Hematological Parameters and Key Cytokines in Patients With Uncomplicated Falciparum Malaria in Hodeidah, Yemen

Immunity to malaria has a major role in controlling disease and pathogenesis with cytokine production being involved in almost each phase of the immune response. The present study aimed to assess hematological variables and to measure plasma levels of TNFα, IFNg and IL10, their ratios, and their relation to parasitemia among patients with uncomplicated falciparum malaria in Hodeidah, Yemen. Forty patients with uncomplicated P. falciparum monoinfection and 40 healthy age and sex matched controls were enrolled in the study. Parasitological diagnosis was conrmed, and parasite density estimated. Hematologic parameters, the presence of gametocytes, and plasma cytokine levels were determined. Results revealed lower Hb levels, RBC, lymphocyte and platelet counts and higher neutrophil and reticulocyte counts among patients compared to controls. TNFα, IFNg and IL10 were higher in patients than controls. A relatively higher IL10 production was demonstrated by the signicantly lower TNFα/IL10 and IFNg/IL10 ratios in patients than controls. TNFα and IL10 correlated positively with parasite density. Reticulocyte count was higher and IFNg level was lower in the presence of gametocytes. Conclusively, uncomplicated falciparum malaria is associated with the ability to regulate the production of the pro-inammatory and anti-inammatory cytokines. This mediates parasite clearance while simultaneously avoiding severe pathology.


Introduction
Malaria is the most important vector-borne parasitic disease in terms of morbidity and mortality. About 228 million cases and 405.000 annual deaths have been recently estimated. In Yemen, more than 65% of the population are exposed to malaria; endemicity varies with the diversity of the topography and climate (World Health Organization, 2019).
It has been reported that three species of malaria exist in Yemen, Plasmodium falciparum (P. falciparum), P. vivax and P. malariae with predominance of P. falciparum (more than 90% of cases). Based on active and passive case detection, a malaria prevalence of 16.2% was reported among the population in Hodeidah, west of Yemen (Al-Maktari et al., 2003) and 8% among school children in the same governorate (Alwajeeh et al., 2020). Efforts towards malaria control in Yemen have been undertaken since the late seventies (Yemeni National Malaria Control Program, 2010). However, it is estimated that about 38% of the population still lives in high transmission areas (World Health Organization, 2019).
Human malaria infections show a wide clinical spectrum ranging from asymptomatic infection to severe life-threatening disease. Immunity to malaria has a major role in controlling disease and pathogenesis. In high transmission settings, most patients have mild malaria, likely due to pre-existing immune protection (Laishram et al., 2012;Pawar, 2014).
Cytokines are a category of signaling molecules used in cellular communication, they are small proteins 5-20 kDa. They include chemokines, interferons, interleukins, lymphokines and tumor necrosis factor.
Based on their functional aspects, they have been classi ed into pro-in ammatory and anti-in ammatory cytokines (Wahab and Hussain, 2013). Pro-in ammatory cytokines are important in cell signaling and they up-regulate in ammatory reactions. These are TNFα, IFNγ with other interleukins as IL2-IL12 (Zhang and An, 2007). Anti-in ammatory cytokines control the pro-in ammatory cytokine response and participate in regulation of the immune response (Opal and De Palo, 2000). IL10 is the most important anti-in ammatory cytokine. It is a potent de-activator of monocyte/macrophage pro-in ammatory cytokine synthesis (Clarke et al., 1998).
Cytokine production is potentially involved in almost each phase of the immune response to malaria (Pawar, 2014). TNFα is produced by numerous cell types mainly by monocytes and tissue macrophages.
TNFα appears to be the most pro-in ammatory cytokine involved in the malaria pathogenesis and cytoadherence of infected erythrocytes (Grau and De Kossodo, 1994).
IFNγ, was known as immune interferon, it is a product of human leucocytes and other antigen-stimulated lymphocytes. Functionally, it heightens both the innate and adaptive immune responses against pathogens and tumors and has the ability to maintain homeostasis. IFNγ was found to mediate protection against pre-erythrocytic malaria infection in murine models. IFNγ level has been linked to reduction of parasitemia and protection from reinfection and complicated disease (McCall and Sauerwein, 2010). However, it is also involved in immunopathology and can exacerbate the malarial disease severity (Dodoo et al., 2002).
IL10 is one of the cytokines that affect malaria infection outcome. IL10 is a fascinating cytokine able to stop immune response by inhibiting the production of a number of cytokines. It suppresses antigen presentation and CD4 + T cell activation and has been linked to high parasitemia. It inhibits the release of pro-in ammatory mediators and thereby inhibits IFNγ-induced secretion of TNFα and protects against tissue damage. (Othoro et al., 1999).
The pattern and extent of hematological parameters and cytokines were reported to vary with the level of malaria, its endemicity, demographic factors and different features of the patients. Therefore, the present study was designed to estimate hematological parameters and to determine the plasma levels of TNFα, IFNγ and IL10, their ratios and their association with parasitemia among patients with falciparum malaria infection as compared to controls in Hodeidah, Yemen.

