key: cord-0833485-zy31jx2a
authors: Wickramasinghe, Wasanthi; Alvitigala, Bhawani Yasassri; Perera, Thisarika; Karunanayake, Panduka; Jayasinghe, Saroj; Rajapakse, Senaka; Weeratunga, Praveen; Wijewickrama, Ananda; Arya, Roopen; Goerlinger, Klaus; Gooneratne, Lallindra Viranjan
title: Rotational thromboelastometry in critical phase of dengue infection: Association with bleeding
date: 2022-04-22
journal: Res Pract Thromb Haemost
DOI: 10.1002/rth2.12704
sha: 625fb8e8172797dbcfa74286999c1b90e977b90f
doc_id: 833485
cord_uid: zy31jx2a

BACKGROUND: The critical phase of dengue carries a high risk of bleeding. Associations of coagulation test parameters and the risk of bleeding in the critical phase is unclear. This study examines the association of rotational thromboelastometry (ROTEM delta and ROTEM platelet) with bleeding risk of patients with dengue in the critical phase. METHODS: A total of 105 patients with confirmed dengue in the critical phase were recruited, with two subsequent prospective time point analyses of ROTEM parameters and platelet count within 24 and 48 hours from the onset of the critical phase. Conventional coagulation tests were performed only at the initial time point. RESULTS: Twenty of 105 patients developed bleeding after onset of the critical phase. Within the first 24 hours of critical‐phase onset, platelet count, coagulation tests, and ROTEM delta were unable to differentiate patients with bleeding manifestations from those without (P < .05). Area under the curve of thrombin receptor activating peptide‐6 assay of ROTEM platelet (TRAPTEM) discriminated patients with bleeding manifestations from those without, at a cutoff value of <12.5 Ω*min at a sensitivity and specificity of 73.7%, and 60.2%. In patients who developed bleeding, the maximum lysis of extrinsic pathway of ROTEM was significantly lower in patients with severe bleeding compared to those with mild to moderate bleeding. (4.3 ± 3.4% vs 9.4 ± 7.5%; P = .01). CONCLUSION: An association with bleeding manifestations and TRAPTEM suggest a potential role for defective platelet aggregation in the pathogenesis of bleeding in the critical phase of dengue.

Dengue is the world's most prevalent mosquito-borne viral infection, transmitted by the Aedes aegypti mosquito. Dengue infection has increased exponentially worldwide and has an incidence of 100 to 400 million cases each year. 1 Sri Lanka is an endemic country for dengue, where 31 162 suspected dengue cases were reported in 2020, and 7833 cases from January to June 2021. 2 There are three defined phases in the natural history of dengue infection. In the febrile phase, patients present with nonspecific, constitutional symptoms and headache, backache, and general malaise. The critical phase of dengue infection is characterized by plasma leakage and carries the highest risk of hemorrhagic complications. Patients in the critical phase of dengue, with evidence of plasma leakage either ultrasonically or with a rise in the hematocrit from baseline with additional bleeding manifestations are classified as dengue hemorrhagic fever. Bleeding manifestations in patients with dengue hemorrhagic fever are heterogenous and range from mild mucocutaneous bleeding to more severe deep bleeding manifestations such as intracranial hemorrhage. 3 Bleeding in dengue is an important determinant of mortality. 4 Platelet dysfunction, thrombocytopenia, derangement of coagulation, and fibrinolysis have been observed in dengue fever and dengue hemorrhagic fever. 5 In addition to the platelet count, conventional coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and serum fibrinogen level are commonly used to investigate hemostatic abnormalities in patients with dengue viral infection.

