Hematological Profile of Children With Malaria in Sorong, West Papua, Indonesia

Background: Malaria remains a major public health problem in Indonesian Papua, with children under five years of age being the most affected group. Hematological changes such as cytopenia that occur during malaria infection have been suggested as potential predictors and can aid in the diagnosis of malaria. This study aimed to assess the hematological alterations associated with malaria infection in children presenting with signs and symptoms of malaria. Methods: A retrospective study was performed by collecting data from the medical records of malaria patients at Sorong Regional General Hospital, Sorong, West Papua, Indonesia, both from outpatient and inpatient clinics, from January 2014 until December 2017. The laboratory profile of children suffering from malaria was evaluated. Results: One hundred and eighty-two children aged 1 month to 18 years old were enrolled. The subjects were mostly male (112, 61.5%) with a mean age of 6.45 years (SD = 4.3 years). Children <5 years suffering from malaria had the highest number at 77 (42.3%). One hundred two subjects (56%) were infected with Plasmodium falciparum . Half of the enrolled subjects (50%) had hemoglobin level (Hb) between 5.1 and 10 gr/dL. A total of 41 children (53.2%) less than 5 years old suffered from P. falciparum infection. In the age group of 5-10 years, there were 34 children (57.6%) who suffered from P. falciparum, and in the age group >10 years, 27 children (58.7%) suffered from P. falciparum infection. Only 4 subjects (5.2%) in the less than 5 years old age group had mixed malaria infection. Of the eight parameters of the hematological profile, there were five parameters that were significantly associated with the diagnostic criteria, namely hemoglobin, hematocrit, leukocytes, platelets and monocytes (p <0.05). clinical are not significantly with Statistical analysis . The data are presented as the means, percentages, standard deviations, medians, and ranges. Statistical analysis was performed using ANOVA, the Kruskal-Wallis test, contingency coefficient correlation, the Mann-Whitney U test, and the independent t-test. The data were analyzed using SPSS version 21 statistical software with appropriate statistical methods. Differences with P-values of less than 0.05 were considered significant. Our study showed that children suffering from malaria in the < 5 years age group had the highest number, at 77 (42.3%). According to WHO data in 2017, children aged under 5 years are most vulnerable group by malaria. 61% (266 000) worldwide This study shows that hematological abnormalities in children with malaria infection are common. Some subjects (50%) had a Hb level between 5.1 and 10 gr/dL. The prevalence of anemia in our study was 77.4%; 14.8% of subjects had mild anemia, 35.7% had moderate anemia, and 26.9% had severe anemia. The rate of anemia in children < 5 years old was higher than that in children 5-10 years old and > 10 years old, with a mean Hb of 8.71 gr/dL. Data from household surveys conducted in 16 high-burden African countries between 2015 and 2017 show that, among children who tested positive for malaria, the prevalence of any anemia was 79%, mild anemia 21%, moderate anemia 50% and severe anemia 8% [2]. A laboratory trial study of 30 patients in Iran observed significantly lower values of Hb/dl, hematocrit (Ht)%, mean corpuscular volume (MCV)/ fl and mean corpuscular hemoglobin (MCH)/ μl, WBC/ μl, and platelet/ μl among malaria-infected children compared to healthy children [9,11,34,35]. Our finding is consistent with this previous reports.

ovale wallikeri) and Plasmodium knowlesi (P. knowlesi) [3]. P. falciparum and P. vivax are the two main causes of human malaria infections. P. falciparum malaria poses a risk of severe complications and contributes to the majority of deaths [4].
In Southeast Asia, Indonesia contributes 9% of all malaria cases and has the second highest burden of disease after India [5]. The WHO estimated that 27% of the 257,563,815 people in the Indonesian population lives in malaria endemic areas. One of the endemic areas of malaria in Indonesia is Papua [6].
Hematological alterations that are thought to characterize malaria are related to the overt biochemical changes that occur during the asexual stage of the life cycle of the malaria parasite [7]. Patients infected with malaria tend to have significantly lower platelet, leukocyte, lymphocyte, eosinophil, red blood cell, and hemoglobin (Hb) counts, while the number of monocytes and neutrophils was significantly higher than that in nonmalaria-infected patients [8][9][10][11]. Anemia [12], leukopenia [13,14] and thrombocytopenia [15,16] are commonly seen in P. falciparum infection, probably as a result of the higher levels of parasitemia found in these patients [17,18]. Thrombocytopenia is most commonly seen in malaria infection [10,19,20].
People with platelet counts <150,000/μL are 12-15 times more likely to develop malaria infection than people with a platelet count >150,000/μL [10]. Pancytopenia and bicytopenia are common hematological problems encountered in clinical practice [8,9] that have multiple causes, and the underlying pathology determines the potential predictors and can aid in the diagnosis, management and prognosis of malaria [21,22]. It plays a major role in fatality [23,24].
The gold standard for malaria examination is microscopic slide examination [25].
Knowledge of changes in various hematological parameters in children suffering from malaria can increase the diagnosis of malaria by increasing suspicion of malaria and encouraging a careful search for parasitemia using a microscope [21]. There has been no research to investigate the effects of malaria on the hematological profile of Indonesian Papuan children.

