Effect of Plasmodium falciparum parasitemia on platelet count in Nigerian patients

Effect of Malaria Parasitemia (Plasmodium Falciparum) on Platelet Count in Man Abstract
Malaria has been determined to be the most common cause of childhood and adult morbidity in Nigeria. It has varied hematological consequences. Plasmodium falciparum is the deadliest Plasmodium species. The hematological abnormalities produced by Plasmodium include thrombocytopenia, anemia, and splenomegaly. For the Nigerian population, we have established that thrombocytopenia could be caused by the parasitemia or in other instances could be due to platelet aggregation. There is considerable platelet count reduction for patients with malaria when compared with patients who do not have malaria. This indicates that platelet loss could be due to the changes that take place in the host caused by the malaria parasites. Identifying the predictors of malaria infection is vital for the effective treatment of the disease. Introduction to Malaria Malaria is still the world's most prevalent, parasitic disease, afflicting approximately 2.2 billion people. Malaria infection remains a major public health problem that causes mortality and morbidity in all age groups. In Africa, south of the Sahara, approximately 90 million clinical cases of malaria occur every year, which cause 1-2 million deaths of the 3.5 million estimated death worldwide. It is estimated that in Nigeria, at least 35,000 children die every year from malaria effects and this accounts for 35% of all the mortalities in infants and children. The deadliness of plasmodium falciparum when compared to other species of plasmodium likely stems from the parasite density it is able to achieve that is typically 100 times higher than of any other species. Plasmodia being a blood parasite produces hematological abnormalities that will include thrombocytopenia, anemia, and splenomegaly. Hematological parameters are affected by factors like genetics and ethnic differences, age, sex, environmental factors like altitude and dietary pattern (Utuk, Ikpeme, Emodi, & Essien, 2014, p. 28). Considering that these factors differ depending on the population and geographical are being studied, it should not be surprising that differences are reported in these parameters worldwide. Genetic factors also contribute significantly to the variance in blood cell lines. Thrombocytopenia is the most common platelet abnormality that is seen in malaria and it occurs in over 80 percent of malaria cases and in most cases it is severe. Nigeria is considered to be a holoendemic country and it has a varying prevalence rate of thrombocytopenia in malaria that has been documented to be as low as 13% and reaches a high of 90% (Utuk et al., 2014, p. 29). Thrombocytopenia with falciparum parasitemia is mostly documented in adult populations and there have been few documentations for children in Nigeria. While documentation on children is limited, it is well understood that falciparum parasitemia mostly infects pregnant women and children. This study aims at identifying the effects of Plasmodium falciparum on platelet count for people living in Nigeria.

History of Plasmodium Falciparum and Malaria Infection Plasmodium falciparum is a protozoan parasite causing malaria and the deadliest species of plasmodium. It has been established that the species originated from the malarial parasite Laverania that is found in gorillas (Liu et al., 2010, p. 421). The first person to identify the parasite was Alphonse Laveran in 1880 and he named it Oscillaria malariae. In 1897, Ronald Ross established that the parasite was transmitted by mosquitoes. Giovanni Battista Grassi comprehensibly determined its transmission from a female anopheline mosquito to humans in 1898 and in 1897 the name Plasmodium falciparum was created by William Welch. The name was formally adopted by the International Commission on Zoological Nomenclature (ICZN) in 1954. During its lifecycle, plasmodium falciparum takes up different forms. The human infective stage is the sporozoites from the salivary gland of a mosquito into the bloodstream. The parasite will then infect the liver and it undergoes asexual reproduction that is followed by its insertion into the red blood cells where the parasite will undergo another round of replication (Liu et al., 2010, p. 421). P. falciparum modifies the surface of an infected red blood cell, which causes it to adhere to blood vessels and to other red blood cells as well. In severe cases, this adherence will lead to obstruction of microcirculation that will result in the dysfunction of many organs. The symptoms of the infection will vary depending on the severity of the infection and will mostly present a range of signs like vomiting diarrhea, flulike symptoms, kidney failure, shock, coma, and death. P. falciparum has been reported to mostly infect pregnant women and children below 5 years.

