DETERMINATION OF HEMATOLOGICAL PARAMETERS AND D-DIMER AMONG SUDANESE CHILDREN INFECTED WITH PLASMODIUM FALCIPARUM IN KHARTOUM STATE – SUDAN

Background: Falciparum malaria is one of the causes of morbidity and mortality in third world , the pathogenesis of the infection results from the sequestration of infected hematological change in vital organs. Aim: this study aim to measuring hematological changes and Ddimer among children affected with P falciparum. Methods: The total study group100 children, 60 as case study infected with Plasmodium Falciparum and 40 as control group healthy children. Hematological parameter measuring by hematological analyzer and D dimer measured by fluorescence Immunoassay used I. Chroma instrument. Results: Statistical analysis results of sixty children infected with malaria falciparum participated in the study the mean±SD had a significantly lower Hb (8.14gm/dl ± 2.2) and haematocrit (24.4% ± 6.6) than control children (p <0.001), Thrombocytopenia was found in 59.3% of enrolled patients. Platelet count (98.53% ± 48.9), D. dimer (7397.58 ng/ml ± 5867.07) (p <0.001). D. dimer showed correlation with parasite density, Platelet count and Haematocrit correlated (r = 0.4, p <0.0001); (p <0.001). Conclusion: Malaria infection by plasmodium falciparum had major effects on hematological parameters such as thrombocytopenia anemia and hyper coagulation have been significantly associated with severity of malaria falciparum.

hyper endemic countries, where mild symptoms of chronic malaria my possibly lead to misdiagnosis.On the contrary, over-diagnosis may also occur.In fact, not all reported malaria cases are confirmed by microscopy or others assay, such as rapid diagnostic tests (RDTs).Furthermore, in hyper endemic areas febrile illnesses from different causes might be misdiagnosed with malaria (2).Plasmodium vivax has unique attributes to support its survival in varying ecologies and climates and very different from P. falciparum., and other related simian species have identical biology, Plasmodium vivax can be grown in non-human primates (NHP), and in short-term ex vivo cultures.(3) Current estimates suggest that approximately 2.4 billion people are at risk of stable or unstable Plasmodium falciparum transmission (2) with 350 to 500 million clinical episodes and 1 million deaths annually (4).Transmission from an infected human host to a susceptible mosquito is mediated through highly specialized sexual-stage parasites, i.e., gametocytes.The gametocytes of P. falciparum hold a prominent place in the history of malaria, in that it was the exflagellating male gametocyte that first led Laveran to describe malaria in the late 19th century, exflagellation is a highly active process whereby motile male microgametes free themselves from the red blood cell in order to locate and fertilize a female macrogamete.Also, on routine microscope blood films, the gametocytes of P. falciparum, with their unique crescent shape, are prominent.Transitioning from the relatively protected and stable environment within the red blood cell of the human host to being an exposed parasite in the lumen of a mosquito mid-gut obviously requires considerably different characteristics (5-6-7-8).Hematological changes are some of the most common complications in malaria and they play a major role in malaria pathology.(9), these changes involve major cell lines including red blood cells (RBC), leukocytes and thrombocytes.Hematological changes in the course of a malaria infection, such as anemia, thrombocytopenia and leukocytosis or leucopoenia are well recognized.These alterations vary with the level of malarial endemicity, background hemoglobinopathy, nutritional status, demographic factors, and also malaria immunity (10-11-12).In malaria-infected patients, especially non-immunes children, prompt and accurate diagnosis is key to effective disease management for a favorable outcome.Clinical diagnosis is widely used for diagnosis of malaria especially in resource-poor countries (13)(14).

Material and method
This Study was designed as a case-control study, carried out in the Ahmed Gasum Pediatric Hospital, in Khartoum State, Sudan.Total study one hundred (100) Sudanese children , 60 of them were infected plasmodium falciparum as case study and 40 were healthy children used as control group, whole blood samples were withdrawn under hygienic conditions, whole blood samples collected in containers with ethylene diamin tetra acetic acid (EDTA) for complete blood count (CBC),which conducted via hematology analyzer KX21, and tri-sodium citrate container for determination of D-dimer level which conducted via i-chroma device which works with fluorescence Immunoassay techniques .Thin and thick blood films stained by Giemsa for each child performed for confirmation and identification of malaria infection under microscopic examination.Thin smears were used for species identification.All blood slides were read by two independent microscopists with discrepancies resolved by a third microscopist.Parasite density was defined as number of Plasmodium parasites per μL of blood, counted against 200 leukocytes assuming a leukocyte count of 8000/μL of blood.If fewer than 10 asexual parasites were detected in the first 200 leukocytes, counting was continued against 500 leukocytes.

Ethical considerations
This study approved by ethical committee of Alzaeim Al Azhari University.Each participant parent signed consent and written forms before undertaking any study-related activities

Statistical analysis:
Data analysis conducted by the statistical package of social science (SPSS) software version 21.Data are expressed as the mean ± standard error of the mean (SEM) unless otherwise stated.All data were compared using analysis of variance (ANOVA) , P values of ≤0.05 were considered statistically significant.How plasmodium falciparum affect coagulation, Hb, PCV and platelet count as well.Considering formation of D dimer and its effect on Hb concentration PCV and platelet count, a negative correlation was found for each, meaning decline of Hb concentration, PCV % and platelet count paralleled with increasing D dimer formation and significant difference was obtained as p value was < 0.001 for Hb, PCV and platelet count as in figures (1 , 2 , 3) respectively.

