Thrombocytopenia a predictor of malaria: how far?

Abstract

Malaria is an acute febrile illness. It is a dangerous disease that contributes to millions of hospital visits and hundreds of thousands of deaths, especially in children residing in sub-Saharan Africa. In a non-immune individual, symptoms usually appear 10–15 days after the infective mosquito bite. The first symptoms—fever, headache, and chills—may be mild and difficult to recognize as malaria. If not treated within 24 h, P. falciparum malaria can progress to severe illness, often leading to death. Children with severe malaria frequently develop one or more of the following symptoms: severe anaemia, respiratory distress in relation to metabolic acidosis, or cerebral malaria. In adults, multi-organ involvement is also frequent. In malaria endemic areas, people may develop partial immunity, allowing asymptomatic infections to occur. Haematological changes are well-recognised with malarial infection however background haemoglobinopathy, nutritional status, demographic factors and malaria immunity play a major role in specific changes in that geographical region. Artemisinin derivatives are new generation antimalarial drugs they are used in the treatment of acute attacks of severe malaria including cerebral malaria. Information on the safety of these new antimalarial drugs on body function is still scanty. Haematological parameters are well studied in P. falciparum infection, but now recent studies have indicated that these changes do occur in P. vivax infection also. Hematological profile together with microscopy will enable rapid diagnosis, prompt treatment and further complications can be avoided. This current review is aimed at providing an up-to-date information on the role of malaria and anti-malarial drugs on haematological parameters especially thrombocytopenia.

Introduction

Malaria is a dangerous disease that contributes to millions of hospital visits and hundreds of thousands of deaths, especially in children residing in sub-Saharan Africa. Although several interventions such as vector control, case detection, and treatment are already in place, there is no substantive reduction in the disease burden (Okagu et al. 2021). It continues to be the major health problem in the tropics with increased morbidity and mortality (Devineni et al. 2015). As most common presentation is fever in endemic region malaria may be considered as a leading differential diagnosis in all patients presenting as acute febrile illness (Khuraiya et al. 2016). Plasmodium vivax malaria is difficult to detect and treat because the parasitemia is typically low in comparison to that of Plasmodium falciparum (Roy et al. 2015). It is believed that Plasmodium falciparum infection is more serious than Plasmodium vivax. However, recently researchers found that Plasmodium vivax is changing its trend and it can also cause serious infections with life threatening complications (Verma et al. 2016). The clinical presentation of malaria is variable among patients and is usually related to the severity of the infection.

Patients present with fever, generalized body weakness (malaise), headache, nausea/vomiting, breathlessness, pain in abdomen, pallor, bleeding manifestations, hepatomegaly, splenomegaly. In all the malaria cases (100%) fever is the common clinical finding and associated with chills and rigors in 76.3% of cases. Typical paroxysms occurr only in few patients. Most of them have daily fever peaking once in a day (Kumbhar et al. 2019). Similar findings were reported by other studies (Patel et al. 2015). The higher percentage of Hepatomegaly and splenomegaly in the study by Khuraiya et al. (2016) might be due to higher percentage Plasmodium falciparum infection or late presentation of clinical symptoms in the patients.

Malaria has been a major human health problem that threatens the lives of about 40% of the World’s population (Gebremeskel and Krogstad 2015) causing morbidity and mortality worldwide (Gayawan et al. 2014). It is endemic in 100 countries making about half of the world’s population to be at risk (Scuracchio et al. 2011). About 50% of Nigerian population is reported to suffer from at least one episode of malaria each year (Imoru et al. 2013). Nigeria has been reported to have the greatest burden of the disease among the endemic countries in the world (Adamu and Jigam 2019). The tropical climate of Nigeria accounts for the high prevalence of the disease, only south of Jos in Plateau State, Nigeria has lower incidence of malaria due to the low temperature of the area (Federal Ministry of Health 2008). Environmental and daily fluctuation of temperature is said to be associated with the infection rate and malaria parasite development (Adamu and Jigam 2019). Malaria affects both sexes and all ages. It is reported to be a common cause of miscarriage in pregnant women, premature delivery, low birth weight, maternal anemia, intrauterine growth retardation and intrauterine death (Aduloju et al. 2013). Plasmodium vivax and Plasmodium falciparum infections can cause complications like severe anemia, cerebral malaria with convulsions, ARDS, renal failure, circulatory collapse, hemoglobinuria, abnormal bleeding, thrombocytopenia, disseminated intravascular coagulation (DIC) and jaundice (Roy et al. 2015).