METHODS
A retrospective study of data from medical records of malaria patients at Sorong Regional General Hospital, Sorong, West Papua, Indonesia Outpatient and Inpatient Clinic from January 2014 until December 2017 was performed. It included 182 children from 1 month to 18 years old with signs and symptoms of malaria at Sorong Regional General Hospital, Sorong, West Papua, Indonesia. Blood samples were collected from children into ethylenediaminetetraacetic acid (EDTA) tubes and used to prepare thin and thick blood films, which were then used for Giemsa microscopy to detect malaria parasites and malaria species. Patients' hematological parameters were determined using an automated hematology analyzer.

Study population.
The study involved male and female children aged one month to 18 years presenting with signs and symptoms with suspected malaria who were referred to the laboratory for investigation after consultation with a physician.

Hematological Profile of Children With Malaria in Sorong, West Papua, Indonesia
The study included children from inpatient and outpatient clinics at Sorong Regional General

Sample Collection
Two to three milliliters of venous blood was collected from each participant using a 5 ml sterile disposable syringe and dispensed into an EDTA anticoagulated test tube, followed by preparation of the thick and thin smears and automated for determination of the complete blood count (CBC). The EDTA test tubes containing the blood samples were gently inverted approximately 8 times to ensure the complete mixture of blood cells. The blood samples were collected by a trained medical laboratory scientist.

Peripheral blood smear examination
Peripheral blood smears were prepared using venous blood samples. We used separate slides for thick and thin smears. For thin film, we brought a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen slide. We waited until the blood spread along the entire width of the spreader slide. While holding the spreader slide at the same angle, we pushed it forward rapidly and smoothly. For thick film, we used the corner of a clean slide and spread the drop of blood in a circle to the size of a dime (diameter 1-2 cm).
We did not make the smear too thick or it would fall off the slide. (We were able to read newsprint through it.) We waited until the thin and thick films were completely dry before staining. We fixed the thin film with methanol (100% or absolute) and allowed it to dry completely before staining. The thick film was not fixed. Thick and thin films of peripheral smear (PS) examination blood slides were prepared and stained with Giemsa. Peripheral blood smear examination for the type of malaria parasite was performed systematically under low power, high power and oil immersion using an Olympus CX21 Microscope.

Definitions and endpoints
 Although a wide and confusing variety of anemia grades have been proposed, the most commonly used definitions in malaria studies, based on hemoglobin concentrations, are as follows: mild anemia if the Hb value is ≤11 g/dL, moderate anemia if the Hb value is ≤ 8 g/dL, and severe anemia if the Hb value is ≤ 5 g/dL [26].
Statistical analysis. The data are presented as the means, percentages, standard deviations, medians, and ranges. Statistical analysis was performed using ANOVA, the Kruskal-Wallis test, contingency coefficient correlation, the Mann-Whitney U test, and the independent t-test. The data were analyzed using SPSS version 21 statistical software with appropriate statistical methods. Differences with P-values of less than 0.05 were considered significant.

