ABSTRACT
An increase level of Acute Phase Reactant Proteins (APRP) including C-reactive protein (CRP) and Antistreptolytin 0 (ASO) are –know markers of acute phase reactions. We have measured the levels of these acute phase reactants in malaria patients during before and after treatment with a view to interpret the level as either markers or predictors of disease severity and/ or prognosis. CRP and ASO were measured in fifty (50) patients (pre and post-treatment) together with - age -and sex- matched controls. Our result showed that the level of RP was statistically significant (P< 0.05 respectively) in both pre and post treated states compared with the controls. The trend was also observed with ASO titres (P < 0.05), but this tend to be reduced in the post-treatment samples. There were no significant differences in the values of pre and post treated states in both CRP and ASO titres (P>0.05 respectively). Conclusively, positive serological reactions of SO and CRP could be associated with malaria parasitaemia. Increase titre of CRP also could indicate a high level of parasitaemia while ASO titre could be regarded as a cross reaction because its titre was kept -low before and after malaria treatment. Determinate titres of ASO may be important to exclude other infection when using it as an index of streptococcus infection.
TABLE OF CONTENTS
TITLE PAGE
DEDICATION
ACKNOWLEDGEMENT
CERTIFICATION
TABLE OF CONTENTS
ABSTRACT
CHAPTER ONE
1-1.1 Introduction 1
1-1.2 History and Incidence 2
1-1.3 Transmission 3
1-1.4 Clinical features and life cycle 4
1-1.5 Mosquito vector 4-5
1-1.6 Pathogenesis 6-10
1-1.7 Sym ptornatic- Diagnosis 10-11
1-1.8 Microcospic examination 11-14
1-1.9 Vector control 14-16
1-1.10 Prophylactic-drugs 16-18
1-2 C-Reactive Protein 18-20
1-3 Tumor necrotic factor 20-22
1-4 Antistretolysin 0 22-24
1-5 Aims 25
1-6 Specific Objective 25
CHAPTER TWO
2.0 Literature Review 26
2.1 Acute phase reactant 26
2.2 Cytokines and the acute phase reactant 27-28
2.3 C-reactive protein and Malaria infection 29-31
2.4 Tumour necrotic factor and malaria infection 31-33
2.5 Cytokines contributions 33-35
CHAPTER THREE
3.0 Materials and Methods 36
3.1 Subjects 36
3.2 Sample Collection 36
3.3 Methodology 37
Procedure 38
Interpretation 38-48
Data management and Analysis 49
CHAPTER FOUR
4.0 Results 50
Table 1 51 Table 2 51
Table 3 52
Figure 1 53
Figure 54
CHAPTER FIVE
5.0 Conclusion & Discussion 55
5.1 Discussion 55-57
5.2 conclusion 57
References 58-62
Appendices 63
CHAPTER ONE
1.1.1 INTRODUCTION
Malaria is a vector-borne infectious disease caused by protozoan parasite (mosquitoes). It was once thought that the disease comes from fetid marshes, hence the malaria (bad air). In 1880, scientists discovered the real causes of ma1aria, one-cell parasite is transmitted from one person to another through the female anopheles mosquito which requires blood to nurture the eggs. Today, approximate by 400% of the world population mostly those living in the world's poorest countries are at risk of malaria. It is widespread in tropical and sub-tropical areas. Each year there are approximately 350-500 million cases of malaria. Ninety percent of death due o malaria occurs in Africa, South of Sahara, mostly among the young children (WHO, 1989).
1.1.2 HISTORY AND INCIDENCE
Historical suggest that malaria has infected human e beginning of human kind. The name "Malaria" bad air") in Italian was .first used in English in H. Walpole when describing the diseases and the shortened to malaria in the 20th century. C. Lavera as the first to identify the parasite in human blood. In 1880 R. Cross discovered mosquito transmitted malaria of four species of malaria, the most serious type is Plasmodium Falciparum
Malaria is a particular problem and a major one In of Asia, Africa and central & South America. Unless precautions are taken anyone leaving or travelling to country where malaria is present can get the disease. Malaria occurs in about 100 countries; approximately about 400/0 of the world population is at risk for contracting malaria. (Coxf et al., 2002).
1.1.3 TRANSMISSION AND CLINICAL SYMPTOMS
Malaria, one of the most health-threatening diseases in the world claims millions of life each year. Malaria parasites are transmitted by the bite of an irifected female mosquito.
