Malaria is a mosquito-borne infectious disease caused by parasitic protozoans of the genus Plasmodium [1]. The parasites causing malaria are members of the Haemosporidiae. These parasites of the genus Plasmodium are transmitted to human beings by a pre-infected female anopheles mosquito bite. Four species are generally recognized in man:

(1) P. falciparum (the malignant tertian malaria parasite)

(2) P. vivax (the benign tertian malaria parasite)

(3) P. ovale (the benign tertian malaria parasite)

(4) P. malariae (the quartan malaria parasite). P.falciparum produces the most serious form of the disease and is responsible for most malaria-related deaths [2].

A variety of hematological alterations like progressively increasing anemia, thrombocytopenia, leucopenia occur in malaria. The derangement in the coagulation profile in malaria is a highly sensitive measure to access the severity and activity of the disease process. There is an accelerated coagulation cascade activity with accelerated fibrinogen turnover, consumption of antithrombin-III (AT-III), and increased concentration of fibrinogen degradation products.

Erythrocytes (RBCs) containing parasites are pro-coagulant. Released cytokines also act as pro-coagulants. Prothrombin time (PT) and activated partial PT (aPTT) is prolonged. Severe hemorrhage is reported in 5% of severe malaria [2]. The patient may develop bleeding gums, epistaxis, petechiae, subconjunctival hemorrhages, melena, and hematemesis [3].

1. To evaluate coagulation abnormalities like- prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen degradation product (D-dimer) in patients of malaria.
2. To study the difference in coagulation parameters between malaria patients and healthy controls and to determine the level of significance of the difference.
3. To understand the severity of coagulation defects and their related complications in patients with malaria.

1. For Hemoglobin, Total WBC count and Platelet count: 2 ml of blood was collected in EDTA vacuttee
2. For PT, aPTT, and D-dimer: 2 ml of venous blood was collected in 3.2% citrate

Processing of sample for coagulation studies:

Samples of the patients were immediately transported to the laboratory and were centrifuged at 3000-4000 revolutions for 15-30 minutes to obtain platelet-poor plasma (<10,000/cumm). Platelet poor plasma was used in PT, aPTT, and D-dimer. Samples were tested within 6 hours of collection of blood samples.

Investigations:

1. Peripheral smear: The clinical diagnosis of malaria was confirmed by peripheral blood smear examination which is the gold standard for the diagnosis of

For microscopic examination, peripheral blood thin and thick films were made on different slides (as per the method described in Practical Hematology- Dacie and Lewis) and stained with field’s stain. Grading of parasitemia (by semi-quantitative scale) was done from thick film examination.

2. Hematological examination: Complete hemogram with CELLDYN Ruby (Abbott) and Abbott CELLDYN 3700. Hemoglobin with less than 12.5 mg/dl was taken altered. Leucopenia with total white blood cell count <4000/cumm was taken altered. Thrombocytopenia with platelet count <1.5 lakh/cumm were taken

Anemia was observed in 55% of all patients, Leukopenia was observed in 22% of patients, and thrombocytopenia was seen in 85.7% of patients who presented with malaria.

Table-2: Changes in coagulation profile in malaria.

PT was prolonged in 41.4% and 43.6% of P. falciparum and P. vivax patients respectively.

Similarly, APTT was prolonged in 31.7% and 33.5% of P. falciparum and P. vivax patients respectively.

D-Dimer was elevated in only 1.7% of all malaria patients.

Table-3: Comparison of coagulation profile between 300 patients with malaria and 300 healthy individuals (controls) were as follows.

Data was presented as Mean±SD, unpaired t-test was applied.

P-value of <0.05 was considered a statistically significant difference.

Thus, there was a statistically significant decrease in platelet count as well as an increase in PT and aPTT in patients with malaria as compared to controls.

Table-4: Comparison of coagulation profile between P. Falciparum and P. Vivax malaria were as follows.

Data was presented as Mean±SD, unpaired t-test was applied. P-value of <0.05 was considered a statistically significant difference.

• Oxidative Stress:

Oxidative stress may, through lipid peroxidation, cause premature platelet death, leading to malaria-associated thrombocytopenia.

• Platelet dysfunction:

During acute, P. falciparum and P. vivax infection, hyper aggregation and enhanced platelet secretory activity are demonstrated. This in vitro study suggested that the interaction between normal platelets and falciparum-infected erythrocytes could induce hypersensitivity of platelets, possibly through the stimulation of ADP released from infected red cells. Antibody bound to platelets as well as the invasion of platelets by malarial parasites may be other response mechanisms.

The other aspect of platelet dysfunction during malarial infection observed in some patients is the defective aggregation of platelets in response to ADP, epinephrine, and collagen and not ristocetin. From electron microscopic study, circulating degranulated platelets were observed during malarial infection. The presentation of the circulating exhausted platelets as a result of persistent in vivo activation is most likely a responsible mechanism causing the platelet hypoactivity [4].

2. Coagulation activation

During severe complicated malarial infection, the activation of the coagulation system leading to in vivo thrombin generation has been demonstrated. The stimulation of the coagulation system is caused by various procoagulants present during malarial infection. The sources of the procoagulants are exposed phosphatidylserine on the cell surface of infected erythrocytes, the lysis of activated platelets together with their secretory products, and the tissue factor (TF) released from damaged vascular endothelial cells. ⁽⁴⁾

Furthermore, certain substances that are released during a severe malarial infection -namely tumor necrosis factor α (TNF α) and histamine - are additional factors that promote fibrin formation. The intrinsic pathway of the coagulation has also been shown to be activated in severe malaria. In turn, this may cause activation of the complement system and release of bradykinin and PMN-derived elastase that could contribute to the pathogenesis of severe malaria. Activation of the coagulation cascade also occurs in mild malaria.

• Endothelial cell activation

The mechanisms involved in vascular endothelial cell damage in severe complicated malaria are multifactorial. A significant rise in plasma levels of both von Willebrand factor (vWF) and its propeptide, indices of chronic and acute endothelial cell perturbation, respectively. The increased levels indicate endothelial damage by the parasitized erythrocytes [7].

In uncomplicated and severe malaria, a coagulation disorder is a common laboratory finding, but bleeding and hemorrhage are observed in very few cases of severe malaria. Therefore, an important distinction should be made between these two pathologic states, one where typical DIC is encountered(e.g., bleeding), and the other where a compensated state (e.g., laboratory changes only) is detectable. DIC is characterized by activation of the coagulation cascade, which leads to the formation of microthrombi in the microcirculation, sometimes localized to a specific organ, but it often presents with an uneven distribution. Two major mechanisms trigger DIC: the release of TF into the circulation or endothelial injury.

While acute DIC is the terminal phase of the coagulation disorder, it is often preceded by a period during which the coagulation cascade is already activated but the increased activation can be compensated by the natural inhibitor systems, a state referred to as compensated DIC. The control mechanisms may effectively prevent severe clinical manifestations such as bleeding and hemorrhage by neutralizing active enzymes and/or by increasing the synthesis of the consumed hemostatic components.

As the trigger for coagulation activation persists in DIC, inhibitors will be gradually exhausted, leading to more coagulation. In this process, many clotting factors—like, fibrinogen and platelets—are consumed, resulting in eventually incomplete impairment of the hemostasis system. This is why the term ‘consumptive coagulopathy’ is often used to denote this process. This results in bleeding tendency or decompensated DIC.

Also, activation of the coagulation cascade is accompanied by compensatory fibrinolysis where an increase in plasminogen-dependent plasmin activity is detected using markers such as D-dimers, as shown in the above table. It also depicts the laboratory profile of acute and ‘compensated’ DIC [8].