Demographic Characteristics and Hematological Profile of Research Subjects
This study included 182 subjects who met the inclusion and exclusion criteria. The subjects were mostly male (112, 61.5%) with a mean age of 6.45 years (SD = 4.3 years).
Children <5 years suffering from malaria had the highest number, 77 (42.3%). The mean body weight of the subjects was 18.95 kg (SD = 11.27 kg). A total of 102 subjects (56%) were infected with P. falciparum. Some subjects (50%) had a Hb level between 5.1 and 10 gr/dL. The results of the hematological profile examination of the subjects are presented in Table 1 below.
Forty-one subjects who were less than five years old suffered from P. falciparum infection (53.2%). Only four subjects (5.2%) less than five years old had mixed infections (Table 2). Table 3 shows the hematological profiles based on the age group of the study subjects. Of the eight parameters in the hematological profile, there are five parameters that were significantly associated with the diagnostic criteria, namely hemoglobin, hematocrit, leukocytes, platelets and monocytes (p <0.05) ( Table 4).
Generally, clinical symptoms are not significantly associated with a malaria diagnosis, and only one variable shows a significant relationship, pale, with a p value of 0.001.

DISCUSSION
In our study, there were more male patients infected with malaria than female patients, which is similar to the results of a study in South Sorong [29]. One hundred two subjects (56%) were infected with P. falciparum. Our study also shows that the most common type of Plasmodium infection in Sorong, West Papua, is P. falciparum. Unlike in other provinces in Indonesia, P. falciparum is the predominant malaria species in Papua.
Malaria is a preventable and treatable condition and remains the most important parasitic disease globally. In 2016, it was still endemic in 91 countries, placing 3.8 billion people at risk. Considerable progress has been made due to aggressive malaria control and elimination efforts since 2000, resulting in a global reduction of 41% in morbidity and 62% in mortality [6]. Many efforts have been made to minimize malaria transmission worldwide; however, this infection remains high among humans [30]. All species of Plasmodium have been documented in Indonesia, including small numbers of human P. knowlesi cases in Kalimantan and Sumatra islands [31,32]. All four species of human malaria parasites are found in Indonesia. P. vivax is the predominant species except in Papua where P. falciparum slightly predominates. P. malariae and P. ovale were mostly found in the eastern part of Indonesia, Nusa Tenggara Timur and Papua. In the past few years, P. knowlesi was documented in humans in Kalimantan [5].  [9,11,34,35]. Our finding is consistent with this previous reports.
Cytopenia is a disorder in which the production of one or more blood cell types ceases or is greatly reduced. The types of cytopenia are anemia, which is a reduction in red blood cells (RBCs); leukopenia, which is a reduction in white blood cells (WBCs); neutropenia (neutrophils make up over half of all WBCs), which is a deficiency in neutrophils; and thrombocytopenia, which is deficiency in platelets. Bicytopenia is defined as a condition in which two out of three cell lines (RBCs, WBCs, and platelets) are reduced. The simultaneous reduction in all three formed cell lines is termed pancytopenia [36]. These kinds of cytopenias are not uncommon in malaria; bone marrow diagnosis of adults with bicytopenia and pancytopenia has shown that 3% of bicytopenia and 6% of pancytopenia were caused by malaria [37,38].
The rate of change varies with malaria level, endemicity, hemoglobinopathy background, nutritional status, demographic factors, and malaria immunity [10,39,40]. The occurrence of cytopenias may be attributed to bone marrow suppression and hemophagocytosis [41]. Bicytopenia and pancytopenia usually result from direct or indirect decreasing effects on hematopoietic cell production in the bone marrow [42,43].
Anemia is also a common manifestation, particularly in infants with P. vivax and in children with P. knowlesi infection [44][45][46]. Anemia is one of the most common complications in malaria infection, especially in younger children and pregnant women in high transmission areas [41,47]. The pathogenesis of anemia during malaria infection is not clearly understood.
However, it is estimated that the main targets of parasites are RBCs, which results in damage to RBCs, acceleration of parasite growth and nonparasitic removal [40], bone marrow dysfunction [48], and the level of parasitemia [49].
Anemia in malaria, however, is associated with a combination of hemolysis of parasitized RBCs, accelerated removal of both parasitized and unparasitized RBCs, depressed and ineffective erythropoiesis due to tumor necrosis factor alpha, anemia of chronic disease, and splenic phagocytosis or pooling [50][51][52].
Thrombocytopenia was seen in 97 children (53.3%) and was highly significant in the age group > 10 years, with a median 105 x10 3 /µL. Thrombocytopenia was observed in malaria-infected children in this study, which is consistent with earlier reports [9,34]. There were both qualitative and quantitative changes in platelet abnormalities in malaria. In this study, platelet counts were significantly reduced in children infected with malaria.