Sporozoites contained in the saliva of the mosquito are inoculated into the blood of human host when the mosquito takes blood meal. Plasmodium falciparum is the malaria parasite that causes the most severe diseases within minutes after been inoculated into the human host by blood sucking mosquito, the parasite in the form of sporozoites target and invade hepatocytes (Sinnis et al., 1994) where they propagate rapidly. Thousand of merozoites as subsequently release from the liver and invade the red blood cell. Severe malaria occur after parasite proliferation inside erythrocytes and the consequence binding of infected red blood cell of the vascular endothelium cytoadherence and non-effected erthrocyte. The most common clinical symptoms of severe Malaria are high fever progression anemia multi-organ Dysfunction and unconsciousness (Miller et al., 1994).
The characteristic feature of malaria is fever caused by the release of toxin (when erythrocitic schizont ruptures which simulate the secretion of cytokines from leucocytes and other cell. The pathogenecity is mainly due to the cytoadherence of falciparum parasitized red cell causing the cell to adhere to one another and to the walls of capillaries in the brain, heart, spleen, intestine, lung's and placenta.
1.1.5 MOSQUITO VECTORS AND THE PLASMODIUM LIFE CYCLE
The parasite's primary (definitive) hosts and transmission vectors are female mosquitoes of the Anopheles genus, while humans and other vertebrates are secondary hosts. Young mosquitoes first ingest the malaria parasite by feeding on an infected human carrier and the infected Anopheles mosquitoes carry Plasmodium sporozoites in their salivary glands. A mosquito becomes infected when it takes a blood meal from an infected human. Once ingested, the parasite gametocytes taken up in the blood will further differentiate into male or female gametes and then fuse in the gametocytes taken up in the blood will further differentiate into male or female gametes and then fuse in the mosquito 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 human host. This type' of transmission is occasionally referred to as anterior station (Talman et al., 2004). The sporozoites are injected into the skin, alongside saliva, when the mosquito takes subsequent blood meal.
Only female mosquitoes feed on blood, thus males 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 (Marcurri et al., 2004).
1.1.6 PATHOGENESIS
The life of cycle of malaria parasites in the human body. A mosquito infects a person by taking a blood meal. First, sporozoites enter the bloodstream, and migrate to the liver. The infect liver cells (hepatocytes), where they multiply into merozoites, rupture the liver cells, and escape back into the bloodstream. Then, the merozoites infect red blood cells where they develop into ring forms, then trophozoites (a reproduction stage), then schizonts (a reproduction stage), then back to merozoites. Sexual forms called gametocytes are produced, which, if taken up by a mosquito, will infect the insect and continue the life cycle.
Malaria in humans develops Via two phases: an exoerythrocytic and an erythrocytic phase. The exoerythrocytic phase involves infection of the hepatic system or liver, whereas the erythrocytic phase involves infection of the erythrocytes, or red blood cells. When an Infected mosquito pierces a person's skin to take blood sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Within 30 minutes of hepatocytes, multiplying asexually and asymptomatically for a period of 6-15 days. Once 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, thus beginning the erythrocytic stage of the life cycle (Bledsoe et al., 2005). The parasite escapes from the liver undetected by 'wrapping itself in the cell membrane of the infected host liver cell (Sturm et al ., 2006).
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 cells.
Some P. vivax and P. ovale 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 and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in these two species of malaria(Cogswell et al., 1992).
The parasite is relatively protected from attack by the immune system because for most of its human life resides within the liver and blood cells and is invisible to Immune surveillance. However"
circulating infected blood cells are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays adhesive protein on the urface 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 [Clin et al., 2000). This “sickness" is the main factor giving rise to hemorrhagic complications of malaria. High endothelial venules (the smallest branches of the circulatory system) can be blocked by the attachment of masses of these infected red blood cells. The blockage of these vessels causes symptoms such as in placental and cerebral malaria. In cerebral malaria the sequestrated red blood can breach the blood brain barrier possibly leading to coma (Adams et al., 2002).
