ASSESSMENT OF SOME HAEMOTOLOGICAL PARAMETER ON MALARIA PATIENT AT GENERAL HOSPITAL OWERRI
ABTRACT
Hematological parameters are measurable indices of the blood that serve as a marker for disease diagnosis. The aim of this study was to evaluate hematological parameters of patients with malaria in Nigeria. This was a prospective study in which the full blood count of patients, with malaria attending the General Hospital Owerri, Nigeria from March to May 2007, were analyzed. Data was analyzed using SPSS version 15.0 software. P value of less than or equal to 0.05 is considered as statistically significant. A total of 100 patients were recruited for the study. Fifty patients had P.falciparum malaria while the remaining was negative and were used as controls. There were more males with malaria (n=30) than females (n=20) and thirty two (64%) were below 5years while 18(36%) were above 5 years. Lymphocyte and monocyte counts were elevated among patients with malaria relative to the control while haemoglobin and platelet levels were significantly decreased (P ≤0.05). The platelet level decreases as the degree of malaria parasitaemia increases. Haemalogical parameters in patients with malaria infection are deranged. Thrombocytopenia could be used to determine presence and severity of malaria.
 CHAPTER ONE
1.0    INTRODUCTION
        Malaria is one of the most prevalent human infections worldwide resulting in 225 million cases each year (WHO, 2010). It is caused by protozoa parasite of the genus plasmodium which infects and destroys red blood cells. Four species of plasmodia (P. falciparum, P. malariae, P. ovale and P. vivax) cause malaria in humans of which P. falciparum is the most common cause of morbidity and mortality (Taylor-Robinson, 1998; Das and Pan, 2006).Malaria kills an average of 1 million Patients in Africa annually, Snow et al., (2005). In Nigeria about 96 millionpeople are exposed to malaria, and out of these 64 million people get infected and almost 300,000 deaths are being reported annually in the general population, of which over 100,000 deaths are of Patients (Alaribe et al.,
2006). Haematological parameters are measurable indices of blood that serve as a marker for disease diagnosis (Petel et al., 2004). Haematological abnormalities such as anaemia and thrombocytopaenia have been observed in patients with malaria (Ladhani et al. 2002; et al. 2007).
The key feature of the biology of the Plasmodium falciparum, the predominant malaria species, is the ability of the infected red blood cells to adhere to the lining of the small blood vessels (Richard et al., 1998). Such sequestered parasites provide considerable obstruction to tissue perfusion. In addition, it is becoming clear that in severe malaria there may be marked reductions in the deformability of uninfected RBCs (Dondorp et al., 2000). RBCs destruction is an inevitable part of malaria, and anaemia further compromises oxygen delivery. Severe anaemia may arise from multiple poorly understood processes including acute haemolysis of uninfected RBCs and dyserythropoeisis, as well as through the interaction of malaria infection with other parasites infection and with nutritional deficiencies (Dondorp et al., 2000). The aim of this study was to determine changes in the haematological parameters of Patients with malaria infection in Nigerian population of Africa. Alterations in the haematological indices may strengthen the suspicion of malaria, prompting more meticulous search for malaria parasite, and timely institution of specific therapy.
Malaria which is the most prevalent infectious disease in the tropical and subtropical regions of the world is of great public health importance (Mishra et al., 2003; Umar et al., 2007; Mia et al., 2011).The World Health Organization reports that malaria, the deadly parasitic disease is responsible for nearly ninety percent of death in Africa (Ogbodo et al., 2010). One-fifth of infants’ death in Africa is caused by the scourge of malaria (Snow et al., 2005; WHO, 2010). In Nigeria, approximately 0.25 million deaths of Patients under the age of five is caused by malaria yearly (UNICEF, 2009). Typhoid fever which is also endemic in Africa is more severe in infants and the elderly (Preston and Boreszyk, 1994; Gatsing et al., 2006). Both malaria and typhoid exhibit close symptomatology and epidemiology (Nsutebu and Ndumbe, 2001; Brian and Wahinuddin, 2006). The first case of malaria-typhoid co-infection occurred among American soldiers in 1862 (Bynum, 2002). The high incidence and prevalence of malaria-typhoid co-infection became popular almost ten years ago whereas the fact that malaria has been prevalently high is already recognized and accepted (Uneke, 2008). The onset and progression of the malaria infection is characterized by vast alterations in hematological and biochemical parameters (Bidaki and Dalimi, 2003). The World health Organization’s (WHO) criteria acknowledges that some biochemical and hematological features should raise the severity of malaria (World Health Organization, 2000).