Thrombocytopenia occurs in 84.9% of patients with malaria infection. This observation may imply that thrombocytopenia can be a marker of Plasmodium infection [9,53,54].
There are various hypotheses about thrombocytopenia that occurs in malaria infections. Thrombocytopenia seems to occur through peripheral damage [55], excessive removal of platelets by spleen pooling [56,57] and platelet consumption by the process of disseminated intravascular coagulopathy (DIC) [58]. Sufficient or increased numbers of megakaryocytes in the bone marrow affect the decrease in thrombopoiesis, which is a possible cause of thrombocytopenia in malaria [56]. The destruction of circulating platelets mediated by immunity has been postulated as the cause of thrombocytopenia seen in malaria infections. Platelets have also been shown to mediate clumping of erythrocytes infected with P. falciparum [59]. This can cause apparent thrombocytopenia. Patients infected with malaria experience increased levels of specific immunoglobulin G (IgG) in the blood that binds to malaria antigens bound to platelets, which may lead to acceleration of platelet destruction [19]. Previous studies revealed that platelet aggregation, which is the clumping of platelets, was incorrectly calculated as a single platelet by the analyzer, causing pseudothrombocytopenia [9]. In addition, during malaria infection, endothelial activation is activated and can contribute to the loss of endothelium barrier function and organ dysfunction. This process can use released platelets and proteins as important regulators of endothelial permeability, resulting in thrombocytopenia [60]. However, thrombocytopenia in malaria infection has also been associated with sequestration and pooling of platelets in the spleen, immune-mediated destruction of circulating platelets, and platelets mediating the clumping of P. falciparum-infected erythrocytes, leading to pseudothrombocytopenia [9,34,59]. Maximum thrombocytopenia occurred on the fifth or sixth day of infection and gradually returned to normal within 5-7 days after parasitemia ceased [61].
This study has shown, however, that there was no significant difference in total white blood cell count in malaria-infected children. A study by Maina et al also showed that there was no significant difference in the total white blood cell count in malaria-infected children compared to control subjects [9]. The difference in values can be related to environmental factors, socioeconomic status, or malaria immunity, among other factors [10,39,40]. Different views have been expressed on the total WBCs in subjects infected with malaria because leukopenia has been reported by several authors [10,34], and leukocytosis has also been documented by other authors [11,55]. Leucopenia is often seen in malaria-infected patients confirmed by other studies showing leucopenia [10,62], in contrast to other studies that show leukocytosis [9].
Our study showed lymphocytopenia in 97 children (53.3%). The study has further revealed that there were no statistically significant differences in granulocyte and lymphocyte counts between malaria-infected and noninfected children, and these findings are in agreement with many earlier reports [9,10,11,39,40,54,63,64] but disagree with the findings of George and Ewelike-Ezeani [34]. In some cases of acute malaria, however, lymphocytopenia has been reported, but this has been associated with redistribution of lymphocytes with sequestration in the spleen [65,66].
Generally, clinical symptoms are not significantly associated with a malaria diagnosis, and only one variable shows a significant relationship, pale, with a p value of 0.001. The first symptoms of malaria are nonspecific and characterized by headache, fatigue, abdominal discomfort, and muscle and joint aches, followed by fever, chills, perspiration, anorexia, vomiting and worsening malaise. These features often lead to overdiagnosis of malaria in developing countries, where diagnosis is frequently based only on clinical judgment with limited resources for parasitological testing [67].
Conversely, in children, these manifestations of uncomplicated malaria can be misinterpreted and attributed to other prevalent infections, such as pneumonia, gastroenteritis, and sepsis [68]. In high transmission areas, high and repeated exposure to parasites has an impact on the acquisition of immunity, resulting in a high proportion of asymptomatic infections, particularly in older children and adults [69].

CONCLUSION
Children infected with malaria revealed changes in some hematological parameters, with anemia, low platelet counts, white blood counts, and lymphocyte counts being the most important predictors of malaria infection in our study area. These parameters could improve malaria diagnosis when used in combination with other clinical diagnoses and microscopy.

Availability of data and materials
The datasets analyzed in this study are available from the corresponding author on request.

Ethics approval and consent to participate
Ethical approval for this study was obtained from the Research Ethics Committee of the Medical School of the Universitas of Sumatera Utara.

Consent for publication
Not applicable.

Competing Interests
The authors have no conflict of interest to declare regarding the publication of this manuscript.

Funding
The authors did not receive any financial support for the research.