Haematological parameters are well studied in Plasmodium falciparum infection, but now recent studies have indicated that these changes do occur in Plasmodium vivax infection also (Srivastava et al. 2011). The nature of hematological abnormalities depends on the time after infection. A recent study has revealed a role of interleukins (IL-4) and interferon’s (IFN-gamma) in erythropoietin suppression. Very recent studies have identified different complex interactions and hematological alterations induced by malaria parasites. However, no studies have hybridized these alterations for a better understanding of Malaria pathogenesis (Okagu et al. 2021). The World Health Organization (WHO) protocol for the evaluation of an antimalarial drug or drug combination includes hematological recovery as an efficacy end-point. Although the treatment of uncomplicated P. falciparum malaria aims at clearing parasite, relieving symptoms and permitting hematological recovery, data on the impact of antimalarial treatment on hematological recovery are few (Huynh et al. 2020).

Diagnosis of malaria

Diagnosis of malaria in pregnancy using blood film detection technique becomes difficult when the parasite sequestrates and replicates in the placenta and therefore may not be found in the film (Public Health England 2014). Placenta malaria parasites can cross placenta wall either during pregnancy or at birth resulting into vertical transmission to the baby. Malaria has resulted to about 11% of maternal and 30% of childhood mortality in Nigeria (Centers for Disease Control and Prevention 2012; Federal Republic of Nigeria 2012). Haematological and biochemical parameters are used as indices to monitor the severity of malaria (Chikezie and Okpara 2013). The degree of changes in haematological and biochemical parameters depend on the level of parasitemia, nutritional status, malaria immunity and the endemicity of the disease (Adamu and Jigam 2019).

Studies have revealed that wide-ranging haematologic changes occur in malaria (Obimba et al. 2015). Alterations in the hematological parameters are also thought to have the capacity to act as an adjuvant tool in strengthening the suspicion of malaria, thereby prompting a more meticulous search for malaria parasites (Sirak et al. 2016). Previous studies involving patients with complicated malaria had demonstrated that a reduced platelet count, reduced white blood cell counts, and decreased red blood cell indices had relatively good sensitivities and specificities in predicting the presence of malaria infection. Changes in physicochemical parameters of P. falciparum infested blood may vary with level of malaria endemicity, presence of haemoglobinopathies, nutritional status, demographic factors and level of malaria immunity (Akaninwor et al. 2013).

In the last 3 decades, big strides have been made in refining, modifying, or inventing highly sensitive and specific diagnostic tools for parasitic infections. For malaria diagnosis, these newer tests are based on serology based assays [ELISA (FAST-ELISA)], and rapid antigen detection systems (RDTs), molecular based approaches [real time polymerase chain reaction, loop-mediated isothermal amplification (Lamp), and luminex], and proteomics technology (mass spectrometry).

The newer serological and molecular based malaria diagnostic approaches provide superior sensitivity and specificity, but it is at a huge cost, in terms of equipment, infrastructure, and personnel which makes most of the newer diagnostic methods inapplicable to many areas in developing countries, where malaria is highly prevalent. Some of the hospitals in the region, however, can afford to carry out a complete blood count for hematological parameters in patients suspected to have an infection. Because microscopy is still considered by many as an imperfect gold standard, efforts have been made to examine role of hematological parameters in the diagnosis of malaria infection. Hematological profile together with microscopy will enable rapid diagnosis, prompt treatment and further complications can be avoided (Kumbhar et al. 2019).

Malaria chemotherapy

One of the major factors that led to the persistence, and indeed explosion of malaria disease despite the availability of very effective antimalarial agents is the emergence of plasmodia that are resistant to one or more classes of antimalarial agents. Chloroquine resistant Plasmodium falciparum is now common in almost all malarious part of the world. Chloroquine resistance is commonly reported as infection originating in Eastern and Western Africa, South America and South East Asia particularly Thailand. Organization shows that resistance to mefloquine is more sporadic, except in the Eastern and Western borders of Thailand and adjacent countries where mefloquine and multidrug resistance is widespread (Madubogwu et al. 2019).

Artemisinins are fast acting and very potent against blood-stage parasites and show activity against early sexual stages of the parasite, which is important for blocking transmission. Heme react with artemisinin much more efficiently than the other iron-containing molecules, supporting the role of redox active heme as the primary activator of artemisinin. Artemisinins have also shown a broad anticancer activities in cell lines and animal models, they have also exhibited anti-schistosomal properties. Artemether (artemisinin derivative) is a lipid soluble methyl ether of dihydro artemisinin with rapid schizonticidal activity against Plamodium falciparium parasites in blood and parasitemia clearance rate of 30–84 h (Elamin and Asiri 2019). Elimination efforts require drugs that alleviate symptoms, prevent transmission and provide a radical cure. In the face of resistance to chloroquine by Plasmodium falciparum, the malarial parasite responsible for the high mortality and morbidity from malarial disease, artemisinin and its derivatives (artesunate, artemether, arteether, and dihydroartemisinin) have given renewed hope for combating resistant strains of Plasmodium falciparum malaria (Okunlola et al. 2013).