In different parts of the world including Nigeria, scientific materials on hematological and biochemical alterations in acute falciparum malaria are available (Mishra et al., 2003; Egwunyenga et al., 2004; Bidaki and Dalimi, 2003; Udosen, 2003), but none have really been reported from Sango-Ota, Ogun State, Nigeria and also scientific information on the impact of malaria-typhoid co-infection on hematological and biochemical parameters are scanty. This study examined the effect of malaria and malaria-typhoid co-infection on some hematological and biochemical indices. The study population includes only the febrile patients that have been clinically said to have malaria and malaria-typhoid co-infection from the results of their malaria and widal tests, respectively.
1.1    AIM AND OBJECTIVE OF THE STUDY
The aim of this study was to determine changes in the hematological parameters of patients with malaria infection in Nigerian population of Africa. Alterations in the hematological indices may strengthen the suspicion of malaria, prompting more meticulous search for malaria parasite, and timely institution of specific therapy. George and Ewelike Ezeani 769
1.2    LITERATURE REVIEW
Malaria is a mosquito-borne infectious disease of humans and other animals caused by protists (a type of microorganism) of the genus Plasmodium. It begins with a bite from an infected female mosquito, which introduces the protists via its saliva into the circulatory system, and ultimately to the liver where they mature and reproduce. The disease causes symptoms that typically include fever and headache, which in severe cases can progress to coma or death. Malaria is widespread in tropical and subtropical regions in a broad band around the equator, including much of Sub-Saharan Africa, Asia, and the Americas.
Five species of Plasmodium can infect and be transmitted by humans. The vast majority of deaths are caused by P. falciparum while P. vivax, P. ovale, and P. malariae cause a generally milder form of malaria that is rarely fatal. The zoonotic species P. knowlesi, prevalent in Southeast Asia, causes malaria in macaques but can also cause severe infections in humans. Malaria is prevalent in tropical regions because the significant amounts of rainfall, consistently high temperatures and high humidity, along with stagnant waters in which mosquito larvae readily mature, provide them with the environment they need for continuous breeding. Disease transmission can be reduced by preventing mosquito bites by distribution of mosquito nets and insect repellents, or with mosquito-control measures such as spraying insecticides and draining standing water.
The World Health Organization has estimated that in 2010, there were 216 million documented cases of malaria. Around 655,000 people died from the disease (roughly 2000 per day), most of whom are Patients in Africa.[1] The actual number of deaths may be significantly higher, as precise statistics are unavailable in many rural areas, and many cases are undocumented. Malaria is commonly associated with poverty and is also a major hindrance to economic development.
Despite a clear need, no vaccine offering a high level of protection currently exists. Efforts to develop one are ongoing. Several medications are available to prevent malaria in travelers to malaria-endemic countries (prophylaxis). A variety of anti-malaria medications are available. Severe malaria is treated with intravenous or intramuscular quinine or, since the mid-2000s, the artemisinin derivative artesunate, which is superior to quinine in both Patients and adults and is given in combination with a second anti-malaria such as mefloquine. Resistance has developed to several anti-malaria drugs, most notably chloroquine and artemisinin.