Although the red blood cell surface adhesive proteins (called PfEMP1, for Plasrnodium 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 effectively limitless versions within parasite populations (Turner et al., 2002). The parasite switches between a broad repertoire of PfEMP 1 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, thereby defining the mosquito as the definitive host of the disease. New sporozoites develop and travel to the mosquito's salivary gland, completing the cycle. Pregnant women are especially attractive to the mosquitoes, (Linds et al., 2000) and malaria in pregnant women is an important cause of stillbirths, infant mortality and low birth weight, (Sutner et al. ,2009) particularly in P. falciparum infection, but also in other species infection, such as P. vivax (Rodriguer et al., 2006).
1.1.7 SYMPTOMATIC DIAGNOSIS
Areas that cannot afford even simple laboratory dignostic tests often use only a history of subjective fever as the indication to treat for malaria. Using Giemsa-stainthat blood smears from children in Malawi, one study showed that when clinical predictors (rectal temperature, nailbed pallor, and splenomegaly) were used as treatment indications, rather than using only a history of subjective fevers, a correct diagnosis increased from 21% to 41% of cases and unnecessary treatment for malaria was
significantly decreased (Red et aI., 2006).
1.1.8 MICROSCOPIC EXAMINATION OF BLOOD FILMS
The most economic, preferred, and reliable diagnosis of malaria is microscopic examination of blood films because each of the four- major parasite species has distinguishing characteristics. Two sorts of blood film are traditionally used. Thin films are similar to usual blood films and allow species identification because the parasite's appearance is best preserved In this preparation. Thick films allow the microscopist to screen a larger volume of blood and are about eleven times more sensitive than the thin film, so picking up low levels of infection is easier on the thick film, but the appearance of the parasite is much more distorted and therefore distinguishing between the different species can be much more difficult. With the pros and cons of both thick and thin smears taken into consideration, it is imperative to utilize both smears while attempting to make a definitive diagnosis (Warhurst et al.,1996).
From the thick film, an experienced microscopist can detect parasite levels. (or parasitemia) down to as low as species look identical and it is never possible to diagnose species on the basis of a single ring form; species identification is always based on several trophozoites
Prevention
Anopheles albimanus mosquito feeding on a human arm. This mosquito is a vector of malaria and mosquito control is a very effective way of reducing the incidence of malaria.
Methods used to prevent the spread of disease, or to protect individuals in areas where malaria is endemic, include prophylactic drugs, mosquito eradication, and the prevention of mosquito bites. The continued existence of malaria in an area requires a combination of high human population density, high mosquito population density, and high rates of transmission from humans to mosquitoes and from mosquitoes to humans. If any of these is lowered sufficiently, the parasite will sooner or later disappear from that area, as happened in North America, Europe and much of Middle East. However, unless the parasite is eliminated from the whole world, it could become re-established if conditions revert to a combination that favors the parasite's reproduction. Many countries are seeing an increasing number of imported malaria cases due to extensive travel and migration.
Many researchers argue that prevention of malaria may be more cost-effective than treatment of the disease in the long run, but the capital costs required are out of reach of many of the world's poorest people. Economic adviser Jeffrey Sachs estimates that malaria can be controlled for US$3 billion in aid per year.
The distribution of funding varies among countries. Countries with large populations do not receive the same amount of support. The 34 countries that received a per capital annual support of less than $1 included some of the poorest countries in Africa.
Brazil, Eritrea, India, and Vietnam have, unlike many other developing nations, successfully reduced the malaria burden. Common success factors included conducive country conditions, a targeted technical approach using a package of effective tools, data-driven decision-making, active leadership at all levels of government, involvement of communities, decentralized implementation and control of finances, skilled technical and managerial capacity at national and sub- national levels, hands-on technical and programmatic support from partner agencies, and sufficient and flexible financing (Barat et al., 2006).
1.1.9 VECTOR CONTROL
Mosquito control
Efforts to eradicate malaria by eliminating mosquitoes have been successful in some areas. Malaria was once common in the United States and southern Europe, but vector control programs, in conjunction with the monitoring and treatment of infected humans, eliminated it from those regions. In some areas, the draining of wetland breeding grounds and better sanitation were adequate. Malaria was eliminated from the northern parts of the USA in the early 20th century by such methods, and the use of the pesticide DDT eliminated it from the South by 1951 (CDC, 2004). In 2002, there were 1,059 cases of malaria reported in the US, including eight deaths, but in only five of those cases was the disease contracted in the United States.
