic dose of heparin in patients who did not have complaints or suspected or confirmed VTE to prevent the progression of COVID-19 disease or death [7].

In COVID-19 infection, acute lung injury was characterized by peripheral distribution of a crazy paving pattern and bilateral ground-glass opacities found on a CT scan. After 2–3 weeks of infection, the severity of the disease is increased, and this is mainly due to the involvement of D-dimer levels. Studies also showed that the D-dimer level was elevated in post-COVID patients after discharge from the hospital. Data from previous studies showed that 62% of patients still had lung abnormalities after 1 week of discharge and that 39% people were suffering from long COVID even after 4 weeks of infection [8]. The mechanism behind the elevation of the D-dimer value is still not clear, but studies showed that careful interpretation of abnormalities in coagulation and extended anticoagulant therapy was needed for these patients after discharge as well [9].

After recovering from COVID-19 infection, a high proportion of patients continue to suffer from fatigue, breathlessness, shortness of breath (SOB), chest pain and muscular pain, among others, for >6 months after infection, which is known as ‘long COVID’ or ‘post-acute sequelae of SARS-CoV-2’ (PASC). About 10% of the population remains unwell beyond 3 weeks after the onset of COVID-19 to longer than than 6 months after the onset [10]. A study conducted in the USA showed that only 65% of patients, including young patients, without any comorbidity returned to normal health in 21 days after the onset of disease [11]. The pathophysiological mechanism behind PASC is not clear, but studies suggest that activation of the coagulation pathway, fibrinolysis and pulmonary microvascular immunothrombosis during the onset of COVID infection may play a role in long COVID [9]. The D-dimer level in the blood is one of the markers for activation of the coagulation pathway, but only limited data has been available about the level of D-dimer in the post-COVID patient. Therefore, the present study aimed to estimate D-dimer levels among patients who continue to have a thrombotic consequence as long COVID manifestation and to correlate between D-dimer levels and severity of long COVID manifestations.

Methodology

This retrospective cross-sectional study was conducted over 10 months by the Department of Biochemistry at the Sree Balaji Medical College and Hospital (SBMCH) from March to December 2021. The data were retrieved from both medical and departmental records.

Subject characterization

A total of 206 patients admitted to the SBMCH intensive care unit (ICU) with real-time PCR-confirmed SARS-CoV-2 infection were enrolled in this study. The patients were asked to come back for a review 2 weeks after discharge.

Inclusion criteria

Patients with primary admission to the ICU with an oxygen requirement and patients referred from COVID-19 wards to the ICU when their requirement for oxygen increased and saturation could not be maintained in the ward were included in the study.

Exclusion criteria

Patients with comorbidities such as diabetes mellitus, hypertension, asthma, bronchitis, ischemic disorders and chronic kidney disease were excluded from the study.

Methodology

The investigation profiles of patients admitted to the ICU at SBMCH were retrieved from both medical and departmental records. Before admission, all patients had to have their D-dimer level checked and estimated periodically as a routine protocol of management. The D-dimer level was measured by an immunoturbidimetry method with the MINI VIDAS^® immunoanalyzer (bioMérieux). The test was carried out in the biochemistry department of a National Accreditation Board for Testing and Calibration Laboratories (NABL)-accredited laboratory. Apart from D-dimer, inflammatory markers such as CRP, serum ferritin and IL-6 were measured using nephelometry, radioimmunoassay and ELISA, respectively.

Patients reporting for follow-up after ICU discharge with long COVID symptoms were followed as per the guidelines and investigated at SBMCH 2 weeks after discharge, at 3 months and at 6 months.

Results

D-dimer elevation

In this study, 206 subjects (65.04% male and 34.95% female) were enrolled in the study. Among the 206 subjects, 9.23% were 70–85 years of age, 47.57% were 50–69 years and 43.2% were 14–49 years (Figure 1). The D-dimer level was checked before patients were admitted to the hospital with COVID-19, after discharge, after 3 months and after 6 months. On admission, patients showed elevated D-dimer values from 0.5 to 10 μg/ml. Among the 206 subjects, only 118 showed a normal D-dimer value (≤0.5 μg/l). Of 88 subjects, 46 showed an elevated D-dimer value from 0.5 to 1 μg/l, 31 showed an elevation from 1.1 to 3.8 μg/l and 11 showed an elevation from 4.7 to >10 μg/l. At discharge or 6 months post COVID-19 infection, the D-dimer was reduced only in minor units, but the difference was not statistically significant (Table 1).

Symptoms of patients

In the PASC duration, patients were observed with varying symptoms, including shortness of breath (SOB), fatiguability, allergy reactions, muscular cramps and vague chest pains, which were not observed in these patients before COVID-19 infection (Figure 2).

