Diagnostic and prognostic value of thrombocytopenia severity in Sudanese children with Falciparum malaria

Malaria remains one of the most significant global public health challenges, with more than 200 million clinical cases worldwide each year. Falciparum malaria accounting for up to 87.6 % of malaria cases in Sudan. Platelets abnormalities as thrombocytopenia especially severe thrombocytopenia have been associated with mortality in patients with P. falciparum infection. The aim of this study was to study the relationships between thrombocytopenia and their severity with falciparum malaria severity, parasitemia and parasite count. This study was case-control hospital based study conducted among 200 Sudanese children admitted to Wad Medani Pediatric Hospital, Wad Medani, Sudan during period September to December 2018. The study population was divided into two groups. Group 1 (UM) was 100 children with uncomplicated falciparum malaria. Group 2 (SM) was 100 children with severe falciparum malaria were diagnosed by WHO criteria. A 2.5 ml venous blood sample was collected from each children. The malaria parasitemia was determined from thick blood films using plus system. The parasite count (% of parasitized red cells counting) was counted from thin blood film. Platelets count was determined using the Sysmex XP 300 N automated hematology analyzer (Sysmex, Kobe, Japan) and confirmed and assessed using stained thin blood film. The data were analyzed using SPSS software (V 20.0) and Stat disk software (V 13.0). The mean age and male: female ratio of Group 1 (UM) were 8.83 ± 4.20 years and 1:1.22 respectively and for Group 2 (SM) were 8.63 ± 3.40 years and 1.56:1 respectively. Group 2 (61 %) associated with thrombocytopenia (mean PLTs 160.91 ± 186.24 × 109/) more than Group 1 (25 %) (mean PLTs 221.10 ± 98.69 × 109/L) (P value = 0.000). The mild, moderate and severe thrombocytopenia account for 19 %, 5 % and 1 % respectively among Group 1; and 22 %, 28 % and 11 % respectively among Group 2 (P value 0.003). Malaria thrombocytopenia was significantly associated (P value = 0.000) and negatively correlated (P value = 0.000; r = 0.341) with malaria parasitemia. The mean parasite count in malaria thrombocytopenia (0.77 ± 0.51 %) was higher than malaria without thrombocytopenia (0.53 ± 0.36 %) (P value 0.000). Thrombocytopenia severity was significantly positive correlated with malaria parasitemia (r = 258; P value = 0.017) and parasite count (r = 0.229; P value = 0.034). The study concluded that the severe thrombocytopenia associated commonly with severe falciparum malaria and falciparum malaria hyperparasitemia.


Introduction
Falciparum malaria is the most virulent and pathogenic form of malaria (1), it is stills an important threat to public health in sub-Saharan Africa, and outside of Africa, particularly in young children, pregnant women and non-immune adults in communities with poor resources (2,3). Falciparum malaria accounting for up to 80% of malaria cases globally (WHO, 2018) and 87.6% in Sudan (4,5).
Falciparum malaria associated with several hematological changes that involve the major blood cell lines such as red blood cells, white blood cells and platelets (6,7), which play a significant role in severity of falciparum malaria (8,9). Malaria hematological changes arising from hemolysis, host immune (inflammatory) response, bone marrow suppression, and splenic pooling (10).
Thrombocytopenia (platelet count less than 150 x 10 9 /L) is common platelets abnormalities and hematological changes (7,11) as well as a common feature of malaria due to all plasmodium species particularly in falciparum malaria (occurs in up to 70% of falciparum malaria patients) (11,12). Platelet survival is reduced to 2-4 days in severe falciparum malaria (7,13). The pathophysiology of malaria associated thrombocytopenia and reduced platelet survival rate are multifactorial. It has been associated with a variety of hematological insults arising from hemolysis, host inflammatory response, hematopoietic suppression, enhanced splenic uptake or sequestration and DIC (platelets may be removed from the circulation at sites of fibrin deposition) (3,13). Some but not all studies have shown that there is strong association between thrombocytopenia and severity of malaria (3,7,14). Severity and mortality of patients with falciparum malaria are increased with severe thrombocytopenia (3).
The present study aimed to study the relationships between thrombocytopenia and their severity with falciparum malaria severity, parasitemia and parasite count among Sudanese children.