Before DDT, malaria was successfully eradicated or controlled also in several tropical areas by removing or poisoning the breeding grounds of the mosquitoes or the aquatic habitats of the larva stages, for example by filling or applying oil to places with standing water. These methods have seen little application in Africa for more than half a century (Killeen et al., 2002). In the 1950s and 1960s, there was a major public health effort to eradicate malaria worldwide by selectively targeting mosquitoes in areas where malaria was rampant (Gladwell et al., 2001).
However, these efforts have so far failed to eradicate malaria in many parts of the developing world-the problem is most prevalent in Africa.
Sterile insect technique is emerging as a potential mosquito control method. Progress towards transgenic, or genetically modified, ill-sects suggest that wild mosquito populations could be made malaria-resistant. Researchers at Imperial College London created the world' s first transgenic malaria mosquito. With the first plasmodium-resistant species announced by a team at Case Western Reserve University in Ohio in 2002 (Wimmer. et al., 2002). Successful replacement of current populations with a new genetically modified population, relies upon a drive mechanism, such as transposable elements to allow for non-Mendelian inheritance of the gene of interest. However, this approach contains many difficulties and success is a distant prospect (knolls et al., 2007). An even more futuristic method of vector control is the idea that lasers could be used to kill flying
mosquitoes (Robert et al.,2009).
1.1.10 PROPHYLACTIC DRUGS
Malaria prophylaxis
Several drugs, most of which are also used for treatment of malaria, can be taken preventively. Generally, these drugs are taken daily or weekly, at a lower dose than would be used for treatment of a person who had actually contracted the disease. Use of prophylactic drugs is seldom practical for full time residents of malaria-endemic areas, and their use is usually restricted to short-term visitors and travelers to malarial regions. This is due to the cost of purchasing the drugs, negative side effects from long-term use, and because some effective anti-malarial drugs are difficult to obtain outside of wealthy nations.
Quinine was used starting in the 17th century as a prophylactic against malaria. The develop men t of more effective alternatives such as quinacrine, chloroquine, and primaquine in the 20th century reduced the reliance on quinine. Today, quinine is still used to treat chloroquine resistant Plasmodium falciparum, as well as severe and cerebral stages of malaria, but is not generally used for prophylaxis.
Modern drugs used preventively include mefloquine (Lariam), doxycycline (available generically), and the combination of atovaquone and proguanil hydrochloride (Malarone). The choice of which drug to use depends on which drugs the parasites in the area are resistant to, as well as side-effects and other considerations. The prophylactic detect does not begin immediately upon starting taking the drugs, so people temporarily visiting malaria-endemic areas usually begin taking the drugs one to two weeks before arriving and must continue taking them for 4 weeks after leaving (with the exception of atovaquone proguanil that only needs be started 2 days prior and continued for 7 days afterwards) .
The use of prophylactic drugs where malaria-bearing mosquitoes are present may encourage the development of partial immunity (Roetsenberj et al., 2009).
1.2 C-REACTIVE PROTEIN
The presence of" c-reactive protein was first demonstrated In the blood of patients with acute inflammatory disease by Tillett and Francis (1930).C-reactive protein has been found to bound to aid in binding of complement proteins to damage cells (Bayazit et al., 2001).
C-reactive protein the first protein to be discovered which behaves as an acute phase reactant. This is so called because it forms a precipitate with the somatic c polysaccharide fraction of the pneumococcal pneumonia (Tillett and Francis, 1930) . .It is no specific for pneumococcal infection (Lotfali et al., 1969). Evidence suggests that the protein is synthesized in the liver, primarily in hepatocytes. In response to injury, local inflammatory cells (neutrophils granulocyte and macrophage secrete a number of cytokines into the blood stream. Most notable of which are the interleukins IL-1, IL-6 and IL-8 and tumour necrosis factor (TNF-a).In the body c-reactive protein plays an important role of interacting with the complement system- and immunologic defense. C-reactive protein acts as one of the main diagnostic markers for inflammation (Wright et al, 1997). Synthesis of C-reactive protein and others acute phase proteins by hepatocytes is modulated by cytokines. Intereukin 1b and 6 and TNF -a are the most important regulators of crp synthesis. The function of crp is felt to be related to its role in the innate system (Duclos, Terry 2000).Similary to immunoglobulin, it activates complement, binds to Fe receptors and acts and acts as opsonin for various pathogens. Interaction of crp with Fe receptors leads to the generation of pro-inflammatory cytokines that enhance the inflammatory response.
