Effects of Malaria Parasite Density on Blood Cell Parameters in Sudanese Patients with Malaria

Malaria is a potentially fatal illness caused by parasites that humans contract through the bites of female Anopheles mosquitoes carrying the infection [1]. From very low or nonexistent signs to severe illness and even death, the infection can cause a wide range of symptoms [1,2]. Human malaria is caused by five parasite types, with Plasmodium falciparum and Plasmodium vivax being the most dangerous [1]. Five WHO areas have malaria infections, and 3.4 billion people worldwide, spread across 91 nations and territories, are at risk of contracting the disease, with an additional 1.1 billion at high risk [3]. Globally, there were an anticipated 247 million malaria cases and 619,000 fatalities in 2021 [1]. Due to inadequate sanitation, malaria is endemic primarily in tropical regions and developing nations [4]. An estimated 3.5 million malaria cases are recorded annually in Ghana [5]. Blood cell count alterations are a recognized characteristic of malaria infections. These alterations affect key cell types, including Red Blood Cells (RBCs), leukocytes, and platelets (also known as thrombocytes). Notable hematological changes associated with malaria infection, including anemia, thrombocytopenia, leukocytosis, and leukopenia, are well-documented. The extent of these changes depends on various factors, including the level of malaria endemicity, existing hemoglobinopathies, nutritional status, demographic variables, and malaria immunity [6-8]. For over twenty years, the World Health Organization (WHO) has recognized hyper parasitemia as a criterion for severe falciparum malaria [9]. Prior research has shown a correlation between the severity of malaria and parasite density [10,11]. The level of parasitemia is also linked to mortality, with death rates significantly higher among patients who have the highest parasite concentrations [12].

Furthermore, anemia resulting from high parasitemia caused by a Plasmodium falciparum infection can pose serious risks [13]. Furthermore, anemia may result from excessive hemolysis of parasitized red blood cells in malarial infections [14]. The majority of malaria patients also experience thrombocytopenia. Additionally, it was observed that the platelet count significantly decreases at high levels of parasitemia. As P. falciparum parasite burdens increase, platelet counts decrease, as reported in earlier studies [15,16]. In Sudan, doctors requested complete blood counts for most febrile patients to aid in the differential diagnosis of infectious and noninfectious diseases. This study aimed to investigate the effects of malaria parasite density on blood cell parameters in Sudanese patients with malaria.

Study design

The study design was a descriptive cross-sectional study.

Study population

Positive samples were collected from malaria patients with Plasmodium falciparum and Plasmodium vivax at the Bahri Health Centers.

Thick blood film

A drop of blood (10μl) was placed at the center of a microscope slide. Place the slide horizontally and stain the unfixed smear with Giemsa’s stain (1 mL of commercial Giemsa + 9mL of phosphatebuffered saline, pH 7.2) for 10 minutes. After washing and drying, examine the smears using light microscopy at a magnification of 100× with oil immersion. The advantage of the thick smear technique is that it concentrates the blood drop into a small area, thereby requiring less time to detect the parasites, which become more visible due to the hemolysis of the unfixed red blood cells.

Thin Blood Film

A drop of blood (3-5μl) was placed at one end of a clean microscope slide and drawn out into a thin film in the usual manner. Briefly air dry and fix in methyl alcohol for 1 minute, then allow to dry. Stain the smears with Giemsa (1ml Giemsa + 9ml PBS, pH 7.2) for 10 minutes. Pour off the stain, wash the slide in tap water, and dry it. Nowadays, fast stains are commonly used, allowing fixation and staining within a few seconds. The slides are then washed in tap water and dried. Examine at a magnification of 100× with oil immersion. This technique enables detailed morphological studies and identification of malaria species, as well as the morphology of red blood cells.

Counting malaria parasites

The count of the asexual stage of the parasite (Plasmodium falciparum and Plasmodium vivax) per microliter of blood was obtained by counting the number of parasites per 200 leukocytes using hand tally counters. The parasite count was then calculated according to the following formula: No. Of asexual stages × Total white blood cell count per microliter / No. Of leukocytes counted. Data were grouped into three categories: high parasitemia (>10 parasites/1 oil field), moderate parasitemia (1–10 parasites/1 oil field), and low parasitemia (1–100 parasites/100 oil fields).