Haematological parameters

Haematological abnormalities are one of the most common complications in malaria as it involves the major cell lines (Imoru et al. 2013). Haematological changes associated with malaria include anaemia, leukocytosis/leucopenia, thrombocytopenia and disseminated intravascular coagulation (Al-Salahy et al. 2016). The degree of these alterations differs with the degree of malaria endemicity, background haemoglobinopathy, nutritional status, environmental factors and malaria immunity (Bhawna et al. 2013).

Changes in blood cell counts are a well-known features of malarial infections. These changes involve major cell lines including red blood cell (RBC), leukocyte and thrombocyte. Hematological changes in the course of a malaria infection, such as anemia, thrombocytopenia and leukocytosis or leucopoenia are well recognized. These alterations vary with the level of malarial endemicity, background hemoglobinopathy, nutritional status, demographic factors, and also malaria immunity (Huynh et al. 2020).

There is significant hematological changes ranging from hemolytic anemia, defective erythropoiesis, and reticulocyte production, leukocytosis, leukocytopenia, thrombocytopenia (TCP) platelets count less than 150 × 103/Microliter, platelet dysfunction in severe malaria, and disseminated intravascular coagulation DIC (Ahamed et al. 2019). Artemether has no effects on hematology parameters except for haematocrit percent and hemoglobin level (Elamin and Asiri 2019).

Effect on haematocrit (hct)

The packed cell volume (pcv) is a measure of the amount of Red Blood Cell in the blood. It is usually higher in neonates and children and decreases with age to adulthood. The level is slightly higher in males than females. The measure of PCV is important in studies involving malaria due to associated haemolysis of red blood cells with attendant anaemia associated with malaria parasitaemia. There is an increase in the PCV after treatment with Arthemether-lumefantrine (Madubogwu et al. 2019).

Effects on haemoglobin (Hb) concentration

Anaemia occurs when the Hb concentration falls below the normal for a person’s age, gender and environment. It is defined as Hb level < 11 mg/dl for both male and female. Artequin (AQ) and other Artemisinin-based therapies have replaced chloroquine (CQ) as the preferred treatment for uncomplicated Plasmodium falciparum malaria, due to the resistance of the parasite to CQ. RBC count, Hb, and PCV are significantly elevated in the CQ-administered group after three days, but after seven days of administration, no significant alterations are observed (Ugwu et al. 2021). This might be attributed to the lack of parasites in the RBCs of the rats used in the study, as CQ only gets protonated—acquiring the ability to lyse RBCs—in the presence of the digestive vacuoles of the plasmodium parasite. On the other hand, the RBC count, Hb, and PCV were significantly reduced in AQ recipients compared with the control. AQ appeared to have no deleterious effect on RBCs, Hb, and PCV when administered at the recommended dose for three days. There is however an indication that prolonged administration of the combination of Artesunate and Mefloquine would possibly lead to anemia. This is in consonance with both the documented pharmacology of artemisinins and reports from preclinical data, suggesting that repeated or prolonged exposure to ACT drugs may affect blood cell counts and predispose one to anemia (Obianine and Ariokujo 2011).

The increase in RBC count, Hb, and PCV observed in the CQ-administered group after three days is in contrast to the results obtained by other researchers, which indicated no changes or reduction in RBC parameters. For instance, one report asserted reductions in RBC, Hb, and anemic tendency after CQ administration, whereas another report did not observe any significant alterations in RBC count and indices when CQ was administered at the recommended duration of three days (Ofem et al. 2013). This deviation from some previous studies stimulates a need for verification of mechanisms. However, this finding could be beneficial in cushioning the anemic conditions that are often associated with malaria. Administration of artemether significantly reduced hemoglobin level (Osonuga et al. 2012). In a study performed by Yin et al. (2014) in dogs, artemether treated dogs showed decrease in both haematocrit and hemoglobin. Reduction in hemoglobin level in artemether treated rats could be due to haemolysis caused by artemether (Rehman et al. 2014).

For continued survival and reproduction, plasmodium parasites need to infect the red blood cells of their human host. Consequently, changes in the red blood cell indices are some of the commonest observations seen in malaria (Muwonge et al. 2013). In endemic areas malaria is the most common cause of severe anaemia (Srivastava et al. 2015). Although malaria caused by PV is thought to be benign, some studies have shown that it can cause severe anaemia as well. In the study of “Comparison of hematological parameters in various acute febrile illnesses” by Chaudhary et al. (2016) found that malaria showed maximum number of cases of anemia. Anaemia in malaria is believed to occur due to haemolysis of parasitised and non-parasitised RBCs, peripheral sequestration of RBCs, and ineffective erythropoiesis. In malaria endemic areas, the prevalence and severity of anaemia are usually determined by a number of interacting factors. These include the level of parasitaemia, age of host, host genetic factors (e.g., co-existing RBC polymorphisms like haemoglobinopathies, G6PD), and non-malarial causes of anaemia e.g., infections, malnutrition (Muwonge et al. 2013). The impact of malaria anemia is greatest in regions of sub-Saharan Africa where underlying anemia and poor nutrition are common. Furthermore, some observers have suggested that malaria-related anemia is more severe in areas of intense malaria transmission and in younger children rather than in older children or adults. Studies especially in south-eastern or eastern Asia have noted Hb decreases or mild anemia among malaria cases may reflect a lower prevalence of underlying anemia, better nutritional status, and/or better access to treatment. This variation might be due to the severity of infection and the level of immunity against the parasite in patients of falciparum and vivax malaria in different countries having endemic and non-endemic pockets (Muwonge et al. 2013).