Study area
The present study was conducted in Hodeidah governorate in the western region of Yemen. Hodeidah has a semi-equatorial climate; warm and humid in summer and moderate in winter. Temperature reaches 40°C during summer and 24°C in winter. The population of the governorate is more than two million people living in rural and urban districts (Yemeni National Malaria Control Program, 2010).

Sample size
Calculation of the required sample size was based on the detection of an average difference of 50 pg/ml in the key cytokines (TNFα, IFNg & IL10) between patients and control groups, with a SD of 0.0 and 20.0 and with 80% power and 1% signi cance level using a two-side two-sample t-test (PASS program version 12). Eighty individuals were enrolled in the study and were divided into two groups as follows: Group 1: Forty patients (6-60 years) of both sexes attending governmental and private hospitals who were diagnosed clinically as having malaria and laboratory-con rmed as P. falciparum monoinfection. Patients having other chronic diseases, associated infections or vital organ dysfunction were excluded from the study.
Group 2: Forty apparently healthy controls, age and sex matched with a negative history of malaria for at least one year. They were selected among persons accompanying the patients and from the workers in the health centers.

Ethical considerations
Ethical considerations and con dentiality were assured for all participants in the study who gave an informed consent. The study was approved by the Research Ethics Committee of the Medical Research Institute, Alexandria University, Egypt and from the hospital administration in Hodeidah.

Data collection
Demographic and clinical data were collected through a predesigned structured questionnaire.

Blood sample collection and preservation
Five milliliters of venous blood were collected into a clean ethylenediaminetetraacetic acid (EDTA) tube. Hematological parameters were studied, and thick and thin blood lms were prepared. Subsequently, plasma samples were separated, labeled, and stored at -20°C till cytokines were measured.
In the same manner, blood samples were obtained from controls and all procedures done for the patients' samples were applied to the control group.

Laboratory procedures
A. Con rmation of diagnosis and estimation of parasitemia Detection of antigen by rapid diagnostic test (RDT) Samples from the collected blood of suspected malaria patients were re-screened for P. falciparum infection by detecting histidine-rich protein 2 (HRP-2) and plasmodium lactate dehydrogenase antigen (pLDH). In this method, CareStar TM Malaria HRP/pLDH (Pf/PAN) Combo (Access Bio Inc., New Jersey, USA) was used according to the manufacturers' instructions.

Giemsa staining and microscopy
Positive samples as diagnosed by RDT were con rmed microscopically after Giemsa staining of thick and thin blood lms (World Health Organization, 1991). Films were examined for asexual and sexual parasite stages.
Parasite density was estimated by counting asexual parasites against WBCs in thick blood lms. One hundred WBCs were counted and parasite density/µl of blood was calculated according to the formula: Parasites per µl were categorized as: low 1-999 parasite/µl, moderate 1000-9999 parasite/µl and high > 10.000 parasite/µl.
Relative reticulocyte count was determined using brilliant cresyl blue as a supravital stain (HiMedia ® Laboratories, India). The number of reticulocytes was expressed as a percentage of the total number of 500 erythrocytes counted (National Committee for Clinical Laboratory Standards, 1997).

