1. Introduction

In the last decade, the incidence of dengue viral infection has increased dramatically, and the disease is becoming more severe. Dengue viral infection can be fatal ^[1]. The World Health Organization has estimated 50 million cases of dengue fever and several hundred thousand cases of dengue hemorrhagic fever occur each year, depending on the epidemic activity. Some 1.8 billion of the population at risk for dengue worldwide ^[2]. Dengue virus is a mosquito-borne Flavivirus that is transmitted by mosquitoes such as Aedes aegypti or Aedes albopictus. Based on the antigenic difference, dengue virus (DENV) can be divided into four different serotypes, DENV 1 – 4 ^[3]. In the last years, Port Sudan faced many outbreaks. The dengue virus serotypes DENV1 and DENV2 were first reported in the 1986 in Port Sudan ^[4], while DENV3 was recently identified in an outbreak ^[5]. Since then, frequent outbreaks reappear in our studied area ^[6]. Mechanisms of bleeding in dengue infection are vasculopathy, thrombocytopenia, coagulopathy, and disseminated intravascular coagulation (DIC). The coagulation system appears to be abnormal during infection manifesting as decreased fibrinogen levels, increase levels of fibrin degradation products (FDP), prolonged partial thromboplastin time and prothrombin time, low levels of coagulation factors VIII and XII ⁷. In addition to the PTT prolongation, the fibrinolytic parameters such as tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in dengue patients are also increased ^{[8, 9]}. This study aimed to identify the impact of dengue infection on the PT and PTT coagulation tests.

2. Material and Method

This study was conducted prospectively for a period from February 2013 to June 2014 during the recent outbreak of dengue in Port Sudan teaching hospital, Red Sea State, Sudan. This study consisted of three hundred thirty four randomly selected patients positive with dengue infection. The inclusion criteria were all patients with clinical features and serologically positive dengue infection included. No hemostatic agents were administrated to the patients. The exclusion criteria were patients with serologically negative dengue, patients with underlying hemostatic disease, and if routine laboratory testing suggested a bacterial, parasite or any viral infection other than dengue infection or any other disease. Hundred and one, apparently healthy normal individuals with no any clinical sign for dengue infection were selected randomly to be the control group. Blood sample were collected from all of the studied population. About 3 ml blood was placed in potassium ethylene diamine tetra acetic acid (EDTA), and 3 ml in citrated buffer.

Patient characteristics of interest included: 1). Demographic: sex, age, residence, tribe, and occupation; 2). Mode of Presentation: presence of fever, joint pain, backache, headache, myalgia, retro-orbital pain and any bleeding episodes; 3) Hematological: Platelet count was done using automated hematology analyzer (Sysmex KX-21N, B 7151, and MF 9/2008 Japan); 4) Coagulation tests: PT and PTT were examined within 4 hours of collection using a semi-automated blood coagulation analyzer (bio bas-1 manufactured by RAL for SPINREACT, SN 536, Spain-European Community). Coagulation tests were determined by (Biomed diagnostic reagent, Germany).

Patients were classified as dengue fever, dengue hemorrhagic fever or dengue shock syndrome according to WHO guidelines and laboratory diagnosis of dengue was established by demonstration of IgM and IgG immune chromatographic Rapid strip test (BioTracer/BioFocus, REF: 17112, Exp.12/2015, Korea), sensitivity 95.6 and specificity 96.

Measurements of laboratory data coagulation tests of patients with DF, DHF were statistically tested by compare mean and Chi-square test which ever was appropriate. A P.value less than 0.05 were considered statistically significant. The Statistical Package for Social Sciences (SPSS 20.0 version, IBN. Chicago, USA) was used for data analysis. The sensitivity and specificity of relevant coagulation tests as predictors of dengue infection were also assessed by performing receptors operating curve (ROC) analysis.

This study was approved by the regional Ethical Review Committee (ERC) and written informed consent was obtained from all of the patients.

3. Results

This is a case control analytical study conducted in Port Sudan teaching hospital, Red Sea State, Sudan. The total number of the confirmed diagnosed dengue patients was 334. The age of the patients in this study was between 3 – 80 years (mean age 30 years). The control individual aged between 6 – 76 years (mean age 22 years). Of the 334 clinical patients, (217) 65% were males and (117) 35% were female. In control group, (64) 63.4% were males and (37) 36.6% were females. Table 1 shows the comparison of different characteristics between patients and controls. It shows that the eastern part of the study area (Selalab) represented the highest incidence (27.2%) region affected by dengue virus infection and the students were the most common segment of occupation affected (34.4%), followed by traders (19.5%), and the house wife (18.0%). Table 1 illustrates that the overwhelming majority of dengue virus infection is among the Northern Sudan tribe (43.1%), followed by the Hadandwa tribe (21%), Bani amer tribe (18.3%), western Sudan tribe (14.7%), and the immigrants tribe (3%). All of the Patients 334 presented was screened for PT and PTT. The normal and altered PT and PTT shows in (Table 2). Median PTT was 32.4" (mean ± SD 33.5 ± 9.7) (range 15.0" – 80.7") and median PT was 13.8" (mean ± SD 14.1 ± 2.1) (range 10.0" – 20.4"). PT and PTT was significantly higher in the DF (90%) and (72.7%) than DHF (10%) and (27.3%) of patients respectively. Prolongations of PT and PTT were observed in DF patients (P.value 0.000) (Table 2). Table 3 shows that prolonged PT and prolonged PTT are comparatively more sensitive, specific, and positive predictive value for DF than DHF (Figure 1).