Effects on WBCs count

Significant decrease in total white blood cell (WBC) count indicates leucopenia. Leucopenia is defined as total white blood cell count < 4000/μl. This could be as a result of sequestration of leukocytes, accelerated destruction or decreased production. This finding is in accordance with the study by Okeke et al. (2016) which reported a significantly lower WBC in infected patients compared to the controls. In another study, Kayode et al. (2011) indicated significantly increased WBC in malaria and malaria typhoid co-infected patients. He attributed the higher WBC count to increased production of leukocytes at the onset of an infection to wade off the invading pathogen/parasite. In a study conducted by Maina et al. (2010) on Kenya children, significant increase in monocyte was recorded in person with malaria infection than in the control. The observed increase in basophil might probably be due to allergic reactions caused by the bite of the mosquito. Report shows that eosinophil, basophil and mast cell as components of innate immune response plays an important role in the pathogenesis of malaria through histamine release (Mecheri 2012). The level of Neutrophils in P. falciparum infected children was found to be significantly lower than that of the controls. This is in line with the report of Senthikumaar and Sarojini (2013) which noted a decreased neutrophil count in malaria infected individual as compared to the control. But incontrast to Maina et al. (2010) increased neutrophil reported which may be due to activated neutrophil production or release from the marrow or suppressed peripheral removal. Total WBC (leukocyte) and lymphocyte count are significantly increased, whereas neutrophil count are significantly reduced in the CQ-treated group after three days of administration (Ugwu et al. 2021). This finding is at variance with an earlier report in which CQ had no significant effect on WBCs throughout the duration of administration (Adeleye et al. 2012). However, another report has it that an ACT drug, Coartem, increases total WBC and lymphocyte count but it decreases neutrophil count; they attributed these changes to the immunological response induced by the drug (Ofem et al. 2013).

There is a significant positive association between leucopenia and P. falciparum malaria. leucopenia and thrombocytopenia occurred with P. falciparum malaria, but these were not associated with death. The leucopenia occurred most probably due to a bone marrow suppression, resulting from cytokines production and/or folate deficiency when associated with pancytopenia (Ahamed et al. 2019).

There is a significant positive association between relative lymphocytosis and P. falciparum. Malaria, this might be a part of the bone marrow failure which is associated with neutropenia because neutrophil has short half-life compared with lymphocytes and its deficiency appears early (Ahamed et al. 2019). Leukocyte changes in malaria are variable and depend on many factors such as acuteness of infection, parasitemia, disease severity, state of the host immunity to malaria, and concurrent infections. Leukocytes play a vital role in the defense against malaria.

Commonly, majority of patients with acute uncomplicated P. falciparum malaria usually have their mean total leukocyte count (TLC) within the normal range. However, in some cases, a mild leucopenia may occur, especially in nonimmune adults or in cases of complicated malaria. In addition, according to previous studies, leucopenia does not appear to be parasite specific (Muwonge et al. 2013). On the contrary a study by Sirak et al. (2016) revealed leucocytosis and neutrophilia as a major feature in malaria infected patients. This is due to increase in the release of WBCs at the initial stage of infection to fight against malaria infection. Further they stated that their study supports that effective immune response to malaria is feature in malaria endemic areas.

Although some discrepancies appear to exist, there have been reports of leukopenia as well as leukocytosis in malarial infection and studies have reported that neutropenia, eosinophilia, neutrophilia, and monocytosis, lymphopenia, are other hematological reactions to malarial infection. This finding is in contrast to previous studies which reported that malaria-induced changes include a reduction in neutrophil counts. In their report, reduction in neutrophil was observed as an important abnormality in patients with severe malaria and associated with a poor prognosis. The reason for this might be the marginalization of neutrophils to the sites of inflammation, splenic localization, and serum lymphotoxic factors (Sirak et al. 2016).

Among these parameters lymphopenia was the commonest abnormality. These findings are supported by other studies (Muwonge et al. 2013) according to the literature lymphopenia, sometimes profound but transient, is a common finding in acute malaria in nonimmune adults. The tissue redistribution of lymphocytes, from the free flowing pool to the marginal pool at the endothelial lining is usually responsible for transient malaria lymphopenia, particularly observed in T lymphocytes. Sometimes lymphocyte destruction as a result of Fas-induced apoptosis is also a factor responsible for lymphopenia (Srivastava et al. 2015). Activation of either phagocytes (neutrophils and macrophages) or natural killer (NK) cells is responsible for the innate immune response to blood borne pathogens. Thus reticuloendothelial hyperplasia involving macrophages is one of the most important early pathological hallmarks in malaria. Hence, monocytosis has been one of the most consistent observations reported from prior similar studies (Akaninwor et al. 2013).