Inflammatory markers

Apart from D-dimer, inflammatory markers such as CRP, serum ferritin and IL-6 were measured. During admission, the inflammatory markers were elevated; however, at discharge and after 3 months, the inflammatory markers were not significantly elevated but were slowly approaching baseline values. Table 2 shows the mean of inflammatory markers within those time intervals (Table 2).

Discussion

The burden created by COVID-19 is enormous, and recovery to the normal state is challenging, even among young patients, and can extend for more than 21 days. A recent study conducted in China showed that 75% of the population surviving COVID-19 infection had any one determined symptom for >6 months [12]. D-dimers are widely used in the diagnosis of deep vein thrombosis, sickle cell anemia, pulmonary embolism and thrombosis of malignancy. In the blood coagulation process, D-dimer formation is mainly due to the activation of three enzymes, namely thrombin, activated factor VIII and plasmin. Once the coagulation process is activated, the thrombin converts the inactivated fibrinogen to an active fibrin. To this fibrin monomer, factor XIIIa covalently cross links and forms an insoluble fibrin gel. To this fibrin gel, plasmin will bind and cleave the fibrin at multiple places, which exposes the D-dimer antigen [13]. In healthy individuals, the conversion of fibrinogen to fibrin is quite limited; therefore, the D-dimer values are much lower. However, in elderly people, D-dimer values are higher than those in healthy individuals [14].

Before the onset of COVID-19 disease, D-dimer was not used as an essential biomarker for the progression of viral or bacterial disease. Due to the intense damage caused by SARS-CoV-2 leading to thromboinflammatory complications, D-dimer levels increased and thus D-dimer became an essential biomarker for the hospitalization of the patients [4]. Many studies have shown D-dimer elevation during COVID-19 infection, but only few data are available for the persistent D-dimer elevation 6 months following infection. The present study showed that D-dimer values were found to be very high even up to three or four follow-up sessions, and this correlated with persistent symptoms and signs of residual or continuing COVID-related complications such as respiratory difficulty, chest pain, palpitations, SOB, fatiguability, insomnia, body aches, myalgias and calf muscle pain, among others. Previous studies also showed the same pattern of symptoms in follow-up patients, and fatigue was yet another persistent symptom correlated with our study [15]. Studies performed in China revealed that nonsurvivors of COVID-19 pneumonia showed a significant increase in the D-dimer value when compared with that of survivors, which might be due to activation of the coagulation pathway and secondary hyperfibrinolysis conditions [16]. The possible mechanism for elevation of the D-dimer level may be due to activation of an inflammatory storm and release of proinflammatory cytokines such as IL-2, IL-7, G-CSF, IP-10, MCP-1, MIP-1A and TNF-α in plasma. This induces the endothelial dysfuction mechanism leading to microvascular system damage and activation of the coagulation system that increases the D-dimer value [17].

A previous study carried out in Dublin, Ireland, also showed elevation of D-dimer in 25.3% of patients after recovery at the range of 504–6726 ng/ml, and 8% of patients showed a markedly increased D-dimer level in their recovery phase that was double the value [9]. He et al. [17] also showed that a D-dimer value >2.025 mg/l is a critical value, and a value greater than this leads to patient mortality. A multicentric study in the UK showed that 30.1% of patients at discharge showed an elevated D-dimer value even after 60 days from discharge [18]. In our study, among the 200 patients who had earlier been admitted with severe COVID-19 and were followed afterward in the outpatient department with variable symptoms had significantly high D-dimer values, although slightly less than the values during admission. Other inflammatory markers such as serum ferritin, CRP and IL-6 were elevated at admission, but levels reduced by time of discharge, and after 3 months, levels were near to the normal range. Previous studies also showed the normalization of inflammatory markers despite elevation of D-dimer levels [9]. Similarly, patients who reported higher lung residual symptoms and signs had elevated D-dimer values compared with convalescing patients with less severe symptoms. Therefore, this study clearly suggests the role of thrombosis as the main pathology during and after COVID-19.

Conclusion

This study showed that normalization of the D-dimer value takes longer after COVID-19 infection. After discharge from the ICU, the D-dimer value remains the same for up to 3 months but reduces slowly by the 6-month follow-up. Patients have complaints such as SOB, extreme fatigue and vague chest pains. Continuous monitoring of the D-dimer value after discharge is essential during anticoagulant therapy. Persistence of long COVID symptoms has a direct correlation with persistent D-dimer values, suggesting an ongoing thromboinflammatory process despite discharge from an acute phase.

Future perspective

The study must be carried out in a large cohort to check the D-dimer values in PASC patients. Considering that an elevated D-dimer value is associated with severe disease such as VTE and sepsis, the recovered COVID-19 patient may succumb to other disease if follow-up is not managed properly. In severely affected COVID-19 patients, the D-dimer level should be checked for up to 6 months or even 1 year.