Discussion
Changes in hematological parameters are likely to be influenced by any disease condition which affects the hemopoetic physiology at any level.This is expected to happen with an endemic disease such as malaria that affects the host homeostasis at various fronts resulting in a many of clinical presentation.Malaria is a major cause of morbidity in the tropical countries.Two hundred and forty seven million cases were reported worldwide in 2006 (15).Hematological changes are some of the most common complications in malaria and they play a major role in malaria pathology.These changes involve the major cell lines such as red blood cells, leucocytes and thrombocytes.In malariainfected patients, especially non-immunes children, early and accurate diagnosis is key to effective disease management for a favorable outcome.Clinical diagnosis is widely used for diagnosis of malaria especially in resource-poor countries.Although fever and other signs and symptoms are known to be fairly sensitive measures of malaria they lack specificity and positive predictive values especially in areas where malaria is less prevalent (10)(11)(12)(13).In this study infected children results in ineffective erythropoiesis and hematological parameters change like Hb concentration, PCV, platelet count and D dimer.All parameters gave significant difference when compared with healthy children; this agrees with a study conducted in in children living in Western Kenya, severe anemia is the predominant severe malaria syndrome peaking in the first two years of life and is attributed to Plasmodium falciparum.(9) Children living in malarial endemic areas repeated attacks of malaria causes of severe anemia due to the red cell destruction and reduced red cell production led to hemolysis and phagocytosis or rupture of infected cells, removal of uninfected cells due to antibody sensitization or other physicochemical membrane changes, and increased reticuloendothelial activity, particularly in organs such as the spleen.Decreased production results from marrow hypoplasia seen in acute infections, and dyserythropoiesis, a morphological appearance, which in functional terms results in ineffective erythropoiesis.(16) The removal of non-parasitized RBCs (nEs) is thought to be the most important, accounting for approximately 90% of the reduction in hematocrit in acute malaria.(17) In previous study had similar results of hematological parameters changes among infected group were compared with those of the controls using t-test.Median values for Hemoglobin and Platelet count were significantly lower for the malaria group compared with the controls (18).As well as data obtained from study in Thailand-Myanmar border, it concerned about platelet count which found low among infected children by 31.8 times (19), indicating agreement with our finding as platelet count was low than healthy subjects count with significant difference and also the same finding data in Ethiopian study targeted Plasmodium falciparum infected subjects (20).
Previous studies study the Mechanisms for thrombocytopenia is dissimilar in malaria species infection, thrombocytopenia in vivax malaria presence to be immune mediated due to nonappearance of blood coagulation activation, while thrombocytopenia in falciparum malaria is most often accompanied by activation of the coagulation cascade among other mechanisms.(21,22) Elevated the D-dimer result may specify the presence of an abnormally high level of fibrin degradation product, an elevated D-dimer does not always indicate the presence of a clot because a number of other factors can cause an increased level, infection P. falciparum malaria infection influences blood coagulation by various interacting pathobiological mechanisms, most important being the cytokine storm, disseminated intravascular coagulation (DIC) and very elevated D-dimer level.The D-dimer in this study showed significant difference with infection of plasmodium falciparum, and increased level of D dimer among case group children, this agreement with a couple of studies concerned about many parameters included D dimer, both bring suggestion that hyper coagulation can be present even parasite density low.(23)(24).Increased fibrinolytic activity and coagulopathy in the malaria patients was reflected by significantly elevated levels of fibrinogen and D-dimer.In non-endemic countries low incidence of P. falciparum malaria infection, diagnosis and treatment are often delayed, potentially leading to complications with an increased mortality due to the lack of physicians experience dealing with the malaria disease.( 25)

Conclusion
Hematological changes are some of the most common complications in malaria and they play a major role in malaria pathology Increase the rate of severity with plasmodium falciparum associate with a variety of hematological complication such as severe anemia, thrombocytopenia, DIC Disseminated intravascular coagulation .
Thrombocytopenia can be taken as a valid marker of development of hypercoagulation dysfunction, irrespective of the severity of the disease.
Complete Blood Count (CBC) and D dimer test can be utilized as an important tool for early detection the complication and management in malaria patients.

Figure 1 :
Figure 1: correlation of D dimer and Hb concentration in infected children with malaria.

Figure 2 :
Figure 2: correlation of D dimer and PCV in infected children with malaria.

Figure 3 :
Figure 3: correlations of D dimer and platelet count in infected children with malaria.

Table 1 : independent T test for case and control group for hematological parameters and D. dimer (Mean ±SD).
were infected with plasmodium falciparum malaria were enrolled in this study, their mean± SD of age and weight were 17.26+15.41months and 8.98+3.89Kg respectively.And 40 subjects were healthy children, set as control group, their mean ± SD of age and weight were 18.00+5.91months and 9.44+3.89Kg respectively.