Effects on RBCs

The artemisinin derivatives which form the backbone of current malaria treatment, exhibit an excellent safety profile. However, in animals, these highly efficacious antimalarial have demonstrated both embryotoxic and reticulocytopenic effects. Reticulocytes are immature red blood cells that originate from orthochromatic normoblast through nuclear exclusion in the bone marrow, are released into the peripheral blood, and undergo further differentiation into mature red blood cells. Acute malaria is associated with red blood cell destruction, resulting in anaemia. Also, a temporary decrease in reticulocyte counts has been demonstrated in paediatric severe malaria patients treated with artemisinin derivatives (Adjei et al. 2020). Several studies have shown a marked suppression of erythropoiesis during malaria. This effect has been variously ascribed to dysregulation of the immune response to the infection. However, in recent years an increasing focus on the direct toxic effect of haemozoin, formed as a residual product by the parasites, and mediated through down regulation of haematopoietic growth factors is being made. Notably, the suppression of erythropoiesis is rapidly reversible as soon as parasites have been cleared from the circulation and demonstrated by the reticulocyte count dynamics. Conversely, it has been shown that low numbers of parasites in asymptomatic malaria, or sub-microscopic parasitaemia, could maintain dyserythropoiesis for prolonged periods, and the relatively low reticulocyte count on day 28 reverting to preillness levels, or sustained bone marrow depression (Adjei et al. 2020).

The artemisinin derivatives are the most rapidly acting antimalarial known, with their antimalarial action partially mediated through an increase in the production of intracellular reactive oxygen intermediates. An initially higher fractional reticulocyte change from baseline, likely reflects the rapid parasite clearance while a higher overall fractional reticulocyte changes between baseline (acute illness) and the presumed steady state day 28 post treatment levels in the amodiaquine-based treatment groups likely reflects a more profound effect of these treatments, or a relatively slower parasite clearance or both. On the other hand, it is also possible that, any such effects of the artemisinin derivatives are not significant beyond early embryonic stages, since it has been shown that any potential effects of artemisinin derivatives, though significant in young embryos, caused only minor changes in reticulocyte counts in later foetal stages (Adjei et al. 2020).

There was a significant positive association between microcytic hypochromic red blood cell and P. falciparum malaria. Microcytic hypochromic red cells might due to either hepcidin production or loss of appetite and poor diet especially in childhood period (Ahamed et al. 2019). The morphological and biochemical alterations on the red cell membrane induced by malaria infection is necessary for the survival of the parasite, its growth, cell differentiation, compartmentalization and nutrient uptake. “The entrance of P. falciparum into red blood cells causes marked increase in inflammatory cytokine secretions (TNFα, IL-I, IL-10 and IFNγ), activation of endothelial cell (due to over expression of cell adhesion molecules; (ICAM-I VCAM-1), coagulation cascade activation (due to platelet consumption and endothelial damage), and parasitized red blood cells Sequestration (Ogbonna et al. 2021).

Effects on thrombocytes

In some studies thrombocytopenia has emerged as predictor of malaria. At high parasitemia the platelets were found to be significantly lower. Similar finding was observed by Sirak et al. (2016). The pathogenesis of thrombocytopenia consists of a myriad of pathogenetic mechanisms involving splenic pooling of platelets, antibody (IgG) mediated platelet destruction, adenosine diphosphate (ADP) release following the haemolysis of parasitised RBCs, dysmegakaryopoiesis, platelet aggregation and activation, parasite invasion of platelets, platelet phagocytosis, platelet adhesion to erythrocytes, and oxidative stress. Nevertheless, thrombocytopenia in malaria is observed to improve with disease resolution, and a normal platelet count is usually reported within 7 days after the initiation of antimalarial treatment (Muwonge et al. 2013; Srivastava et al. 2015).

In an attempt to compensate for the low absolute platelet count, the bone marrow increases the formation of megakaryocytes, which usually escape from the bone marrow as megaplatelets during an acute malaria infection. Evidence to support this hypothesis comes from a study by Kreil et al. that found a marked elevation in the level of thrombopoietin, a key platelet growth factor in patients with malaria. Because of an increase in the amount of mega platelets, the mean platelet volume is increased during an acute malaria infection. These findings may suggest that uncomplicated malaria is associated with mild or nonsignificant changes in the platelet profile (Muwonge et al. 2013). The major haematological change seen in the Kumbhar et al. (2019) research was thrombocytopenia. Several other studies observed similar finding (Khuraiya et al. 2016).