Methods
This is study was case control hospital based study, conducted in Wad Medani Pediatric Hospital in collaboration with Faculty of Medical Laboratory Sciences, University of Gezira, Wad Medani, Sudan as part of a wider research project studying the association between TNF-α levels and TNF-α 238 alleles polymorphisms and falciparum malaria anemia. The study population was divided into two groups. Group 1 (UM) was 100 subjects previously diagnosed as uncomplicated falciparum malaria by blood film or ICT. Group 2 (SM) was 100 subjects previously diagnosed as severe falciparum malaria by blood film and WHO criteria (18).
All study procedures were approved by the Ethics Committees of Ministry of Health, Gezira State and Faculty of Medical Laboratory Sciences, University of Gezira, Sudan. Informed consent was written from each participant parents. A 2.5 ml venous blood samples were collected by clean venipuncture in K3EDTA containers. Thin and thick films were prepared immediately. Malaria parasitemia was determined from thick blood films using plus system (19). Parasite count (% of parasitized red cells counting) was counted from thin blood film (18). Platelets count was determined using the Sysmex XP 300 N automated hematology analyzer (Sysmex, Kobe, Japan). The platelets count was confirmed and assessed using stained thin blood film. Thrombocytopenia was defined as a platelets count of less than 150 × 10 9 /L (20). Mild thrombocytopenia was defined as a platelets count between 100 and 150 × 10 9 /μl, moderate thrombocytopenia was defined as between 50 and 99 × 10 9 /μl and severe thrombocytopenia was defined as below 50 × 10 9 /μl (20). The data were analyzed using SPSS software (V 20.0) and Stat disk software (V 13.0)..

Figure 1
Association between malaria severity and malaria thrombocytopenia (P value = 0.000).

Discussion
Falciparum malaria is still a major health problem in Sudan accounts for up to 80% of malaria cases globally (21) and about 87.6% of malaria cases in Sudan (4,5). Poor sanitation and absence of majors protective is significantly leading to increased prevalence of the disease. Children suffer more malaria episodes and are more prone to severe malaria compared to adults and accounted for 61% (266 000) of all malaria deaths. In fact, about 285,000 children died before their fifth birthdays in 2016 in Africa According to the World Health Organization (21). therefore malaria remains the largest cause of children death in Africa (22). The association of thrombocytopenia (Low Platelets count) with all plasmodium malaria types generally and falciparum malaria particularly is an equivocal. The current study aimed to throw a light on thrombocytopenia severity association with malaria severity, parasitemia and parasite count among Sudanese children.
In the present study male: female ratio was higher in Group 2 (1.56: 1) than Group 1 (1: 1.122) (P value = 0.023). Furthermore 81 (40.5%) patients from Urban; while 119 (59.5%) patients from rural (P value = 0.002). Similarly, survey was done in Sudan in 21,988 individuals to show the prevalence of malaria and results showed infection was higher in male more than female, and also higher in ruler area compare to urban (26).
Thrombocytopenia had strong negative correlation with parasitemia (r -0.335, P value 0.000), and parasite count (r -0.268, P value 0.000). Previous studies concluded that the thrombocytopenia is associated with peripheral parasitemia levels (14,30,32,(34)(35)(36). The average of platelets count among Group 2 was lower than Group 2 (P value 0.000). The study done in Kenya showed the platelets count were significantly lower in malaria-infected children (30).
Children with thrombocytopenia were 24.4 times (odds ratio) more likely to have falciparum malaria. Maina et al., reported that children with platelets count of <150,000/uL were 13.8 times (odds ratio) more likely to have malaria. Similar previous study concluded that Individuals with platelet counts < 150,000/μL are 12-15 times more likely to have malaria infection than persons with platelet counts > 150,000/μL. The risk of thrombocytopenia was 36.0% higher for SM (2.44 times) compared to UM, and 38.9% higher for hyperparasitemia (3.08 times) compared to non hyperparasitemia. The risk of severe thrombocytopenia is 14.0% higher for SM (4.50 times) compared to UM, and 16.2% higher for hyperparasitemia compared to non hyperparasitemia. Previous study demonstrated that malaria parasite has a direct lytic effect on the platelets (33).
Thrombocytopenia is a frequent finding in falciparum malaria and possible causes are due to decreased platelets production, increased levels of cytokines and immunological destruction due to antibody and cellular immune responses, Oxidative stress damage of platelets, removal of platelets from circulation through enhanced splenic uptake and sequestration or by of fibrin deposition. In addition may result from antibodies, produced against the antimalarial drugs quinine and quinidine, interacting with platelets (7,13,15).

Conclusion
The study concluded that the thrombocytopenia associated commonly with severe falciparum malaria and falciparum malaria hyperparasitemia particularly severe thrombocytopenia (PLTs > 50 × 10 9 /μl ). So thrombocytopenia and their severity may help to assess the disease severity and to improve the management of falciparum malaria among patients.