Evaluation of markers of thrombosis and brinolysis in malaria patients in tertiary care hospital in Central India

Background: Cases of malaria are known to be associated with variable degrees of coagulopathy as evident from abnormalities of screening coagulation assays in a large number of patients during the illness. However, attempts to use laboratory parameters as indicators of the severity of the underlying coagulopathy and for monitoring disease progression have met with only partial success. Methods: This prospective study was conducted in the department of Pathology in Ruxmaniben Deepchand Gardi Medical College, Ujjain. Blood samples of all the patients diagnosed positive with malaria were collected in vials containing ethylene tetra-acetic acid tri potassium salt for hematological parameters and in trisodium citrate tubes for coagulation assays. Result: Out of the total positive cases, PT was prolonged in 65% of total cases, Percentage positivity for prolonged PT for falciparum and vivax is 60% and 68.5% respectively. APTT has raised 47% of malaria positive cases with percentage positivity for falciparum and vivax 48% and 45.7% respectively. 55% of total cases showed raised levels of fibrin degradation products, which included 48% of falciparum cases and 60% of vivax cases. Out of 120 positive cases of malaria, D- Dimer was raised 41.7% cases. Conclusion: As blood is the chief component affected by malaria, not only proper examination of peripheral smears is required for detection and species identification, but a careful evaluation of other laboratory parameters should be done irrespective of the malarial species for the early diagnosis of compensatory and non-compensatory consumptive coagulopathy.


Introduction
William Osler has said that "Humanity" has but 3 great enemies; fever, famine, and war; of these by far the greatest, by far the most terrible is fever".Malaria is the most important protozoal parasitic disease of humans.The word malaria derives its origin from the Italian "mal-aria" meaning bad air [1].WHO estimated worldwide prevalence of malaria is about 300-500 million per annum.It is present in more than 100 countries around the world.Most of these are developing countries or underdeveloped countries.[2] Cases of malaria are known to be associated with variable degrees of coagulopathy as evident from abnormalities of screening coagulation assays in a large number of patients during the illness.This study aimed to see if the elevation in D-Dimer levels and fibrin degradation products act as a marker of thrombosis and fibrinolysis respectively.
Malaria affects almost all the organs of the body but the chief component affected is blood.Therefore, hematological changes play a major role in malaria pathology.The present study is an effort to correlate the laboratory changes in blood, and if they are the indicator of its clinical outcome.

Result
During the study period, 120 patients were diagnosed as malaria positive in the hospital, of which 70(58%) patients were of Plasmodium vivax (P.v) and 50 (42%) patients were of Plasmodium falciparum (P.f).Out of total cases, 72 (60%) patients were males whereas 48 (40%) patients were females i.e, more number of males were affected.
P.v was seen more in males whereas, P.f was seen more in females.Of all the cases 71.6% cases were observed in the age group of 0-40 years and 28.3% cases were observed in the later ages the frequency decreases with increasing age.In the age group of 0 to 20 years equal prevalence of vivax and falciparum was found, however with increasing age the prevalence of falciparum malaria decreased (Table 1).

Chouhan S. et al: Evaluation of markers of thrombosis and fibrinolysis
01.All proven patients with malaria admitted in our tertiary care hospital were included.
02.Both male and female patients of any age were included.
03. Cases with mixed malarial infection were also included in the study.
01. Cases of co-infection with malaria and dengue will be excluded from the study.
02. Pregnant women will be excluded.83% of the total study cases had thrombocytopenia with platelet counts < 1,50,000/cumm, 97% of P.
vivax and 64% patients of P. falciparum showed thrombocytopenia.
38 of total malaria positive patients, platelet counts ranged from 25,000 to <50,000/ The P value is 0.000683 which is significant (Figure1).

Fig-1: Degree of thrombocytopenia.
Out of the total positive cases, PT was prolonged in 65% of total cases, Percentage positivity for prolonged PT for P.f and P.v is 60% and 68.5% respectively.
APTT was raised in 47% of malaria positive cases (P value> 0.05, not significant) with percentage positivity for P.f and P.v 48% and 45.7% respectively.
24 (18 P.v and 6 P.f) of the total malaria cases had isolated prolongation of PT, however, only 2 cases of P.v showed isolated prolongation of APTT.This shows that the extrinsic pathway of coagulation is affected more in malaria as compared to the intrinsic pathway.
The common pathway is affected giving rise to both prolongations in PT and APTT in a significant number of 54 cases (Figure 2).In the present study, Platelets were raised in 70 cases (65%), and APTT was raised in 56 cases (47%).Our findings are comparable to the studies conducted by Rajniti Prasad et al [10], Dutta et al [11].A study conducted by Dutta et al [11]