However, several limitations of conventional coagulation tests such as the inability to determine fibrinolysis, clot stability, and hypercoagulability and to explain in vivo changes responsible for hemostatic dysfunction have made rotational thromboelastometry (ROTEM) delta and whole blood impedance aggregometry (ROTEM platelet) more widely used in the recent past. 6 8 Two studies performed on Brazilian and Sri Lankan patients with dengue fever have revealed that ROTEM viscoelastic testing has higher sensitivity than conventional coagulation tests in determining coagulation abnormalities in patients with dengue. 8, 9 However, these studies did not evaluate the correlation of ROTEM abnormalities with clinically observed bleeding manifestations in patient with dengue infection. Nor did they focus on abnormalities of these tests in patients in the critical phase of illness when the risk of major bleeding is highest. 10 It is currently unclear why some patients in the critical phase of dengue go on to manifest bleeding and progress to dengue hemorrhagic fever. It is also currently unclear if these bleeding manifestations are primarily driven by platelet dysfunction or by coagulation disturbance. Furthermore, there is a paucity of reliable clinical and laboratory predictors to determine the risk and severity of bleeding complications in patients in the critical phase of dengue. This is an important therapeutic need in dengue management with implications for triage, monitoring of patients, and early identification of bleeding complications to limit the morbidity and mortality of the disease. To this end, we evaluated the utility of ROTEM delta and ROTEM platelet as a tool to differentiate patients with bleeding manifestations from those without in the critical phase of dengue infection. | 3 of 10 WICKRAMASINGHE Et Al.

Patients with dengue fever admitted to the above hospitals within the first 3 days of symptom onset were serially followed up and recruited soon after they entered the critical phase of dengue, which is characterized by plasma leakage.

A diagnosis of dengue was identified by the presence of a suggestive clinical syndrome including fever, nausea, rash, arthralgia, myalgia, leukopenia, and warning signs such as abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy, restlessness, liver enlargement, and postural hypotension. 11 Informed and written consent was obtained from consecutive patients with suspected dengue infection. All patients who gave consent underwent dengue virus nonstructural protein 1 (NS1) and dengue IgM testing for confirmation of the diagnosis. A case of confirmed dengue was defined as a patient with a suggestive clinical syndrome with a positive dengue NS1 antigen test and/or positive dengue IgM antibody test using ELISA methodology. 11 Plasma leakage and the onset of the critical phase of illness was diagnosed by (i) a >15% rise in hematocrit or leakage identified by ultrasound scan (U/S) or X-ray or (2) a >20% rise in hematocrit with or without U/S or X-ray evidence of leakage. 12 All patients thus selected (n = 105) were monitored for evidence of bleeding, with the severity of bleeding categorized into moderate and severe based on the following criteria.

Patients with mild to moderate bleeding have either (i) no evidence of hemodynamic compromise with the need of intervention, (ii) bleeding at injection site, (iii) nose or gum bleeding, (iv) gastrointestinal tract bleeding without shock or hemodynamic instability with need of blood transfusion, (v) macroscopic hematuria, (vi) vaginal bleeding requiring hormonal therapy, or (vii) eye bleeding. 12 

Severe bleeding is any bleeding (i) at a critical organ, (ii) resulting in hemodynamic instability, (iii) resulting in death or disability, (iv) in need of transfusion, or (v) that persists after taking measures to stop bleeding. 12 Patients with a prior diagnosis of a bleeding disorder, liver disease, or chronic kidney disease were excluded. Furthermore, those with an estimated glomerular filtration rate (eGFR) <90 mL/min/1.73 m 2 F I G U R E 1 Process of recruiting patients and sample collection at two time points in the study. NS1, dengue virus nonstructural protein 1; ROTEM, rotational thromboelastometry or criteria fulfilling acute kidney injury 13 at baseline or at the stage of blood sampling for coagulation studies and ROTEM were excluded.

A flowchart demonstrating patient recruitment is presented in 

Analysis was performed with all 105 patients. No missing data were found. Descriptive data were reported as mean ± standard deviation and/or range. All data were statistically analyzed using SPSS 23 

Approval was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC-19-033).

The outcome of bleeding occurred in 20 patients (19%). Samples were drawn as described above, and this timing corresponded to 4 to 7 days from the onset of fever (mean, 5 ± 1 days). Bleeding occurred within 6 to 12 hours of the onset of the critical phase. The mean age of the population was 32.6 ± 12.5 years. No deaths were reported. The majority were men (70%). Elevated ALT and AST was seen in 96.9% and 89.6%, respectively. Each patient with bleeding had one or more Platelets × 10 9 /L (day 1) 32 ± 5 (9-98) 38 ± 2 (4-98) .26

Platelets × 10 9 /L (day 2) 28 ± 3 (5-62) 37.0 ± 3 (4-105)

.09

Note: Data are n (%) and mean ± standard deviation (range).

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase. P < .05; statistically significant in differentiating patients with bleeding manifestations from without.

