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 significantly lower in patients than in controls. This is in agreement with previous studies (Erhabor et al., 2014; Sakzabre et al., 2020). In endemic areas, anemic individuals are more likely to be malaria positive than the non-anemic counterparts (Nlinwe and Nange, 2020). The etiology of anemia in malaria is known to be multifactorial, and due to hemolysis of RBCs, their autoimmune destruction and accelerated removal, depressed erythropoiesis, and splenic phagocytosis or pooling (Bashawri et al., 2002). Removal of non-parasitized RBCs was explained as the most important mechanism of anemia (Imoru et al., 2013).
Considering the leucocytes, there was a significant increase in neutrophils and a significant decrease of lymphocytes in patients with no difference in total WBCs, monocytes, and eosinophils. Malaria-related neutrophilia and/or lymphocytopenia were reported in several studies (Maina et al., 2010; Kotepui et al., 2014; Al-Salahy et al., 2016) Neutrophilia is related to an acute inflammatory response and an early release of neutrophils from the bone marrow in response to infection (Al-Salahy et al., 2016). The decrease in lymphocytes was explained to be due to their sequestration in the spleen (Wickramasinghe and Abdalla, 2000). Unlike the reduction in RBC counts, the changes in WBC total and differential counts were inconsistently reported in malaria patients. Total WBC counts are generally low to normal during malaria, this is widely thought to reflect their localization away from the peripheral circulation (McKenzie et al., 2005). Contrary to the present study findings, leukocytosis, neutropenia, monocytosis were observed in some patients (Maina et al., 2010; Sakzabre et al., 2020). Lymphocytosis was associated with falciparum malaria mortality among children (Ladhani et al., 2002). Increased eosinophil counts 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-significant 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 significantly higher in patients than controls. These findings 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 significantly 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 finding is in line with previous reports (Nwanjo and Opara, 2005; Maina et al., 2010), others reported that the alterations of one or more parameters were more obvious in patients with high parasitemia (Leowattana et al., 2008, Erhabor et al., 2014, Kotepui et al., 2015). Discrepant findings may be attributed to the effect of several confounding factors such as malnutrition, duration of infection, and the degree of adaptive immunity (Berens-Riha et al., 2014; Kotepui et al., 2015; Sakzabre et al., 2020).
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 influenced by physiological changes that accompany malaria infection as well as by hemolysis of RBCs and specific 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 significant 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-inflammatory cytokines and chemokines, followed by the production of anti-inflammatory cytokines (Lyke et al., 2004). Data from animal models have indicated that the pro-inflammatory 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 pro-inflammatory 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-inflammatory 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 correlated with the degree of parasitemia. This is in agreement with other studies (Day et al., 1999; Medina et al., 2011).
The early production of IFNγ from dendritic cells or monocytes, appears to be a pivotal component of the pro-inflammatory 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 significantly 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, inflammatory cytokines were found to enhance gametocyte destruction through the production of toxic nitric oxides at the peak of infection (Naotunne et al., 1991, 1993).
As to IL10, it is known to down-regulate anti-inflammatory cytokines preventing detrimental immune reactions. In the present study, the level of IL10 was significantly 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-inflammatory 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 significantly 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. These findings are supported by some previous reports while they disagree with others (Goncalves et al., 2012; Mbengue et al., 2016).
In conclusion, the main hematological findings 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-inflammatory cytokines, IFNγ and TNFα and the anti-inflammatory cytokine, IL10 with a relatively higher production of the latter. This pattern of cytokine regulation probably mediates parasite clearance while simultaneously avoiding severe pathology.