1.3    MEANING OF MALARIA
The word malaria comes from 18th century Italian mala meaning "bad" and aria meaning "air". Most likely, the term was first used by Dr. Francisco Torti, Italy, when people thought the disease was caused by foul air in marshy areas. It was not until 1880 that scientists discovered that malaria was a parasitic disease which is transmitted by the anopheles mosquito. The mosquito infects the host with a one-cell parasite called plasmodium. Not long after they found out that Malaria is transmitted from human-to-human through the bite of the female mosquito, which needs blood for her eggs.  According to Medilexicon's medical dictionary, Malaria is "A disease caused by the presence of the sporozoan Plasmodium in human or other vertebrate erythrocytes, usually transmitted to humans by the bite of an infected female mosquito of the genus Anopheles that previously sucked blood from a person with malaria. Malaria is also known as Jungle fever, Marsh fever, Paludal fever Approximately 40% of the total global population is at risk of Malaria infection. During the 20th century the disease was effectively eliminated in the majority of non-tropical countries. Today Malaria causes over 350 million human acute illnesses, as well as at least one million deaths annually. The anopheles mosquito exists in most tropical and many sub-tropical countries of Latin America and the Caribbean, Africa, Oceania, and Asia.  According to WHO (World Health Organization), the majority of Malaria deaths occur among Patients in sub-Saharan Africa, killing an African child every 30 seconds. Not only is Malaria associated with poverty, it is also a cause of poverty and an important obstacle to economic development.
1.4    HISTORY OF MALARIA
Malaria has infected humans for over 50,000 years, and Plasmodium may have been a human pathogen for the entire history of the species. Close relatives of the human malaria parasites remain common in chimpanzees. Some evidence suggests that the most virulent strain of human malaria may have originated in gorillas.
References to the unique periodic fevers of malaria are found throughout recorded history, beginning in 2700 BC in China. Malaria may have contributed to the decline of the Roman Empire, and was so pervasive in Rome that it was known as the "Roman fever". Several regions in ancient Rome were considered at-risk for the disease because of the favourable conditions present for malaria vectors. This included areas such as southern Italy, the island of Sardinia, the Pontine Marshes, the lower regions of coastal Etruria and the city of Rome along the Tiber River. The presence of stagnant water in these places was preferred by mosquitoes for breeding grounds. Irrigated gardens, swamp-like grounds, runoff from agriculture, and drainage problems from road construction led to the increase of standing water
 The term malaria originates from Medieval Italian: mala aria — "bad air"; the disease was formerly called ague or marsh fever due to its association with swamps and marshland. Malaria was once common in most of Europe and North America, where it is no longer endemic, though imported cases do occur. Malaria was the most important health hazard encountered by U.S. troops in the South Pacific during World War II, where about 500,000 men were infected. According to Joseph Patrick Byrne, "Sixty thousand American soldiers died of malaria during the African and South Pacific campaigns." Scientific studies on malaria made their first significant advance in 1880, when Charles Louis Alphonse Laveran—a French army doctor working in the military hospital of Constantine in Algeria—observed parasites inside the red blood cells of infected people for the first time. He therefore proposed that malaria is caused by this organism, the first time a protist was identified as causing disease.
For this and later discoveries, he was awarded the 1907 Nobel Prize for Physiology or Medicine. The malarial parasite was called Plasmodium by the Italian scientists Ettore Marchiafava and Angelo Celli. A year later, Carlos Finlay, a Cuban doctor treating people with yellow fever in Havana, provided strong evidence that mosquitoes were transmitting disease to and from humans. This work followed earlier suggestions by Josiah C. Nott,  and work by Sir Patrick Manson, the "father of tropical medicine", on the transmission of filariasis.
In April 1894, a Scottish physician Sir Ronald Ross visited Sir Patrick Manson at his house on Queen Anne Street, London. This visit was the start of four years of collaboration and fervent research that culminated in 1898 when Ross, who was working in the Presidency General Hospital in Calcutta, proved the complete life-cycle of the malaria parasite in mosquitoes. He thus proved that the mosquito was the vector for malaria in humans by showing that certain mosquito species transmit malaria to birds. He isolated malaria parasites from the salivary glands of mosquitoes that had fed on infected birds.