Table 1. Characteristics of patients and control in the study

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Figure 1. Receptor Operating Characteristic (ROC) curve of PT (prothrombin time) and PTT (partial thromboplastin time) for dengue virus infection

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Table 2. PTT and PT results in comparison to DF/DHF

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Table 3. Statistical analysis of PT and PTT for diagnosis of Dengue fever (DF) and Dengue hemorrhagic fever (DHF)

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279 (83.5%) of the patients had thrombocytopenia. Thrombocytopenia was found more severe with bleeding individuals (P.value 0.000) (Table 4).

Table 4. Association of bleeding with thrombocytopenia

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4. Discussion

The exact causes that can complicate the dengue infection are plasma leakage due to increased capillary permeability, and hemostasis impairment ^[7]. Bleeding manifestations in dengue infection are highly variable and ranging from a positive tourniquet test, mild bleeding from the skin such as petechiae, ecchymoses, purpura, or hematoma at the site of injection, gum bleed, epistaxis, hematuria, to severe bleeding such as haemoptysis and gastrointestinal tract bleeding. Gastrointestinal bleed (Haematamesis) is the most frequent severe bleeding that lead to mortality. This study shows that 35 patients (10.5%) had bleeding. Patients with mild bleeding manifestations such as epistaxis (5/35:14.2%), hematuria (19/35:54.3%), and gum bleed (9/35:25.7%), patients with severe bleeding such as haematamesis and haemoptysis (1/35:2.9%). A study by Phuong et al who performed a prospective study of DHF in Vietnam 1998 with total 319 DHF cases showed regarding to bleeding manifestations similar results to this study. In that study 11% showed spontaneous bleeding ^[10]. Hemostasis defects are caused by multiple factors such as a vasculopathy, thrombopathy, and defects in the coagulation system ^[11]. Hemostasis is maintained by the balance between the coagulation and fibrinolysis systems. During dengue infection, there is imbalance between these systems. Abnormal PT/PTT can occur as a consequence of imbalance in the coagulation system ^[12]. Plasma PT and PTT are indicators of extrinsic and intrinsic coagulation system during the cascade. This study found that there was a significant relation between prolonged PT /PTT and dengue fever (P.value 0.000). PT prolonged was seen in (30/334:9%) of patients with or without hemorrhage, while PTT prolongation was observed in (42/334:12.6%) of patients.

A prolonged PT/PTT values during the dengue infection indicates that there is a defect in coagulation cascade pathway. Accordingly, our study hypothesized that the coagulation system activated; we attribute this to the presence of dengue virus. Recently, we found nonstructural protein-1 (NS1) of dengue virus can bind to both thrombin and prothrombin. The thrombin activity is not altered when NS1 bind to thrombin, the binding of NS1 to prothrombin can inhibit it is activation which may contribute to prolongation of PTT in dengue patients ^[13]. This may explain why PTT abnormality occurs within 1^st week of fever onset when antibodies are still under developed ^[14]. In addition, NS1 may also contribute to plasma leakage by mechanism without antibody involved. These results suggest that DENV secreted NS1 plays a direct and important role in vascular leakage and haemorrhage in DHF/DSS ^[15]. Regardless of the causes of the coagulopathy, our study showed that the coagulation activity may be impaired during dengue infection and that this disorder may associate with bleeding.

During the acute phase of the dengue viral infection, there is an increase in capillary permeability due to release of inflammatory mediators such as cytokines, Complement (C3a, C5a), tumor necrosis factor (TNF-α), interleukin (IL-2), IL-6, IL-10, interferon-α, and histamine. Thrombocytopenia and plasma leakage during dengue infection cause a decrease in fibrinogen and coagulation factors (factor II, V, VII, VIII, IX, X, and XII). In addition, liver cells can be damage by inflammation; this can be seen by an increase in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels. Damage of liver cells furthermore, decrease coagulation factors synthesis. AST/ALT may increases in dengue infection as well as the coagulation factors influenced due to low synthesis or high consumption; this will prolong PT/PTT during the acute stage of dengue infection ^[16]. Abnormal fibrinolysis can occur as a consequence of imbalance in the production of tissue-type plasminogen activator (tPA) and plasminogen activators inhibitors (PAI-1). Defect in coagulation and fibrinolysis system will appear as bleeding, intravascular thrombosis or both ^{[12, 16]}. If the dengue infection accompanied by bleeding complication and there is possibility of DIC, this indicate that there are defect in intrinsic and extrinsic cascade.

Previous study that showed similar results was reported by Krishnamurit et al who conducted a prospective study on the bleeding mechanism during DHF without shock. Their study found that prolonged PTT had significant relationship with the dengue infection ^[17]. In addition, a study by Lui et al in Taiwan also showed similar results; a prolonged PTT was found in more than 20% dengue cases ^[18]. Wills et al studied coagulation defects during DHF in 167 children diagnosed with DSS in Vietnam between July 1998 and February 1999. In that study found prolonged PT and PTT in DSS patients with bleeding ^[7]. Their results different from ours finding because all cases included in their study had DSS with DIC complication. Further studies are needed to confirm our finding and to know specifically which factors may be involved.

Prolonged PT and prolonged PTT are too late to be used as prognostic indicators. Furthermore, PT and PTT are not routinely investigated in many hospitals, limiting their use in prognostic indicators.

5. Conclusion

In conclude the finding of prolonged PT/PTT in patients with dengue infection proved that the coagulation was impaired. These parameters may be used to develop a future scoring system to predict and manage dengue infection severity early in the course of the illness.

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