Rapid Diagnosis Test (RDTs)

Blood collected from the patient was applied to the sample pad on the test card along with specific reagents. After 15 minutes, the presence of specific bands in the test card window indicates whether the patient is infected with Plasmodium falciparum or Plasmodium vivax of human malaria. It is recommended that the laboratory maintain a supply of blood containing P. falciparum and P. vivax for Use as a positive control.

Complete Blood Count (CBC)

Blood was drawn from the vein (venipuncture) using a disposable plastic syringe from the antecubital vein or the back of the hand. After cleaning the skin with 70% alcohol, the needle was inserted into the vein, and blood was slowly withdrawn until a sufficient amount was collected. The blood was slowly poured into EDTA anticoagulant (about 2.5ml) and gently mixed without frothing, then put in the Sysmex machine for reading. Examinations included (HB, WBC, Neutrophil, Lymphocyte, Monocyte, Eosinophil, and Basophil (mixed), Red Blood Cell, Hematocrit, MCV, MCH, MCHC, RDW, Platelet, MPV).

The statistical software used for this study’s data analysis was the Statistical Package for Social Sciences (SPSS) version 17. The data was collected on a hard drive and analyzed using chisquare tests with 95% confidence intervals. P-values less than 0.05 and t-test tabulation correlation were considered statistically significant.

Between December 2015 and May 2016, data from 60 cases of patients with malaria, for which all parameters were available, were collected for this study. Of these cases, 36 were caused by P. falciparum, while P. vivax caused 24. Analysis indicated a significant association between malaria parasites (P. falciparum, P. vivax) and Residence (P<0.05) (X²=17.093, DF=9, P=0.047).

The highest percentage was noted in Bahari at 64% (P. falciparum 52% compared to P. vivax 38%) (Table 1). Analysis indicated an insignificant association between malaria parasites (P. falciparum, P. vivax) and nationality (X²=1.525, DF=1, P=0.217) (Table 2). Similarly, the analysis indicated a trend of association between malaria parasites (P. falciparum, P. vivax) and season (X²=3.333, DF=1, P=0.068) (Table 3). Furthermore, the analysis revealed a significant association between malaria parasites (P. falciparum, P. vivax) and gender (X²= 5.884, DF =1, P = 0.015) (Table 4-13).

Table 1:Relationship between malaria parasites P. falciparum, P. vivax, and residence.

Table 2:Relationship between malaria parasites P. falciparum, P. vivax, and nationality.

Table 3:Malaria parasites P. falciparum and P. vivax association with season.

Table 4:Malaria parasites P. falciparum and P. vivax association with gender.

Table 5:Malaria parasites P. falciparum and P. vivax association with age groups.

Table 6:Malaria parasites P. falciparum and P. vivax are associated with fever.

Table 7:Malaria parasites P. falciparum and P. vivax associated with the use of bed nets.

Table 8:Malaria parasites P. falciparum and P. vivax are associated with the use of indoor residual spraying.

Table 9:The relationship between malaria parasites P. falciparum and P. vivax and Parasitaemia.

Table 10:Malaria parasites P. falciparum and P. vivax associate with the Cytoplasm of red blood cells.

Table 11:Malaria parasites P. falciparum and P. vivax are associated with the size of red blood cells.

Table 12:Malaria parasites P. falciparum and P. vivax are associated with the age of red blood cells.

Table 13:Malaria parasites P. falciparum and P. vivax are associated with blood parameters.