For this work, Ross received the 1902 Nobel Prize in Medicine. After resigning from the Indian Medical Service, Ross worked at the newly established Liverpool School of Tropical Medicine and directed malaria-control efforts in Egypt, Panama, Greece and Mauritius.[103] The findings of Finlay and Ross were later confirmed by a medical board headed by Walter Reed in 1900. Its recommendations were implemented by William C. Gorgas in the health measures undertaken during construction of the Panama Canal. This public-health work saved the lives of thousands of workers and helped develop the methods used in future public-health campaigns against the disease.
The first effective treatment for malaria came from the bark of cinchona tree, which contains quinine. This tree grows on the slopes of the Andes, mainly in Peru. The indigenous peoples of Peru made a tincture of cinchona to control malaria. The Jesuits noted the efficacy of the practice and introduced the treatment to Europe during the 1640s, where it was rapidly accepted.  It was not until 1820 that the active ingredient, quinine, was extracted from the bark, isolated and named by the French chemists Pierre Joseph Pelletier and Joseph Bienaimé Caventou.[106][107]
Quinine become the predominant malarial medication until the 1920s, when other medications began to be developed. In the 1940s, chloroquine replaced quinine as the treatment of both uncomplicated and severe malaria until resistance supervened, first in Southeast Asia and South America in the 1950s and then globally in the 1980s. Artemisinins, discovered by Chinese scientist Tu Youyou in the 1970s from the plant Artemisia annua, became the recommended treatment for P. falciparum malaria, administered in combination with other anti-malarias as well as in severe disease.
The first pesticide used for indoor residual spraying was DDT.  Although it was initially used exclusively to combat malaria, its use quickly spread to agriculture. In time, pest control, rather than disease control, came to dominate DDT use, and this large-scale agricultural use led to the evolution of resistant mosquitoes in many regions. The DDT resistance shown by Anopheles mosquitoes can be compared to antibiotic resistance shown by bacteria. During the 1960s, awareness of the negative consequences of its indiscriminate use increased, ultimately leading to bans on agricultural applications of DDT in many countries in the 1970s.
Malaria vaccines have been an elusive goal of research. The first promising studies demonstrating the potential for a malaria vaccine were performed in 1967 by immunizing mice with live, radiation-attenuated sporozoites, which provided significant protection to the mice upon subsequent injection with normal, viable sporozoites. Since the 1970s, there has been a considerable effort to develop similar vaccination strategies within humans.
1.5    FIVE TYPES OF MALARIA
Plasmodium vivax (P. vivax) - milder form of the disease, generally not fatal. However, infected people still need treatment because their untreated progress can also cause a host of health problems. This type has the widest geographic distribution globally. About 60% of infections in India are due to P. vivax. This parasite has a liver stage and can remain in the body for years without causing sickness. If the patient is not treated, the liver stage may re-activate and cause relapses - malaria attacks - after months, or even years without symptoms.
Plasmodium malariae (P. malariae) - milder form of the disease, generally not fatal. However, the infected human still needs treatment because no treatment can also lead to a host of health problems. This type of parasite has been known to stay in the blood of some people for several decades.
Plasmodium ovale (P. ovale) - milder form of the disease, generally not fatal. However, the infected human still needs to be treated because it may progress and cause a host of health problems. This parasite has a liver stage and can remain in the body for years without causing sickness. If the patient is not treated, the liver stage may re-activate and cause relapses - malaria attacks - after months, or even years without symptoms.
Plasmodium falciparum (P. faliparum) - the most serious form of the disease. It is most common in Africa, especially sub-Saharan Africa. Current data indicates that cases are now being reported in areas of the world where this type was thought to have been eradicated.
Plasmodium knowlesi (P. knowlesi) - causes malaria in macaques but can also infect humans.