Platelets play an important role in the integrity of normal homeostasis; Mean Platelet Volume (MPV) is an indicator for its functions, including aggregation, release of thromboxane A₂, platelet factor 4, β-thromboglobulin, and expression of glycogen 1b and glycogen IIb/IIIa receptors. After three days of administration of chloroquine (CQ) and amodiaquine (AQ), no significant alterations were observed in platelet count and platelet indices. However, after seven days, platelet count and prothrombin clotting time (PCT) reduced significantly in the AQ- and CQ-treated groups compared with the control (Ugwu et al. 2021). Hence, prolonged ingestion of these drugs could affect platelet function negatively. A work carried out in the past had reported little or no adverse effect on platelet count after CQ administration (Ofem et al. 2013). In another study, contrary to this, a low platelet count followed CQ administration, but not so with artesunate (Omotosho et al. 2014). It has been reported that malaria is a major health problem in the tropics with high morbidity and mortality, and it is associated with different degrees of low platelet count with increased bleeding tendency (Shaikh et al. 2011). Platelet indices are routinely reported as part of the complete blood count, but their use is generally restricted to narrowing the differential diagnosis of anemia. It is also elevated in thrombotic thrombocytopenic purpura, a disease of unknown origin, characterized by abnormally low levels of platelets in the blood, formation of blood clot in the arterioles and capillaries of many organs, and neurological damage. MPV is not significantly altered after administration of the two anti-malaria drugs (AQ and CQ). MPV a determinant of platelet function, is a newly emerging risk factor for atherothrombosis. Increase in MPV has been documented in patients with metabolic syndrome, stroke, and diabetes mellitus. Many studies have shown that increased MPV is one of the risk factors for myocardial infarction, cerebral ischemia, transient ischemic attacks, and chronic vascular disease (Ugwu et al. 2021).

In a previous study on effects of chloroquine on the complement and coagulation systems, it was noted that the activation of the classical pathway of complement by antibody coated sheep erythrocytes and aggregated 1 gG was blocked by. Similarly, chloroquine prevented normal rabbit erythrocytes and Zymosan from activating the alternative pathway of complement. The activation ofC3 by immune complexes of different solubilities also was inhibited by chloroquine. Chloroquine abrogated the clotting of plasma by calcium chloride and thrombin. This effect can be reversed by the addition of excess thrombin but not of calcium chloride. Chloroquine was also found to enhance degradation of FXIII in fibroblasts (Etukudoh et al. 2020).

Another study on effect of p6304 (2-chloro-3-(4-hexylphenyl)-amino-1,4-naphthoquinone) as a novel antithrombotic agent was investigated, alongside a naphthoquinone NQ304. The NQ304 was found not to affect the coagulation system, since it did not change the APTT and Thrombin Time (TT). The agent prevented death due to pulmonary thrombosis by the platelet aggregates in mice in vivo. In the mouse tail bleeding time test, NQ304 showed a significant prolongation of the tail bleeding time in conscious mice (Etukudoh et al. 2020).

Naphthoquione and chloroquine are drugs used in the treatment of malaria parasitaemia. Malaria is a disease caused by the parasites of Plasmodium spp. It is a serious problem particularly in the tropical and third world countries. The disease is typically transmitted by the bite of an infected mosquito. The persistence of malaria as a public health problem, which is partly as a result emergence of Plasmodium spp. resistance to antimalarial drugs is leading to increased morbidity and mortality (Abdulrahaman and Dallatu 2012; Etukudoh et al. 2019a, b), but according to literature, these drugs are no longer choice drugs for malaria treatment since they are used in the treatment of other diseases.

Although allergic reaction from supplementation is possible, there is no known toxicity associated with high doses of the phylloquinone or menaquinone forms of vitamin K and therefore no tolerable intake level has been set. However, vitamin K3 (menadione), a synthetic form of the vitamin K is demonstrably toxic. The FDA has banned this from over the counter supplements because large doses have been shown to cause allergic reactions, hemolytic anemia and cytotoxicity in liver cells, but the comparison between chloroquine and naphthoquinone has not been considered on the basis of its effects on APTT and PT. Phylloquinone and menaquinone are capable of blocking the blood thinning action of anticoagulants like warfarin, which work by interfering with the action of vitamin K, they also reverse the tendency of these drugs to cause arterial calcification in the long term. Calcium and phospholipids are required for the kinase and thrombokinase complexes to function. Calcium mediates the binding of complexes via the gamma-carboxyl residues on FX and FIX to the phospholipid surfaces expressed by platelets, as well as procoagulant microparticles or microvesicles shed from them (Etukudoh et al. 2020).