Malaria is endemic throughout Sudan, with varying levels of endemicity in different regions. It accounts for 16.6% of outpatient visits and 31.2% of inpatient admissions at various public health service facilities [17]. The main objective of this study was to examine the effects of malaria parasite species Plasmodium falciparum and Plasmodium vivax density on blood cell parameters in Sudanese patients. Differences in parasite positivity rates and the density of Plasmodium infections (geometric mean of parasite counts) were analyzed in Bahri, Aldewim, Alhalfaia, Alkadru, Alsalama, Elsawra, Om Alqura South, Ombada, Omdurman, and Shambat. Such variations can be attributed to differing environmental conditions in each area, which impact mosquito habitats and breeding, alongside other socio-economic factors such as education, financial status, family size, and water storage systems. Babiker and his colleagues found that within a given area, the abundance and dispersal of mosquitoes- and thus, exposure to infectious bites- are not uniform [18]. Consequently, significant heterogeneity in parasite rates and malaria morbidity may be observed. This finding aligns with Beier and his colleagues, who noted that entomological inoculation rates correlate with both parasite rates and parasite density [19]. Additionally, differences in parasite positivity rates between males and females were noted, with females exhibiting higher parasite positivity rates and parasite densities than males, likely due to a weakened immune system during pregnancy. Rogier [20] reported that gender influences tumor necrosis factors, immune response, and parasite density [20]. No significant association was found between age and the positivity rate or density of Plasmodium falciparum and Plasmodium vivax infections, which could be linked to socioeconomic factors such as education and bed nets. These observations are at odds with Christophers’ results [21]. The clinical symptoms commonly associated with malaria, including sweating and pale convulsions, were similar among individuals infected with Plasmodium parasites; this was corroborated by Sayonara, along with a stronger correlation observed between parasite densities and the morphology of red blood cells, including Size, Cytoplasm, and age, due to malaria infection, a finding supported by Cooke BM [22]. Regarding differential white blood cell counts, individuals infected with Plasmodium showed comparable results, but a notable difference in neutrophil and lymphocyte counts was identified. This aligns with expected immune system responses: higher neutrophil counts were observed in infected individuals, while lymphocyte counts were noted by Coller and his colleagues [23]. The study indicated a significant difference in Mean Corpuscular Hemoglobin (MCH) and mean corpuscular hemoglobin concentration between those infected with Plasmodium falciparum and those with Plasmodium vivax; lower levels were present in the former group. This was anticipated due to the parasites’ destruction of infected red blood cells and the immune response. Malaria causes anemia through various complex mechanisms, including the rupture of infected erythrocytes, autoimmune destruction of both parasitized and normal erythrocytes, reticuloendothelial hyperactivity, and dyserythropoietic attribute the reduction in hemoglobin concentration with Plasmodium falciparum infection primarily to the direct destruction of red blood cells by parasites; even low levels of parasitemia can theoretically lead to long-term severe anemia due to the hemolysis of parasitized cells. Thus, red blood cell hemolysis can occur during acute malaria attacks with high parasite density and during high-density asymptomatic infections. However, there was a tendency for hemoglobin concentration to decrease as infection density increased, a trend also noted by Kitua et al. [24], who found no significant correlation between malaria and anemia in their study [24]. This research also revealed a significant difference in Red cell Distribution Width (RDW), with a higher mean in Plasmodium falciparum infections compared to Plasmodium vivax, which is an expected result given the destruction of infected red blood cells by the parasites Dondorp et al. [25]. A significant decrease in platelets is one of the more well-known hematologic changes observed in patients with malaria. This study supports the finding that lower platelet counts are present among patients infected with P. falciparum compared to those with P. vivax due to the presence of P. falciparum in deep vessels and the consumption of platelets caused by partial blockage by the parasite. A previous study revealed that the prevalence of thrombocytopenia was similar between infections of vivax and falciparum malaria. However, patients with severe falciparum malaria had a significantly lower platelet count compared to those with non-severe falciparum malaria. These findings align with those of Saravu K, et al. [26]. Results from other studies indicate that thrombocytopenia appears to occur through peripheral destruction Ladhani S, et al. [27]. Immune-mediated destruction of circulating platelets may contribute to thrombocytopenia in malaria infections.

This study finds that patients infected with various malarial parasites demonstrate significant changes in their hematological parameters. The two most prominent alterations observed during malarial infection are in neutrophil and lymphocyte counts. Furthermore, patients with different densities of malaria parasites, specifically Plasmodium falciparum and lasmodium vivax, experience marked decreases in platelet count (thrombocytopenia) and also in Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC).

The patient’s written consent has been collected.

Omdurman Islamic University granted permission for this investigation. All study participants were informed about the study’s purpose. Permission for this study was obtained from the local authorities in the area. This study’s aims and benefits were explained with the assurance of confidentiality. All protocols in this study were done according to the Declaration of Helsinki (1964).

No specific grant was awarded for this research from any funding organization in the public, private, or nonprofit sectors.

The authors have declared that no competing interests exist.

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