1.6    HOW HUMAN BECOME INFECTED WITH MALARIA
The female Anopheles mosquito transmits the parasite to a human when it takes a blood meal - it bites the human in order to feed on blood. Only the female Anopheles mosquito can transmit malaria, and it must have been infected through a previous blood meal taken from an infected human. When the mosquito bites an infected person a minute quantity of the malaria (plasmodium) parasite in the blood is taken. Approximately one week later that same infected mosquito takes its next blood meal. The plasmodium parasites mix with the mosquito's saliva and are injected into the host (human being).
1.6.1 Human-to-human transmission of Malaria
As the parasite exists in human red blood cells, malaria can be passed on from one person to the next through organ transplant, shared use of needles/syringes, and blood transfusion. An infected mother may also pass malaria on to her baby during delivery (birth) - this is called 'congenital malaria'.
1.7    SIGNS AND SYMPTOMS OF MALARIA
The signs and symptoms of malaria typically begin 8–25 days following infection;  however, symptoms may occur later in those who have taken anti-malaria medications as prevention. Initial manifestations of the dieases—common to all malaria species—are similar to flu-like symptoms. The presentation may include headache, fever, shivering, arthralgia (joint pain), vomiting, hemolytic anemia, jaundice, hemoglobinuria, retinal damage,[5] and convulsions. Approximately 30% of people however will no longer have a fever upon presenting to a health care facility.
The classic symptom of malaria is paroxysm—a cyclical occurrence of sudden coldness followed by rigor and then fever and sweating, occurring every two days in P. vivax and P. ovale infections, and every three days (tertian fever) for P. malariae. P. falciparum infection can cause recurrent fever every 36–48 hours (quartan fever) or a less pronounced and almost continuous fever.
Severe malaria is usually caused by P. falciparum, and typically arises 6–14 days after infection. Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly (enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur. Renal failure is a feature of blackwater fever, where hemoglobin from lysed red blood cells leaks into the urine. Cerebral malaria (encephalopathy specifically related to P. falciparum infection) is associated with retinal whitening, which may be a useful clinical sign in distinguishing malaria from other causes of fever. Individuals with severe malaria frequently exhibit neurological symptoms, including abnormal posturing, nystagmus, disconjugate gaze (failure of the eyes to turn together in the same direction), opisthotonus, seizures, or coma.
1.8    CAUSES OF MALARIA
Malaria parasites are from the genus Plasmodium (phylum Apicomplexa). In humans, malaria is caused by P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi. Among those infected, P. falciparum is the most common species identified (~75%) followed by P. vivax (~20%). P. falciparum accounts for the majority of deaths; non-falciparum species have been found to be the cause of about 14% of cases of severe malaria in some groups. P. vivax proportionally is more common outside of Africa.  There have been documented human infections with several species of Plasmodium from higher apes; however, with the exception of P. knowlesi—a zoonotic species that causes malaria in macaques—these are mostly of limited public health importance.
1.9    LIFE CYCLE
The definitive hosts for malaria parasites are female mosquitoes of the Anopheles genus, which act as transmission vectors to humans and other vertebrates, the secondary hosts. Young mosquitoes first ingest the malaria parasite by taking a blood meal from an infected vertebrate carrier. Once ingested, the parasite gametocytes taken up in the blood differentiate into male or female gametes and fuse in the mosquito's gut.
This produces an ookinete that penetrates the gut lining and produces an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that migrate through the mosquito's body to the salivary glands, where they are then ready to infect a new vertebrate host. The sporozoites are injected into the skin, alongside saliva, when the mosquito takes a subsequent blood meal. This type of transmission is occasionally referred to as anterior station transfer.
Only female mosquitoes feed on blood; male mosquitoes feed on plant nectar, and thus do not transmit the disease. The females of the Anopheles genus of mosquito prefer to feed at night. They usually start searching for a meal at dusk, and will continue throughout the night until taking a meal. Malaria parasites can also be transmitted by blood transfusions, although this is rare.