Epidemiology of Malaria Plasmodium falciparum is found across the globe in all continents apart from Europe. The World Health Organization (WHO) reported that 219 million people suffered from malaria in 2017, which was an increase from the 2016 number of 216 million. Of the infected number 435,000 people died. In Nigeria, within the same year, it is estimated that the confirmed cases were 11 million and out of this, it is estimated that there were 79,000 deaths. This clearly indicates that the infection is prevalent in Africa where 92% of malaria death occur (Adedapo, Falade, Kotila, & Ademowo, 2007, p. 267). The most affected age group if children below 58 years and 61 percent of malaria deaths occur in this age group. Nigeria is reported to have the highest incidence rate in Africa accounting for 27 percent of the total global cases. Europe is a malaria-free region and this shows that it is possible to eradicate the disease (Olasehinde et al., 2019, p. 1941). The parasite and its disease was well known in Europe and was eradicated by the use of medical programmes like insecticide spraying, environmental engineering, and drug therapy since the early 20th century. This resulted in the eradication of the disease in the 1970s. If it was eradicated in Europe, then even in Africa and globally it can be eradicated as well.

Life Cycle of Malaria Parasite (Plasmodium Falciparum) The life cycle of the malaria parasite involves two hosts namely the mosquito and humans. Humans act as the intermediate hosts where asexual reproduction takes place. The anopheline mosquito is the definitive host that harbors the sexual reproduction stage. The process begins with a blood meal, where a malaria-infected Anopheles mosquito transfers saliva and sporozoites into the human circulatory system. The saliva contains antihemostatic and anti-inflammatory enzymes, which disrupt blood clotting and also inhibit pain reaction (Adedapo et al., 2007, p. 269). Normally, the immune system will clear the sporozoites from the blood circulation in 30 minutes. However, a few will escape and they will invade the liver cells. Gliding is the mode used by the sporozoites to move in the bloodstream. Once in the liver, the parasite will lose its apical complex and transform into a trophozoite. Asexual reproduction will take place in the liver through exoerythrocytic schizogony that results in merozoites. The merozoites are eventually released into the bloodstream. During the multiplication via asexual reproduction, the parasites will cause no symptoms and this process takes 7 to 10 days. The merozoites will invade the red blood cells (erythrocytes) (McMorran, 2019, p. 68). Here they will multiply again until the cell bursts, which results in them invading more erythrocytes. This process is repeated and it causes fever each time the parasites break free and invade the erythrocytes. It is during this stage that the symptoms of malaria-like fever, anemia, and neurological disorder will be manifested. Some of the erythrocytes will leave the cycle of asexual multiplication and instead of them replicating, the merozoites will develop into sexual forms of the parasite that is called gametocytes, which circulate in the bloodstream. The gametocytes will take about 7-15 days for them to reach full maturity in the gametocytogenesis process. The gametocytes will be ingested by another Anopheles mosquito during a blood meal. The parasite will then undergo a sporogonic cycle when in the mosquito. The microgametes will penetrate the macrogametes resulting in zygotes while in the mosquito's stomach. The zygotes will then become elongated and motile (ookinetes) that invade the midgut wall of the mosquito where they will develop into oocysts. The oocysts will grow, rupture, and release sporozoites. The sporozoites make their way into the mosquito's salivary glands where they will be inoculated into another human host thereby perpetuating the malaria life cycle.

Figure 1: CDC illustration of the life cycles of malaria parasites Signs and Symptoms of Malaria If health providers are not alert to the possibility of malaria, they can miss to diagnose uncomplicated malaria. Considering that malaria can progress to severe forms rapidly and become fatal, malaria should be considered in patients who are located in malaria-prone areas like Nigeria. The most frequent symptoms of malaria include fever and chills that can in some cases be accompanied by headache, vomiting and diarrhea, myalgias, and arthralgias (McMorran, 2019, p. 67). For rare uncomplicated infection, the symptoms are flu-like and the attack lasts about 6-10 hours, with a presentation of a hot stage, cold stage, and sweat stage. During these stages, the infected person will show symptoms of sweats, chills, headache, nausea, body aches, vomiting, and malaise (Adedapo et al., 2007, p. 266). Other clinical features that appear are thrombocytopenia, hypoglycemia, anemia, splenomegaly, pulmonary or renal dysfunction, and neurologic changes. The symptoms can vary depending on the infecting species, immune status of the individual, and the level of parasitemia (Olasehinde et al., 2019, p. 1942). In most cases, infections caused by plasmodium falciparum will progress to severe, potentially fatal forms that will involve the central nervous system (cerebral malaria), severe anemia, acute renal failure, or acute respiratory distress syndrome.