C-reactive protein IS a sensitive indicator of the early phase of inflammatory or tissue process. C-reactive protein testing has been allocated for the assessment of general well being in both human and veterinary studies.
1.3 TUMOUR NECROSIS FACTOR
In 1970, researchers took sarcoma cells in culture and exposed them to a protein produced by white blood cells .The protein caused necrosis (death) of the sarcoma cells but had little effect on normal cells in the culture. Hence the protein was called "tumour necrosis factor"
TNF-a was formerly known as cachetin, cytotoxic factor differentiation including factor haemorrhagic factor, macrophage cytotoxin and necrosis. It is a cytokine involved in systemic inflammation and it is a member of a group of cytokines that stimulate the acute phase reaction. The primary role is in regulation of immune cells. (Abbas et aI., 1994).
TNF -Q is produced primarily by activated monocytes and macrophages and to a lesser extend by others cells type such as activated T cells ,B cells , NK (non killers cells).
(Gallard et al,.200 1). TNF-Q are mediators of both specific and non-specific biological responses and an important link between immune and inflammatory reactions. TNF-Q is released in response to bacteria (endotoxin) and produced within some bacteria that is released only when the bacteria disintegrate. TNF -Q is the principal mediator of the host response and may play role in multiple biological activities anti-parasitic, antiviral etc (Maciazet et al., 1997). TNF-Q is also have wide spread effects in inflammation and healing and are in involved in granuloma formation and fibrosis in many organ system and when produced in excess vascular shock (Playfair 1996).
Although TNF is valuable in killing cells in myeloma and sarcoma tumour. It can promote growth of others kind of cancers, therefore the action of TNF is continually under the research with the hope increasing its effectiveness on killing cancer while decreasing the toxic side effects on healthy tissue.
ANTISTREPTOLYSIN 0 (ASO)
This is also known as ASO. It is Antistreptolysin O. This test helps to determine whether a person has had a recent group A streptococcal infection; to' help diagnose post streptococcal sequelae of rheumatic fever and erulonephitis.
This test (ASO) IS carried out when a patient has symptoms of rheumatic fever (cardiac function abnormalities) symptoms suggestive of a streptococcal infection but no culture was done to confirm a Group A streptococcal infection. In most cases streptococcal infections are identified and treated with antibiotics and the infections resolve. In case where they do not cause identifiable symptoms and/or go untreated, however post-streptococcal sequelae, namely rheumatic fever and gtomerulonephrities, can develop in some patients especially young children. The ASO test, therefore is ordered if a patient presents with symptoms suggesting either of these conditions.
The ASO test is ordered when a patient has symptoms that the doctor suspects may be due to an illness caused by previous streptococcal infection. It is ordered when the symptoms emerge, usually in the weeks following a sore throat or skin infection. The test may be ordered twice over a period of 10 - 14 days to determine if the antibody level is rising, falling, or remaining the same.
The ASO "'test results can be reported In several ways, however, the interpretation is generally the same: the higher the result, the more antibody that is present in the blood (unless a titre is performed, which is a ratio and therefore is interpreted differently).
The ASO antibody is either absent or present in very low concentrations in patients who have not had a recent streptococcal (strep) infection. Antibodies are produced about a week to a month after the initial streptococcal infection. ASO levels peak at about 4 to 6 weeks after the illness and then taper off but may remain at detectible levels for several months after the strep infection has resolved.
If the test is negative or if ASO is present in very low concentrations, then the patient most likely has not had a recent streptococcal infection. This is especially true if a sample taken 10 to 14 days later is also negative minimal.
If the ASO level is high or is rising, then it is likely that a recent streptococcal infection has occurred. ASO levels that re initially high and then decline suggest that an infection has occurred and may be resolving.
The ASO test does not predict if complications will occur following a streptococcal infection, nor do they predict the severity of the disease. If symptoms of rheumatic fever or glomerulonephrities are present, an elevated ASO level may be used to confirm the diagnosis.
1-5 AIMS
To determine the levels of acute phase reactant proteins especially C-reactive protein (CRP) and antistreptolysin O (ASO) in Nigerians with malaria infection.
1-6 SPECIFIC OBJECTIVE
To examine the response of this proteins during and post-treated malaria. With a view of adopting them as markers of:-
1. Diseases severity
2. Prognosis
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