Data for naphthoquinone shows that for the control, the activated thromboplastin time (APTT) was higher than the prothrombin time (PT). The second group of Swiss Mice treated with 0.1 mg/kg of naphthoquinone, showed a decrease in APTT and PT. This shows that there may be a likelihood of elevated factor VII which could be the result of an acute phase reaction, the bloods reaction to acute tissue inflammation or trauma. It could also mean that there are circulating anti-coagulants which tends to shorten APTT, a situation known as disseminating intravascular co-agulation (DIC). The PT only increased slightly staying within range of that obtained with the normal control. In administering 0.5 mg/kg of naphthoquinone, there was a further decrease in APTT and PT, which agrees with earlier studies (Etukudoh et al. 2019b) which noted a decrease in some haematological parameters of plasmodium infected mice administered with chloroquine and naphthoquinone. This shows a further intense acute tissue inflammation with a sustained PT showing apart from the presence of Lopus anti-coagulant (LA), a defect in (factors VII, IX, or XI) their functions being quite intertwined, factor VII (f VII) serves to initiate the process of co-agulation in conjunction with the Tissue Factor (TF) which is factor III. This occurs only when there is a vessel injury; tissue factor at this point is exposed to the blood and circulating factor VII. While tissue factor is a transmembrane glycoprotein expressed in the perivascular tissue as main initiators of in vivo blood clotting, it forms a complex with factor VII to activate factor IX. Both pathways interlace with the activation of factor IX which leads to the cleavage of prothrombin, (factor II) to thrombin (factor IIa). Factor XI is a zymogen of a blood co-agulation protease, factor Xia, which contribute to homeostasis through activation of factor XI. It was previously reported that a newly synthesized naphthoquinone NQ304 had no effect on PT and APTT but prevented thrombosis due to its ability to prevent platelet aggregation. Mice given 1.0 mg/kg dose of naphthoquinone, showed an increase in both APTT and PT. This slight increase in both APTT and PT could be indicative of likely Willebrand disease, liver disease, defects in factors (I, II, V, and X) as well as DIC (Etukudoh et al. 2020).

The group of mice that were given 2.0 mg/kg of naphthoquinone showed a remarkable increase in APTT as well as PT with the differences being very significant when compared to those of the control. This shows most likely, the presence of spontaneous bleeding as well as advanced stage of DIC. Summarily, naphthoquinone doses below 2.0 mg/kg may not be toxic, reduces APTT while sustaining PT, but doses of 2.0 mg/kg and above may be toxic and cause spontaneous bleeding as well as liver damage among other conditions. Administering 0.1 mg/kg of chloroquine in mice decreases in APTT, with a slight increase in PT, but the difference is not so significant. This could be the result of elevated factor VIII. Administering 0.5 mg/kg of chloroquine in mice showed an increase in APTT, while the PT also increased. This could be a pointer to Decreased or defective factor I (fibrinogen), II (prothrombin), V or X, severe liver disease, acute DIC (Etukudoh et al. 2020).

These mean there is likelyhood of spontaneous bleeding, indicating that at this dosage, chloroquine had begun to exhibit toxicity. The group of mice with on 1.0 mg/kg dose of chloroquine, showed a significant increase in APTT and an increase in PT, this shows defective factor I (fibrinogen), II (prothrombin), V or X, severe liver disease, acute DIC, with most likely, the presence of nonspecific inhibitors which may be Lupus anti-coagulants which binds to phospholipids found on the surface of platelets. The mice that were given 2.0 mg/kg of chloroquine showed increase in APTT and PT. At this dose, APTT value doubles the reference value of the control, while the PT is six times its reference value. Bleeding at this point may be more severe and may lead to thrombosis with life threatening complications such as stroke or a heart attack. Chloroquine may be toxic at a dosage as low as 0.5 mg/kg with graded life threatening complications (Etukudoh et al. 2020).

Severe TCP is frequently noticed with P. falciparum Malaria. TCP during malarial infection may appear even before fever, anemia, and splenomegaly become manifest. During early stages of malaria, platelet agglutination as a result of endothelial cell activation and release of activated von Willebrand factor occur which may cause thrombocytopenia. Direct invasion of platelets by malarial parasites may occur. Thrombocytopenia in malaria is usually mild and treated by eradication of malaria parasite. Thrombocytopenia is an early and consistent feature of malaria, but its pathogenesis remains incompletely understood. The causes of thrombocytopenia in falciparum malaria are increased platelet consumption as evidenced by shortened survival of platelets and the finding of increased megakaryocytes in patient’s bone marrow and elevated plasma thrombopoietin levels, and systemic microvascular sequestration and endothelial activation may play a pathophysiological role, a hypothesis supported by the observation that the radiolabeled platelets of patients with falciparum malaria are diffusely sequestered rather than pooling in the liver or spleen. Population studies have shown an association between thrombocytopenia and outcome, and a recent study from India proposed that thrombocytopenia should be added to the World Health Organization (WHO) criteria for the definition of severe malaria (Ahamed et al. 2019).

There is a significant positive association between thrombocytopenia and P. falciparum malaria. The thrombocytopenia is usually not associated with bleeding even in patients with very severe form and there is a complete recovery after treatment, so thrombocytopenia is a significant indicator for malaria, especially in correlation with typical fever and hypotension (Ahamed et al. 2019).