The Effects of Malaria Parasitemia on Platelet-Count Thrombocytopenia is the most commonly reported condition for patients with malaria caused by plasmodium falciparum. It has also been established that in Nigeria patients who are positive for malaria have a high prevalence of thrombocytopenia as compared to those who are malaria negative (Yusuf, Abdullahi, Idris, & Jobbi, 2019, p. 47). According to (Yusuf et al., 2019, p. 47), the mechanisms that lead to thrombocytopenia in malaria are well known and there are prognostic significance and the link with poor outcomes. Platelets do play a vital protective role in fighting erythrocytic infection by malaria parasites. Thrombocytopenia is a condition where a patient will have a low blood platelet count. This condition is mainly caused by an infection. In other cases, it can also be caused by some medications. Thrombocytopenia affects both adults and children. In malaria cases, thrombocytopenia occurs due to the continued destruction and attack of red blood cells during the multiplication of merozoites (del Portillo et al., 2012, p. 345). While it is not well known what happens to platelet when a patient has malaria, del Portillo et al. (2012, p. 345) posits that it has been well observed that in most cases the levels of platelet reduce considerably and they return to normal once the patient is not infected. The biological workings are being studied and numerous pieces of literature exist that show that thrombocytopenia is also linked to malaria infection. Multiple works of literature have shown that there might be some accelerated clearance mechanism of platelets during a malarial infection. This could be the reason why there is a reduction in platelet count. Other studies have linked the reduce number to platelet death, which can be mediated through the analogous to apoptosis process in nucleated cells that are involved in the activation of platelet caspases.
Having a high malaria parasitemia count is not an indication of the severity of malaria. There are individuals who have a high parasite count and they are not severely ill while others will have a low parasite count and be fatally ill. Therefore, using this model might not be appropriate especially considering that it is assumed that having a high parasitemia count is also an indication of low platelet count. What is most certain is that malaria can be directly linked to the reduction of platelet count in humans. The severity of malaria, however, does not seem to have an underlying impact on the platelet count. Relationship Between Platelet Aggregate Flag and Platelet-Count Platelet clumping has been found to exist in malaria infection cases and has been described as the most vital platelet abnormality observed in most studies. There have been a large number of small platelets that have been seen to be mixing up or clamping with a few giant platelets. This could be due to cytokine interference of megakaryopoiesis (Maina et al., 2010, p. 8). The peripheral blood smears from samples obtained have shown that samples with platelet aggregate flag indicated small platelet aggregates mixed with giant platelet. This could explain the reason why there is a low platelet count. The combination of large and small platelets results in the reduction in the number of platelets counted and this could explain why in some of the cases there have been reports that patients have low platelet count. According to Maina et al. (2010, p. 8) The platelet clumps are mostly made up of groups of three to twelve platelets and this would falsely be counted as a single platelet by most analyzers. In essence and to a trained one could be able to differentiate the clumping and instead count the platelets as individual platelets clumped together instead of assuming, they are one. Pseudo-thrombocytopenia is what results from the clumping of platelets and the resultant miscounting. What this means is that while there is a likelihood that patients with malaria are likely to develop thrombocytopenia, having a reduced platelet count for some patients could be attributed to pseudo-thrombocytopenia. This could also explain the lack of bleeding. Thrombocytopenia is linked to bleeding and for most patients, there has to be uncontrollable bleeding. However, in the case of malaria, bleeding has not been observed in all patients. This has led to most scientists questioning the reason for a reduced platelet count in patients.
There is another counter-argument for pseudo-thrombocytopenia in that a patient with giant platelets and increased mean platelet volume (MPV) indicates that pseudo-thrombocytopenia might not be the cause of thrombocytopenia in a patient infected with malaria. Not all samples will have microscopically detectable platelet aggregates even if they have thrombocytopenia. Increased MPV and giant platelets could be an indicator of premature release of platelets from the bone marrow and this will be in line with how true thrombocytopenia occurs. Platelet Hypersensitivity It has been noted that patients suffering from malaria their platelets become hypersensitive. The hypersensitivity is caused by the addition of ADP between 1.0 and 5.0 micro M. Having hypersensitive platelets does enhance hemostatic responses and this could be the reason why patients with malaria rarely have bleeding episodes even though there is a clear indication of the presence of thrombocytopenia (Maina et al., 2010, p. 8). When platelets are hypersensitive, they tend to react to the slightest triggers. This increased hypersensitivity increases the number of platelets produced and circulating in the bloodstream, which also increases the platelet removal by splenic pooling. An increase in platelet removal results in a reduction of platelet and this leads to thrombocytopenia. The immune system could also be destructing circulating platelets and this can be a leading cause of thrombocytopenia as seen in malaria. Hypersensitive platelets respond much faster than would be normal and this could be triggered by the malaria parasites during erythrocytes (del Portillo et al., 2012, p. 347). With the continued destruction of red blood cells, there is also a likelihood that platelets are destroyed and this would increase their production to restore the decreased platelets in the blood. However, this increment could be counterproductive since the platelet is destroyed by the splenic process.