TA B L E 1 Baseline characteristics and routine laboratory parameters in patients with and without bleeding manifestations bleeding manifestations: hematuria (n = 6), gum bleeding (n = 3), melena (n = 5), hematemesis (n = 1), and hemoptysis (n = 2), and eight patients who had no overt bleeding but red cell transfusion was done (Table S1) . Of all 20 patients with bleeding manifestations, 16 patients received red blood cell transfusions. No significant difference was observed in platelet count, hematocrit, AST, and ALT between patients with bleeding manifestations and patients without bleeding manifestations (Table 1) . No thrombotic complications were reported.

Increased PT, aPTT, and TT was observed in 2.8%, 27.6%, and 37.1%, respectively, and reduced fibrinogen in only 8.6% of patients ( 

We then sought to examine if there was any variation of the (Table S3 ).

TRAPTEM in the ROTEM platelet module measures platelet aggregation activated by the agonist thrombin receptor activating peptide-6. 7 Although ROTEM platelet parameters are affected by platelet count, 7 this effect was alleviated by our recruitment criteria, ensuring that platelet counts were uniform across the groups. Overall Table 2) . (see Table S4 for percentage derangement of ROTEM parameters between the patients with and without bleeding manifestations). and those without (see Table S5 ).

Patients with bleeding manifestations were categorized into (i) severe bleeding and (ii) mild to moderate bleeding, as described earlier.

Significant association of bleeding severity as defined above was seen only with the EXTEM ML (P = 0.01) ( Table 3) . (See Table S6 for the association of all ROTEM parameters with type of bleeding.)

NATEM is a more sensitive method in detecting true endogenous coagulopathy than EXTEM. NATEM does not use any activators except for recalcification while EXTEM is an activated method. 

To our knowledge, this is the first viscoelastometry and impedance aggregometry study done on patients who are in the critical phase of dengue comparing those with bleeding and those without. Note: Data are n (count) and mean (range).

Abbreviations: CT, clotting time; EXTEM, extrinsically activated ROTEM delta assay; MCF, maximum clot firmness; ML, maximum lysis; NATEM, nonactivated ROTEM assay.

*P < .05; statistically significant association.

The observation that bleeding occurs in patients with dengue fever with normal platelet counts also provides evidence that alteration in platelet function, activation, and aggregation is probably the key factor in the pathogenesis of bleeding in dengue. 20 Furthermore, we failed to demonstrate statistically significant 

Though this was an explorative study, we report a new finding, which is an association of platelet dysfunction and bleeding manifes- 

ROTEM delta parameters are more sensitive to changes in coagulation in patients with dengue in the critical phase with plasma leakage than conventional coagulation tests. However, they could not differentiate those with bleeding manifestations from those without bleeding manifestations in the initial part of the critical phase. The only ROTEM parameter that was able to discriminate those with bleeding manifestations from those without was TRAPTEM AUC on ROTEM platelet. This association suggests that platelet function may contribute to bleeding in patients with dengue with plasma leakage more than thrombocytopenia and coagulation derangements.

NATEM was more sensitive in detecting fibrinolysis than EXTEM, but there was no clear evidence suggesting that fibrinolysis has an impact on bleeding in these patients with thrombocytopenia. Future studies should focus on the cross talk between platelet function and the fibrinolytic system in both critical phase and febrile phase and include a larger number of patients with bleeding manifestations to provide more robust results.

The authors thank all the consultant physicians at National Hospital Sri Lanka for permitting this study to be carried out in their wards. A special thanks to the staff of the medical wards at National Hospital Sri Lanka for their support.

KG is the medical director of TEM Innovations, Munich, Germany, and supported the study with ROTEM reagents, ROTEM cuvettes, and hirudin blood sampling tubes free of charge. All other authors declared no competing interests.

KG conceptualized the study and provided resources.

LG was involved in project conceptualization and administration.

LG and PW were involved in funding acquisition. WW was involved in data collection, investigation, formal analysis, and methodology. TP collected data and investigated. PK and AW supervised the project. YA was involved in data curation and writing of the original draft. SJ, SR, RA, and all other authors were involved in reviewing and editing of the manuscript. All the authors viewed the manuscript and provided approval for submission of this article.

Relevant data will be made available on request. 

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