Effects on other blood parameters

Thrombocytopenia, defined as platelet level < 150,000/μl is notably the most important change seen in P. falciparum infection (Shiraz and Mumtaz 2012) It due to peripheral destruction or consumption by disseminated intravascular coagulation female based on WHO cut-off value (Manas et al. 2014). The pathogenesis of anaemia is obscure though it is thought to be caused by a combination of haemolysis of parasitized red cell, accelerated removal of both parasitized and un-parasitized red cells, ineffective erythropoiesis and splenic phagocytosis/pooling (Akhtar et al. 2012). Normocytic Normochromic anaemia is mostly seen in P. falciparum infection. The significant decrease in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) could possibly be due to microcytosis resulting from nutritional or iron deficiency anaemia (Quintero et al. 2011). This finding is in contrast with Manas et al. (2014) significant higher MCV and MCH in malaria infected patients in comparison to non-malaria infected patients. In conclusion, P. falciparum malaria infection demonstrated a significant impact on haematological parameters. Plasmodium falciparum malaria infected children exhibit important haematological changes with low PCV, Hb, WBC and platelet being the most important predictors of malaria infection. The artemisinin derivatives show variable effects on a series of blood cell indices including, inhibition of lymphocyte proliferation and depression of neutrophil phagocytic ability (Adjei et al. 2020).

Administration of the anti-malaria drugs AQ and CQ for three days does not alter the values of MCV, MCH, and MCHC significantly; however, after seven days of administration, significant reductions in MCV and MCHC are observed. This is a possible indication that prolonged administration (for more than three days) of AQ and CQ could lead to a microcytic variety of anemia (Ugwu et al. 2021). As hemoglobin, the other red blood indices i.e. RBC count, PCV, MCV, MCH, MCHC, RDW not much altered. Muwonge et al. (2013). This could probably be because uncomplicated malaria is associated with milder biochemical changes, for example, a lower production of cytokines, less endothelial cell activation, milder changes in the coagulation profile, less sequestration, and less hemolysis as opposed to complicated/severe malaria. Chandra and Chandra (2013) also observed the similar findings.

Increase in mean corpuscular volume (MCV) in malaria-infected patients might be due to anemia associated with malaria that causes increased rate of RBC production leading to the release of immature RBCs into blood circulation. Furthermore due to abnormalities in the nitric oxide (NO) levels that occur in malarial infection, the mean corpuscular volume might be increased. In addition, NO can inhibit the enzyme methionine synthase, so functional vitamin B12 deficiency state may occur, which can lead to megaloblastic anemia, and studies suggested that NO is associated with the low serum level of vitamin B12 (Sirak et al. 2016).

Anemia in this study was mainly normocytic normochromic. Similar finding was there in the study by Saurabh Srivastava (Srivastava et al. 2015). In the majority of cases, anaemia does not require any treatment and improves gradually; but in a few cases, blood transfusion (packed red blood cells) may be required. Elevation of ESR was observed in 78% of cases in concordance with the study by Akaninwor et al. (2013). Elevation of ESR has been reported in acute and chronic infections, chronic inflammatory disorders, malignancies especially Hodgkin's disease tissue necrosis and pregnancy. ESR is used by some researchers as basis for the diagnosis and monitoring of therapeutic intervention of malaria. They suggested that ESR was elevated during acute malaria infection and declined with recovery. However, measurement of ESR is often used as a non-specific test for acute illness and may reflect the acute process of the disease (Akaninwor et al. 2013).

Limitations of the study and recommendation

The limitations of some of these studies were cross-sectional, small sample size and confounding factors that may affect hematological parameters, such as nutritional deficiencies and genetic backgrounds of patients, common bacterial, viral, and helminth infections. We have not excluded these confounding factors. We recommend studies on larger sample size and using experimental designs (case control, etc.) and with exclusion of the confounding factors (Kumbhar et al. 2019).

Conclusion

Thrombocytopenia is a significant indicator for malaria, especially in correlation with typical fever and hypotension. Hematological profile together with microscopy will enable rapid diagnosis, prompt treatment and further complications can be avoided. The administration of AQ or CQ at their recommended duration (three days) is relatively safe. On prolonged administration, AQ (and not CQ) could predispose the body to anemia and bleeding tendencies; however, both drugs could cause thrombocytopenia when administered beyond the recommended duration of three days. This, however, may not be a demerit, as there are beneficial effects of regulation of platelet count and activity. The reality of the safety margin of CQ is quite evident in this study. More studies need to be conducted along this line to delineate such mechanisms. Treatment of children with acute uncomplicated malaria showed an overall marginally lower reticulocyte count change dynamics in those treated with artesunate-amodiaquine or artemether-lumefantrine, compared with those treated with amodiaquine. However, the discrepancy between this study and the only available report on this subject call for additional studies, and post-marketing surveillance to confirm that any such changes are minimal and reversible, even in potential vulnerable patient sub-groups, is indicated.

Funding

No funding was received.

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.