Causes of Thrombocytopenia in Malaria Infection Antibody-Mediated Platelet Destruction
Antibody-mediated platelet destruction occurs when there is a dysfunctional proliferation of autoreactive T cells. These T cells will induce uncontrollable development of autoantibody producing B cells that leads to the continued anti-platelet autoimmunity. The autoimmune response will result in the increased destruction of platelets (Wassmer et al., 2008, p. 73). The destruction will be taking place at a much faster rate thus exceeding the compensatory capacity of bone marrow. If the bone marrow is not able to produce platelets at the same rate as they are being destroyed, then there is going to be a reduced platelet count and this continued reduction will result in the development of thrombocytopenia. The malaria causing parasites are assumed to cause the dysfunction.
Splenic Sequestration
Infections caused by malaria parasites will cause dramatic and abnormal splenic responses that are characterized by splenomegaly. The size of the spleen has been used to determine how intense malaria transmission is within the endemic regions of Nigeria. The spleen is the main organ that is charged with the development of immune responses and the elimination of parasite RBC during the erythrocytic stage of malaria infection (del Portillo et al., 2012, p. 347). However, it has been established that the parasites have formulated a method for counteracting by building chronic infections by evading ad modulating the immune responses, which results in the remodeling of the spleen that will at times provoke abnormal immune responses. These abnormal immune responses result in the elimination of vital platelet as the body is fighting the infection, which results in the reduction in platelet count leading to thrombocytopenia.
Oxidative Stress
Oxidative stress is defined as an imbalance in the body of free radicals and antioxidants. As a response to the malaria infection that is caused by the Plasmodium falciparum parasite, the body's natural defense mechanism will be activated with the involvement of phagocytes (Postma, Zuidema, Mommérs, & Eling, 1996, p. 122). Percário et al. (2012, p. 16347) state that these will, in turn, generate large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that will cause an imbalance between the establishment of oxidizing species and antioxidants activity. The imbalance created is what triggers oxidative stress. Oxidative stress is a vital mechanism of human hosts as a response to infections (Ebrahim, 2018, p. 25). In the case of malaria, this response can result in the death of the parasites. Oxidative stress has been shown by recent studies to cause thrombocytopenia that is associated with malaria.
Increased Platelet Aggregation
Malaria infection is likely to cause an increased platelet aggregation. Platelet aggregation is the clumping of small platelet with giant platelets. These clumps result in the reduction of platelet count and this causes thrombocytopenia (Klei, Meinderts, van den Berg, & van Bruggen, 2017, p. 75). Platelet aggregation reduces the number of platelets in the bloodstream. The giant platelets indicate a compensatory premature release of platelets from the spleen (Sarr et al., 2017, p. 33). This release is triggered by the host's response to a reduction in platelet count to ensure that there are enough platelets in the bloodstream. However, this results in the absorption of small platelets into the giant platelets leading to a further reduction in the count. Laboratory Diagnosis Laboratory diagnosis of malaria is done using blood smears. A blood smear is a drop of blood that is applied to and spread on a glass slide. The blood smear is then treated with a special stain and examined under a microscope for the parasite and morphology of infected blood cells. Clinical presentation of malaria is hard to determine conclusively and this is why it is recommended that a blood smear be done. Low platelet count has already been established to be a characteristic of malarial infection. Thrombocytopenia might be more common than an anemia in cases of acute malaria infection (Saravu, Docherla, Vasudev, & Shastry, 2011, p. 596). A patient with a platelet count of <100,000/mm3 and presenting with the symptoms of malaria should be assumed to be suffering from malaria (Utuk et al., 2014, p. 29). People who live in endemic areas like Nigeria presenting with the clinical signs of malaria and are positive for a low platelet count should be placed under treatment for malaria. A low platelet count is indicative of increased peripheral destruction, platelet consumption by disseminated intravascular coagulation (DIC) process or excessive removal of platelets (Klei et al., 2017, p. 74). All these are indications of abnormal responses that can be caused by malaria parasites. Platelet aggregation can also be detected via microscopic evaluation. An experienced laboratorian is able to distinguish platelet aggregation when making microscopic observations of the blood cells. This is vital for the determination of the processes and abnormalities in the host. In Nigeria, it has been established that most patients will have platelet aggregation, which reduces their overall platelet count.