C. Measurement of cytokines
The assays of cytokines were carried out in the laboratory of the University of Science and Technology hospital in Sana'a. Plasma samples were examined for TNFα, IFNg, and IL10 using the Quantikine ® capture ELISA kits (R&D Systems, Minneapolis, MN, USA). Each sample was examined for the three cytokines on the same day to avoid repeated sample freezing and thawing. All assay procedures were performed according to the manufacturer's instructions and concentrations of cytokines were expressed as pg/ml.

Statistical analysis
Coded questionnaire and laboratory data were entered, veri ed and analyzed using Statistical Package for the Social Sciences (SPSS) software, version 23.0 (IBM SPSS Inc., IL, USA). Differences between continuous/interval variables were compared using parametric independent t-test or one-way ANOVA and the non-parametric Mann-Whitney or Kruskal Wallis tests whichever suitable. Differences were considered statistically signi cant at p≤ 0.05.

Results
Characteristics of the study population A total of 80 individuals, 40 patients and 40 controls participated in the present study. Their demographic characteristics, parasitological and clinical indices are presented in table (1). Thirty-eight of the 40 patients had fever and 30 had paroxysms. None had complications. The median asexual parasite density of patients was 5,286 parasites/µl of blood with a range of 412-72,200 parasites/µl. It is observed that the majority of patients (65%) were of medium grade. Gametocytes were detected in ve patients (12.5%). There was no signi cant difference in total WBCs, monocytes and eosinophil counts. None of the hematological parameters showed signi cant difference in relation to the grades of parasitemia (Table  3). Patients with gametocytemia had signi cantly lower Hb levels and RBC counts as well as signi cantly higher reticulocyte counts (Table 4).      The levels of key cytokines, TNFα, IFNg, and IL10 were measured in P. falciparum-infected participants and compared to controls. There was a signi cant increase in the three cytokines in patients than in controls. The cytokine ratios, TNFα/ IL 10 and IFNg/ IL 10 ratios were signi cantly lower in patients than controls, whereas TNFα/ IFNg ratio was not signi cantly different (Table 5).
Regarding parasite density, signi cantly high TNFα and IL10 levels were found in patients with high parasite density. IFNg level did not show signi cant difference in relation to the grade of infection. The ratios TNFα/ IL 10, IFNg/ IL 10, and TNFα/ IFNg were not signi cantly different in infected participants in relation to parasitemia grades (Table 6). Among the three studied cytokines, only IFNg was signi cantly low in patients with gametocytemia (Table 7).