 1.10    PATHOGENESIS
Malaria infection develops via two phases: one that involves the liver or hepatic system (exoerythrocytic), and one which involves red blood cells, or erythrocytes (erythrocytic). When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver where they infect hepatocytes, multiplying asexually and asymptomatically for a period of 8–30 days.
After a potential dormant period in the liver, these organisms differentiate to yield thousands of merozoites, which, following rupture of their host cells, escape into the blood and infect red blood cells to begin the erythrocytic stage of the life cycle.The parasite escapes from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell.
Within the red blood cells, the parasites multiply further, again asexually, periodically breaking out of their hosts to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cell.
Some P. vivax sporozoites do not immediately develop into exoerythrocytic-phase merozoites, but instead produce hypnozoites that remain dormant for periods ranging from several months (6–12 months is typical) to as long as three years. After a period of dormancy, they reactivate and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in P. vivax infections, although their existence in P. ovale is uncertain.
The parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance. However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels, thereby sequestering the parasite from passage through the general circulation and the spleen.The blockage of the microvasculature causes symptoms such as in placental and cerebral malaria. In cerebral malaria the sequestrated red blood cells can breach the blood–brain barrier possibly leading to coma. Micrograph of a placenta from a stillbirth due to maternal malaria. H&E stain. Red blood cells are anuclear; blue/black staining in bright red structures (red blood cells) indicate foreign nuclei from the parasites
Although the red blood cell surface adhesive proteins (called PfEMP1, for P. falciparum erythrocyte membrane protein 1) are exposed to the immune system, they do not serve as good immune targets, because of their extreme diversity; there are at least 60 variations of the protein within a single parasite and even more variants within whole parasite populations.The parasite switches between a broad repertoire of PfEMP1 surface proteins, thus staying one step ahead of the pursuing immune system.
Some merozoites turn into male and female gametocytes. If a mosquito pierces the skin of an infected person, it potentially picks up gametocytes within the blood. Fertilization and sexual recombination of the parasite occurs in the mosquito's gut. New sporozoites develop and travel to the mosquito's salivary gland, completing the cycle. Pregnant women are especially attractive to the mosquitoes, and malaria in pregnant women is an important cause of stillbirths, infant mortality and low birth weight, particularly in P. falciparum infection, but also in other species infection, such as P. vivax.
1.11    GENETIC RESISTANCE
Due to the high levels of mortality and morbidity caused by malaria—especially the P. falciparum species—it is thought to have placed the greatest selective pressure on the human genome in recent history. Several diseases may provide some resistance to it including sickle cell disease, thalassaemias, glucose-6-phosphate dehydrogenase deficiency as well as the presence of Duffy antigens on the subject's red blood cells.
The impact of sickle cell anemia on malaria immunity is of particular interest. Sickle cell anemia causes a defect to the hemoglobin molecule in the blood. Instead of retaining the biconcave shape of a normal red blood cell, the modified hemoglobin S molecule causes the cell to sickle or distort into a curved shape. Due to the sickle shape, the molecule is not as effective in taking or releasing oxygen, and therefore malaria parasites cannot complete their life cycle in the cell. Individuals who are homozygous for sickle cell anemia seldom survive this defect, while those who are heterozygous experience immunity to the disease. Although the potential risk of death for those with the homozygous condition seems to be unfavourable to population survival, the trait is preserved because of the benefits provided by the heterozygous form.
1.13    MALARIAL HEPATOPATHY
Hepatic dysfunction as a result of malaria is rare and is usually a result of a coexisting liver condition such as viral hepatitis and chronic liver disease. Hepatitis, which is characterised by inflammation of the liver, is not actually present in what is called malarial hepatitis; the term as used here invokes the reduced liver function associated with severe malaria.While traditionally considered a rare occurrence, malarial hepatopathy has seen an increase in malaria-endemic areas, particularly in Southeast Asia and India.Liver compromise in people with malaria correlates with a greater likelihood of complications and death.