Figure 2: A peripheral blood smear showing a ring form of Plasmodium falciparum (arrow) on the first day of hospitalization

Figure 3: Normal Platelet Count and Low Platelet Count
Result
We have established that hematological abnormalities are the hallmarks of malaria infection. These abnormalities are common and more pronounced in malaria infection caused by Plasmodium falciparum. This could be due to the high levels of parasitemia that is found in these patients (De Niz et al., 2016, p. 5). Nigeria is a high transmission area, we have established that anemia is one of the complications in malaria especially for pregnant women and young children. Anemia is caused by the increased removal of both non-parasitized and parasitized red blood cells. Children who are infected with Plasmodium falciparum have a significant reduction in hemoglobin when they are compared to children who are not infected (Saravu et al., 2011, p. 594). Peripheral blood smears that were examined under a microscope confirmed the presence of small platelet aggregation and this triggered the platelet aggregate flag. Platelet aggregation is detected based on their size. Worth noting is that a majority of the people infected with malaria had a platelet aggregation. This indicates that the low platelet count may be seen is as a result of platelet aggregation and not just platelet reduction in the body. Other researchers have indicated that it is possible for people or children with normal platelet counts to also have platelet aggregation. This counters the argument that platelet aggregation does reduce the total platelet count in the body. Therefore, platelet aggregation cannot be a conclusive indicator of malaria infection.
Thrombocytopenia cannot be ruled out for patients with malaria infection (Kehinde et al., 2005, p. 11). There has been a considerable number of patients who have thrombocytopenia and they have been discovered to also have acute malaria. This demonstrates that malaria parasites are the leading cause of thrombocytopenia in malaria-endemic areas. Within the Nigerian population, there could be other causes of thrombocytopenia but the leading cause is a malarial infection. For most studies, thrombocytopenia is defined as having a platelet count <150 x 103/?L or <100 x 103/?L. It is defined as severe if the platelet count is <50 x 103/?L. Clinical Management The World Health Organization (WHO) has indicated that malaria is a preventable and treatable disease. The main goal of treatment is ensuring the complete cure of the individual. This means that there should be a rapid and full elimination of the Plasmodium parasite from the individual's blood. This will prevent the progression of the infection to severe disease or death. Reducing the spread and transmission of the disease is another goal. As has been indicated in this paper, the lifecycle of malaria is dependent on two hosts the human and the anopheles’ mosquito. If we are able to cure and treat malaria in humans then the anopheles’ mosquito will not be able to spread the disease to other humans. Without the human host, the malaria parasite will not progress and we will manage to eradicate malaria completely (Shaikh, Ahmed, & Diju, 2011, p. 145). In the meantime, we need to ensure that people living in endemic areas like Nigeria are well educated and receive the necessary treatment for malaria. The WHO recommends that patients with uncomplicated malaria caused by Plasmodium falciparum be treated with artemisinin-based combination therapies (ACTs). ACTs combine two active ingredients that have different mechanisms of action (Bankole et al., 2016, p. 301). This makes the drugs most effective in the market today. To treat Plasmodium falciparum, the WHO recommends the patient be administered 5 ACTs.
Severe malaria should be treated using injectable artesunate for at least 24 hours. This should be followed with a complete 3-day treatment with an ACT once the patient is able to tolerate oral medications. In cases where injectable treatment cannot be administered and for children below 6 years of age who have severe malaria, then a pre-referral treatment should be administered with rectal artesunate before they are referred to a health care facility that will offer the patient the care needed (Falade et al., 2016, p. 294). This is mostly applicable for a rural area in Nigeria where some of the facilities do not have the requisite treatments or drugs for handling severe malaria cases. Care should be taken to ensure that a patient with severe malaria is not administered artemisinin-based injectables or artesunate suppositories as monotherapies. Initial malaria treatment using these medications can cause resistance and result in an incomplete cure. Discussions The best predictors in malaria infection remain to be thrombocytopenia and anemia. This means that laboratory diagnosis gives us the best tool for diagnosing malaria, especially in endemic areas. Since malaria has symptoms that are similar to other diseases, we must ensure we are not treating negative malaria. Laboratory diagnosis also ensures that the disease can be discovered early and this will assist in treating and preventing its transmission. Early detection will allow for treatment before the infection spreads and becomes severe. Although severe malaria can also be treated the impact it has on the patient's body can result in other complications. Having a low platelet count increases the diagnosis of malaria and it has a sensitivity of 80 percent and specificity of 84 percent. This will ensure that there is a reduction of malaria negative patients being given antimalaria drugs. In malaria-endemic regions, the combination of sensitivity and specificity ensures there is an increase in the probability of detecting malaria.