Discussion
Malaria is endemic in Yemen with P. falciparum being the predominant species. The current study was conducted on forty patients with P. falciparum monoinfection and forty healthy age and sex-matched individuals as controls. None of the patients had complications and most of them had a medium parasitemia grade.
The current study revealed that the mean Hb concentration and RBC counts were signi cantly lower in patients than in controls. This is in agreement with previous studies ( were reported in children with asymptomatic falciparum malaria, but low counts and increased eosinophil activity were found in cerebral malaria (Dunyo et al., 1998). A previous study linked the changes in differential WBC counts to the level of host immunity with non-immune patients displaying pronounced changes (Berens-Riha et al., 2014). In the present study, the non-signi cant changes in monocytes and eosinophils might be attributed to the patients being of uncomplicated malaria.
Regarding reticulocyte counts, this parameter is widely used to evaluate bone marrow erythropoietic activity and it is essential with diagnosis and prognosis of anemia (Peebles et al., 1981). In the present study, although the median reticulocyte count was within the normal range, the count was signi cantly higher in patients than controls. These ndings are in contrast with previous studies (Roberts et al., 2005). Low reticulocyte counts were attributed to the inhibition of erythropoiesis by the malaria parasites and their products (Dormer et al., 1983). It was postulated that sinusoidal obstruction by parasitized RBCs together with kidney involvement may lead to bone marrow hypoxia and consequently to low reticulocyte counts (Abdalla, 1990).
In the present study, platelet counts in parasitized patients were signi cantly reduced compared to controls. Thrombocytopenia was reported in many previous studies (Erhabor et al., 2014;Sakzabre et al., 2020; Omarine Nlinwe and Nange, 2020). This was explained to be due to sequestration and pooling of the platelets in the spleen as well as to their immune-mediated destruction and coagulation disturbances. There is increasing advocacy for including thrombocytopenia as a severe malaria criterion (Tanwar et al., 2012).
Regarding parasite density in the present study, parasitemia grade was medium in most patients, high in 25% of them and low in 10%. There was no relation between parasitemia grades and any of the hematological parameters. Although this nding is in line with previous reports (Nwanjo and  Gametocytogenesis may be simply programmed to occur after a number of cycles of asexual replication. Yet it has been suggested that gametocyte production may be in uenced by physiological changes that accompany malaria infection as well as by hemolysis of RBCs and speci c antibodies (Sinden, 1983). In the present study, low mean values of Hb and RBCs and high reticulocyte counts were observed in the presence of gametocytes.
Regarding the pattern of the key cytokines in P. falciparum infection, there was a signi cant elevation in the levels of TNFα, IFNγ as well as IL10 in patients compared to controls. Immune response to P. falciparum infection is mediated by the production of pro-in ammatory cytokines and chemokines, followed by the production of anti-in ammatory cytokines (Lyke et al., 2004). Data from animal models have indicated that the pro-in ammatory cytokines are essential to parasite clearance but must be regulated at the appropriate point to prevent pathology (Dodoo et al., 2002). High levels of the proin ammatory cytokines TNFα and IFNγ have been associated with severe pathology, while low levels of regulatory cytokines such as IL10 were associated with acute malaria (Couper et al., 2008). The balance between the anti-parasitic and the immunopathogenic effects of cytokines is a mark of clinical immunity to malaria (Pawar, 2014).
TNFα is the most famous pro-in ammatory cytokine marker of severe malaria (Armah et al., 2005). In the present study, although its level was high in patients, its effect was probably controlled by the consequent increase of IL10 which led to alleviation of the severity of the disease. TNFα in the present study The early production of IFNγ from dendritic cells or monocytes, appears to be a pivotal component of the pro-in ammatory responses leading to upregulation of TNFα. IFNγ was reported as an important determinant of the wellbeing of the patients being generally associated with protective mechanisms (Mbengue et al., 2016). In the present study, there was no relation between IFNγ and the parasitemia grades, while its level was signi cantly higher in the absence of gametocytes. Although gametocytes do not cause any clinical manifestation in malaria, it has been shown that immunity against their antigens is elicited early and could reduce the number of gametocytes achieving maturity in the peripheral blood.
Late gametocyte immunity may affect their number and infectivity (de Jong et al., 2020). In splenectomized macaques infected with Plasmodium cynomolgi, in ammatory cytokines were found to enhance gametocyte destruction through the production of toxic nitric oxides at the peak of infection (Naotunne et al., 1991(Naotunne et al., , 1993. As to IL10, it is known to down-regulate anti-in ammatory cytokines preventing detrimental immune reactions. In the present study, the level of IL10 was signi cantly higher in patients than controls and its level was positively related to the parasitemia grades. Yet. there was no relation with the presence of gametocytes. This is in agreement with previous results (Medina et al., 2011;Goncalves et al., 2012;Moncunill et al., 2013).
Balanced pro-and anti-in ammatory cytokines play a pivotal role in the regulation of malaria. The severity of malaria is related to the balance between IL10 and TNFα-concentration (Othoro et al., 1999). It has been found that the ratios IL10/TNFα and IL10/IFNγ returned to the normal range after chemotherapy as found in controls (Goncalves et al., 2012). In the present study, both TNFα/IL10 and IFNγ/IL10 were signi cantly lower in patients than controls, while the ratio TNFα /IFNγ was similar to that in controls.
This denotes a relatively higher IL10 production in the patients.
All cytokine ratios did not show a relation with parasitemia grade nor with the presence of gametocytes. In conclusion, the main hematological ndings in patients with uncomplicated malaria in Hodeidah are low Hb level, low RBC, lymphocyte and platelet counts, and high neutrophil and reticulocyte counts.
Individuals displaying such hematological changes should undergo malaria testing. Clinical immunity to malaria is characterized by upregulated levels of the pro-in ammatory cytokines, IFNγ and TNFα and the anti-in ammatory cytokine, IL10 with a relatively higher production of the latter. This pattern of cytokine regulation probably mediates parasite clearance while simultaneously avoiding severe pathology.

Declarations
Funding Not applicable.
Declaration of Competing Interest The authors declare that they have no competing interests.