Conclusion
There are many national like (National Vector Borne Disease Control Programme) implemented in India for the control of parasitic diseases like malaria.But, the inability to completely control and eliminate malaria, and it still exists as a burden, especially to economically reproductive ages.
What the study to the existing knowledge?
However, attempts to use laboratory parameters as indicators of the severity of the underlying coagulopathy and for monitoring disease progression have met with only partial success [3].Therefore, the authors wanted to revisit this subject in a prospective study involving a large number of malaria patients admitted at our tertiary care hospital in Ujjain and see if the platelet count is a good indicator of the underlying vasculopathy and of the hemorrhagic tendency and that normal screening coagulation assays are associated with the absence of hemorrhagic complications.
of the study: Department of Pathology in Ruxmaniben Deepchand Gardi Medical College, Ujjain.Duration and type of the study: The present prospective study was conducted over one and a half years i.e.January 2015 to July 2016.Ethical considerations and permission: Ethical considerations and permission were met through the Institutional ethical committee.A total of 120 patients were included in the study.Blood samples of all the patients diagnosed positive with malaria were collected in vials containing ethylene tetra-acetic acid tri potassium salt (K3EDTA) for hematological parameters and in trisodium citrate tubes (3.8%) for coagulation assays.Each blood sample underwent Peripheral blood smear examination, Complete blood counts, Prothrombin time (PT), International normalized ratio(INR), Activated partial thromboplastin time(APTT), Fibrin degradation product (FDP), and D-Dimer.A prestructured proforma was used to collect baseline data.

03 .
Patients with acute hepatitis, leptospirosis, salmonella, and those with other bacterial and viral infections; immuno-compromised individuals, e.g.AIDS and patients having other causes of coagulopathy will be excluded.

Fig- 2 :
Fig-2: Coagulation parameters in malaria cases.55% of total cases showed raised levels of fibrin degradation products, which included 48% of falciparum cases and 60% of vivax cases.Table 2 shows the relationship between different coagulation parameters in cases of malaria.Out of 120 positive cases of malaria, D-Dimer was raised in 50 (41.7%)cases which consisted of 45.8 % of the vivax and 35.7% of falciparum cases that were tested.The mean hemoglobin of study cases is 9.74 g/dl, which shows that malaria causes a mild degree of anemia, however, mean hemoglobin of 8.4 g/dl was observed in falciparum malaria cases.(The t-value is 3.63596, P-value is 0.000295, the result is significant) .The average MCV and MCHC remain at the lower limit of normal irrespective of the species of malaria.The average platelet count in malaria shows thrombocytopenia and was 88.9x 10^3/μl, but the average platelet count in patients with P.vivax was lower (64.9 x 10^3/μl) and in the patients with P. falciparum, it was 122.3x 10^3/ μl.(The t value is -2.31398,P-value is 0.012116.The result is significant at p<0.05) our results were not comparable to the study by Himanshu Shekar et al [6] where the incidence of thrombocytopenia was more in falciparum cases and may be due to the high endemicity of P. falciparum in the study population.Various screening tests were used as indicators of coagulation in malarial patients which included PT, APTT, FDP, and D-Dimer in the present study.Prothrombin time is the measure of the integrity of extrinsic and the final common pathway of coagulation.Whereas activated partial thromboplastin time is the indictor for intrinsic and the common pathway of coagulation.
Products of degradation of fibrin meshwork by the action of plasmin lead to the formation of crosslinked fibrin degradation products.D-Dimer is the smallest plasmin resistant unit of cross-linked fibrin degradation products.It comprises two D fragments which are linked together.Estimation of raised FDP and D-Dimer are the markers of thrombotic events such as DIC.The presence of D-Dimer represents the activation of coagulation cascade followed by activation of the fibrinolytic system resulting in the fatal complication of DIC in malaria.
As blood is the component affected by malaria, not only proper of peripheral smears is required for detection and species identification, but a careful evaluation of other laboratory parameters including platelet count, PT, APTT, FDP, and if required D-Dimer level estimation should be done irrespective of the malarial species for the early diagnosis of compensatory and non-compensatory consumptive coagulopathy and the life-threatening bleeding manifestations.

et al: Evaluation of markers of thrombosis and fibrinolysis Tropical Journal of Pathology and Microbiology 2020;6(8)
83% of cases in the present study suffered from thrombocytopenia.Studies conducted by Sharma et al [7] who showed thrombocytopenia in 90% of their cases, Himanshu Shekhar Das et al [6] reported thrombocytopenia in 68% of cases, and Kini Reshma G et al [5] in 96% of their cases.

et al: Evaluation of markers of thrombosis and fibrinolysis Tropical Journal of Pathology and Microbiology 2020;6(8)
Quantitative estimation of D-Dimer levels were found above the normal levels (>200ng/dl) in 41.7% in the present study.Chouhan S.